The purpose of this study was to elucidate the mechanisms by which histone acetylation participates in transcriptional regulation of hsp70 (heat-shock protein 70) genes SSA3 and SSA4 in yeast. Our results indicated that histone acetylation was required for the transcriptional activation of SSA3 and SSA4. The HATs (histone acetyltransferases) Gcn5 (general control non-derepressible 5) and Elp3 (elongation protein 3) modulated hsp70 gene transcription by affecting the acetylation status of histone H3. Although the two HATs possessed overlapping function regarding the acetylation of histone H3, they affected hsp70 gene transcription in different ways. The recruitment of Gcn5 was Swi/Snf-dependent and was required for HSF (heat-shock factor) binding and affected RNAPII (RNA polymerase II) recruitment, whereas Elp3 exerted its roles mainly through affecting RNAPII elongation. These results provide insights into the effects of Gcn5 and Elp3 in hsp70 gene transcription and underscore the importance of histone acetylation for transcriptional initiation and elongation in hsp genes.
- chromatin remodelling
- heat-shock protein (hsp)
- histone acetyltransferase (HAT)
- histone modification
The eukaryotic genome is packaged into chromatin, which acts as a constant barrier to transcription and other cellular processes that require access to DNA. Therefore the structure of chromatin has to be modified before active transcription can proceed. The first group of chromatin-remodelling factors identified is composed of enzymes that covalently modify the N-terminus of histones, such as HATs (histone acetyltransferases) and histone methyltransferases. Modification of chromatin by HATs and HDACs (histone deacetylases) plays a key role in transcriptional regulation [1–4]. Changes to histone acetylation, both at the gene promoter and coding regions, correlates with transcriptional activation and repression. A study has suggested that transcript elongation is required to form an unfolded structure of transcribing nucleosomes, and histone acetylation is needed to maintain this unfolded structure .
Gcn5 (general control non-derepressible 5) is the catalytic subunit of SAGA (Spt-Ada-Gcn5-acetyltransferase), a major transcription-related HAT complex in yeast , and Elp3 (elongation protein 3) is the catalytic subunit of Elongator, a HAT complex originally isolated with hyperphosphorylated, elongating RNAPII (RNA polymerase II) . Histone H3 is the primary target for both of these complexes, and the complexes display significant functional redundancy [6,8–11]. Previous genetic studies demonstrated that individual mutations in GCN5 and ELP3 resulted only in fairly mild phenotypic changes [12,13], whereas deletion of both genes conferred a number of severe growth defects. These defects include slow growth, temperature sensitivity and an inability to ferment galactose and sucrose . Similar phenotypes are conferred by point mutations which debilitate the HAT activities of these enzymes without affecting their incorporation into the SAGA and Elongator complexes, suggesting that growth defects in gcn5Δelp3Δ cells occur due to the loss of the HAT activities of both SAGA and Elongator complexes . It has been reported that a significantly lower level of histone acetylation in the coding region of several genes correlated with reduced transcription of the gene in gcn5Δelp3Δ yeast cells . Together, these studies argue that histone acetylation catalysed by Gcn5 and Elp3 may play an important role in transcription in vivo, yet the mechanisms underlying this role are unclear.
The HSP (heat-shock protein) 70 family is the most highly conserved of the HSP families, and its members can be found in organisms ranging from bacteria to plants and animals . Intracellular Hsp70 has several identified functions, including stabilization of protein structure and prevention of protein aggregation by binding to unfolded proteins, and regulation of protein activity, availability and/or transport [15–17]. Hsp70 can also protect cells from apoptotic stimuli, including DNA damage, UV irradiation, serum withdrawal and exposure to chemotherapeutic agents [18–20]. The hsp70 heat-shock-response gene is one of the best studied examples of eukaryotic transcription elongation regulation. Dramatic domain-wide nucleosomal disassembly has been shown to be associated with heat-shock-inducible genes, including hsp82, hsp12, hsp26 and SSA4 in yeast . This raised the question of whether histone acetylation is required for the activation of hsp genes, and if so, how histone acetylation regulates the transcription of these hsp genes. To address these questions, we chose SSA3 and SSA4 as target genes for biochemical and molecular studies. Both SSA3 and SSA4 are inducible hsp70 genes in yeast. The promoter of SSA3 is weakly bound by HSF (heat-shock factor), whereas the promoter of SSA4 is tightly bound by HSF. Moreover, it has been reported that the domain-wide displacement of histones is activated by HSF .
