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Previous Article | Next Article 
Molecular and Cellular Biology, November 2003, p. 8323-8333, Vol. 23, No. 22
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.22.8323-8333.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
Received 20 June 2003/ Returned for modification 7 August 2003/ Accepted 11 August 2003
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ABSTRACT |
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INTRODUCTION |
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In Saccharomyces cerevisiae cells, the general initiation factors localize specifically to promoter regions (22-25, 37). Association of these initiation factors is mutually interdependent and is strongly correlated with transcriptional activity (23, 25). Thus, stable association of the preinitiation complex with promoters is the major, although not exclusive, limiting step for transcription in yeast cells. Unlike the initiation factors, putative elongation factors such as TFIIS, Spt4/5, Spt6, TREX, and the Paf complex associate with coding region of active genes (18, 37, 48). Kinetic analysis involving regulated expression of a long gene indicates that the Hpr1 and Tho2 subunits of the TREX complex travel with Pol II through the coding region (48). As TFIIS, Spt4/5, Spt6, and the Paf complex associate with Pol II (18, 45, 46, 51), Pol II-associated factors are exchanged during the transition between initiation and elongation.
The mammalian FACT complex was identified as a biochemical activity that acts subsequent to initiation to facilitate elongation on chromatin templates (34). Mammalian FACT is composed of two proteins that are homologous to yeast Spt16(Cdc68) and Pob3 (36). FACT specifically interacts with nucleosomes and H2A/H2B dimers, and FACT activity in vitro is blocked by cross-linking nucleosomal histones, suggesting that FACT might promote nucleosome disassembly upon transcription (36). In addition, FACT can affect elongation on naked DNA templates by acting in concert with P-TEFb CTD kinase to counteract the negative elongation activities of DSIF and NELF in vitro (50).
Yeast Spt16(Cdc68) was identified by its effects on transcription and cell cycle control (30, 41), whereas Pob3 was identified by copurification with DNA polymerase I (52). Spt16 and Pob3 form a heterodimer (4, 53) that interacts with the high-mobility group protein Nhp6 (3, 10), and both FACT subunits are essential for viability. Yeast FACT interacts with TFIIE (15) and with Sas3, the catalytic subunit of the NuA3 histone acetylase complex (14). Strains with spt16 or pob3 mutations show increased or decreased expression of various genes (3, 9, 28, 30, 41, 42), although the bases for these effects are unknown. Spt16 genetically interacts with TFIIS, Spt4/5, Spt6, and the Paf complex (7, 11, 27, 34, 47), proteins believed to be involved in elongation. Taken together, the results of these experiments indicate that FACT is an elongation factor whose activity is particularly relevant in the context of nucleosomal templates.
In accord with the idea that FACT is an elongation factor, we show that yeast FACT travels with elongating Pol II at transcriptionally active genes in vivo. Unexpectedly, FACT-deficient cells show decreased levels of preinitiation complexes at promoters and increased Pol II density and transcription in the 3' portion of certain coding regions. Thus, in addition to associating with elongating Pol II, FACT contributes to transcriptional initiation at normal promoters and suppresses initiation within coding regions. We suggest that FACT contributes to transcriptional fidelity by linking the processes of initiation and elongation.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chromatin immunoprecipitation. Chromatin immunoprecipitation in yeast cells was performed essentially as described previously (23, 48) using antibodies against the nonphosphorylated CTD of Pol II (8WG16; Covance), Rpb3 (NeoClone Biotechnology), HA epitope (12CA5), Myc epitope (06-549; Upstate Biotechnology), TATA-binding protein (TBP), TFIIB, Tfb3 (kindly provided by Michael Keogh and Steve Buratowski), Spt16, and Pob3 (the latter two were kindly provided by Tim Formosa). Comparable results for Pol II occupancy were obtained with the 8WG16, Rpb3, and 12CA5 (for strains containing HA-Rpb3) antibodies; presumably, this is due to the presence of nonphosphorylated heptad repeats even when the CTD is phosphorylated during the elongation process. For all experiments involving the YLR454 coding region, PCRs contained multiple primer pairs such that relative occupancy levels for the different regions were directly determined in the same reaction. Quantitated data are presented in arbitrary units that are directly related to the apparent immunoprecipitation efficiency (i.e., the amount of material immunoprecipitated relative to that of the input sample), as determined from PhosphorImager analysis.
