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. 2009 Dec;29(24):6473-87.
doi: 10.1128/MCB.01033-09. Epub 2009 Oct 12.

NuA4 lysine acetyltransferase Esa1 is targeted to coding regions and stimulates transcription elongation with Gcn5

Daniel S Ginsburg  1 , Chhabi K Govind, Alan G Hinnebusch
Affiliations

NuA4 lysine acetyltransferase Esa1 is targeted to coding regions and stimulates transcription elongation with Gcn5

Daniel S Ginsburg et al. Mol Cell Biol. 2009 Dec.

Abstract

NuA4, the major H4 lysine acetyltransferase (KAT) complex in Saccharomyces cerevisiae, is recruited to promoters and stimulates transcription initiation. NuA4 subunits contain domains that bind methylated histones, suggesting that histone methylation should target NuA4 to coding sequences during transcription elongation. We show that NuA4 is cotranscriptionally recruited, dependent on its physical association with elongating polymerase II (Pol II) phosphorylated on the C-terminal domain by cyclin-dependent kinase 7/Kin28, but independently of subunits (Eaf1 and Tra1) required for NuA4 recruitment to promoters. Whereas histone methylation by Set1 and Set2 is dispensable for NuA4's interaction with Pol II and targeting to some coding regions, it stimulates NuA4-histone interaction and H4 acetylation in vivo. The NuA4 KAT, Esa1, mediates increased H4 acetylation and enhanced RSC occupancy and histone eviction in coding sequences and stimulates the rate of transcription elongation. Esa1 cooperates with the H3 KAT in SAGA, Gcn5, to enhance these functions. Our findings delineate a pathway for acetylation-mediated nucleosome remodeling and eviction in coding sequences that stimulates transcription elongation by Pol II in vivo.

