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.