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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.
Evolutionarily conserved histone chaperones play important roles in chromatin biogenesis and remodeling (1, 2). Histone chaperones bind histones off chromatin and shield them from nonspecific interactions with DNA and other proteins (1, 2). Histone chaperones can assemble histones onto a DNA template in vitro and may have important regulatory roles in replication and transcription (1, 2). Yeast Nap1 is a member of a family of evolutionarily conserved histone chaperones (3). Nap1 primarily binds to histones in the cytoplasm and promotes the formation of import complexes with distinct karyopherins (4–6). Although the mechanism is not well elucidated, in the nucleus, Nap1 presumably supplies new histones for the assembly of nucleosomes, as well as binding histones from disassembled nucleosomes and reassembling them into new nucleosomes after DNA replication (3). Recent evidence suggests that Nap1 can interact with histones H2A, H2B, and H2A.Z, and H3 and H4 although the H3-H4 interaction is less well studied (7–9). Outside of replication, Nap1 has important roles in transcription and in the maintenance of chromatin integrity (10). We have shown that Nap1 can be recruited to both promoters and open reading frames (ORFs), and this recruitment is upregulated during transcription (10). Recruitment of Nap1 to ORFs is partially mediated by interaction with TREX family members although other mechanisms likely exist (10). Nap1 is required to prevent the misincorporation and overassembly of H2A and H2B dimers into chromatin (11). Nap1 has also recently been shown to stimulate the activity of the ATP-dependent remodeler RSC in in vitro transcription assays (12). Taken together, these data suggest that Nap1 plays important roles in nucleosome remodeling during transcription.
In yeast, the second Nap1 family histone chaperone is Vps75, which plays an important role as a chaperone for the histone acetyltransferase (HAT) Rtt109 (4, 13). Rtt109 is a cell cycle-regulated HAT that is essential for the acetylation of histone H3 K56, and it also acetylates H3 K4, K9, and K27, a function that it shares with Gcn5 (13–19). Vps75 is essential for Rtt109 stability and for Rtt109-mediated acetylation of H3 K9 (16, 19–21). Vps75 is not essential for the acetylation of H3 K56 by Rtt109 although it may play a role in this activity, whereas the chaperone Asf1 is essential for all Rtt109 HAT activities (16, 22, 23). Acetylation of H3 K56 (H3 K56Ac) is mostly linked to functions during replication and DNA repair where new H3 is acetylated prior to nucleosome assembly (13, 23). H3 K56Ac is also important during transcription (13, 23). It has been shown that although the levels of H3 decrease in the ORF when GAL1 expression is induced, the relative level of H3 K56Ac increases (23). As Rtt109 is not thought to acetylate H3 K56 in chromatin, the relative increase in this acetylation mark is likely due to histone exchange leading to the incorporation of acetylated histones. The Rtt109 chaperone, Vps75, is recruited to elongating genes, and loss of Vps75 leads to a decrease in H3 K56Ac at these genes (23). Deletion of Vps75 also results in changes in nucleosome remodeling during transcription (23). Taken together these data suggest that Vps75 likely plays important roles as a histone chaperone during transcription elongation and is important for Rtt109 activity.
The packaging of histones into chromatin creates a barrier for the elongating polymerase (24). This necessitates chromatin disassembly in front of the polymerase to allow for its passage along the DNA. Chromatin is rapidly reassembled behind the polymerase to prevent initiation from cryptic transcription start sites within the ORF (25). As described above, histones can also be dynamically exchanged during transcription to allow for the incorporation of modified histones such as H3 K56Ac (23). How this disassembly and reassembly are regulated and coordinated with polymerase passage is not well understood. Phosphorylation of the repetitive C-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit and posttranslational modifications of histones likely plays a central role (24). After the elongating polymerase has escaped the promoter, it is phosphorylated by the kinase Ctk1 on Ser 2 of the repetitive CTD (26). The CTD phosphorylated at Ser 2 serves to recruit the methyltransferase Set2 to coding regions of elongating genes (25). Set2 methylates H3 K36 (H3 K36Me), and it has been shown that the methylation of H3 K36 allows the recruitment of the small Rpd3 (Rpd3S) complex to chromatin through direct interaction of subunits Eaf3 and Rco1 with H3 K36Me (25). The recruitment of this histone deacetylase (HDAC) complex leads to deacetylation of H3 and H4, thereby promoting chromatin reassembly and compaction after polymerase passage. Experiments that measure the production of transcripts from intragenic “cryptic” transcriptional start sites in the FLO8 and STE11 genes have demonstrated that Ctk1, Set2, and Eaf3 play a role in this process (27–32). Deletion of CTK1, SET2, and EAF3 leads to an increase in shorter cryptic transcripts, indicating that the polymerase has initiated transcription within the coding region (27–32). In addition there is a commensurate increase in acetylation of histones where transcription from cryptic sites is observed (27–32). It has also been demonstrated that there is a second pathway for Rpd3S recruitment, i.e., through direct interaction with the phosphorylated CTD, thus bypassing the need for Set2. However, H3 K36Me remains necessary for Rpd3S HDAC activity (33, 34). Numerous studies have indicated that HATs, as well as HDACs, are cotranscriptionally recruited to coding regions (33–36). In general, it is thought that acetylation contributes to histone eviction and RNAP II processivity, whereas HDACs promote decreased acetylation and increased histone occupancy.
