Figure 6.

p18^Hamlet phosphorylation is required for its interaction with Arp6, and regulation of H2A.Z binding to YL-1 during myogenesis. (A) Proposed interactions between essential components of SWR1, the yeast homolog of the SRCAP complex. (B) U2OS cells were transfected with 1 μg of HA-tagged H2A.Z and Myc-p18^Hamlet expression vectors, and 48 h later, the total lysates were immunoprecipitated (IP) with HA antibodies to evaluate the interaction of H2A.Z with both overexpressed (Myc) and endogenous (End) p18^Hamlet. Input represents 10% of the total lysate used for the IPs. (C) U2OS cells were transfected with 1 μg of either empty vector or a Myc-tagged Arp6 expression vector, and the total lysates were analysed by IP of p18^Hamlet or H2A.Z endogenous proteins followed by Myc immunoblotting. Input corresponds to 5% of the total lysates used for the IPs. (D) C2C12 cells were transfected with p18^Hamlet wt and mutant 4A alone or in combination with Myc-Arp6, and 48 h later, the total lysates were IP with a p18^Hamlet antibody followed by Myc immunoblotting. Input corresponds to 10% of the total lysate used for IPs. (E) C2C12 cells were cultured in GM or in DM for the indicated times, and the total lysates were analysed by IP with an YL-1 antibody followed by H2A.Z immunoblotting. Input corresponds to 5% of the total lysates used for the IPs. (F) The presence of SRCAP in the myogenin TATA box-containing region was analysed by ChIP using chromatin obtained from C2C12 cells grown in GM or after 14 h in DM and either treated with p18^Hamlet siRNA or SB203580 as indicated. Both quantitative and semiquantitative PCR analysis are shown.