**1. Introduction**

Epigenetic changes in living organisms can basically be grouped under two headings. One is protein acetylation, which is an epigenetic modification at the protein level. The other is DNA methylation, an epigenetic modification that occurs at the DNA level. In living species, all macro-molecular structures are determined by nucleotide sequences in the genome, and there is a different mechanism that can be transferred to cell from cell, which has inherent

© 2016 The Author(s). Licensee InTech. 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. © 2018 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.

ability to determine gene expression. It is called epigenetic code. During creation of this code, DNA sequence does not undergo any change. The genetic and epigenetic alterations mentioned above result in the activation of oncogenes or the inactivation of tumour suppressor genes. Methylation may occur in any living organism from bacteria to complex species such as humans. The most common type of methylation is the methylation of gene promoters. This is followed by exon methylation, intron methylation and exon-intron methylations, which may be observed quite frequently.

changes to square may cause differentiation by altering histone function. For example, it can be seen that one, two and three bases of methyl group are added in methylation of arginine [1]. Histone changes are carried out by various enzymes. These include histone acetyl transferases (HATs), histone deacetylases (HDACs) and histone methyl transferases. The equilibrium in activity of these enzymes and their associated proteins is important when they can perform functions of normal cells. These equilibrium distortions can cause problems that can occur from

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The underlying transcriptional silencing mechanism of DNA methylation is based on the overmethylation of cytosine in CpG-rich islands in the promoter region of a gene. This mechanism cooperates with histone deacetylation to suppress the chromatin structure. GC-rich DNA sequences in the human genome are often found in the promoter region and exon 1 of about 50% of all genes [2]. DNA methylation is the main underlying mechanism that regulates gene expression in mammalian cells, as it happens to be one of the major mechanisms for

The most widely studied and the most well-established epigenetic mechanism is DNA methylation. It is an enzymatic change where cytosines are converted to 5′-methylcytosine. The cytosine-end methylation seen in mammalian genome often occurs at the 5'-CpG-3′ dinucleo-

Methylation occurs by means of DNA methyltransferase (DNMT) enzymes. The DNMT family consists of four members, namely DNMT1, DNMT2, DNMT3A and DNMT3B. These enzymes are stratified into two groups: those that protect the methylated region and the ones that add new methyl groups. About 70% of all CpG dinucleotides of the human genome are methylated [5]. The remaining are the CpG-rich promoter regions of about 200 base pairs or are the first exons of genes. These regions are also called CpG islands and are found in 60% of all genes [6]. CpG methylation is programmed during the early embryonic period and preserved in later periods. CpG methylation is highly important with regard to normal functions of a given cell, as it affects the regulation of gene expression. For example, DNA methylation plays an important role in gene silencing of the inactive X chromosome as well as the regulation of age-related

Although the structural changes that occur in DNA are usually termed as mutations, not every alteration is actually a mutation. A mutation refers to any change at base level such as purineto-pyrimidine (G-A) or pyrimidine-to-pyrimidine (C-T) changes; single or multiple alterations; insertions, deletions and even single nucleotide polymorphisms (SNP). Yet, SNPs differ from mutations due to their structure. When methylation is compared with other changes in DNA, the methylation process may be considered as another type of mutation, with a change in the structure of the base resulting from a chemical change in DNA. However, mutations are rare changes compared to methylation, and they may or may not be repaired by DNA repair mechanisms [8]. They can be inherited from any ancestor or parent, and they may also occur as germline changes. On the other hand, SNPs can be called DNA alterations, which are more common in the population and which manifest themselves as susceptibility to disease, rather

the silencing of genes involved in cell cycle as well as cell growth and death [3].

loss of cell function to cancer formation.

**1.2. DNA modifications—methylations**

tides, which are also called CpG dinucleotides [4].

or tissue-specific gene expression [7].

