Epigenomics Sequencing: DNA Methylation Analyses Between Eukaryotes and Prokaryotes

Introduction to DNA Methylation

For more than decades, bases altered by methylation had been thought to exist at a low frequency in DNA. Methyltransferases (DNA methylases) that transmit the chemically active methyl group from S-adenosylmethionine to either carbon 5 of the cytosine solvents or the exocyclic amino group that is affixed to carbon 6 of the adenine residues (m6A) of the DNA chain are involved in this DNA alteration.

The DNA methylation sequence is particular to species. A tiny proportion of the cytosine residues are methylated in the DNA of some prokaryotes, while only adenine residues are methylated in some.  The DNA contains both 5-methylcytosine (m5Cyt) and N6-methyladenine (m6Ade) in the third cluster of prokaryotic organisms. Overall, eukaryotic DNA is methylated solely by cytosine residues.

DNA Methylation in Prokaryotes

DNA methylation is utilized in bacteria as a signal for the regulation of a particular DNA-protein interaction. Usually, methylation processes consist of a DNA methylase and one or more proteins binding to DNA that can coincide with the target methylation site on DNA, thereby obstructing that site's methylation.

The target site's methylation restricts protein binding, which can lead to two methylated and nonmethylated alternative methylation states of the target site. This tends to lead to a sequence of DNA methylation that governs which genes are displayed, and hence how the ecosystem engages with a microorganism.

DNA Methylation in Eukaryotes

DNA methylation in eukaryotes is frequently used to turn off the genes' signaling function. In many eukaryotic mechanisms, such as embryonic growth, genome imprinting, X-chromosome inactivation, and usually maintaining the consistency of chromosomes, DNA methylation has been displayed to have a vital function. In mammals, where the methylation phase for 75% of all CpG dinucleotides is in somatic cells, the process is very precise.

Considering the magnitude of the influence of DNA methylation, it is not strange that several illnesses, such as those noticed in humans are also linked to these effects. The global methylation trend in mammals makes it difficult to assess if methylation is a default situation or is aimed towards particular gene sequences. The CpG islands, however, typically occur near transcription starting sites, implying that there is an identification framework.

DNA Methylation: Eukaryotes VS Prokaryotes

DNA methylation happens in eukaryotes only on the residues of cytosine and especially for CpG sequences. Whereas in prokaryotes, the main epigenetic signal is the methylation of adenine residues. Only a couple of DNA methyltransferases are used by eukaryotes; in bacteria, where many of them have high sequence precision, the figures are much greater. The significant human gastric pathogen Helicobacter pylori, for instance, has a large DNA methyltransferase gene repertoire, with various strains involving distinct and rather special sequences.

Even so, across prokaryotes and eukaryotes, the defense mechanism of DNA methylation is comparable. In humans and rodents, for instance, embedded viral sequences can be methylated to suppress the genes implemented. In mice as well, the same processes have been discovered to suppress transgenes. The recognition and eradication features of DNA methylation machines, therefore, appear to be preserved.

As the eukaryotic genome is much more intricate in comparison to the prokaryotic genome, the impact of methylated cytosine as a "fifth base" in the eukaryotic genome has been suggested by many research. In addition, many studies showed that the methylation of cytosine is implicated in the eukaryotic genome's functional reorganization.

DNA Methylation Sequencing

DNA methylome analysis has been performed using microarrays for many years. Sequencing DNA methylation is a newly developed innovation-based primarily on bisulfite conversion to distinguish between methylated cytosines and unmethylated cytosines. Unmethylated cytosines are transformed to uracils upon bisulfite diagnosis, while 5mCs are non-reactive and maintained. Unmethylated cytosines are read as thymine in the sequencing phase, while methylated cytosines are still read as cytosine.

Bisulfite conversion-based sequencing can be carried out as either Whole-Genome Bisulfite Sequencing (WGBS) or Reduced Representation Bisulfite Sequencing, based on genomic coverage (RRBS). WGBS charges more and there is much greater involvement in the affiliated data evaluation.  Instead, RRBS offers a cost-effective method to surveying DNA methylation by sampling genome areas rich in CpG. Genomic DNA is processed with a methylation-insensitive restriction enzyme, such as MspI, to conduct RRBS. To create a library for sequencing, the digested DNA fragments are then confined to adapter ligation, bisulfite conversion, and PCR.