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Snf1p-dependent Spt-Ada-Gcn5-acetyltransferase (SAGA) Recruitment and Chromatin Remodeling Activities on the HXT2 and HXT4 Promoters

2005; Elsevier BV; Volume: 281; Issue: 7 Linguagem: Inglês

10.1074/jbc.m509330200

ISSN

1083-351X

Autores

Chris J.C. van Oevelen, Hetty A.A.M. van Teeffelen, Folkert J. van Werven, H. T. Marc Timmers,

Tópico(s)

Peptidase Inhibition and Analysis

Resumo

We previously showed that the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex is recruited to the activated HXT2 and HXT4 genes and plays a role in the association of TBP-associated factors. Using the HXT2 and HXT4 genes, we now present evidence for a functional link between Snf1p-dependent activation, recruitment of the SAGA complex, histone H3 removal, and H3 acetylation. Recruitment of the SAGA complex is dependent on the release of Ssn6p-Tup1p repression by Snf1p. In addition, we found that the Gcn5p subunit of the SAGA complex preferentially acetylates histone H3K18 on the HXT2 and HXT4 promoters and that Gcn5p activity is required for removal of histone H3 from the HXT4 promoter TATA region. In contrast, histone H3 removal from the HXT2 promoter does not require Gcn5p. In conclusion, although similar protein complexes are involved, induction of HXT2 and HXT4 displays important mechanistic differences. We previously showed that the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex is recruited to the activated HXT2 and HXT4 genes and plays a role in the association of TBP-associated factors. Using the HXT2 and HXT4 genes, we now present evidence for a functional link between Snf1p-dependent activation, recruitment of the SAGA complex, histone H3 removal, and H3 acetylation. Recruitment of the SAGA complex is dependent on the release of Ssn6p-Tup1p repression by Snf1p. In addition, we found that the Gcn5p subunit of the SAGA complex preferentially acetylates histone H3K18 on the HXT2 and HXT4 promoters and that Gcn5p activity is required for removal of histone H3 from the HXT4 promoter TATA region. In contrast, histone H3 removal from the HXT2 promoter does not require Gcn5p. In conclusion, although similar protein complexes are involved, induction of HXT2 and HXT4 displays important mechanistic differences. The yeast Saccharomyces cerevisiae utilizes glucose fermentation to generate metabolic energy. To optimize this process, glucose concentrations are carefully monitored, and gene expression is tightly linked to glucose availability. When glucose is present, genes involved in the uptake and fermentation of glucose are actively transcribed. However, glucose can also mediate repressive signals for genes involved in processing non-fermentable carbon sources and genes involved in the uptake and processing of alternative carbon sources. This latter process is known as glucose repression (1.Carlson M. Curr. Opin. Microbiol. 