Methylation Mechanisms in Embryonic Period

**21**

**Chapter 2**

**Abstract**

Demethylation in Early Embryonic

DNA repair processes arose early in evolution. During evolution, DNA base excision repair apparently acquired additional roles in demethylation of cytosines in DNA. Demethylation is central to two mammalian fundamental processes. Embryonic reprogramming and neuronal memory require rapid gene expression alterations depending in part on demethylations. The active demethylation reactions in both processes primarily depend, first, on the family of 5-methylcytosine oxidases sharing the acronym ten-eleven translocation (TET methylcytosine dioxygenases) and, second, on DNA base excision repair enzymes. In mice, within 6 h of fertilization, the paternal chromosomes are close to 100% actively demethylated through TET and repair activity. (Methylation of maternal DNA is blocked during subsequent cycles of replication, so methyl groups on maternal DNA, passively, becomes highly diluted over the next 4 days.) Rats subjected to one instance of contextual fear conditioning create an especially strong long-term memory. At 24 h after training, 9.2% of the genes in the rat genomes of hippocampus neurons are differentially methylated, including over 500 genes with demethylation. The emergence of embryonic development in evolution depended on preexisting DNA methylation/demethylation pathways to modify gene expression. The further emergence of memory likely evolved from the earlier set of methylation/demethylation

Development and Memory

*Carol Bernstein and Harris Bernstein*

capabilities associated with embryonic development.

neuroepigenetics, neurogenesis, brain evolution

**1. Introduction**

**Keywords:** TET enzymes, OGG1, epigenetic, base excision repair, DNA repair,

DNA repair processes have a central role in epigenetic demethylation reactions that are employed in both early embrylonic development and in memory. DNA likely emerged as the genetic material as long as 3.5 billion years ago [1]. From its inception as the genetic material, DNA was likely subject to damage. In present day organisms damage to DNA is frequent and occurs due to both metabolic and hydrolytic processes [2] as well as a result of environmental agents such as UV light and ionizing radiation. Thus, enzymes promoting DNA repair likely have been retained based on their adaptive benefit since early evolution. Currently, in humans, about 169 different DNA repair proteins have been identified [3]. During the course of evolution, many of these DNA repair proteins developed more than one enzymatic capability. For instance, at least 17 DNA repair proteins act in both a DNA repair pathway and in an apoptosis pathway [4]. These dual role proteins are required

#### **Chapter 2**

## Demethylation in Early Embryonic Development and Memory

*Carol Bernstein and Harris Bernstein*

#### **Abstract**

DNA repair processes arose early in evolution. During evolution, DNA base excision repair apparently acquired additional roles in demethylation of cytosines in DNA. Demethylation is central to two mammalian fundamental processes. Embryonic reprogramming and neuronal memory require rapid gene expression alterations depending in part on demethylations. The active demethylation reactions in both processes primarily depend, first, on the family of 5-methylcytosine oxidases sharing the acronym ten-eleven translocation (TET methylcytosine dioxygenases) and, second, on DNA base excision repair enzymes. In mice, within 6 h of fertilization, the paternal chromosomes are close to 100% actively demethylated through TET and repair activity. (Methylation of maternal DNA is blocked during subsequent cycles of replication, so methyl groups on maternal DNA, passively, becomes highly diluted over the next 4 days.) Rats subjected to one instance of contextual fear conditioning create an especially strong long-term memory. At 24 h after training, 9.2% of the genes in the rat genomes of hippocampus neurons are differentially methylated, including over 500 genes with demethylation. The emergence of embryonic development in evolution depended on preexisting DNA methylation/demethylation pathways to modify gene expression. The further emergence of memory likely evolved from the earlier set of methylation/demethylation capabilities associated with embryonic development.

**Keywords:** TET enzymes, OGG1, epigenetic, base excision repair, DNA repair, neuroepigenetics, neurogenesis, brain evolution

#### **1. Introduction**

DNA repair processes have a central role in epigenetic demethylation reactions that are employed in both early embrylonic development and in memory. DNA likely emerged as the genetic material as long as 3.5 billion years ago [1]. From its inception as the genetic material, DNA was likely subject to damage. In present day organisms damage to DNA is frequent and occurs due to both metabolic and hydrolytic processes [2] as well as a result of environmental agents such as UV light and ionizing radiation. Thus, enzymes promoting DNA repair likely have been retained based on their adaptive benefit since early evolution. Currently, in humans, about 169 different DNA repair proteins have been identified [3]. During the course of evolution, many of these DNA repair proteins developed more than one enzymatic capability. For instance, at least 17 DNA repair proteins act in both a DNA repair pathway and in an apoptosis pathway [4]. These dual role proteins are required

for DNA repair when DNA damages are at relatively low levels but are active and required for apoptosis when DNA damages are at high levels.

In addition to the multiple roles of some DNA repair proteins, some endogenously produced DNA damaging agents also appear to have multiple roles. Reactive oxygen species (ROS) are produced by mitochondria during oxidative metabolism, and a small proportion are released from the mitochondria and interact with proteins, lipids and DNA to alter their structures. ROS can damage DNA in ways that are mutagenic or disruptive to expression. Thus, excessive ROS can cause mutations and other alterations leading to cancer [5]. However, ROS can interact with DNA to serve important positive roles. A large body of literature has shown the necessary roles of appropriate levels of ROS in embryonic development [6, 7] and in learning and memory [8, 9].

#### **2. Demethylation in embryogenesis**

During early embryogenesis of mammals, pathways of rapid demethylation are employed at multiple DNA sites to form totipotent cells. Subsequently, locally deposited methylations enable formation of subsets of cells that became specialized tissue types, such as primordial germ cells and neuronal stem cells [10]. Such rapid demethylations and subsequent methylations have also now been found to occur in the formation of memories and learning [11] and in both cases the mechanism of methyl group removal occurs by similar pathways involving TET enzymes and base excision DNA repair.

In embryogenesis, rapid and large scale demethylations occur at two stages [12]. One extensive set of demethylations occurs within a few hours after the sperm enters the egg, forming the zygote. Almost all methyl groups are removed from the paternal-origin chromosomes within 6 h of forming the zygote, before any replication has occurred [13]. Another extensive demethylation occurs early in embryogenesis, in the nuclei of the primordial germ cells shortly after they devolve from the other cells which are forming somatic tissues [14]. This stage of demethylation occurs in two phases. There is a first phase of rapid proliferation without methylation, causing dilution of methylation with a loss of methylation at almost all genomic sequences. Then there is a second phase, involving specific sites including germ-line and meiosis specific genes, where the demethylation is active and proceeds by pathways involving TET enzymes and base excision DNA repair.

Methylation of sites (which can be demethylated) in mammalian DNA are usually restricted to cytosines, forming 5-methylcytosine (5mC) (**Figure 1**). In this figure, the addition of a methyl group at the 5 position of cytosine is shown within a red oval. Of all the cytosines in DNA, the 5mCs occur primarily at "CpG" sites [16]. A CpG site is where a cytosine in a DNA strand is followed by a guanine nucleotide in the linear sequence of bases along the 5′ to 3′ direction. There are 28 million CpG sites in the human genome [17]. In humans, about 60% of the 28 million CpG sites are methylated in most somatic tissues [18]. CG dinucleotides (CpG sites) represent about 1% of total bases in the mammalian genome [19]. Three DNA methyltransferases in humans can methylate a base in DNA. These enzymes show a strong preference for methylating cytosines in CpG sites [20].

Mouse DNA is very similar to human DNA, with about 99% of mouse genes having a homolog in the human genome, and mice and humans having about the same number of genes [21]. However, the mouse sequence is about 14% shorter than the human sequence [21]. The mature mouse sperm genome has 80–90% overall methylation of its CpG sites, the highest global DNA methylation level of

**23**

**Figure 2.**

*Methylation levels during mouse early embryonic development.*

*Demethylation in Early Embryonic Development and Memory*

any cell in the mouse [12]. Because of its shorter sequence, we can speculate that there may be fewer than 28 million CpG sites in the mouse genome, perhaps 86% as many as in the human genome, or about 24 million CpG sites. Thus, of the likely 24 million CpG sites, there are about 19–22 million methylated sites in mouse sperm DNA. In mouse zygotes, partial demethylation of the paternal nucleus is already evident 3 h after formation of the zygote [13]. By 6 h, demethylation of the paternal nucleus appears to be complete (**Figure 2**). During the subsequent first mitosis, there is just a small but significant residual methylation signal in some but not all of the paternally derived chromosomes [13]. By 3–4 days after fertilization, after replication to generate 16 cells, the embryo has formed a morula (a round body of cells with no differentiation) (**Figure 2**). By this time both the paternal and maternal chromosomes have mixed together in a single nuclear area and all have very low levels of methylation (In **Figure 2**, the methylation levels of the paternal and maternal chromosome are approximately represented by the blue lines during the period they can be distinguished. When the chromosomes become mixed, after two mitoses, the methylation level of the mixed chromosomes is represented by a

The almost compete demethylation of the zygote DNA in the paternal chromosomes at 22–25 million CpG sites occurs before any DNA replication. Thus, it occurs by an active process not connected to replication. The demethylation of the maternal chromosomes appears to largely take place by blockage of the methylating enzymes from acting on maternal-origin DNA and dilution of the methylated

*DNA methylation most often is the addition of a methyl group to cytosine in DNA. The image shows cytosine and 5-methylcytosine. In mammals, DNA methylation most frequently occurs at a cytosine followed by guanine* 

*DOI: http://dx.doi.org/10.5772/intechopen.90306*

brown line).

**Figure 1.**

*in the DNA [15].*

#### *Demethylation in Early Embryonic Development and Memory DOI: http://dx.doi.org/10.5772/intechopen.90306*

any cell in the mouse [12]. Because of its shorter sequence, we can speculate that there may be fewer than 28 million CpG sites in the mouse genome, perhaps 86% as many as in the human genome, or about 24 million CpG sites. Thus, of the likely 24 million CpG sites, there are about 19–22 million methylated sites in mouse sperm DNA. In mouse zygotes, partial demethylation of the paternal nucleus is already evident 3 h after formation of the zygote [13]. By 6 h, demethylation of the paternal nucleus appears to be complete (**Figure 2**). During the subsequent first mitosis, there is just a small but significant residual methylation signal in some but not all of the paternally derived chromosomes [13]. By 3–4 days after fertilization, after replication to generate 16 cells, the embryo has formed a morula (a round body of cells with no differentiation) (**Figure 2**). By this time both the paternal and maternal chromosomes have mixed together in a single nuclear area and all have very low levels of methylation (In **Figure 2**, the methylation levels of the paternal and maternal chromosome are approximately represented by the blue lines during the period they can be distinguished. When the chromosomes become mixed, after two mitoses, the methylation level of the mixed chromosomes is represented by a brown line).

The almost compete demethylation of the zygote DNA in the paternal chromosomes at 22–25 million CpG sites occurs before any DNA replication. Thus, it occurs by an active process not connected to replication. The demethylation of the maternal chromosomes appears to largely take place by blockage of the methylating enzymes from acting on maternal-origin DNA and dilution of the methylated

#### **Figure 1.**

*DNA Methylation Mechanism*

and memory [8, 9].

excision DNA repair.

**2. Demethylation in embryogenesis**

for DNA repair when DNA damages are at relatively low levels but are active and

In addition to the multiple roles of some DNA repair proteins, some endogenously produced DNA damaging agents also appear to have multiple roles. Reactive oxygen species (ROS) are produced by mitochondria during oxidative metabolism, and a small proportion are released from the mitochondria and interact with proteins, lipids and DNA to alter their structures. ROS can damage DNA in ways that are mutagenic or disruptive to expression. Thus, excessive ROS can cause mutations and other alterations leading to cancer [5]. However, ROS can interact with DNA to serve important positive roles. A large body of literature has shown the necessary roles of appropriate levels of ROS in embryonic development [6, 7] and in learning

During early embryogenesis of mammals, pathways of rapid demethylation are employed at multiple DNA sites to form totipotent cells. Subsequently, locally deposited methylations enable formation of subsets of cells that became specialized tissue types, such as primordial germ cells and neuronal stem cells [10]. Such rapid demethylations and subsequent methylations have also now been found to occur in the formation of memories and learning [11] and in both cases the mechanism of methyl group removal occurs by similar pathways involving TET enzymes and base

In embryogenesis, rapid and large scale demethylations occur at two stages [12]. One extensive set of demethylations occurs within a few hours after the sperm enters the egg, forming the zygote. Almost all methyl groups are removed from the paternal-origin chromosomes within 6 h of forming the zygote, before any replication has occurred [13]. Another extensive demethylation occurs early in embryogenesis, in the nuclei of the primordial germ cells shortly after they devolve from the other cells which are forming somatic tissues [14]. This stage of demethylation occurs in two phases. There is a first phase of rapid proliferation without methylation, causing dilution of methylation with a loss of methylation at almost all genomic sequences. Then there is a second phase, involving specific sites including germ-line and meiosis specific genes, where the demethylation is active and proceeds by pathways involving TET enzymes and base excision DNA

Methylation of sites (which can be demethylated) in mammalian DNA are usually restricted to cytosines, forming 5-methylcytosine (5mC) (**Figure 1**). In this figure, the addition of a methyl group at the 5 position of cytosine is shown within a red oval. Of all the cytosines in DNA, the 5mCs occur primarily at "CpG" sites [16]. A CpG site is where a cytosine in a DNA strand is followed by a guanine nucleotide in the linear sequence of bases along the 5′ to 3′ direction. There are 28 million CpG sites in the human genome [17]. In humans, about 60% of the 28 million CpG sites are methylated in most somatic tissues [18]. CG dinucleotides (CpG sites) represent about 1% of total bases in the mammalian genome [19]. Three DNA methyltransferases in humans can methylate a base in DNA. These enzymes show a strong prefer-

Mouse DNA is very similar to human DNA, with about 99% of mouse genes having a homolog in the human genome, and mice and humans having about the same number of genes [21]. However, the mouse sequence is about 14% shorter than the human sequence [21]. The mature mouse sperm genome has 80–90% overall methylation of its CpG sites, the highest global DNA methylation level of

ence for methylating cytosines in CpG sites [20].

required for apoptosis when DNA damages are at high levels.

**22**

repair.

*DNA methylation most often is the addition of a methyl group to cytosine in DNA. The image shows cytosine and 5-methylcytosine. In mammals, DNA methylation most frequently occurs at a cytosine followed by guanine in the DNA [15].*

**Figure 2.** *Methylation levels during mouse early embryonic development.*

maternal DNA during replication. At the second metaphase after fertilization, maternal chromosomes showed methylation on only one of the two sister chromatids. This sister chromatid differentiation is consistent with mostly replicationdependent passive maternal chromosome demethylation [22]. Consequently, four-cell embryos have a much lower methylation density over the maternal nuclear compartment. Methylation of the maternal genome further decreases with every additional replication cycle. The morula (at the 16 cell stage), overall, has much reduced methylation of DNA.

High levels of *de novo* DNA methylation then occur in the cells of the inner cell mass of the blastocyst, to establish the specific methylation patterns of principal cell lineages in the early embryo [13]. Afterwards, by day 5 of mouse embryogenesis, the epiblast is formed, followed by implantation of the epiblast in the uterine epithelium (**Figure 2**). By day seven after fertilization, the newly formed primordial germ cells (PGC) in the implanted embryo devolve from the remaining somatic cells. At this point the PGCs have high levels of methylation. These cells migrate from the epiblast along the hindgut toward the genital ridges starting about day 7.8. By day 8.5 they are rapidly proliferating and beginning demethylation in two waves. In the first wave, demethylation is by replicative dilution, but in the second wave demethylation is by an active process. The second wave, during days 9.5–13.5, leads to demethylation of specific loci. At day 13.5, the PGC genomes display the lowest levels of DNA methylation of any cells of the mouse in the entire life cycle [14].

#### **2.1 Mechanisms of demethylation**

The demethylation of methylated CpG sites of DNA occurs in three stages: (1) recruitment of a TET enzyme to initiate demethylation (although there is one minor mechanism that does not utilize a TET enzyme); (2) intermediate steps of oxidation or oxidative deamination (forming intermediate products of demethylation); and (3) culminating steps of DNA base excision repair resulting in final replacement of 5-methylcytosine with cytosine.

The pathways by which demethylation can occur [23] are shown in outline in **Figure 3**. This figure indicates two types of oxidation reactions that may occur in demethylation. One occurs by oxidation of the added methyl group at the 5 position of cytosine. The other occurs through oxidative deamination of the amine group at the 4 position of cytosine. The pathway on the left depends on oxidation of each of the adducts on the 5 position of cytosine, sequentially, by a TET enzyme, followed by action of base excision repair (BER) enzymes. TET enzymes (ten-eleven translocation methylcytosine dioxygenases) oxidize adducts on cytosine in an iron and alpha-ketoglutarate dependent process. This TET-type dependent pathway likely carries out the bulk of the demethylations discussed here. However, as reviewed [25], two other pathways involving AID/APOBEC and base excision repair enzymes can occur. In one pathway there is an initial TET reaction. The other pathway involving AID/APOBEC results in oxidative deamination of 5mC directly to thymine followed by base excision repair. The activity of AID/APOBEC appears to cooperate with a TET enzyme in neuronal functions [26]. It is notable that demethylation, in all its pathways, employs the enzymes of the base excision repair pathway.

#### **3. Base excision repair**

In **Figure 3**, base excision repair is indicated by the highlighted acronym "BER". To complete the description of the mechanism shown above, we include a

**25**

**Figure 3.**

ligase to seal the strand.

remain in most steps of this diagram.

*Demethylation in Early Embryonic Development and Memory*

diagram illustrating the base excision repair pathway used in the latter stages of the conversion of 5mC to C (**Figure 4**). In this diagram the two strands of DNA are represented by parallel horizontal lines. With the first downward arrow we show thymine DNA glycosylase (TDG) removing 5-formylcytosine (5fC) from the DNA backbone, leaving an *apyrimidinic site.* Then AP endonuclease cleaves the 5′ deoxyribose-phosphate in the DNA backbone of a single strand, leaving a 3′ hydroxy end and a 5′ deoxyribose phosphate end (second downward arrow). This is followed by either short patch or long patch repair. In short patch repair, 5′ dRP lyase trims the 5′ dRP end to form a phosphorylated 5′ end. This is followed by DNA polymerase β adding a single cytosine to pair with the pre-existing guanine in the complementary strand and then DNA ligase to seal the cut strand. In long patch repair, DNA synthesis is thought to be mediated by polymerase δ and polymerase ε performing displacing synthesis to form a flap. Pol β can also perform long-patch displacing synthesis. Long-patch synthesis typically inserts 2–10 new nucleotides. Then flap endonuclease removes the flap, and this is followed by DNA

*excision repair (BER) enzymes to yield cytosine (Cyt). Figure by [24].*

*Demethylation of 5-Methylcytosine (5mC) in neuron DNA. As reviewed in [23], in brain neurons 5mC is oxidized by the ten-eleven translocation (TET) family of dioxygenases (TET1, TET2, TET3) to generate 5-hydroxymethylcytosine (5hmC). In successive steps TET enzymes further hydroxylate 5hmC to generate 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Thymine-DNA glycosylase (TDG) recognizes the intermediate bases 5fC and 5caC and excises the glycosidic bond resulting in an apyrimidinic site (AP site). In an alternative oxidative deamination pathway, 5hmC can be oxidatively deaminated by activityinduced cytidine deaminase/apolipoprotein B mRNA editing complex (AIP/APOBEC) deaminases to form 5-hydroxymethyluracil (5hmU) or 5mC can be converted to thymine (Thy). 5hmU can be cleaved by TDG, single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), Nei-Like DNA Glycosylase 1 (NEIL1), or methyl-CpG binding protein 4 (MBD4). AP sites and T:G mismatches are then repaired by base* 

In an example below (see "Targeting TET to 5-methylcytosine") we show that, in at least one well documented case, the ROS-induced damage of 8-OHdG at a CpG site initiates demethylation. In the base excision pathways shown in **Figure 4**, it is not clear at what stage 8-OHdG itself may be removed. Thus, 8-OHdG is allowed to

*DOI: http://dx.doi.org/10.5772/intechopen.90306*

*Demethylation in Early Embryonic Development and Memory DOI: http://dx.doi.org/10.5772/intechopen.90306*

#### **Figure 3.**

*DNA Methylation Mechanism*

reduced methylation of DNA.

**2.1 Mechanisms of demethylation**

5-methylcytosine with cytosine.

**3. Base excision repair**

maternal DNA during replication. At the second metaphase after fertilization, maternal chromosomes showed methylation on only one of the two sister chromatids. This sister chromatid differentiation is consistent with mostly replicationdependent passive maternal chromosome demethylation [22]. Consequently, four-cell embryos have a much lower methylation density over the maternal nuclear compartment. Methylation of the maternal genome further decreases with every additional replication cycle. The morula (at the 16 cell stage), overall, has much

High levels of *de novo* DNA methylation then occur in the cells of the inner cell mass of the blastocyst, to establish the specific methylation patterns of principal cell lineages in the early embryo [13]. Afterwards, by day 5 of mouse embryogenesis, the epiblast is formed, followed by implantation of the epiblast in the uterine epithelium (**Figure 2**). By day seven after fertilization, the newly formed primordial germ cells (PGC) in the implanted embryo devolve from the remaining somatic cells. At this point the PGCs have high levels of methylation. These cells migrate from the epiblast along the hindgut toward the genital ridges starting about day 7.8. By day 8.5 they are rapidly proliferating and beginning demethylation in two waves. In the first wave, demethylation is by replicative dilution, but in the second wave demethylation is by an active process. The second wave, during days 9.5–13.5, leads to demethylation of specific loci. At day 13.5, the PGC genomes display the lowest levels of DNA methylation of any cells of the mouse in the entire life cycle [14].

The demethylation of methylated CpG sites of DNA occurs in three stages: (1) recruitment of a TET enzyme to initiate demethylation (although there is one minor mechanism that does not utilize a TET enzyme); (2) intermediate steps of oxidation or oxidative deamination (forming intermediate products of demethylation); and (3) culminating steps of DNA base excision repair resulting in final replacement of

The pathways by which demethylation can occur [23] are shown in outline in **Figure 3**. This figure indicates two types of oxidation reactions that may occur in demethylation. One occurs by oxidation of the added methyl group at the 5 position of cytosine. The other occurs through oxidative deamination of the amine group at the 4 position of cytosine. The pathway on the left depends on oxidation of each of the adducts on the 5 position of cytosine, sequentially, by a TET enzyme, followed by action of base excision repair (BER) enzymes. TET enzymes (ten-eleven translocation methylcytosine dioxygenases) oxidize adducts on cytosine in an iron and alpha-ketoglutarate dependent process. This TET-type dependent pathway likely carries out the bulk of the demethylations discussed here. However, as reviewed [25], two other pathways involving AID/APOBEC and base excision repair enzymes can occur. In one pathway there is an initial TET reaction. The other pathway

involving AID/APOBEC results in oxidative deamination of 5mC directly to thymine followed by base excision repair. The activity of AID/APOBEC appears to cooperate with a TET enzyme in neuronal functions [26]. It is notable that demethylation, in

all its pathways, employs the enzymes of the base excision repair pathway.

