Association of HCF-1 with the human homologs of components of the Saccharomyces cerevisiae Set1/Ash2 HMT complex and HMT activity. (A) Similarity between components of the S. cerevisiae Set1/Ash2 complex and human proteins identified among the HIPs. Schematic representations of yeast Set1/Ash2 complex proteins, and related HIPs (hSet1, accession no. gi 6683126; hAsh2, accession no. gi 4757790; and WDR5, accession no. gi 16554627) are shown on the left and right, respectively. Structural domains and motifs are shown in each protein and defined by the key on the bottom right. RRM, RNA recognition motif; SET, Set domain; postSET, Set domain-associated cysteine-rich motif; HBM, HCF-1-binding motif; PHD, PHD zinc-finger motif; SPRY, domain in SPla and the RYanodine receptor; WD40, WD40 repeats; RIIa, protein kinase A regulatory subunit dimerization domain motif. (B) Two-step purified f-HCF-1N (lane 2) and mock (lane 3) samples were analyzed by immunoblot with anti-human Set1 (panel a), Ash2 (panel b), WDR5 (panel c), and SUV39H1 (panel d) antibodies. Labeled arrowheads indicate polypeptides of interest; NE, HeLa nuclear extract (lane 1) corresponding to 5% of the samples in lanes 1 and 2 was used as a positive control. (C) HCF-1 associates with HMT activity. Two-step f-HCF-1N and mock HeLa-cell purified samples were incubated with core histones or recombinant histone H3 in the presence of [3H]AdoMet. Proteins were resolved by 15% SDS-PAGE and examined by Coomassie blue staining (lower panel) or fluorography (upper panel). The positions of histones are indicated on the right. NE, HeLa nuclear extract used as a positive control. (D) Histone H3-K4 specificity of human Set1/Ash2 HMT. Recombinant histone H3 was in vitro methylated using f-HCF-1N two-step purified material and subjected to 16 Edman degradation cycles. The radioactivity released in each cycle is indicated. (E) Interplay between K9 methylation and K4 methylation by Set1/Ash2 HMT. Two-step f-HCF-1N and mock-purified samples were incubated with a histone H3 peptide corresponding to amino acids 1–15, or the corresponding peptide trimethylated at K4 or K9. 3H incorporation for each substrate peptide was defined as the difference in 3H CPM obtained with the f-HCF-1N and mock samples. The average of five experiments is shown.