Figure 4

Preferential binding of WDR5 and BPTF to H2A.Z nucleosomes. (A) LD611 cells were transfected with H2A and H2A.Z expression vectors, and mononucleosomes were prepared by MNase digestion as recently described [25]. Mononucleosomes containing ectopic H2A (lane 1) and H2A.Z (lane 2) were sequentially immunoprecipitated from total mononucleosomes with anti-Flag and anti-HA antibodies. Histone compositions of the purified nucleosomes were analyzed by 15% SDS-PAGE followed by Coomassie brilliant blue (CBB) staining. The relative levels of endogenous/ectopic H2A and H2A.Z in the purified nucleosomes were determined by Western blotting using H2A and H2A.Z antibodies. (B) The proteins co-purified with H2A and H2A.Z nucleosomes were resolved in 4 to 20% SDS-polyacrylamide gels, and then visualized by silver staining. (C) Venn diagram depicts the number of proteins associated with H2A nucleosomes (65), H2A.Z nucleosomes (85) and those in common (58). (D) The proteins specifically co-purified with H2A.Z nucleosomes were functionally classified by gene ontology analysis. The number of proteins in each functional group is shown. (E) The preferential binding of WDR5 and BPTF to H2A.Z nucleosomes were analyzed by Western blotting. (F) Mononucleosomes containing ectopic H2A or H2A.Z were purified as in (A), and subjected to Western blotting with antibodies specific for the indicated histone modifications.