In **Figure 3**, base excision repair is indicated by the highlighted acronym "BER". To complete the description of the mechanism shown above, we include a

**24**

*Demethylation of 5-Methylcytosine (5mC) in neuron DNA. As reviewed in [23], in brain neurons 5mC is oxidized by the ten-eleven translocation (TET) family of dioxygenases (TET1, TET2, TET3) to generate 5-hydroxymethylcytosine (5hmC). In successive steps TET enzymes further hydroxylate 5hmC to generate 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Thymine-DNA glycosylase (TDG) recognizes the intermediate bases 5fC and 5caC and excises the glycosidic bond resulting in an apyrimidinic site (AP site). In an alternative oxidative deamination pathway, 5hmC can be oxidatively deaminated by activityinduced cytidine deaminase/apolipoprotein B mRNA editing complex (AIP/APOBEC) deaminases to form 5-hydroxymethyluracil (5hmU) or 5mC can be converted to thymine (Thy). 5hmU can be cleaved by TDG, single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), Nei-Like DNA Glycosylase 1 (NEIL1), or methyl-CpG binding protein 4 (MBD4). AP sites and T:G mismatches are then repaired by base excision repair (BER) enzymes to yield cytosine (Cyt). Figure by [24].*

diagram illustrating the base excision repair pathway used in the latter stages of the conversion of 5mC to C (**Figure 4**). In this diagram the two strands of DNA are represented by parallel horizontal lines. With the first downward arrow we show thymine DNA glycosylase (TDG) removing 5-formylcytosine (5fC) from the DNA backbone, leaving an *apyrimidinic site.* Then AP endonuclease cleaves the 5′ deoxyribose-phosphate in the DNA backbone of a single strand, leaving a 3′ hydroxy end and a 5′ deoxyribose phosphate end (second downward arrow). This is followed by either short patch or long patch repair. In short patch repair, 5′ dRP lyase trims the 5′ dRP end to form a phosphorylated 5′ end. This is followed by DNA polymerase β adding a single cytosine to pair with the pre-existing guanine in the complementary strand and then DNA ligase to seal the cut strand. In long patch repair, DNA synthesis is thought to be mediated by polymerase δ and polymerase ε performing displacing synthesis to form a flap. Pol β can also perform long-patch displacing synthesis. Long-patch synthesis typically inserts 2–10 new nucleotides. Then flap endonuclease removes the flap, and this is followed by DNA ligase to seal the strand.

In an example below (see "Targeting TET to 5-methylcytosine") we show that, in at least one well documented case, the ROS-induced damage of 8-OHdG at a CpG site initiates demethylation. In the base excision pathways shown in **Figure 4**, it is not clear at what stage 8-OHdG itself may be removed. Thus, 8-OHdG is allowed to remain in most steps of this diagram.

**Figure 4.**

*An example of base excision repair of 5-formylcytosine (5fC) (adjacent to 8-OHG, an oxidized guanine) by short patch repair or long patch repair.*

#### **4. TET enzymes**

As described by Jin et al. [27] and Melamed et al. [28], there are a number of TET enzymes, including at least two isoforms of TET1, one of TET2 and three isoforms of TET3. As reviewed [28], the full-length canonical TET1 isoform appears virtually restricted to early embryos, embryonic stem cells and PGCs. The dominant TET1 isoform in most somatic tissues, at least in the mouse, arises from alternative promoter usage which gives rise to a short transcript and a truncated protein designated TET1s. The isoforms of TET3 are the full length form TET3FL, a short form splice variant TET3s, and a form that occurs in oocytes and neurons designated TET3o. TET3o is created by alternative promoter use and contains an additional first N-terminal exon coding for 11 amino acids. TET3o only occurs in oocytes and neurons and was not expressed in embryonic stem cells or in any other cell type or adult mouse tissue tested [27]. Whereas TET1 expression can barely be detected in oocytes and zygotes, and TET2 is only moderately expressed, the TET3 variant TET3o shows extremely high levels of expression in oocytes and zygotes, but is nearly absent at the 2-cell stage [29].

The TET enzymes generally do not specifically bind to 5-methylcytosine except under particular conditions, such as the two conditions described below, in "Targeting TET1 to 5-methylcytosine" and in "TET in learning and memory." Without targeting, TET1 predominantly binds to high CG promoters and CpG islands (CGIs) genome-wide by its CXXC domain that can recognize un-methylated CGIs [30]. TET2 does not have an affinity for 5-methylcytosine in DNA [31]. The CXXC domain of the full-length TET3, which is the predominant form expressed in neurons, binds most strongly to CpGs modified by 5-carboxycytosine (5caC) (**Figure 3**), although it does also bind to un-methylated CpGs [28].

**27**

**Figure 5.**

*Demethylation in Early Embryonic Development and Memory*

One mode of recruitment of a TET enzyme to 5-methylcytosine in DNA, in order to initiate demethylation, was investigated by Zhou et al. [32]. In this mode, recruitment was found to depend on ROS treatment of cells. This finding is significant because appropriate levels of ROS are known to be needed in both embryogenesis [6, 7] and in learning and memory [8, 9]. ROS cause oxidative damages to DNA, but these damages are not random. Because electron "hole" pausing at the sites of the lowest ionization potential increases the probability of stable adduct formation, DNA oxidation tends to be sequence dependent [19]. As reviewed by Ming et al. [19], cytosine methylation increases the reactivity of guanine bases in 5mCpG dinucleotides toward electrophiles and oxidants. This is likely due to the transmission of an electronic effect from the 5mC to its partner guanine through hydrogen bonding within the 5mC:G base pair. Ming et al. [19] experimentally showed that oxidation of guanines was enhanced within endogenously methylated 5mCpG

There are many types of oxidative DNA damage, but the most common endogenous oxidative damage in DNA is 8-OHdG [33]. The molecular structure of 8-OHdG is shown as part of **Figure 5**. In **Figure 5**, the structure labeled in red as "8-OHdG" is a guanine with the oxidative damage, an added OH group at the 8 position of the pentane (5-sided) ring, shown in red. 8-OHdG can be experimentally increased in cells by treatment with Hoechst dye followed by micro-irradiation with 405 nm light [34]. The irradiation can be performed along a narrow line.

*Initiation of DNA demethylation at a CpG site. In adult somatic cells DNA methylation typically occurs in the context of CpG dinucleotides (CpG sites), forming 5-methylcytosine-pG, or 5mCpG. Reactive oxygen species (ROS) may attack guanine at the dinucleotide site, forming 8-hydroxy-2*′*-deoxyguanosine (8-OHdG), and resulting in a 5mCp-8-OHdG dinucleotide site. The base excision repair enzyme OGG1 targets 8-OHdG and binds to the lesion without immediate excision. OGG1, present at a 5mCp-8-OHdG site recruits TET1 and TET1* 

*oxidizes the 5mC adjacent to the 8-OHdG. This initiates demethylation of 5mC [37].*

*DOI: http://dx.doi.org/10.5772/intechopen.90306*

**4.1 Targeting TET to 5-methylcytosine**

dinucleotides.

#### **4.1 Targeting TET to 5-methylcytosine**

*DNA Methylation Mechanism*

**4. TET enzymes**

*short patch repair or long patch repair.*

**Figure 4.**

but is nearly absent at the 2-cell stage [29].

As described by Jin et al. [27] and Melamed et al. [28], there are a number of TET enzymes, including at least two isoforms of TET1, one of TET2 and three isoforms of TET3. As reviewed [28], the full-length canonical TET1 isoform appears virtually restricted to early embryos, embryonic stem cells and PGCs. The dominant TET1 isoform in most somatic tissues, at least in the mouse, arises from alternative promoter usage which gives rise to a short transcript and a truncated protein designated TET1s. The isoforms of TET3 are the full length form TET3FL, a short form splice variant TET3s, and a form that occurs in oocytes and neurons designated TET3o. TET3o is created by alternative promoter use and contains an additional first N-terminal exon coding for 11 amino acids. TET3o only occurs in oocytes and neurons and was not expressed in embryonic stem cells or in any other cell type or adult mouse tissue tested [27]. Whereas TET1 expression can barely be detected in oocytes and zygotes, and TET2 is only moderately expressed, the TET3 variant TET3o shows extremely high levels of expression in oocytes and zygotes,

*An example of base excision repair of 5-formylcytosine (5fC) (adjacent to 8-OHG, an oxidized guanine) by* 

The TET enzymes generally do not specifically bind to 5-methylcytosine except under particular conditions, such as the two conditions described below, in "Targeting TET1 to 5-methylcytosine" and in "TET in learning and memory." Without targeting, TET1 predominantly binds to high CG promoters and CpG islands (CGIs) genome-wide by its CXXC domain that can recognize un-methylated CGIs [30]. TET2 does not have an affinity for 5-methylcytosine in DNA [31]. The CXXC domain of the full-length TET3, which is the predominant form expressed in neurons, binds most strongly to CpGs modified by 5-carboxycytosine (5caC)

(**Figure 3**), although it does also bind to un-methylated CpGs [28].

**26**

One mode of recruitment of a TET enzyme to 5-methylcytosine in DNA, in order to initiate demethylation, was investigated by Zhou et al. [32]. In this mode, recruitment was found to depend on ROS treatment of cells. This finding is significant because appropriate levels of ROS are known to be needed in both embryogenesis [6, 7] and in learning and memory [8, 9]. ROS cause oxidative damages to DNA, but these damages are not random. Because electron "hole" pausing at the sites of the lowest ionization potential increases the probability of stable adduct formation, DNA oxidation tends to be sequence dependent [19]. As reviewed by Ming et al. [19], cytosine methylation increases the reactivity of guanine bases in 5mCpG dinucleotides toward electrophiles and oxidants. This is likely due to the transmission of an electronic effect from the 5mC to its partner guanine through hydrogen bonding within the 5mC:G base pair. Ming et al. [19] experimentally showed that oxidation of guanines was enhanced within endogenously methylated 5mCpG dinucleotides.

There are many types of oxidative DNA damage, but the most common endogenous oxidative damage in DNA is 8-OHdG [33]. The molecular structure of 8-OHdG is shown as part of **Figure 5**. In **Figure 5**, the structure labeled in red as "8-OHdG" is a guanine with the oxidative damage, an added OH group at the 8 position of the pentane (5-sided) ring, shown in red. 8-OHdG can be experimentally increased in cells by treatment with Hoechst dye followed by micro-irradiation with 405 nm light [34]. The irradiation can be performed along a narrow line.

#### **Figure 5.**

*Initiation of DNA demethylation at a CpG site. In adult somatic cells DNA methylation typically occurs in the context of CpG dinucleotides (CpG sites), forming 5-methylcytosine-pG, or 5mCpG. Reactive oxygen species (ROS) may attack guanine at the dinucleotide site, forming 8-hydroxy-2*′*-deoxyguanosine (8-OHdG), and resulting in a 5mCp-8-OHdG dinucleotide site. The base excision repair enzyme OGG1 targets 8-OHdG and binds to the lesion without immediate excision. OGG1, present at a 5mCp-8-OHdG site recruits TET1 and TET1 oxidizes the 5mC adjacent to the 8-OHdG. This initiates demethylation of 5mC [37].*

Within 6 s of the irradiation with 405 nm light, there is half-maximum recruitment of OGG1 to the irradiated line. OGG1 (8-oxoguanine DNA glycosylase) is an enzyme that removes the oxidative damage 8-OHdG from DNA [35]. Removal of 8-OHdG, during base excision repair, occurs with a half-life of 11 min [36]. Thus, OGG1 protein rapidly complexes with 8-OHdG (6 s) but the OGG1-8-OHdG complex has a relatively long half-life (11 min).

H2O2 is a reactive oxygen species. Zhou et al. [32] treated cells in culture with 500 μM H2O2 for 6 h and this caused a more than 3-fold increase in 8-OHdG. The cells treated with H2O2 also became substantially demethylated, with methylation reduced to less than 1/4th the original methylation level. They then used cells in which OGG1 was inhibited, either by applying siRNA or by using OGG1 mutant knockout cells. In cells with inhibited or absent OGG1, treatment with H2O2 did not cause demethylation. These first experiments indicate that OGG1 has a role in H2O2 -induced demethylation.

Zhou et al. [32] examined the interaction between OGG1 and the TET enzymes that are involved in demethylation [23]. OGG1 did not interact with TET2 or TET3. However, OGG1 interacted with TET1. They found that the two proteins co-immunoprecipitated, and this co-immunoprecipitation did not depend on interactions with DNA or with 8-OHdG. Thus, OGG1 can attract or "recruit" TET1. They then used a double-stranded oligonucleotide containing 8-OHdG in solution in a pull-down assay using streptavidin beads. They found that OGG1 added to the assay could be pulled down by oligonucleotides containing 8-OHdG. TET1 could not be pulled down by oligonucleotides containing 8-OHdG, but TET1 could be pulled down if in the presence of OGG1. Their results imply that OGG1 attaches to 8-OHdG and then recruits TET1 to 8-OHdG lesions. They indicated that this could allow TET1 to initiate DNA demethylation of methylated CpGs after 8-OHdG lesions are formed (**Figure 5**). As shown in this figure, TET1 first interacts with OGG1 and then is close enough to the methyl group CH3 (shown in red) on the 5 position of the cytosine, to initiate the oxidation of the methyl group. This mechanism is notable for likely using two co-opted elements of DNA base excision repair (BER). First, OGG1 is an initiating enzyme in BER of 8-OHdG, but acts here to recruit TET1. Second, once the intermediate products of demethylation are formed by TET1, such as 5fC or 5caC as shown in **Figure 3**, then thymine DNA glycosylase (TDG) can initiate BER as shown in **Figure 4**, and complete the demethylation of 5mC to C.

OGG1 knockout mice seem to undergo a fairly normal embryogenesis, and the young new mice appear to be mostly normal [38], though they have a deficit in learning and memory as shown by a passive avoidance test [39] and a deficiency in immune responses (reviewed in [40]). TET1 knockout mice are also viable and fertile, with no discernible morphological or growth abnormality. However, TET1 knockout mice have an impairment in spatial learning and short-term memory [41] as well as deficiencies in fear memory extinction and spacial memory extinction [42]. On the other hand, over-expression of TET1 impairs hippocampus-dependent long-term associative memory [43]. A TET3 homozygous mutation, unlike a TET1 knockout, leads to neonatal lethality [44]. Thus TET3 is essential in embryogenesis. As pointed out above, TET3 (but not TET1 and TET2) is highly expressed in oocytes and zygotes (also shown in [45]).

#### **5. Demethylation in neurogenesis**

Neurogenesis in mouse takes place starting about day 10.5 after fertilization of the egg. Early in neurogenesis, some embryonic stem cells (ESCs) begin

**29**

*Demethylation in Early Embryonic Development and Memory*

**6. Demethylation in learning and memory**

of methylation (or demethylation) of DNA.

types, and 30 such sites could have up to 230, or 1.07 × 109

differentiating into neural stem cells (NSCs) and neural progenitor cells (NPCs) [46]. At this point, 8% of CpGs unmethylated in ESCs become largely methylated in NPCs, whereas approximately 2% of CpGs methylated in ESCs become unmethylated [46]. These data suggest that 5mC undergoes significant dynamic changes during ESC differentiation into NSCs. As shown by Pilz et al. [47], NPCs generate neurons throughout life in the dentate gyrus of the hippocampus of mice. Zhang et al. [41] examined adult NPCs purified from wild type and TET1 knockout mice. They found that 478 genes showed elevated promoter methylation levels in TET1-null NPCs compared to the wild-type control, while only 32 genes had lower methylation. Thus, TET1 appears to function in demethylation during neurogenesis

Learning and memory have levels of permanence, differing from other mental processes such as thought, language, and consciousness, which are temporary in nature. Learning and memory can be either slowly accumulated (multiplication tables) or rapidly (touching a hot stove), but once attained, can be recalled into conscious use for a long time. As pointed out by Alberini [48], humans can generally recall a painful fact or trauma in detail for a lifetime. Similarly, humans remember a very happy day for a long time afterwards. At least two early proposals were presented, indicating, on theoretical grounds, that the methylation and demethylation of DNA in neurons is the physical basis of memories. In 1969 Griffith and Mahler [49] published an article that made a number of salient points. They noted that, at least in man, memories may survive for periods of almost the entire lifetime. Further, DNA is the one molecule which, apart from possible minor effects due to genetic damage and repair, is surely present in neurons for the whole of the lifetime of the organism. This led them to the suggestion that the physical basis of memory could lie in the enzymatic modification of the DNA of nerve cells. They further indicated that a plausible suggestion would be that the modification consists

In 1999 Holliday [50] noted that long-term human memory can be retained for many decades. The exceptional stability required suggests that essential memory components may be based on chemical changes. He proposed that the enzymatic modification of cytosine in DNA to 5-methylcytosine may provide this necessary stability. The general model proposed is that specific sites in the DNA of neurons required for memory can exist in alternative methylated or non-methylated states. The initial signal, which is to be memorized, switches the DNA from a modified to an unmodified state, or vice versa. It should be noted that the presence or absence of DNA methylation at a particular sequence of DNA can be thought of as a 0, 1 binary code. Thus, 10 such sites have 210 (1024) epigenotypes and potential pheno-

such a set of control mechanisms has enormous potential for neuronal specificity. One form of long-term memory, associative learning, is contextual fear conditioning [51]. As an example of contextual fear conditioning, a rodent is placed in a novel environment (a new context) and is then subjected to an electric shock (e.g. a footshock). The rodent then experiences robust fear learning, shown by a strong fear response, when the rodent is placed in that context again. Contextual fear conditioning occurs very rapidly (it can occur with a single event) and it has a lasting effect [51]. Kim and Jung [51] reviewed the evidence that the hippocampus region of the brain is where contextual fear memories are first stored, and that this storage is transient and does not remain in the hippocampus (**Figure 6**). (Note that

epigenotypes. Clearly,

*DOI: http://dx.doi.org/10.5772/intechopen.90306*

in the adult brain.

*Demethylation in Early Embryonic Development and Memory DOI: http://dx.doi.org/10.5772/intechopen.90306*

*DNA Methylation Mechanism*

H2O2 -induced demethylation.

complex has a relatively long half-life (11 min).

Within 6 s of the irradiation with 405 nm light, there is half-maximum recruitment of OGG1 to the irradiated line. OGG1 (8-oxoguanine DNA glycosylase) is an enzyme that removes the oxidative damage 8-OHdG from DNA [35]. Removal of 8-OHdG, during base excision repair, occurs with a half-life of 11 min [36]. Thus, OGG1 protein rapidly complexes with 8-OHdG (6 s) but the OGG1-8-OHdG

H2O2 is a reactive oxygen species. Zhou et al. [32] treated cells in culture with 500 μM H2O2 for 6 h and this caused a more than 3-fold increase in 8-OHdG. The cells treated with H2O2 also became substantially demethylated, with methylation reduced to less than 1/4th the original methylation level. They then used cells in which OGG1 was inhibited, either by applying siRNA or by using OGG1 mutant knockout cells. In cells with inhibited or absent OGG1, treatment with H2O2 did not cause demethylation. These first experiments indicate that OGG1 has a role in

Zhou et al. [32] examined the interaction between OGG1 and the TET enzymes

OGG1 knockout mice seem to undergo a fairly normal embryogenesis, and the young new mice appear to be mostly normal [38], though they have a deficit in learning and memory as shown by a passive avoidance test [39] and a deficiency in immune responses (reviewed in [40]). TET1 knockout mice are also viable and fertile, with no discernible morphological or growth abnormality. However, TET1 knockout mice have an impairment in spatial learning and short-term memory [41] as well as deficiencies in fear memory extinction and spacial memory extinction [42]. On the other hand, over-expression of TET1 impairs hippocampus-dependent long-term associative memory [43]. A TET3 homozygous mutation, unlike a TET1 knockout, leads to neonatal lethality [44]. Thus TET3 is essential in embryogenesis. As pointed out above, TET3 (but not TET1 and TET2) is highly expressed in oocytes

Neurogenesis in mouse takes place starting about day 10.5 after fertilization of the egg. Early in neurogenesis, some embryonic stem cells (ESCs) begin

that are involved in demethylation [23]. OGG1 did not interact with TET2 or TET3. However, OGG1 interacted with TET1. They found that the two proteins co-immunoprecipitated, and this co-immunoprecipitation did not depend on interactions with DNA or with 8-OHdG. Thus, OGG1 can attract or "recruit" TET1. They then used a double-stranded oligonucleotide containing 8-OHdG in solution in a pull-down assay using streptavidin beads. They found that OGG1 added to the assay could be pulled down by oligonucleotides containing 8-OHdG. TET1 could not be pulled down by oligonucleotides containing 8-OHdG, but TET1 could be pulled down if in the presence of OGG1. Their results imply that OGG1 attaches to 8-OHdG and then recruits TET1 to 8-OHdG lesions. They indicated that this could allow TET1 to initiate DNA demethylation of methylated CpGs after 8-OHdG lesions are formed (**Figure 5**). As shown in this figure, TET1 first interacts with OGG1 and then is close enough to the methyl group CH3 (shown in red) on the 5 position of the cytosine, to initiate the oxidation of the methyl group. This mechanism is notable for likely using two co-opted elements of DNA base excision repair (BER). First, OGG1 is an initiating enzyme in BER of 8-OHdG, but acts here to recruit TET1. Second, once the intermediate products of demethylation are formed by TET1, such as 5fC or 5caC as shown in **Figure 3**, then thymine DNA glycosylase (TDG) can initiate BER as shown in **Figure 4**, and complete the demethylation of

**28**

5mC to C.

and zygotes (also shown in [45]).

**5. Demethylation in neurogenesis**

differentiating into neural stem cells (NSCs) and neural progenitor cells (NPCs) [46]. At this point, 8% of CpGs unmethylated in ESCs become largely methylated in NPCs, whereas approximately 2% of CpGs methylated in ESCs become unmethylated [46]. These data suggest that 5mC undergoes significant dynamic changes during ESC differentiation into NSCs. As shown by Pilz et al. [47], NPCs generate neurons throughout life in the dentate gyrus of the hippocampus of mice. Zhang et al. [41] examined adult NPCs purified from wild type and TET1 knockout mice. They found that 478 genes showed elevated promoter methylation levels in TET1-null NPCs compared to the wild-type control, while only 32 genes had lower methylation. Thus, TET1 appears to function in demethylation during neurogenesis in the adult brain.

#### **6. Demethylation in learning and memory**

Learning and memory have levels of permanence, differing from other mental processes such as thought, language, and consciousness, which are temporary in nature. Learning and memory can be either slowly accumulated (multiplication tables) or rapidly (touching a hot stove), but once attained, can be recalled into conscious use for a long time. As pointed out by Alberini [48], humans can generally recall a painful fact or trauma in detail for a lifetime. Similarly, humans remember a very happy day for a long time afterwards. At least two early proposals were presented, indicating, on theoretical grounds, that the methylation and demethylation of DNA in neurons is the physical basis of memories. In 1969 Griffith and Mahler [49] published an article that made a number of salient points. They noted that, at least in man, memories may survive for periods of almost the entire lifetime. Further, DNA is the one molecule which, apart from possible minor effects due to genetic damage and repair, is surely present in neurons for the whole of the lifetime of the organism. This led them to the suggestion that the physical basis of memory could lie in the enzymatic modification of the DNA of nerve cells. They further indicated that a plausible suggestion would be that the modification consists of methylation (or demethylation) of DNA.

