Figure 4
Specific association of ATRX and H3K9 me3 with lack of H3K4 me2 at repetitive DNA sequences on the Y chromosome. A) Metaphase spreads of peripheral lymphocytes (top panel) and primary fibroblasts (lower panel). Thin arrows indicate the characteristic association of ATRX (green) at pericentric heterochromatin in the autosomes of both cell lineages. ATRX consistently labels the Y chromosome (red) in mouse embryonic fibroblasts (bold arrow) but is undetectable in the Y chromosome of peripheral lymphocytes. Scale bars = 10 μm. B) Chromatin immunoprecipitation (ChIP) analysis of histone modifications associated with a Y-chromosome specific repetitive sequence. ChIP assays were performed on male embryonic fibroblasts (upper panel) and peripheral lymphocytes (lower panel) using antibodies directed against ATRX, H3K4me2 and H3K9me3. Representative gel images are depicted above the corresponding graphs. A rabbit IgG (lanes 3 and 4) and an anti-TFIIB antibody (lanes 11 and 12) served as negative and positive control, respectively. In primary fibroblasts, the specific association of H3K9me3 (lanes 9 and 10) and ATRX (lanes 7 and 8) with repetitive sequences on the Y chromosome results in a significant enrichment of co-precipitated DNA fragments corresponding to the Y chromosome-specific repeat (Y666) compared to a non-specific IgG negative control (lanes 3 and 4) and the H3K4me2 antibody (lanes 5 and 6). However, although H3K9me3 is also enriched at these sequences in peripheral lymphocytes (lower panel), ATRX associations are not significantly different from samples precipitated with the negative control (IgG) or H3K4me2. PCR amplification was conducted using increasing amounts of precipitated template (input) to ensure that the PCR reaction was within the linear range. Error bars represent the SEM of three independent experiments and different superscripts indicate significant differences (p < 0.05).