Specific Loss of Histone H3 Lysine 9 Trimethylation and HP1γ/Cohesin Binding at D4Z4 Repeats Is Associated with Facioscapulohumeral Dystrophy (FSHD)
Figure 6
SUV39H1 HMTase is solely responsible for H3K9me3 at D4Z4, which is necessary, but not sufficient, for the recruitment of HP1γ and cohesin.
(A) SUV39H1 is responsible for H3K9me3 and HP1γ/cohesin association at D4Z4. HeLa cells were treated with siRNA specific for SUV39H1, G9a, or control siRNA, and ChIP analysis using 4qHox primers was performed for the presence of cohesin, HP1γ and H3K9me3 (lanes 1–16). Preimmune IgG serves as a negative control. The rDNA (445/446) and c-Myc regions were used for comparison. Western-blot analysis of G9a and SUV39H1 siRNA depletion is also shown (lanes 17–21). Depleted proteins are indicated at the top and proteins detected by western blot analysis are indicated on the left. α-tubulin serves as a loading control. (B) HP1γ and cohesin binding to D4Z4 is cell type-specific. ChIP analysis of D4Z4 and rDNA regions was performed using normal and 4qF lymphoblasts (lanes 1–10). Western blot analysis comparing the level of H3K9me3 between HeLa and lymphoblasts (256 (normal) and B8-1 (4qF)) is also shown (lanes 11–13). Coomassie staining of core histones is included as a loading control. (C) Not all H3K9me3-positive repeats are bound by HP1γ and cohesin. Six different repeat sequences (as in Figure S1) were tested for cohesin and HP1γ binding in HeLa cells. While H3K9me3 was detected at all six repeat sequences tested, cohesin and HP1γ binding was found at only three repeats (α-sat and sat2 on chromosome 1 and DXZ4).