In 1999 Holliday [50] noted that long-term human memory can be retained for many decades. The exceptional stability required suggests that essential memory components may be based on chemical changes. He proposed that the enzymatic modification of cytosine in DNA to 5-methylcytosine may provide this necessary stability. The general model proposed is that specific sites in the DNA of neurons required for memory can exist in alternative methylated or non-methylated states. The initial signal, which is to be memorized, switches the DNA from a modified to an unmodified state, or vice versa. It should be noted that the presence or absence of DNA methylation at a particular sequence of DNA can be thought of as a 0, 1 binary code. Thus, 10 such sites have 210 (1024) epigenotypes and potential phenotypes, and 30 such sites could have up to 230, or 1.07 × 109 epigenotypes. Clearly, such a set of control mechanisms has enormous potential for neuronal specificity.

One form of long-term memory, associative learning, is contextual fear conditioning [51]. As an example of contextual fear conditioning, a rodent is placed in a novel environment (a new context) and is then subjected to an electric shock (e.g. a footshock). The rodent then experiences robust fear learning, shown by a strong fear response, when the rodent is placed in that context again. Contextual fear conditioning occurs very rapidly (it can occur with a single event) and it has a lasting effect [51]. Kim and Jung [51] reviewed the evidence that the hippocampus region of the brain is where contextual fear memories are first stored, and that this storage is transient and does not remain in the hippocampus (**Figure 6**). (Note that while this diagram shows a single hippocampus in a human brain, humans have two hippocampi, one in each hemisphere of the brain.) They point out, in rats, that contextual fear conditioning is abolished when the hippocampus is subjected to hippocampectomy just 1 day after conditioning. However, the rats retain a considerable amount of contextual fear when a long delay of 28 days is imposed between the time of conditioning and the time of hippocampectomy. Using localized lidocaine injections to impede brain functions, Frankland et al. [53] showed that much of the long term storage of contextual fear conditioning memory appears to take place in the anterior cingulate cortex (**Figure 6**) (Note that there is a single anterior cingulate cortex of the human brain and it *resides in the medial wall of the two cerebral hemispheres*).

When methods to detect DNA methylation at specific locations on chromosomes became available, early experiments focused on particular genes known to be important for memory. One such gene is *PP2B* (*protein phosphatase 2B*), also known as *calcineurin* (*CaN*). This gene is of particular interest because it is the only Ca++ activated protein phosphatase in the brain and a major regulator of key proteins essential for synaptic transmission and neuronal excitability [54]. Miller et al. [55] found that persistent, specific hypermethylation of the *CaN* gene in the anterior cingulate cortex was induced in rats by a single contextual fear conditioning event at a time when a long-term memory was formed. Demethylation at a specific locus also has been investigated. Brain-derived neurotrophic factor (BDNF) is known to be important in memory [56]. As reviewed by Lubin et al. [57], the *bdnf* gene consists of eight 5′ exons each linked to individual promoter regions, and a 3′ exon (IX). Lubin et al. [57] subjected rats to contextual fear conditioning. Their sequencing data confirmed active demethylation of *bdnf* exon IV after fear conditioning along with a strong increase in expression of exon IV in the hippocampus at 2 h after fear conditioning. As noted above [51], the hippocampus region of the brain is where contextual fear memories are first stored, but this storage is transient. In the experiments of Lubin et al. [57] the RNA expression of exon IV of the *bdnf* gene returned to baseline level by 24 h after the fear conditioning.

More recently, methods became available to identify differentially methylated genes in entire genomes. In 2016, Halder et al. [58] used mice subjected to contextual fear conditioning and evaluated whole neuron genomes for differentially methylated genes and for differentially expressed genes. In one part of their study they looked at

**31**

*Demethylation in Early Embryonic Development and Memory*

the hippocampal CA1 region, a region that is crucial for short-term memory formation during contextual fear conditioning. In the hippocampus 1 h after contextual fear conditioning, there were 675 demethylated genes and 613 hypermethylated genes. The consolidation of memory at 1 h after contextual fear conditioning was accompanied by the differential methylation of genes coding for ion channels, transcription factors, and constituents of the CREB and PKA signaling cascades, all of which have been shown to contribute to the early phases of learning and memory processes. These changes were transient in the hippocampal neurons, and almost none were present after 4 weeks. This also implies that the hypermethylated genes at 1 h then underwent active demethylation during the 4 weeks after contextual fear conditioning. Halder et al. [58], in addition, examined the anterior cingulate cortex, a brain region important for associative memory acquisition and maintenance of long-term memory. In the anterior cingulate cortex, at 1 h after contextual fear conditioning, there were 6250 differentially methylated genes, including 2423 demethylated genes. At 4 weeks after training 1223 differentially methylated genes persisted, including 118 demethylated genes. In addition, at 4 weeks after training they found 1700 differentially expressed genes in the anterior cingulate cortex. Their findings suggest that long-term memory (4 weeks) is associated with differential methylation of DNA and altered expression of more than a thousand genes in mouse neurons. In 2017, Duke et al. [59], working with rats, studied neuron genomes in the hippocampus after contextual fear conditioning. At 24 h after contextual fear conditioning there were 2097 differentially methylated genes, with about 40% being demethylated. There were also 564 genes with upregulated expression and 1048 genes with downregulated expression. Hypermethylated regions overlapping differentially expressed genes were associated with decreased gene expression, consistent with the concept that cytosine methylation is often a mechanism for suppressing transcription. At 24 h after training, 9.2% of the genes in the rat genome of hippocampus neurons were differentially methylated. Gene Ontology term analysis was performed, and differentially expressed gene enrichment analysis revealed that many of the genes involved in synaptic functions were up-regulated 24 h after

In 2011, Guo et al. [26] were the first to show that TET1 is involved in neuronal activity-induced DNA demethylation and increased expression of memory-related genes in the mouse hippocampal dentate neurons. Demethylation of neuronal genes by TET1 appears to depend on TET1 being recruited to relevant genes. One mechanism of recruitment of TET appears to be by complexing with a specific "immediate early gene." The immediate early genes (IEGs) are a class of genes that are rapidly and transiently activated by a variety of signaling cascades and phosphorylation events, usually in a protein synthesis-independent manner, in response to neuronal activation [60]. ERG1 (Krox-24, Zif268) is an IEG product and is a neuronal activity-induced transcription factor. ERG1 appears to play an important role in learning and memory [60]. ERG1 is required specifically for the consolidation of long-term memory (while the related transcription factor ERG3 is primarily essential for short-term memory). As reviewed by Sun et al. [61], the short form of TET1, TET1s, is present in the brain. Sun et al. [61] experimentally showed that EGR1 and TET1s form a complex, independently of attachment to DNA. ERG1 undergoes rapid induction and appears to attach to binding sites at many genes upon neuronal activation. When ERG1 binds to a site, it is able to recruit a TET1s enzyme to that site. This allows TET1s to cause demethylation of a gene downstream

of the binding site of EGR1, with upregulation of that gene's expression.

*DOI: http://dx.doi.org/10.5772/intechopen.90306*

contextual fear conditioning in rats.

**6.1 TET in learning and memory**

**Figure 6.** *Some regions of the brain involved in memory [52].*

#### *Demethylation in Early Embryonic Development and Memory DOI: http://dx.doi.org/10.5772/intechopen.90306*

*DNA Methylation Mechanism*

*hemispheres*).

while this diagram shows a single hippocampus in a human brain, humans have two hippocampi, one in each hemisphere of the brain.) They point out, in rats, that contextual fear conditioning is abolished when the hippocampus is subjected to hippocampectomy just 1 day after conditioning. However, the rats retain a considerable amount of contextual fear when a long delay of 28 days is imposed between the time of conditioning and the time of hippocampectomy. Using localized lidocaine injections to impede brain functions, Frankland et al. [53] showed that much of the long term storage of contextual fear conditioning memory appears to take place in the anterior cingulate cortex (**Figure 6**) (Note that there is a single anterior cingulate cortex of the human brain and it *resides in the medial wall of the two cerebral* 

When methods to detect DNA methylation at specific locations on chromosomes

became available, early experiments focused on particular genes known to be important for memory. One such gene is *PP2B* (*protein phosphatase 2B*), also known as *calcineurin* (*CaN*). This gene is of particular interest because it is the only Ca++ activated protein phosphatase in the brain and a major regulator of key proteins essential for synaptic transmission and neuronal excitability [54]. Miller et al. [55] found that persistent, specific hypermethylation of the *CaN* gene in the anterior cingulate cortex was induced in rats by a single contextual fear conditioning event at a time when a long-term memory was formed. Demethylation at a specific locus also has been investigated. Brain-derived neurotrophic factor (BDNF) is known to be important in memory [56]. As reviewed by Lubin et al. [57], the *bdnf* gene consists of eight 5′ exons each linked to individual promoter regions, and a 3′ exon (IX). Lubin et al. [57] subjected rats to contextual fear conditioning. Their sequencing data confirmed active demethylation of *bdnf* exon IV after fear conditioning along with a strong increase in expression of exon IV in the hippocampus at 2 h after fear conditioning. As noted above [51], the hippocampus region of the brain is where contextual fear memories are first stored, but this storage is transient. In the experiments of Lubin et al. [57] the RNA expression of exon IV of the *bdnf* gene

returned to baseline level by 24 h after the fear conditioning.

More recently, methods became available to identify differentially methylated genes in entire genomes. In 2016, Halder et al. [58] used mice subjected to contextual fear conditioning and evaluated whole neuron genomes for differentially methylated genes and for differentially expressed genes. In one part of their study they looked at

**30**

**Figure 6.**

*Some regions of the brain involved in memory [52].*

the hippocampal CA1 region, a region that is crucial for short-term memory formation during contextual fear conditioning. In the hippocampus 1 h after contextual fear conditioning, there were 675 demethylated genes and 613 hypermethylated genes. The consolidation of memory at 1 h after contextual fear conditioning was accompanied by the differential methylation of genes coding for ion channels, transcription factors, and constituents of the CREB and PKA signaling cascades, all of which have been shown to contribute to the early phases of learning and memory processes. These changes were transient in the hippocampal neurons, and almost none were present after 4 weeks. This also implies that the hypermethylated genes at 1 h then underwent active demethylation during the 4 weeks after contextual fear conditioning. Halder et al. [58], in addition, examined the anterior cingulate cortex, a brain region important for associative memory acquisition and maintenance of long-term memory. In the anterior cingulate cortex, at 1 h after contextual fear conditioning, there were 6250 differentially methylated genes, including 2423 demethylated genes. At 4 weeks after training 1223 differentially methylated genes persisted, including 118 demethylated genes. In addition, at 4 weeks after training they found 1700 differentially expressed genes in the anterior cingulate cortex. Their findings suggest that long-term memory (4 weeks) is associated with differential methylation of DNA and altered expression of more than a thousand genes in mouse neurons.

In 2017, Duke et al. [59], working with rats, studied neuron genomes in the hippocampus after contextual fear conditioning. At 24 h after contextual fear conditioning there were 2097 differentially methylated genes, with about 40% being demethylated. There were also 564 genes with upregulated expression and 1048 genes with downregulated expression. Hypermethylated regions overlapping differentially expressed genes were associated with decreased gene expression, consistent with the concept that cytosine methylation is often a mechanism for suppressing transcription. At 24 h after training, 9.2% of the genes in the rat genome of hippocampus neurons were differentially methylated. Gene Ontology term analysis was performed, and differentially expressed gene enrichment analysis revealed that many of the genes involved in synaptic functions were up-regulated 24 h after contextual fear conditioning in rats.

#### **6.1 TET in learning and memory**

In 2011, Guo et al. [26] were the first to show that TET1 is involved in neuronal activity-induced DNA demethylation and increased expression of memory-related genes in the mouse hippocampal dentate neurons. Demethylation of neuronal genes by TET1 appears to depend on TET1 being recruited to relevant genes. One mechanism of recruitment of TET appears to be by complexing with a specific "immediate early gene." The immediate early genes (IEGs) are a class of genes that are rapidly and transiently activated by a variety of signaling cascades and phosphorylation events, usually in a protein synthesis-independent manner, in response to neuronal activation [60]. ERG1 (Krox-24, Zif268) is an IEG product and is a neuronal activity-induced transcription factor. ERG1 appears to play an important role in learning and memory [60]. ERG1 is required specifically for the consolidation of long-term memory (while the related transcription factor ERG3 is primarily essential for short-term memory). As reviewed by Sun et al. [61], the short form of TET1, TET1s, is present in the brain. Sun et al. [61] experimentally showed that EGR1 and TET1s form a complex, independently of attachment to DNA. ERG1 undergoes rapid induction and appears to attach to binding sites at many genes upon neuronal activation. When ERG1 binds to a site, it is able to recruit a TET1s enzyme to that site. This allows TET1s to cause demethylation of a gene downstream of the binding site of EGR1, with upregulation of that gene's expression.

*TET1* knockout mice [62] and *ERG1* knockout mice [63] are viable. Both have some developmental deficiencies [62, 63], and *TET1* knockouts [41, 43] and *ERG1* knockouts [64] each have some learning and memory deficiencies. Sun et al. [61] examined where differentially methylated regions occurred in the two types of knockout mice. Compared to wild-type mice, 322 and 2373 differentially methylated regions were identified in the brain frontal cortices (**Figure 6**) of *EGR1* knockout and *TET1* knockout mice respectively. There were 184 of these differentially methylated regions overlapping in the two types of knockout mice. This indicated that while ERG1 can bring TET1 to a DNA site to promote demethylation, TET1 is also brought too many other sites as well, presumably by other factors.

#### **7. Conclusions**

In evolutionary biology, the term exaptation refers to an evolutionary shift in the function of a trait over the course of natural selection [65]. For instance, a trait may evolve initially because it serves a particular function, but during the course of further evolution it may come to serve another function or an additional function. Such shifts in function are thought to be common in evolutionary history. As one example, bird feathers likely evolved initially for temperature regulation, and were later adapted for flight [65].

The idea that the function of a trait may shift during evolution was for many decades referred to as "preadaptation". However, this term suggests teleology in biology in conflict with natural selection and thus the term "preadaptation" has been replaced in the literature by "exaptation." This concept has recently been applied to the cognitive neurosciences [66]. It was proposed that substantial changes in function such as development of contemporary complex cognition including grammatical language, reading, writing and calculation abilities have occurred without evident changes in brain morphology over the past 150,000 years.

The evolutionary emergence of embryonic development also appears to have depended on an early exaptation. Enzymatic pathways that repair damage to the DNA genome likely existed very early in the history of life [67]. Processes that repair DNA, such as base excision repair, can also facilitate epigenetic modifications, particularly demethylation reactions, that alter gene expression and hence the function of cell lineages. Such epigenetic modifications play a central role in embryonic development including neurogenesis. Epigenetic alterations such as 5-methylcytosine are structurally similar to unwanted damages that are the primary target of DNA repair processes. Thus acquiring the new function of recognizing epigenetically methylated bases may have been enabled by this similarity. However, in the case of epigenetic demethylations, the effect of removing methyl groups and restoring the genome is to allow expression of genes that had been previously epigenetically silenced by methylation. Methylation and demethylation are reciprocal processes that appear to act coordinately to direct gene expression during embryonic development. DNA methylation reactions often cause silencing of gene expression, while demethylation reactions can reverse this process to allow expression. These mechanisms for controlling gene expression and the consequent facilitation of cell differentiation leading to embryonic development may have emerged in evolution as early as the origin of multicellular organisms more than 1 billion years ago [68].

Just as the evolutionary shift in the function of DNA repair appears to be central to the emergence of embryonic development and neurogenesis, this derived capability likely also gave rise to memory and learning. The molecular processes of epigenetic methylation and demethylation that underlie embryonic development

**33**

**Author details**

*Demethylation in Early Embryonic Development and Memory*

will contribute to disease prevention and treatment.

\*Address all correspondence to: bernstein324@yahoo.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Carol Bernstein\* and Harris Bernstein University of Arizona, Tucson, Arizona USA

provided the original work is properly cited.

also appear to underlie memory and learning. Thus the capacity for memory and learning may have evolved from a set of earlier epigenetic capabilities whose func-

Parkinson disease patients treated with levodopa are subject to dyskinesia, a persistent behavioral sensitization that develops after levodopa exposure. Reorganization of DNA methylation patterns in the genome due to aberrant expression of DNA demethylation enzymes appears to have a pivotal role in the development of levodopa-induced dyskinesia [70]. Modification of DNA methylation is considered to be a promising novel therapeutic target for use in preventing or reversing dyskinetic behaviors [70]. Huntington's disease is a neurodegenerative disease that typically becomes apparent in midlife. This disease is associated with substantial changes in brain DNA methylation levels [71]. Aicardi-Goutieres syndrome (AGS) is a neurodegenerative condition characterized by early onset, often in infancy. Cells deficient in AGS proteins display a substantial 5–20% reduction in genomic methylation levels overall, and this reduction is distributed widely in the genome [72]. The fragile X syndrome is a prevalent form of mental retardation. This condition is caused by loss of expression of the *FMR1* gene, usually due to expansion of a CGG repeat sequence (>200 repeats) in the first exon of *FMR1*. This sequence expansion leads to abnormal methylation of the promoter region that then causes transcriptional silencing of the *FMR1* gene and an absence of the fragile X mental retardation protein [73]. Several studies have described methylation alterations in various regions of the brain in Alzheimer's disease, as reviewed by Yokoyama et al. [74]. The results of these studies, so far, appear to be somewhat contradictory and additional studies will be needed to provide clear conclusions. These various studies of DNA methylation alterations are still at an early stage, but nevertheless suggest that as our basic understanding of how epigenetic DNA methylation patterns influence neurodegenerative disease advances, this understanding

In several neurodegenerative diseases epigenetic alterations appear to underlie characteristic features of the disease phenotype [69]. Proper functioning of the nervous system likely depends on DNA repair processes that not only restore DNA sequence information, but also facilitate normal gene expression by maintaining an appropriate set of epigenetic markers, particularly DNA methylation patterns. Understanding changes in DNA methylation patterns during early development and neurogenesis may contribute to the prevention or treatment of particular neurode-

tion was to promote embryonic reprogramming and neurogenesis.

*DOI: http://dx.doi.org/10.5772/intechopen.90306*

generative diseases.

#### *Demethylation in Early Embryonic Development and Memory DOI: http://dx.doi.org/10.5772/intechopen.90306*

*DNA Methylation Mechanism*

**7. Conclusions**

later adapted for flight [65].

*TET1* knockout mice [62] and *ERG1* knockout mice [63] are viable. Both have some developmental deficiencies [62, 63], and *TET1* knockouts [41, 43] and *ERG1* knockouts [64] each have some learning and memory deficiencies. Sun et al. [61] examined where differentially methylated regions occurred in the two types of knockout mice. Compared to wild-type mice, 322 and 2373 differentially methylated regions were identified in the brain frontal cortices (**Figure 6**) of *EGR1* knockout and *TET1* knockout mice respectively. There were 184 of these differentially methylated regions overlapping in the two types of knockout mice. This indicated that while ERG1 can bring TET1 to a DNA site to promote demethylation, TET1 is

In evolutionary biology, the term exaptation refers to an evolutionary shift in the function of a trait over the course of natural selection [65]. For instance, a trait may evolve initially because it serves a particular function, but during the course of further evolution it may come to serve another function or an additional function. Such shifts in function are thought to be common in evolutionary history. As one example, bird feathers likely evolved initially for temperature regulation, and were

The idea that the function of a trait may shift during evolution was for many decades referred to as "preadaptation". However, this term suggests teleology in biology in conflict with natural selection and thus the term "preadaptation" has been replaced in the literature by "exaptation." This concept has recently been applied to the cognitive neurosciences [66]. It was proposed that substantial changes in function such as development of contemporary complex cognition including grammatical language, reading, writing and calculation abilities have occurred without evident changes in brain morphology over the past 150,000 years. The evolutionary emergence of embryonic development also appears to have depended on an early exaptation. Enzymatic pathways that repair damage to the DNA genome likely existed very early in the history of life [67]. Processes that repair DNA, such as base excision repair, can also facilitate epigenetic modifications, particularly demethylation reactions, that alter gene expression and hence the function of cell lineages. Such epigenetic modifications play a central role in embryonic development including neurogenesis. Epigenetic alterations such as 5-methylcytosine are structurally similar to unwanted damages that are the primary target of DNA repair processes. Thus acquiring the new function of recognizing epigenetically methylated bases may have been enabled by this similarity. However, in the case of epigenetic demethylations, the effect of removing methyl groups and restoring the genome is to allow expression of genes that had been previously epigenetically silenced by methylation. Methylation and demethylation are reciprocal processes that appear to act coordinately to direct gene expression during embryonic development. DNA methylation reactions often cause silencing of gene expression, while demethylation reactions can reverse this process to allow expression. These mechanisms for controlling gene expression and the consequent facilitation of cell differentiation leading to embryonic development may have emerged in evolution as early as the origin of multicellular organisms more than 1

Just as the evolutionary shift in the function of DNA repair appears to be central to the emergence of embryonic development and neurogenesis, this derived capability likely also gave rise to memory and learning. The molecular processes of epigenetic methylation and demethylation that underlie embryonic development

also brought too many other sites as well, presumably by other factors.

**32**

billion years ago [68].

also appear to underlie memory and learning. Thus the capacity for memory and learning may have evolved from a set of earlier epigenetic capabilities whose function was to promote embryonic reprogramming and neurogenesis.

In several neurodegenerative diseases epigenetic alterations appear to underlie characteristic features of the disease phenotype [69]. Proper functioning of the nervous system likely depends on DNA repair processes that not only restore DNA sequence information, but also facilitate normal gene expression by maintaining an appropriate set of epigenetic markers, particularly DNA methylation patterns. Understanding changes in DNA methylation patterns during early development and neurogenesis may contribute to the prevention or treatment of particular neurodegenerative diseases.

Parkinson disease patients treated with levodopa are subject to dyskinesia, a persistent behavioral sensitization that develops after levodopa exposure. Reorganization of DNA methylation patterns in the genome due to aberrant expression of DNA demethylation enzymes appears to have a pivotal role in the development of levodopa-induced dyskinesia [70]. Modification of DNA methylation is considered to be a promising novel therapeutic target for use in preventing or reversing dyskinetic behaviors [70]. Huntington's disease is a neurodegenerative disease that typically becomes apparent in midlife. This disease is associated with substantial changes in brain DNA methylation levels [71]. Aicardi-Goutieres syndrome (AGS) is a neurodegenerative condition characterized by early onset, often in infancy. Cells deficient in AGS proteins display a substantial 5–20% reduction in genomic methylation levels overall, and this reduction is distributed widely in the genome [72]. The fragile X syndrome is a prevalent form of mental retardation. This condition is caused by loss of expression of the *FMR1* gene, usually due to expansion of a CGG repeat sequence (>200 repeats) in the first exon of *FMR1*. This sequence expansion leads to abnormal methylation of the promoter region that then causes transcriptional silencing of the *FMR1* gene and an absence of the fragile X mental retardation protein [73]. Several studies have described methylation alterations in various regions of the brain in Alzheimer's disease, as reviewed by Yokoyama et al. [74]. The results of these studies, so far, appear to be somewhat contradictory and additional studies will be needed to provide clear conclusions. These various studies of DNA methylation alterations are still at an early stage, but nevertheless suggest that as our basic understanding of how epigenetic DNA methylation patterns influence neurodegenerative disease advances, this understanding will contribute to disease prevention and treatment.