The purpose of this study was to investigate the roles of histone acetylation in transcriptional regulation of these hsp70 genes and to explore the molecular mechanisms underlying this process. We used yeast strains lacking GCN5 and ELP3, which encode two transcription-related HATs, as a model to investigate the effects of histone acetylation on induced transcription of hsp70 genes. We discovered that histone acetylation indeed plays a key role in both transcriptional activation and elongation of the SSA3 and SSA4 genes. Our results shed light on to the mechanisms underlying regulation of hsp70 gene transcription by Gcn5 and Elp3, and highlight the importance of histone acetylation in transcriptional initiation and elongation of hsp genes.
Yeast strains and media
All Saccharomyces cerevisiae strains used are listed in Table 1. Cells were grown at 26 °C to mid-log phase in rich YPD (yeast extract, peptone, dextrose) medium. Yeast cells transformed with pRS313, pRS314 and pRS316 plasmids were grown under similar conditions in SC (synthetic complete) medium (lacking histidine, tryptophan and uracil respectively). Heat-shock induction was performed by transferring the culture to a 37 °C water bath and shaking vigourously; once the temperature reached 37 °C, the incubation was allowed to continue for an additional 10 min. Cells carrying the rpb1-1 mutation and its wild-type counterpart  were grown in YPD medium at 25 °C to mid-log phase and transferred to 37 °C for 30 min to induce heat-shock.
The GCN5 coding sequence was amplified from yeast genomic DNA by PCR using Pfu polymerase and primers G1 and G4 containing EcoRV and XhoI sites respectively. The PCR products were cloned into the EcoRV and XhoI sites of the pRS314 vector to create pRS314-GCN5. The gcn5HAT− insert, which lacks the sequence encoding the catalytic motif II-III-IV of the HAT domain of GCN5 (amino acid residues 170–253), was amplified from pRS314-GCN5 using primers G1/G2 and G3/G4 and the two products were mixed together and amplified using primers G1 and G4. The PCR products were cloned into the EcoRV and XhoI sites in pRS314 to create pRS314-gcn5HAT−. The oligonucleotide primer sequences used are listed in Table 2. After cloning, the entire coding region of all cloned fragments was subjected to sequencing analysis before introduction into yeast. The constructs pRS316-ELP3 and pRS316-yhelp3HAT− were amplified as described previously . Yeast cells were transformed according to Hill et al. .
RNA extraction, RT–PCR (reverse transcription–PCR), and real-time PCR analyses
Total RNA was extracted from yeast cells using the method of Trotter et al. . RT–PCR was performed using the Access RT–PCR System supplied by Promega. Real-time PCR was performed using an ABI Prism 7000 Sequence Detection System (Applied Biosystems), following the manufacturer's protocol, with SYBR Green (TaKaRa, Osaka, Japan) used as a double-stranded DNA-specific fluorescent dye. ACT1 was used as an internal reference for standardizing SSA3 and SSA4 mRNA expression. The PCR primers used to amplify SSA3, SSA4 and ACT1 are listed in Table 2. The data were analysed by calculating 2−ΔΔCt .