We set out to determine how the chaperones Nap1 and Vps75 function during transcription and whether they play a role in the regulation of histone acetylation and chromatin structure during transcription elongation. Nap1 genetically interacted with several transcription elongation factors, and both Nap1 and Vps75 interacted with the kinase CTK1. We show that Nap1 and Vps75 are both necessary for efficient transcription in the absence of Ctk1 and that loss of either Nap1 or Vps75 serves to suppress the cryptic transcription observed in the absence of Ctk1. We show that Vps75 likely functions with Rtt109 and that both Vps75 and Nap1 promote acetylation of H3 in the ORF. Due to their distinct interactions with SET2, Nap1 and Vps75 may also play separate roles during transcription elongation.
All yeast strains are haploid Saccharomyces cerevisiae strains and derived from S288C/BY4741 except where noted. The following strains, in which ORFs were replaced with KanMX, were obtained from the Open Biosystems deletion collection: the vps75Δ, gcn5Δ, rtt109Δ, asf1Δ, rad6Δ, ctk1Δ, cdc73Δ, rtf1Δ dst1Δ, set2Δ, elp3Δ, elp1Δ, and nap1Δ strains. The vps75::NatMX and nap1::NatMX strains were generated from Y2454 (MATα ura3Δ0 leu2Δ0 his3Δ1 lys2Δ0 MFA1pr-HIS3 can1Δ0) and mated with the strains noted above to produce the following haploid strains (37): the nap1::NatMX ctk1::KanMX, nap1::NatMX cdc73::KanMX, nap1::NatMX rtf1::KanMX, nap1::NatMX dst1::KanMX, nap1::NatMX elp3::KanMX, nap1::NatMX elp1::KanMX, cdc73::KanMX vps75::NatMX, rtf1::KanMX vps75::NatMX, set2::KanMX vps75::NatMX, and set2::KanMX nap1::NatMX strains. The ctk1::URA3 strain was created by direct integration of URA3 into the CTK1 gene. The ctk1::URA3 vps75::NatMX, ctk1::URA3 asf1::KanMX, ctk1::URA3 rtt109::KanMX, and ctk1::URA3 gcn5::KanMX strains were made by mating and dissection. H3 K56 and K9 mutants were expressed from plasmids in a strain where both HHT1 and HHT2 had been deleted, kindly provided by C. Logie, Radboud University, Netherlands. CTK1 was deleted in this strain by integration of the NatMX cassette into the gene (38). Ctk1-WT (where WT is wild type) and Ctk1-D324N and Ctk1-T338A mutants were expressed from plasmids in the ctk1Δ strain background and were kindly provided by M. J. Solomon Lab, Yale University (39). Strains were grown under standard conditions, and for spot assays overnight cultures were diluted and grown to an optical density (OD) of 1.0. Culture ODs were normalized and serially diluted 10-fold. Yeast strains were grown on the plates indicated in the figures and figure legends for 2 to 3 days.
Yeast were grown overnight in yeast extract-peptone-dextrose (YPD) medium at 30°C, sonicated, and observed by differential interference contrast (DIC) microscopy using a 100× objective and a Nikon Microphot microscope. Images were captured using OpenLab software. To quantitate abnormal bud phenotypes, >170 budded cells from an asynchronous population were scored, after sonication, to determine the percentage of those with abnormal buds. Abnormal buds were defined as cells with multiple buds and/or buds whose lengths exceeded the mother cell diameter. The experiment was repeated three times (10).
Total RNA was extracted from 250 ml of yeast at an OD of 1.0 by acid phenol (pH 4.3). For induction of GAL1, cells were grown in glucose and then switched to galactose-containing medium at 30°C for the time indicated. For PHO5 induction, cells were grown in phosphate-containing medium and then switched to phosphate-free medium. The mixtures were heated at 65°C for 1 h and vortexed every 15 min, and RNA was precipitated after chloroform extraction using ethanol (EtOH). Five micrograms of RNA was used for each reverse transcription using an Invitrogen Superscript III first-strand reverse transcription-PCR (RT-PCR) kit following the manufacturer's instruction. Real-time PCR was done in triplicate using Bioline SensiMix on a Bio-Rad real-time PCR machine and analyzed by IQ5 software. For Northern blotting, mRNAs were selected by oligo(dT) cellulose (GE Healthcare) following published protocols (40). Approximately 8 μg of mRNA was loaded on a 1% formaldehyde agarose gel and run at 120 V for 3.5 h; 20 × SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) buffer was used to transfer RNA onto nitrocellulose membranes. The membrane was cross-linked by Stratagene cross-linker at total 1,200-mJ energy level. Probes to actin and STE11 mRNA were made by PCR using the following primers: STE11 primers STE11 (5′-CAAATTATGTGTGCATCCAGCCATGG-3′) and 5′-TTACGGTCCATTTGAGGAATCACTG-3′ and actin primers ACT1 (5′-CTATGTTACGTCGCCTTGGACT-3′) and 5′-CACTTGTGGTGAACGATAGATGG-3′. The products were labeled with [γ-32P]dCTP by random priming using an Invitrogen Random Primers DNA labeling system. The small cryptic transcripts of STE11 and ACT1 signals were quantified from Northern blots using a phosphorimager and ImageQuant software.