#### **Author details**

Carol Bernstein\* and Harris Bernstein University of Arizona, Tucson, Arizona USA

\*Address all correspondence to: bernstein324@yahoo.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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**34**

*DNA Methylation Mechanism*

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Neuroscience Letters. 2016;**625**:40-46. DOI: 10.1016/j.neulet.2016.01.023

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[44] Gu TP, Guo F, Yang H, Wu HP, Xu GF, Liu W, et al. The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature. 2011;**477**(7366):606-610. DOI: 10.1038/ nature10443

[45] Zhao H, Chen T. Tet family of 5-methylcytosine dioxygenases in mammalian development. Journal of Human Genetics. 2013;**58**(7):421-427. DOI: 10.1038/jhg.2013.63

[46] Wang Z, Tang B, He Y, Jin P. DNA methylation dynamics in neurogenesis. Epigenomics. 2016;**8**(3):401-414. DOI: 10.2217/epi.15.119

[47] Pilz GA, Bottes S, Betizeau M, Jörg DJ, Carta S, Simons BD, et al. Live imaging of neurogenesis in the adult mouse hippocampus. Science. 2018;**359**(6376):658-662. DOI: 10.1126/ science.aao5056

[48] Alberini CM. Long-term memories: The good, the bad, and the ugly. Cerebrum. 2010;**2010**:21. PMID: 23447766

[49] Griffith JS, Mahler HR. DNA ticketing theory of memory. Nature. 1969;**223**(5206):580-582. DOI: 10.1038/223580a0

[50] Holliday R. Is there an epigenetic component in long-term memory? Journal of Theoretical Biology. 1999;**200**(3):339-341. DOI: 10.1006/ jtbi.1999.0995

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Neuroscience and Biobehavioral Reviews. 2006;**30**(2):188-202. PMID: 16120461

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[61] Sun Z, Xu X, He J, Murray A, Sun MA, Wei X, et al. EGR1 recruits TET1 to shape the brain methylome during development and upon neuronal activity. Nature Communications. 2019;**10**(1):3892. DOI: 10.1038/ s41467-019-11905-3

[62] Dawlaty MM, Ganz K, Powell BE, Hu YC, Markoulaki S, Cheng AW, et al. Tet1 is dispensable for maintaining pluripotency and its loss is compatible with embryonic and postnatal development. Cell Stem Cell. 2011;**9**(2):166-175. DOI: 10.1016/j. stem.2011.07.010

[63] Topilko P, Schneider-Maunoury S, Levi G, Trembleau A, Gourdji D, Driancourt MA, et al. Multiple pituitary and ovarian defects in Krox-24 (NGFI-A, Egr-1)-targeted mice. Molecular Endocrinology. 1998;**12**(1):107-122. DOI: 10.1210/mend.12.1.0049

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[65] Gould SJ, Vrba ES. Exaptation—A missing term in the science of form. Paleobiology. 1982;**8**(1):4-15. DOI: 10.1017/S0094837300004310

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[70] Figge DA, Eskow Jaunarajs KL, Standaert DG. Dynamic DNA methylation regulates levodopainduced dyskinesia. The Journal of Neuroscience. 2016;**36**:6514-6524. DOI: 10.1523/JNEUROSCI.0683-16.2016

[71] Horvath S, Langfelder P, Kwak S, Aaronson J, Rosinski J, Vogt TF, et al. Huntington's disease accelerates epigenetic aging of human brain and disrupts DNA methylation levels. Aging (Albany NY). 2016;**8**:1485-1512. DOI: 10.18632/aging.101005

[72] Lim YW, Sanz LA, Xu X, Hartone SR, Chetin F. Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi-Goutieres syndrome. eLife. 2015;**4**:08007. DOI: 10.7554/eLife.08007

[73] Serrano M. Epigenetic cerebellar diseases. Handbook of Clinical Neurology. 2018;**155**:227-244. DOI: 10.1016/B978-0-444-64189-2.00015-9

[74] Yokoyama AS, Rutledge JC, Medici V. DNA methylation alterations in Alzheimer's disease. Environmental Epigenetics. 2017;**3**:dvx008. DOI: 10.1093/eep/dvx008

**39**

**Chapter 3**

**Abstract**

Development

*Aleksandra Stanković*

folate intake, genotype

**1. Introduction**

Global DNA Methylation

as a Potential Underlying

Mechanism of Congenital Disease

During the last decade, quantitative measurement of the methylation status in white blood cells (WBCs) has been used as a potential biomarker in a variety of diseases. Long interspersed nucleotide element-1 (LINE-1) has been used widely as a surrogate marker of global DNA methylation. Altered maternal DNA methylation is suggested to be an underlying mechanism in the trisomy 21 and the development of birth defects, including congenital heart defects (CHDs). The molecular mechanisms that underlie the epigenetic regulation of gene transcription are independent of DNA sequence, but they do depend on environmental stimuli, which are especially important in fetal development in utero environment. Folic acid deficiency and genetic variations of folate pathway genes, such as the methylenetetrahydrofolate reductase gene (*MTHFR*), are foremost among these maternal risk factors. Also, there are exogenous risk factors (cigarette smoking, alcohol intake, medication use, periconceptional maternal supplementation, body mass index, and dietary habits) with impact on maternal LINE-1 methylation. *MTHFR* C677T genotype/ diet and other environmental factors as significant predictors of LINE-1 DNA methylation in regard to congenital diseases will be discussed in the chapter.

**Keywords:** DNA methylation, LINE-1, congenital anomaly, development, nutrition,

According to WHO, congenital anomalies (CAs) are birth defects that can be defined as structural or functional malformations [1]. CAs occur during intrauterine life and can be identified prenatally or at birth or later in infancy. CAs are important causes of infant and childhood deaths and chronic illness/disability. Long-term disability may have significant impacts on patients, families, healthcare systems, and societies. Some CAs can be prevented by adequate intake of folic acid (FA) through fortification of staple foods or supplementation. Among severe congenital anomalies, the most common ones are congenital heart defects (CHDs), neural tube defects (NTD), and Down syndrome (DS). For renal dysplasia an increasing trend was observed recently in Europe [2]. Several factors have been proposed to have a significant role in the development of CAs: one or more genes;

#### **Chapter 3**

*DNA Methylation Mechanism*

lm.045112.117

neuro.01.002.2008

s41467-019-11905-3

stem.2011.07.010

2017;**24**(7):278-288. DOI: 10.1101/

[67] DiRuggiero J, Brown JR, Bogert AP, Robb FT. DNA repair systems in archaea: Mementos from the last universal common ancestor? Journal of Molecular Evolution. 1999;**49**(4):474- 484. DOI: 10.1007/pl00006570

[68] Strother PK, Battison L, Brasier MD, Wellman CH. Earth's earliest nonmarine eukaryotes. Nature.

2011;**473**(7348):505-509. DOI: 10.1038/

[69] Bernstein C, Bernstein H. The role of DNA repair and the epigenetic markers left after repair in neurologic functions, including memory and learning. In: Mognato M, editor. DNA Repair—An Update. Rijeka, Croatia: IntechOpen; 2018. pp. 105-126. DOI:

[70] Figge DA, Eskow Jaunarajs KL, Standaert DG. Dynamic DNA methylation regulates levodopainduced dyskinesia. The Journal of Neuroscience. 2016;**36**:6514-6524. DOI: 10.1523/JNEUROSCI.0683-16.2016

[71] Horvath S, Langfelder P, Kwak S, Aaronson J, Rosinski J, Vogt TF, et al. Huntington's disease accelerates epigenetic aging of human brain and disrupts DNA methylation levels. Aging (Albany NY). 2016;**8**:1485-1512. DOI:

10.5772/intechopen.83477

10.18632/aging.101005

[72] Lim YW, Sanz LA, Xu X,

DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi-Goutieres syndrome. eLife. 2015;**4**:08007. DOI: 10.7554/eLife.08007

Hartone SR, Chetin F. Genome-wide

[73] Serrano M. Epigenetic cerebellar diseases. Handbook of Clinical Neurology. 2018;**155**:227-244. DOI: 10.1016/B978-0-444-64189-2.00015-9

[74] Yokoyama AS, Rutledge JC, Medici V. DNA methylation alterations in Alzheimer's disease. Environmental Epigenetics. 2017;**3**:dvx008. DOI:

10.1093/eep/dvx008

nature09943

[60] Poirier R, Cheval H, Mailhes C, Garel S, Charnay P, Davis S, et al. Distinct functions of egr gene family members in cognitive processes. Frontiers in Neuroscience. 2008;**2**(1):47-55. DOI: 10.3389/

[61] Sun Z, Xu X, He J, Murray A, Sun MA, Wei X, et al. EGR1 recruits TET1 to shape the brain methylome during development and upon neuronal activity. Nature Communications. 2019;**10**(1):3892. DOI: 10.1038/

[62] Dawlaty MM, Ganz K, Powell BE, Hu YC, Markoulaki S, Cheng AW, et al. Tet1 is dispensable for maintaining pluripotency and its loss is compatible

[63] Topilko P, Schneider-Maunoury S, Levi G, Trembleau A, Gourdji D, Driancourt MA, et al. Multiple pituitary and ovarian defects in Krox-24 (NGFI-A,

Egr-1)-targeted mice. Molecular Endocrinology. 1998;**12**(1):107-122. DOI: 10.1210/mend.12.1.0049

[64] Jones MW, Errington ML, French PJ, Fine A, Bliss TV, Garel S, et al. A requirement for the immediate early gene Zif268 in the expression of late LTP and long-term memories. Nature Neuroscience. 2001;**4**(3):289-

296. DOI: 10.1038/85138

[65] Gould SJ, Vrba ES. Exaptation—A missing term in the science of form. Paleobiology. 1982;**8**(1):4-15. DOI: 10.1017/S0094837300004310

[66] Ardila A. The evolutionary concept of "preadaptation" applied to cognitive neurosciences. Frontiers in Neuroscience. 2016;**10**:103. DOI:

10.3389/fnins.2016.00103

with embryonic and postnatal development. Cell Stem Cell. 2011;**9**(2):166-175. DOI: 10.1016/j.

**38**

## Global DNA Methylation as a Potential Underlying Mechanism of Congenital Disease Development

*Aleksandra Stanković*

### **Abstract**

During the last decade, quantitative measurement of the methylation status in white blood cells (WBCs) has been used as a potential biomarker in a variety of diseases. Long interspersed nucleotide element-1 (LINE-1) has been used widely as a surrogate marker of global DNA methylation. Altered maternal DNA methylation is suggested to be an underlying mechanism in the trisomy 21 and the development of birth defects, including congenital heart defects (CHDs). The molecular mechanisms that underlie the epigenetic regulation of gene transcription are independent of DNA sequence, but they do depend on environmental stimuli, which are especially important in fetal development in utero environment. Folic acid deficiency and genetic variations of folate pathway genes, such as the methylenetetrahydrofolate reductase gene (*MTHFR*), are foremost among these maternal risk factors. Also, there are exogenous risk factors (cigarette smoking, alcohol intake, medication use, periconceptional maternal supplementation, body mass index, and dietary habits) with impact on maternal LINE-1 methylation. *MTHFR* C677T genotype/ diet and other environmental factors as significant predictors of LINE-1 DNA methylation in regard to congenital diseases will be discussed in the chapter.

**Keywords:** DNA methylation, LINE-1, congenital anomaly, development, nutrition, folate intake, genotype

#### **1. Introduction**

According to WHO, congenital anomalies (CAs) are birth defects that can be defined as structural or functional malformations [1]. CAs occur during intrauterine life and can be identified prenatally or at birth or later in infancy. CAs are important causes of infant and childhood deaths and chronic illness/disability. Long-term disability may have significant impacts on patients, families, healthcare systems, and societies. Some CAs can be prevented by adequate intake of folic acid (FA) through fortification of staple foods or supplementation. Among severe congenital anomalies, the most common ones are congenital heart defects (CHDs), neural tube defects (NTD), and Down syndrome (DS). For renal dysplasia an increasing trend was observed recently in Europe [2]. Several factors have been proposed to have a significant role in the development of CAs: one or more genes;

infectious, maternal diabetes or obesity; and nutritional and environmental factors [2]. Identification of the exact cause/causes recently became even more complicated with addition of new factors. Epigenetic factors, as it is DNA methylation, have been shown to have an impact on the gene expression, through modulation by nutrition or environmental stimuli that occur during intrauterine development, but could even be a consequence of maternal or paternal lifestyle factors. Altered DNA methylation was suggested to be an underlying mechanism in the development of CAs, CHDs, NTD, congenital anomaly of the kidney and urinary tract (CAKUT), and autism spectrum disorders (ASD) and in imprinting genetic disorders [3–12]. Congenital heart defects (CHDs) are the most common birth defects in humans with a prevalence of 0.8% [13, 14]. Only about 15–20% of CHDs can be attributed to known causes, whereas chromosomal abnormalities occur in 5–10% of cases [14]. The highest association with major heart abnormalities is observed in DS [15]. CHDs are reported in approximately 40% of DS cases, typically involving septal defects such as atrial septal defect, ventricular septal defect, and complete atrioventricular canal [16]. The etiology of most CHDs remains largely unknown, but it is considered to involve multiple genetic, epigenetic, environmental, and lifestyle factors [13, 14, 17]. Risk factors, including aging, body mass index (BMI), cigarette smoking, alcohol intake, folate deficiency, MTHFR polymorphisms, and hyperhomocysteinemia, have been proposed to be the modulators of DNA methylation patterns [3–6, 18–20]. Maternal intrauterine milieu, such as maternal environment during pregnancy (hypoxia, stress, obesity, diabetes, toxins, altered nutrition, inflammation, and reduced utero-placental blood flow) could affect fetal methylation programming, thereby affecting fetal growth and the lifelong health of the fetus [21, 22]. It was reported that the maternal LINE-1 hypomethylation is linked with the increased risk for non-syndromic CHD, particularly septal defects [4, 5].

#### **2. DNA gene-specific methylation and global DNA methylation**

DNA methylation is a key factor of the epigenetic machinery that is responsible for regulating gene expression and, therefore, cell function. This component is one of the most important in mammalian embryonic development, differentiation, and many of congenital and complex diseases [3–6, 23–25]. The DNA methylation has nonrandom, well-regulated, and tissue-specific patterns [26]. Abnormal genespecific demethylation and global hypomethylation (involving repeat sequences throughout the genome) can potentially lead to overexpression of genes and activation of transposable elements contributing to disease. Regulation of gene expression through methylated or unmethylated human genome can exist at approximately 3 × 107 CpG short sequences of 5–10 CpG dinucleotides [27, 28].

DNA methylation is required in many processes such as X chromosome inactivation, imprinting, embryogenesis, gametogenesis, and silencing of repetitive DNA elements [29]. It refers to the covalent addition of a methyl group to the cytosine located at the 5′-position to guanosine in a CpG dinucleotide, catalyzed by the activity of three DNA methyltransferases (DNMTs) [30]. Recent findings of tissue-specific expression of ten-eleven translocation (TET) proteins revealed that this epigenetic event is not irreversible and, even more, TET was shown to be able to modify methylcytosine and potentially erase DNA methylation [31].

Each of the three DNMT genes was found to be mutated in specific and diverse human syndromes [32]. DNA methylation is required to protect chromosomal integrity, by preventing reactivation of endoparasitic sequences that cause chromosomal instability, translocations, increased mutation events, loss of imprinting, and gene disruption [29]. Genome-wide methylation profiling has recently become

**41**

*Global DNA Methylation as a Potential Underlying Mechanism of Congenital Disease…*

dependent processes and their associated biological functions [27].

Gene-specific DNA methylation analysis does not provide a global picture of DNA methylation changes within a genome. Global DNA hypomethylation occurs mainly at heavily methylated noncoding regions of DNA, particularly repeat

In humans, nearly 80% CpG islands occur in transposon-derived sequences, throughout the genome, such as long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs) [42]. LINE-1 is the largest member of the LINE family with more than 500,000 copies comprising approximately 17% of the genome [43]. CpG islands within LINE-1 sequences and their methylation levels correlate with the global genomic DNA methylation level [44, 45], so LINE-1 methylation has been widely used as a surrogate marker of global genomic DNA methylation [46], and methylation status of LINE-1 in white blood cells (WBC) is a potential biomarker in a variety of diseases [4, 45–48] in research on cancer, cardiovascular, neurodegenerative, and CAs [3–6, 48–51]. Human genome has on average 80–100 active LINE-1, and it has been estimated that new LINE-1 insertion in genome occurs in at least 1 in every 50 humans within a parental germ cell or during early fetal development [40]. Thus, LINE-1 hypomethylation in the parental germline, along with altered miRNA expression, might also significantly affect

**2.1 LINE-1 DNA methylation**

sequences and transposable elements [40, 41].

genome stability during the fetal development [52, 53].

**3. DNA methylation during gametogenesis and embryogenesis**

DNA methylation changes are particularly dynamic in gametogenesis and early

embryogenesis. During the course of mammalian differentiation and development, DNA methylation undergoes remodeling to eventually generate the cell type-specific methylation patterns, found in somatic cells of adults. During the

possible and revealed genes of interest that were enriched in multiple biological processes involved in fetal development [3], and specific hypermethylation was linked to gene silencing in some pediatric disorders [33, 34]. Moreover, epigenetic mechanisms including parent of origin-specific DNA methylation include genomic imprinting as restriction of gene expression [35]. Moreover, imprinting in embryos was found to be parentally sex-specific, and this effect could be more complex than previously suggested [36]. Hypomethylation of imprinted loci (HIL) throughout the genome was observed in patients with imprinted disorders. Among approximately 70 known imprinted genes, there are some that are causing disorders affecting growth, including one in the DS critical region [35]. Aberrant methylation in four maternally methylated regions was observed at whole genome methylation analysis. However, methylation of a CpG island does not necessarily lead to gene silencing. For example, the gene for telomerase has been shown to be activated by methylation [37]. Telomerases are crucial elements in maintaining cell life, could possibly reverse an aging mechanism, and rejuvenate cell viability. Enzyme telomerase modulates elongation of telomeres, by adding repeating DNA sequences to the ends of the chromosomes, and telomere serves as a bioprotective mechanism of chromosome attrition at each cell division [38]. Telomeres could become too short to allow replication or dysfunctional in some congenital disease which may lead to chromosome instability or cell death [39]. Besides DNA coding region, studies have shown that DNA methylation of noncoding DNA plays an important role in modulating structure and dynamics of chromatin, as well as many other chromatin-

*DOI: http://dx.doi.org/10.5772/intechopen.90996*

*Global DNA Methylation as a Potential Underlying Mechanism of Congenital Disease… DOI: http://dx.doi.org/10.5772/intechopen.90996*

possible and revealed genes of interest that were enriched in multiple biological processes involved in fetal development [3], and specific hypermethylation was linked to gene silencing in some pediatric disorders [33, 34]. Moreover, epigenetic mechanisms including parent of origin-specific DNA methylation include genomic imprinting as restriction of gene expression [35]. Moreover, imprinting in embryos was found to be parentally sex-specific, and this effect could be more complex than previously suggested [36]. Hypomethylation of imprinted loci (HIL) throughout the genome was observed in patients with imprinted disorders. Among approximately 70 known imprinted genes, there are some that are causing disorders affecting growth, including one in the DS critical region [35]. Aberrant methylation in four maternally methylated regions was observed at whole genome methylation analysis. However, methylation of a CpG island does not necessarily lead to gene silencing. For example, the gene for telomerase has been shown to be activated by methylation [37]. Telomerases are crucial elements in maintaining cell life, could possibly reverse an aging mechanism, and rejuvenate cell viability. Enzyme telomerase modulates elongation of telomeres, by adding repeating DNA sequences to the ends of the chromosomes, and telomere serves as a bioprotective mechanism of chromosome attrition at each cell division [38]. Telomeres could become too short to allow replication or dysfunctional in some congenital disease which may lead to chromosome instability or cell death [39]. Besides DNA coding region, studies have shown that DNA methylation of noncoding DNA plays an important role in modulating structure and dynamics of chromatin, as well as many other chromatindependent processes and their associated biological functions [27].

#### **2.1 LINE-1 DNA methylation**

*DNA Methylation Mechanism*

infectious, maternal diabetes or obesity; and nutritional and environmental factors [2]. Identification of the exact cause/causes recently became even more complicated with addition of new factors. Epigenetic factors, as it is DNA methylation, have been shown to have an impact on the gene expression, through modulation by nutrition or environmental stimuli that occur during intrauterine development, but could even be a consequence of maternal or paternal lifestyle factors. Altered DNA methylation was suggested to be an underlying mechanism in the development of CAs, CHDs, NTD, congenital anomaly of the kidney and urinary tract (CAKUT), and autism spectrum disorders (ASD) and in imprinting genetic disorders [3–12]. Congenital heart defects (CHDs) are the most common birth defects in humans with a prevalence of 0.8% [13, 14]. Only about 15–20% of CHDs can be attributed to known causes, whereas chromosomal abnormalities occur in 5–10% of cases [14]. The highest association with major heart abnormalities is observed in DS [15]. CHDs are reported in approximately 40% of DS cases, typically involving septal defects such as atrial septal defect, ventricular septal defect, and complete atrioventricular canal [16]. The etiology of most CHDs remains largely unknown, but it is considered to involve multiple genetic, epigenetic, environmental, and lifestyle factors [13, 14, 17]. Risk factors, including aging, body mass index (BMI), cigarette smoking, alcohol intake, folate deficiency, MTHFR polymorphisms, and hyperhomocysteinemia, have been proposed to be the modulators of DNA methylation patterns [3–6, 18–20]. Maternal intrauterine milieu, such as maternal environment during pregnancy (hypoxia, stress, obesity, diabetes, toxins, altered nutrition, inflammation, and reduced utero-placental blood flow) could affect fetal methylation programming, thereby affecting fetal growth and the lifelong health of the fetus [21, 22]. It was reported that the maternal LINE-1 hypomethylation is linked with the increased risk for non-syndromic CHD, particularly septal defects [4, 5].

**2. DNA gene-specific methylation and global DNA methylation**

CpG short sequences of 5–10 CpG dinucleotides [27, 28].

modify methylcytosine and potentially erase DNA methylation [31].

