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. 2013 Apr;33(8):1645-56.
doi: 10.1128/MCB.01121-12. Epub 2013 Feb 11.

Histone chaperones Nap1 and Vps75 regulate histone acetylation during transcription elongation

Yu-Ming Xue  1 , Anna K Kowalska, Kamila Grabowska, Katarzyna Przybyt, Magda A Cichewicz, Brian C Del Rosario, Lucy F Pemberton
Affiliations

Histone chaperones Nap1 and Vps75 regulate histone acetylation during transcription elongation

Yu-Ming Xue et al. Mol Cell Biol. 2013 Apr.

Abstract

Histone chaperones function in chromatin assembly and disassembly, suggesting they have important regulatory roles in transcription elongation. The Saccharomyces cerevisiae proteins Nap1 and Vps75 are structurally related, evolutionarily conserved histone chaperones. We showed that Nap1 genetically interacts with several transcription elongation factors and that both Nap1 and Vps75 interact with the RNA polymerase II kinase, CTK1. Loss of NAP1 or VPS75 suppressed cryptic transcription within the open reading frame (ORF) observed when strains are deleted for the kinase CTK1. Loss of the histone acetyltransferase Rtt109 also suppressed ctk1-dependent cryptic transcription. Vps75 regulates Rtt109 function, suggesting that they function together in this process. Histone H3 K9 was found to be the important lysine that is acetylated by Rtt109 during ctk1-dependent cryptic transcription. We showed that both Vps75 and Nap1 regulate the relative level of H3 K9 acetylation in the STE11 ORF. This supports a model in which Nap1, like Vps75, directly regulates Rtt109 activity or regulates the assembly of acetylated chromatin. Although Nap1 and Vps75 share many similarities, due to their distinct interactions with SET2, Nap1 and Vps75 may also play separate roles during transcription elongation. This work sheds further light on the importance of histone chaperones as general regulators of transcription elongation.