For STE11 experiments, cells were grown in YPD medium, and chromatin immunoprecipitation (ChIP) assays were performed as previously described (10). For PHO5 experiments, cells were grown in phosphate-containing medium and then transferred to low-phosphate medium. Immunoprecipitations were carried out with anti-H3 antibody (Millipore), anti-H3 K9 acetyl (Abcam), and anti-H3 K56 acetyl antibody (Millipore) coupled to protein A-agarose or protein A-coupled magnetic beads. Oligonucleotide sets were designed to amplify the STE11 ORF at bp +190, +990, and +1790 (midprobe) from STE11 ATG as previously described (41). The PHO5 promoter and ORF primers were previously described (10). Real-time PCR was performed using SensiMix Plus SYBR and fluorescein (Quantace) in a MiniOpticon real-time PCR System (Bio-Rad). All experiments were performed in duplicate or triplicate. ChIP signal is expressed as a ratio of acetylated H3 to total H3 signal for each strain and is displayed whereby the signal from WT cells (STE11) or WT cells grown under noninducing conditions (PHO5) is set to 100%.
We have shown that Nap1 is recruited to the ORFs of some activated genes during transcription (10). This recruitment is consistent with a role in transcription elongation. In order to investigate the role of Nap1 in this process, we analyzed the genetic interactions between NAP1 and factors involved in transcription elongation. We first analyzed the interaction of NAP1 with the ELP genes (ELP1-4 and ELP6), which encode components of the Elongator complex (42). A genetic interaction between NAP1 and ELP3 was previously reported in a high-throughput screening approach (43). We determined that cells that lack any component of Elongator have mild growth defects on caffeine- and hydroxyurea (HU)-containing media; this defect was greatly exacerbated in cells that lack both the NAP1 and ELP genes (Fig. 1A and data not shown). Cells lacking NAP1 have a delay in activating Clb2-Cdc28, resulting in cells with aberrantly long and/or branched buds (44). This phenotype is observed in only 2 to 10% of cells in the population in the single-deletion strain, depending on the strain background (Fig. 1B). When we observed BY 4741 cells lacking NAP1 and different ELP genes (ELP1-4 and ELP6), we saw a huge increase in multibudded cells in the double mutants (~35% abnormal) compared to numbers in the single mutants (~2 to 5% abnormal) (Fig. 1B). These data suggest a genetic interaction between NAP1 and Elongator components. We analyzed whether NAP1 genetically interacts with other factors involved in transcription elongation. We tested Dst1, which is the general transcription elongation factor TFIIS, the PAF complex, which is a five-protein complex involved in transcription elongation and regulation of RNAP II, and Rad6, which is an E2 ubiquitin-conjugating enzyme important for transcription elongation (24, 45, 46). We tested the relevant single and double mutants for growth defects. We observed that cells lacking both NAP1 and RTF1 or CDC73 (both PAF1 complex components) grew better than the relevant single-deletion strains. This was particularly evident on plates lacking inositol or with HU (Fig. 1C). This phenotype was not apparent with deletion of the other PAF1 complex component LEO1 (data not shown). Cells lacking NAP1 and RAD6 gene grew slightly better than the single mutants under some conditions (Fig. 1C and data not shown). These results suggest that NAP1 has genetic interactions with several elongation factors, including PAF1 complex components.
We also determined whether NAP1 genetically interacted with CTK1. Ctk1 is a kinase that phosphorylates the Rpb1 protein of RNAP II and is required for efficient transcription elongation (26). We observed a strong negative synthetic growth defect in cells lacking both Nap1 and Ctk1 (Fig. 2A). We analyzed this growth defect further by growing cells on different media. We observed that the synthetic growth defect was exacerbated on media lacking inositol or containing hydroxyurea and when grown at 37°C (Fig. 2A). The growth defect was particularly clear on HU, where the ctk1Δ nap1Δ cells were barely viable (Fig. 2A). This suggests that cells lacking Ctk1 require Nap1 for normal transcription and possibly replication or DNA repair. We therefore determined whether Nap1 was required for transcription of inducible genes in strains that lacked Ctk1. WT, ctk1Δ, nap1Δ, and ctk1Δ nap1Δ cells were grown in YPD medium and then switched to galactose-containing medium. RNA was purified and used to make cDNA by reverse transcription. Real-time PCR revealed that transcription of the GAL1 gene was reduced about 5-fold in the ctk1Δ nap1Δ cells compared to wild-type levels (Fig. 2B). This effect was dependent on the loss of NAP1 as cells lacking only CTK1 expressed approximately 3-fold more GAL1 RNA than the double mutant. We carried out a similar experiment looking at the role of NAP1 in the induction of the PHO5 gene in medium lacking phosphate. As with GAL1, we observed a clear decrease in transcription in cells lacking NAP1 and CTK1 compared to cells deleted for CTK1 (Fig. 2B). Interestingly, as has been previously noted by others and us, in strains where NAP1 alone was deleted, we observed that transcription of PHO5 and GAL1 was slightly increased compared to WT levels under inducing conditions (10, 11).