DNA methylation is required in many processes such as X chromosome inactivation, imprinting, embryogenesis, gametogenesis, and silencing of repetitive DNA elements [29]. It refers to the covalent addition of a methyl group to the cytosine located at the 5′-position to guanosine in a CpG dinucleotide, catalyzed by the activity of three DNA methyltransferases (DNMTs) [30]. Recent findings of tissue-specific expression of ten-eleven translocation (TET) proteins revealed that this epigenetic event is not irreversible and, even more, TET was shown to be able to

Each of the three DNMT genes was found to be mutated in specific and diverse human syndromes [32]. DNA methylation is required to protect chromosomal integrity, by preventing reactivation of endoparasitic sequences that cause chromosomal instability, translocations, increased mutation events, loss of imprinting, and gene disruption [29]. Genome-wide methylation profiling has recently become

DNA methylation is a key factor of the epigenetic machinery that is responsible for regulating gene expression and, therefore, cell function. This component is one of the most important in mammalian embryonic development, differentiation, and many of congenital and complex diseases [3–6, 23–25]. The DNA methylation has nonrandom, well-regulated, and tissue-specific patterns [26]. Abnormal genespecific demethylation and global hypomethylation (involving repeat sequences throughout the genome) can potentially lead to overexpression of genes and activation of transposable elements contributing to disease. Regulation of gene expression through methylated or unmethylated human genome can exist at approximately

**40**

3 × 107

Gene-specific DNA methylation analysis does not provide a global picture of DNA methylation changes within a genome. Global DNA hypomethylation occurs mainly at heavily methylated noncoding regions of DNA, particularly repeat sequences and transposable elements [40, 41].

In humans, nearly 80% CpG islands occur in transposon-derived sequences, throughout the genome, such as long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs) [42]. LINE-1 is the largest member of the LINE family with more than 500,000 copies comprising approximately 17% of the genome [43]. CpG islands within LINE-1 sequences and their methylation levels correlate with the global genomic DNA methylation level [44, 45], so LINE-1 methylation has been widely used as a surrogate marker of global genomic DNA methylation [46], and methylation status of LINE-1 in white blood cells (WBC) is a potential biomarker in a variety of diseases [4, 45–48] in research on cancer, cardiovascular, neurodegenerative, and CAs [3–6, 48–51]. Human genome has on average 80–100 active LINE-1, and it has been estimated that new LINE-1 insertion in genome occurs in at least 1 in every 50 humans within a parental germ cell or during early fetal development [40]. Thus, LINE-1 hypomethylation in the parental germline, along with altered miRNA expression, might also significantly affect genome stability during the fetal development [52, 53].

#### **3. DNA methylation during gametogenesis and embryogenesis**

DNA methylation changes are particularly dynamic in gametogenesis and early embryogenesis. During the course of mammalian differentiation and development, DNA methylation undergoes remodeling to eventually generate the cell type-specific methylation patterns, found in somatic cells of adults. During the

gametogenesis, DNA is demethylated within each developing germ cell and then remethylated/reset to the methylation patterns specific to gametogenesis. The differentially methylated regions (DMRs) are sperm and egg specific [54, 55]. This process establishing the specific methylation of imprinted loci before fertilization, as well as other non-imprinted loci, may also be subject to at least partial erasure of methylation during gametogenesis [56–58]. The zygotic DNA demethylation after fertilization in mouse embryogenesis affects parental genome on a genome-wide level including single gene loci and repetitive elements. The maternal genome-wide methylation is unaffected [59]. This process changes the methylation patterns of the gametes and establishes the DNA methylation patterns found in somatic differentiated cells in adults through induced expression of DNMT and de novo methylation of genome in post-implantation mouse embryos [60–62]. It has been shown that in small studies of human embryos, there is a demethylation process at the 4-cell stage followed by remethylation at late morula [63]. Even more, expression patterns of DNMTs after cryopreservation of human embryos could be disturbed and could have long-term developmental consequences [64] that suggest the importance of DNA methylation program maintenance during development. Periods during gametogenesis and embryogenesis may also present windows of opportunity for environmental influences on DNA methylation pattern. The DMRs are established during gametogenesis at imprinted and non-imprinted loci and are susceptible to environmental factors [65, 66]. LINE-1 methylation in sperm could be a risk marker of infertility in man at nicotine/alcohol exposure [67]. It is also possible to alter DNA methylation levels and patterns within intact mammalian cells by treatment with various chemical inhibitors, DNA-demethylating drugs, which have recently been introduced as potential therapeutic agents for the treatment of human diseases, particularly myelodysplastic syndromes [68].

The dynamic reprogramming and other epigenetic patterns which could affect normal patterns of gene expression/genome stability during development could lead to an increased risk of CAs or complex diseases later in life [65–67].

#### **4. LINE-1 DNA methylation and environmental influences (e.g., diet and nutrition)**

Previous research was focused on the effect of specific foods on the DNA methylation process, but there is currently growing interest in determining how dietary patterns may affect global and local DNA methylation in humans. There are some studies that suggest that frequent use of vegetables and/or fruits decreased the risk of LINE-1 hypomethylation [69–71]. Biological explanation could be in beneficial modulation of pathways involved in epigenetic mechanisms by intake of high variety of nutritive and bioactive substances included in fruit- and vegetablerich food. These components were polyphenols; flavonoids; carotenoids; folates; vitamins C, E, and A; minerals; and fibers [72, 73]. As it is known that many crucial cellular processes depend on folate, including DNA methylation [74], low folate intake in daily food could be supplemented by synthetic form as folic acid (FA) and through fortification programs [75]. Even more, harmful effect of particulate matter exposure on LINE-1 methylation level could be counteracted by healthy food consumption such as Mediterranean diet [76]. Also, fatty acids can modify DNA methylation in vitro, but limited information is available from human studies. Some studies observed that intake of vegetable oil/dietary fat seemed to be negatively correlated with LINE-1 methylation [69, 77]. Others show no changes in methylation profile after supplementation with grape seed flavanols [78]. The interindividual variation in blood cell DNA methylation in interventional studies, which are usually

**43**

*Global DNA Methylation as a Potential Underlying Mechanism of Congenital Disease…*

**4.1 Methylenetetrahydrofolate reductase (MTHFR), folate metabolism,** 

**4.2 Epigenetic, genetic, and nutrigenomic risk factors for congenital** 

**diseases: DNA methylation, global DNA methylation, miRNA, MTHFR** 

Low folate status (as defined by various measures including blood folate concentrations, folate intake, and/or FA intake) has been associated with an increased risk of cardiovascular disease, cancers, CAs, CHD, and NTD [5, 6, 89–94]. Also, this deficiency is clearly detrimental to the embryo and shows possible longer-term risks of diabetes or other health outcomes and health problems associated with child mortality and morbidity [95]. Periconceptional supplementation of FA also reduces the risk of congenital heart diseases (previous ref) and preterm birth and low birth weight [96, 97]. The prevalence of neural tube defects (NTDs) has been significantly lowered in more than 70 countries worldwide by applying fortification with FA, but in all European governments there is still an issue with FA fortification of centrally processed and widely eaten foods in prevention of unwanted birth outcomes [98]. The mechanisms by which low folate status contributes to these disorders have not been understood completely but, to a certain extent, could be explained by different molecular pathways. Folate depletion could be a destabilizing factor during DNA replication. If inadequate folate availability is present during cell division, the production of

Folate can be a limiting factor in many biological reactions. The methylene tetrahydrofolate reductase (MTHFR) is an enzyme important for the folate metabolism which is in the basis of the DNA, RNA, and protein methylation. Genomic DNA methylation directly correlates with folate status and inversely with plasma homocysteine (tHcy) levels [79–82]. The one-carbon pathway and thus DNA methylation function under tight regulatory controls. S-Adenosyl methionine (SAM) is the major regulator of folate-dependent Hcy remethylation because it is a potent inhibitor of MTHFR. When the SAM concentration is high, MTHFR is inhibited and hence remethylation of homocysteine. Conversely, if SAM concentrations are low, remethylation of homocysteine is favored. Hyperhomocysteinemia is an emerging risk factor for various cardiovascular diseases, and, with the increasing significance of this genetic variant in the view of morbidity and mortality impact on the patients, further prevention strategies and nutritional recommendations with the supplementation of folate would be necessary as part of future health education. Other essential nutrients that are naturally present in some foods or as dietary supplement, like vitamin B6, B12, B2, and choline, are necessary in addition to folate to maintain DNA methylation [83]. It is also recognized that S-adenosylhomocysteine (SAH) functions as a potent product inhibitor of SAMdependent methyltransferases [84]. For this reason, continual hydrolysis of SAH to homocysteine is important for DNA methylation [85]. Plasma homocysteine elevation has been associated with increased concentration of SAH, and increased SAH was in correlation with global DNA hypomethylation [86]. Methionine is the substrate for SAM, a cofactor and methyl group donor for numerous methylation reactions including the methylation of DNA, RNA, and histones [87]. A number of SAM-dependent reactions have regulatory roles by affecting both, genome stability

subtle changes in DNA methylation in response to dietary factors.

rather small, demands studies with larger sample size to avoid masking the possibly

*DOI: http://dx.doi.org/10.5772/intechopen.90996*

**and its role in DNA methylation**

and gene transcription [88].

**polymorphism, and low folate status**

*Global DNA Methylation as a Potential Underlying Mechanism of Congenital Disease… DOI: http://dx.doi.org/10.5772/intechopen.90996*

rather small, demands studies with larger sample size to avoid masking the possibly subtle changes in DNA methylation in response to dietary factors.

#### **4.1 Methylenetetrahydrofolate reductase (MTHFR), folate metabolism, and its role in DNA methylation**

Folate can be a limiting factor in many biological reactions. The methylene tetrahydrofolate reductase (MTHFR) is an enzyme important for the folate metabolism which is in the basis of the DNA, RNA, and protein methylation. Genomic DNA methylation directly correlates with folate status and inversely with plasma homocysteine (tHcy) levels [79–82]. The one-carbon pathway and thus DNA methylation function under tight regulatory controls. S-Adenosyl methionine (SAM) is the major regulator of folate-dependent Hcy remethylation because it is a potent inhibitor of MTHFR. When the SAM concentration is high, MTHFR is inhibited and hence remethylation of homocysteine. Conversely, if SAM concentrations are low, remethylation of homocysteine is favored. Hyperhomocysteinemia is an emerging risk factor for various cardiovascular diseases, and, with the increasing significance of this genetic variant in the view of morbidity and mortality impact on the patients, further prevention strategies and nutritional recommendations with the supplementation of folate would be necessary as part of future health education. Other essential nutrients that are naturally present in some foods or as dietary supplement, like vitamin B6, B12, B2, and choline, are necessary in addition to folate to maintain DNA methylation [83]. It is also recognized that S-adenosylhomocysteine (SAH) functions as a potent product inhibitor of SAMdependent methyltransferases [84]. For this reason, continual hydrolysis of SAH to homocysteine is important for DNA methylation [85]. Plasma homocysteine elevation has been associated with increased concentration of SAH, and increased SAH was in correlation with global DNA hypomethylation [86]. Methionine is the substrate for SAM, a cofactor and methyl group donor for numerous methylation reactions including the methylation of DNA, RNA, and histones [87]. A number of SAM-dependent reactions have regulatory roles by affecting both, genome stability and gene transcription [88].

#### **4.2 Epigenetic, genetic, and nutrigenomic risk factors for congenital diseases: DNA methylation, global DNA methylation, miRNA, MTHFR polymorphism, and low folate status**

Low folate status (as defined by various measures including blood folate concentrations, folate intake, and/or FA intake) has been associated with an increased risk of cardiovascular disease, cancers, CAs, CHD, and NTD [5, 6, 89–94]. Also, this deficiency is clearly detrimental to the embryo and shows possible longer-term risks of diabetes or other health outcomes and health problems associated with child mortality and morbidity [95]. Periconceptional supplementation of FA also reduces the risk of congenital heart diseases (previous ref) and preterm birth and low birth weight [96, 97]. The prevalence of neural tube defects (NTDs) has been significantly lowered in more than 70 countries worldwide by applying fortification with FA, but in all European governments there is still an issue with FA fortification of centrally processed and widely eaten foods in prevention of unwanted birth outcomes [98]. The mechanisms by which low folate status contributes to these disorders have not been understood completely but, to a certain extent, could be explained by different molecular pathways. Folate depletion could be a destabilizing factor during DNA replication. If inadequate folate availability is present during cell division, the production of

*DNA Methylation Mechanism*

gametogenesis, DNA is demethylated within each developing germ cell and then remethylated/reset to the methylation patterns specific to gametogenesis. The differentially methylated regions (DMRs) are sperm and egg specific [54, 55]. This process establishing the specific methylation of imprinted loci before fertilization, as well as other non-imprinted loci, may also be subject to at least partial erasure of methylation during gametogenesis [56–58]. The zygotic DNA demethylation after fertilization in mouse embryogenesis affects parental genome on a genome-wide level including single gene loci and repetitive elements. The maternal genome-wide methylation is unaffected [59]. This process changes the methylation patterns of the gametes and establishes the DNA methylation patterns found in somatic differentiated cells in adults through induced expression of DNMT and de novo methylation of genome in post-implantation mouse embryos [60–62]. It has been shown that in small studies of human embryos, there is a demethylation process at the 4-cell stage followed by remethylation at late morula [63]. Even more, expression patterns of DNMTs after cryopreservation of human embryos could be disturbed and could have long-term developmental consequences [64] that suggest the importance of DNA methylation program maintenance during development. Periods during gametogenesis and embryogenesis may also present windows of opportunity for environmental influences on DNA methylation pattern. The DMRs are established during gametogenesis at imprinted and non-imprinted loci and are susceptible to environmental factors [65, 66]. LINE-1 methylation in sperm could be a risk marker of infertility in man at nicotine/alcohol exposure [67]. It is also possible to alter DNA methylation levels and patterns within intact mammalian cells by treatment with various chemical inhibitors, DNA-demethylating drugs, which have recently been introduced as potential therapeutic agents for the treatment of human dis-

The dynamic reprogramming and other epigenetic patterns which could affect normal patterns of gene expression/genome stability during development could

lead to an increased risk of CAs or complex diseases later in life [65–67].

Previous research was focused on the effect of specific foods on the DNA methylation process, but there is currently growing interest in determining how dietary patterns may affect global and local DNA methylation in humans. There are some studies that suggest that frequent use of vegetables and/or fruits decreased the risk of LINE-1 hypomethylation [69–71]. Biological explanation could be in beneficial modulation of pathways involved in epigenetic mechanisms by intake of high variety of nutritive and bioactive substances included in fruit- and vegetablerich food. These components were polyphenols; flavonoids; carotenoids; folates; vitamins C, E, and A; minerals; and fibers [72, 73]. As it is known that many crucial cellular processes depend on folate, including DNA methylation [74], low folate intake in daily food could be supplemented by synthetic form as folic acid (FA) and through fortification programs [75]. Even more, harmful effect of particulate matter exposure on LINE-1 methylation level could be counteracted by healthy food consumption such as Mediterranean diet [76]. Also, fatty acids can modify DNA methylation in vitro, but limited information is available from human studies. Some studies observed that intake of vegetable oil/dietary fat seemed to be negatively correlated with LINE-1 methylation [69, 77]. Others show no changes in methylation profile after supplementation with grape seed flavanols [78]. The interindividual variation in blood cell DNA methylation in interventional studies, which are usually

**4. LINE-1 DNA methylation and environmental influences** 

eases, particularly myelodysplastic syndromes [68].

**(e.g., diet and nutrition)**

**42**

thymidine could be compromised and may be substituted in the DNA sequence by uracil. This mutagenic event may trigger the defect in an effort to repair DNA and increase the frequency of chromosomal breaks [90]. Low FA in tissue culture has been shown to result in the formation of micronuclei (chromosome breakage) and that the presence of MTHFR C677T polymorphism (TT genotype) increases the micronuclei formation, under the low folate conditions [99]. This MTHFR polymorphism was associated with various diseases, and allele frequencies vary depending on ethnicity (reviewed in [100]). This gene is mapped on chromosome 1 (1p36.6), and the genetic variant assigned as C677T (rs1801133) is located in exon 4 in this gene. This polymorphism results in the conversion at codon 222, valine to alanine. Carriers of the T allele have lower enzyme activity [101]. The MTHFR 677TT homozygous subjects have higher homocysteine levels than the normal, non-mutated controls. To date, most studies have shown that the *MTHFR* C677T genotype is related to biomarkers, such as serum folate, tHcy concentration, and folate intake. Elevated blood tHcy is a well-recognized and modifiable risk factor for cerebral and cardiovascular disease [101, 102]. Reduction of the enzyme activity leads to elevated Hcy concentrations [103]. The TT genotype has been associated with elevated tHcy levels in populations with low folate intake [104]. Previous tHcy-lowering trials have not considered whether and to what extent these factors could modify the efficacy of folic acid (FA) treatment. In some countries with folate fortification like America, Australia, and New Zealand, the effect of TT genotype is not so obvious like in Asia region where folate intake is low [94]. In those who are homozygous for the mutation (TT genotype), enzyme function is only 30% of normal, and data provide evidence that nutrition can counteract genetic susceptibility. Recently, large, randomized trial in a population without mandatory FA fortification demonstrated that the adverse effect of the TT genotype on tHcy levels can be ameliorated by raising serum folate levels above the threshold (15 ng/mg or 34 nmol/L) via FA treatment and it provides new evidence to support a personalized FA treatment [94]. The gene-nutrient interaction between MTHFR C677T variant and folate status was also observed on the risk of anencephaly. Mothers with 677TT genotype with serum folate levels in the upper tercile (>14.1 ng/ml) had a 95% lower risk to have a child with anencephaly than mothers with serum folate levels in the first and second terciles [92]. Results about DS and MTHFRC677T polymorphism as a risk factor of its occurrence are still conflicting. The recent meta-analysis suggested that MTHFR 677T is a major risk factor for DS birth [105], while previous smaller studies did not recognize such risk [106, 107]. Studies performed analyzing peripheral lymphocytes of women with DS offspring revealed several markers of global genome instability, including an increased frequency of micronuclei, shorter telomeres, and impaired DNA methylation at MTHFR promoter [108, 109]. Hypermethylation of MTHFR promoter may lead to CHD in DS subjects [109]. Functional inactivation of MTHFR gene expression could be a mechanism of impaired folate metabolism, which is known to play a role in chromosomal breakage, abnormal chromosomal segregation, and genomic instability and therefore a developmental defect in the CHD in DS. Another suggested mechanism is lower LINE-1 methylation, the surrogate marker for global methylation levels, in young mothers of DS compared to controls, suggesting the possibility of impaired DNA methylation causing maternally derived trisomy 21 [6]. Also, there is evidence from intervention studies of the effects of dietary factors, where FA was the most common intervention agent (33%). Meta-regression analysis showed that the dose of supplementary FA was the only identified factor (p < 0.001) showing a positive relationship with DNA methylation patterns in humans [93]. MTHFR genotype-dependent association between lower global DNA methylation and lower plasma folate concentration

**45**

ASD [126].

CHD<sup>+</sup>

**and diet**

later life [118–121].

twins were observed [112].

nutrition) [21, 113] and paternal diet [114, 115].

*Global DNA Methylation as a Potential Underlying Mechanism of Congenital Disease…*

was detected in observational studies in healthy subjects [81, 82, 110]. Global DNA methylation at maternal front (p = 0.04) and hypomethylation of MTHFR gene at fetal front (p = 0.001) might be a characteristic of preeclampsia [111]. The combination of MTHFR C677T genotype and diet significantly influenced global DNA methylation in mothers with DS children. The lowest values of global DNA methylation were observed in mothers with MTHFR 677 CT+TT genotype and low dietary folate [6]. Even more, recently the association between maternal LINE-1 methylation and the occurrence of CHD in children with DS was shown, as well as the impact of endogenous maternal factors (*MTHFR* C677T polymorphism) and exogenous maternal factors (body mass index and dietary habits such as folate intake) on maternal LINE-1 methylation and on the occurrence of CHD in children with DS. Study showed that the *MTHFR* genotype/diet combination and BMI were significantly associated with LINE-1 methylation in mothers of children with DS/

[5]. Recently, micro-RNA signatures discordant for CHD in monozygotic

**4.3 DNA methylation in developmental exposure to the maternal environment** 

It has been suggested that disease risk of long-term health outcomes may be in part determined by maternal (in utero effects of environmental exposures, toxins/

Birth defects occur in 6–10% of babies born to mothers with pregestational diabetes, which is a significant health problem. It has been demonstrated that exposure to maternal diabetes during pregnancy changes gene expression levels in the mouse embryo, disrupting essential cellular activities [116], and could lead to disruption of crucial epithelial and mesenchymal cell interactions in developing kidney, leading to kidney and urinary tract malformation [117]. Underlying mechanisms are still unknown. There is a proposed lack of precision in the developmental program, which is essential for organogenesis induced by hyperglycemia effects on oxidative stress. That exposure to a diabetic intrauterine environment during pregnancy could be teratogenic by leading to defects like CAKUT in the fetus and associate with metabolic or cardiovascular diseases in

Changes in maternal dietary FA can affect the DNA methylation patterns of offspring in mice [61]. The *agouti* mouse is a best-studied example [122]. Recently, in the human genome, loci were found to show differential methylation in response to season of birth that is similar to the *agouti* locus, but the identity of the causative agent for the changes in DNA methylation is unclear [123]. Recent study examined the prospective association between multivitamin supplementation during pregnancy and maternal plasma folate/vitamin B12 levels at birth and child's autism spectrum disorder (ASD) risk. Moderate (3–5 times/week) self-reported supplementation during pregnancy was associated with decreased risk of ASD, consistent with previous findings. But, extremely high maternal plasma folate and B12 levels at birth were associated with ASD risk. This study raises new questions about the impact of extremely elevated levels of plasma folate and B12 exposure in utero on early brain development [124]. However, study on postmortem cortical brain samples reveals that global DNA methylation was markedly enriched in ASD brains [125]. In some diseases, methylation mosaicism was found to be present. This is a common phenomenon in Fragile X syndrome (FXS). A decreased gene expression was found to be a main contributor to the cognitive impairment observed in the study of 12 FXS males with atypical mosaicism, seven of whom presented with

*DOI: http://dx.doi.org/10.5772/intechopen.90996*

*Global DNA Methylation as a Potential Underlying Mechanism of Congenital Disease… DOI: http://dx.doi.org/10.5772/intechopen.90996*

was detected in observational studies in healthy subjects [81, 82, 110]. Global DNA methylation at maternal front (p = 0.04) and hypomethylation of MTHFR gene at fetal front (p = 0.001) might be a characteristic of preeclampsia [111]. The combination of MTHFR C677T genotype and diet significantly influenced global DNA methylation in mothers with DS children. The lowest values of global DNA methylation were observed in mothers with MTHFR 677 CT+TT genotype and low dietary folate [6]. Even more, recently the association between maternal LINE-1 methylation and the occurrence of CHD in children with DS was shown, as well as the impact of endogenous maternal factors (*MTHFR* C677T polymorphism) and exogenous maternal factors (body mass index and dietary habits such as folate intake) on maternal LINE-1 methylation and on the occurrence of CHD in children with DS. Study showed that the *MTHFR* genotype/diet combination and BMI were significantly associated with LINE-1 methylation in mothers of children with DS/ CHD<sup>+</sup> [5]. Recently, micro-RNA signatures discordant for CHD in monozygotic twins were observed [112].

#### **4.3 DNA methylation in developmental exposure to the maternal environment and diet**

It has been suggested that disease risk of long-term health outcomes may be in part determined by maternal (in utero effects of environmental exposures, toxins/ nutrition) [21, 113] and paternal diet [114, 115].