Transcriptional analysis. Total RNA was prepared by the hot phenol method (13), followed by treatment with DNase I (Promega) and removal of DNase I by phenol extraction. To determine RNA levels, RNA was randomly (for MDN1 and FLO8) or gene specifically (for YLR454) primed to generate cDNA that was subjected to PCR amplification and detection by ethidium bromide staining. For Northern blotting analysis, poly(A)-containing RNA was enriched from total RNA by using an Oligotex kit (Qiagen), electrophoretically separated on a 1% agarose gel containing formaldehyde, and transferred to a nylon membrane. RNA was detected by hybridization with random-primed probes comprising the 5'-most or 3'-most 800 bp of the YLR454 coding region.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FACT travels with elongating Pol II throughout the mRNA coding region. To examine whether FACT travels with elongating Pol II, we developed an approach that kinetically monitors association during the first or last "waves" of transcription under conditions where gene expression is rapidly induced or repressed. This approach relies on an allele in which the GAL1 promoter drives expression of the long (8 kb) YLR454 open reading frame at its natural locus (Fig. 2A) and has been used for analysis of the Hpr1 and Tho2 components of the TREX complex (48). To visualize the first wave of transcription, we analyzed FACT and Pol II occupancy at 2-min intervals after galactose addition (Fig. 2B). Pol II began to accumulate at the promoter by 4 min, whereas accumulation at the 3' end began roughly 10 min after induction. The 6-min difference in Pol II occupancy at the 5' and 3' ends of this 8-kb gene indicates that the Pol II elongation rate is approximately 1.3 kb/min, in accord with previous results determined by other methods (13, 33). FACT association is similar to Pol II association, indicating that FACT enters the gene at or near the promoter and proceeds to the 3' end of the gene during the first round of transcription.
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FACT association appears to occur just downstream of the preinitiation complex. To address when FACT associates with the transcription complex, we compared its promoter-proximal distribution to Pol II and other transcription factors at the GAL1-YLR454 locus (Fig. 3A). For Rpb3, Spt16, and Hpr1 (a component of the TREX complex), we arbitrarily defined occupancy at a downstream location (region D) as 1.0 and directly measured the relative occupancy at three overlapping locations near the promoter (region A) and immediate 5' portion of the coding sequence (regions B and C).
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TFIIH is required for FACT association in vivo. In yeast cells, TFIIH is localized at the promoter (37), where it plays a critical role in the transition between transcriptional initiation and elongation. The Kin28 subunit of TFIIH is required for phosphorylation of the Pol II CTD at serine 5 in the vicinity of the promoter (17, 44), although Kin28-mediated CTD phosphorylation per se is not essential for transcriptional activity (16, 17, 31). Thermal inactivation of Kin28 does not affect TBP association at promoters (23), although it significantly reduces association of Pol II (44).
To address the mechanism of FACT recruitment, we examined Spt16 association in kin28-ts16 cells shifted to the restrictive temperature (Fig. 4). Under these conditions, TFIIH (assayed with antibodies against the Tfb3 subunit) association with the PYK1 and TDH3 promoters is drastically reduced. Interestingly, inactivation of Kin28 significantly reduces Spt16 and Pob3 association at the promoter and mRNA coding region in a manner similar to that of Pol II. In contrast, loss of Kin28 function does not affect the association of TBP at the promoter. Thus, TFIIH is required for FACT association in vivo, probably as a consequence of a functional connection (direct or indirect) between FACT and Pol II.
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As FACT is essential for cell viability, we examined two conditional Spt16 alleles to assess the role of FACT in TBP occupancy in vivo. First, we generated a copper-inducible, double-shutoff allele (32) in which SPT16 transcription was repressed and Spt16 protein was rapidly degraded upon addition of copper (Fig. 5A). Second, we analyzed TBP occupancy in a strain containing spt16-197, a temperature-sensitive allele (30), that was shifted to the restrictive temperature (Fig. 5B). When Spt16 was inactivated by either of these two approaches, TBP occupancy at all three promoters tested was approximately threefold lower in the strain containing the conditional spt16 allele than in the isogenic strain containing the wild-type SPT16 allele. In addition, occupancy of TFIIB at these promoters was reduced to a comparable extent upon inactivation of Spt16. Thus, these results indicate that Spt16 contributes to TBP and TFIIB occupancy, and hence the level of preinitiation complexes, at normal promoters.
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Loss of FACT results in increased Pol II occupancy within the 3' portions of certain mRNA coding regions. Given that FACT travels with elongating Pol II, we examined Pol II density in the conditional spt16 mutant strains. In accord with the decreased occupancy of TBP at promoters, copper-inducible depletion (Fig. 6A) or thermal inactivation (Fig. 6B) of Spt16 resulted in reduced Pol II density at both the proximal and distal regions of a variety of genes such as TDH3, RPS5, PYK1, and FBA1.
