A Poised Initiation Complex Is Activated by SNF1
2007; Elsevier BV; Volume: 282; Issue: 52 Linguagem: Inglês
10.1074/jbc.m707363200
ISSN1083-351X
AutoresChristine Tachibana, Rhiannon Biddick, G. Lynn Law, Elton T. Young,
Tópico(s)Plant Gene Expression Analysis
ResumoSnf1, the yeast AMP kinase homolog, is essential for derepression of glucose-repressed genes that are activated by Adr1. Although required for Adr1 DNA binding, the precise role of Snf1 is unknown. Deletion of histone deacetylase genes allowed constitutive promoter binding of Adr1 and Cat8, another activator of glucose-repressed genes. In repressed conditions, at the Adr1-and Cat8-dependent ADH2 promoter, partial chromatin remodeling had occurred, and the activators recruited a partial preinitiation complex that included RNA polymerase II. Transcription did not occur, however, unless Snf1 was activated, suggesting a Snf1-dependent event that occurs after RNA polymerase II recruitment. Glucose regulation persisted because shifting to low glucose increased expression. Glucose repression could be completely relieved by combining the three elements of 1) chromatin perturbation by mutation of histone deacetylases, 2) activation of Snf1, and 3) the addition of an Adr1 mutant that by itself confers only weak constitutive activity. Snf1, the yeast AMP kinase homolog, is essential for derepression of glucose-repressed genes that are activated by Adr1. Although required for Adr1 DNA binding, the precise role of Snf1 is unknown. Deletion of histone deacetylase genes allowed constitutive promoter binding of Adr1 and Cat8, another activator of glucose-repressed genes. In repressed conditions, at the Adr1-and Cat8-dependent ADH2 promoter, partial chromatin remodeling had occurred, and the activators recruited a partial preinitiation complex that included RNA polymerase II. Transcription did not occur, however, unless Snf1 was activated, suggesting a Snf1-dependent event that occurs after RNA polymerase II recruitment. Glucose regulation persisted because shifting to low glucose increased expression. Glucose repression could be completely relieved by combining the three elements of 1) chromatin perturbation by mutation of histone deacetylases, 2) activation of Snf1, and 3) the addition of an Adr1 mutant that by itself confers only weak constitutive activity. The general model for eukaryotic transcription activation is that a preinitiation complex forms by ordered recruitment of specific and general transcription factors to a promoter (see reviews in Refs. 1Featherstone M. Curr. Opin. Genet. Dev. 2002; 12: 149-155Crossref PubMed Scopus (71) Google Scholar, 2Cosma M.P. Mol. Cell. 2002; 10: 227-236Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 3Fry C.J. Peterson C.L. Science. 2002; 295: 1847-1848Crossref PubMed Scopus (77) Google Scholar). Early in activation, promoter-specific factors bind, possibly with the aid of general chromatin remodeling factors (4Adkins M.W. Williams S.K. Linger J. Tyler J.K. Mol. Cell Biol. 2007; 27: 6372-6382Crossref PubMed Scopus (71) Google Scholar, 5Cosma M.P. Panizza S. Nasmyth K. Mol. Cell. 2001; 7: 1213-1220Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Bound activators recruit coactivators including histone modifiers and chromatin remodeling complexes such as SAGA 2The abbreviations used are: SAGASpt3-Ada1-Gcn5-acetyltransferase complexSWI/SNFSWI/SNF complexCENcentromereHDAChistone deacetylases Hda1 and Rpd3TFIIBgeneral transcription factor IIBqPCRreal-time quantitative PCRChIPchromatin immunoprecipitationPol IIRNA polymerase IICTDC-terminal domain of Pol II large subunit Rbp1TBPTATA-box binding proteinNuSAnucleosome scanning assayHAhemagglutininSer-5-Pphosphorylated Ser-5 2The abbreviations