Vps75 is a member of the Nap1 family of histone chaperones in yeast and predominantly interacts with H3 and H4 (19, 22, 47). We determined whether Vps75 similarly interacted with PAF1 complex components and CTK1. In contrast to the synthetic growth effects observed in nap1Δ cdc73Δ and nap1Δ rtf1Δ cells, we did not observe that vps75Δ rtf1Δ or vps75Δ cdc73Δ cells grew significantly differently from the single mutants (Fig. 3A). This suggests that genetic interactions with the PAF1 complex are unique to NAP1.
Ctk1 interacts with different factors involved in transcription elongation, but little is known about its interactions with histone chaperones. We constructed strains deleted for CTK1 and VPS75 and for CTK1 and ASF1 and tested growth as before. Similar to loss of Nap1, loss of Vps75 in strains that were lacking Ctk1 resulted in growth defects on complete synthetic medium (CSM) and on medium lacking inositol and containing HU (Fig. 3B). We also observed a similar synthetic growth defect with a ctk1Δ asf1Δ strain, particularly on plates lacking inositol (Fig. 3B). To determine whether, like Nap1, Vps75 was also important for transcription in cells lacking CTK1, we analyzed GAL1 RNA expression after induction. As with the nap1Δ strain, we observed that strains lacking CTK1 and VPS75 showed a significant reduction in mRNA expression compared to strains with single deletions (Fig. 3C). This indicates that Vps75 is required for the normal transcription of inducible genes in strains that lacked Ctk1.
Ctk1 functions both as a CTD kinase and as a scaffolding protein; the latter function is independent of Ctk1 kinase activity (48). This raised the question of whether the loss of Ctk1, or Ctk1 kinase activity, is important in the absence of histone chaperones. We determined that the Ctk1 kinase domain is important for the genetic interaction with NAP1 and VPS75. Expression of wild-type CTK1 from a plasmid restored the growth defect observed in ctk1Δ nap1Δ and ctk1Δ vps75Δ strains. A mutant version of Ctk1 which is kinase deficient (Ctk1-D324N) did not rescue growth, and another mutant, Ctk1-T338A, which has partial kinase activity, rescued the growth defect but less efficiently than the WT (Fig. 3D) (39). This effect was more pronounced when cells were grown on galactose-containing medium. This result suggests that phosphorylation by Ctk1 is important in the absence of Nap1 or Vps75, rather than its activity as a scaffold to recruit proteins to the promoter.
Ctk1 plays an important role in the pathway that prevents intragenic transcription initiation (25). Mutations in several factors involved in transcription elongation result in the inability to deacetylate and/or reassemble nucleosomes in the ORF. This allows RNAP II to initiate transcription at cryptic transcriptional start sites within certain ORFs such as STE11 (25, 27). These shorter cryptic transcripts can be observed by Northern blotting, and we used this assay to determine whether histone chaperones play important roles in regulating chromatin structure during transcription elongation. We made mRNA from ctk1Δ vps75Δ and ctk1Δ nap1Δ strains and compared the levels of cryptic STE11 transcripts with those from ctk1Δ strains. As expected, we observed an abundant smaller transcript that initiated from the cryptic transcriptional start site in RNA from ctk1Δ cells (Fig. 4A). We quantified our Northern blots and detected very low levels of STE11 cryptic transcript with nap1Δ and vps75Δ cells, similar to the levels observed from wild-type strains (Fig. 4B). Surprisingly, compared to the levels observed with the ctk1Δ strain, in cells with deletions of both ctk1Δ and vps75Δ or of ctk1Δ and nap1Δ, we observed a significant decrease in the amount of cryptic transcripts (Fig. 4). These results suggest that at STE11, loss of Vps75 and Nap1 reverses the ctk1Δ defect. We hypothesize that this is due to altered acetylation or chromatin structure in the absence of these histone chaperones.
Vps75 and Asf1 regulate the activity of the HAT Rtt109 (13, 23). Our cryptic transcription assay suggests that histone chaperones could affect acetylation of ORF nucleosomes; we therefore decided to investigate the role of the HAT Rtt109 in this process. Rtt109 is in a complex with Vps75, and Vps75 is necessary for Rtt109-mediated acetylation of the H3 tail (K9 and K27) (14, 16, 19–21). Rtt109 also acetylates H3 K56, but this activity is mostly dependent on Asf1 although there is some evidence that Vps75 is also important (13, 23). We hypothesized that in the absence of Ctk1, the important function of Vps75 is to regulate Rtt109 activity and increase or maintain H3 acetylation. In this case, we would expect the ctk1Δ rtt109Δ strain to phenocopy the ctk1Δ vps75Δ strain. Observation of the growth of the ctk1Δ rtt109Δ strain compared to that of the ctk1Δ and rtt109Δ strain by dilution spot assay suggests that this is indeed the case. The double mutant grew more slowly than either single mutant, and this growth defect was exacerbated on different media, including media lacking inositol (Fig. 5A; compare with Fig. 3B). We also tested whether the ctk1Δ rtt109Δ strain gave a result to similar that of the ctk1Δ vps75Δ strain when transcripts of the STE11 gene were analyzed. We observed that, as before, there was a high level of shorter cryptic transcripts with the ctk1Δ strain, but these were drastically reduced with the ctk1Δ rtt109Δ strain (Fig. 5B). These results are consistent with the idea that the important function of Vps75 during transcription is to regulate Rtt109 acetylation.
