**4. Epigenetic modifications in context of chromatin**

The organization of DNA into chromatin, although very necessary, imposes constraints on all the nuclear processes which require DNA as a template like replication, transcription and repair. Therefore, in order to gain access to the underlying DNA, chromatin structure is dynamically regulated through various mechanisms. This flexibility is permitted by mechanisms like histone modifications, incorporation of histone variants and chromatin remodeling [2].

Histone modifications act as binding platforms for various effectors for appropriate downstream signaling. Histone variants are incorporated by replacing canonical histones under specified conditions into nucleosomes. The variants possess different bio-physical properties compared to their canonical counterparts and hence play crucial roles in cellular processes like DNA repair. Chromatin remodeling leads to sliding of nucleosomes along chromatin, exposing regions of genome which could be acted upon by trans-acting factors for specified outcomes.

## **4.1 Histone modifications**

Histone proteins undergo a variety of covalent modifications which can either lead to compaction or relaxation of the underlying DNA within chromatin. The outcome of these modifications is dictated by the type of modification, degree of modification as well as stage of the cell cycle. Histone proteins consist of a highly structured C-terminal globular domain and an unstructured N-terminal tail. Globular domains are generally involved in mediating histone-histone and histone-DNA interactions while as N-terminal tails act as sites for covalent modifications. Among the different classes of histone proteins, histone H3 and H4 generally undergo covalent modifications in their tails. Though recently, H2A and H2B have also been observed to undergo certain modifications [55, 56]. Similarly, many modifications have been observed in the globular domain of histone H3 as well [1]. Histone modifications play role in numerous biological processes like gene regulation, DNA repair, chromosome condensation and spermatogenesis [57]. Some of the well-recognized histone modifications include acetylation and ubiquitination of lysine (K) residues, phosphorylation of serine (S) and threonine (T) residues, methylation of arginine (R) and lysine (K) residues as well as other less known modifications [58, 59]. These modifications are largely postulated to affect chromatin function through two distinct mechanisms: By altering the electrostatic charge of histones, these could alter the structural properties or the binding of histones to DNA. As against the first mechanism, some of the modifications create binding surfaces for the recruitment of specific functional complexes to their sites of action e.g., proteins containing bromodomains recognize acetylated residues while those containing chromodomains recognize methylated residues [60, 61]. It was, In fact, the potential specificity of these interactions which prompted Struhl and Allis to propose the *'histone code hypothesis'* according to which "*specific combinatorial sets of histone modification signals dictate the recruitment of particular trans-acting factors to accomplish specific functions"* [62]. Initially, it was thought that histone proteins undergo covalent modifications after translation (post translational modifications) in a manner dictated by nucleosomal context. But recently, it has been observed that histones can undergo co-translational modifications as well, depending upon the cellular context. This observation has added an additional layer into the role of histones in regulation of cellular homeostasis and clearly calls for more research in the field. Perturbations in histone modifications is associated with many physiological disturbances, including carcinogenesis [5].

### *4.1.1 Histone acetylation and deacetylation*

Acetylation is the most widely studied post translational modification in histones. This modification involves transfer of an acetyl group from N-acetyl-Co-A to the € amino group of lysine with the help of histone acetyltransferases (HATs). Histone acetylation is associated with loosing of chromatin structure due to neutralization of the positive charge on histones with the negative charge on acetyl group which is responsible for increase in transcription. In fact, various transcription activator or co-activator complexes contain HAT activity such as CBP 300, TAF II 250. Reversal of acetylation is carried out by another class of enzymes known as histone deactylases (HDACs). Both HATs and HDACs have been studied extensively in relation to various diseases, including neurodegeneration and cancer [4]. Depending upon the gene/s being involved (oncogenes or tumor suppressor genes), HATs and HDACs can have different effects on the cancer outcomes.