Birth defects occur in 6–10% of babies born to mothers with pregestational diabetes, which is a significant health problem. It has been demonstrated that exposure to maternal diabetes during pregnancy changes gene expression levels in the mouse embryo, disrupting essential cellular activities [116], and could lead to disruption of crucial epithelial and mesenchymal cell interactions in developing kidney, leading to kidney and urinary tract malformation [117]. Underlying mechanisms are still unknown. There is a proposed lack of precision in the developmental program, which is essential for organogenesis induced by hyperglycemia effects on oxidative stress. That exposure to a diabetic intrauterine environment during pregnancy could be teratogenic by leading to defects like CAKUT in the fetus and associate with metabolic or cardiovascular diseases in later life [118–121].

Changes in maternal dietary FA can affect the DNA methylation patterns of offspring in mice [61]. The *agouti* mouse is a best-studied example [122]. Recently, in the human genome, loci were found to show differential methylation in response to season of birth that is similar to the *agouti* locus, but the identity of the causative agent for the changes in DNA methylation is unclear [123]. Recent study examined the prospective association between multivitamin supplementation during pregnancy and maternal plasma folate/vitamin B12 levels at birth and child's autism spectrum disorder (ASD) risk. Moderate (3–5 times/week) self-reported supplementation during pregnancy was associated with decreased risk of ASD, consistent with previous findings. But, extremely high maternal plasma folate and B12 levels at birth were associated with ASD risk. This study raises new questions about the impact of extremely elevated levels of plasma folate and B12 exposure in utero on early brain development [124]. However, study on postmortem cortical brain samples reveals that global DNA methylation was markedly enriched in ASD brains [125]. In some diseases, methylation mosaicism was found to be present. This is a common phenomenon in Fragile X syndrome (FXS). A decreased gene expression was found to be a main contributor to the cognitive impairment observed in the study of 12 FXS males with atypical mosaicism, seven of whom presented with ASD [126].

*DNA Methylation Mechanism*

thymidine could be compromised and may be substituted in the DNA sequence by uracil. This mutagenic event may trigger the defect in an effort to repair DNA and increase the frequency of chromosomal breaks [90]. Low FA in tissue culture has been shown to result in the formation of micronuclei (chromosome breakage) and that the presence of MTHFR C677T polymorphism (TT genotype) increases the micronuclei formation, under the low folate conditions [99]. This MTHFR polymorphism was associated with various diseases, and allele frequencies vary depending on ethnicity (reviewed in [100]). This gene is mapped on chromosome 1 (1p36.6), and the genetic variant assigned as C677T (rs1801133) is located in exon 4 in this gene. This polymorphism results in the conversion at codon 222, valine to alanine. Carriers of the T allele have lower enzyme activity [101]. The MTHFR 677TT homozygous subjects have higher homocysteine levels than the normal, non-mutated controls. To date, most studies have shown that the *MTHFR* C677T genotype is related to biomarkers, such as serum folate, tHcy concentration, and folate intake. Elevated blood tHcy is a well-recognized and modifiable risk factor for cerebral and cardiovascular disease [101, 102]. Reduction of the enzyme activity leads to elevated Hcy concentrations [103]. The TT genotype has been associated with elevated tHcy levels in populations with low folate intake [104]. Previous tHcy-lowering trials have not considered whether and to what extent these factors could modify the efficacy of folic acid (FA) treatment. In some countries with folate fortification like America, Australia, and New Zealand, the effect of TT genotype is not so obvious like in Asia region where folate intake is low [94]. In those who are homozygous for the mutation (TT genotype), enzyme function is only 30% of normal, and data provide evidence that nutrition can counteract genetic susceptibility. Recently, large, randomized trial in a population without mandatory FA fortification demonstrated that the adverse effect of the TT genotype on tHcy levels can be ameliorated by raising serum folate levels above the threshold (15 ng/mg or 34 nmol/L) via FA treatment and it provides new evidence to support a personalized FA treatment [94]. The gene-nutrient interaction between MTHFR C677T variant and folate status was also observed on the risk of anencephaly. Mothers with 677TT genotype with serum folate levels in the upper tercile (>14.1 ng/ml) had a 95% lower risk to have a child with anencephaly than mothers with serum folate levels in the first and second terciles [92]. Results about DS and MTHFRC677T polymorphism as a risk factor of its occurrence are still conflicting. The recent meta-analysis suggested that MTHFR 677T is a major risk factor for DS birth [105], while previous smaller studies did not recognize such risk [106, 107]. Studies performed analyzing peripheral lymphocytes of women with DS offspring revealed several markers of global genome instability, including an increased frequency of micronuclei, shorter telomeres, and impaired DNA methylation at MTHFR promoter [108, 109]. Hypermethylation of MTHFR promoter may lead to CHD in DS subjects [109]. Functional inactivation of MTHFR gene expression could be a mechanism of impaired folate metabolism, which is known to play a role in chromosomal breakage, abnormal chromosomal segregation, and genomic instability and therefore a developmental defect in the CHD in DS. Another suggested mechanism is lower LINE-1 methylation, the surrogate marker for global methylation levels, in young mothers of DS compared to controls, suggesting the possibility of impaired DNA methylation causing maternally derived trisomy 21 [6]. Also, there is evidence from intervention studies of the effects of dietary factors, where FA was the most common intervention agent (33%). Meta-regression analysis showed that the dose of supplementary FA was the only identified factor (p < 0.001) showing a positive relationship with DNA methylation patterns in humans [93]. MTHFR genotype-dependent association between lower global DNA methylation and lower plasma folate concentration

**44**

#### **5. Epigenetic pattern transmission from parent to offspring: understanding disease inheritance**

The heritability of epigenetic modifications, including histone modifications and DNA methylation, provides a memory of cell function and identity. Transmission of epigenetic information to subsequent generations may provide evolutionary mechanisms that impact on adaptation to changed environment. Defining the mechanisms that establish and regulate the transmission of epigenetic information from parent to offspring is critical for understanding disease heredity. Detection of modified methylation patterns is important in inappropriate imprinting of certain either maternal or paternal genes, which are "turned on" by epigenetic phenomenon that leads to diseases such as Angelman syndrome and Prader-Willi syndrome. Methylation patterns with detrimental effects on development have been established for disorders of methylation, by several groups of researchers [127, 128]. One of the developed blood tests (EpiSign) claims to diagnose 19 congenital diseases [129]. Also, it is important to establish the potential for epigenomic drugs that have an impact on the germline epigenome and subsequent offspring [130, 131]. Currently, the molecular pathways that regulate epigenetic information in the germline and its transmission to offspring are poorly understood. Recent study reveals a novel role for the histone-modifying complex, PRC2, in maternal intergenerational transmission of epigenetic effects on offspring, with important implications for understanding disease inheritance [115]. PRC2 is involved in the regulation of many fundamental biological processes and is especially essential for embryonic stem cells. However, how the formation and function of PRC2 are regulated is mostly unknown. Recent findings identify miR-323-3p as a new regulator for PRC2, providing a new approach for regulating PRC2 activity via microRNAs [132]. Specific epigenetic pattern was observed to be essential in the development of CHD and CAKUT. Impaired transcriptional profiles in individuals with CHDs [133] and CAKUT [134, 135] were shown to be affected by epigenetic regulators of gene expression, using bioinformatical analysis and integrated prediction algorithms [136]. The miRNA-145 expression was confirmed in infants with CHD that negatively regulates gene expression important for heart development [133]. The altered hsa-miR-144 expression was, for the first time, identified in CAKUT and could be connected with biological processes crucial for normal development of kidney and urinary tract [135]. Although the importance of mothers' health prior to conception and during pregnancy is now well accepted, recent data also implicate fathers' health/nutritional status (overnutrition, undernutrition, and other forms of stress) in contribution to the risk of metabolic disease and obesity in offspring. Epigenetic paternal inheritance of chronic disease provides novel opportunities for multigenerational disease prevention [137]. Germ cell-dependent mechanisms have recently been linked to these intergenerational effects. There is increasing evidence that disruptions in male germ cell epigenetic reprogramming are associated with offspring abnormalities. Adequate supply of methyl donors is required in the fetal period, which is the critical time of DNA methylation pattern acquisition for developing male germ cells. In addition, DNA methylation patterns continue to be remodeled postnatal during spermatogenesis. Previous studies have shown that lifetime (prenatal and postnatal) folic acid deficiency and high-dose supplementation can alter the DNA methylation in sperm [138]. Recent study examined the genome-wide DNA methylation patterns in placentas and embryos in correlation with maternal FA supplementation in the prevention of CAs associated with assisted reproductive technologies (ART). Results demonstrate dose-dependent and sex-specific effects of FA intake; moderate dose of FA supplements may be optimal in ART for both sexes [139]. Even more, recent data suggest that genome-wide DNA methylation in the

**47**

**7. Conclusion**

*Global DNA Methylation as a Potential Underlying Mechanism of Congenital Disease…*

consequence of epigenomic regulation of gene expression [141].

for a diagnostic and decreasing cost of patients' treatment.

The heritability of epigenetic modifications, including histone modifications and DNA methylation, provides a memory of cell function and identity. The dynamic reprogramming and other epigenetic patterns which could affect normal patterns of gene expression/genome stability during development could lead to an increased risk of CAs or complex diseases later in life. The sperm- and egg-specific DMT established during gametogenesis at imprint and non-imprint loci are susceptible to environmental factors. Embryogenesis may also present a window of opportunity for environmental influences on DNA methylation pattern. Changes in maternal dietary FA can affect the DNA methylation of offspring that could affect CA development. LINE-1 hypomethylation in the parental germline might also significantly affect genome stability during the fetal development. The *MTHFR* T carriers have lower enzyme activity, and dose of supplementary FA shows a positive relationship with DNA methylation patterns in humans. The lowest values of LINE-1 methylation, the surrogate marker for global DNA methylation,

placentas from preterm infants could be associated with maternal socioeconomic status [140]. On the other hand, genomic information was identical in monozygotic twins, but they could be discordant for congenital renal agenesis which could be a

CAs are complex traits with polygenic, epigenetic, and environmental components. Advances in human DNA methylation research and growing epigenetic data offer a new avenue for the translation of research to clinical applications. Current methylome analysis has been helpful in major human diseases revealing an epigenetic influence, but current approaches are inadequate for the translation of these advances to clinical diagnostics. There is a need to deal with big data in modern genomic medicine, so bioinformatics and applied mathematics are of a fundamental help in simulation studies and tests of methylome datasets. Signal detection theory and machine learning approaches applied on methylome datasets from ASD patients demonstrate high discriminatory power for the methylation signal induced by disease [142]. Even more, advanced machine learning analysis includes a combination of active learning and imbalanced class learning and deep learning to develop a more efficient feature selection process and for the generation and simultaneous computation of any genomic or biological dataset applied to medicine [143]. This approach demonstrates the feasibility in clinical diagnostics. Genetic risk scores (GRS) are widely used for risk prediction in complex diseases. Evidence is growing that methylation risk scores (MRS) may be constructed for multiple health purposes. MRS is defined as weighted sums of the individual's methylation markers' beta values of a preselected number of CpG sites and can be useful in interaction and mediation analyses, for environmental exposures as biomarker, and for prediction of individual risks of disease predisposition or treatment success [144]. As we know that methylation data is specific (for different tissues) and sensitive to confounding factor, e.g., by age or sex, adaption of current GRS approaches is complex and needs deep profiling in construction of such risk scores. The analysis of whole biomarker genomic and epigenomic regions and prediction of disease predisposition, course and therapy response by risk scores could in future suffice

*DOI: http://dx.doi.org/10.5772/intechopen.90996*

**6. Future perspectives**

placentas from preterm infants could be associated with maternal socioeconomic status [140]. On the other hand, genomic information was identical in monozygotic twins, but they could be discordant for congenital renal agenesis which could be a consequence of epigenomic regulation of gene expression [141].

#### **6. Future perspectives**

*DNA Methylation Mechanism*

**understanding disease inheritance**

**5. Epigenetic pattern transmission from parent to offspring:** 

The heritability of epigenetic modifications, including histone modifications and DNA methylation, provides a memory of cell function and identity. Transmission of epigenetic information to subsequent generations may provide evolutionary mechanisms that impact on adaptation to changed environment. Defining the mechanisms that establish and regulate the transmission of epigenetic information from parent to offspring is critical for understanding disease heredity. Detection of modified methylation patterns is important in inappropriate imprinting of certain either maternal or paternal genes, which are "turned on" by epigenetic phenomenon that leads to diseases such as Angelman syndrome and Prader-Willi syndrome. Methylation patterns with detrimental effects on development have been established for disorders of methylation, by several groups of researchers [127, 128]. One of the developed blood tests (EpiSign) claims to diagnose 19 congenital diseases [129]. Also, it is important to establish the potential for epigenomic drugs that have an impact on the germline epigenome and subsequent offspring [130, 131]. Currently, the molecular pathways that regulate epigenetic information in the germline and its transmission to offspring are poorly understood. Recent study reveals a novel role for the histone-modifying complex, PRC2, in maternal intergenerational transmission of epigenetic effects on offspring, with important implications for understanding disease inheritance [115]. PRC2 is involved in the regulation of many fundamental biological processes and is especially essential for embryonic stem cells. However, how the formation and function of PRC2 are regulated is mostly unknown. Recent findings identify miR-323-3p as a new regulator for PRC2, providing a new approach for regulating PRC2 activity via microRNAs [132]. Specific epigenetic pattern was observed to be essential in the development of CHD and CAKUT. Impaired transcriptional profiles in individuals with CHDs [133] and CAKUT [134, 135] were shown to be affected by epigenetic regulators of gene expression, using bioinformatical analysis and integrated prediction algorithms [136]. The miRNA-145 expression was confirmed in infants with CHD that negatively regulates gene expression important for heart development [133]. The altered hsa-miR-144 expression was, for the first time, identified in CAKUT and could be connected with biological processes crucial for normal development of kidney and urinary tract [135]. Although the importance of mothers' health prior to conception and during pregnancy is now well accepted, recent data also implicate fathers' health/nutritional status (overnutrition, undernutrition, and other forms of stress) in contribution to the risk of metabolic disease and obesity in offspring. Epigenetic paternal inheritance of chronic disease provides novel opportunities for multigenerational disease prevention [137]. Germ cell-dependent mechanisms have recently been linked to these intergenerational effects. There is increasing evidence that disruptions in male germ cell epigenetic reprogramming are associated with offspring abnormalities. Adequate supply of methyl donors is required in the fetal period, which is the critical time of DNA methylation pattern acquisition for developing male germ cells. In addition, DNA methylation patterns continue to be remodeled postnatal during spermatogenesis. Previous studies have shown that lifetime (prenatal and postnatal) folic acid deficiency and high-dose supplementation can alter the DNA methylation in sperm [138]. Recent study examined the genome-wide DNA methylation patterns in placentas and embryos in correlation with maternal FA supplementation in the prevention of CAs associated with assisted reproductive technologies (ART). Results demonstrate dose-dependent and sex-specific effects of FA intake; moderate dose of FA supplements may be optimal in ART for both sexes [139]. Even more, recent data suggest that genome-wide DNA methylation in the

**46**

CAs are complex traits with polygenic, epigenetic, and environmental components. Advances in human DNA methylation research and growing epigenetic data offer a new avenue for the translation of research to clinical applications. Current methylome analysis has been helpful in major human diseases revealing an epigenetic influence, but current approaches are inadequate for the translation of these advances to clinical diagnostics. There is a need to deal with big data in modern genomic medicine, so bioinformatics and applied mathematics are of a fundamental help in simulation studies and tests of methylome datasets. Signal detection theory and machine learning approaches applied on methylome datasets from ASD patients demonstrate high discriminatory power for the methylation signal induced by disease [142]. Even more, advanced machine learning analysis includes a combination of active learning and imbalanced class learning and deep learning to develop a more efficient feature selection process and for the generation and simultaneous computation of any genomic or biological dataset applied to medicine [143]. This approach demonstrates the feasibility in clinical diagnostics. Genetic risk scores (GRS) are widely used for risk prediction in complex diseases. Evidence is growing that methylation risk scores (MRS) may be constructed for multiple health purposes. MRS is defined as weighted sums of the individual's methylation markers' beta values of a preselected number of CpG sites and can be useful in interaction and mediation analyses, for environmental exposures as biomarker, and for prediction of individual risks of disease predisposition or treatment success [144]. As we know that methylation data is specific (for different tissues) and sensitive to confounding factor, e.g., by age or sex, adaption of current GRS approaches is complex and needs deep profiling in construction of such risk scores. The analysis of whole biomarker genomic and epigenomic regions and prediction of disease predisposition, course and therapy response by risk scores could in future suffice for a diagnostic and decreasing cost of patients' treatment.

#### **7. Conclusion**

The heritability of epigenetic modifications, including histone modifications and DNA methylation, provides a memory of cell function and identity. The dynamic reprogramming and other epigenetic patterns which could affect normal patterns of gene expression/genome stability during development could lead to an increased risk of CAs or complex diseases later in life. The sperm- and egg-specific DMT established during gametogenesis at imprint and non-imprint loci are susceptible to environmental factors. Embryogenesis may also present a window of opportunity for environmental influences on DNA methylation pattern. Changes in maternal dietary FA can affect the DNA methylation of offspring that could affect CA development. LINE-1 hypomethylation in the parental germline might also significantly affect genome stability during the fetal development. The *MTHFR* T carriers have lower enzyme activity, and dose of supplementary FA shows a positive relationship with DNA methylation patterns in humans. The lowest values of LINE-1 methylation, the surrogate marker for global DNA methylation,

were observed in mothers with MTHFR 677 CT+TT genotype and low dietary folate, suggesting the possibility of impaired DNA methylation causing maternally derived trisomy 21. Also, *MTHFR* genotype/diet and BMI combination influence LINE-1 methylation in mothers that could be a risk factor for DS/CHD<sup>+</sup> development in children. The studies discussed in this chapter provide new evidence to support nutrigenomic personalized FA treatment of mothers with risk genotype to prevent global DNA hypomethylation as potential underlying mechanism of CA development.

### **Acknowledgements**

The work was funded by Grant No. OI175085, Ministry of Education, Science and Technological Development, Republic of Serbia.

### **Author details**

Aleksandra Stanković

Laboratory for Radiobiology and Molecular Genetics, "Vinca" Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia

\*Address all correspondence to: alexas@vinca.rs

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**49**

*Global DNA Methylation as a Potential Underlying Mechanism of Congenital Disease…*

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731. DOI: 10.1038/nrn1986

to a histone H3, lysine 4 methyltransferase complex. Developmental Cell. 2007;**13**(4):

580-592. DOI: 10.1016/j. devcel.2007.09.004

[10] Chen S, Bellew C, Yao X,

10.1074/jbc.M111.248278

pcl.2014.11.005

10.4161/epi.6.7.16537

10.1016/j.jacc.2011.08.025

[14] Dolk H, Loane M, Garne E, European Surveillance of Congenital Anomalies (EUROCAT) Working Group. Congenital heart defects in Europe: Prevalence and perinatal mortality, 2000 to 2005. Circulation.

Stefkova J, Dipp S, Saifudeen Z, et al. Histone deacetylase (HDAC) activity is critical for embryonic kidney gene expression, growth, and differentiation. The Journal of Biological Chemistry. 2011;**286**(37):32775-32789. DOI:

[11] Adams DJ, Clark DA. Common genetic and epigenetic syndromes. Pediatric Clinics of North America. 2015;**62**(2):411-426. DOI: 10.1016/j.

[12] Skinner MK. Environmental

[13] van der Linde D, Konings EE, Slager MA, Witsenburg M, Helbing WA, Takkenberg JJ, et al. Birth prevalence of congenital heart disease worldwide: A systematic review and meta-analysis. Journal of the American College of Cardiology. 2011;**58**(21):2241-2247. DOI:

epigenetic transgenerational inheritance and somatic epigenetic mitotic stability. Epigenetics. 2011;**6**(7):838-842. DOI:

[9] Patel SR, Kim D, Levitan I, Dressler GR. The BRCT-domain containing protein PTIP links PAX2

*DOI: http://dx.doi.org/10.5772/intechopen.90996*

[1] WHO. Available from: https://www. who.int/news-room/fact-sheets/detail/

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The work was funded by Grant No. OI175085, Ministry of Education, Science

Laboratory for Radiobiology and Molecular Genetics, "Vinca" Institute of Nuclear

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

develop-

LINE-1 methylation in mothers that could be a risk factor for DS/CHD<sup>+</sup>

and Technological Development, Republic of Serbia.

**48**

**Author details**

Aleksandra Stanković

Sciences, University of Belgrade, Belgrade, Serbia

\*Address all correspondence to: alexas@vinca.rs

provided the original work is properly cited.

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2011;**123**(8):841-849. DOI: 10.1161/ CIRCULATIONAHA.110.958405

[21] Dimasuay KG, Boeuf P, Powell TL, Jansson T. Placental responses to changes in the maternal environment determine fetal growth. Frontiers in Physiology. 2016;**7**:12. DOI: 10.3389/

[22] Argyraki M, Damdimopoulou P, Chatzimeletiou K, Grimbizis GF, Tarlatzis BC, Syrrou M, et al. In-utero stress and mode of conception: Impact on regulation of imprinted genes, fetal development and future health. Human Reproduction Update. 2019;**25**(6):777-801. DOI: 10.1093/

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[129] EpiSign Complete South Carolina: Greenwood Genetic Center. 2019. Available from: https://www.ggc.org/ test-finder-item/episign-complete)/

[130] Jarred EG, Bildsoe H, Western PS. Out of sight, out of mind? Germ cells and the potential impacts of epigenomic

Stringer JM. Transgenerational epigenetic inheritance: Adaptation through the germline epigenome? Epigenomics. 2015;**7**:829-846. DOI: 10.2217/epi.15.36

[132] Zhang Y, Teng F, Luo GZ, Wang M, Tong M, Zhao X, et al. MicroRNA-323-3p regulates the activity of

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Xing H, Sun M, Wang J, et al. MiRNA-145 regulates the development of congenital heart disease through targeting FXN. Pediatric Cardiology. 2016;**37**:629-636. DOI: 10.1007/

[134] Jovanovic I, Zivkovic M, Kostic M, Krstic Z, Djuric T, Licastro D, et al. Transcriptome-driven integrative exploration of functional state of ureter tissue affected by CAKUT. Life Sciences. 2018;**212**:1-8. DOI: 10.1016/j.

