Specific Loss of Histone H3 Lysine 9 Trimethylation and HP1γ/Cohesin Binding at D4Z4 Repeats Is Associated with Facioscapulohumeral Dystrophy (FSHD) (original) (raw)
Figure 7
D4Z4-specific co-recruitment of HP1γ, cohesin, and cohesin loading factor Scc2.
(A) Binding of HP1γ and cohesin to D4Z4 is interdependent. ChIP analysis of HeLa cells after individual depletion of the cohesin subunit hSMC1, HP1γ, or the cohesin loading factor Scc2 by siRNA as indicated (lanes 1–12). Cohesin and HP1γ binding was compared between D4Z4 and rDNA (445/446). Real-time PCR analysis using Q–PCR primers is shown underneath. Western blot analysis of hSMC1, HP1γ and Scc2 depletion is also shown (lanes 13–16). (B) HP1γ and cohesin binding do not affect each other at other repeat sequences. Realtime PCR analysis of Rad21 (“cohesin”), HP1γ and H3K9me3 ChIP DNA from HeLa cells treated with control, SMC1, HP1γ, or Scc2 siRNA as indicated using Q-PCR primers specific for D4Z4, α-sat and sat2 repeat sequences on chromosome 1, and DXZ4 (as in Figure S2). (C) Scc2 binding to D4Z4 is compromised by HP1γ depletion. Realtime PCR analysis of Scc2 ChIP DNA from HeLa cells treated with control, HP1γ, or Scc2 siRNA as indicated using Q-PCR primers specific for D4Z4. (D) Coimmunoprecipitation (co-IP)–western blot analysis of cohesin and Scc2 interaction with HP1γ. HeLa nuclear extracts were used for co-IP using antibody specific for Scc2 or cohesin (Rad21) as previously described [53],[61]. After low-salt washes, precipitated materials were eluted with 1.0 M KCl (“wash”) and further eluted with 2.0 M guanidine-HCl (“eluate”). Eluted proteins were analyzed by SDSPAGE and western blotting using antibody specific for HP1γ. For comparison, a similar co-IP analysis was performed and probed with antibody specific for CTCF.