### *4.1.2 HATs, HDACs and cancer*

Relationship between histone acetylation status and cancer has been demonstrated in various studies. For example, a loss of acetylation on lysine 16 of histone H4 (H4K16) has been observed in cancer cell lines and primary human tissues by Fraga et al. [63]. Decrease in promoter acetylation and consequent decline in expression of P21 gene has been observed in some forms of cancer with subsequent rescue of expression upon treatment of cells with HDAC inhibitors under similar conditions [64]. Another study has linked decrease in histone acetylation with tumor invasiveness and metastasis [65]. Accumulating data also shows that HDACs are involved in hematological malignancies like acute promyelocytic leukemia (APL) due to aberrant recruitment to non-target promoters, as a result of interaction with translocation-induced fusion proteins like RAR-PML [66]. Downregulation of E-cadherin due to decrease in promoter acetylation levels has been implicated in the invasive potential by carcinomas [67, 68]. A number of studies have also linked levels of specific classes of HDAC enzymes with different forms of cancer like increase in HDAC1 expression in gastric [69], prostate [70], colon [71], breast carcinoma [72], increase in HDAC2 expression in cervical [73], gastric [74] and colorectal carcinoma [75]; increase in HDAC3 expression in colon carcinoma [76] and increase in HDAC 6 in breast carcinoma [71]. Mutations in HDAC2 gene has also been reported in sporadic colorectal carcinomas [77].

Various mechanisms are responsible for the role of specific forms of enzymes in specific cancer types, largely depending upon their interaction partners and the pathways involved. For example, HDAC1 has been shown to play a role in transcriptional repression of various oncogenic targets of retinoblastoma gene (Rb). Therefore, loss of HDAC1 activity leads to compromise in efficiency of Rb in downregulation of target oncogenes [78]. HDAC3 has also been seen to interact with retinoblastoma protein (Prb) in cancer, Perhaps the most important HDAC III enzyme in cancer is SIRT1 due to its role in regulation of protein factors like P53 [79], androgen receptor [80], p300 [81], E2F1 [82], DNA repair factor ku70 [83] and most importantly, NF-KB [84].

### *4.1.3 Histone methylation*

Histone methylation involves transfer of methyl group(s) from S-adenosylmethionine to lysine or arginine residues on histones. The enzymes catalyzing histone methylation are known as histone methyltransferases (HMTs). Depending on the

### *Evolution of Epigenome as the Blueprint for Carcinogenesis DOI: http://dx.doi.org/10.5772/intechopen.97379*

target residue, histone methyltransferases are of two kinds 1. Histone lysine methyltransferases (HKMTs) and histone arginine methyltransferases (HRMTs). Also, lysine residues have three replaceable amino groups on the β-carbon. Therefore, lysine can undergo mono, di or tri-methylation whereas arginine can undergo only mon and di methylation.

Histone methylation is most commonly observed on lysine residues of H3 and H4 tails [85]. It is the most diverse histone modification in terms of complexity and is involved in various functions, depending on the physiological context. Histone methylations commonly associated with gene activation include H3K4, H3K36 and H3K79 and those associated with gene inactivation include H3K9, H3K27 and H4K20 [86]. Furthermore, variations in the degree of methylation on a single residue can also amplify the histone code further. For example, monomethylated H4K20 (H4K20me1) is involved in the compaction of chromatin and therefore transcriptional repression. However, H4K20me2 is associated with repair of DNA damage [63].

Histone methylation is involved in several cellular functions like maintenance of chromatin structure, DNA repair, gene silencing, prevention of hyper-recombination, maintenance of genome integrity et cetera. It is also involved in maintenance of X-chromosome integrity and silencing through excessively methylation of H3K9 on the second copy of human X chromosome in female cells. This provides a binding surface for methyl domain binding (MDB) protein and heterochromatin protein (HP1) to heterochromatinize and silence the second copy of X-chromosome [87, 88]. Since histone methylation plays a paramount role in regulation of gene expression and represents the most stable and complex histone modification, even slight changes to the methylation pattern can have deleterious effects on the organism. In *Saccharomyces cerevisiae*, a lethal mutation that leads to H3K4, H3K36 and H3K79 methylation inactivates many genes required for cell cycle progression and hence causes a delay in mitosis. It has been discovered that deletion of the methyltransferase genes which play role in the above-mentioned methylations allows this organism to live since the lysine residues in question are not methylated [89].

