Figure 6. HP1α recruits JHDM3A to Lbx1 gene and knockdown of JHDM3A in myoblasts impairs Lbx1, MyoD and myogenin expression.

A. C2C12 myoblasts were transfected with NSsiRNA or HP1αsiRNA, 48 hours after transfection ChIP assay was performed with anti-JHDM3A antibody. Lbx1 exon 2 and myogenin gene promoter were amplified. B. C2C12 myoblasts were transfected with the indicated siRNA, 48 hours after transfection total cell lysates were subjected to Western blotting with the specified antibodies. C, D Immunoprecipitation (IP) was performed using nuclear extracts prepared from NIH3T3 cells over-expressing Flag-JHDM3A and GFP-HP1α with either anti-Flag (C) or anti-GFP antibodies (D). Immunoprecipitated complexes were blotted (IB) with indicated antibodies. E, F. Endogenous HP1 and JHDM3A interaction was examined using C2C12 myoblasts nuclear extract. Nuclear extracts were immunoprecipitated with anti-HP1α (E) or anti-JHDM3A (F) antibody, and western blotted for HP1α and JHDM3A. G, In vitro translated ³⁵S-JHDM3A protein was incubated with GST or GST-HP1α immobilized on the glutathione beads. Bound proteins were separated on SDS-PAGE and visualized by autoradiography. H. C2C12 myoblasts cultured in GM were transfected with NSsiRNA or JHDM3AsiRNA. After 48 hours, total RNA was isolated and expression of the indicated genes determined by semiquantitative PCR. I. C2C12 myoblasts were transfected with NSsiRNA or JHDM3AsiRNA, 48 hours after transfection cross-linked chromatin was extracted and immunoprecipitated with anti-H3K9me3 antibody. Lbx1 exon 2 genomic sequences were amplified. J. C2C12 myoblasts were transfected with the indicated siRNA, 48 hours after transfection total cell lysates were subjected to Western blotting with the specified antibodies.