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Figures

FIG. 1.
FIG. 1.
H3 and H4 acetylation stimulate transcription elongation. (A) Effect of esa1 on H4 acetylation in bulk histones was analyzed by culturing WT and esa1-L254P strains for the indicated times at 36°C and subjecting WCEs to Western analysis with antibodies against tetra-acetylated H4 or the H4 C terminus. (B) 6-AUs of WT, esa1, gcn5Δ, and gcn5Δ esa1 strains. Cells were grown to stationary phase, and serial dilutions were spotted on SC-Ura plates lacking or containing 100 μg/ml 6-AU and incubated at 30° or 36°C for 7 days. (C) GLAM assays were conducted on strains harboring the plasmid-borne reporters PGAL1-PHO5::LAC4 or PGAL1-PHO5, shown schematically. Cells were grown in SCGal-Ura at 30°C and transferred to 36°C for 1 h, and Pho5 specific activity was measured. GLAM ratios were calculated as the ratio of Pho5 activity for the long versus short reporter. The gcn5Δ esa1Δ GLAM ratio was determined to be significantly less than WT by Student's t test (* P < 0.01). (D) Northern analysis of transcripts from PGAL1-YLR454w and GAL1. Strains were grown in SC medium containing 2% raffinose (SCRaf) at 30°C, transferred to 36°C for 1 h, and treated with 2% galactose for 1 h at 36°C. Total RNA was extracted and subjected to Northern analysis with probes to the GAL1 ORF and the 5′-half of the YLR454w ORF. A sample blot is shown with quantitation of multiple experiments below, with the amount of YLR454w mRNA normalized to that of GAL1. Lane 5 is a longer exposure of lane 4. The esa1, gcn5Δ, and gcn5 esa1Δ ratios were determined to be significantly less than WT by Student's t test (*, P < 0.01; **, P < 0.001).
FIG. 2.
FIG. 2.
NuA4 and SAGA mutants reduce transcription elongation rate. ChIP analysis results are shown for Pol II (Rpb3) runoff from the 8-kb PGAL1-YLR454w ORF during glucose repression. (A) ChIP PCR primer locations at PGAL1-YLR454w. The nucleotide coordinates of all primers for ChIP analysis are in Table 2. (B to E) WT (B), esa1 (C), gcn5Δ (D), and gcn5Δ esa1 (E) strains were grown as described for Fig. 1D and treated with 4% glucose for the indicated times before cross-linking, and ChIP was performed with anti-Rpb3 antibodies and primers to amplify an intergenic region on chromosome I and PGAL1-YLR454w in the presence of [α-33P]dATP. Relative occupancy was calculated, taking the ratio of radioactivities of PCR products for PGAL1-YLR454w versus the chromosome I reference and dividing by the same ratio for input samples. The background values at 40 min were subtracted, and values in glucose were normalized to values in galactose.
FIG. 3.
FIG. 3.
NuA4 and SAGA mutants reduce histone eviction at GAL1. (A) ChIP PCR primer locations in GAL1 and other loci. (B to E) ChIP analysis of H4-Ac (B and C), H3 (D), and Rpb3 (E) at GAL1. WT, KAT mutant, and HDA mutant strains were grown in SCRaf at 30°C, transferred to 36°C for 1 h (B, D, and E) or 4 h (C), and treated with 2% galactose for 30 min at 36°C. ChIP was performed with anti-H4-Ac (B and C), anti-H3 (D), or anti-Rpb3 (E) antibodies and primers to PCR amplify the GAL1 UAS, TATA, or 3′ ORF sequences and the POL1 promoter (D), or an intergenic region of Chr V (E). (B and C) H4-Ac/H3 occupancy was calculated as the ratio of GAL1 PCR products for immunoprecipitated versus input samples divided by H3 occupancies in panel C. The H4-Ac/H3 ratio in galactose was determined to be significantly lower than WT by Student's t test (*, P < 0.01; **, P < 0.001). (D) H3 occupancy was calculated as the ratio of PCR products for GAL1 versus POL1 for the immunoprecipitated samples divided by the same ratio for input chromatin samples. H3 occupancy in galactose was determined to be significantly greater than WT by Student's t test (*, P < 0.01; **, P < 0.001). (E) Rpb3 occupancy was calculated as the ratio of PCR products for GAL1 versus chromosome V for immunoprecipitated samples divided by the same ratio for input chromatin samples. Rpb3 occupancy in galactose was determine to be significantly lower than WT by Student's t test (*, P < 0.01).
FIG. 4.
FIG. 4.