Rtt109 can acetylate several lysines in H3, and whereas acetylation of H3 K9 and K27 is redundant with Gcn5, Rtt109 is the only HAT responsible for K56Ac (13). This suggested that H3 K56 was a possible candidate for the residue that Rtt109 must acetylate to allow transcription from cryptic sites. Consistent with this model, Vps75 is important for the activation-associated recruitment of H3 K56Ac to the GAL10 ORF (23). In addition there is a statistically significant overlap between genes that are enriched for K56 acetylation and Vps75 recruitment (23). Using ChIP, we analyzed the recruitment of H3 K56Ac, relative to total H3, to the PHO5 promoter and ORF in cells grown under noninducing and inducing conditions. The relative amount of H3 K56 acetylation increased in both the promoter and ORF when WT cells were shifted to medium lacking phosphate (Fig. 6A). We observed that this increase was dependent on the presence of Vps75. In addition, the relative amount of H3 K56 acetylation was decreased in the in the ORF in the absence of Vps75 compared to levels in wild-type cells grown in either medium (Fig. 6A). These data further suggest that Vps75 plays an important role in regulating K56 acetylation during transcription and in the ORF of inducible genes.
As both Vps75 and Rtt109 appear to regulate cryptic transcription in the absence of CTK1, it was possible that this was due to a shared function in the assembly of chromatin acetylated at H3 K56. We investigated whether acetylation of H3 K56 was important for transcription elongation. We expressed WT or mutant H3 from a plasmid in a strain where both genomic copies of H3 were deleted (38). In addition, CTK1 was deleted in this strain. We compared growth of strains expressing H3 with that of strains expressing H3 K56R or H3 K56Q, which would be acetylation deficient or mimic acetylation at this residue, respectively. We observed that all the strains lacking CTK1 grew more slowly than strains with CTK1 (Fig. 6B). As expected H3 K56R strains grew very slowly on HU, whereas H3 K56Q strains had a very mild defect. These defects were exacerbated in the ctk1Δ strains: the ctk1Δ H3 K56Q strain grew more slowly than the ctk1Δ H3 WT strain, and the ctk1Δ H3 K56R strain was inviable on HU (Fig. 6B). H3, H3 K56Q, and H3 K56R strains grew similarly on CSM and CSM lacking inositol in the presence and absence of CTK1. As the ctk1Δ H3 K56R strain did not phenocopy the ctk1Δ rtt109Δ strain on medium lacking inositol (compare Fig. 6B and and5A),5A), this suggests that the growth defect on this medium is due to loss of an Rtt109 function other than K56 acetylation.
We next analyzed the expression of the STE11 genes in the H3, H3 K56Q, and H3 K56R strains by Northern blotting to determine the effect of these mutations on the production of cryptic transcripts. In all strains expressing CTK1, we saw a low level of cryptic transcripts, including both histone mutants (Fig. 6C). In the ctk1Δ strains we did not observe a difference between the ctk1Δ H3 WT and ctk1Δ H3 K56R strains, but we did observe increased cryptic transcripts in the ctk1Δ H3 K56Q strain (Fig. 6C). This mutation is expected to mimic acetylation of H3 K56. This suggests that constitutive acetylation at this residue increases cryptic transcription, which is consistent with a model where acetylated chromatin is not properly reassembled at the STE11 ORF. As the ctk1Δ H3 K56R strain did not suppress cryptic transcription, in contrast to the ctk1Δ rtt109Δ strain, it suggests that there are additional Rtt109 acetylation sites important for this process (compare Fig. 6C and and5B5B).
Vps75 also regulates the ability of Rtt109 to acetylate H3 K9, and in the absence of Gcn5, Vps75 is required for H3 K9 acetylation (14, 16, 19). It has also been shown by ChIP that there is a significant overlap in the occupancy of H3 K9Ac and Vps75 throughout the genome (23). We therefore examined whether reversible acetylation of H3 K9 was important for the production of cryptic transcripts, explaining the important roles of Vps75 and Rtt109 in this process. We constructed strains lacking endogenous H3 that expressed H3, H3 K9R, and H3 K9Q from a plasmid and were also deleted for CTK1. All strains lacking CTK1 grew more slowly than the wild-type strain, but no significant additional genetic interactions were noted (Fig. 7A). We analyzed these strains for the production of cryptic STE11 transcripts. In the presence of Ctk1, very low levels of cryptic transcripts were observed in all strains (Fig. 7B). In the absence of CTK1, strains expressing WT H3 and the constitutive acetylation mimic H3 K9Q had high levels of cryptic transcripts (Fig. 7B). This phenotype was reversed in ctk1Δ strains expressing H3 K9R, which could not be acetylated (Fig. 7B). Loss of acetylation of this residue phenocopied loss of Vps75 and Rtt109, suggesting that acetylation of H3 K9 plays an important role in modifying chromatin structure and the regulation of transcription (compare Fig. 7B with with4B4B and and5B5B).
