**2. DNA methylation**

DNA methylation is a well conserved process that occurs in eukaryotes and prokaryotes (Klose & Bird, 2006). DNA methylation refers to the covalent addition of a methyl group to carbon number five in the nitrogenous base cytosine at the DNA strand (Fuks, 2005;Szyf et al., 2008). However, methylation does not occur in every cytosine, but only those adjacent to guanine are targets for the methylation by the methyltransferases enzymes. The CpG may occur in multiple repeats which are known as CpG islands (Fuks, 2005). These regions are often associated with the promoter regions of genes. Almost half of the genes in our genome have CpG rich promoter regions. In the whole genome, about 80% of the CpG dinucleutides not associated with CpG islands are heavily methylated (Robertson & Jones, 2000). In contrast the CpG islands associated with gene promoters are usually unmethylated (Singal & Ginder, 1999). There are a number of factors that may maintain the undermethylated state of CpG islands, such as sequence feature, SP1 binding sites, specific acting enhancer elements, as well as specific histone methylation mark H3K4me3, which prevents the binding of de novo methylation complexes (Straussman et al., 2009). Methylation of the CpG islands in the promoter region silences gene expression, and the absence of methylation is associated with active transcription. Thus unmethylated CpG islands are associated with the promoters of transcriptionally active genes, such as housekeeping genes and many regulated genes, such as genes showing tissue specific expression (Bird, 1986;Song et al., 2005).

CpG dinucleotides are under-represented in the genome except for small clusters, referred to as CpG islands, located in or near the promoter of greater than 70% of all genes (Balch et al, 2007; Brena et al, 2006; Hellebrekers et al, 2007). Promoter methylation is known to participate in reorganizing chromatin structure and also plays a role in transcriptional inactivation. It is believed that the chromatin surrounding an active promoter containing an unmethylated CpG island is "open" and allows for the access of transcription factors and other coactivators. An inactive promoter containing methylated CpG dinucleotides is associated with a "closed" chromatin configuration and results in transcription factors unable to access the promoter (Dworkin et al., 2009).

#### **2.1 DNA methylation and breast cancer**

There are well understood genetic alterations associated with breast carcinogenesis, including specific gene amplifications, deletions, point mutations, chromosome rearrangements, and aneuploidy. In addition to these highly characterized mutations, epigenetic alterations resulting in aberrant gene expression are key contributor to breast tumorigenesis (Campan et al., 2006; Giacinti et al., 2006; Mirza et al., 2007; Sharma et al., 2005; Sui et al., 2007; Vincent-Salomon et al., 2007; Visvanathan et al., 2006; Zhou et al., 2006. Decreased methylation of repetitive sequences in the satellite DNA of the pericentric region of chromosomes is associated with increased chromosomal rearrangements, mitotic recombination, and aneuploidy (Eden et al., 2003,Karpf and Matsui, 2005). Intragenomic endoparasitic DNA, such as L1 (long interspersed nuclear elements) (Schulz, 2006) and Alu (recombinogenic sequence)

including cancer have been found to be associated with aberrant DNA methylation. Therefore we will summarize the, in this chapter the current knowledge on mechanisms of

DNA methylation is a well conserved process that occurs in eukaryotes and prokaryotes (Klose & Bird, 2006). DNA methylation refers to the covalent addition of a methyl group to carbon number five in the nitrogenous base cytosine at the DNA strand (Fuks, 2005;Szyf et al., 2008). However, methylation does not occur in every cytosine, but only those adjacent to guanine are targets for the methylation by the methyltransferases enzymes. The CpG may occur in multiple repeats which are known as CpG islands (Fuks, 2005). These regions are often associated with the promoter regions of genes. Almost half of the genes in our genome have CpG rich promoter regions. In the whole genome, about 80% of the CpG dinucleutides not associated with CpG islands are heavily methylated (Robertson & Jones, 2000). In contrast the CpG islands associated with gene promoters are usually unmethylated (Singal & Ginder, 1999). There are a number of factors that may maintain the undermethylated state of CpG islands, such as sequence feature, SP1 binding sites, specific acting enhancer elements, as well as specific histone methylation mark H3K4me3, which prevents the binding of de novo methylation complexes (Straussman et al., 2009). Methylation of the CpG islands in the promoter region silences gene expression, and the absence of methylation is associated with active transcription. Thus unmethylated CpG islands are associated with the promoters of transcriptionally active genes, such as housekeeping genes and many regulated genes, such as genes showing tissue specific expression (Bird, 1986;Song et al.,