used are: SAGASpt3-Ada1-Gcn5-acetyltransferase complexSWI/SNFSWI/SNF complexCENcentromereHDAChistone deacetylases Hda1 and Rpd3TFIIBgeneral transcription factor IIBqPCRreal-time quantitative PCRChIPchromatin immunoprecipitationPol IIRNA polymerase IICTDC-terminal domain of Pol II large subunit Rbp1TBPTATA-box binding proteinNuSAnucleosome scanning assayHAhemagglutininSer-5-Pphosphorylated Ser-5 or SWI/SNF. The resulting alterations to chromatin and interactions between bound activator, Mediator complex, and general transcription factors ultimately leads to the recruitment of TBP and RNA Pol II (5Cosma M.P. Panizza S. Nasmyth K. Mol. Cell. 2001; 7: 1213-1220Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 6Bryant G.O. Ptashne M. Mol. Cell. 2003; 11: 1301-1309Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 7Govind C.K. Yoon S. Qiu H. Govind S. Hinnebusch A.G. Mol. Cell Biol. 2005; 25: 5626-5638Crossref PubMed Scopus (90) Google Scholar). Spt3-Ada1-Gcn5-acetyltransferase complex SWI/SNF complex centromere histone deacetylases Hda1 and Rpd3 general transcription factor IIB real-time quantitative PCR chromatin immunoprecipitation RNA polymerase II C-terminal domain of Pol II large subunit Rbp1 TATA-box binding protein nucleosome scanning assay hemagglutinin phosphorylated Ser-5 Spt3-Ada1-Gcn5-acetyltransferase complex SWI/SNF complex centromere histone deacetylases Hda1 and Rpd3 general transcription factor IIB real-time quantitative PCR chromatin immunoprecipitation RNA polymerase II C-terminal domain of Pol II large subunit Rbp1 TATA-box binding protein nucleosome scanning assay hemagglutinin phosphorylated Ser-5 Although the model implies transcription when all components of the preinitiation complex are in place, several cases are known in which Pol II is recruited to a promoter but does not proceed through the open reading frame without an additional signal (8Rougvie A.E. Lis J.T. Cell. 1988; 54: 795-804Abstract Full Text PDF PubMed Scopus (464) Google Scholar, 9Rougvie A.E. Lis J.T. Mol. Cell Biol. 1990; 10: 6041-6045Crossref PubMed Scopus (134) Google Scholar, 10Boehm A.K. Saunders A. Werner J. Lis J.T. Mol. Cell Biol. 2003; 23: 7628-7637Crossref PubMed Scopus (188) Google Scholar). The classic example is the Drosophila hsp70 heat shock gene. In the absence of an activating heat shock and with very little bound heat shock factor, Pol II binds the promoter and initiates transcription, only to pause ∼25 nucleotides from the initiation site. Within seconds after heat shock, additional heat shock factor binds, elongation proceeds through the open reading frame (11O'Brien T. Lis J.T. Mol. Cell Biol. 1993; 13: 3456-3463Crossref PubMed Scopus (131) Google Scholar), and more Pol II is recruited (10Boehm A.K. Saunders A. Werner J. Lis J.T. Mol. Cell Biol. 2003; 23: 7628-7637Crossref PubMed Scopus (188) Google Scholar). This mechanism allows for a rapid transcriptional response to a potentially lethal environmental challenge. Reacting to a change in carbon source does not have the urgency of responding to heat shock, and activation of the glucose-repressed genes does not involve a paused polymerase (see review of glucose repression in Ref. 12Schuller H.J. Curr. Genet. 