**2. Cytosine modifications**

To maintain the normal life process, any base modification must be dynamically and tightly regulated in accordance with stages of the growth, development, and reproduction, including modification generation by methyltransferase complexes (writers), removal by demethyltransferases (erasers), as well as the preferential binding protein components (readers), to get the related epigenetic markers into the biochemical effects.

### **2.1. Methyltransferases of cytosine methylation**

DNA methylation, particularly the most abundant CpG methylation marker 5-mC, is an essential modification of DNA in the mammalian genome, typically linked with gene silencing and involved in gene regulation, development, genome defense, and disease. A family of DNA methyltransferases named (DNMTs) is responsible for the addition of methyl groups to the 5-position of the carbon, including DNMT1, DNMT2, DNMT3a, DNMT3b, and DNMT3L. The five members are structurally and functionally distinct. The three methyltransferase enzymes DNMT1, DNMT3a, and DNMT3b serve as writers for the de novo CpG methylation pattern during embryogenesis [28, 29], while DNMT1 could confer the maintenance of parent DNA methylation patterns to the new daughter strand DNA during DNA replication [30].

Traditionally, DNMT1 was regarded as the maintenance methyltransferase copying methylation marks of hemimethylated DNA to the newly synthesized daughter strand during DNA replication, making the enzyme indispensable for dividing progenitor cells [29, 39, 40]. This is supported by the finding that DNMT1 has higher affinity to hemimethylated DNA [41, 42] and that gene knockout of Dnmt1 in the central nervous system leads to lethal in mice [43]. While Dnmt1 deletion in all dividing somatic cells is also lethal [43–47], mouse embryonic stem cells are viable, despite the resulting global loss of DNA methylation [48]. Notably, human embryonic stem cells (ESCs) also displayed a global demethylation upon Dnmt1 deletion [49].

However, in accordance with the special requirement, the DNMT1 and DNMT3A are functionally correlated. For example, in the adult brain, both methyltransferases could carry out cytosine methylation in the promoter and gene body regions, leading to transcription repression [31].

While DNMT1 is believed to function mainly for the maintenance of established patterns of DNA methylation in normal living cells, in the diseased cells such as cancer cells, DNMT1 alone is not sufficient to maintain the programmed normal gene hypermethylation. As such, the collaboration of DNMT1 and DNMT3b is indispensable for the maintenance function.

Dnmt3l is believed to function as a stimulator of the Dnmt3A and Dnmt3B, and has related function with DNMT2 [32, 36–38].

Sirt1 regulates DNA methylation and differentiation potential of embryonic stem cells by antagonizing Dnmt3l. DNMT2, a tRNA methyltransferase and the most conserved member of the DNMTs methylates tRNAs to protect them from ribonuclease digestion. More importantly, DNMT2 is functionally related to the sperm small RNA (sncRNAs) mediated essentially in writing the "paternal epigenetic signature" to sperm RNA [32]. The mechanism is that the DNMT2-conferred m5C in sncRNAs regulates the secondary structure and biological properties of sncRNAs, suggesting that sperm RNA modifications could serve as one of the carriers for paternally imprinted epigenetic memories [33].

Coordination of the DNA methylation by DNMTs as well as histone modifications contributes to the regulation of cell death through development, aging, and disease [34, 35].

### **2.2. Demethylation and demethylases**

**1. Introduction**

188 Chromatin and Epigenetics

5-cytosine (5-C).

TET1 in the regulation of tumorigenesis.

the related epigenetic markers into the biochemical effects.