CpG dinucleotides are under-represented in the genome except for small clusters, referred to as CpG islands, located in or near the promoter of greater than 70% of all genes (Balch et al, 2007; Brena et al, 2006; Hellebrekers et al, 2007). Promoter methylation is known to participate in reorganizing chromatin structure and also plays a role in transcriptional inactivation. It is believed that the chromatin surrounding an active promoter containing an unmethylated CpG island is "open" and allows for the access of transcription factors and other coactivators. An inactive promoter containing methylated CpG dinucleotides is associated with a "closed" chromatin configuration and results in transcription factors

There are well understood genetic alterations associated with breast carcinogenesis, including specific gene amplifications, deletions, point mutations, chromosome rearrangements, and aneuploidy. In addition to these highly characterized mutations, epigenetic alterations resulting in aberrant gene expression are key contributor to breast tumorigenesis (Campan et al., 2006; Giacinti et al., 2006; Mirza et al., 2007; Sharma et al., 2005; Sui et al., 2007; Vincent-Salomon et al., 2007; Visvanathan et al., 2006; Zhou et al., 2006. Decreased methylation of repetitive sequences in the satellite DNA of the pericentric region of chromosomes is associated with increased chromosomal rearrangements, mitotic recombination, and aneuploidy (Eden et al., 2003,Karpf and Matsui, 2005). Intragenomic endoparasitic DNA, such as L1 (long interspersed nuclear elements) (Schulz, 2006) and Alu (recombinogenic sequence)

epigenetic and its potential application in breast cancers.

unable to access the promoter (Dworkin et al., 2009).

**2.1 DNA methylation and breast cancer** 

**2. DNA methylation** 

2005).

repeats, are silenced in somatic cells and become reactivated in human cancer (Berdasco & Esteller, 2010). Furthermore, aberrations in DNA methylation patterns of the CpG islands in the promoter regions of tumor-suppressor genes are accepted as being a common feature of human cancer (Esteller, 2008). CpG island promoter hypermethylation affects genes from a wide range of cellular pathways, such as cell cycle, DNA repair, toxic catabolism, cell adherence, apoptosis, and angiogenesis, among others (Esteller, 2008), and may occur at various stages in the development of cancer. (Berdasco & Esteller, 2010).

Fig. 1. Hypothetical model that explain how CpG island promoter hypermethylation. Multiple genes are hypermethylated in breast cancer compared to non-cancerous tissue which are affected genes from a wide range of cellular pathways induced by epigenetic changes.

Therefore, DNA methylation not only participates in cancer but has been found to regulate the histone modifications involved in tumor formation. The presence of certain histone modifications such as H4 R3 me2 is a marker of prostate cancer and increased expression of HDAC6 in breast cancer (Kurdistani, 2007). In addition the prognosis of certain malignancies can be affected by epigenetic status (Sakuma et al., 2007) . In normal cells, repetitive genomic sequences (e.g., centromeric satellite α-DNA and juxtacentromeric satellite DNA) are heavily methylated (Esteller, 2007; Jones & Baylin, 2002). The maintenance of methylation in this repetitive DNA could be important for the protection of chromosomal integrity by preventing chromosomal rearrangements, translocations and gene disruption through the reactivation of transposable elements (Eden et al., 2003; Ehrlich, 2002; Jones & Baylin, 2002). Besides hypermethylation of gene-associated CpG islands, hypomethylation of repetitive genomic DNA has also been identified as a specific feature in

Epigenetics and Breast Cancer 295

mediated transcriptional repression, and in turn causing the DNA methylation defect (global hypomethylation) (Sinkkonen et al., 2008; Benetti et al., 2008). Regarding the role of DNMTs in breast tumorigenesis, it has been reported that DNMT3b mRNA is overexpressed in breast cancer, a finding that correlates well with the hypermethylator phenotype and poor prognosis