2003; 43: 139-160Crossref PubMed Scopus (349) Google Scholar). We have, however, detected a polymerase complex bound to a glucose-repressed promoter in a strain deleted for the histone deacetylase (HDAC) genes, HDA1 and RPD3. In HDAC mutants, the Adr1 and Cat8 activators, which normally bind to glucose-repressed promoters only in low glucose conditions, bound constitutively. SNF1, which encodes the yeast AMP kinase homolog that is regulated by environmental stresses, was required for the constitutive activator binding as it is for binding in glucose-derepressing conditions (13Young E.T. Kacherovsky N. Van Riper K. J. Biol. Chem. 2002; 277: 38095-38103Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 14Randez-Gil F. Bojunga N. Proft M. Entian K.D. Mol. Cell Biol. 1997; 17: 2502-2510Crossref PubMed Scopus (105) Google Scholar, 15Charbon G. Breunig K.D. Wattiez R. Vandenhaute J. Noel-Georis I. Mol. Cell Biol. 2004; 24: 4083-4091Crossref PubMed Scopus (40) Google Scholar). At the Adr1-and Cat8-regulated ADH2 promoter, a complex was assembled that contained Pol II and components of SAGA, SWI/SNF, TFIIB, and Mediator. Despite the presence of Pol II, transcription was very low. We used the opportunity of a Pol II complex that appeared to be poised for transcription at a glucose-regulated promoter to determine that there are two subsequent steps in the relief of glucose repression, at least one of which can be triggered by activation of Snf1. Strains and Primers—Strains are in supplemental Table 1. Yeast strains were grown as described in Sherman (16Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2543) Google Scholar). Repressing medium contained 5% glucose; derepressing medium contained 0.05% glucose. Epitope tagging, gene deletion, and marker swapping were according to Knop et al., Cross, and Guldener et al. (17Knop M. Siegers K. Pereira G. Zachariae W. Winsor B. Nasmyth K. Schiebel E. Yeast. 1999; 15: 963-972Crossref PubMed Scopus (812) Google Scholar, 18Cross F.R. Yeast. 1997; 13: 647-653Crossref PubMed Scopus (140) Google Scholar, 19Guldener U. Heck S. Fielder T. Beinhauer J. Hegemann J.H. Nucleic Acids Res. 1996; 24: 2519-2524Crossref PubMed Scopus (1357) Google Scholar) respectively, with the exception of SNF1, which was deleted with EcoRI-cut pST70 (20Thompson-Jaeger S. Francois J. Gaughran J.P. Tatchell K. Genetics. 1991; 129: 697-706Crossref PubMed Google Scholar). A multicopy SPT15 (TBP) plasmid was a gift from Steve Hahn's laboratory. ChIP, β-Galactosidase Activity Assay, and Real-time Quantitative PCR—Chromatin immunoprecipitation and gene-specific PCR with gel electrophoresis were performed as described (21Young E.T. Dombek K.M. Tachibana C. Ideker T. J. Biol. Chem. 2003; 278: 26146-26158Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Antibodies were from Santa Cruz Biotechnology Inc. (9E10 anti-Myc and F7 anti-HA) and Abcam (8WG16 anti-Pol II and Ab5131 anti-CTD phosphoserine 5). For expression analysis, RNA was isolated by hot phenol extraction (22Collart M.A. Oliviero S. Ausubel F.M. Brent R. Kingston R.T. Moode D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene/Wiley Interscience, New York1993: 13.12.1-13.12.5Google Scholar) and converted to cDNA with a SuperScript III kit (Invitrogen) according to the manufacturer's directions. ChIP and cDNA were quantified by real-time quantitative PCR (qPCR) with an MJResearch Chromo4 system, using ABI or Quantace SYBR Master Mix, according to the manufacturers' instructions. Primer sequences are available on request. ChIP data were analyzed using the method of Steger et al. (23Steger D.J. Haswell E.S. Miller A.L. Wente S.R. O'Shea E.K. Science. 2003; 299: 114-116Crossref PubMed Scopus (313) Google Scholar) or Bryant and Ptashne (6Bryant G.O. Ptashne M. Mol. Cell. 2003; 11: 1301-1309Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). β-Galactosidase assays were performed as described in Guarente (24Guarente L. Methods Enzymol. 