Figure 8.

E12/E47 and HEB exchange at the leiomodin 2 (Lmod2) and desmin (Des) promoters. (A) E12/E47 binds to the promoters of Des and Lmod2 in myoblasts. Cross-linked extracts from proliferating myoblasts (UD, undifferentiated cells) were immunoprecipitated with antibodies against the E2A gene products. Immunoprecipitated DNA was purified and amplified with primers specific to the promoters of Ckm, Des, Tnni2, Lmod2 and Tcap. (B) E12/E47 and HEB exchange at the Lmod2 promoter. Cross-linked extracts from proliferating myoblasts and myofibers in differentiation media for two days were immunoprecipitated with antibodies against the E2A gene products, HEB or IgG. Immunoprecipitated DNA was purified and amplified with primers specific to the promoter of Lmod2. (C) E12/E47 and HEB exchange at the Des promoter. Cross-linked extracts from myofibers in differentiation media for two or three days were immunoprecipitated with antibodies against the E2A gene products, HEB or IgG. Immunoprecipitated DNA was purified and amplified with primers specific to the promoter of Des. Relative enrichment at the IgH locus was used to normalize the data. The fold enrichment values were calculated as described in Methods. (D) Gene expression analysis of HEB in cells expressing a small hairpin RNA (shRNA) construct targeting HEB or a scrambled control (scr). (E) Western blot analysis of the cells described in Figure 8D. The Western blot was probed with antibodies against HEB. (F) HEB is not required to displace E12/E47 at promoters. Results of chromatin immunoprecipitation assays performed after two days of differentiation on HEB-depleted cells and the scr control are shown.