**4. Histone conformation**

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 removed by the histone deacetylases (HDACs).

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.

Epigenetics and Breast Cancer 297

As a result of that, cancer was thought to be exclusively a consequence of genetic changes in key tumor-suppressor genes and oncogenes that regulate cell proliferation, DNA repair, cell differentiation, and other homeostatic functions. During the last decade, the study of epigenetic mechanisms in cancer, such as DNA methylation, histone modification, nucleosome positioning, and micro RNA expression, has provided extensive information about the mechanisms that contribute to the neoplastic phenotype through the regulation of expression of genes critical to transformation pathways. Regarding DNA methylation, the low level of CpG methylation in tumors compared with that in their normal-tissue counterparts was one of the first epigenetic alterations to be found in human cancer (Feinberg & Vogelstein, 1983;Goelz et al., 1985) this let us to think that the cancer cells have a specific epigenome. hypomethylation in cancer cells is associated with a number of adverse products, including chromosome instability, activation of transposable elements, and loss of

As well documented, about 80 % of human transcribed RNA is not translated into protein. This RNA was thought to be either functionless (Mattick, 2001), or transcriptional noise (Dennis, 2002). From this population, micro RNAs (miRNA) have an established epigenetic role with the potential to be implicated in programming. micro RNA (miRNA) are small untranslated RNAs generally 21-25 mucleotides in length (Bartel, 2004), they regulate gene expression by affecting the stability or the translation efficiency of target mRNA. They bind their complementary mRNA and thus dsRNA is formed, this recognized as foreign RNA and cleaved to be degraded. Matching between the miRNAs and mRNA doesn't have to be perfect as even incomplete binding can block translation (Mattick & Makunin, 2005). Nearly 30% of genes expression is probably regulated by miRNA via the interaction between miRNAs and their target mRNA. Individual miRNA may regulate 200 targets by partial base pairing to mRNA, sugessting that one miRNA may control numerous biological or pathological signaling pathway by affecting the expressions and functions of their targets. It has been reported that miRNA has a role in the development process (He & Hannon, 2004), including a role in the process of stem cell differentiation (Houbaviy et al., 2003). Also it has been shown in cancer studies of miRNA that DNA methylation and histone modification control the expression of these small RNAs. This was achieved by studying the effect of DNA demethylating agents and hisdtone deacetylases inhibitors on the expression of miRNA expression particularly the miR-127 which is embedded in CpG island (Saito &

Genomic imprinting is a developmental phenomenon that describes a unique form of gene regulation that leads to only one parental allele being expressed depending on its parental origin (Delaval & Feil, 2004;Surani, 1991). Insulin-like growth factor 2 (IGF2) and its receptor IGF2R are two of the first reported genes subjected to imprinting regulation (Barlow et al., 1991;DeChiara et al., 1991). In mouse genome there are 600 predicted imprinted genes (Luedi et al., 2005). These identified imprinted genes have a major common feature in that they are associated with at least one regulatory DNA element, often referred to as imprinted control region (ICR). The ICR region is essential in regulating the parental origin-specific

genomic imprinting (Berdasco & Esteller, 2010).

**6. Micro RNA and epigenetic** 

Jones, 2006;Saito et al., 2006).

**7. Genomic imprinting** 

The acetyltransferases catalyse the addition of the acetyl group from acetyl coenzyme A (acetyl-CoA) to the epsilon-amino group of specific lysine residues (it-Si-Ali et al., 1998;Kim et al., 2000), where deacetylases reverse the reaction (Kuo & Allis, 1998). There are eighteen HDAC enzymes in mammalian cells which are divided into two families: a) zinc metalloenzymes that catalyses the hydrolysis of acetylated specific residues on histone tails and include class I, II and 1V HDACs, and b) NAD-dependent Sir2 deactylases which are considerd as class III HDACs (Glaser, 2007;Vigushin et al., 2001).

Class I is a group of four enzymes known as HDAC1, 2, 3 and 8 and this class is associated with gene regulation. They are expressed ubiquitously and they function exclusively in the nucleus (Brehm et al., 1998;Glaser, 2007). Class II is subdivided into class IIA, which includes HDAC 4, 5, 7 and 9 and class IIB that includes HDAC 6 and 10. Class II enzymes shuttle between cytoplasm and nucleus, and they involve mainly in cell differentiation and are highly expressed in certain tissues such as heart, skeletal muscle and brain (de Ruijter et al., 2003;Glaser, 2007;Grozinger et al., 1999;Vigushin et al., 2001). Class III includes the NADdependent deacetylases which is a group of seven enzymes that are involved in maintaining the chromatin stability. They can remove the acetyl groups from histones as will as other proteins (Kyrylenko et al., 2003). Class IV contains one member which is HDAC 11. It is closely related to class I thus some reviewers consider it as a member of that class. The function of HDAC 11 has not been characterized yet (Crabb et al., 2008;de Ruijter et al., 2003).

