Figure 5.

Knockdown of Mef 2d and Mef 2c in C2C12 cells decreases Ash2L at the Myog and Ckm promoters. (a) RNA was isolated from differentiating C2C12 cells (48 h) that were untransfected or transfected with siRNA targeting Mef 2d or Mef 2c or with untargeted control siRNA. RNA was reverse-transcribed and subjected to qPCR analysis of gene expression with primers specific for Myog, Ckm or Acta1 transcripts along with primers specific for 18S RNA. Expression is reported relative to the control 18S RNA signal. Average values of triplicate qRT-PCR reactions are shown; error bars represent s.d. Each experiment was performed at least twice with independently isolated RNA. (b) cDNA was prepared as in a and analyzed by semiquantitative PCR with primers specific to the genes indicated. (c,d) C2C12 cells were transfected with siRNA targeting both Mef2c and Mef2d (such that both family members would be knocked down) or with an untargeted control siRNA. Cells were then differentiated for 48 h and analyzed by ChIP to measure enrichment of H3K4 methylation at the Myog promoter (c) or relative recruitment of transcriptional activators and coactivators (d). Immunopurified DNA was quantified by qRT-PCR with hydrolysis probes. Average values of duplicate qRT-PCR reactions are shown; error bars represent s.d. Each experiment was performed at least twice with independent chromatin samples and yielded similar results both times.