**2.1. Methyltransferases of cytosine methylation**

**2. Cytosine modifications**

Epigenetics is defined as the investigation on gene expression alterations heritable to next generations caused by nongenetic but heritable cellular memory other than DNA sequence variations [1]. The epigenetic memories including dynamic base modifications (DNA methylation/ demethylation), histone modifications, chromatin architecture, and noncoding RNAs maintain all the biological processes in the programmed tracks. Any aberrant alterations could lead to development of abnormality and initiation of diseases such as neurological disorders and cancers as reviewed in [2–8]. A micro-event in base modification could lead to strong "earthquake" in the signaling pathways and the consequent alteration of organism phenotypes, even diseases. The most extensively studied modifications are methylation and demethylation of

DNA base modifications such as methylation of 5-mC [9–14] and 5-hydroxymethylcytosine (5-hmC) [15–21] have been acknowledged as the best characterized epigenetic markers in mammalian brains [20, 22–24] and ES cells [25–27], essentially regulating chromatin structure and consequently gene expression with the potential mechanisms. This review article mainly focuses on the recent advances in methylation/demethylation modifications of 5-C in mammalian genomes, including methylation/demethylation machineries, methyltransferase complexes (writers) and demethylase complexes (erasers), as well as the distinct functions of

To maintain the normal life process, any base modification must be dynamically and tightly regulated in accordance with stages of the growth, development, and reproduction, including modification generation by methyltransferase complexes (writers), removal by demethyltransferases (erasers), as well as the preferential binding protein components (readers), to get

DNA methylation, particularly the most abundant CpG methylation marker 5-mC, is an essential modification of DNA in the mammalian genome, typically linked with gene silencing and involved in gene regulation, development, genome defense, and disease. A family of DNA methyltransferases named (DNMTs) is responsible for the addition of methyl groups to the 5-position of the carbon, including DNMT1, DNMT2, DNMT3a, DNMT3b, and DNMT3L. The five members are structurally and functionally distinct. The three methyltransferase enzymes DNMT1, DNMT3a, and DNMT3b serve as writers for the de novo CpG methylation pattern during embryogenesis [28, 29], while DNMT1 could confer the maintenance of parent DNA

methylation patterns to the new daughter strand DNA during DNA replication [30].

Traditionally, DNMT1 was regarded as the maintenance methyltransferase copying methylation marks of hemimethylated DNA to the newly synthesized daughter strand during DNA The dynamic DNA methylation/demethylation is tightly regulated during the whole life span. DNA demethylation, the removal of a methyl group, is not just a reverse process of methylation, but rather very complicated metabolic pathways indispensable for reactivation of genes and directly involved in pathogenesis of diseases such as cancers and neurological disorders. Either passive, active, or combination of both, leads to demethylation of DNA. The passive mechanism renders the automatic demethylation in a way that dilution and gradual loss of methylation in the newly synthesized DNA strands during successive replication rounds. In contrast, the active demethylation is believed to be the most important mechanism for active DNA demethylation via 5-mC oxidation catalyzed by the 10-11 translocation proteins (TETs) in alpha-ketoglutarate (a-KG) and Fe(II) dependent manner [22]. In addition to TETs, several other enzymes are acknowledged to be involved in the active mechanisms for demethylation, such as activation-induced cytidine deaminase (AID) [50], TET [51, 52], and thymine DNA glycosylase (TDG) [53–55].

5-hmC is generated by oxidation of 5-mC by TET, and the 5-hmC faces several fates once it is generated. First, the 5-hmC could be directly converted to regular cytosine through mechanisms involving the base excision repair pathway. Second, stepwise, a small percentage (~10%) of the 5-hmC is converted to 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC), respectively [56, 57]. The 5-fC and 5-caC are finally converted into regular cytosine [58] with the help of Thymine-DNA glycosylase (TDG). Finally, in some tissues such as stem cells and adult neuron cells, high 5-hmC levels could be detected particularly in transcribed regions adjacent to the promoter and enhancers, positively correlating with gene expression. The low turnover rates of 5-hmC in some tissues suggest that besides serving as an intermediate of active demethylation, the stable accumulation of the 5-hmC forms a dynamic 5-hmC landscape to serve as special epigenetic markers, potentially altering the local chromatin structures via recruiting or repelling some special protein components with high affinity to or low even repellent to 5-hmC-harboring DNA [59, 60]. For example, 5-hmC loss has become a hall marker for cancer cells [61–66]. In addition, the TET members are acknowledged as the tumor suppressors as Tet gene mutations or deletions have been identified in some tumor tissues [67].

