DNMT3B3 modulates DNA methylation activity without affecting DNA methylation patterns.

<p>(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 <i>in vitro</i> 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 <i>in vitro</i> assays. Results in panels C and D are from triplicate experiments and shown with average and standard deviations.