H3 and H4 acetylation stimulate RSC and SWI/SNF recruitment. (A) ChIP analysis of Sth1 occupancy at the GAL1 3′ ORF. STH1-myc strains were grown in SCRaf at 30°C, transferred to 36°C for 1 h, and treated with 2% galactose for 30 min at 36°C. ChIP was conducted using anti-Myc antibodies and PCR primers to amplify the GAL1 3′ ORF and chromosome V. Sth1 occupancy was calculated as described for Fig. 3D. Sth1 occupancy in the mutants in galactose was determined to be significantly less than WT by Student's t test (*, P < 0.01). (B) Effect of KAT mutants on Myc-Sth1 and Myc-Snf6 protein levels was analyzed by culturing strains for 1.5 h at 36°C and subjecting WCEs to Western analysis with anti-Myc or anti-Gcd6 antibodies. (C) ChIP analysis of Snf6 occupancy at the GAL1 3′ ORF was conducted as described for panel A. Occupancy in galactose was normalized to that in raffinose. Snf6 occupancy in esa1 was determined to be significantly less than WT by Student's t test (**, P < 0.001).
FIG. 5.
FIG. 5.
RSC stimulates efficient transcription elongation. (A) 6-AUs was measured as described for Fig. 1B, except 75 μg/ml 6-AU was used. (B) GLAM assays were conducted as described for Fig. 1C. GLAM ratios in the mutants were determined to be significantly less than in WT by Student's t test (*, P < 0.01). (C) Northern analysis of transcripts from PGAL1-YLR454w and GAL1 was conducted in strains of the indicated genotype as described for Fig. 1D. The ratio in the mutants was determined to be significantly lower than in WT by Student's t test (*, P < 0.01). (D to F) ChIP analysis of Pol II (Rpb3) in the 8-kb PGAL1-YLR454w ORF during galactose induction. Strains were grown and ChIP was conducted as described for Fig. 2B, except the occupancy values in galactose were normalized to those after 40 min in glucose. Occupancy values across the ORF were normalized to those at 2 kb. At 8 kb, the Rpb3 occupancy in the mutant is significantly lower than occupancy in WT (*, P < 0.01).
FIG. 6.
FIG. 6.
NuA4 is recruited to Gcn4 target gene promoters and coding sequences during transcription activation. (A) Diagram of NuA4 with relevant subunits labeled. Adapted from reference . (B) Representative ChIP data for Eaf1 occupancy at ARG1. EAF1-myc strains were grown in SC at 30°C, treated with 0.6 μM SM for 30 min, and ChIP was performed using anti-Myc antibodies and PCR primers to amplify the ARG1 UAS or 3′ ORF and chromosome V (Chr V). (C) Summary of ChIP data for Eaf1 at ARG1. Occupancy was calculated as described for Fig. 3D. (D and E) ChIP analysis of Eaf5 (D) and Eaf7 (E) at ARG1. ChIP was performed as described for panel B, and occupancy was calculated as described for Fig. 3D. (F to H) ChIP analysis of Eaf1 (F), Eaf5 (G), and Eaf7 (H) at ARG4. ChIP was performed as described for B, and occupancy was calculated as described for Fig. 3D. NuA4 occupancy was determined to be significantly greater in WT than in gcn4Δ (**, P < 0.001).
FIG. 7.
FIG. 7.
NuA4 is recruited to GAL1 coding sequences during transcription activation. (A to C) ChIP analysis of Eaf1 (A), Epl1 (B), and Esa1 (C) at GAL1. EAF1-myc strains were grown at 30°C in SCRaf and treated with 2% galactose for 30 min. ChIP was performed as described for Fig. 6B using primers to the GAL1 UAS or 3′ ORF and chromosome V (Chr V). NuA4 occupancy was determined to be significantly greater in galactose than in raffinose (**, P < 0.001).
FIG. 8.
FIG. 8.
NuA4 occupancy at promoters is stimulated by its Eaf1 and Tra1 subunits. (A to C) ChIP analysis of Epl1 occupancies was conducted as described for Fig. 6B with the addition of PCR primers to amplify the ARG4 (B) and HIS4 (C) UAS and 3′ ORF, normalizing occupancy in WT and eaf1Δ strains to that in gcn4Δ. (D and E) ChIP analysis of Eaf5 occupancies was conducted as described for Fig. 6B with the addition of PCR primers to amplify the ARG4 UAS and 3′ ORF (E), normalizing occupancy in WT and eaf1Δ strains to that in gcn4Δ. NuA4 occupancy was determined to be significantly greater in WT than in eaf1Δ (**, P < 0.001). (F) CoIP of Esa1 with myc- or FLAG-tagged NuA4 subunits. Strains with the indicated tagged genes were grown in yeast extract-peptone-dextrose, and WCEs were immunoprecipitated with anti-myc or anti-FLAG antibodies and subjected to Western analysis with anti-Esa1 antibodies.
