[3] 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.
Epigenetic modifications play an important role in gene expression and regulation, and are involved in numerous cellular processes such as in differentiation/development[7] and tumorigenesis.
In eukaryotes, methylation is most commonly found on the carbon 5 position of cytosine residues (5mC) adjacent to guanine, termed CpG dinucleotides.
For example, in mouse primordial germ cells, a genome wide de-methylation even occurs; by implantation stage, methylation levels return to their previous somatic values.
Binding of these proteins recruit histone deacetylases (HDACs) enzyme which initiate chromatin remodeling such that the DNA becoming less accessible to transcriptional machinery, such as RNA polymerase, effectively repressing gene expression.
The basic and repeating units of chromatin, nucleosomes, consist of an octamer of histone proteins (H2A, H2B, H3 and H4) and a 146 bp length of DNA wrapped around it.
Note that several modification types including methylation, phosphorylation and ubiquitination can be associated with different transcriptional states depending on the specific amino acid on the histone being modified.
[14] Histone modifications regulate gene expression by two mechanisms: by disruption of the contact between nucleosomes and by recruiting chromatin remodeling ATPases.
An example of the first mechanism occurs during the acetylation of lysine terminal tail amino acids, which is catalyzed by histone acetyltransferases (HATs).
[14][16] The second process involves the recruitment of chromatin remodeling complexes by the binding of activator molecules to corresponding enhancer regions.
[13][1] As in the other genomics fields, epigenomics relies heavily on bioinformatics, which combines the disciplines of biology, mathematics and computer science.
It had been known that certain regions within chromatin were extremely susceptible to DNAse I digestion, which cleaves DNA in a low sequence specificity manner.
Such hypersensitive sites were thought to be transcriptionally active regions, as evidenced by their association with RNA polymerase and topoisomerases I and II.
[20] Histone modification was first detected on a genome wide level through the coupling of chromatin immunoprecipitation (ChIP) technology with DNA microarrays, termed ChIP-Chip.
The cells are next lysed, allowing for the chromatin to be extracted and fragmented, either by sonication or treatment with a non-specific restriction enzyme (e.g., micrococcal nuclease).
This analysis step was done by amplifying the restriction fragments via PCR, separating them through gel electrophoresis and analyzing them via southern blot with probes for the region of interest.
[9] DNA methylation profiling on a large scale was first made possible through the Restriction Landmark Genome Scanning (RLGS) technique.
While this now allows for methylation pattern to be determined on the highest resolution possible, on the single nucleotide level, challenges still remain in the assembly step because of reduced sequence complexity in bisulphite treated DNA.
Increases in read length seek to address this challenge, allowing for whole genome shotgun bisulphite sequencing (WGBS) to be performed.
[30] Changes in chromatin accessibility are important epigenetic regulatory processes that govern cell- or context-specific expression of genes.
[32] MNase-seq and DNase-seq both follow the same principles, as they employ lytic enzymes that target nucleic acids to cut the DNA strands unbounded by nucleosomes or other proteic factors, while the bounded pieces are sheltered, and can be retrieved and analysed.
This technique has been used to such an extent that nucleosome-free regions have been labelled as DHSs, DNase I hypersensitive sites,[34] and has been ENCODE consortium's election method for genome wide chromatin accessibility analyses.
The free and linked fragments are separated with a traditional phenol-chloroform extraction, since the proteic fraction is stuck in the interphase while the unlinked DNA shifts to the aqueous phase and can be analysed with various methods.
[37] Sonication produces random breaks, and therefore is not subject to any kind of bias, and is also the bigger length of the fragments (200-700 nt) makes this technique suitable for wider regions, while it's unable to resolve the single nucleosome.
[32] Unlike the nuclease-based methods, FAIRE-seq allows the direct identification of the transcriptionally active sites, and a less laborious sample preparation.
Discrimination between hydroxymethylation and methylation is possible thanks to solid-state nanopores even if the current while passing through the high-field region of the pore may be slightly influenced in it.
These metrics, defined as pulse width and interpulse duration (IPD), add valuable information about DNA polymerase kinetics.
In 2010 a team of scientists demonstrated the use of single-molecule real-time sequencing for direct detection of modified nucleotide in the DNA template including N6-methyladenosine, 5-methylcytosine and 5-hydroxylcytosine.
[48] In 2017, another team proposed a combined bisulfite conversion with third-generation single-molecule real-time sequencing, it is called single-molecule real-time bisulfite sequencing (SMRT-BS), which is an accurate targeted CpG methylation analysis method capable of a high degree of multiplying and long read lengths (1.5 kb) without the need for PCR amplicon sub-cloning.
[50] In the next several years, high-throughput data have indeed uncovered the abundance of epigenetic modifications and their relation to chromatin functioning which has motivated new theoretical models for the appearance, maintaining and changing these patterns,.