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FACT inhibits inappropriate transcriptional initiation from cryptic promoters within mRNA coding regions. There are two possible explanations for how inactivation of FACT causes increased Pol II density at the 3' portions of certain coding regions. One explanation is that loss of Spt16 affects the release of Pol II from the distal end of the affected genes to generate a "clogged pipe" in which Pol II molecules pile up behind the nonreleased Pol II molecule. We do not favor this explanation because increased Pol II density at the 3' end of YLR454 is observed even when the GAL1 promoter driving the gene is nearly inactive, and because it is difficult to account for gene-specific effects. In addition, increased Pol II density occurred over at least a 4-kb region at the 3' end of the YLR454 gene (Fig. 6A; see below), a result that differs from the relatively localized increase in Pol II distribution under conditions when Pol II elongation is arrested at a particular location in vivo (20). Alternatively, loss of Spt16 might result in inappropriate initiation at one or more sites within the mRNA coding region. This internal initiation model explains why increased Pol II density can occur over a relatively large region, and it can easily account for gene-specific effects.
Three additional lines of evidence indicate that loss of Spt16 results in internal initiation within the normal mRNA coding region. First, for all three genes tested, inactivation of Spt16 caused increased RNA levels at the 3' portions and decreased RNA levels at the 5' portions (Fig. 7A). This observation is consistent with the internal initiation model but is not easily explained by the clogged-pipe model.
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Third, the sequence TATAAAAT, a functionally optimal TATA element (54), lies within an AT-rich region (68% over 200 bp) just upstream of this 5.3-kb transcript. TBP occupancy (defined as the ratio in spt16-197 versus wild-type strains) in the vicinity of the TATAAAAT sequence was 1.4-fold higher than at other locations within the YLR454 coding region (Fig. 7C). Although this effect is quantitatively modest, it is significant (P = 0.0008 in four independent experiments in which relative TBP occupancies at the YLR454 regions were directly determined in an internally controlled manner). In addition, Pol II occupancy in the TATAAAAT region was about 1.8-fold higher than at the 5' portion of the YLR454 coding region. The presence of TBP in the vicinity of an optimal TATA element just upstream of the novel 5.3-kb transcript demonstrates that loss of Spt16 results in internal initiation with the YLR454 coding region. Thus, FACT inhibits inappropriate initiation from cryptic promoters within mRNA coding regions.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FACT was characterized biochemically as a factor that acts subsequent to transcriptional initiation (34), and we show here that FACT travels with elongating Pol II in vivo. Unexpectedly, analysis of Spt16-defective cells indicated that FACT is also important for preventing internal initiation from cryptic promoters, and hence for fidelity of transcriptional initiation in vivo. First, Pol II density increased to abnormally high levels over extended 3' portions of some, but not all, genes. Second, RNA levels increased at the 3' portions and decreased at the 5' portions of all three affected genes tested. Third, in the case of YLR454, we observed a novel and discrete-sized poly(A)-containing transcript as well as an optimal TATA element within an AT-rich region in the immediate vicinity of the presumed 5' end of the novel transcript. Fourth, TBP occupancy increased in the vicinity of the optimal TATA element. These four lines of evidence convincingly demonstrate that initiation from cryptic Pol II promoters within mRNA coding regions occurs when FACT is inactivated; there is no other plausible interpretation that is consistent with all the data. Internal initiation within other genes has been observed in spt16 mutant strains (Craig Kaplan and Fred Winston, personal communication).
In addition to increased initiation within certain mRNA coding regions, loss of Spt16 activity resulted in reduced association of two essential components of the preinitiation complex, TBP and TFIIB, at all normal promoters tested. Furthermore, the level of de novo TBP recruitment to the GRE2 promoter upon osmotic stress was comparably reduced in FACT-deficient cells. The decreased TBP and TFIIB occupancy at normal promoters reflects a specific function of FACT and does not arise indirectly from a defect on transcription or Pol II occupancy in the vicinity of the promoter. Decreased association of preinitiation complex components was observed with two different kinds of conditional Spt16 alleles under inactivation conditions identical to those used to analyze the direct functions of many other essential transcription factors. More importantly, decreased TBP occupancy was not observed in Kin28-deficient cells, a condition in which Pol II transcription is essentially abolished (12) and Pol II occupancy in the vicinity of the promoter is significantly reduced (44) (Fig. 4). Thus, FACT is important for wild-type levels of preinitiation complexes and presumably for transcriptional initiation at natural promoters.