On the other hand, TET1 also confers transcription repression of its target genes. It is accepted that the TET1-mediated transcription repression does not require the catalytic activity of the TET1 in conversion of 5-mC to 5-hmC, but rather the interaction between TET1 and some other protein components that contain repressor complexes [79]. Several mechanisms for TET1-mediated transcription repression have been proposed. First of all, TET1 binds a large number of polycomb target genes and interacts with SIN3A, the core component of the SIN3A co-repressor complex, leading to the transcription repression of their co-target genes via the

Cytosine Modifications and Distinct Functions of TET1 on Tumorigenesis

http://dx.doi.org/10.5772/intechopen.83709

191

The second mechanism of the TET1 conferred transcription repression is involved in TET1 interaction with recruitment of MBD repression complexes such as MBD3 [78, 81] at least in ES cells. The evidence of the mechanism includes the co-localization of TET1 and MBD3 in ESCs, higher affinity to 5-hmC than 5-mC, and association of the MBD3 knockdown with reduced level of 5-hmC as well as the enhanced expression of the 5-hmC-modified genes.

Several other mechanisms that TET1 represses the transcription have been also uncovered. It is convinced that TET1 is involved in the repression of polycomb-targeted regulator genes in accordance with the development stage by recruiting polycomb repressive complex 2 (PRC2) to the CpG-rich promoters of these genes [82]. Further study indicated requirement of the catalytic activity in oxidation of 5-mC to 5-hmC for the PRC repressive complex-mediated repression, evidenced by the fact that the PRC2 was co-localized with 5-hmC [80], while TET1

During the early stages of epiblast differentiation, repression of TET1 target genes was conferred by the interaction between TET1 and the JMJD8 and enhancement of the JMJD8 demethylase transcriptional repressor expression [83], but does not require the TET1 oxidation activity. Although TET1, TET2, and TET3 are all expressed in gonadotrope-precursor cells, the TET1 expression was dramatically decreased in the differentiated cells. Differentiation with according increase in the expression of the luteinizing hormone gene (Lhb). The short isoform of TET1 with deletion of the N-terminal CXXC-domain binds the H3K27me2/3 enriched region located at the upstream promoter of the Lhb gene, downregulating its expression and

Initially, given the mutations and the deletions as predominant variation of TET proteins, particularly TET1, in human cancer genomes, it was accepted that TET1 functions as a tumor suppressor [61, 65, 66]. Indeed, TET1 and TET3 bear the predominant mutations in some tumors including colorectal cancer, melanoma, and cutaneous squamous cell carcinoma [88–90]. However, emerging evidences are connecting the TET1 overexpression and tumorigenesis as well, most likely attributed to activation of cancer-specific oncogenic pathways mediated by

recruits the EZH2 DNMT-containing PRC complex targeting H3K27 methylation.

SIN3A conferred histone deacetylation [76, 80].

leading to differentiation deficiency [73].

**3. Distinct functions of TET1 on tumorigenesis**

TET1 conferred hypomethylation [72, 84] (**Figures 1** and **2**).

**3.1.** *Tet1* **functions as an oncogene in some cancers**

In mammalian genome, the TET family is consisted of three members, including TET1, TET2, and TET3. While all three TET members could function as hydroxylases for conversion of 5-mC to 5-hmC and further stepwise from 5-hmC to 5-fC and 5-fC to 5-caC, their functions involved in diverse biological pathways are in the development stage and specifically in tissue-dependent manners [25, 68].