Histones are five basic nuclear proteins that form the core of the nuclesome. The histone octamer contains two molecules each of histones H2A, H2B, H3 and H4. Histone H1 the linker histone is located outside the core and involve in the packing of DNA (Kornberg & Lorch, 1999). DNA wraps around the octamer in two turns of 146 base pairs (Luger et al., 1997), and the adjacent nucleosomes are connected and wrapped on each other by H1. Consequently histone modifications play a major role in regulating gene expression and extend the information potential of the DNA which explains the growing interest of the 'Histone Code' (Jenuwein & Allis, 2001;Zhang & Reinberg, 2001a). Modifications to amino acids on the N-terminal tails of histones protruding from the nucleosome core can induce both an open or closed chromatin structure and these affect the ability of transcription factors to access promoter regions to activate transcription. The covalent modification can be acetylation, methylation, phosphorylation and ubiquitination. Methylation of some residues is associated with both transcriptional repression, such as methylation of histone 3 lysine 9 (H3 K9) (Nakayama et al., 2001a)and others with transcriptional activation, such as methylation of histone 3 lysine 4 (H3 K4) (Strahl et al., 1999). Histone methylation is performed by histone methltransferase (HMTs) which can transfer up to three methyl groups to lysine residues within the tails of the histones with different effects on gene activity. Acetylation which occurs at lysine residue is associated with transcriptional activation (Turner, 2000). This modification is performed by histone acetylases (HATs) and

Other important regulators of chromatin conformation include the polycomb group (PcG) and trithorax group (trxG) proteins, which have key role in developmental gene regulation (Schuettengruber et al., 2007). They are recruited to response elements near proximal promoters to direct histone modifications, which induce both an active chromatin structure (trxG) and an inactive chromatin structure (PcG). Trithorax group proteins methylate H3 K4 to induce an active chromatin configuration (Schuettengruber et al., 2007), while PcG proteins direct the methylation of H3 K27 to induce a repressive chromatin configuration. The effect of PcG protein are however reversible, as removal of PcG during development leads to gene activation. PcG protein have been found to be implicated in regulation of developmental transcription factors, genomic imprinting and X chromosome inactivation (Heard, 2005). Acetylation of histones has been extensively studied as one of the key regulatory mechanisms of gene expression (Grant, 2001). Histone acetylation was found to affect RNA transcription as early as the 1960s (Allfrey et al., 1964). The highly conserved lysine residue at the N-terminal of H3 at position 9, 14, 18 and 23, and H4 lysine 5,8,12 and 16, are frequently targeted for modification (Roth et al., 2001). Acetylations of the lysine residues neutralize the positive charge of the histone tails. And therefore, decrease their affinity for DNA which results in open chromatin conformation allowing the transcriptional machinery to reach its target (Hong et al., 1993). Additionally, many histone acetylases (HATs) (Brownell & Allis, 1996; Parthun et al., 1996) and histone deacetylases (HDACs) (Taunton et al., 1996) have been described previously.

in breast tumors (Girault et al., 2003; Roll et al.,2008).

removed by the histone deacetylases (HDACs).

**4. Histone conformation** 

human cancers (Feinberg & Vogelstein, 1983; Narayan et al., 1998; Jones & Baylin, 2002). Although less well studied than DNA hypermethylation, several lines of investigation indicate that the global DNA hypomethylation identified in cancer cells might contribute to structural changes in chromosomes, loss of imprinting (LOI), micro satellite and chromosome instability through aberrant DNA recombination, aberrant activation of protooncogene expression and increased mutagenesis (Chen et al.,1998; Eden et al., 2003; Kaneda & Feinberg, 2005; Jones & Baylin, 2002). Global genomic hypomethylation in breast cancer has been known to correlate with some clinical features such as disease stage, tumor size and histological grade (Soares et al., 1999). Some proto-oncogenes implicated in proliferation and metastasis (e.g., synuclein γ and urokinase genes) or drug resistance to endocrine therapy (e.g., N-cadherin, ID4, annexin A4, β-catenin and WNT11 genes) have been found to be upregulated in breast cancer through the hypomethylation of their promoters (Fan et al., 2006; Gupta et al., 2003; Pakneshan et al., 2004).