1999; 2: 202-207Crossref PubMed Scopus (458) Google Scholar, 2.Rolland F. Winderickx J. Thevelein J.M. FEMS Yeast Res. 2002; 2: 183-201Crossref PubMed Google Scholar). A central component in the glucose repression pathway is the Tup1p-Ssn6p complex. The Ssn6p-Tup1p complex has no DNA binding activity and depends on sequence-specific factors such as Mig1p for association to its target promoters (3.Treitel M.A. Carlson M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3132-3136Crossref PubMed Scopus (337) Google Scholar, 4.Smith R.L. Johnson A.D. Trends Biochem. Sci. 2000; 25: 325-330Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). The Ssn6p-Tup1p repressor complex genetically interacts with components of the Mediator complex and is associated with histone deacetylation (HDAC) 2The abbreviations used are: HDAC, histone deacetylation; SAGA, Spt-Ada-Gcn5-acetyltransferase; HAT, histone acetyltransferase; HA, hemagglutinin; TBP, TATA box-binding protein; UAS, upstream activating sequence.2The abbreviations used are: HDAC, histone deacetylation; SAGA, Spt-Ada-Gcn5-acetyltransferase; HAT, histone acetyltransferase; HA, hemagglutinin; TBP, TATA box-binding protein; UAS, upstream activating sequence. enzymes (5.Wahi M. Johnson A.D. Genetics. 1995; 140: 79-90Crossref PubMed Google Scholar, 6.Carlson M. Annu. Rev. Cell Dev. Biol. 1997; 13: 1-23Crossref PubMed Scopus (179) Google Scholar, 7.Kuchin S. Carlson M. Mol. Cell. Biol. 1998; 18: 1163-1171Crossref PubMed Scopus (110) Google Scholar, 8.Watson A.D. Edmondson D.G. Bone J.R. Mukai Y. Yu Y. Du W. Stillman D.J. Roth S.Y. Genes Dev. 2000; 14: 2737-2744Crossref PubMed Scopus (129) Google Scholar, 9.Davie J.K. Edmondson D.G. Coco C.B. Dent S.Y. J. Biol. Chem. 2003; 278: 50158-50162Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Moreover, deletion of either SSN6 or TUP1 results in an altered chromatin organization of RNR3, FLO1, SUC2, and a-cell-specific genes (10.Cooper J.P. Roth S.Y. Simpson R.T. Genes Dev. 1994; 8: 1400-1410Crossref PubMed Scopus (166) Google Scholar, 11.Gavin I.M. Simpson R.T. EMBO J. 1997; 16: 6263-6271Crossref PubMed Scopus (72) Google Scholar, 12.Ducker C.E. Simpson R.T. 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To ensure efficient influx of glucose, yeast cells can express different hexose transporter (HXT) genes. The HXT2 and HXT4 genes encode high affinity glucose transporters, whose expression is repressed by high levels of glucose (18.Ozcan S. Johnston M. Mol. Cell. Biol. 1995; 15: 1564-1572Crossref PubMed Google Scholar). Repression of the HXT2 and HXT4 genes is mediated by both subunits of the Ssn6p-Tup1p complex, because deletion of either SSN6 or TUP1 results in promoter activation of these genes under non-inducing conditions (18.Ozcan S. Johnston M. Mol. Cell. Biol. 1995; 15: 1564-1572Crossref PubMed Google Scholar, 19.Ozcan S. Johnston M. Mol. Cell. Biol. 1996; 16: 5536-5545Crossref PubMed Scopus (84) Google Scholar). To release Ssn6p-Tup1p-mediated repression under inducing conditions (i.e. low glucose), the HXT2 and HXT4 genes require Snf1p function (18.Ozcan S. Johnston M. Mol. Cell. Biol. 1995; 15: 1564-1572Crossref PubMed Google Scholar). Snf1p encodes a serine/threonine kinase and is required for the expression of glucose-repressed genes (20.Celenza J.L. Carlson M. Mol. Cell. Biol. 1984; 4: 49-53Crossref PubMed Scopus (98) Google Scholar, 21.Celenza J.L. Carlson M. Science. 