#### *2.2.1. TET1 and regulation of its target gene expression*

Highly expressed in ESCs, PGCs, and inner cell mass of blastocyst, TET1 protein has been proven to be mainly responsible for the initial oxidation of 5-mC to 5-hmC, and to establish the paradoxically dual distinct epigenetic patterns in transcriptional activation and repression in accordance with life processes of growth and development. Alternative splicing mechanism leads to several TET1 isoforms, including the full-length canonical and the short transcripts [69–73]. TET1 expression is regulated by very complicated factors including the reprogramming factors such as Oct3/4, Nanog, and Myc [68, 70] in early embryos, ESCs and PGCs [69], the transcription factors in the differentiated cells, and STAT3/STAT5 in acute myeloid leukemia (AML) [74].

The full length of TET1 protein is believed to have multiple functions in regulation of gene expression. In general, TET1 catalyzes the oxidation of 5-mC to 5-hmC, which serves as an epigenetic marker and intermediate for active demethylation, leading to transcription activation. The more emerging evidence has supported the TET1 conferred transcription activation and repression of its direct target genes [75–77] at the transcriptional level. At the molecular level, the interaction between TET1 and SIN3a facilitates transcription activation of their target genes at the transcription level. More importantly, the interaction has been detected between TET1/ TET2 and E26 transformation-specific or E-twenty-six (ETS) family, one of the largest transcription factor families. For example, ETS variant 2 (ETV2), an ETS family transcription factor, interacts with TET1/TET2 to recruit the demethylases to the Robo4 promoter for demethylationmediated transcription activation during endothelial differentiation. More recently, the Methyl-CpG-binding domain (MBD) protein, such as MBD1, through its CXXC domain recruits TET1 other than TET2 and TET3 to the heterochromatin for oxidation of 5-mC to 5-hmC, whereas the resulting 5-hmC releases the MBD1 from the binding sites by affinity-based displacement [78].

On the other hand, TET1 also confers transcription repression of its target genes. It is accepted that the TET1-mediated transcription repression does not require the catalytic activity of the TET1 in conversion of 5-mC to 5-hmC, but rather the interaction between TET1 and some other protein components that contain repressor complexes [79]. Several mechanisms for TET1-mediated transcription repression have been proposed. First of all, TET1 binds a large number of polycomb target genes and interacts with SIN3A, the core component of the SIN3A co-repressor complex, leading to the transcription repression of their co-target genes via the SIN3A conferred histone deacetylation [76, 80].

The second mechanism of the TET1 conferred transcription repression is involved in TET1 interaction with recruitment of MBD repression complexes such as MBD3 [78, 81] at least in ES cells. The evidence of the mechanism includes the co-localization of TET1 and MBD3 in ESCs, higher affinity to 5-hmC than 5-mC, and association of the MBD3 knockdown with reduced level of 5-hmC as well as the enhanced expression of the 5-hmC-modified genes.

Several other mechanisms that TET1 represses the transcription have been also uncovered. It is convinced that TET1 is involved in the repression of polycomb-targeted regulator genes in accordance with the development stage by recruiting polycomb repressive complex 2 (PRC2) to the CpG-rich promoters of these genes [82]. Further study indicated requirement of the catalytic activity in oxidation of 5-mC to 5-hmC for the PRC repressive complex-mediated repression, evidenced by the fact that the PRC2 was co-localized with 5-hmC [80], while TET1 recruits the EZH2 DNMT-containing PRC complex targeting H3K27 methylation.

During the early stages of epiblast differentiation, repression of TET1 target genes was conferred by the interaction between TET1 and the JMJD8 and enhancement of the JMJD8 demethylase transcriptional repressor expression [83], but does not require the TET1 oxidation activity. Although TET1, TET2, and TET3 are all expressed in gonadotrope-precursor cells, the TET1 expression was dramatically decreased in the differentiated cells. Differentiation with according increase in the expression of the luteinizing hormone gene (Lhb). The short isoform of TET1 with deletion of the N-terminal CXXC-domain binds the H3K27me2/3 enriched region located at the upstream promoter of the Lhb gene, downregulating its expression and leading to differentiation deficiency [73].