ChIP (chromatin immunoprecipitation)
After the appropriate heat-shock treatment, cells were cross-linked with 1% (v/v) formaldehyde for 10 min at 37 °C, and then lysed in 700 μl of lysis buffer [50 mM Hepes/KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100 and 0.1% (v/v) sodium deoxycholate] with protease inhibitors. The sonicated lysates were processed using a ChIP assay kit (Upstate Biotechnology), essentially as described by the manufacturer. Antibodies against RNAPII (05-623) and Ac-H3 (acetyl-histone H3; 06-599) were purchased from Upstate Biotechnology; anti-Gcn5 antibody (sc-9078) was purchased from Santa Cruz Biotechnology. The antibody against the C-terminus of histone H3 was a gift from Dr Alain Verreault (Institute for Research in Immunology and Cancer, University of Montreal, Montreal, Canada), the antibody against Elp3 was a gift from Dr Jesper Q. Svejstrup (Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire, U.K.) and the antibody against HSF was a gift from Dr Gross (Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, SD, U.S.A.). Input and immunoprecipitated DNA were analysed by real-time PCR using the ABI Prism 7000 sequence detector. To calculate the relative abundance of a given gene promoter or coding sequence present in an immunoprecipitate, the following formula was used: Qgene=IPgene/inputgene to calibrate the variation between different samples and primer pairs. The constitutively expressed ACT1 gene was used as an internal control for normalization of all RNAPII, histone H3 and Ac-H3 ChIP results. The data from ChIP assays with Ac-H3 antibodies were normalized to the total level of H3 present at the corresponding time point. The primers used are shown in Table 2.
Histone H3 acetylation affects SSA3 and SSA4 gene transcription
Previous research suggested the removal of nucleosomes as a mechanism to achieve high levels of transcription . This theory raised the question of whether histone acetylation is required for the activation of genes that have lost the histone–DNA contact. In the present study, we first tested the histone H3 levels at SSA3 and SSA4 genes in response to heat-shock induction. Cells were subjected to heat-shock treatment for 10 min at 37 °C, and the abundance of histone H3 associated with the promoter and coding regions of SSA3 and SSA4 was assessed by ChIP using an antibody recognizing the C-terminus of histone H3. The results showed that histone H3 associated with SSA4 was displaced, whereas that associated with SSA3 was not (Figure 1A). In order to evaluate the roles of histone acetylation in transcription of the two hsp70 genes, we analysed the histone H3 acetylation state at SSA3 and SSA4 during heat induction. Because of the observed loss of histone–DNA contacts (Figure 1A), the data from ChIP assays using acetyl-histone H3 antibodies were normalized to the total level of H3 present at the corresponding time point. As can be seen from Figure 1(B), on induction of transcription, the level of acetylated histone H3 was increased at the promoter and coding regions of both SSA3 and SSA4 genes. To further determine whether the modification of histone H3 and histone H4 is important for hsp70 gene transcription, we used yeast strains carrying point mutations in sequences encoding histone H3 at Lys14 (K14R mutant; LFY14), histone H4 at Lys8 (K8R mutant; LFY8) or both histone H3 K14R and histone H4 K8R (LFY814), and tested the mRNA levels of SSA3 and SSA4 on induction of transcription. We found that expression of both genes was decreased in the histone mutant yeast strains (Figure 1C). These results indicated that the lysine residues in the N-termini of histone H3 and histone H4 were important for transcription, and that transcription-associated histone acetylation occurred in the coding regions of these genes.
Gcn5 and Elp3 regulated SSA3 and SSA4 transcription and the function was dependent on their HAT activities
It has been reported that cells which lack Gcn5 and Elp3 have widespread and severe histone H3 hypoacetylation in chromatin, and hypoacetylation of the coding region correlates with inhibition of transcription . To determine the specific HAT(s) responsible for H3 acetylation in the regulation of SSA3 and SSA4, we used strains which were null mutants for the genes encoding the two transcription-related HATs GCN5 and ELP3. First, we performed temperature-sensitive experiments and the results indicated that the gcn5Δelp3Δ yeast strain and the strains transformed with only one of the HATs exhibited growth defects at 39 °C (Figure 2A). When Gcn5 and Elp3 were deleted, the level of SSA3 and SSA4 mRNA was decreased (Figure 2B). This indicated that Gcn5 and Elp3 play a role in SSA3 and SSA4 transcription. To determine whether the function of Gcn5 and Elp3 is dependent on their HAT activities, functional compensation experiments were performed using plasmids containing mutated forms of the GCN5 and ELP3 genes pRS314-gcn5HAT− and pRS316-elp3HAT−, and the consequences for growth were tested by examination of temperature sensitivity. The results showed that the HAT activities of Gcn5 and Elp3 were important for compensating for the temperature sensitive phenotype (Figure 2A), and were apparently important for efficient transcription of SSA3 and SSA4 (Figure 2B). These results indicated that the HAT activities of Gcn5 and Elp3 were required for their function.