FIG. 9.
FIG. 9.
NuA4 recruitment to the ORF is stimulated by active transcription and Ser5 CTD phosphorylation. (A and B) ChIP analysis of Eaf1 (A) or Epl1 (B) occupancies of the ARG1 UAS and 3′ ORF was conducted as described for Fig. 6B, normalizing occupancy in WT and arg1-ΔTATA strains to that in gcn4Δ. The reductions in occupancies conferred by ΔTATA were judged significant by Student's t test (*, P < 0.01; **, P < 0.001). (C and D) ChIP analysis of Ser5P (C) or Rpb3 (D) occupancies at ARG1. Cells were grown at 30°C in SC, transferred to 36°C for 30 min, and treated with 0.6 μM SM for 30 min at 36°C, and ChIP was conducted as described for Fig. 6B with addition of PCR amplification of ARG1 5′ ORF and TATA (for Rpb3) and POL1 (reference for Ser5P). Occupancy in WT and kin28-ts was normalized to that in gcn4Δ. (E) Ratio of Eaf1 occupancy to Rpb3 occupancy at ARG1. The decreased ratio for the 3′ ORF conferred by kin28-ts was judged significant by Student's t test (**, P < 0.001). (F) ChIP analysis of Epl1 and Eaf1 occupancies at the ADH1 3′ ORF. Cells were grown in SC at 30°C and transferred to 36°C for 30 min, and ChIP was performed with anti-Myc or anti-Rpb3 antibodies and primers to PCR amplify the ADH1 3′ ORF. Shown is the ratio of NuA4 occupancy to Rpb3 occupancy. The decreased ratios conferred by kin28-ts were judged significant by Student's t test (**, P < 0.001). (G) ChIP analysis of Epl1 and Eaf1 occupancies at the PMA1 3′ ORF was conducted as described for panel F, except using primers to amplify PMA1 3′ ORF. The decreased occupancies in kin28-ts were determined to be significantly reduced compared to WT by Student's t test (*, P < 0.01). (H) Coimmunoprecipitation of Pol II with myc-Eaf1. WCEs were immunoprecipitated with anti-myc antibodies and subjected to Western analysis with anti-Ser5P, anti-Ser2P, anti-Rpb1, and anti-Rpb3 antibodies.
FIG. 10.
FIG. 10.
NuA4 interaction with nucleosomes, but not Pol II, is stimulated by H3 methylation. (A and B) ChIP analysis of Eaf1 (A) and Epl1 (B) occupancies at ARG1 was conducted with WT, set1Δ set2Δ, or gcn4Δ EPL1-Myc or EAF1-Myc strains as described for Fig. 6B. Occupancy in WT and set1Δ set2Δ strains was normalized to that in gcn4Δ. (C) ChIP analysis of Eaf1 occupancy at the GAL1 3′ ORF was conducted as described for Fig. 7A. The decreased occupancy in set1Δ set2Δ was determined to be significantly reduced compared to WT by Student's t test (*, P < 0.01). (D) ChIP analysis of Eaf1 occupancy at the ADH1 3′ ORF. EAF1-myc strains were grown at 30°C, and ChIP was performed using anti-myc antibodies and PCR primers to amplify the ADH1 3′ ORF. The decreased occupancy in set1Δ set2Δ was determined to be significantly reduced compared to WT by Student's t test (*, P < 0.01). (E) Coimmunoprecipitation of myc-Eaf1 with H3 and Pol II. WCEs were immunoprecipitated with anti-myc or anti-H3 antibodies and subjected to Western analysis with anti-Ser2P (anti-myc IP) and anti-Myc (anti-H3 IP) antibodies. (F) ChIP analysis of H4-Ac at the ARG1 3′ ORF was conducted as described for Fig. 6B with antibodies to H4-Ac or H3 and primers to PCR amplify the chromosome VI telomere (H4-Ac) or POL1 (H3). H4-Ac occupancy was normalized to H3 occupancy. The decreased H4-Ac/H3 ratio in the mutant was determined to be significant by Student's t test (*, P < 0.01).
FIG. 11.
FIG. 11.
Model for two-stage cotranscriptional recruitment of NuA4 and its role in promoting elongation. (A) NuA4 is recruited to promoters by interaction of its Tra1 subunit with activators. Tra1 association with NuA4 depends on Eaf1. NuA4 acetylates H4 in promoter nucleosomes to stimulate PIC assembly. NuA4 occupies coding sequences cotranscriptionally stimulated by interaction with Pol II phosphorylated on Ser5 of the CTD. (B) NuA4 subsequently engages coding sequence nucleosomes in a manner stimulated by H3 methylation, and NuA4 acetylates H4 in these nucleosomes. (C) H4 acetylation by NuA4 stimulates RSC recruitment to coding sequences. (D) RSC helps to displace nucleosomes in the coding sequence to stimulate efficient transcription elongation.
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