5-Methylcytosine is a methylated form of the DNA base cytosine (C) that regulates gene transcription and takes several other biological roles. When cytosine is methylated, the DNA maintains the same sequence, but the expression of methylated genes can be altered. 5-Methylcytosine is incorporated in the nucleoside 5-methylcytidine.
Transcription is the process of copying a segment of DNA into RNA. The segments of DNA transcribed into RNA molecules that can encode proteins are said to produce messenger RNA (mRNA). Other segments of DNA are copied into RNA molecules called non-coding RNAs (ncRNAs). mRNA comprises only 1–3% of total RNA samples. Less than 2% of the human genome can be transcribed into mRNA, while at least 80% of mammalian genomic DNA can be actively transcribed, with the majority of this 80% considered to be ncRNA.
The CpG sites or CG sites are regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5' → 3' direction. CpG sites occur with high frequency in genomic regions called CpG islands.
In biochemistry, the DNA methyltransferase family of enzymes catalyze the transfer of a methyl group to DNA. DNA methylation serves a wide variety of biological functions. All the known DNA methyltransferases use S-adenosyl methionine (SAM) as the methyl donor.
Regulation of gene expression, or gene regulation, includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products. Sophisticated programs of gene expression are widely observed in biology, for example to trigger developmental pathways, respond to environmental stimuli, or adapt to new food sources. Virtually any step of gene expression can be modulated, from transcriptional initiation, to RNA processing, and to the post-translational modification of a protein. Often, one gene regulator controls another, and so on, in a gene regulatory network.
DNA methylation is a biological process by which methyl groups are added to the DNA molecule. Methylation can change the activity of a DNA segment without changing the sequence. When located in a gene promoter, DNA methylation typically acts to repress gene transcription. In mammals, DNA methylation is essential for normal development and is associated with a number of key processes including genomic imprinting, X-chromosome inactivation, repression of transposable elements, aging, and carcinogenesis.
DNA glycosylases are a family of enzymes involved in base excision repair, classified under EC number EC 3.2.2. Base excision repair is the mechanism by which damaged bases in DNA are removed and replaced. DNA glycosylases catalyze the first step of this process. They remove the damaged nitrogenous base while leaving the sugar-phosphate backbone intact, creating an apurinic/apyrimidinic site, commonly referred to as an AP site. This is accomplished by flipping the damaged base out of the double helix followed by cleavage of the N-glycosidic bond.
Base excision repair (BER) is a cellular mechanism, studied in the fields of biochemistry and genetics, that repairs damaged DNA throughout the cell cycle. It is responsible primarily for removing small, non-helix-distorting base lesions from the genome. The related nucleotide excision repair pathway repairs bulky helix-distorting lesions. BER is important for removing damaged bases that could otherwise cause mutations by mispairing or lead to breaks in DNA during replication. BER is initiated by DNA glycosylases, which recognize and remove specific damaged or inappropriate bases, forming AP sites. These are then cleaved by an AP endonuclease. The resulting single-strand break can then be processed by either short-patch or long-patch BER.
Bisulfitesequencing (also known as bisulphite sequencing) is the use of bisulfite treatment of DNA before routine sequencing to determine the pattern of methylation. DNA methylation was the first discovered epigenetic mark, and remains the most studied. In animals it predominantly involves the addition of a methyl group to the carbon-5 position of cytosine residues of the dinucleotide CpG, and is implicated in repression of transcriptional activity.
Epigenomics is the study of the complete set of epigenetic modifications on the genetic material of a cell, known as the epigenome. The field is analogous to genomics and proteomics, which are the study of the genome and proteome of a cell. Epigenetic modifications are reversible modifications on a cell's DNA or histones that affect gene expression without altering the DNA sequence. Epigenomic maintenance is a continuous process and plays an important role in stability of eukaryotic genomes by taking part in crucial biological mechanisms like DNA repair. Plant flavones are said to be inhibiting epigenomic marks that cause cancers. Two of the most characterized epigenetic modifications are DNA methylation and histone modification. Epigenetic modifications play an important role in gene expression and regulation, and are involved in numerous cellular processes such as in differentiation/development and tumorigenesis. The study of epigenetics on a global level has been made possible only recently through the adaptation of genomic high-throughput assays.
The Illumina Methylation Assay using the Infinium I platform uses 'BeadChip' technology to generate a comprehensive genome-wide profiling of human DNA methylation. Similar to bisulfite sequencing and pyrosequencing, this method quantifies methylation levels at various loci within the genome. This assay is used for methylation probes on the Illumina Infinium HumanMethylation27 BeadChip. Probes on the 27k array target regions of the human genome to measure methylation levels at 27,578 CpG dinucleotides in 14,495 genes. The Infinium HumanMethylation450 BeadChip array targets > 450,000 methylation sites. In 2016, the Infinium MethylationEPIC BeadChip was released, which interrogates over 850,000 methylation sites across the human genome.