1983; 101: 181-191Crossref PubMed Scopus (873) Google Scholar). Immunoprecipitations and Western Blots—Anti-HA and anti-Myc antibodies were from Santa Cruz Biotechnology. Anti-Adr1 was from Dombek et al. (25Dombek K.M. Camier S. Young E.T. Mol. Cell Biol. 1993; 13: 4391-4399Crossref PubMed Google Scholar). Immunoprecipitations were carried out as in Strahl-Bolsinger et al. (26Strahl-Bolsinger S. Hecht A. Luo K. Grunstein M. Genes Dev. 1997; 11: 83-93Crossref PubMed Scopus (594) Google Scholar), without DNase I treatment and using 2 μg of monoclonal anti-HA (F-7) or 6 μg of monoclonal anti-Myc (9E10) per mg of lysate. The method of Kushnirov (27Kushnirov V.V. Yeast. 2000; 16: 857-860Crossref PubMed Scopus (669) Google Scholar) was used for non-immunoprecipitated Western blot samples. Western blots were performed with the Odyssey system (Licor), using 1:500–1:1000 diluted polyclonal anti-HA (Y-11) or monoclonal anti-Myc (9E10) as primary antibody. Supercoiling Assay—The–640 to +135 region of the ADH2 gene was cloned into the multiple cloning site of pALTL, a modified version of pALT (28Ducker C.E. Simpson R.T. EMBO J. 2000; 19: 400-409Crossref PubMed Scopus (71) Google Scholar), resulting in pLLTY1. pLLTY1 was digested with EcoRI, and the resulting ∼2.5-kbp DNA fragment (which contained the cloned promoter, TRP1 and ARS1) was ligated and used to transform the different yeast strains to Trp prototrophy. Mid-log phase yeast cells from 50-ml cultures grown in either repressing or derepressing conditions were pelleted and washed once in cold water, and the cell pellet was frozen on dry ice and stored at–70 °C. DNA was isolated from cell pellets using glass beads and phenol as outlined by Hoffman (29Hoffman C. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene/Wiley Interscience, New York1997: 13.11.2-13.11.4Google Scholar). DNA was electrophoresed on a 1% agarose, 1× Tris-borate-EDTA gel. Both gel and running buffer contained 15 μg/ml chloroquine. Gels were run at a constant 30 V for 22–36 h. Standard Southern analysis techniques were used for gel blotting and probing with a P32-labeled 400-bp fragment from pLLTY1. Imaging of blots and the quantitation of band intensities employed a GE Healthcare Storm 840 PhosphorImager and ImageQuant 5.4. Nucleosome Scanning Assay (NuSA)—200 ml of repressed or derepressed cell cultures was processed using the procedures in the yeast culture, micrococcal nuclease digestion, protein degradation, and DNA purification steps as outlined in Liu et al. (30Liu C.L. Kaplan T. Kim M. Buratowski S. Schreiber S.L. Friedman N. Rando O.J. PLoS Biol. 2005; 3: e328Crossref PubMed Scopus (397) Google Scholar). Specifically, repressed cells were incubated with zymolyase for 15 min at 30 °C, whereas derepressed cells were incubated for 45 min. After the RNase A digestion, samples were analyzed on 2% agarose gel to determine the extent of digestion, and only samples that were highly enriched in mononucleosomal DNA were used. The DNA representing the mononucleosomal fraction was isolated using a Qiagen gel extraction kit. DNA samples were diluted 1:300–1:500 and used in qPCR reactions to quantify the presence of a specific amplicon. The protection value set for each amplicon corresponds to the -fold enrichment of that amplicon in the mononucleosomal DNA when compared with the undigested sample and normalized to CEN3 values. qPCR primers were designed to cover the promoter of ADH2 and FBP1 with amplicons averaging 100 bp in size (sequences available upon request). Mutations in HDACs Cause Activator Binding in Non-activating Conditions—In glucose-starved cells, Adr1 and Cat8 contribute to the activation of dozens of glucose-repressed genes (21Young E.T. Dombek K.M. Tachibana C. Ideker T. J. Biol. Chem. 2003; 278: 26146-26158Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 31Haurie V. Perrot M. Mini T. Jeno P. Sagliocco F. Boucherie H. J. Biol. Chem. 2001; 276: 76-85Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Neither factor is