Volume 12, Issue 5, Pages 1325-1332 (November 2003) Methylation of Histone H3 K4 Mediates Association of the Isw1p ATPase with Chromatin  Helena Santos-Rosa, Robert Schneider, Bradley E. Bernstein, Nickoletta Karabetsou, Antonin Morillon, Christoph Weise, Stuart L. Schreiber, Jane Mellor, Tony Kouzarides  Molecular Cell  Volume 12, Issue 5, Pages 1325-1332 (November 2003) DOI: 10.1016/S1097-2765(03)00438-6

Figure 1 hSNF2H and Isw1p Are Binding to Methylated K4 H3 (A) H3 tail peptides un-, di-, or trimethylated on K4 were coupled to Sulfolink beads and used as affinity columns for hSNF2H. hSNF2H was detected by Western blot. 20% of the input nuclear extract was loaded. (B) Protein extracts from an Isw1-myc and a Chd1p-myc yeast strain were prepared and affinity purifications were performed as in (A). Recombinantly expressed Gcn5-His bromodomain was used as binding control. Bound proteins were detected by Western blot. 25% of the input yeast extract and 10% of the recombinant Gcn5 input was loaded. (C) Isw1-myc was immunoprecipitated from wt and set1Δ extracts, and the precipitate was probed in a Western assay with an H3-specific antibody. 5% of the Input yeast extract was loaded. (D) As in (C), but mono-me K4, di-me K4, or tri-me K4 specific antibodies were used, to detect the methylation state of H3 associated with Isw1p. 5% of the Input was loaded. (E) Di-me and tri-me K4 peptides can elute Isw1-myc from a crude chromatin fraction. Eluted Isw1p-myc was detected by Western blot. (F) Purified Flag-Isw1p monomer was used in pull-down assays. No binding to di- or trimethylated K4 H3 peptide is detectable. Addition of protein extract (lanes 3 and 5) prepared from an isw1Δ strain restores the binding to methylated K4 H3 peptide. 50% of the Input was loaded. Molecular Cell 2003 12, 1325-1332DOI: (10.1016/S1097-2765(03)00438-6)

Figure 2 Isw1p Association with Chromatin Depends on Set1p (A and B) SET1 dependency for the association of Isw1p with chromatin. Isogenic wt and set1Δ strains expressing Isw1-myc were crosslinked and the chromatin prepared was precipitated with anti-myc antibody and analyzed by quantitative PCR. “Isw1p and Set1p dependent genes” (A) are genes whose transcription levels are downregulated in ISW1 and SET1 deleted strains, as obtained from microarray analysis. “Isw1p dependent genes” (B) are genes whose transcription levels are downregulated in ISW1 but not in SET1 deleted strains, as obtained from microarray analysis. Relative fluorescence units are calculated as described in Experimental Procedures. (C and D) Chromatin from wt-1 and isogenic set1Δ, wt and isogenic set1N1016Q mutant (set1Δ complemented with wt SET1, set1 N1016Q, and set1 C1068A alleles on plasmids), wt-2 and isogenic H3K4A mutant strains, expressing Isw1-myc, was immunoprecipitated with anti-myc antibody and analyzed for the presence of Isw1p with primers from within the MPP10 coding region. (D) As in (C) but analyzed with primers from within the YOL106 coding region. In each panel the region amplified by PCR is underlined. Input, 1:50 diluted input chromatin; No ab, no antibody control; α-myc, anti-myc antibody. (E and F) Isw1p and trimethylated K4 H3 distribution within coding regions of Isw1p- and Set1p-dependent genes. (E) Isw1p and trimethylated K4H3 distribution within MPP10 ORF. (F) Isw1p and trimethylated K4H3 distribution within YOL106 ORF. Primers at the 5′ (ATG region) are marked in red and at the 3′ end of the gene in green. The diagrams represent relative fluorescence units (IP/input). Molecular Cell 2003 12, 1325-1332DOI: (10.1016/S1097-2765(03)00438-6)

Figure 3 Set1p and Isw1p Regulate Common Processes In Vivo (A) Growth properties of a set1Δ isw1Δ strain complemented with plasmids harboring SET1 and ISW1 wt alleles, SET1 wt and isw1K227R mutant, set1N1016Q mutant and ISW1 wt, or isw1K227R set1N1016Q mutants (upper panel) and isogenic wt, dot1Δ, and set1N1016Q (lower panel). Culture serial dilutions were spotted to minimal complete medium (SDC), and minimal medium lacking methionine (SDC-met). Plates were incubated for 3 days at 30°C. (B) MET16 poly(A)+ mRNA level is downregulated in isw1K227R mutant as determined by Northern analysis. Poly(A)+ mRNA was prepared from wt, isw1Δ, and isw1K227R strains grown in the absence of methionine (SDC−met). (C) Upper: Schematic representation of MET16 nucleosome position as obtained from micrococcal nuclease (MNase) accessibility. The numbers between nucleosomes correspond to cleavage sites relative to the mRNA start site at (+1), which is (−60) with respect to the ATG. Lower: Isogenic wt and set1Δ strains, expressing Isw1-myc, were grown in SDC-methionine. The crosslinked chromatin was precipitated with anti-myc antibodies and analyzed by quantitative PCR with the primers to nucleosome +1 (92/253). (D) Set1p- and Isw1p-dependent change in micrococcal nuclease (MNase) accessibility in yeast chromatin at the MET16 locus mapped by indirect end label analysis. DNA was subject to digestion with EcoRI and a Southern blot of DNA hybridized to an EcoRI (+796)/Msc1 (+472) fragment is shown (probe). The position of the Set1p- and Isw1p-dependent structures is marked with asterisks. Lane 1 shows wt chromatin prepared in SDC+methionine; lanes 2–4 show wt, isw1K227R, and set1C1068A chromatin prepared in minimal medium SDC−methionine. All lines show chromatin in permeabilized cells digested with 150 units/ml MNase. Molecular Cell 2003 12, 1325-1332DOI: (10.1016/S1097-2765(03)00438-6)

Figure 4 RNAPII and Rna15p Recruitment and Distribution Are Altered in a set1 N1016Q Mutant Cells with the genotypes indicated were grown overnight in minimal medium lacking methionine (SDC−met), and crosslinked chromatin was precipitated with antibodies specific to RNAPII (A); Rna15p (B) and DNA were detected using real-time PCR to amplify regions corresponding to nucleosomes +1 and + 5 at MET16. Molecular Cell 2003 12, 1325-1332DOI: (10.1016/S1097-2765(03)00438-6)