Kolic I, et al. Transcriptome-wide based identification of miRs in congenital anomalies of the kidney and urinary tract (CAKUT) in children: The significant upregulation of tissue miR-144 expression. Journal of Translational

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[131] Prokopuk L, Western PS,

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**61**

**Chapter 4**

in Mammals

*Motoko Unoki*

**Abstract**

summarized.

chromatin remodeling

**1. Introduction**

Recent Insights into the

Mechanisms of *De Novo* and

Maintenance of DNA Methylation

DNA methylation is one of the key epigenetic mechanisms essential for transcriptional regulation, silencing of transposable elements, and genome stabilization. Under physiological conditions, DNA methylation is erased and then established genome-wide during gametogenesis and embryogenesis. *De novo* DNA methylation by the enzymatic reaction of the *de novo* DNA methyltransferases (DNMTs), DNMT3A and DNMT3B, occurs during the establishment of DNA methylation patterns specific to each germ cell type or somatic cell type after the erasure. Once cell type-specific DNA methylation patterns are established during embryogenesis, which can extend to early childhood, the maintenance of DNA methyltransferase DNMT1 and its cofactor UHRF1 cooperatively maintain the pattern throughout the individual's lifetime. Recently, our group found that UHRF1 is also involved in *de novo* DNA methylation during oogenesis. Moreover, our group has identified two genes, *CDCA7* and *HELL*S, to be the causative genes of ICF syndrome, characterized by hypomethylation of centromeric and pericentromeric repetitive sequences. Because CDCA7/HELLS comprise a chromatin remodeling complex, there are evidently certain regions where chromatin remodeling is required to achieve maintenance of DNA methylation. In this chapter, the current situation with respect to our understanding of *de novo* and maintenance of DNA methylation mechanisms under physiological conditions in mammals is

**Keywords:** *de novo* DNA methylation, maintenance of DNA methylation, embryogenesis, gametogenesis, oocyte, PGC, ubiquitylation, UHRF1, DNMT1,

Methylation at the C5 positions of cytosine (i.e., 5mC) in the CpG context (hereafter called DNA methylation) plays a major role in the transcriptional regulation of gene expression, the silencing of transposable elements (TEs), and genome integrity. The enzymatic activities catalyzing DNA methylation can be classified into two types. One is *de novo* DNA methylation, which is an activity by which

DNMT3A, DNMT3B, DNMT3L, ICF syndrome, CDCA7, HELLS,

#### **Chapter 4**

## Recent Insights into the Mechanisms of *De Novo* and Maintenance of DNA Methylation in Mammals

*Motoko Unoki*

### **Abstract**

DNA methylation is one of the key epigenetic mechanisms essential for transcriptional regulation, silencing of transposable elements, and genome stabilization. Under physiological conditions, DNA methylation is erased and then established genome-wide during gametogenesis and embryogenesis. *De novo* DNA methylation by the enzymatic reaction of the *de novo* DNA methyltransferases (DNMTs), DNMT3A and DNMT3B, occurs during the establishment of DNA methylation patterns specific to each germ cell type or somatic cell type after the erasure. Once cell type-specific DNA methylation patterns are established during embryogenesis, which can extend to early childhood, the maintenance of DNA methyltransferase DNMT1 and its cofactor UHRF1 cooperatively maintain the pattern throughout the individual's lifetime. Recently, our group found that UHRF1 is also involved in *de novo* DNA methylation during oogenesis. Moreover, our group has identified two genes, *CDCA7* and *HELL*S, to be the causative genes of ICF syndrome, characterized by hypomethylation of centromeric and pericentromeric repetitive sequences. Because CDCA7/HELLS comprise a chromatin remodeling complex, there are evidently certain regions where chromatin remodeling is required to achieve maintenance of DNA methylation. In this chapter, the current situation with respect to our understanding of *de novo* and maintenance of DNA methylation mechanisms under physiological conditions in mammals is summarized.

**Keywords:** *de novo* DNA methylation, maintenance of DNA methylation, embryogenesis, gametogenesis, oocyte, PGC, ubiquitylation, UHRF1, DNMT1, DNMT3A, DNMT3B, DNMT3L, ICF syndrome, CDCA7, HELLS, chromatin remodeling

#### **1. Introduction**

Methylation at the C5 positions of cytosine (i.e., 5mC) in the CpG context (hereafter called DNA methylation) plays a major role in the transcriptional regulation of gene expression, the silencing of transposable elements (TEs), and genome integrity. The enzymatic activities catalyzing DNA methylation can be classified into two types. One is *de novo* DNA methylation, which is an activity by which

methyl groups are added to cytosine at unmethylated DNA; *de novo* DNA methyltransferases DNMT3A and DNMT3B, together with their coactivator DNMT3L, are known to catalyze this reaction [1]. The other DNA methylation type is maintenance of DNA methylation, an activity by which unmethylated cytosine residues of hemi-methylated DNA (cytosine methylation on only one strand of the CG dyad) are methylated after DNA replication; DNA methyltransferase 1 (DNMT1) and its cofactor, ubiquitin-like, containing PHD and RING finger domains 1 (UHRF1), are responsible for this function [2]. In this chapter, the current status of knowledge of the two types of DNA methylation, including recent findings from our group, is summarized. In addition, possible mechanisms underlying the control of regionspecific methylation by *de novo* DNMTs as well as possible maintenance of DNA methylation mechanisms, with or without chromatin remodeling, are discussed.

#### **2.** *De novo* **DNA methylation**

#### **2.1 Timing of physiological** *de novo* **DNA methylation**

*De novo* DNA methylation is a process by which methyl groups are added to unmethylated DNA at specific CpG sites, catalyzed by DNMT3A and DNMT3B [1]. In most differentiated cells, *de novo* DNA methylation is basically undesirable, since precise maintenance of the DNA methylation pattern, once established, is essential to sustain the appropriate functions of each cell type. Under physiological conditions, DNA methylation is widely erased, and then cell-type specific DNA methylation patterns are established during gametogenesis and embryogenesis [3] (**Figure 1**). During oogenesis and spermatogenesis, *de novo* DNA methylation occurs to establish oocyte- and sperm-specific DNA methylation patterns, following its elimination in primordial germ cells (PGCs). During post-implantation embryogenesis, which can extend to early childhood, *de novo* DNA methylation occurs to establish DNA methylation patterns specific to each cell type after they are erased in preimplantation embryos.

#### **Figure 1.**

*Known factors involved in dynamic physiological demethylation and de novo DNA methylation during mouse gametogenesis and embryogenesis. DNA methylation is widely erased, and then sex-specific or cell typespecific DNA methylation patterns are established during gametogenesis and embryogenesis. Levels of DNA methylation of imprinting control regions (ICRs) and some transposable elements (TEs) are maintained in the face of genome-wide demethylation in preimplantation embryos. DOHaD, developmental origins of health and disease; SCMC, subcortical maternal complex; E6.5, embryonic day 6.5; E10.5, embryonic day10.5; E12.5, embryonic day 12.5.*

**63**

*Recent Insights into the Mechanisms of* De Novo *and Maintenance of DNA Methylation…*

The erasure of DNA methylation in PGCs is probably the result of a defect in maintenance of DNA methylation, caused by the diminished expression of UHRF1 in the cells [4]. After the demethylation, DNMT3A establishes the methylation pattern in combination with DNMT3L, which itself does not possess enzymatic activity but is indispensable for the activity of DNMT3A [5–7] in oocytes arrested at an early stage of the first meiotic division or in prospermatogonia arrested at the G1 phase [8]. Although the major role of UHRF1 is in the maintenance of DNA methylation (Section 2.2), our group has recently found that UHRF1 is involved in 25% of the genome-wide *de novo* DNA methylation in oocytes [9]. The absence of the UHRF1 protein preferentially decreased DNA methylation levels at transcriptionally inactive regions without histone H3 trimethylation at lysine 36 (H3K36me3) mark. Given that only a small percentage decrease in DNA methylation was observed in *DNMT1* KO oocytes [10] and that UHRF1 has the potential to interact with *de novo* DNMTs [11], UHRF1 may cooperate with DNMT3A for the establishment of methylation patterns. Despite the involvement of UHRF1 in *de novo* DNA methylation in oocytes, our group found that the localization of UHRF1 in oocytes is mainly in the cytoplasm [9]. Recently, cytoplasmic Stella (also known as DPPA3 and PGC7), which is localized in both the cytoplasm and the nucleus, is reported to contribute to the cytoplasmic localization of UHRF1 in oocytes to prevent aberrantly excessive *de novo* DNA methylation by the UHRF1 protein complex [12]. Nuclear Stella is also reported to inhibit the association of UHRF1 with chromatin, resulting in a possible double-layer mechanism to prevent aberrant *de novo* DNA

During post-implantation embryogenesis and early childhood, not only DNMT3A but also DNMT3B proves to be essential for establishing the characteristic methylation pattern [14]. These enzymes may work together or independently to establish specific DNA methylation patterns in each cell type. However, it still has to be determined when the establishment of the methylation pattern is completed, although it probably depends on the cell type. The "developmental origins of health and disease" (DOHaD) is a concept that has emerged over the past three decades, linking the risk of diseases in later childhood and adult life with the environmental conditions of the early life, including nutrient availability to the mothers. Accumulating evidence suggests that the environment can change the epigenetic state, including DNA methylation of the fetus and infant, with the state being maintained throughout the lifetime of the individual [15]. A well-known experiment showed that early experience in childhood permanently alters behavior and physiology; interactions between rat mothers and their offspring, including the licking and grooming of the pups by their mother in the first week of life, altered the DNA methylation status of the *glucocorticoid receptor* promoter in the hippocampus of the offspring, resulting in differential stress tolerance among the offspring [16]. This indicates that the establishment of DNA methylation is not complete by

the first week after birth, at least in the hippocampal neurons of the rat.

The mechanisms underlying the specification of the genomic regions targeted by *de novo* DNMTs have remained largely elusive. In oocytes, a significant positive correlation between transcription and highly methylated regions has been reported [17]. It is known that transcriptionally active regions are marked with H3K36me3 and that the histone methyltransferase SET domain containing 2 (SETD2) is responsible for the histone methylation in oocytes [18]. Since SETD2 is reported to interact with the phosphorylated C-terminal domain of RNA polymerase II (RNA pol II) [19], SETD2 appears to methylate histones at regions actively transcribed

**2.2 Specification of** *de novo* **DNA methylation sites**

*DOI: http://dx.doi.org/10.5772/intechopen.89238*

methylation by the complex [13].

*Recent Insights into the Mechanisms of* De Novo *and Maintenance of DNA Methylation… DOI: http://dx.doi.org/10.5772/intechopen.89238*

The erasure of DNA methylation in PGCs is probably the result of a defect in maintenance of DNA methylation, caused by the diminished expression of UHRF1 in the cells [4]. After the demethylation, DNMT3A establishes the methylation pattern in combination with DNMT3L, which itself does not possess enzymatic activity but is indispensable for the activity of DNMT3A [5–7] in oocytes arrested at an early stage of the first meiotic division or in prospermatogonia arrested at the G1 phase [8]. Although the major role of UHRF1 is in the maintenance of DNA methylation (Section 2.2), our group has recently found that UHRF1 is involved in 25% of the genome-wide *de novo* DNA methylation in oocytes [9]. The absence of the UHRF1 protein preferentially decreased DNA methylation levels at transcriptionally inactive regions without histone H3 trimethylation at lysine 36 (H3K36me3) mark. Given that only a small percentage decrease in DNA methylation was observed in *DNMT1* KO oocytes [10] and that UHRF1 has the potential to interact with *de novo* DNMTs [11], UHRF1 may cooperate with DNMT3A for the establishment of methylation patterns. Despite the involvement of UHRF1 in *de novo* DNA methylation in oocytes, our group found that the localization of UHRF1 in oocytes is mainly in the cytoplasm [9]. Recently, cytoplasmic Stella (also known as DPPA3 and PGC7), which is localized in both the cytoplasm and the nucleus, is reported to contribute to the cytoplasmic localization of UHRF1 in oocytes to prevent aberrantly excessive *de novo* DNA methylation by the UHRF1 protein complex [12]. Nuclear Stella is also reported to inhibit the association of UHRF1 with chromatin, resulting in a possible double-layer mechanism to prevent aberrant *de novo* DNA methylation by the complex [13].

During post-implantation embryogenesis and early childhood, not only DNMT3A but also DNMT3B proves to be essential for establishing the characteristic methylation pattern [14]. These enzymes may work together or independently to establish specific DNA methylation patterns in each cell type. However, it still has to be determined when the establishment of the methylation pattern is completed, although it probably depends on the cell type. The "developmental origins of health and disease" (DOHaD) is a concept that has emerged over the past three decades, linking the risk of diseases in later childhood and adult life with the environmental conditions of the early life, including nutrient availability to the mothers. Accumulating evidence suggests that the environment can change the epigenetic state, including DNA methylation of the fetus and infant, with the state being maintained throughout the lifetime of the individual [15]. A well-known experiment showed that early experience in childhood permanently alters behavior and physiology; interactions between rat mothers and their offspring, including the licking and grooming of the pups by their mother in the first week of life, altered the DNA methylation status of the *glucocorticoid receptor* promoter in the hippocampus of the offspring, resulting in differential stress tolerance among the offspring [16]. This indicates that the establishment of DNA methylation is not complete by the first week after birth, at least in the hippocampal neurons of the rat.

#### **2.2 Specification of** *de novo* **DNA methylation sites**

The mechanisms underlying the specification of the genomic regions targeted by *de novo* DNMTs have remained largely elusive. In oocytes, a significant positive correlation between transcription and highly methylated regions has been reported [17]. It is known that transcriptionally active regions are marked with H3K36me3 and that the histone methyltransferase SET domain containing 2 (SETD2) is responsible for the histone methylation in oocytes [18]. Since SETD2 is reported to interact with the phosphorylated C-terminal domain of RNA polymerase II (RNA pol II) [19], SETD2 appears to methylate histones at regions actively transcribed

*DNA Methylation Mechanism*

**2.** *De novo* **DNA methylation**

erased in preimplantation embryos.

**2.1 Timing of physiological** *de novo* **DNA methylation**

methyl groups are added to cytosine at unmethylated DNA; *de novo* DNA methyltransferases DNMT3A and DNMT3B, together with their coactivator DNMT3L, are known to catalyze this reaction [1]. The other DNA methylation type is maintenance of DNA methylation, an activity by which unmethylated cytosine residues of hemi-methylated DNA (cytosine methylation on only one strand of the CG dyad) are methylated after DNA replication; DNA methyltransferase 1 (DNMT1) and its cofactor, ubiquitin-like, containing PHD and RING finger domains 1 (UHRF1), are responsible for this function [2]. In this chapter, the current status of knowledge of the two types of DNA methylation, including recent findings from our group, is summarized. In addition, possible mechanisms underlying the control of regionspecific methylation by *de novo* DNMTs as well as possible maintenance of DNA methylation mechanisms, with or without chromatin remodeling, are discussed.

*De novo* DNA methylation is a process by which methyl groups are added to unmethylated DNA at specific CpG sites, catalyzed by DNMT3A and DNMT3B [1]. In most differentiated cells, *de novo* DNA methylation is basically undesirable, since precise maintenance of the DNA methylation pattern, once established, is essential to sustain the appropriate functions of each cell type. Under physiological conditions, DNA methylation is widely erased, and then cell-type specific DNA methylation patterns are established during gametogenesis and embryogenesis [3] (**Figure 1**). During oogenesis and spermatogenesis, *de novo* DNA methylation occurs to establish oocyte- and sperm-specific DNA methylation patterns, following its elimination in primordial germ cells (PGCs). During post-implantation embryogenesis, which can extend to early childhood, *de novo* DNA methylation occurs to establish DNA methylation patterns specific to each cell type after they are

*Known factors involved in dynamic physiological demethylation and de novo DNA methylation during mouse gametogenesis and embryogenesis. DNA methylation is widely erased, and then sex-specific or cell typespecific DNA methylation patterns are established during gametogenesis and embryogenesis. Levels of DNA methylation of imprinting control regions (ICRs) and some transposable elements (TEs) are maintained in the face of genome-wide demethylation in preimplantation embryos. DOHaD, developmental origins of health and disease; SCMC, subcortical maternal complex; E6.5, embryonic day 6.5; E10.5, embryonic day10.5; E12.5,* 

**62**

**Figure 1.**

*embryonic day 12.5.*

#### **Figure 2.**

*A model for transcription-coupled and transcription-uncoupled de novo DNA methylation in oocytes. SETD2 methylates H3K36 accompanied with transcription by RNA polymerase II, and DNMT3A recognizes the histone mark and methylates DNA, resulting in the establishment of DNA methylation pattern specific to oocytes. Long terminal repeat (LTR)-retrotransposons activated in PGCs may be partially involved in triggering the transcription. Although UHRF1 is involved in the de novo DNA methylation of regions without the H3K36me3 mark, there could be additional mechanisms for transcription-uncoupled de novo DNA methylation.*

by the polymerase. On the other hand, the PWWP domain of DNMT3A recognizes H3K36me3 [20], and mutations in this domain, which disrupt this recognition, cause microcephalic dwarfism with aberrant DNA methylation in humans and in a mouse model [21, 22]. Oocyte-specific *SETD2* KO also causes aberrant DNA methylation [23]. Taken together, it appears that SETD2 methylates H3K36 accompanied by transcription by RNA pol II and DNMT3A recognizes the histone mark and methylates the DNA, resulting in the establishment of DNA methylation patterns specific to oocytes (**Figure 2**). However, there are exceptions. For example, as described above, UHRF1 is involved in 25% of the genome-wide *de novo* DNA methylation, mostly at transcriptionally inactive regions lacking the H3K36me3 mark [9]. It is still unknown which factors trigger transcription in oocytes, although transcription from long terminal repeat (LTR)-retrotransposons, whose methylation is erased in PGCs, could be one such trigger [24].

During embryogenesis, transcription factors probably define certain transcribed regions in each cell type as only four transcriptional factors (OCT3/4, SOX2, KLF4, and MYC), together known as OSKM or Yamanaka factors, can drive drastic transcriptional change and define epigenetically active regions in differentiated cells, resulting in induced pluripotent stem (iPS) cells [25]. DNMTs can access regions, where the transcription factors are absent, to passively specify regions for DNA methylation (**Figure 3**). Noncoding RNAs, such as PIWI-interacting RNAs (piRNAs) and long noncoding RNAs (lncRNAs), can also contribute to the specification of regions for DNA methylation (**Figure 3**). piRNAs are the largest class (26–31 nucleotides) of small noncoding RNA expressed in animal cells, which were first discovered in *Drosophila* as RNAs interacting with the PIWI protein; human and mouse homologs are HIWI and MIWI, respectively. In most cases, precursor piRNAs are derived from piRNA clusters in the genome composed of mutated TEs. The precursor piRNAs are processed by several steps and matured by the addition of a methyl group at their 3′ ends [26]. Then, the maturated piRNAs interact with Argonaute (AGO) family proteins and cleave the TEs, which are undesirably transcribed by the erasure of DNA methylation in PGCs [26]. Although the underlying mechanisms are unknown, piRNAs silence these TEs by epigenetic modifications, including DNA methylation, especially during spermatogenesis [27]. In addition, lncRNAs can specify *de novo* DNA methylation-acquired regions. *X-inactive specific transcript* (*XIST*) is one of the best-studied lncRNAs. *XIST* RNA is randomly expressed from one of two X-chromosomes in mammalian female cells during

**65**

**Figure 3.**

*silenced regions.*

*Recent Insights into the Mechanisms of* De Novo *and Maintenance of DNA Methylation…*

embryogenesis and covers the X-chromosome *in cis* to trigger silencing of most genes on it by several layers of epigenetic modifications, including DNA methyla-

*A model for formation of transcriptionally active and silenced regions during embryogenesis.* 

*Transcription factors (TFs) could define transcriptionally active regions, while small RNAs and lncRNAs could define transcriptionally silenced regions in addition to transcription-coupled de novo DNA methylation. After transcriptionally active and silenced regions could be actively determined, suppressive mark modifiers, such as de novo DNMTs, may add suppressive epigenetic marks to accessible regions, which transcriptional machineries do not occupy, resulting in the passive formation of transcriptionally* 

**3.1 Maintenance of DNA methylation by the DNMT1/UHRF1 complex**

the SRA-mediated recognition of hemi-methylated DNA [40, 41].

Once DNA methylation patterns specific to each cell type are established, the pattern is maintained by the DNMT1/UHRF1 complex throughout the individual's lifetime [2]. UHRF1 (also known as Np95 or ICBP90) is a multidomain protein, which contains a ubiquitin-like (UBL) domain, a tandem Tudor domain (TTD), a plant homeodomain (PHD) finger, a SET and RING-associated (SRA) domain, and a really interesting new gene (RING) domain. The TTD recognizes di−/tri-methylated H3K9 (H3K9me2/me3) and also LIG1 (LIG1K126me2/me3) [30, 31], the PHD recognizes the unmethylated N-terminus of histone H3 and LIG1 [32], the SRA domain recognizes hemi-methylated DNA at the replication fork [33–35], and the RING domain mono-ubiquitylates multiple lysines of histone H3 at K14, K18, and K23 and those of the PCNA-associated factor 15 (PAF15) at K15 and K24 [36–39]. The UBL domain facilitates both the RING-mediated ubiquitylation and

Current consensus has it that the process of maintenance of DNA methylation operates as follows. After DNA replication, UHRF1 directly recognizes hemi-methylated DNA and mono-ubiquitylates histone H3K14, K18, and K23, to recruit DNMT1 to the hemi-methylation sites. Then, DNMT1 recognizes two of the three ubiquitylated histone lysine residues through the replication foci targeting sequence (RFTS)

tion, to achieve dosage compensation [28, 29].

**3. Maintenance of DNA methylation**

*DOI: http://dx.doi.org/10.5772/intechopen.89238*

*Recent Insights into the Mechanisms of* De Novo *and Maintenance of DNA Methylation… DOI: http://dx.doi.org/10.5772/intechopen.89238*

#### **Figure 3.**

*DNA Methylation Mechanism*

**Figure 2.**

*methylation.*

by the polymerase. On the other hand, the PWWP domain of DNMT3A recognizes H3K36me3 [20], and mutations in this domain, which disrupt this recognition, cause microcephalic dwarfism with aberrant DNA methylation in humans and in a mouse model [21, 22]. Oocyte-specific *SETD2* KO also causes aberrant DNA methylation [23]. Taken together, it appears that SETD2 methylates H3K36 accompanied by transcription by RNA pol II and DNMT3A recognizes the histone mark and methylates the DNA, resulting in the establishment of DNA methylation patterns specific to oocytes (**Figure 2**). However, there are exceptions. For example, as described above, UHRF1 is involved in 25% of the genome-wide *de novo* DNA methylation, mostly at transcriptionally inactive regions lacking the H3K36me3 mark [9]. It is still unknown which factors trigger transcription in oocytes, although transcription from long terminal repeat (LTR)-retrotransposons, whose methyla-

*A model for transcription-coupled and transcription-uncoupled de novo DNA methylation in oocytes. SETD2 methylates H3K36 accompanied with transcription by RNA polymerase II, and DNMT3A recognizes the histone mark and methylates DNA, resulting in the establishment of DNA methylation pattern specific to oocytes. Long terminal repeat (LTR)-retrotransposons activated in PGCs may be partially involved in triggering the transcription. Although UHRF1 is involved in the de novo DNA methylation of regions without the H3K36me3 mark, there could be additional mechanisms for transcription-uncoupled de novo DNA* 

During embryogenesis, transcription factors probably define certain transcribed

regions in each cell type as only four transcriptional factors (OCT3/4, SOX2, KLF4, and MYC), together known as OSKM or Yamanaka factors, can drive drastic transcriptional change and define epigenetically active regions in differentiated cells, resulting in induced pluripotent stem (iPS) cells [25]. DNMTs can access regions, where the transcription factors are absent, to passively specify regions for DNA methylation (**Figure 3**). Noncoding RNAs, such as PIWI-interacting RNAs (piRNAs) and long noncoding RNAs (lncRNAs), can also contribute to the specification of regions for DNA methylation (**Figure 3**). piRNAs are the largest class (26–31 nucleotides) of small noncoding RNA expressed in animal cells, which were first discovered in *Drosophila* as RNAs interacting with the PIWI protein; human and mouse homologs are HIWI and MIWI, respectively. In most cases, precursor piRNAs are derived from piRNA clusters in the genome composed of mutated TEs. The precursor piRNAs are processed by several steps and matured by the addition of a methyl group at their 3′ ends [26]. Then, the maturated piRNAs interact with Argonaute (AGO) family proteins and cleave the TEs, which are undesirably transcribed by the erasure of DNA methylation in PGCs [26]. Although the underlying mechanisms are unknown, piRNAs silence these TEs by epigenetic modifications, including DNA methylation, especially during spermatogenesis [27]. In addition, lncRNAs can specify *de novo* DNA methylation-acquired regions. *X-inactive specific transcript* (*XIST*) is one of the best-studied lncRNAs. *XIST* RNA is randomly expressed from one of two X-chromosomes in mammalian female cells during

tion is erased in PGCs, could be one such trigger [24].