Methylation-specific PCR (MSP) and methylation-sensitive restriction fragment length polymorphism (MS-RFLP) are the two most widely utilized methods in DNA methylation studies. Also, modified DNA sequencing with bisulphite treatment, known as bisulphite sequencing, may also be employed to investigate the conformation of the region of interest. These 3 are considerably successful methods. With the advances in technology and reduced costs, methylation-specific DNA sequencing has become a frequently used method to investigate the methylated regions identified by means of these methods. Whether a methylation region affects the expression of the gene of interest is another aspect to take into account as some genes may not yield any products although they are not methylated. In that case, one should consider that the gene in question may be activated by other mechanisms. With a better understanding of such histone and DNA modifications, they now attract attention as therapeutic targets in cancer and various diseases. They have started to create new alternatives especially in cancer treatments. Various computer programs have begun to be developed for methylation analysis. This section discusses all of the aforementioned conditions separately.

### **1.1. Histone modifications**

The most basic unit of the structure called chromatin is nucleosomes. A nucleosome is a unit of 146 bp stretch of DNA over H2A, H2B, H3 and H4 central histone proteins and binding of H1 protein to the structure as a lock. In addition, these constructs provide necessary packaging for the DNA molecule, which is quite large, to fit in a small area (**Figure 1**).

Covalent changes in amino acids are found in tail parts of central histone proteins form the epigenetic code. As a result of these changes, chromosome structure constitutes expression control constructs in DNA by acquiring heterochromatin (expressionally inactive) or by forming regions euchromatin (expressionally active regions). Histone modifications can be classified as acetylation, methylation and phosphorylation. The modifications are mostly visible and reversible at the amino (NH<sup>3</sup> -) and carboxyl (COO-) ends of central histone proteins. Each of these

**Figure 1.** Core histone and nucleosome structure.

changes to square may cause differentiation by altering histone function. For example, it can be seen that one, two and three bases of methyl group are added in methylation of arginine [1]. Histone changes are carried out by various enzymes. These include histone acetyl transferases (HATs), histone deacetylases (HDACs) and histone methyl transferases. The equilibrium in activity of these enzymes and their associated proteins is important when they can perform functions of normal cells. These equilibrium distortions can cause problems that can occur from loss of cell function to cancer formation.

#### **1.2. DNA modifications—methylations**

ability to determine gene expression. It is called epigenetic code. During creation of this code, DNA sequence does not undergo any change. The genetic and epigenetic alterations mentioned above result in the activation of oncogenes or the inactivation of tumour suppressor genes. Methylation may occur in any living organism from bacteria to complex species such as humans. The most common type of methylation is the methylation of gene promoters. This is followed by exon methylation, intron methylation and exon-intron methylations, which

Methylation-specific PCR (MSP) and methylation-sensitive restriction fragment length polymorphism (MS-RFLP) are the two most widely utilized methods in DNA methylation studies. Also, modified DNA sequencing with bisulphite treatment, known as bisulphite sequencing, may also be employed to investigate the conformation of the region of interest. These 3 are considerably successful methods. With the advances in technology and reduced costs, methylation-specific DNA sequencing has become a frequently used method to investigate the methylated regions identified by means of these methods. Whether a methylation region affects the expression of the gene of interest is another aspect to take into account as some genes may not yield any products although they are not methylated. In that case, one should consider that the gene in question may be activated by other mechanisms. With a better understanding of such histone and DNA modifications, they now attract attention as therapeutic targets in cancer and various diseases. They have started to create new alternatives especially in cancer treatments. Various computer programs have begun to be developed for methylation analysis. This section

The most basic unit of the structure called chromatin is nucleosomes. A nucleosome is a unit of 146 bp stretch of DNA over H2A, H2B, H3 and H4 central histone proteins and binding of H1 protein to the structure as a lock. In addition, these constructs provide necessary packaging for

Covalent changes in amino acids are found in tail parts of central histone proteins form the epigenetic code. As a result of these changes, chromosome structure constitutes expression control constructs in DNA by acquiring heterochromatin (expressionally inactive) or by forming regions euchromatin (expressionally active regions). Histone modifications can be classified as acetylation, methylation and phosphorylation. The modifications are mostly visible and revers-


may be observed quite frequently.