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Figures

Fig 1
Fig 1
NAP1 genetically interacts with other transcription elongation factors. (A) Tenfold serial dilutions of the indicated strains were spotted onto YPD or CSM plates with the indicated additives and grown at 30°C or 37°C for 2 days. (B) The indicated strains were visualized by DIC microscopy (left). The percentage of budding cells with abnormal buds from the indicated strains was calculated (right). (C) Tenfold serial dilutions of the indicated strains were spotted onto CSM plates with HU or lacking inositol (−ino) and grown at 30°C or, where indicated, at 37°C for 2 days.
Fig 2
Fig 2
Strains lacking CTK1 require NAP1 for normal growth and transcription. (A) Tenfold serial dilutions of the indicated strains were spotted onto CSM plates with the indicated additives or lacking inositol (−ino) and grown at 30°C or 37°C for 2 days. (B) The indicated strains were grown in galactose-containing medium for 2 h (left) or medium lacking phosphate for 4 h (right). RNA was isolated from the indicated strains and expression of the GAL1 or PHO5 transcript relative to ACT1 transcript was determined by reverse transcription real-time PCR and expressed as a percentage of WT levels.
Fig 3
Fig 3
Strains lacking CTK1 require VPS75 for normal growth and transcription. (A and B) Tenfold serial dilutions of the indicated strains were spotted onto CSM plates with the indicated additives or lacking inositol and grown at 30°C for 2 days. (C) The indicated strains were grown in galactose-containing medium for 2 h. RNA was isolated from the indicated strains, and expression of GAL1 transcript relative to ACT1 was determined by reverse transcription real-time PCR and expressed as a percentage of WT levels. (D) The ctk1Δ nap1Δ and ctk1Δ vps75Δ strains containing empty plasmid (−) or a plasmid encoding Ctk1, Ctk1-T338A, or Ctk1-D324N were spotted onto CSM plates lacking leucine and with dextrose [−Leu (dex)] or galactose [−Leu(gal)] and grown at 30°C for 2 days.
Fig 4
Fig 4
Nap1 and Vps75 regulate transcription from cryptic transcriptional start sites. mRNA was isolated from the indicated strains and separated by Northern blotting. Blots were probed for STE11 and ACT1 and 32P quantified by phosphorimager. (A) Northern blot showing full-length and cryptic STE11 transcript and actin transcript of indicated strains. (B)The amount of the smaller cryptic STE11 transcript relative to actin was calculated, and the result is shown as a percentage of the value observed with the ctk1Δ strain (set as 100%).
Fig 5
Fig 5
Rtt109 regulates transcription from cryptic transcriptional start sites. (A) Tenfold serial dilutions of the indicated strains were spotted onto CSM plates or CSM lacking inositol and grown at 30°C for 2 days. (B) mRNA was produced from the indicated strains and separated by Northern blotting. Blots were probed for STE11 and ACT1, and the signal was quantified by phosphorimager. The relative levels of the smaller cryptic STE11 transcript compared to actin were calculated, and the results are shown as percentages of the values observed with the ctk1Δ strain (set as 100%).
Fig 6
Fig 6
Acetylation of H3 K56 is not required for elevated cryptic transcription in ctk1Δ cells. (A) H3 K56Ac occupancy relative to total H3 occupancy at the PHO5 promoter and ORF was determined by ChIP assay with real-time PCR. Chromatin was isolated from WT and vps75 strains prior to (uninduced [un]) and after (induced [ind]) growth for 7 h in medium lacking phosphate. Occupancy is calculated relative to the signal from an equal amount of protein from the input sample. Occupancy is displayed as relative to that observed in WT (uninduced), set at 100%. (B) Strains lacking HHT1 and HHT2 expressed WT and mutant versions of H3 from a plasmid as indicated on the left. Indicated strains were also deleted for ctk1. Tenfold serial dilutions were spotted onto CSM plates with the indicated additives or lacking inositol and grown at 30°C for 3 days. (C) mRNA was produced from strains expressing H3 mutants as described in panel B and separated by Northern blotting. Blots were probed for STE11 and ACT1, and the signal was quantified by phosphorimager. The relative levels of the smaller cryptic STE11 transcript compared to actin were calculated, and results are shown as percentages of those observed with the ctk1Δ strain expressing WT H3 (set as 100%).
Fig 7
Fig 7
Vps75 and Nap1 regulate H3 K9 acetylation. (A) Strains lacking HHT1 and HHT2 expressed WT and mutant versions of H3 from a plasmid, as indicated on the left. Indicated strains were also deleted for ctk1. Tenfold serial dilutions were spotted onto CSM plates with the indicated additives or lacking inositol and grown at 30°C for 2 days. (B) mRNA from strains expressing H3 mutants as described in panel A was subjected to Northern blotting. The relative levels of the smaller cryptic STE11 transcript compared to actin were calculated, and results are shown as percentages of those observed with the ctk1Δ strain expressing WT H3 (set as 100%). (C) H3 K9Ac occupancy relative to total H3 occupancy at the STE11 ORF was determined by ChIP assay with real-time PCR using the primer sets indicated on the schematic (numbers equal midprobe distances from STE11 ATG in base pairs). H3 K9 acetylation is displayed as relative to that observed in WT strains, set at 100%. (D) Tenfold serial dilutions of the indicated strains were spotted onto CSM plates with the indicated additives or lacking inositol and grown at 30°C for 2 days. (E) mRNA from indicated strains was subjected to Northern blotting. The relative levels of the smaller cryptic STE11 transcript compared to actin were calculated, and results are shown as percentages of those observed with the ctk1Δ strain (set as 100%).
Fig 8
Fig 8
Nap1 and Vps75 regulate cryptic transcription by different pathways. (A) Tenfold serial dilutions of the indicated strains were spotted onto CSM plates with the indicated additives or lacking inositol and grown at 30°C for 2 days. (B) mRNA from indicated strains was subjected to Northern blotting. The relative levels of the smaller cryptic STE11 transcript compared to actin were calculated, and results are shown as percentages of those observed with the set2Δ strain (set as 100%).
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