Methylated DNA immunoprecipitation is a large-scale purification technique in molecular biology that is used to enrich for methylated DNA sequences. It consists of isolating methylated DNA fragments via an antibody raised against 5-methylcytosine (5mC). This technique was first described by Weber M. et al. in 2005 and has helped pave the way for viable methylome-level assessment efforts, as the purified fraction of methylated DNA can be input to high-throughput DNA detection methods such as high-resolution DNA microarrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq). Nonetheless, understanding of the methylome remains rudimentary; its study is complicated by the fact that, like other epigenetic properties, patterns vary from cell-type to cell-type.
Yippee-like 3 (Drosophila) is a protein that in humans is encoded by the YPEL3 gene. YPEL3 has growth inhibitory effects in normal and tumor cell lines. One of five family members (YPEL1-5), YPEL3 was named in reference to its Drosophila melanogaster orthologue. Initially discovered in a gene expression profiling assay of p53 activated MCF7 cells, induction of YPEL3 has been shown to trigger permanent growth arrest or cellular senescence in certain human normal and tumor cell types. DNA methylation of a CpG island near the YPEL3 promoter as well as histone acetylation may represent possible epigenetic mechanisms leading to decreased gene expression in human tumors.
Combined Bisulfite Restriction Analysis is a molecular biology technique that allows for the sensitive quantification of DNA methylation levels at a specific genomic locus on a DNA sequence in a small sample of genomic DNA. The technique is a variation of bisulfite sequencing, and combines bisulfite conversion based polymerase chain reaction with restriction digestion. Originally developed to reliably handle minute amounts of genomic DNA from microdissected paraffin-embedded tissue samples, the technique has since seen widespread usage in cancer research and epigenetics studies.
Cancer epigenetics is the study of epigenetic modifications to the DNA of cancer cells that do not involve a change in the nucleotide sequence, but instead involve a change in the way the genetic code is expressed. Epigenetic mechanisms are necessary to maintain normal sequences of tissue specific gene expression and are crucial for normal development. They may be just as important, if not even more important, than genetic mutations in a cell's transformation to cancer. The disturbance of epigenetic processes in cancers, can lead to a loss of expression of genes that occurs about 10 times more frequently by transcription silencing than by mutations. As Vogelstein et al. points out, in a colorectal cancer there are usually about 3 to 6 driver mutations and 33 to 66 hitchhiker or passenger mutations. However, in colon tumors compared to adjacent normal-appearing colonic mucosa, there are about 600 to 800 heavily methylated CpG islands in the promoters of genes in the tumors while these CpG islands are not methylated in the adjacent mucosa. Manipulation of epigenetic alterations holds great promise for cancer prevention, detection, and therapy. In different types of cancer, a variety of epigenetic mechanisms can be perturbed, such as the silencing of tumor suppressor genes and activation of oncogenes by altered CpG island methylation patterns, histone modifications, and dysregulation of DNA binding proteins. There are several medications which have epigenetic impact, that are now used in a number of these diseases.
Reduced representation bisulfite sequencing (RRBS) is an efficient and high-throughput technique for analyzing the genome-wide methylation profiles on a single nucleotide level. It combines restriction enzymes and bisulfite sequencing to enrich for areas of the genome with a high CpG content. Due to the high cost and depth of sequencing to analyze methylation status in the entire genome, Meissner et al. developed this technique in 2005 to reduce the amount of nucleotides required to sequence to 1% of the genome. The fragments that comprise the reduced genome still include the majority of promoters, as well as regions such as repeated sequences that are difficult to profile using conventional bisulfite sequencing approaches.
Whole genome bisulfite sequencing is a next-generation sequencing technology used to determine the DNA methylation status of single cytosines by treating the DNA with sodium bisulfite before high-throughput DNA sequencing. The DNA methylation status at various genes can reveal information regarding gene regulation and transcriptional activities. This technique was developed in 2009 along with reduced representation bisulfite sequencing after bisulfite sequencing became the gold standard for DNA methylation analysis.
Generally, in progression to cancer, hundreds of genes are silenced or activated. Although silencing of some genes in cancers occurs by mutation, a large proportion of carcinogenic gene silencing is a result of altered DNA methylation. DNA methylation causing silencing in cancer typically occurs at multiple CpG sites in the CpG islands that are present in the promoters of protein coding genes.
An epigenome-wide association study (EWAS) is an examination of a genome-wide set of quantifiable epigenetic marks, such as DNA methylation, in different individuals to derive associations between epigenetic variation and a particular identifiable phenotype/trait. When patterns change such as DNA methylation at specific loci, discriminating the phenotypically affected cases from control individuals, this is considered an indication that epigenetic perturbation has taken place that is associated, causally or consequentially, with the phenotype.
CpG island hypermethylation is a phenomenon that is important for the regulation of gene expression in cancer cells, as an epigenetic control aberration responsible for gene inactivation. Hypermethylation of CpG islands has been described in almost every type of tumor.
