Epigenetics is the study of heritable change in gene activity despite alteration of the hosts DNA sequence. Change in gene activity done independently of the DNA sequence is achieved by way of histone and DNA methylation. Gene silencing in DNA methylation is carried out by DNA methyltransferases 1, 2 and 3a/b (DNMT1, DNMT2, DNMT3A/B). On a broad level, DNMTs methylate the fifth carbon of cytosine residues in DNA within CG dinucleotides. However, DNMT3 on its own is a de novo methyltransferase required for the establishment of genomic methylation patterns during development and reproduction. Studies on the structure of DNMT3 have revealed that DNMT3A and DNMT3B are highly similar in that they both contain a PWWP domain, a PHD-like ADD domain and a catalytic domain. What’s more, the catalytic activities of DNMT3A and DNMT3B have been shown to be attracted to sequences alongside CG sites versus within CG dinucleotides.
DNMT3A Antibody (64B1446) [NB120-13888] - Ntera2 cells were fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X TBS + 0.5% Triton-X100. The cells were incubated with anti-DNMT3A (68B1446) [NB120-13888] at a 1:200 dilution overnight at 4C and detected with an anti-mouse Dylight 488 (Green) at a 1:500 dilution. Actin was detected with Phalloidin 568 (Red) at a 1:200 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.
Epigenetic regulation is a key factor in chromatin dynamics and participates in cellular differentiation, oncogenic transformation, development and long-term memory. Sheikh et al used a DNMT3A antibody in their research to discover that DNMT3 regulated Dpp6 causing the inhibition of RA induced neuronal differentiation of P19 cells. First, Sheikh’s group tested the mRNA activity of DNMT1, DMNT2 and DNMT3A/B using primary antibodies on real time PCR after RA treatment in P19 neurons. They found that DNMT3a expression was reduced after 6 days and remained low from there on out. Next, they studied the interaction between DNMT3B and Dpp6 by using primary antibodies to measure the expression level of Dpp6 once DNMT3B was knocked down using shRNA. They concluded that absence of DNMT3B did in fact increase Dpp6 expression 3.5 fold, thus resulting in inhibition of P19 differentiation.
In addition to this development study, Leitch et al took a closer look at DNA methylation in pluripotent embryonic stem cells (ESCs) and embryonic germ cells (EGCs) using a DNMT3A antibody. What’s interesting about these two cell lines is that EGC’s display programmed DNA methylation while ESC’s do not. Yet overall, this group’s findings revealed that both EGCs and ESCs reach a transcriptionally similar identity despite their distinct embryonic origins. With this finding it is clear that the level of DNA methylation in pluripotent cells is independent of their origin as well. Overall, this suggests that a cells ability to be pluripotent is the protection from de novo methylation.
All in all, the activity of DNMT3A can greatly alter developmental processes by targeting genes involved with differentiation and pluripotency. The mechanism behind why certain methyltransferases target developmental genes and at which stages is still undetermined.
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