### *4.1.4 HMTs and cancer*

Cancer cells use a diverse range of molecular mechanisms to alter histone methylation landscape. These include mis-regulation of histone methyltransferases and/ or demethylases, mistargeting of histone methyltransferases and mutations in methyltransferases. For example, if areas around oncogenes become unmethylated, these genes will attain the potential of being transcribed at an alarming rate. On the contrary, if areas around tumor suppressor genes become highly methylated, these genes will lose their activity and therefore cancer will be more likely to occur [90]. Accumulating data suggests that histone methylation is mis-regulated in various forms of cancer [91, 92]. Fraga et al. [63] have observed that loss of H4K20 trimethylation that leads to hypomethylation of repetitive sequences is a common event in human cancers which occurs at a early stage during tumorigenesis. Mutations on the genes encoding histone proteins are also linked with cancers. 30% of paediatric glioblastomas have mutations at key post translational modification sites in histone genes [93]. Recently, mutations in metabolic enzymes have also been observed to have a role in histone methylation status alteration. The mutated metabolic enzymes produce altered metabolites (popularly known as oncometabolites) which jeopardize the function of methylase enzymes. For instance, inhibition of histone demethylation Jumonji C enzymes by the oncometabolite d-2-hydroxyglutarate [94–97].

### *4.1.5 Histone phosphorylation*

Phosphorylation of histones takes place on serine, threonine, tyrosine and histidine residues, predominantly in the N-terminal tails of all nucleosomal histones by histone kinase enzymes which transfer a phosphate group from ATP to the hydroxyl group of the target amino-acid side chain. Phosphate group contains significant negative charge and therefore phosphorylation is generally associated with transcriptional upregulation. Various proteins have been identified which contain phosphor-binding domains [98, 99]. Histone phosphorylation changes dynamically with the transcriptional profile of the cell [100]. For example, H3Ser10 phosphorylation correlates with gene activation in mammalian cells and heat shock response induced transcription in *Drosophila* [101]. However, the same phosphorylation is associated with chromosome condensation and segregation during mitosis and meiosis [102]. Histone phosphorylations also play a pivotal role in response to DNA damage e.g., phosphorylation of H2A(X) on serine 139 in mammalian cells (referred to as γ H2AX) and S129 of H2A in yeast [103].

### *4.1.6 Histone phosphorylation and cancer*

Regulation of the level of histone H3 phosphorylation by an interplay of the activities of kinases and phosphatases serves as a means of promoting chromosomal condensation and segregation in mitosis [104]. Phosphorylation of H3S10 has also been linked to the expression of proto-oncogenes like *c-fos* [105–107]. It has been detected with the aid of ChIP assay that phosphoacetylation of H3 tails exist at the promoters of several MAP- kinase activated genes as well as the promoters of *c-fos* and *c-jun* [108]. H2A(X) phosphorylation is involved breast cancer [109] and colon cancer [110]. Histidine phosphorylation on histone H4 has been shown to be involved in liver regeneration and cancer [111]. Phosphoacetylation of histones, involving phosphorylation of histone H3 on residue serine 10 and acetylation of histone H4 on lysine 12 has been shown to have a role prognosis of oral squamous cell carcinoma [112].

### *4.1.7 Histone ubiquitination*

It is a process in which ubiquitin molecules are added to lysine residues of histones. Monoubiquitination is the major form of ubiquitination in histones. However, histones H2A and H2B can also be modified by polyubiquitination. The first ubiquitinated histone to be identified was H2A [113]. H2A and H2B also hold the distinction of being the most abundantly ubiquitinated proteins in the nucleus [113, 114]. In addition, H3, H4 as well as H1 have been reported to be modified by ubiquitin but the biological function of these ubiquitinations has not been well characterised [115, 116]. Histone ubiquitinations perform a number of important nucleosomal functions. Chromatin immunoprecipitation (ChIP) experiments have revealed enrichment of monoubiquitinated H2A (H2Aub) in the satellite regions of genome and of H2Bub in transcriptionally active genes [117, 118].

### *4.1.8 Histone ubiquitination and cancer*

Several recent studies have linked ubiquitination, especially H2Bub with inflammation and cancer [119–121]. Histone H2Bub1 predominantly resides downstream to transcription start sites (TSS), a position which allows association with highly

## *Evolution of Epigenome as the Blueprint for Carcinogenesis DOI: http://dx.doi.org/10.5772/intechopen.97379*

transcribed genes and therefore makes this protein a likely target in cancer [117]. RNF20/RNF40 has been shown to negatively regulate cancer- related inflammation in mice and humans through increased recruitment of repressive NF-κB subunit p50 to various gene targets to downregulate their transcription [121]. RNF40 is also known to modulate NF-κB activity in colorectal cancer in mice [122] while as RNF20 and H2B ubiquitylation have also been shown to be involved in breast cancer [123]. Loss of H2B monoubiquitination has also been shown to activate immune pathways by alteration of chromatin accessibility in ovarian cancer [124–126].
