Figure 2: DNMT3B contributes to SAHH-mediated, H19-dependent methylation change in CME.

(a) Myotubes were transfected with siCon or siH19, followed by RNA extraction and RT–qPCR analysis 48 h later. Relative RNA levels of H19 and Sahh are presented. (b) In vivo SAHH activity assessed 48 h post-siRNA transfection, as described in a. (c) Relative DMR methylation assessed using QMSP. (d) Sequence of the DMR amplified using the QMSP primers, with the differentially methylated cytosine highlighted in red. Numbers above the sequence mark positions of the indicated nucleotides in the chromosome. Numbers below indicate percentage of methylation in siH19 (left) versus siCon (right) cells, as determined using genome-wide methylation analysis. (e) Myotubes were transfected with siCon, siH19 or siH19 plus siDnmt3b, followed by RNA extraction and RT–qPCR analysis 48 h post transfection. Relative RNA levels of H19 and Dnmt3b are presented. (f) Western blot results representative of three independent transfection experiments (performed as described in e) are shown. Antibodies specific for DNMT3B and beta-tubulin were used on the top and bottom blots, respectively. Numbers underneath the blots indicate DNMT3B protein levels after normalization against TUBB loading controls with that in siCon cells arbitrarily set as 100%. (g) Myotubes were transfected with siCon, siH19 or siH19 plus siDnmt3b. DMR methylation was assessed 48 h post transfection using QMSP. (h) DNMT3B occupation at CME. Myotubes were transfected with siCon or siH19. ChIP analysis was performed 48 h later using anti-DNMT3B antibody to evaluate binding of DNMT3B at the DMR. ns, not statistically different. (i) Association of activated RNAP with Nctc1 and H19. Myotubes were transfected as described in h. ChIP analysis was performed using anti-RNAP antibody. (j) Relative RNA levels of H19 and Nctc1 following siRNA transfection. Student’s t-tests were performed to compare differences between quantitative variables. Numbers are mean±s.d. (n=3). *P<0.05, **P<0.01.