Figure 4.

p18^Hamlet mediates histone H2A.Z binding to chromatin and induces muscle-specific gene expression. (A) C2C12 myoblasts were cultured in growing conditions and transfected with control (C) or p18^Hamlet siRNA, and 72 h later H2A.Z binding to the indicated regions of the myogenin promoter (see Figure 2A) was analysed by ChIP and qPCR. Semiquantitative PCR analysis was also performed with samples of the same regions. (B) C2C12 myoblasts were incubated in DM for 14 h and the effect of p18^Hamlet downregulation on H2A.Z binding to the myogenin promoter was analysed by ChIP and both semiquantitative and quantitative PCRs. (C) C2C12 myoblasts were transfected with a Myc-tagged p18^Hamlet expression construct and clones were selected with G418. Myogenin and both endogenous and Myc-tagged p18^Hamlet protein levels were analysed by immunoblotting in the indicated clones. (D) Binding of H2A.Z to the myogenin TATA box-containing region was determined by ChIP assays and both semiquantitative and quantitative PCR in control (number 1) and p18^Hamlet overexpressing (number 6) C2C12 clones (left and middle panels). Myogenin mRNA levels were estimated by semiquantitative RT–PCR (right panel).