1986; 233: 1175-1180Crossref PubMed Scopus (508) Google Scholar). Snf1p kinase phosphorylates Mig1p, which results in a functional disruption of the Mig1p-Ssn6p interaction on repressed genes (22.Treitel M.A. Kuchin S. Carlson M. Mol. Cell. Biol. 1998; 18: 6273-6280Crossref PubMed Scopus (258) Google Scholar, 23.Ozcan S. Johnston M. Microbiol. Mol. Biol. Rev. 1999; 63: 554-569Crossref PubMed Google Scholar, 24.Papamichos-Chronakis M. Gligoris T. Tzamarias D. EMBO Rep. 2004; 5: 368-372Crossref PubMed Scopus (95) Google Scholar). Furthermore, Snf1p has also been reported to phosphorylate serine 10 of histone H3 (H3S10), which is important for SAGA recruitment to the INO1 promoter (25.Lo W.S. Gamache E.R. Henry K.W. Yang D. Pillus L. Berger S.L. EMBO J. 2005; 24: 997-1008Crossref PubMed Scopus (79) Google Scholar). The SAGA complex is a multisubunit complex with distinct transcriptional activities (26.Grant P.A. Sterner D.E. Duggan L.J. Workman J.L. Berger S.L. Trends Cell Biol. 1998; 8: 193-197Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 27.Timmers H.T. Tora L. Trends Biochem. Sci. 2005; 30: 7-10Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). It is composed of different classes of proteins, which were identified in independent genetic screens. The Spt3p and Spt8p proteins regulate TBP activity, whereas the Gcn5p subunit has histone acetyltransferase (HAT) activity (28.Eisenmann D.M. Arndt K.M. Ricupero S.L. Rooney J.W. Winston F. Genes Dev. 1992; 6: 1319-1331Crossref PubMed Scopus (188) Google Scholar, 29.Eisenmann D.M. Chapon C. Roberts S.M. Dollard C. Winston F. Genetics. 1994; 137: 647-657Crossref PubMed Google Scholar, 30.Sterner D.E. Grant P.A. Roberts S.M. Duggan L.J. Belotserkovskaya R. Pacella L.A. Winston F. Workman J.L. Berger S.L. Mol. Cell. Biol. 1999; 19: 86-98Crossref PubMed Scopus (288) Google Scholar, 31.Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Edmondson D.G. Roth S.Y. Allis C.D. Cell. 1996; 84: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1273) Google Scholar, 32.Grant P.A. Duggan L. Cote J. Roberts S.M. Brownell J.E. Candau R. Ohba R. Owen-Hughes T. Allis C.D. Winston F. Berger S.L. Workman J.L. Genes Dev. 1997; 11: 1640-1650Crossref PubMed Scopus (870) Google Scholar). Furthermore, subunits, including Spt7p and Spt20p, comprise the structural backbone of the SAGA complex supporting the Spt3p-Spt8p-TBP and Gcn5p-HAT modules (30.Sterner D.E. Grant P.A. Roberts S.M. Duggan L.J. Belotserkovskaya R. Pacella L.A. Winston F. Workman J.L. Berger S.L. Mol. Cell. Biol. 1999; 19: 86-98Crossref PubMed Scopus (288) Google Scholar, 32.Grant P.A. Duggan L. Cote J. Roberts S.M. Brownell J.E. Candau R. Ohba R. Owen-Hughes T. Allis C.D. Winston F. Berger S.L. Workman J.L. Genes Dev. 1997; 11: 1640-1650Crossref PubMed Scopus (870) Google Scholar, 33.Wu P.Y. Ruhlmann C. Winston F. Schultz P. Mol. Cell. 2004; 15: 199-208Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The SAGA complex is specifically recruited via its Tra1p subunit by activators like Gal4p and Gcn4p (34.Brown C.E. Howe L. Sousa K. Alley S.C. Carrozza M.J. Tan S. Workman J.L. Science. 