detected at target promoters in repressed cells by ChIP, but binding is detected after derepression in low glucose (32Tachibana C. Yoo J.Y. Tagne J.B. Kacherovsky N. Lee T.I. Young E.T. Mol. Cell Biol. 2005; 25: 2138-2146Crossref PubMed Scopus (118) Google Scholar). In strains deleted for HDA1 and RPD3, Adr1 binds to the ADH2 promoter, even in repressing conditions, without activating transcription (see below and see Ref. 33Verdone L. Wu J. van Riper K. Kacherovsky N. Vogelauer M. Young E.T. Grunstein M. Di Mauro E. Caserta M. EMBO J. 2002; 21: 1101-1111Crossref PubMed Scopus (49) Google Scholar). We extended this analysis and found that in the HDAC mutants, both Adr1 and Cat8 bound constitutively to the promoters of several Adr1-and Cat8-regulated genes, specifically ACS1, ADH2, ADY2, ATO3, FBP1, JEN1, ICL1, MDH2, MLS1, and PUT4 (Fig. 1 and supplemental Fig. 1). Increased Activator Levels in HDAC Mutants Do Not Account for Binding—For both Adr1 and Cat8, binding under repressed conditions was unexpected. CAT8 expression is dependent on Snf1 (14Randez-Gil F. Bojunga N. Proft M. Entian K.D. Mol. Cell Biol. 1997; 17: 2502-2510Crossref PubMed Scopus (105) Google Scholar, 34Rahner A. Scholer A. Martens E. Gollwitzer B. Schuller H.J. Nucleic Acids Res. 1996; 24: 2331-2337Crossref PubMed Scopus (72) Google Scholar), which is inactive in vitro when isolated from glucose-grown cells (35Wilson W.A. Hawley S.A. Hardie D.G. Curr. Biol. 1996; 6: 1426-1434Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). Expression of ADR1 is not SNF1-dependent (36Dombek K.M. Young E.T. Mol. Cell Biol. 1997; 17: 1450-1458Crossref PubMed Scopus (37) Google Scholar), but Adr1 binding during derepression requires SNF1 (13Young E.T. Kacherovsky N. Van Riper K. J. Biol. Chem. 2002; 277: 38095-38103Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). HDAC mutations might increase Adr1 and Cat8 protein levels, resulting in binding by simple mass action, so we examined Adr1 and Cat8 levels by Western blot. Adr1 and Cat8 were present in glucose-grown cells, although as expected from previous results (34Rahner A. Scholer A. Martens E. Gollwitzer B. Schuller H.J. Nucleic Acids Res. 1996; 24: 2331-2337Crossref PubMed Scopus (72) Google Scholar, 37Sloan J.S. Dombek K.M. Young E.T. J. Biol. Chem. 1999; 274: 37575-37582Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 38Hedges D. Proft M. Entian K.D. Mol. Cell Biol. 1995; 15: 1915-1922Crossref PubMed Scopus (153) Google Scholar), their levels increased dramatically upon derepression (Fig. 2, note that Cat8 samples were concentrated by immunoprecipitation before blotting). Levels of both factors were elevated in Δhda1Δrpd3 strains (Fig. 2, A and B). Previous work showed that a multicopy ADR1 strain overproduces Adr1 but does not show binding in repressed conditions (13Young E.T. Kacherovsky N. Van Riper K. J. Biol. Chem. 2002; 277: 38095-38103Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Fig. 2C shows that although Adr1 in HDAC mutants was elevated, by comparison with the multicopy strain, these levels alone were not sufficient for binding. Snf1 Is Required for Activator Binding in HDAC Mutants—The Δhda1Δrpd3 mutants appeared to overcome the regulation of Adr1 and Cat8 in two ways: they increased protein levels, and they allowed DNA binding in repressing conditions. Normally, SNF1 is part of Adr1 and Cat8 regulation, so we quantified the DNA binding of Adr1 and Cat8 in the absence of SNF1. In all cases tested, binding in repressed Δhda1Δrpd3 strains was reduced when an additional Δsnf1 mutation was introduced (Fig. 1 and supplemental Fig. 1). The effect of Δsnf1 on Adr1 and Cat8 binding in Δhda1Δrpd3 strains was comparable with the effect on binding in