USF1 associates with H3K4 methyltransferase hSET1- and nucleosome-remodeling NURF complexes. (A) Partial list of USF1-associated polypeptides identified by LC-MS/MS is shown. (B) Endogenous USF1 associates with hSET1 and NURF complexes in erythroid cells. Nuclear extracts from K562 cells were immunoprecipitated with USF1 and hSET1 antibodies and analyzed by Western blotting. (C) Nuclear extracts from HeLa cells expressing Flag-USF1 were fractionated through a Sephacryl S-300 HR column. The peak fractions from the 1.8-MDa complex (I) corresponding to hSET1, the 300 to 670-kDa complex (II) corresponding to PRMT1 and HATs, and the < 50-kDa complex (III) were collected, respectively. The pooled fractions were immunoprecipitated with Flag antibody. The precipitates were then analyzed by Western blot analysis using antibodies against hSET1, ASH2L, RBBP5, BPTF, and SNF2. (D) USF1 associates with H3K4-specific methyltransferase activity. The purified USF1-associated complexes were incubated with histone H3 peptide or H3K4me3 peptide in the presence of [³H]AdoMet as a cofactor. The proteins were resolved by SDS-PAGE and visualized by fluorography. (E) ATP-dependent nucleosome sliding by the USF1-associated protein complex. A 194-bp DNA fragment containing a strong nucleosome positioning sequence was PCR amplified and assembled with purified HeLa core histones. The nucleosome sliding reactions were carried out with increasing amounts of the purified USF1-associated complexes in the presence or absence of ATP. Mobilized mononucleosomes were analyzed on a native 6% polyacrylamide gel and stained with ethidium bromide. The purified recombinant Drosophila NURF complex was used as a positive control.