FIG. 1.
Histone tail modifications in ES cells, hybrid cells, and thymocytes. (A) Experimental scheme with intersubspecific hybrid cells between M. musculus domesticus (dom) ES cells and M. musculus molossinus (mol) thymocytes. Asterisks represent DNA sequence-polymorphic sites. Histone modifications in ES cells, hybrid cells, and thymocytes were examined by Western blotting and ChIP assays with antibodies specific to acetylated H3 (AcH3), acetylated H4 (AcH4), dimethylated H3 lysine 4 (H3-K4me2), trimethylated H3-K4 (H3-K4me3), dimethylated H3 lysine 9 (H3-K9me2), trimethylated H3-K9me3, and trimethylated H3 lysine 27 (H3-K27me3). (B) Immunocytochemical analysis of histone modifications in ES cells and thymocytes. The cells were immunostained with antibodies (red) against AcH3, AcH4, H3-K4me2, H3-K4me3, H3-K9me2, H3-K9me3, and H3-K27me3. ES cell nuclei were immunostained with antibody against OCT4 (green). Nuclei were counterstained with DAPI (blue). Circles indicate thymocytes. (C) Western blotting analysis of histone modifications in ES cells, hybrid cells, and thymocytes. Total protein extracted from the cells was immobilized onto nitrocellulose membrane and hybridized with antibodies against AcH3, AcH4, H3-K4me2, and H3-K9me2. Total H3 was used as loading control. (D) Slot blot hybridization analysis of histone modifications with a sonicated genomic DNA probe from mouse embryonic fibroblasts. DNA immunoprecipitated with antibodies against AcH3, AcH4, H3-K4me2, H3-K4me3, H3-K9me2, H3-K9me3, and H3-K27me3 was slot blotted and hybridized. The relative levels of the histone modifications detected in ES cells, hybrid cells, and thymocytes are summarized in histogram form. (E) Slot blot hybridization analysis of histone modifications with a B2 repeat probe. DNA immunoprecipitated with antibodies against AcH3, AcH4, H3-K4me2, and H3-K9me2 was analyzed as detected in panel D.