Epigenomics Dna Methylation And Histone Acetylation

One relevant aspect of the methylation process, which was already known to operate in the inacti-vation of the X chromosome42 and loss of imprinting,43 is that it can be pharmacologically reversed. If DNA MTase, the enzyme responsible for methyl-ating the DNA, can be inhibited by a methyltrans-ferase inhibitor, the overmethylation can be reversed, thereby switching the tumor suppressor gene back on. This approach will provide a powerful platform for the development of anticancer drugs (Figure 1.4).

In summary, four different features of DNA methy-lation make this process relevant to understanding the molecular bases of tumor development and potential new approaches in cancer treatment:

1. DNA methylation is a functional and hence "epi-genetic" process (no mutation is involved).

2. It, therefore, affects the function of genes, but not their structure, as mutation does.

3. It explains functional phenomena involved in tumor development, such as loss of imprinting or "silencing" of TSGs.

4. It can be reversed with drugs such as 5-azacyti-dine.

Methylation of DNA of certain control regions in our genome can cause genes to be inappropriately "si-lenced."37 DNA methylation is a chemical modification of cytosine, one of DNA's four bases. This modification consists of the addition of a methyl group (CH3-) to the cytosine residues of the DNA double helix. The altered cytosine is called methyl-cytosine, and it represents a critical factor in gene regulation. The enzyme responsible for DNA methylation in humans is known as DNA cytosine methyltransferase (DNA MTase). Abnormal methylation events occur during aging and in the development of many cancers. It is now well known that up to 65% of all cancers originate from inappropriate DNA methylation of some key genes rather than from mutations.

In many instances, hypermethylation of DNA is the suspected cause of the cancer. Hypermethylation inappropriately switches off critical genes, thus allowing cancer to develop. Studies on DNA methyla-tion in cancer have revealed that aberrant methyla-tion of normally unmethylated CpG islands (portions of DNA containing an elevated percentage of cytosine on one side and guanine, the complementary base, on the other) located in the promoter region of genes (the regions promoting or enhancing gene transcription, and hence, expression), is associated with transcrip-tional inactivation of defined TSGs.38,39 Other genes, such as those involved in the apoptosis process (e.g., CSP8)40 or DNA mismatch repair,41 may also be involved, leading to their "silencing" or lack of expression, and hence to cancer initiation and progression.

Normally methylated TSG promoter

Normally methylated TSG promoter

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FIGURE 1.4. Schematic illustration of how DNA methylation of the promoter region of genes affects the transcription and hence the expression of a TSG. It is evident that hypermethylation of the promoter region turns the gene off, thus inducing, when both copies of the gene are involved, uncontrolled cell proliferation. The normal gene expression can be restored by inhibiting the enzyme responsible for DNA methylation (DNA methyltransferase).

In eukaryotic cells, genes are complexed with core histones and other chromosomal proteins to form chromatin. The basic repeating unit of chromatin is called a nucleosome. Each nucleosome is made of two copies of each of the four core histones (H2A, H2B, H3, and H4) wrapped by 146 base pairs (bp) of DNA (Figure 1.5). With the aid of additional proteins, including histone H1, the nucleosomes are further packaged into 30 nm fibers. When these fibers unfold, DNA becomes accessible for transcription; on the contrary, when they are packaged together, DNA becomes inaccessible. The unfolding involves posttranslational modifications of the core histone amino-terminal tails. Each core histone is composed of a structured, three-helix domain and two unstructured tails. These tails are susceptible to a variety of covalent modifications, such as acetylation, phosphorylation, and methylation, whose roles are now beginning to be unveiled.44

At present, both genetic and biochemical studies support an important role for histone tail acetylation in transcriptional regulation. The acetylation status of histones, in turn, depends on the activity of two enzymes with opposite actions: histone acetylase (HAT) and histone deacetylase (HDAC). Both these enzy matic activities are required for the activation or repression of transcription. Because of their important roles in the regulation of such events, enzymes that affect histone acetylation status are increasingly being associated with tumors.45 Histone deacetylation, as operated by HDAC, involves the removal, through hydrolysis, of the acetyl groups (COO-) from the e-amino group of the histone's lysine side chain, with an overall increase of histone's positive charge and affinity for the negatively charged DNA, thus making the DNA itself relatively inaccessible to transcription factors (reduced gene expression). When HDAC is inhibited, the amount of counterenzyme, HAT, becomes excessive, and hyperacetylation occurs. Hyperacetyl-ation, in turn, leads to an increase in the negative charge of histones, disrupting the structure of the his-tone and allowing its DNA to unfold. The unfolded state of histone then permits transcription factors to reach previously hidden genetic information, with a consequent increase in gene expression.46

The anticancer potential of HDAC inhibitors stems from their ability to affect several cellular processes that are unregulated in cancer cells. For example, assuming that one or more TSGs are silenced in cancer cells, the theoretical role of HDAC inhibitors

Dna Methylation Inhibitors

FIGURE 1.5. Schematic illustration of how the degree of acetylation of histones modulates gene expression, based on a hypothetical TSG. In the normally acetylated histone, the acetyl groups add a negative charge to histone proteins, thus allowing the negatively charged DNA to unfold to become accessible to transcription factors for regular gene expression. When histone deacetylase (HDAC) removes acetyl groups from histone proteins, the global charge of histones becomes positive, thus attracting the negatively charged DNA, which wraps around histone proteins, thus becoming inaccessible to transcription factors. This process will silence the normal expression of TSGs, thus pushing the cell toward an uncontrolled condition of proliferation. This condition can be reversed and a normal TSG expression restored by using HDAC inhibitors.

consists of promoting the transcription of the silenced gene or genes, thus reversing the cancer phenotype. Preliminary reports indicate that HDAC inhibitors activate differentiation programs, inhibit the cell cycle, and induce apoptosis; but they also seem to activate the host immune response and play a role in inhibition of angiogenesis. Hence the mechanisms whereby HDAC inhibitors induce tumor cell growth arrest, differentiation, and/or apoptosis are presumably more complex than those suggested by this short review. Intensive research in phase I and II clinical trials using several HDAC inhibitors has been initiated. HDAC inhibitors seem to possess minimal toxicity and are well tolerated at the doses needed to hyperacetylate histones and achieve clinical outcomes.47,48 It is worthwhile, when mentioning the potentialities of histone acetylation status in regulating gene expression, to note that histone methyltranferases have been found to contain a potential methyl-CpG-binding domain, raising the important possibility that histone methylation, in a manner similar to histone deacetyl-ation, might function in concert with DNA methyla-tion to switch off tumor suppressor or DNA mismatch repair genes in cancer.

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