wild-type RPD3 and HDA1; some Snf1-independent binding of Adr1 remained (data not shown and (13Young E.T. Kacherovsky N. Van Riper K. J. Biol. Chem. 2002; 277: 38095-38103Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), whereas Cat8 binding was dramatically diminished, possibly because of its dual dependence on Snf1 for transcription and for post-translational modification. Initiation Complex Components Bind ADH2 in HDAC Mutants—Since activator binding is an early step in the recruitment of coactivators such as SAGA, SWI/SNF, and Mediator, we tested for binding of representative subunits of these complexes in repressed Δhda1Δrpd3 cells using ADH2 as our model. ChIP analysis detected components of SAGA (Gcn5), SWI/SNF (Snf2 and Snf5), TFIIB (Sua7), Mediator (Med15, Med18, and Med14), and Pol II (Rbp1) at the ADH2 promoter in repressed HDAC knock-out strains (Fig. 3A, compare background levels in striped bars with binding in Δhdac in stippled bars). Comparable results for other genes known to be bound by Adr1 and Cat8 (data not shown for ADY2, FBP1, JEN1, and MLS1) suggested that these complexes were also present at other Adr1-and Cat8-dependent promoters. TBP did not appear to be in the complex, consistent with earlier findings (33Verdone L. Wu J. van Riper K. Kacherovsky N. Vogelauer M. Young E.T. Grunstein M. Di Mauro E. Caserta M. EMBO J. 2002; 21: 1101-1111Crossref PubMed Scopus (49) Google Scholar), although binding above background was difficult to detect. Consistent with the TBP results, binding of the TBP-interacting protein Sua7 was also relatively low in repressed HDAC mutants. Deletion of ADR1 or SNF1 reduced Pol II occupancy at ADH2 in HDAC mutants by 50% relative to ADR1 or SNF1 wild-type strains (Fig. 3A, last four bars). Binding of Activators in Repressed HDAC Mutants Does Not Fully Activate Transcription—Despite the binding of activators, coactivators and Pol II, Northern blots had shown little or no ADH2 RNA from repressed Δhda1Δrpd3 cells (33Verdone L. Wu J. van Riper K. Kacherovsky N. Vogelauer M. Young E.T. Grunstein M. Di Mauro E. Caserta M. EMBO J. 2002; 21: 1101-1111Crossref PubMed Scopus (49) Google Scholar, 39Agricola E. Verdone L. Di Mauro E. Caserta M. Mol. Microbiol. 2006; 62: 1433-1446Crossref PubMed Scopus (24) Google Scholar). Quantitation with a sensitive qPCR technique detected some transcript from ADH2 in a repressed HDAC mutant, but expression was less than 1% of wild-type derepressed levels. Expression from other Adr1-and Cat8-dependent promoters was generally less than 10% of wild-type derepressed levels (Fig. 4) with the exception of POT1 (29%) and ICL2 (36%). Fig. 4 shows data for full-length transcripts, but the same results were obtained when one gene (ADY2) was analyzed for production of shorter transcripts using primers to detect the 3′ end, middle, and 5′ end of the RNA (supplemental Table 2). RNA abundance was unchanged regardless of the primers used to detect it, suggesting that Pol II was not pausing or stalling in the middle of the gene. Expression of ADR1 and CAT8 was consistent with the protein levels seen in Fig. 2. Since TBP is a very late addition to the preinitiation complex (6Bryant G.O. Ptashne M. Mol. Cell. 2003; 11: 1301-1309Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) and ChIP assays showed no difference between TBP at the ADH2 promoter in wild-type and Δhda1Δrpd3 strains, we supplied the cells with an excess of TBP from a high copy plasmid with SPT15 (TBP) under ADH1 promoter control. This resulted in high levels of TBP by Western blot 3R. Chang, unpublished. but did not increase transcription from an ADH2-lacZ reporter in a Δhda1Δrpd3 strain (supplemental Fig. 2). To evaluate the state of the polymerase