### **5. DNA methylation and histone modification**

Besides to the promoter methylation, chromatin modification may also contribute to silencing genes in cancer cells. Post-translational modifications to histone proteins occur after translation primarily in the NH2 terminal tail of histones and include acetylation, methylation, phosphorylation, or ubiquitination (Dworkin et al., 2009). Three decade ago Razin and Cedar (1977) have reported the presence of tight correlation between DNA and chromatin structure (Razin & Cedar, 1977). It was believed the relationship is a unidirectional relationship i.e the state of DNA methylation defines chromatin structure; methylated DNA results in closed chromatin configuration while unmethylated DNA results in open chromatin configuration. This hypothesis was supported by research finding that showed that methylated DNA binding proteins recruits chromatin modification enzymes to methylated genes such as MeCP2 (Meehan et al., 1992;Nan et al., 1997). There is increasing evidence showing that changes in chromatin structure would alter DNA methylation patterns. Furthermore, the targeting of DNA methylation enzymes to genes promoters is guided by chromatin modifying enzymes. The fact that is chromatin configuration is dynamic and that is chromatin modifying enzymes activated by cellular signaling pathways. This provides a link between the extracellular environment and the state of DNA methylation (Szyf, 2007). One of the evidence of the link between chromatin modiling and DNA methylation in humans and mice mutation of the SWI-SNF proteins which are involved in chromatin remidling, result in defect in DNA methylation (Szyf, 2007). A number of histone methyltransferases, such as G9a, SUV39H1 and EZH2, a member of the multiprotein polycomb complex PRC2 can regulate DNA methylation by either recruiting or regulating the stability of DNMTs. DNMTs in turn can recruit HDACs and MBPs to achieve chromatin condensation and gene silencing (Sharma et al., 2010). This relationship between the epigenetic machinery makes the epigenetic mechanisms of genome expression a tightly regulated process.

The acetyltransferases catalyse the addition of the acetyl group from acetyl coenzyme A (acetyl-CoA) to the epsilon-amino group of specific lysine residues (it-Si-Ali et al., 1998;Kim et al., 2000), where deacetylases reverse the reaction (Kuo & Allis, 1998). There are eighteen HDAC enzymes in mammalian cells which are divided into two families: a) zinc metalloenzymes that catalyses the hydrolysis of acetylated specific residues on histone tails and include class I, II and 1V HDACs, and b) NAD-dependent Sir2 deactylases which are

Class I is a group of four enzymes known as HDAC1, 2, 3 and 8 and this class is associated with gene regulation. They are expressed ubiquitously and they function exclusively in the nucleus (Brehm et al., 1998;Glaser, 2007). Class II is subdivided into class IIA, which includes HDAC 4, 5, 7 and 9 and class IIB that includes HDAC 6 and 10. Class II enzymes shuttle between cytoplasm and nucleus, and they involve mainly in cell differentiation and are highly expressed in certain tissues such as heart, skeletal muscle and brain (de Ruijter et al., 2003;Glaser, 2007;Grozinger et al., 1999;Vigushin et al., 2001). Class III includes the NADdependent deacetylases which is a group of seven enzymes that are involved in maintaining the chromatin stability. They can remove the acetyl groups from histones as will as other proteins (Kyrylenko et al., 2003). Class IV contains one member which is HDAC 11. It is closely related to class I thus some reviewers consider it as a member of that class. The function of

Besides to the promoter methylation, chromatin modification may also contribute to silencing genes in cancer cells. Post-translational modifications to histone proteins occur after translation primarily in the NH2 terminal tail of histones and include acetylation, methylation, phosphorylation, or ubiquitination (Dworkin et al., 2009). Three decade ago Razin and Cedar (1977) have reported the presence of tight correlation between DNA and chromatin structure (Razin & Cedar, 1977). It was believed the relationship is a unidirectional relationship i.e the state of DNA methylation defines chromatin structure; methylated DNA results in closed chromatin configuration while unmethylated DNA results in open chromatin configuration. This hypothesis was supported by research finding that showed that methylated DNA binding proteins recruits chromatin modification enzymes to methylated genes such as MeCP2 (Meehan et al., 1992;Nan et al., 1997). There is increasing evidence showing that changes in chromatin structure would alter DNA methylation patterns. Furthermore, the targeting of DNA methylation enzymes to genes promoters is guided by chromatin modifying enzymes. The fact that is chromatin configuration is dynamic and that is chromatin modifying enzymes activated by cellular signaling pathways. This provides a link between the extracellular environment and the state of DNA methylation (Szyf, 2007). One of the evidence of the link between chromatin modiling and DNA methylation in humans and mice mutation of the SWI-SNF proteins which are involved in chromatin remidling, result in defect in DNA methylation (Szyf, 2007). A number of histone methyltransferases, such as G9a, SUV39H1 and EZH2, a member of the multiprotein polycomb complex PRC2 can regulate DNA methylation by either recruiting or regulating the stability of DNMTs. DNMTs in turn can recruit HDACs and MBPs to achieve chromatin condensation and gene silencing (Sharma et al., 2010). This relationship between the epigenetic machinery makes the epigenetic mechanisms of genome

HDAC 11 has not been characterized yet (Crabb et al., 2008;de Ruijter et al., 2003).

considerd as class III HDACs (Glaser, 2007;Vigushin et al., 2001).

**5. DNA methylation and histone modification** 

expression a tightly regulated process.

As a result of that, cancer was thought to be exclusively a consequence of genetic changes in key tumor-suppressor genes and oncogenes that regulate cell proliferation, DNA repair, cell differentiation, and other homeostatic functions. During the last decade, the study of epigenetic mechanisms in cancer, such as DNA methylation, histone modification, nucleosome positioning, and micro RNA expression, has provided extensive information about the mechanisms that contribute to the neoplastic phenotype through the regulation of expression of genes critical to transformation pathways. Regarding DNA methylation, the low level of CpG methylation in tumors compared with that in their normal-tissue counterparts was one of the first epigenetic alterations to be found in human cancer (Feinberg & Vogelstein, 1983;Goelz et al., 1985) this let us to think that the cancer cells have a specific epigenome. hypomethylation in cancer cells is associated with a number of adverse products, including chromosome instability, activation of transposable elements, and loss of genomic imprinting (Berdasco & Esteller, 2010).