Loss of H3 K9 acetylation suppressed ctk1Δ-dependent cryptic transcription similarly to loss of Vps75 and Nap1. We therefore hypothesized that both Ctk1 and the histone chaperones Nap1 and Vps75 may differentially regulate the levels of H3 K9 acetylation in the STE11 ORF. Using ChIP, we analyzed the occupancy of H3 K9Ac, relative to total H3, at different regions of the STE11 ORF. We compared several primer sets in the ORF, including two that overlap the major known cryptic transcriptional start sites (41). In all cases we observed that relative H3 K9 acetylation increased in the ctk1Δ strains compared to levels in the wild-type strains (Fig. 7C). We also observed that relative H3 K9 acetylation was reduced back to wild-type levels in the double deletion strains. We did not observe any difference between the ctk1Δ nap1Δ and the ctk1Δ vps75Δ strains (Fig. 7C). These findings correlate with our data on cryptic transcription and suggest that both Vps75 and Nap1 regulate H3 K9 acetylation.
Although Rtt109 acetylates H3 K9 in a Vps75-dependent manner, studies have determined that Gcn5 is the major HAT for H3 K9 (35). From our H3 K9R mutational studies we could not rule out that Gcn5 also played a role in the regulation of cryptic transcription. Therefore, we determined whether, in the absence of CTK1, loss of Gcn5 also reversed the cryptic phenotype. We constructed a ctk1Δ gcn5Δ strain, which grew very slowly compared to the single deletions and was inviable on HU (Fig. 7D). Surprisingly, when we examined cryptic transcripts from these strains, the ctk1Δ and ctk1Δ gcn5Δ strains gave similarly high levels of cryptic transcripts, and we observed no cryptic transcripts with the Gcn5 deletion (Fig. 7E). This suggests that Rtt109, rather than Gcn5, is the important HAT that regulates cryptic transcription, most likely through K9 acetylation. This also suggests that the severe synthetic growth defect observed in the ctk1Δ double deletion strains tested here is not necessarily an indicator of an effect on cryptic transcription.
Both NAP1 and VPS75 can be recruited to ORFs and have a synthetic growth defect with CTK1, and their deletion reverses the increase in cryptic transcription observed in a ctk1Δ strain. This suggests that they function similarly in transcription elongation. To further explore these similarities, we tested whether Vps75 and Nap1 genetically interacted with the H3 K36 methyltransferase, SET2. Like the ctk1Δ strain, the set2Δ strain also has increased transcription from cryptic start sites in STE11 (25). We created nap1Δ set2Δ and vps75Δ set2Δ strains and determined that these strains did not have any apparent growth defects (Fig. 8A). As set2Δ cells have increased transcription from cryptic start sites, we tested whether loss of NAP1 or VPS75 would suppress this effect. We observed that loss of VPS75 and SET2 together reduced the amount of STE11 cryptic transcript observed in the set2Δ strain by at least 3-fold (Fig. 8B). In contrast, when we examined STE11 transcription in the set2Δ nap1Δ strain, we did not see any reduction in the abundant cryptic transcript that was observed with the set2Δ mutation alone (Fig. 8B). This suggests that the pathways by which Nap1 and Vps75 regulate chromatin acetylation or reassembly during transcription elongation are different.
We set out to try to understand the role of histone chaperones in transcription elongation. We first focused on Nap1 as we have previously shown that Nap1 is recruited to promoters and ORFs (10). However, the function of Nap1 in transcription is unclear as Nap1 is mostly cytoplasmic at steady state, and the most obvious defect in a Nap1 deletion is elongated buds (5). The elongated buds are indicative of a delay in the activation of Clb2-Cdc28 complexes that lead to the switch from polar to isotropic growth of the emerging bud (44). Our growth and morphology assays identified genetic interactions between NAP1 and several different transcription elongation factors. We found that the combined deletion of Nap1 and Elongator components leads to a growth defect and an increase in the number of aberrantly budded cells. This suggests either that loss of both Nap1 and Elongator affects the transcription of important cell cycle factors or possibly that loss of Nap1 directly affects bud neck signaling, which is exacerbated by changes in transcription. Our data suggest that the functions of Nap1 and Elongator intersect. Although Elongator has more than one function, it has HAT activity, which may explain its putative shared function with Nap1 in transcription elongation (42).