To investigate whether the effect of Gcn5 and Elp3 on SSA3 and SSA4 transcription was a direct effect, we used ChIP to detect if Gcn5 and Elp3 could interact with the genes, and the results showed that Gcn5 and Elp3 do interact with SSA3 and SSA4 (Figures 2C and 2D). Additionally, Gcn5 was found to be recruited mainly at the promoter regions of the genes (Figure 2C), whereas Elp3 was located in the coding region (Figure 2D). When the genes were heat-shock-induced, the recruitment of these two HATs was enhanced (Figures 2C and 2D). These experiments suggested that the expression phenotypes associated with the GCN5- and ELP3-deletion mutations could be attributed to a direct role for Gcn5 and Elp3 in regulating SSA3 and SSA4 transcription.
Gcn5 and Elp3 affected histone H3 acetylation in SSA3 and SSA4 genes
It has suggested that transcription-dependent histone acetylation occurred at SSA3 and SSA4, and that both Gcn5 and Elp3 were the key HATs involved in SSA3 and SSA4 gene transcription. It is therefore possible to speculate that Gcn5 and Elp3 may affect SSA3 and SSA4 transcription by acetylating histones associated with these genes on induction. To experimentally test this assumption, we investigated the effect of Gcn5 and Elp3 on the displacement of histones and on histone H3 acetylation at SSA3 and SSA4. We first tested whether Gcn5 and Elp3 could affect the displacement of histones. ChIP analysis of the histone H3 levels in gcn5Δ (JSY142), elp3Δ (JSY131) and gcn5Δelp3Δ (JSY144) yeast indicated that Gcn5 and Elp3 had a more severe effect on SSA3 than on SSA4 (Figure 3A). The nucleosome density in the coding region of SSA3 decreased to 50% on heat-shock induction in gcn5Δelp3Δ cells, whereas for SSA4, the loss of nucleosomes occurred to a similar extent both in wild-type and mutant yeast strains (Figure 3A). We then examined the level of acetylation of histone H3 at SSA3 and SSA4 in response to heat induction. The elp3Δgcn5Δ strain was shown to be deficient in the ability to acetylate histone H3 associated with the promoter and coding regions of the two genes (Figure 3B). This result implied that Gcn5 and Elp3 regulated SSA3 and SSA4 expression by acetylating histone H3 associated with these genes.
RNAPII is required for the recruitment of Elp3
To address whether the recruitment of Gcn5 and Elp3 to SSA3 and SSA4 was transcription-dependent, we used a yeast strain expressing the temperature-sensitive allele of the largest RNAPII subunit, Rpb1 (rpb1-1; Z4). Under the restrictive temperature, the rpb1-1 expressing strain rapidly ceases transcription due to the loss of RNAPII polymerization activity , as well as loss of recruitment of the polymerase to promoter regions . The rpb1-1 strain and its wild-type counterpart were induced at 25 °C and 37 °C respectively, and the amount of Elp3 and Gcn5 recruitment was monitored by ChIP. As shown in Figure 4(B), when wild-type yeast cells were heat-shocked, the binding of Elp3 to SSA3 and SSA4 increased, whereas in rpb1-1 cells, recruitment of Elp3 was decreased at 37 °C. The recruitment of Elp3 was apparently dependent on active RNAPII transcription. However, Gcn5 recruitment to SSA3 and SSA4 was RNAPII-independent, because similar amounts of Gcn5 were found to be present in SSA3 and SSA4 promoters in both wild-type and rpb1-1 yeast strains grown at 37 °C (Figure 4A). These results suggested that although the two HATs had overlapping roles in transcription regulation, they might exert their functions via distinct mechanisms.
