Introduction

The term ‘epigenetics’ was first coined by British embryologist Conrad Washington, who described it as 'the branch of biology that studies the casual interaction between genes and their products which bring phenotypes into being.’ Epigenetics is the study often mechanisms through which cells control gene activity without inducing any change in the structure or sequence of primary deoxyribonucleic acid (DNA). These heritable and stable changes in gene expression are an implication of alterations in chromosomes rather than the DNA sequence. These cells regulate gene expression through chemical modifications in the chromosomal superstructure which comprises DNA (histones). Since epigenetics are an interface between the genome and environment, whether the genes are turned off and on is influenced by environmental factors including diet and pollutants which impacts the epigenome. Ultimately, this affects the phenotype and health outcomes.

These modifications have influenced many biochemical conditions which have offered interventions that could possibly alleviate or prevent certain diseases. Epigenetics in disease depends on two cases of gene involvement. Genes that are epigenetically regulated: this depends on the degree to which an inherited gene (normal or mutated) is expressed or not. The other gene is the one whose products are participants of epigenetic machinery, contributing to the expression of other genes. For instance, the gene MECP2 contributes to the silencing of the sequence (methylated regions of DNA) by encoding proteins that bind to said regions. Mutations that impair the gene can lead to Rett syndrome.

Epigenetic Mechanisms

Epigenetic mechanisms form a regulatory layer within cells, controlling gene expression and gene silencing. This regulation varies across tissues and is crucial in cell differentiation. During early development, genome-wide patterns of DNA and histone modifications are established and persist through multiple cell divisions. However, in cancer, normal epigenetic patterns are disrupted, resulting in the expression of anti-apoptotic genes and silencing of tumor-suppressing genes, CDKN2A, for example. Moreover, there are three different types of epigenetic mechanisms, namely DNA methylation, histone modification and non-coding RNA associated gene silencing.

1.   DNA methylation

i.              Intro

DNA methylation is catalyzed by DNA methyltransferase enzymes (Dnmts) which function by adding methyl groups from S-adenyl methionine (SAM) directly on theC5 position of cytosine nucleotide within a cytosine-guanine sequence (which are often surrounded by other CpG’s forming a CpG island) to form5methylcytosine (5mC). CpG islands are common targets for epigenetic DNA methylation, particularly within promoter regions. Dnmt3a and Dnmt3b are classified as de novo DNA (they establish new methylation pattern to unmodified DNA). Dnmt1, however, functions during DNA replication to copy the DNA methylation pattern from the parent strand onto the newly synthesized daughter strand. This is the ‘maintenance’ Dnmt and also maintains DNA methylation pattern during replication. When it undergoes semiconservative replication, the parental DNA returns the original DNA methylation patterns.

ii.            Locations

The majority of DNA methylation occurs on CpG sites or cytosine that follow up a guanine nucleotide. One particular location are the ‘intergenic regions’ that possess viral elements which undergo silencing by methylation. Oftentimes these elements are deactivated by mutations or methylation. The major function of DNA methylation is to inhibit the expression of potentially dangerous genetic elements. Dnmt1 for example, maintains the repression of Intracisternal A particle (IAP), an aggressive retrovirus present in mouse genomes by methylation. Furthermore, CpG islands are another location comprising of at least 200 base pairs. They have higher CpG density than the rest of the genome and promote gene expression by controlling the chromatin structure and ‘transcription factor binding.’ A transcription factor is any protein involved in altering gene expression levels and involved in transcription. DNA is wrapped around histones forming packaged structure called nucleosomes. Their tight association with each other directly corresponds to decreasing permissiveness for gene expression. Around 50% of CpG islands contain known transcription start sites, but mostly devoid of promoter elements.

iii.         Basic Mechanism of DNA methylation

This mechanism is divided into three classes of catalysts,

Writers: catalyze the addition of methyl groups to cytosine residues

Erasers: modify and remove the methyl group

Readers: recognize and bind to methyl groups, thereby influencing gene expression

·     The 3 key members of Dnmt family are involved. Dnmt1 methylates hemi methylated DNA during replication, preserving the original methylation process in the process. Dnmt3a is broadly expressed while Dnmt3b is quite restricted. Dnmt3L lacks catalytic activity and associates with the above methyltransferases, stimulating their function during early development. De novo DNA methylation, carried out by Dnmt3a and Dnmt3b, remains an intriguing process with multiple proposed mechanisms. While the exact targeting mechanisms are not fully understood, two main theories have emerged. First, RNA interference (RNAi) might guide Dnmts to silence specific DNA sequences, although evidence for this in mammalian cells is weak. Second, transcription factors play a critical role. They can either recruit Dnmts to specific gene promoters or protect CpG sites from de novo methylation. CpG islands, in particular, seem to rely on transcription factor binding forprotection. Overall, these mechanisms work together to establish de novo DNA methylation patterns.

