(A–D) ChIP analyses of Ser5-phosphoryated Rpb1 (S5P-Rpb1) (A–B), Rpd3-myc (C), and Rco1-myc (D) occupancies in WT, kin28-ts16 or gcn4Δ strains harboring RPD3-myc (A & C) or RCO1-myc (B & D). Cells were cultured in SC at 25°C, transferred to 37 °C for 30 min prior to treatment with SM for another 30 min, and then subjected to ChIP. (E) ChIP analyses of Rco1-myc and Rpb3 in KIN28 RCO1-myc and kin28-as RCO1-myc strains cultured in SC medium lacking Ile and Val and treated with NA-PP1 at 12 µM for 42 min and with SM for the last 30 min of the NA-PP1 treatment. (F) WCEs of WT or set2Δ strains harboring RLI1-myc or RCO1-myc cultured in YEPD were immunoprecipitated with anti-myc antibodies and immune complexes were probed by Western blot (WB) with antibodies against Ser5P (H14), hypophosphorylated Rpb1 (8WG16), or histone H3. Input (In), supernatant (S) and pellet (P) fractions are marked. Two exposures are shown for the H14 blots. (G) Top two panels: WCEs of isogenic KIN28 or kin28-ts16 strains harboring RCO1-myc, cultured in YEPD at 25°C and transferred to 37 °C for 1 h, were immunoprecipitated with anti-myc antibodies and immune complexes probed with the indicated antibodies. Bottom panels: same as the top except that anti-Rpb3 antibodies were used for immunoprecipitation. (H) WCEs from KIN28 RCO1-myc or kin28-ts16 RCO1-myc cells cultured as in (G) were immunoprecipitated with anti-myc antibodies and probed with the indicated antibodies. (I) Biotinylated CTD peptides (1.5µg) unphosphorylated (CTD) or phosphorylated on Ser5 (S5P), Ser2 (S2P), or both residues (S2P S5P) were adsorbed to streptavidin-coated magnetic beads and incubated at 4°C with reconstituted Rpd3C(S) expressed in insect cells. Mock: incubations with beads alone. Bound (B) and unbound, supernatant proteins (S) were subjected to SDS-PAGE and Western analysis with anti-Flag or Rpd3 antibodies. (J) Same as (H) except including CTD peptides with Asp replacing Ser2 or Ser5. All error bars represent the SEM. For Rpb3 occupancies in kin28-ts see Fig. S1B–C.

(A–B & F–G) Occupancies of Hda1-myc at ARG1 measured by ChIP. **; p value calculated using student’s t test is < 0.005. (C–E) Occupancies of Hda1 (C) Rpb3 (Pol II; D) and S5P-Rpb1 (E) measured by ChIP. All error bars represent the SEM.

(A) H3Ac/H3 ratios (left) or H4Ac/H3 ratios (right) at ARG1 determined by ChIP for WT and the rpd3Δ hos2Δ hda1Δ triple mutant. *; p value < 0.01 and **; p value < 0.005. (B) H3Ac/H3 and H4Ac/H3 ratios in (A) were normalized to the values in WT cells at each location at ARG1. (C) H3 occupancies at ARG1 measured by ChIP. (D) H3Ac/H3 and H4Ac/H3 ratios determined in (C) and normalized to the WT ratios plotted as a histogram (Acetylation), along with the fold increase in H3 eviction as a line graph (H3 eviction) calculated as the reciprocal of the mutant total H3 occupancies normalized to the WT H3 occupancy. (E) Rpb3 (Pol II) occupancies at ARG1 determined by ChIP. All error bars represent the SEM.

(A) Location of primers for ChIP analysis of ARG1. (B–F) Occupancies of Rpd3-myc, Dep1-myc and Rco1-myc at ARG1 were measured by ChIP in the indicated strains cultured in SC medium and treated with sulfometuron methyl (SM) for 30 min to induce Gcn4. Cross-linked chromatin was immunoprecipitated with anti-myc antibodies and DNA from immunoprecipitated (IP) and input samples was subjected to PCR in the presence of [³³P]-dATP to amplify radiolabeled fragments of ARG1 or the POL1 ORF, analyzed as a control, which were resolved by PAGE and quantified by phosphorimaging. Ratios of ARG1 to POL1 signals in IP samples were normalized to the ratios for Input samples to yield occupancy values. (G) Occupancies of Rco1-myc at the 3’ ORF regions of STE11 and FLO8 measured by ChIP in the indicated strains. (H–I) Occupancies of acetylated H4 (H4Ac) and total H3 at the 3’ ORFs of STE11, FLO8 and ARG1 were measured by ChIP in cells grown in YEPD, using primers for these genes and non-transcribed sequences at the right-arm telomere of VI (TEL VI) (H) or POL1 (I). (J) H4Ac occupancies from (H) normalized for total H3 (I). All error bars represent the standard error of mean (SEM). For Rpb3 occupancies in TATAΔ and set2Δ see Fig. S1A.

(A–C) Occupancies of Hos2-myc and Rpb3 at ARG1 measured by ChIP under Gcn4-inducing conditions (D–E) WCEs were immunoprecipitated with anti-myc antibodies and immune complexes probed with the antibodies listed on the left. All error bars represent the SEM.

(A–B) Occupancies of H3Ac, H4Ac or total H3 at ARG1 determined by ChIP in WT cells uninduced (UI) or induced (I) for Gcn4 and the ratio of H3Ac to H3 (A) or H3 (B) occupancies were calculated. H3Ac/H3 ratios (C) or H4Ac/H3 ratios (D) were determined by ChIP for WT and the indicated single HDA mutants. POL1 and TEL VI regions were used to normalize total H3, and acetylated H3 and H4 occupancies at ARG1, respectively. All error bars represent the SEM. Occupancies of H3Ac and H4Ac in double deletion mutants are shown in Fig. S2.

(A–D) Occupancies of H3Ac/H3 (A and C, **; p value < 0.005) and H3 (B and D) at PMA1 (A and B) and ADH1 (C and D) determined by ChIP. (E) Model for two-stage co-transcriptional recruitment of Rpd3C(S). (1) Pol II is phosphorylated on Ser5 of the CTD by Kin28 during promoter clearance. Previous rounds of transcription leave ORF nucleosomes methylated on H3-K4 by Set1, more heavily towards the 5’ end, and on H3-K36 by Set2, more heavily towards the 3’ end. (2) Elongating Ser5-phosphorylated Pol II is phosphorylated on Ser2 by Bur1 and Ctk1, enabling efficient recruitment of Rpd3C(S) by direct interaction with Ser2-, Ser5-diphosphorylated CTD repeats. Subsequently, Rpd3C(S) is transferred from the phospho-CTD to H3 tails by interaction of H3-K36me₂ with the chromodomain in Eaf3 and PHD finger in Rco1 (not depicted) for subsequent histone deacetylation. All error bars represent the SEM.