Gcn5 affected the recruitment of RNAPII and Elp3 affected RNAPII-mediated transcription elongation
We then intended to investigate the mechanisms by which Gcn5- and Elp3-catalysed histone acetylation affects the RNAPII-based transcription of SSA3 and SSA4. As members of the HSP70 family, SSA3 and SSA4 genes may be expressed both at a low (basal) level under normal growth conditions, and at a high (induced) level after heat shock. Under non-heat shock conditions, the promoter sequences of hsp70 genes are occupied by transcription factors, such as GAF (GAGA factor), TBP (TATA-box-binding protein) and RNAPII. On heat-shock treatment, the inducible expression of the hsp70 genes is mediated by the interaction of HSFs with HSEs (heat-shock elements). To determine whether Gcn5 and Elp3 influenced the recruitment of RNAPII, ChIP assays were performed using an antibody against RNAPII. As shown in Figure 5(A), induction of SSA3 and SSA4 in the elp3Δ yeast strain resulted in nearly the same levels of RNAPII recruitment to the promoter regions of the genes as demonstrated for the wild-type strain; this implied that Elp3 did not affect RNAPII recruitment. However, the amount of RNAPII associated with the promoter regions of SSA3 and SSA4 in gcn5Δ and elp3Δgcn5Δ yeast strains was decreased, and this was more pronounced in elp3Δgcn5Δ cells, where RNAPII was decreased to 20–30% (Figure 5A). This result indicated that the recruitment of RNAPII required Gcn5.
To further determine whether Gcn5 and Elp3 influenced the RNAPII-mediated transcription elongation along the hsp70 genes, we measured the density of RNAPII at the promoters, ORFs (open reading frames) and 3′ UTRs (untranslated region) of hsp70 genes by using ChIP assays with an antibody against RNAPII. Previous work has reported that a significant proportion of RNAPII molecules initiating transcription never progress to the end of the gene . This experiment was based on the assumption that if the HATs are involved in transcriptional elongation, the density of RNAPII would be expected to be lower (in relative terms) at the 3′ end of the gene than at the promoter of the gene after depletion of Gcn5 and Elp3. Remarkably, we found that RNAPII density was progressively decreased across the SSA3 and SSA4 genes in elp3Δ and elp3Δgcn5Δ cells (Figure 5B), with an RNAPII density similar to untreated cells observed at the promoter, but less than 50–60% density in elp3Δ cells and 10–20% density in elp3Δgcn5Δ cells was observed at the 3′ end of the genes (Figure 5B). Moreover, when the hsp70 genes were induced by heat-shock, this decrease in RNAPII density was more striking (Figure 5C). However, the RNAPII density did not change noticeably from the promoter regions to the 3′UTR of SSA3 and SSA4 in gcn5Δ cells (Figures 5B and 5C). The double mutant had a more severe effect on RNAPII transcription (Figures 5B and 5C). We propose that Gcn5 affects the recruitment of RNAPII, whereas Elp3 influences RNAPII elongation along the genes.
Gcn5 is Swi/Snf-dependent and is required for HSF recruitment
Histone acetylation has been shown to facilitate HSF-induced transcription of an in vitro-reconstituted heat-shock gene . HSF-mediated transactivation of target genes in response to stress is thought to occur principally through the SAGA pathway . We were interested in determining whether Gcn5 affected the binding of HSF. ChIP assays were performed in elp3Δ, gcn5Δ and elp3Δgcn5Δ cells, using an antibody against HSF. The results showed that the recruitment of HSF to gene promoters in gcn5Δ and elp3Δgcn5Δ yeast were decreased, and recruitment was unchanged in elp3Δ cells (Figure 6A). This implied that Gcn5, but not Elp3, affected the binding of HSF.
