FIGURE 6.

Modulating the levels of HP1 isoforms affects muscle terminal differentiation. A and B, C2C12 cells stably expressing HA-FLAG tagged HP1 isoforms as indicated or control cells (ctrl) were cultured under proliferation conditions or in differentiation medium. Proliferating cells were tested by immunofluorescence using anti-HA antibody (Roche Applied Science) and 4′,6′-diamino-2-phenylindole (DAPI) to stain DNA (A) or analyzed by Western blotting with isoform-specific anti-HP1, anti-myogenin, anti-MCK, and anti-α-tubulin as a loading control (B). Note that the kinetic studies were carried out in the same 10-cm-diameter cell culture dish for each sample. C, FLAG-HA tagged HP1 isoforms are recruited to MyoD target promoters regardless of differentiation. ChIP experiments using anti-FLAG resin were performed from myoblasts stably expressing the tagged HP1 isoforms (or from the control cell line, ctrl) either proliferating (black bars) or cultured in differentiation conditions (gray bars). We quantified copy numbers of the MCK, ItgA7, and p21 promoter regions harboring the MyoD target sequence. The 36B4 gene was used as a negative control. The results are the means of three independent experiments.