Specific Loss of Histone H3 Lysine 9 Trimethylation and HP1γ/Cohesin Binding at D4Z4 Repeats Is Associated with Facioscapulohumeral Dystrophy (FSHD) (original) (raw)
Figure 2
D4Z4 chromatin contains both euchromatic and heterochromatic histone modifications.
(A) Antibodies specific for H3K4me2, H3K4me3, H3K9me3, H3K27me3, H3Ac, and acetylated H4 (H4Ac), as well as control preimmune IgG, were used for ChIP in HeLa cells. The ChIP DNA was amplified using 4qHox primers and primers specific for regions on chromosomes 10 and 19 containing short Alu repeat sequences. The presence of H3K9me3 was confirmed by two different antibodies (lanes 10–14) [18]. (B) Double-ChIP analysis of D4Z4 histone modifications. H3K9me3 ChIP (1st ChIP) was eluted and followed by the second (2nd) ChIP reactions using antibodies specific for H3K4me2, H3K9me3, H3K27me3, or preimmune IgG. The ChIP DNA was amplified using 4qHox primers. (C) The proximal region of the D4Z4 cluster is euchromatic. ChIP analysis of the first proximal D4Z4 repeat using the 4qA161-1 primer pair (See Figure 1D for sequence amplification specificity) was performed in HeLa, normal human fibroblasts (FB), myoblasts (MB), and lymphoblasts (LB). (D) Histone ChIP in human ES cells. ChIP DNA derived from the ES cell lines H1 and H9 was amplified by 4qHox primers. Antibodies used for ChIP are indicated at the top.