Our results indicate that FACT has an important role in the fidelity of transcriptional initiation. We suspect that increased internal initiation and decreased preinitiation complexes and transcription at normal promoters in FACT-deficient cells represent two sides of the same molecular phenomenon. Specifically, we suggest that the number of functional preinitiation complexes is limiting in vivo, such that increased internal initiation will be directly linked to decreased initiation at normal promoters due to competition for preinitiation complexes. mRNA coding regions are approximately five times larger than promoters (1,500 versus 300 bp), and the vast majority of yeast genes are poorly expressed (12, 13) and presumably have weak promoters. If internal initiation in FACT-deficient cells occurs at 20% of the level observed at most yeast promoters, the number of Pol II initiation events would effectively double. This estimate is plausible (and perhaps even low) given that internal initiation was observed at a significant proportion of the genes tested and often occurs at a level higher than that observed at normal promoters (as defined by Pol II density and RNA levels). In this view, FACT does not directly contribute to transcriptional initiation per se but rather affects transcriptional fidelity and hence the distribution of preinitiation complexes throughout the genome.
The model that FACT contributes to the fidelity, but not the mechanism, of transcriptional initiation is consistent with the results of biochemical experiments indicating a role after preinitiation complex formation (34) and the suggestion that FACT associates with active genes just downstream from the promoter (Fig. 3) (John Lis and Danny Reinberg, personal communication). In addition, this model explains the apparent contradiction that TBP occupancy decreases upon inactivation of FACT, but not Kin28, even though FACT association with active genes is drastically reduced in Kin28-deficient cells. In the absence of Kin28, transcription is blocked at an early step; hence, if FACT is important at a later step in the transcription process, its association with the gene is irrelevant under Kin28-deficient conditions. In contrast, inactivation of FACT does not disrupt preinitiation complexes but rather redistributes them towards mRNA coding regions and away from normal promoters.
FACT contributes to transcriptional fidelity by linking initiation and elongation. As FACT specifically associates with active Pol II genes, we favor the idea that inhibition of internal initiation is linked to the function of FACT during the elongation process. Together with the biochemical (34, 36) and genetic (11) interactions of FACT with chromatin, we suggest a model in which FACT-containing Pol II elongation complexes generate or maintain a chromatin structure that inhibits preinitiation complex formation or stability within mRNA coding regions. In the absence of FACT, elongating Pol II complexes disrupt chromatin structure, thereby making mRNA coding regions more permissive for initiation. As exemplified by the novel YLR454 transcript, internal initiation sites would depend on the presence and relative location of otherwise cryptic TATA elements, initiator elements, and activator binding sites (and perhaps other sequence-dependent features) within the coding region. The likelihood of such cryptic promoter elements should increase in accord with the length of the coding region, and indeed, the internal transcripts observed here were generally in large mRNA coding regions.
It is important to note that increased internal initiation in spt16 mutant strains occurs at the expense of initiation at promoters, and it can occur at poorly expressed genes. We therefore suggest that, in the absence of FACT, the disruption of chromatin structure by elongating Pol II persists long enough to permit preinitiation complex formation and initiation within mRNA coding regions. Based on measurements of initiation frequency in vivo (13), we estimate that weak promoters initiate transcription once every 5 to 30 min. In addition, the GAL1 promoter used here is not completely inactive in glucose medium, because strains containing GAL1 promoter fusions to essential genes are often viable in glucose medium. Nevertheless, although we favor the view that FACT-dependent suppression of internal initiation is related to FACT's function during Pol II transcription, we cannot eliminate the possibility that it reflects a genome-wide, nontranscriptional function of FACT.
It is inevitable that the large and complex Pol II elongation machinery will disrupt nucleosome-DNA interactions as it travels across the gene. In principle, DNA in nucleosomes disrupted by elongating Pol II might be relatively accessible to nuclear proteins; hence, transcriptional elongation might undermine transcriptional fidelity. We suggest that FACT is important in reversing the nucleosome disruption that occurs upon elongation by Pol II, thereby preventing inappropriate access of transcription factors to mRNA coding regions. In this sense, elongation-related functions of FACT are linked to subsequent initiation events. If the number of functional preinitiation complexes is limiting yeast cells, an increase in internal initiation would concomitantly lead to a decrease in initiation at normal promoters by a simple competition model. Thus, by linking the processes of initiation and elongation, FACT increases transcription from the correct initiation site while minimizing initiation from internal sites, thereby improving the fidelity of Pol II transcription.
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ACKNOWLEDGMENTS |
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This work was supported by an NIH postdoctoral fellowship to P.B.M. and a research grant from the National Institutes of Health to K.S. (GM 30186).
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FOOTNOTES |
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