We next sought to test the contribution of the chromatin remodelling complex Swi/Snf in SSA3 and SSA4 expression, because of its critical roles in the transcriptional activation of inducible yeast genes such as HO, SUC2, INO1 and PHO8 , as well as its known roles in transcriptional regulation, particularly in elongation, of the mammalian hsp70 genes [32,33]. As shown in Figure 6(B), heat-shock-induced SSA3 and SSA4 expression was reduced by 2-fold in cells which were either SNF2 or SNF5 null strains. This required further investigation into whether the Swi/Snf complex interacted with Gcn5 and/or Elp3. As shown in Figures 6(C) and 6(D), the amount of Elp3 associated with SSA3 and SSA4 did not change, whereas the amount of Gcn5 was sharply reduced in snf2Δ and snf5Δ cells. This indicated that the Swi/Snf complex had significant effects on the recruitment of Gcn5, but not on Elp3. These results suggested that the recruitment of Gcn5 and its roles in hsp70 transcription was dependent on the presence of the Swi/Snf complex.
The information arising from this study provides a further insight into understanding the connection between histone acetylation and hsp70 gene transcription regulation. First, histone acetylation was required for both SSA3 and SSA4 transcription, regardless of the occurrence of nucleosome disassembly. Secondly, the HATs Gcn5 and Elp3 modulated SSA3 and SSA4 gene transcription through altering histone H3 acetylation. Although both HATs studied were able to acetylate histone H3, they regulated hsp70 gene transcription through different mechanisms. The interaction of Gcn5 was Swi/Snf-dependent and was required for HSF recruitment and affected RNAPII recruitment, whereas Elp3 exerted its roles mainly through affecting RNAPII elongation.
Nucleosome displacement and transcription-associated histone acetylation
In an attempt to understand the potential diversity in chromatin-based mechanisms of transcription regulation, we investigated transcription-dependent nucleosome density and acetylation level in the SSA3 and SSA4 genes. Our ChIP results indicated that histones lost contact with DNA in both the promoter and coding regions of the SSA4 gene (Figure 1A, right-hand panel). Interestingly, an apparent contrast between SSA4 and SSA3 seemed to exist, as in the former gene, histones lost contact with DNA, whereas in the latter, no net loss of histone–DNA contact occurred (Figure 1A, left-hand panel). These results suggested the existence of distinct mechanisms for enabling transcription through the SSA3 and SSA4 coding regions. However, our results showed that histone H3 associated with both SSA3 and SSA4 was acetylated in a transcription-dependent manner (Figure 1B). This implied that histone acetylation was important for transcription regardless of whether nucleosome disassembly occurred. Our ChIP results for the SSA4 gene were in accordance with previously published work , where the loss of nucleosomes also occurred to a similar extent in both the wild-type and the mutant yeast strains (Figure 3A, right-hand panel). This indicated that loss of the histone–DNA contacts detected in this region may have occurred in an acetylation-independent manner. However, the result from the ChIP assays for SSA3 was surprisingly. The amount of histone H3 associated with SSA3 in elp3Δgcn5Δ cells was decreased to 50% of that detected in wild-type cells (Figure 3A, left-hand panel). This indicated that either nucleosome eviction or histone acetylation was required for transcription through chromatin. Previously published results suggested that Gcn5 had no effect on nucleosome loss . Presumably, the absence of a detectable role for Gcn5 may be the result of its functional redundancy with other proteins (such as Elp3). The evidence presented here suggested that RNAPII transcription along SSA3 might proceed through two distinct mechanisms: (i) a histone acetylation-dependent mechanism, little associated with the net loss of nucleosomes, and (ii) a pathway based on loss of the histone–DNA contact. Furthermore, we demonstrated that mutation of Lys8 (K8R) in histone H4 also influenced SSA3 and SSA4 expression (Figure 1C). The levels of SSA3 and SSA4 transcription in histone H3 K14R and histone H4 K8R mutant strains were similar because there was functional redundancy between different lysine residues, and so individual lysine residues may not be essential. However, the results in Figure 1(C) support the theory that the two lysine residues tested in this experiment, histone H3 Lys14 and histone H4 Lys8, are important because when these sites are mutated to arginine residues, the transcription of both SSA3 and SSA4 is significantly reduced as a result of heat-shock induction compared with the wild-type control. It should be noted that although we have focused on histone H3, there is evidence that mutations in Gcn5 and Elp3 also affected the acetylation of histone H4 [9,11,34], and this might contribute to some of the effects described in this present study.