2 Chromatin and Epigenetics

**1.1. Histone modifications**

ible at the amino (NH<sup>3</sup>

**Figure 1.** Core histone and nucleosome structure.

discusses all of the aforementioned conditions separately.

the DNA molecule, which is quite large, to fit in a small area (**Figure 1**).

The underlying transcriptional silencing mechanism of DNA methylation is based on the overmethylation of cytosine in CpG-rich islands in the promoter region of a gene. This mechanism cooperates with histone deacetylation to suppress the chromatin structure. GC-rich DNA sequences in the human genome are often found in the promoter region and exon 1 of about 50% of all genes [2]. DNA methylation is the main underlying mechanism that regulates gene expression in mammalian cells, as it happens to be one of the major mechanisms for the silencing of genes involved in cell cycle as well as cell growth and death [3].

The most widely studied and the most well-established epigenetic mechanism is DNA methylation. It is an enzymatic change where cytosines are converted to 5′-methylcytosine. The cytosine-end methylation seen in mammalian genome often occurs at the 5'-CpG-3′ dinucleotides, which are also called CpG dinucleotides [4].

Methylation occurs by means of DNA methyltransferase (DNMT) enzymes. The DNMT family consists of four members, namely DNMT1, DNMT2, DNMT3A and DNMT3B. These enzymes are stratified into two groups: those that protect the methylated region and the ones that add new methyl groups. About 70% of all CpG dinucleotides of the human genome are methylated [5]. The remaining are the CpG-rich promoter regions of about 200 base pairs or are the first exons of genes. These regions are also called CpG islands and are found in 60% of all genes [6]. CpG methylation is programmed during the early embryonic period and preserved in later periods. CpG methylation is highly important with regard to normal functions of a given cell, as it affects the regulation of gene expression. For example, DNA methylation plays an important role in gene silencing of the inactive X chromosome as well as the regulation of age-related or tissue-specific gene expression [7].

Although the structural changes that occur in DNA are usually termed as mutations, not every alteration is actually a mutation. A mutation refers to any change at base level such as purineto-pyrimidine (G-A) or pyrimidine-to-pyrimidine (C-T) changes; single or multiple alterations; insertions, deletions and even single nucleotide polymorphisms (SNP). Yet, SNPs differ from mutations due to their structure. When methylation is compared with other changes in DNA, the methylation process may be considered as another type of mutation, with a change in the structure of the base resulting from a chemical change in DNA. However, mutations are rare changes compared to methylation, and they may or may not be repaired by DNA repair mechanisms [8]. They can be inherited from any ancestor or parent, and they may also occur as germline changes. On the other hand, SNPs can be called DNA alterations, which are more common in the population and which manifest themselves as susceptibility to disease, rather than resulting in a direct disease phenotype. At this point, methylation is not considered as a mutation, despite the fact that it prevents cytosine behaviour by adding a methyl group from CpG dinucleotides to cytosine [9, 10].

Several bacteria contain an N4-methylcytosine base whose function has not been fully characterised. There are studies indicating that these N4C modifications affect global gene expres-

Logic of Epigenetics and Investigation of Potential Gene Regions

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5

Methylation in bacteria is different from eukaryotes in that it is seen in the fourth carbon of the cytosine as well as methylated adenine (N6-methyladenine) in addition to the fourth carbon of cytosine. DNA methylation occurs in bacteria by methyl binding to cytosine C-5 or N-4 and N6-adenine on the DNA methyltransferase enzyme side. N6-methyladenine is found only in bacteria and in less complex eukaryotes, and not in vertebrates [11]. Interestingly, bacteria also contain a restriction modification system that digests DNA methylase to provide protection against foreign DNA. These consist of the restriction enzyme systems called DcM, which recognises the 5-C cytosine, and Dam, which recognises methylated adenine. Of these, the Dam family is the most well-known protein group. The functional domain of Dam is a DNA MTase

Similar to eukaryotes, bacteria also have rRNA methylation. The most important aspect of this methylation is that it creates targets for bacterial infections that can cause infection in humans. While promoter methylation is associated with negative expression, this may not always be the case for exon methylation. Still, sometimes exon methylation shows no effect on gene expression. Investigation on genetic mechanisms affecting cardiomyocyte differentiation includes some studies, which show that intragenic methylations create cellular memory through this

