Inactive DNMT3B Splice Variants Modulate De Novo DNA Methylation (original) (raw)
Figure 2
DNMT3B3 modulates DNA methylation activity without affecting DNA methylation patterns.
(A) HEK293c18 cells were transfected with the pFC19 target episome and combinations of DNMT3 expression vectors, as indicated. DNA methylation was assessed by Southern blot after digestion of episomal DNA with a methylation-sensitive restriction enzyme. Higher molecular weight bands are indicative of DNA methylation. (B) Bisulfite sequencing was performed on a 500 base pair region of harvested episomal DNA. Co-expression of DNMT3B2 with DNMT3B3 does not lead to a change in DNA methylation patterns as judged by the fact that the ordered ranks of the 48 methylation sites in the region do not shift significantly. (C) Pre-incubation of DNMT3B3 with active DNMT3A2 leads to a stimulation of catalytic activity. The graph displays the result of quantitative in vitro activity assays in which the incorporation of labeled methyl groups into DNA was measured at increasing ratios of DNMT3B3ct to DNMT3A2. (D) DNMT3B3 hinders the stimulatory effect of DNMT3L. Pre-incubation of increasing concentrations of DNMT3B3ct to constant amounts of DNMT3A2 and DNMT3L leads to a progressive decline in DNA methylation activity as measured in quantitative in vitro assays. Results in panels C and D are from triplicate experiments and shown with average and standard deviations.