Nap1 interacted with some components of the PAF1 complex. This complex consists of Paf1, Ctr9, Cdc73, Rtf1, and Leo1 (49). We were unable to construct the paf1Δ nap1Δ double mutant but were able to test cdc73Δ, rtf1Δ, and leo1Δ mutants. It is not clear why deletion of NAP1 rescued the growth defects observed with some components of the PAF1 complex, but it argues that this complex and Nap1 may have opposite functions. Strains lacking PAF1 components are sensitive to 6-azauracil (6-AU), and the Paf1 complex associates with RNAP II in open reading frames; both of these observations are consistent with a role in elongation (49). Interestingly loss of Paf1 and Ctr9 leads to a decrease in Set2 activity (H3 K36 methylation), while there is a moderate reduction with Cdc73 and no defect with Rtf1 and Leo1 deletions (49). This suggests that there is a precedent for genetic interactions with some but not all members of the PAF complex. Taken together, the genetic interactions with Elongator, the PAF1 complex, and Rad6 provide more evidence that Nap1 functions in transcription elongation, and future experiments will determine mechanistically how the functions of these factors intersect.
Ctk1 phosphorylates the CTD of Rbp1 during the elongation phase of transcription. The methyltransferase Set2 is recruited to the phosphorylated CTD, and therefore Ctk1 is necessary for full activity of Set2 (25, 50). In the absence of Ctk1, cells have an alteration in chromatin structure allowing transcription from cryptic start sites present within genes, including STE11 (25, 50). We used genetic interaction with CTK1 to uncover the function of histone chaperones during transcription elongation. Four genes that we tested, NAP1, VPS75, ASF1, and the HAT RTT109 all had similar genetic interactions with CTK1. All double-deletion strains grew poorly, and this was exacerbated in the absence of inositol and with HU. In addition, of those that we tested, the ctk1Δ vps75Δ and ctk1Δ nap1Δ strains had strong defects in inducible transcription. Surprisingly, simultaneous loss of vps75, nap1, or rtt109 reversed the phenotype observed with the ctk1Δ strains whereby STE11 is transcribed from a cryptic transcription start site. This suggests that Nap1, Vps75, and Rtt109 have similar functions in transcription and may function in opposition to Ctk1.
Mutations that result in transcription from cryptic sites have been observed in genes encoding proteins that affect chromatin structure during transcription elongation (27, 29). This can be through changes in acetylation status, for example, loss of Rpd3 recruitment (25). It is also thought that some mutations, including loss of the histone chaperone Rtt106, may directly affect chromatin structure (41, 51). Most of the factors tested previously, including all of the histone chaperones, increase cryptic transcription when they are deleted (52). We show for the first time a related group of proteins that have the opposite effect. Deletion of the histone chaperones under study here, Nap1 and Vps75, reverse the cryptic transcription phenotype observed with the ctk1Δ strains to nearly wild-type levels of transcriptional suppression. This suggests a model whereby Nap1 and Vps75 promote histone acetylation or changes in nucleosome structure during transcription elongation.
Vps75 is necessary for Rtt109 acetylation of H3 K9 and also plays a role in the acetylation of H3 K56 (14, 16, 19). The interactions of VPS75 and RTT109 with CTK1 were very similar; loss of both in conjunction with a ctk1Δ mutation led to a similar growth defect and restored the cryptic transcription defect. The fact that Vps75 interacts with Rtt109 and regulates its function, coupled with the finding that Rtt109 has the same effect on STE11 transcription, strongly suggests that Rtt109 and Vps75 together regulate acetylation at STE11. The acetylation sites recognized by Rtt109 are known, and we first analyzed H3 K56 as Rtt109 is the only HAT that can acetylate this residue. We found that mimicking constitutive acetylation of H3 K56 actually exacerbated the ctk1Δ mutation-dependent cryptic transcription phenotype. Surprisingly loss of acetylation of this lysine, which would be the same as deletion of Rtt109, did not suppress the level of cryptic site transcription to wild-type levels. This suggests that while acetylation of H3 K56 may play a role and while mimicking constitutive H3 K56 acetylation may exacerbate cryptic transcription, Rtt109 also has other important functions. Genome-wide studies have indicated higher levels of H3 K9Ac in regions occupied by Vps75, and Vps75 is necessary for Rtt109-mediated acetylation of H3 K9 (23). We analyzed H3 K9 acetylation and observed that the H3 K9R mutation reversed the cryptic site transcription phenotype. This suggests that the H3 K9R mutation phenocopies loss of Rtt109 and of Vps75, which makes sense as H3 K9 is a Vps75-dependent Rtt109 substrate. However, if H3 K9 is the important acetylation site in this pathway, it is surprising that the other major H3 K9 HAT, Gcn5, cannot replace Rtt109 function in rtt109Δ ctk1Δ cells (35). We observed that ctk1Δ gcn5Δ cells had a severe growth defect, but we saw no change in cryptic transcription beyond that which we observed with ctk1Δ cells alone. These findings point to the idea that Rtt109-dependent acetylation of H3 K9 is not functionally redundant with Gcn5. As Rtt109 acetylates H3 K9 out of chromatin, it is possible that during transcription histones are first acetylated by Rtt109 and then loaded at specific genes or specific nucleosomes (53).