2001; 292: 2333-2337Crossref PubMed Scopus (282) Google Scholar). SAGA is essential for the Spt3p-dependent TBP recruitment to the TATA box region of the GAL1 promoter (35.Dudley A.M. Rougeulle C. Winston F. Genes Dev. 1999; 13: 2940-2945Crossref PubMed Scopus (171) Google Scholar, 36.Bhaumik S.R. Green M.R. Genes Dev. 2001; 15: 1935-1945Crossref PubMed Scopus (238) Google Scholar, 37.Larschan E. Winston F. Genes Dev. 2001; 15: 1946-1956Crossref PubMed Scopus (243) Google Scholar). In addition, Spt3p and Gcn5p were shown to recruit Mot1p to the GAL1 gene and to play a role in nucleosomal arrangement of the GAL1 promoter after activation (38.Topalidou I. Papamichos-Chronakis M. Thireos G. Tzamarias D. EMBO J. 2004; 23: 1943-1948Crossref PubMed Scopus (29) Google Scholar). However, deletion of GCN5 only has a mild effect on GAL1 transcription (35.Dudley A.M. Rougeulle C. Winston F. Genes Dev. 1999; 13: 2940-2945Crossref PubMed Scopus (171) Google Scholar, 36.Bhaumik S.R. Green M.R. Genes Dev. 2001; 15: 1935-1945Crossref PubMed Scopus (238) Google Scholar). Surprisingly, SAGA recruitment to the GAL1 gene involves Cti6p, a protein that physically bridges Ssn6p to the SAGA complex (39.Papamichos-Chronakis M. Petrakis T. Ktistaki E. Topalidou I. Tzamarias D. Mol. Cell. 2002; 9: 1297-1305Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). We have shown recently that SAGA is specifically recruited to the upstream regions of the HXT2 and HXT4 genes (40.van Oevelen C.J. van Teeffelen H.A. Timmers H.T. Mol. Cell. Biol. 2005; 25: 4863-4872Crossref PubMed Scopus (20) Google Scholar). Although the SAGA complex is required for the recruitment of both Mot1p and the TFIID subunit, Taf1p, to the TATA box regions via different modules, SAGA is only essential for HXT4 expression (40.van Oevelen C.J. van Teeffelen H.A. Timmers H.T. Mol. Cell. Biol. 2005; 25: 4863-4872Crossref PubMed Scopus (20) Google Scholar). Here, we describe a functional link between release of repression by Snf1p, SAGA recruitment, and its consequences for chromatin regulation of the HXT2 and HXT4 promoters. Acetylation of histone H3 depends on the HAT activity of Gcn5p but does not play a role in transcription activation. Together, these experiments show a novel relation between Snf1p-dependent activation, release of Ssn6p-Tup1p repression, SAGA recruitment, and chromatin regulation on the HXT2 and HXT4 genes. Yeast Strains—The S. cerevisiae strains used in this study are listed in Table 1. All procedures were performed according to standard methods (41.Burke D. Dawson D. Stearns T. Cold Spring Harbor LaboratoryMethods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. 2000 Ed. Cold Spring Harbor Laboratory Press, Plainview, NY2000Google Scholar). The HA3-MOT1 (DP107) and HA3-TAF1 (YBY838) strains have been described previously (42.Poon D. Campbell A.M. Bai Y. Weil P.A. J. Biol. Chem. 1994; 269: 23135-23140Abstract Full Text PDF PubMed Google Scholar, 43.Bai Y. Perez G.M. Beechem J.M. Weil P.A. Mol. Cell. Biol. 1997; 17: 3081-3093Crossref PubMed Scopus (61) Google Scholar). Single deletion strains were constructed as described (44.Reid R.J. Lisby M. Rothstein R. Methods Enzymol. 2002; 350: 258-277Crossref PubMed Scopus (84) Google Scholar). However, the gcn5Δ strain was constructed using a GCN5 disruption cassette kindly provided by Dr. G. Thireos (45.Syntichaki P. Topalidou I. Thireos G. Nature. 