poised at the ADH2 gene, we did ChIP for phosphorylation of serine 5 of the CTD, a modification suggestive of initiation (40Komarnitsky P. Cho E.J. Buratowski S. Genes Dev. 2000; 14: 2452-2460Crossref PubMed Scopus (798) Google Scholar). The paused polymerase at the Drosophila hsp70, hsp26, GAP, TUB, and Actin5C genes is Ser-5-P-modified (10Boehm A.K. Saunders A. Werner J. Lis J.T. Mol. Cell Biol. 2003; 23: 7628-7637Crossref PubMed Scopus (188) Google Scholar). In repressed Δhda1Δrpd3 cells, Ser-5-P was detected at the ADH2 promoter at 17% of derepressed levels (Fig. 3B). Hypophosphorylated Pol II, detected with an antibody against unphosphorylated CTD, was present at 42% of derepressed levels, comparable with the total Pol II seen in Fig. 3A. Since different antibodies have different precipitation efficiencies, we could not compare ChIP values directly, but the poised polymerase appeared to be a mixture in which only a fraction was in the Ser-5-P state, measured relative to derepressed levels. By the same criterion, a greater fraction was hypophosphorylated. As a control, CTD Ser-5-P was examined in repressed and derepressed wild-type cells, at the 5′ end of the housekeeping gene ACT1. Levels decreased in low glucose, consistent with reduced transcription during slower cell growth (Fig. 3B). ADH2 Promoter Chromatin Structure Is Altered in Repressed HDAC Mutants—Transcription of Adr1-dependent genes upon activation in low glucose is associated with an Adr1-dependent chromatin remodeling event at the promoter (41Verdone L. Camilloni G. Di Mauro E. Caserta M. Mol. Cell Biol. 1996; 16: 1978-1988Crossref PubMed Scopus (85) Google Scholar, 42Agricola E. Verdone L. Xella B. Di Mauro E. Caserta M. Biochemistry. 2004; 43: 8878-8884Crossref PubMed Scopus (18) Google Scholar). We used two techniques, supercoiling analysis and NuSA, to determine whether activator binding without low glucose signaling in a Δhda1Δrpd3 strain affected the ADH2 promoter chromatin structure. The supercoiling analysis relies on the fact that the amount of negative supercoiling on an isolated DNA plasmid is proportional to the number of nucleosomes assembled on the DNA in vivo (43Simpson R.T. Thoma F. Brubaker J.M. Cell. 1985; 42: 799-808Abstract Full Text PDF PubMed Scopus (373) Google Scholar). Thus by determining the change in the distribution of topoisomers between samples, one can determine the change in nucleosome density. The–640 to +135 region (relative to the ATG initiation codon) of the ADH2 gene was cloned into a multicopy TRP1/ARS1 yeast plasmid (28Ducker C.E. Simpson R.T. EMBO J. 2000; 19: 400-409Crossref PubMed Scopus (71) Google Scholar). In this plasmid, the cloned promoter, TRP1 and ARS1, can be released by digestion with EcoRI, ligated, and used to transform yeast to Trp prototrophy. The 2.5-kbp episome has the same chromatin architecture as the chromosomal ADH2 promoter, and Adr1-dependent loss of nucleosome density is observed in derepressing conditions. 4G. L. Law, unpublished. When the topoisomer distribution was compared in the wild-type strain between repressed, 2.5-h derepressed, or 5-h derepressed, a downward shift representing loss of one nucleosome occurred between 2.5 and 5 h of derepression (Fig. 5, A and B). This shift in the distribution of topoisomers was not observed when DNA from a Δsnf1 mutant was examined after growth in repressing and derepressing conditions (Fig. 5A, lanes 7 and 8). However, using a Δhda1Δrpd3 strain, the topoisomer distribution seen in the repressed and 2.5-h derepressed samples was similar to the distribution seen at 5 h of derepression in the wild-type strain, indicating that the nucleosome density in the Δhda1Δrpd3 strain in high glucose resembles