Histone H4 facilitates histone H3 methylation by Set2 in vitro. (A) HMT assays were performed using purified chicken mononucleosomes (Nuc), recombinant histone H3 (rH3), recombinant histone H4 (rH4), a mixture of rH3 and rH4, or core histones (Core His) isolated from human 293T cells incubated with or without recombinant, purified CBP-Set2 protein. HMT reactions were analyzed by filter-binding assays and scintillation counting to measure total ³H-methyl incorporation. (B) The same reaction samples in A were resolved by SDS-PAGE and analyzed by fluorography (³H-methyl) and Coomassie blue staining. Asterisks represent breakdown products of histone H3 or CBP-Set2.

Amino acids 31–39 in the N terminus of Set2 contain a histone H4 interaction motif. (A) Schematic representation of CBP-Set2 constructs used for in vitro binding assays. The SET domain (SET) along with the AWS, the post-SET domain (PS), WW domain (WW), coiled-coil motif (CC), and the SRI are shown. (B) The N terminus of Set2 containing the SET domain binds to GST-H4_24–50. Bacterial extracts from cells expressing CBP-Set2_1–261, CBP-Set2_262–475, and CBP-Set2_476–733 were incubated with GST-H4_24–50. (C) Amino acids 31–39 in the N terminus of Set2 interact with GST-H4_24–50. Bacterially expressed and purified CBP-Set2_1–119, CBP-Set2_120–261, CBP-Set2 _{120–261ΔAWS}, CBP-Set2_1–63, CBP-Set2_{1–261Δ11–15}, CBP-Set2_1–50, CBP-Set2_1–30, and CBP-Set2_Δ31–39 were incubated in the presence of GST-H4_24–50 beads. In the in vitro binding assays described in B and C, bound and input amounts of CBP-Set2 and CBP-Set2 mutant protein were detected by immunoblotting with an anti-CBP antibody. The amount of GST or GST-H4_24–50 fusion protein used was analyzed by Coomassie blue staining. (D) Set2 binds unmodified H3 K36 peptide. Western blot analysis of CBP-Set2 after pull-down assays using biotinylated histone H3 (21–44), mono-, di-, and trimethyl-histone H3 K36 peptides.

Set2 lacking the histone H4 interaction motif is targeted to gene-specific loci by associating with the phosphorylated CTD of RNAPII. (A) Yeast set2Δ cells were transformed with empty vector or the indicated Set2-3XFlag plasmid constructs. Whole-cell extracts were prepared and immunoprecipitated with the α-Flag antibody followed by immunoblot analysis with the H5 [α-Ser2P] or H14 [α-Ser5P] antibodies. All input extracts showed similar levels of Set2 as indicated by an α-Flag immunoblot. (B) The Ctk1 complex does not regulate Set2-mediated H3 K36 monomethylation in vivo. Whole-cell extracts prepared from wild-type or ctk1Δ, ctk2Δ, and ctk3Δ strains were immunoblotted with H3 K36 methyl-specific antibodies. Immunoblots histone H3 serve as a loading control. Asterisk indicates a nonspecific band. (C) The indicated Set2 strains were examined for H3 K36 trimethylation, H4 acetylation, and the presence of Set2 at STE11 and FLO8 loci by ChIP. Input and histone H3 levels were used as loading controls.

Location of H4 K44 within the nucleosome and a step-wise model of Set2-mediated methylation. (A) The structure of the yeast nucleosome core particle: Histone H4 (green), Histone H3 (blue), H2A (gray), H2B (yellow), and DNA (orange). The crystal structure of the yeast nucleosome shows that H4 K44, marked in red, is located in the L1 loop region linking α-helix 1 and α-helix 2 of H4. (B) The structure of H3/H4 heterotetramer in the absence of H2A and H2B. (C) A step-wise model of Set2-mediated H3 K36 methylation. During transcription elongation, the CTD of RNAPII is phosphorylated by Ctk1 kinase. The SRI domain in the C terminus of Set2 binds to the phosphorylated CTD of RNAPII and recruits Set2 to the 3′ ends of ORFs. Once Set2 is recruited to chromatin, the N-terminal histone H4 interaction motif of Set2 will dock on chromatin by binding histone H4, allowing Set2 to efficiently di- and trimethylate H3 K36. After H3 K36 methylation, the Rpd3S complex is recruited to deacetylate histone H3 and H4 to prevent cryptic intragenic transcription. Blue nucleosomes represent acetylated histones and red nucleosomes represent deacetylated histones.

H4 K44 is needed for H3 K36 methylation in vivo and Set2 interaction in vitro. (A) Alignment of amino acid sequences of histone H3 (33–37), histone H4 (13–17) and histone H4 (41–45). Identical amino acids are shaded gray. (B) Yeast whole-cell extracts generated from cells expressing wild-type (WT) histones or the indicated histone mutants were immunoblotted with H3 K4, H3 K36, and H3 K79 methyl-specific or H4 acetylation-specific (H4 Acetyl) antibodies. Immunoblots for histone H3 and H4 were used as loading controls. Asterisks indicate a nonspecific band. (C) In vitro HMT assays were performed using yeast chromatin substrates isolated from cells expressing wild-type histones or histone mutants in the absence or presence of recombinant CBP-Set2. HMT reactions were analyzed by filter-binding assays and scintillation counting. (D) In vitro binding assays were performed to examine if Set2 interacts with GST-H4_24–50. Bacterial expressed CBP-Set2 cell extracts were incubated with GST, GST-H4_24–50, or GST-H4_24–50 lacking residues G₄₂VKR₄₅ (GST-H4_{24–50ΔGVKR}) or GST-H4_24–50 with K44 mutations (K44R, K44Q, and K44E). Bound and input amounts of CBP-Set2 protein were detected by immunoblotting with an anti-CBP antibody. The amount of GST or GST-H4 fusion protein used was analyzed by Coomassie blue staining.

The histone H4 interaction motif of Set2 is needed for proper H3 K36 di- and trimethylation and binding nucleosomes. (A) Yeast whole-cell extracts of set2Δ cells expressing the indicated Set2-HA constructs were generated and immunoblotted using the indicated H3 K36 methyl-specific antibodies. Histone H3 and Set2-HA proteins were detected by immunoblotting with anti-H3 or anti-HA antibodies. All Set2 constructs were C-terminally tagged with a single HA tag and expressed by the endogenous Set2 promoter. Asterisk indicates a nonspecific band. (B,C) In vitro HMT assays were performed using recombinant-purified CBP-Set2_1–733, CBP-Set2_262–733, CBP-Set2_Δ31–39, or CBP-Set2_1–618 incubated with chicken nucleosomes and analyzed at various time points. (D) CBP-Set2 proteins described in B were used for nucleosome-binding assays at three different protein concentrations: 1.0, 2.0, and 4.0 μM.

Defects in Set2-mediated H3 K36 di- and trimethylation increase resistance to 6-AU. (A) Cells with H3 K36 methylation defects exhibit 6-AU resistance. Yeast cells expressing wild-type histones or the indicated histone mutants were assayed for growth in the presence or absence of 6-AU (150 μg/mL). (B) Set2 mutant strains that have H3 K36 methylation defects exhibit 6-AU resistance. Yeast cells expressing wild-type or Set2 mutant strains were assayed for growth in the presence or absence of 6-AU (150 μg/mL). A Set2 deletion strain (set2Δ) was used in A and B for a positive control for 6-AU resistance.