**64**

*A model for formation of transcriptionally active and silenced regions during embryogenesis. Transcription factors (TFs) could define transcriptionally active regions, while small RNAs and lncRNAs could define transcriptionally silenced regions in addition to transcription-coupled de novo DNA methylation. After transcriptionally active and silenced regions could be actively determined, suppressive mark modifiers, such as de novo DNMTs, may add suppressive epigenetic marks to accessible regions, which transcriptional machineries do not occupy, resulting in the passive formation of transcriptionally silenced regions.*

embryogenesis and covers the X-chromosome *in cis* to trigger silencing of most genes on it by several layers of epigenetic modifications, including DNA methylation, to achieve dosage compensation [28, 29].

#### **3. Maintenance of DNA methylation**

#### **3.1 Maintenance of DNA methylation by the DNMT1/UHRF1 complex**

Once DNA methylation patterns specific to each cell type are established, the pattern is maintained by the DNMT1/UHRF1 complex throughout the individual's lifetime [2]. UHRF1 (also known as Np95 or ICBP90) is a multidomain protein, which contains a ubiquitin-like (UBL) domain, a tandem Tudor domain (TTD), a plant homeodomain (PHD) finger, a SET and RING-associated (SRA) domain, and a really interesting new gene (RING) domain. The TTD recognizes di−/tri-methylated H3K9 (H3K9me2/me3) and also LIG1 (LIG1K126me2/me3) [30, 31], the PHD recognizes the unmethylated N-terminus of histone H3 and LIG1 [32], the SRA domain recognizes hemi-methylated DNA at the replication fork [33–35], and the RING domain mono-ubiquitylates multiple lysines of histone H3 at K14, K18, and K23 and those of the PCNA-associated factor 15 (PAF15) at K15 and K24 [36–39]. The UBL domain facilitates both the RING-mediated ubiquitylation and the SRA-mediated recognition of hemi-methylated DNA [40, 41].

Current consensus has it that the process of maintenance of DNA methylation operates as follows. After DNA replication, UHRF1 directly recognizes hemi-methylated DNA and mono-ubiquitylates histone H3K14, K18, and K23, to recruit DNMT1 to the hemi-methylation sites. Then, DNMT1 recognizes two of the three ubiquitylated histone lysine residues through the replication foci targeting sequence (RFTS)

#### **Figure 4.**

*A model of maintenance of DNA methylation where the CDCA7/HELLS complex is unrequired or required. After DNA replication, UHRF1 directly recognizes hemi-methylated DNA, and mono-ubiquitylates multiple histone lysines, H3K14, K18, and K23, to recruit DNMT1 to the hemi-methylation sites. Consequently, DNMT1 recognizes two of the three ubiquitylated lysine residues through the replication foci targeting sequence (RFTS) domain and methylates the nascent strand in hemi-methylated DNA, resulting in the maintenance of the methylation pattern. LIG1 methylated by G9a/GLP helps UHRF1 to maintain DNA methylation. In addition, the CDCA7/HELLS complex is required for maintaining the DNA methylation of centromeric and pericentromeric regions. The complex may also be required for maintaining regions that are heterochromatic, late replicating, and histone H1 rich, all these regions being nucleosome dense.*

domain and methylates the nascent strand in hemi-methylated DNA, resulting in the maintenance of the methylation patterns (**Figure 4**). Immediately prior to the methylation of hemi-methylated DNA by DNMT1, it has been reported that the deubiquitylation of histones by ubiquitin specific peptidase 7 (USP7) is required [42]. DNA ligase 1 (LIG1), which is critical for the joining together of Okazaki fragments [43], is also involved in this process [31]. Euchromatic histone lysine methyltransferase 2 (EHMT2, also called G9a) and EHMT1 (also called GLP) methylate K126 of LIG1. UHRF1 recognizes the methylated LIG1, and this interaction facilitates the recruitment of UHRF1 to DNA replication sites. Since LIG1 is indispensable for completing the lagging strand synthesis, the interaction between UHRF1 and LIG1 may be especially important for maintenance of DNA methylation of the strand (**Figure 4**).

#### **3.2 Maintenance of DNA methylation by the CDCA7/HELLS chromatin remodeling complex**

The cell division cycle-associated 7 (CDCA7)/helicase lymphoid-specific (HELLS) chromatin remodeling complex is also involved in maintenance of DNA methylation. Recently, an international group including us identified *CDCA7*

**67**

*Recent Insights into the Mechanisms of* De Novo *and Maintenance of DNA Methylation…*

[53], the same pathway seems to be disrupted in ICF2, ICF3, and ICF4.

complex may be required for such regions (**Figure 4**).

A recent study revealed that, in addition to centromeric and pericentromeric repeats, DNA methylation levels of other heterochromatic late-replicating regions are affected in ICF2, ICF3, and ICF4 patients, though not in ICF1 patients [54]. As *UHRF1* KO and *DNMT1* KO cause hypomethylation of the entire genome, including centromeric and pericentromeric repeats [2], the DNMT1/UHRF1 complex is surely essential for maintaining these regions. However, the CDCA7/HELLS complex seems to be required for assisting the DNMT1/UHRF1 complex to methylate hemimethylated DNA, possibly by sliding nucleosomes in a region-specific manner [53]. Supporting this idea, our group detected an interaction between CDCA7 and UHRF1 [55]. Late-replicating regions tend to be heterochromatic regions, where the nucleosome density is high. Therefore, the CDCA7/HELLS chromatin remodeling

Using human embryonic kidney 293 cells, our group reported that *DNMT3B* KO caused a slight decrease in DNA methylation of pericentromeric repeats after 4 months of KO by the CRISPR/Cas9 system, while *CDCA7* KO and *HELLS* KO caused drastic decreases in DNA methylation even after only 2 months [55], indicating that the CDCA7/HELLS chromatin remodeling complex is essential for maintaining the DNA methylation of the repeats, whereas the requirement of DNMT3B for the maintenance is limited in differentiated cells. In the *CDCA7* KO and *HELLS* KO cells, DNA methylation levels of centromeric repeats were also decreased, but the level of decrease was much less than that of pericentromeric repeats. This indicates that the CDCA7/HELLS complex is less essential for maintenance of DNA methylation of centromeric repeats. Because the chromatin structure, density of nucleosomes, and histone variants are different between centromeric and pericentromeric regions, these differences may determine the levels of requirement for the chromatin remodeling complex. In addition, it has been reported that nucleosomes and the linker histone H1 are barriers to access of DNMTs to DNA and that HELLS and deficient in DNA methylation 1 (DDM1), a plant homolog of HELLS, are required for the methylation of DNA wrapped around nucleosomes [56, 57]. Consistent with these reports, the most abundant proteins co-immunoprecipitated with human CDCA7 were histone H1 and core histones in our group's report [55]. The interaction between the CDCA7/HELLS complex and histone H1 may also be

and *HELLS* (also known as *LSH*) to be causative genes of the immunodeficiency, centromeric instability, facial anomalies (ICF) syndrome type-3 and type-4 (hereafter ICF3 and ICF4), respectively [44]. The syndrome is a rare autosomal recessive disorder characterized by reduced immunoglobulin levels in the serum and recurrent infection [45]. Centromeric instability manifests as stretched heterochromatin, chromosome breaks, and multiradial configurations involving the centromeric/pericentromeric regions of chromosomes 1, 9, and 16 in activated lymphocytes [46], and the cytological defects are accompanied by DNA hypomethylation in pericentromeric satellite-2 and -3 repeats of these chromosomes. Patients with the ICF syndrome are classified into two groups [47]. One group includes ICF syndrome type-1 (ICF1), which shows DNA hypomethylation only at the pericentromeric repeats. A causative gene for this group is *DNMT3B* [1, 48, 49]. The second group includes ICF syndrome type-2, type-3, and type-4 (ICF2, ICF3, and ICF4, respectively), which shows DNA hypomethylation at centromeric α-satellite repeats in addition to the pericentromeric repeats. As described above, causative genes for ICF3 and ICF4 are *CDCA7* and *HELLS,* respectively [44]. The causative gene for ICF2 is *zinc finger and BTB domain containing 24* (*ZBTB24*) [50]. As ZBTB24 is a transcriptional activator of *CDCA7* [51, 52], and CDCA7 and HELLS constitute a chromatin remodeling complex, in which CDCA7 stimulates the nucleosome remodeling activity of HELLS

*DOI: http://dx.doi.org/10.5772/intechopen.89238*

#### *Recent Insights into the Mechanisms of* De Novo *and Maintenance of DNA Methylation… DOI: http://dx.doi.org/10.5772/intechopen.89238*

and *HELLS* (also known as *LSH*) to be causative genes of the immunodeficiency, centromeric instability, facial anomalies (ICF) syndrome type-3 and type-4 (hereafter ICF3 and ICF4), respectively [44]. The syndrome is a rare autosomal recessive disorder characterized by reduced immunoglobulin levels in the serum and recurrent infection [45]. Centromeric instability manifests as stretched heterochromatin, chromosome breaks, and multiradial configurations involving the centromeric/pericentromeric regions of chromosomes 1, 9, and 16 in activated lymphocytes [46], and the cytological defects are accompanied by DNA hypomethylation in pericentromeric satellite-2 and -3 repeats of these chromosomes.

Patients with the ICF syndrome are classified into two groups [47]. One group includes ICF syndrome type-1 (ICF1), which shows DNA hypomethylation only at the pericentromeric repeats. A causative gene for this group is *DNMT3B* [1, 48, 49]. The second group includes ICF syndrome type-2, type-3, and type-4 (ICF2, ICF3, and ICF4, respectively), which shows DNA hypomethylation at centromeric α-satellite repeats in addition to the pericentromeric repeats. As described above, causative genes for ICF3 and ICF4 are *CDCA7* and *HELLS,* respectively [44]. The causative gene for ICF2 is *zinc finger and BTB domain containing 24* (*ZBTB24*) [50]. As ZBTB24 is a transcriptional activator of *CDCA7* [51, 52], and CDCA7 and HELLS constitute a chromatin remodeling complex, in which CDCA7 stimulates the nucleosome remodeling activity of HELLS [53], the same pathway seems to be disrupted in ICF2, ICF3, and ICF4.

A recent study revealed that, in addition to centromeric and pericentromeric repeats, DNA methylation levels of other heterochromatic late-replicating regions are affected in ICF2, ICF3, and ICF4 patients, though not in ICF1 patients [54]. As *UHRF1* KO and *DNMT1* KO cause hypomethylation of the entire genome, including centromeric and pericentromeric repeats [2], the DNMT1/UHRF1 complex is surely essential for maintaining these regions. However, the CDCA7/HELLS complex seems to be required for assisting the DNMT1/UHRF1 complex to methylate hemimethylated DNA, possibly by sliding nucleosomes in a region-specific manner [53]. Supporting this idea, our group detected an interaction between CDCA7 and UHRF1 [55]. Late-replicating regions tend to be heterochromatic regions, where the nucleosome density is high. Therefore, the CDCA7/HELLS chromatin remodeling complex may be required for such regions (**Figure 4**).

Using human embryonic kidney 293 cells, our group reported that *DNMT3B* KO caused a slight decrease in DNA methylation of pericentromeric repeats after 4 months of KO by the CRISPR/Cas9 system, while *CDCA7* KO and *HELLS* KO caused drastic decreases in DNA methylation even after only 2 months [55], indicating that the CDCA7/HELLS chromatin remodeling complex is essential for maintaining the DNA methylation of the repeats, whereas the requirement of DNMT3B for the maintenance is limited in differentiated cells. In the *CDCA7* KO and *HELLS* KO cells, DNA methylation levels of centromeric repeats were also decreased, but the level of decrease was much less than that of pericentromeric repeats. This indicates that the CDCA7/HELLS complex is less essential for maintenance of DNA methylation of centromeric repeats. Because the chromatin structure, density of nucleosomes, and histone variants are different between centromeric and pericentromeric regions, these differences may determine the levels of requirement for the chromatin remodeling complex. In addition, it has been reported that nucleosomes and the linker histone H1 are barriers to access of DNMTs to DNA and that HELLS and deficient in DNA methylation 1 (DDM1), a plant homolog of HELLS, are required for the methylation of DNA wrapped around nucleosomes [56, 57]. Consistent with these reports, the most abundant proteins co-immunoprecipitated with human CDCA7 were histone H1 and core histones in our group's report [55]. The interaction between the CDCA7/HELLS complex and histone H1 may also be

*DNA Methylation Mechanism*

domain and methylates the nascent strand in hemi-methylated DNA, resulting in the maintenance of the methylation patterns (**Figure 4**). Immediately prior to the methylation of hemi-methylated DNA by DNMT1, it has been reported that the deubiquitylation of histones by ubiquitin specific peptidase 7 (USP7) is required [42]. DNA ligase 1 (LIG1), which is critical for the joining together of Okazaki fragments [43], is also involved in this process [31]. Euchromatic histone lysine methyltransferase 2 (EHMT2, also called G9a) and EHMT1 (also called GLP) methylate K126 of LIG1. UHRF1 recognizes the methylated LIG1, and this interaction facilitates the recruitment of UHRF1 to DNA replication sites. Since LIG1 is indispensable for completing the lagging strand synthesis, the interaction between UHRF1 and LIG1 may be especially important for maintenance of DNA methylation of the strand (**Figure 4**).

*late replicating, and histone H1 rich, all these regions being nucleosome dense.*

*A model of maintenance of DNA methylation where the CDCA7/HELLS complex is unrequired or required. After DNA replication, UHRF1 directly recognizes hemi-methylated DNA, and mono-ubiquitylates multiple histone lysines, H3K14, K18, and K23, to recruit DNMT1 to the hemi-methylation sites. Consequently, DNMT1 recognizes two of the three ubiquitylated lysine residues through the replication foci targeting sequence (RFTS) domain and methylates the nascent strand in hemi-methylated DNA, resulting in the maintenance of the methylation pattern. LIG1 methylated by G9a/GLP helps UHRF1 to maintain DNA methylation. In addition, the CDCA7/HELLS complex is required for maintaining the DNA methylation of centromeric and pericentromeric regions. The complex may also be required for maintaining regions that are heterochromatic,* 

**3.2 Maintenance of DNA methylation by the CDCA7/HELLS chromatin** 

The cell division cycle-associated 7 (CDCA7)/helicase lymphoid-specific (HELLS) chromatin remodeling complex is also involved in maintenance of DNA methylation. Recently, an international group including us identified *CDCA7*

**66**

**Figure 4.**

**remodeling complex**

a cue to identify regions where the complex is required for maintenance of DNA methylation (**Figure 4**).

#### **3.3 Maintenance of DNA methylation by the proteins associated with multi-locus imprint disorder**

It is reported that mutations in genes encoding zinc finger protein 57 (ZFP57) and components of subcortical maternal complex (SCMC), including NLRP2, NLRP5, NLRP7, PADI6, OOEP, and TLE6, cause the multi-locus imprint disorder, which exhibits DNA hypomethylation at multiple imprinting control regions (ICRs) [58–61]. Since the hypomethylation is observed in both paternally and maternally methylated ICRs, these factors are thought to be involved in maintenance of DNA methylation against genome-wide DNA demethylation in preimplantation embryos (**Figure 1**). Mutations in *ZFP57* cause transient neonatal diabetes mellitus [61]. As ZFP57 is a nuclear protein, which recognizes the methylated TGCCGC hexanucleotide found in almost all ICRs and which acts together with ZNF445, KRAB-associated protein-1 (KAP1), DNMTs, SET domain bifurcated histone lysine methyltransferase 1 (SETDB1), and heterochromatin protein 1 (HP1) [62, 63], ZFP57 is considered to maintain DNA methylation by directly binding to ICRs with such proteins. However, the mechanism by which SCMC components, which are localized adjacent to the oocyte membrane, can maintain DNA methylation at ICRs remains elusive [59]. Among the multi-locus imprint disorder cases, just one case, who has a heterozygous mutation (V159 M in isoform 1, V172 M in isoform 2) in the TTD of UHRF1, has been reported [60].

#### **4. Conclusions**

I identified UHRF1 as a novel methyl-CpG binding protein in 2004 by biotinavidin pulldown assay using biotin-labeled methylated DNA mixed with nuclear extracts and subsequent mass spectrometric analysis [64, 65]. Since then, an understanding of the mechanism by which maintenance of DNA methylation is achieved has quickly expanded and deepened, progress that I would never have imagined at that time. When the involvement of UHRF1 in maintenance of DNA methylation was reported [2], the recognition of hemi-methylated DNA by UHRF1 was reported [32, 34, 35], and the ubiquitylation of histone H3 by UHRF1 was reported [36], each time I felt that the mechanism of maintenance of DNA methylation had been resolved. However, the mechanism is more complicated than expected, and more factors could still be involved to assist the DNMT1/UHRF1 complex, depending on context such as replication timing, replication strand, and higher-order chromatin structure. We still cannot take our eyes off advances in this field.

#### **Acknowledgements**

This work was supported by JSPS KAKENHI Grant number JP18K06961. I would like to thank Enago (www.enago.jp) for the English language review.

**69**

**Author details**

Kyushu University, Fukuoka, Japan

provided the original work is properly cited.

Motoko Unoki

*Recent Insights into the Mechanisms of* De Novo *and Maintenance of DNA Methylation…*

I would like to thank Drs. Kyohei Arita (Yokohama City University), Jafar Sharif (RIKEN), and Atsuya Nishiyama (the University of Tokyo) for involving me in the UHRF1 community and discussing many interesting topics. I would like to thank Dr. Hisato Kobayashi (Nara Medical University) for notifying me of a possible role of LTR-retrotransposons in oocytes. I also would like to thank Profs. Hiroyuki Sasaki (Kyushu University) and Hironori Funabiki (Rockefeller University) for deepening my insights into epigenetics and chromatins. Finally, I would like to thank Prof. Yusuke Nakamura (Japanese Foundation for Cancer Research) for encouraging me with challenging experiments, which resulted in the identification

Division of Epigenomics and Development, Medical Institute of Bioregulation,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: unokim@bioreg.kyushu-u.ac.jp

*DOI: http://dx.doi.org/10.5772/intechopen.89238*

of UHRF1, when I was a PhD student.

**Thanks**

#### **Conflict of interest**

The author has declared that no conflict of interest exists.

*Recent Insights into the Mechanisms of* De Novo *and Maintenance of DNA Methylation… DOI: http://dx.doi.org/10.5772/intechopen.89238*

#### **Thanks**

*DNA Methylation Mechanism*

methylation (**Figure 4**).

**4. Conclusions**

our eyes off advances in this field.

**Acknowledgements**

**Conflict of interest**

**with multi-locus imprint disorder**

a cue to identify regions where the complex is required for maintenance of DNA

It is reported that mutations in genes encoding zinc finger protein 57 (ZFP57) and components of subcortical maternal complex (SCMC), including NLRP2, NLRP5, NLRP7, PADI6, OOEP, and TLE6, cause the multi-locus imprint disorder, which exhibits DNA hypomethylation at multiple imprinting control regions (ICRs) [58–61]. Since the hypomethylation is observed in both paternally and maternally methylated ICRs, these factors are thought to be involved in maintenance of DNA methylation against genome-wide DNA demethylation in preimplantation embryos (**Figure 1**). Mutations in *ZFP57* cause transient neonatal diabetes mellitus [61]. As ZFP57 is a nuclear protein, which recognizes the methylated TGCCGC hexanucleotide found in almost all ICRs and which acts together with ZNF445, KRAB-associated protein-1 (KAP1), DNMTs, SET domain bifurcated histone lysine methyltransferase 1 (SETDB1), and heterochromatin protein 1 (HP1) [62, 63], ZFP57 is considered to maintain DNA methylation by directly binding to ICRs with such proteins. However, the mechanism by which SCMC components, which are localized adjacent to the oocyte membrane, can maintain DNA methylation at ICRs remains elusive [59]. Among the multi-locus imprint disorder cases, just one case, who has a heterozygous mutation (V159 M in isoform 1, V172 M in

I identified UHRF1 as a novel methyl-CpG binding protein in 2004 by biotinavidin pulldown assay using biotin-labeled methylated DNA mixed with nuclear extracts and subsequent mass spectrometric analysis [64, 65]. Since then, an understanding of the mechanism by which maintenance of DNA methylation is achieved has quickly expanded and deepened, progress that I would never have imagined at that time. When the involvement of UHRF1 in maintenance of DNA methylation was reported [2], the recognition of hemi-methylated DNA by UHRF1 was reported [32, 34, 35], and the ubiquitylation of histone H3 by UHRF1 was reported [36], each time I felt that the mechanism of maintenance of DNA methylation had been resolved. However, the mechanism is more complicated than expected, and more factors could still be involved to assist the DNMT1/UHRF1 complex, depending on context such as replication timing, replication strand, and higher-order chromatin structure. We still cannot take

This work was supported by JSPS KAKENHI Grant number JP18K06961. I would like to thank Enago (www.enago.jp) for the English language review.

The author has declared that no conflict of interest exists.

**3.3 Maintenance of DNA methylation by the proteins associated** 

isoform 2) in the TTD of UHRF1, has been reported [60].

**68**

I would like to thank Drs. Kyohei Arita (Yokohama City University), Jafar Sharif (RIKEN), and Atsuya Nishiyama (the University of Tokyo) for involving me in the UHRF1 community and discussing many interesting topics. I would like to thank Dr. Hisato Kobayashi (Nara Medical University) for notifying me of a possible role of LTR-retrotransposons in oocytes. I also would like to thank Profs. Hiroyuki Sasaki (Kyushu University) and Hironori Funabiki (Rockefeller University) for deepening my insights into epigenetics and chromatins. Finally, I would like to thank Prof. Yusuke Nakamura (Japanese Foundation for Cancer Research) for encouraging me with challenging experiments, which resulted in the identification of UHRF1, when I was a PhD student.

### **Author details**

Motoko Unoki Division of Epigenomics and Development, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan

\*Address all correspondence to: unokim@bioreg.kyushu-u.ac.jp

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 3

Methylation Mechanisms

in Process of Cancer

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Section 3