the wild-type density in low glucose (Fig. 5A, lanes 4 and 5, and B). Upon derepression, there is a greater loss of nucleosomes in the Δhda1Δrpd3 strain (Fig. 5A, lane 6, and B). The second technique used to detect changes in the chromatin structure was a NuSA. NuSA quantifies the micrococcal nuclease sensitivity of a DNA sequence in vivo and was used to detect nucleosome positioning and density at the ADH2 promoter region. The monosomal DNA resulting from micrococcal nuclease digestion was analyzed using an array of tiled amplicons spanning the ADH2 promoter region and qPCR. The NuSA for wild-type repressed cells showed the three previously described nucleosomes: N-2 at the 5′ end of the nucleosome free region, N-1 covering the TATA box, and N+1 covering the translational start site (42Agricola E. Verdone L. Xella B. Di Mauro E. Caserta M. Biochemistry. 2004; 43: 8878-8884Crossref PubMed Scopus (18) Google Scholar) (Fig. 5C). When wild-type cells were shifted to derepressing conditions, two changes in the chromatin were seen. An overall reduction of nucleosome density was represented by a decrease in the amplitude of the nucleosome protection peaks and a small but reproducible 3′ shift in the position of N-1 (Fig. 5C, inset). Nucleosome density in repressed Δhda1Δrpd3 mutants was comparable with derepressed wild type; however, the small shift in N-1 position was not seen (Fig. 5C). This agrees with data from Verdone et al. (33Verdone L. Wu J. van Riper K. Kacherovsky N. Vogelauer M. Young E.T. Grunstein M. Di Mauro E. Caserta M. EMBO J. 2002; 21: 1101-1111Crossref PubMed Scopus (49) Google Scholar), showing increased acetylation and micrococcal nuclease sensitivity of the ADH2 promoter in the HDAC mutants. The FBP1 promoter was also analyzed by NuSA (data not shown), and the same trend was seen. There was an overall decrease in nucleosome density in derepressed chromatin when compared with repressed chromatin, and the nucleosome density in the repressed Δhda1Δrpd3 mutant more closely resembled the wild-type derepressed than the wild-type repressed samples. Snf1 Activation and a Non-phosphorylatable Adr1 Contribute to Activation—The repressed Δhda1Δrpd3Δ strains presented a unique opportunity for investigating late stage steps in activation of glucose-repressed genes. RNA Pol II and associated transcription factors appeared to be poised at ADH2 and other Adr1-and Cat8-dependent promoters. Some chromatin remodeling had occurred, and possibly some initiation, but glucose repression still persisted. We hypothesized that one or more final steps, perhaps involving the kinase activity of Snf1, were required for full derepression. Snf1 is kept inactive by the Reg1.Glc7 phosphatase complex (44Tu J. Carlson M. EMBO J. 1995; 14: 5939-5946Crossref PubMed Scopus (195) Google Scholar, 45Sanz P. Alms G.R. Haystead T.A. Carlson M. Mol. Cell Biol. 2000; 20: 1321-1328Crossref PubMed Scopus (179) Google Scholar), so REG1 was knocked out in Δhda1Δrpd3 and isogenic HDA1 RPD3 strains, and RNA was measured by qPCR. As seen previously (25Dombek K.M. Camier S. Young E.T. Mol. Cell Biol. 1993; 13: 4391-4399Crossref PubMed Google Scholar, 46Neigeborn L. Carlson M. Genetics. 1984; 108: 845-858Crossref PubMed Google Scholar, 47Neigeborn L. Carlson M. Genetics. 1987; 115: 247-253Crossref PubMed Google Scholar), 5J. J. Infante, unpublished. in a Δreg1 strain, the Snf1-dependent SUC2 gene was almost fully activated on glucose (80–115% of wild-type derepressed), and ADH2 was expressed to ∼7% of wild-type derepressed levels (Fig. 6A and data not shown). Low glucose increased ADH2 expression nearly 5-fold, to 33% of wild-type derepressed levels. The generatio
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