Figure 5.

Accumulation and phosphorylation of p18^Hamlet are necessary for H2A.Z chromatin enrichment and myogenin expression. (A) C2C12 myoblasts were transfected with plasmids encoding either wt or a non-phosphorylatable version (4A) of Myc-tagged p18^Hamlet. Expression of p18^Hamlet, myogenin induction and myocyte morphological changes were analysed by immunofluorescence (left panel) in at least 300 cells. The efficiency of transfection was 28 and 20% for the wt and 4A p18^Hamlet constructs, respectively. The percentage of myogenin-positive cells for each transfection is represented (right panel). (B) C2C12 myoblasts were transfected with either MKK6DD alone or in combination with Myc-tagged p18^Hamlet wt and 4A mutant, and 24 h later, the total lysates were analysed by immunoblotting using the indicated antibodies. Tubulin was used as loading control. Transfection efficiency was about 50%. (C) Chromatin from C2C12 myoblasts transfected as in (B) was collected 24 h after transfection, and H2A.Z binding to the myogenin TATA box-containing region was assayed by ChIP. Both semiquantitative and quantitative PCRs are shown. (D) Upregulation of p18^Hamlet expression during myogenic differentiation in vivo correlates with p38 MAPK activation and myogenin induction. Extracts of plantaris muscles either in basal state or undergoing de novo myogenesis (3 days after overloading) were analysed by immunoblotting using the indicated antibodies. Myogenin is a marker of differentiating satellite cells/myoblasts (left panel). Sections of mouse plantaris muscles as above were immunostained with the indicated antibodies and detected with peroxidase (H2A.Z and p18^Hamlet) or immunofluorescence (Myogenin and Pax7). Nuclei were detected with DAPI. Pax7 identifies satellite cells/myoblasts present both in basal and overloading conditions. Myogenin-positive satellite cells are exclusively detected in muscle with ongoing myogenesis/differentiation (right panel). Bar=50 μm.