In all eukaryotic cells, mitochondrion is the most important organelle for cellular energy and the only organelle containing genomic material apart from the nucleus. Owing to its unique and small genome, this organelle exerts certain proteins and RNAs needed for respiratory reactions and cell growth. Together with the nucleus, it is one of the two genetic systems found in the cell. Mitochondrial DNA (mtDNA) has a circular structure and is located inside mitochondrial matrix, bound to the internal membrane. The mtDNA consists of 16,569 base pairs in a loop form, containing a heavy chain (H) and a light chain. This chain structure contains 2 rRNA molecules, 22 tRNA molecules, and 13 genes necessary for oxidative phosphorylation and electron transport (**Figure 2**). A healthy mitochondrion exerts adequate functions by means of certain proteins that are present in the mechanism of oxidative phosphorylation. This genome is about 16.5 kb in humans, and 13 proteins and rRNAs are synthesised from the mitochondrial genome in mammals [19, 20]. Therefore, the slightest change in mitochondrial

As is the case with mutations, methylation is a mechanism that alters the way the genes work together with the diet, drugs and oxidative stress. Methylation profile of human mtDNA starts from the intrauterine period. With the aid of foetal thyroid hormones, mtDNA copy number and mtDNA methylation are regulated by a thyroid-dependent pathway [23]. In addition, mtDNA is also affected by airway pollutants. The elemental carbon present in benzene and exhaust gas in traffic may influence the number of mtDNA copies by means of ribosomal

sion in *Helicobacter pylori*, an example of carcinogenic bacteria [17].

with an alpha molecule consisting of a polypeptide of 10 amino acids [18].

genome can potentially affect the life of the cell, and thus the organism [21].

mechanism, particularly in pluripotent cells [11].

**3. Mitochondrial methylation**

RNA methylation [24].

The most appropriate means of this option are the CpG sequences within the DNA, which are bound to their conjugates through an enzymatic process in a stronger manner compared to the A-T pairs. This is because ApTs are bound to their complementary pairs in the corresponding chain by means of two hydrogen bonds, while CpGs are bound with three. Such binding characteristics are expected to provide stability to CpGs compared to ApTs. This may explain the greater frequency of methylation in CpGs rather than ApTs in the organism.

Methylation usually occurs through the addition of a methyl group to CpG sequence or to the C base in these CpG islands. Although such a change normally appears as a mutation, it is understood that, unlike mutations, this change is a highly functional mechanism in terms of cellular development and quite common across living organisms from bacteria to highly complex multicellular species. In this way, the organism can adapt to environmental changes by changing the activation of the desired genes in response to external influences when necessary, thereby maintaining vitality and survival. During a methylation reaction, 5-methylcytosine is formed with the addition of a methyl group to the fifth carbon of the cytosine in CpG base pairs by the DNA methyltransferase enzyme (**Figure 1**). Potentially, any CpG base pair or island may undergo methylation. In addition, the fourth nitrogen of cytosine and sixth nitrogen of adenine, which are usually not found in multicellular organisms, may also be methylated in addition to the 5-methylcytosine formation in bacteria [11].

Genomic imprinting is another example of DNA methylation that is involved in single-allele gene expression. Approximately 80 loci are suppressed in this way. The tissue-specific and condition-specific expressions of these genes occur through the regulation of methylation [12]. At the end of 1970s, a decrease in methylcytosine numbers was observed in the genome of tumour cells [13]. This was referred to as hypomethylation of DNA and was demonstrated in benign and malignant tumours [14]. Hypomethylation of DNA may also activate oncogenes. Studies have shown hypomethylation in SI00A4, a metastasis-associated gene in colorectal cancers and the genes, cyclin-D2 and maspin, in gastric carcinomas [14, 15]. Hypomethylation may cause loss of imprinting (LOI), thereby promoting cell proliferation. One of the best examples of this process is the loss of imprinting in the IGF2/H19 region, which is seen in about 40% of colorectal cancers [1].