·     Erasing DNA methylation: TET enzymes play a crucial role in DNA demethylation. They oxidize 5-methylcytosine to form5-hydroxymethylcytosine (5hmC). This is further oxidized to 5-formylcytosine(5fC) and finally to 5-carboxylcytosine (5caC). Thymine DNA glycosylase (TDG)then removes the oxidized bases leading to demethylation. Furthermore, active transcription can also inhibit DNA methylation. Transcription factors may form DNA nascent-RNA helices giving rise to R-loops of ssDNA (single strand) that excludes de novo methylation.

·     Reading DNA methylation: this involves two key mechanisms, physical impediment, in which methylation physically hinders transcriptional protein binding to the gene and MBDs action (methyl CpG binding domain proteins). Methylated DNA can be bound by MBD proteins influencing gene regulation. MBD containing proteins recruit HDAC complexes (histone deacetylase) and chromatin remodeling factors that lead to chromatin compaction and consequentially, transcriptional repression. Transcriptionalrepression is where MBD proteins bind to methylated DNA contributing to gene silencing by recognizing methylated CpG sites and recruiting co-repressing complexes that alter the chromatin structure. Ultimately, this prevents the transcription from non-methylated CpG promoters.

2.   Histone Modification

i.              Intro

Histone modifications or post-translational modifications to histones are enzyme catalyzed and include the processes acetylation, methylation, phosphorylation and ubiquitylation, which affect DNA-histone interactions in nucleosomes and recruiting histone modifications, thereby impacting gene expression. In this article, we will solely discuss histone acetylation and methylation. Histone modifications actin diverse biological processes which include transcriptional activation/inactivation, chromosome packages and DNA repair. Histone H3 is acetylated at lysine 9, 14, 18, 23, 56 (this correlates with transcription activation) and methylated at arginine 2 and lysine 4, 9, 27, 36, 79. Phosphorylated at ser10, ser28, Thr3, Thr11. Histone H4 is primarily acetylated at lysine 5, 8, 12 and 16, methylated at arginine 3 and lysine 20, and phosphorylated at serine 20.

ii.            Histone Acetylation

Histone acetylation occurs at positively charged lysine residues which deteriorate DNA-histone interactions, thus altering or opening chromatin structure and facilitating transcription. This involves the enzymatic addition of an acetyl group (COCH₃) from acetyl co-enzyme A. Furthermore, the modifying enzyme involved in histone acetylation are called histone acetyltransferase (HATs) which also control H3and H4acetylation. The HATs recognized can be categorized into 5 families, namelyGNAT1 and MYST and ACTR (a nuclear receptor co-activator). Histone H3acetylation may be increased by the repression of HDACs (histone deacetylases) and decreased by HAT inhibition.

iii.         Histone Methylation

The transfer of methyl groups from S adenosyl-L-methionine to lysine or arginine residues of histones by HMTs (histone methyltransferases). They control DNA methylation through chromatic dependent transcriptional repression or activation. There are specific genes within the DNA-histone association that may be silenced or activated. Different HMTs are specific for lysine and arginine residues which they modify. BothH3-K9 and H3-K27 (histone 3 at lysine 9 and lysine 27) methylation oversees heterochromatin formation and participates in gene silencing. Arginine methylation of H3 and H4 promotes transcriptional activation, which is mediated by PRMTs (protein arginine methyltransferases). About 7 members are involved in methylation of histones. It may oversee mono or dimethylation of arginine residues. PRMTs are strongly linked to diseases, particularly the type IIPRMTs. For example, PRMT5 plays a role in the suppression of certain tumor suppressor genes such as RB tumor. Detection of the activity of type II PRMTs is crucial in understanding and benefitting cancer diagnostics.

3.   Non-coding RNA associated Gene Silencing

An ncRNA is a functional RNA molecule that undergoes transcription but is not translated into proteins. This plays a crucial role in epigenetic gene expression. Many postulations about their accountability for the difference in phenotype between species within human population despite the similarity in encoded proteins have been proposed. Some ncRNAs include micro RNAs (miRNA) and small interfering RNA (siRNA) which includes less than 30 nucleotides. On the other hand, long non-coding RNA (lncRNA) include more than 200 nucleotides. These RNA regulate gene expression by formation of heterochromatin.

RNA silencing mechanisms include RNA interference (RNAi). Endogenous miRNAs or exogenously derived siRNAs induce the degradation of complementary mRNA. Why is this process so important? It has a diverse range of functions. Antiviral protection, maintenance of heterochromatin structure. Small RNAs even regulate various processes like cell fate, proliferation and insulin secretion.  

Written by: Iman Zahra

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