These data suggest that the important role of Vps75 in this process is to mediate Rtt109-dependent acetylation during transcription. We analyzed the level of H3 K9 acetylation in the STE11 gene compared with the level of total H3 by ChIP. We determined that acetylation of H3 K9 increased in the absence of ctk1 compared to levels in wild-type cells, which is consistent with the increase in cryptic transcription. Surprisingly, we also observed that this increase was reversed in both the ctk1Δ vps75Δ and ctk1Δ nap1Δ strains. This suggests that both Vps75 and Nap1 regulate H3 K9 acetylation. As discussed above, Vps75 likely accomplishes this by regulating Rtt109 activity. We do not yet know if the important role of Vps75 is to target Rtt109 to the relevant pool of histones or to stimulate the exchange of Rtt109-modified histones with chromatin. We do not know whether Nap1 also regulates Rtt109 in some way, or whether Nap1 can regulate chromatin acetylation at a different point in the pathway such as through the assembly of acetylated histones.
While the manuscript was under review, the Venkatesh et al., using genome-wide approaches, showed that loss of SET2 results in increased histone exchange and enrichment of H3 K56 acetylation over different ORFs (54). They also showed that increased acetylation of both H3 and H4 correlates with increased exchange using set2Δ cells and that increases in acetylation observed in set2Δ cells could be reversed by deletion of Rtt109 or Asf1 (54). They proposed that the function of Set2 was to suppress histone exchange and thus maintain H3 K36 methylation. Conversely, histone exchange would allow the introduction of specifically acetylated histones which promote transcription. Similar to our study with the ctk1Δ strain, they observed that the set2Δ rtt109Δ strain reversed cryptic transcription phenotype of the set2Δ strain (54). This is most likely due to decreased acetylation and exchange. In the case of Set2, they found that the important Rtt109 substrate was H3 K56, and they did not observe an important role for H3 K9 acetylation. Although there are parallels with our experiments, these data would suggest that the set2Δ- and ctk1Δ-dependent cryptic transcription pathways are dependent on different acetylation sites. However, they are both dependent on the HAT Rtt109, further emphasizing its role as a HAT that acetylates histones out of chromatin. Future experiments will determine whether, like Set2, Ctk1 suppresses histone exchange. The increase in H3 K9 acetylation in the absence of Ctk1 we observe is consistent with this model, as H3 K9 is acetylated out of chromatin by Rtt109 and must subsequently be incorporated by histone exchange.
The role of histone chaperones Vps75 and Nap1 in the set2Δ-dependent pathway was not investigated (54). We observed that loss of Vps75 reversed the cryptic transcription phenotype observed in set2Δ cells, which would be consistent with its interaction with Rtt109. We did not observe that Nap1 deletion reversed the set2Δ-dependent cryptic transcription phenotype, further emphasizing that Set2 and Ctk1 may regulate cryptic transcription through different pathways. This is consistent with previously published reports where Ctk1 appears to be able to affect cryptic transcription through Rpd3 activity in a SET2-dependent and SET2-independent manner (33, 34).
We do not know whether Vps75 and Nap1 share similar functions during transcription elongation. Genetic experiments were not very revealing; the ctk1Δ nap1Δ vps75Δ strain grew similarly to the double-deletion strains (data not shown). However, VPS75 did not share the same genetic interactions with the PAF1 complex and Elongator as NAP1, and loss of NAP1 did not reverse the cryptic transcription phenotype observed with the set2 deletion. It is likely that Vps75 and Rtt109 function together in a pathway that involves histone acetylation and histone exchange; however, the function of Nap1 in the regulation of chromatin structure is less clear. In vitro, whereas Vps75 has been shown to stimulate Rtt109 function, Nap1 does not appear to do so (47, 55). Surprisingly, we observed that deletion of Nap1 suppresses the ctk1Δ-dependent increase in H3 K9 acetylation, indicating that Nap1 does regulate chromatin acetylation either directly or perhaps indirectly through chromatin assembly. It is possible that Nap1 acts with ATP-dependent remodeling complexes such as RSC: Nap1 is a potent and specific histone acceptor in RSC disassembly assays, and Nap1 stimulates RSC-dependent in vitro transcription assays (12, 56). Importantly, like loss of Nap1, loss of Rsc1 has been shown to suppress cryptic transcription observed with deletion of Rco1, a component of Rpd3S (41). This is one of the only other examples of a factor suppressing cryptic transcription (41). Interestingly, Smolle et al. recently showed that two other ATP-dependent remodelers, Isw1 and Chd1, like Set2 suppress histone exchange and cryptic transcription (57). We hypothesize therefore that Nap1 may stimulate transcription elongation by promoting histone exchange or chromatin remodeling through its interactions with the ATP-dependent remodeler RSC.
In summary, we show that Nap1 and Vps75 regulate cryptic transcription observed in the absence of Ctk1. Rtt109 acetylation of H3 K9 also plays a role in this pathway, and both Vps75 and Nap1 promote acetylation of H3 in the ORF. This suggests that these histone chaperones can orchestrate changes in chromatin acetylation, likely through their roles in chromatin assembly and exchange, and so play important roles in the regulation of transcription elongation.
We thank Anoosha Kasanagottu and Korinna Straube for technical help and C. Logie, Radboud University, Netherlands, and M. J. Solomon, Yale University, for strains.
This work was supported by research grant R01 GM65385 from the National Institutes of Health.
Published ahead of print 11 February 2013