Functional redundancy of Gcn5 and Elp3: similarities and differences
Experiments that determined the acetylation level of histone H3 in cells lacking the transcription-related HATs Gcn5 and/or Elp3 demonstrated the functional redundancy of these two HATs in the regulation of hsp70 gene expression. Histone acetylation at the promoter and coding regions of hsp70 genes was reduced by deletion of Elp3 or Gcn5, and the acetylation in elp3Δgcn5Δ double mutant strain was much lower than that observed in the individual single mutant yeast strains (Figure 3B). Moreover, the elp3Δgcn5Δ double mutant had a more severe effect on hsp70 expression, suggesting that Gcn5 and Elp3 were at least partly functionally redundant in hsp70 transcription regulation, as the absence of one of the proteins was compensated for by the activity of the other protein. This demonstration of the functional redundancy of the two HATs is consistent with previously published work [8,9].
In this present study, it has been implied that Gcn5 and Elp3 might play distinct roles in SSA3 and SSA4 regulation. Our studies with the elp3Δ and gcn5Δ deletion mutants revealed that Gcn5 was the major HAT responsible for promoter acetylation, and Elp3 was the major HAT responsible for coding region acetylation (Figures 2C and 3B). Specifically, our results support the view that the Elp3-containing Elongator complex may function via its interaction with the elongating RNAPII, as proposed by Wittschieben et al. . This is in apparent contradiction to the model suggested by Pokholok et al. , which described a cytoplasm-based function for the Elongator complex. However, we showed that Gcn5 exerted its role via affecting the recruitment of RNAPII, and probably other transacting factors, in the promoter regions of SSA3 and SSA4 (Figures 5 and 6).
Relationship between Swi/Snf, Gcn5 and HSF
The abundance of Swi/Snf complex is positively correlated with that of HSF, affects the rate of RNAPII elongation at HSP82 , and mediates the release of paused RNAPII within the 5′ UTR of the hsp70 gene . These reports prompted us to investigate the roles of Swi/Snf complex in regulating SSA3 and SSA4. Our results showed that Swi/Snf was required for the recruitment of Gcn5 to SSA3 and SSA4 (Figure 6D). However, other studies have suggested that Gcn5 and Swi/Snf were recruited by HSF to wild-type HSP82, but were not recruited to the 2 bp HSE1 mutant hsp82-P2 . In this present study, we showed that Gcn5 was important for HSF binding to the promoter regions of SSA3 and SSA4 (Figure 6A). Based on these results, we propose a model in which a certain degree of chromatin acetylation is necessary for the recruitment of HSF to the promoter region of a gene, and this in turn triggers the recruitment of chromatin remodelling factors, such as Swi/Snf, to the chromatin to facilitate the association of co-activators to the promoter.
We thank Dr Jesper Q. Svejstrup (Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire, U.K.), Dr Richard Young (Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA, U.S.A.) and Dr Jinqiu Zhou (Institute of Cell Biology, Chinese Academy of Sciences, Shanghai, China) for their gifts of yeast strains. We also thank Dr Jesper Q. Svejstrup, Dr Gross (Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, SD, U.S.A.) and Dr Alain Verreault (Institute for Research in Immunology and Cancer, University of Montreal, Montreal, Canada) for their gifts of antibodies. This work was supported by the grants from the National Natural Science Foundation of China (30370316), the National Basic Research Program of China (2005CB522404, 2006CB910506), and the Program for Changjiang Scholars and Innovative Research Team (PCSIRT) in Universities (IRT0519).
Abbreviations: Ac-H3, acetyl-histone H3; ChIP, chromatin immunoprecipitation; Elp, elongation protein; Gcn, general control non-derepressible; HAT, histone acetyltransferase; HSE, heat-shock element; HSF, heat-shock factor; HSP, heat-shock protein; ORF, open reading frame; RNAP, RNA polymerase; RT–PCR, reverse transcription–PCR; SAGA, Spt-Ada-Gcn5-acetyltransferase; SC, synthetic complete; UTR, untranslated region; YPD, yeast extract, peptone, dextrose
- © The Authors Journal compilation © 2008 Biochemical Society