2000; 404: 414-417Crossref PubMed Scopus (167) Google Scholar). All deletions were verified by PCR analysis with primers corresponding to both wild-type and disruption alleles. The TUP1-HA3 and SPT20-TAP alleles have been described previously (16.Davie J.K. Trumbly R.J. Dent S.Y. Mol. Cell. Biol. 2002; 22: 693-703Crossref PubMed Scopus (63) Google Scholar, 40.van Oevelen C.J. van Teeffelen H.A. Timmers H.T. Mol. Cell. Biol. 2005; 25: 4863-4872Crossref PubMed Scopus (20) Google Scholar). Sequences of oligonucleotides used in this study are available upon request.TABLE 1Yeast strains and genotypes used in this studyStrainGenotypeReferenceDY151MATα leu2–3,112 his3–11,15 trp1–1 can1–100 ade2–1 ura3–1(16.Davie J.K. Trumbly R.J. Dent S.Y. Mol. Cell. Biol. 2002; 22: 693-703Crossref PubMed Scopus (63) Google Scholar)DY11Isogenic to DY151 except TUP1::3 x HA(16.Davie J.K. Trumbly R.J. Dent S.Y. Mol. Cell. Biol. 2002; 22: 693-703Crossref PubMed Scopus (63) Google Scholar)COY003Isogenic to DY151 except tup1Δ::URA3This reportCOY005Isogenic to DY151 except mig1Δ::URA3This reportCOY007Isogenic to DY151 except rgt1Δ::URA3This reportCOY011Isogenic to DY151 except ssn6Δ::URA3This reportCOY013Isogenic to DY151 except snf1Δ::URA3This reportCOY026Isogenic to DY151 except gcn5Δ::KANAThis reportCOY189Isogenic to COY026 except pPRS414-GCN5This reportCOY190Isogenic to COY026 except pRSS414-GCN5 F221AThis reportCOY044Isogenic to DY11 except snf1Δ::URA3This reportCOY052Isogenic to DY11 except spt20Δ::URA3This reportCOY142MATa leu2–3,112 his3–11,15 trp1–1 can1–100 ade2–1 ura3–1 SPT20::TAP::TRP1(40.van Oevelen C.J. van Teeffelen H.A. Timmers H.T. Mol. Cell. Biol. 2005; 25: 4863-4872Crossref PubMed Scopus (20) Google Scholar)COY119Isogenic to COY142 except snf1Δ::URA3This reportCOY125Isogenic to COY142 except tup1Δ::URA3This reportCOY127Isogenic to COY142 except ssn6Δ::URA3This reportDPY107MATa ura3–52 lys2–801 ade2–101 trp1-Δ1 his3-Δ200 leu2-Δ1 mot1::TRP1::flu3::MOT1(42.Poon D. Campbell A.M. Bai Y. Weil P.A. J. Biol. Chem. 1994; 269: 23135-23140Abstract Full Text PDF PubMed Google Scholar)COY089Isogenic to DPY107 except spt3Δ::URA3(40.van Oevelen C.J. van Teeffelen H.A. Timmers H.T. Mol. Cell. Biol. 2005; 25: 4863-4872Crossref PubMed Scopus (20) Google Scholar)COY091Isogenic to DPY107 except spt8Δ::URA3(40.van Oevelen C.J. van Teeffelen H.A. Timmers H.T. Mol. Cell. Biol. 2005; 25: 4863-4872Crossref PubMed Scopus (20) Google Scholar)COY094Isogenic to DPY107 except spt20Δ::URA3This reportJCA001Isogenic to DPY107 except gcn5Δ::KANA(40.van Oevelen C.J. van Teeffelen H.A. Timmers H.T. Mol. Cell. Biol. 2005; 25: 4863-4872Crossref PubMed Scopus (20) Google Scholar)YBY838MATa ura3–52 lys2–801 ade2–101 trp1-Δ1 his3-Δ200 leu2-Δ taf11Δ::TRP1 pRS313–3Flu-TAF130-His6-FLAG (HIS CEN)(43.Bai Y. Perez G.M. Beechem J.M. Weil P.A. Mol. Cell. Biol. 1997; 17: 3081-3093Crossref PubMed Scopus (61) Google Scholar)JCA002Isogenic to YBY838 except gcn5Δ::KANA(40.van Oevelen C.J. van Teeffelen H.A. Timmers H.T. Mol. Cell. Biol. 2005; 25: 4863-4872Crossref PubMed Scopus (20) Google Scholar)JHY90MATa leu2–3,112 his3Δ1 trp1–289 ade2–101 ura3–52 lys2–801 Δ(hht1-hhf1) Δ(lyhht2-hhf2) pJH18 [CEN TRP1 HHT2-HHF2](57.Lo W.S. Trievel R.C. Hojas J.R. Duggan L. Hsu J.Y. Allis C.D. Marmorstein R. Berger S.L. Mol. Cell. 2000; 5: 917-926Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar)JHY91Isogenic to JHY90 but pJH15 [CEN TRP1 hht2–3(S10A) HHF2](57.Lo W.S. Trievel R.C. Hojas J.R. Duggan L. Hsu J.Y. Allis C.D. Marmorstein R. Berger S.L. Mol. Cell. 2000; 5: 917-926Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar)LPY7091MATa 1 his3-Δ200 leu2-Δ1 ura3–52 trp1ΔhisG snf1::TRP1(25.Lo W.S. Gamache E.R. Henry K.W. Yang D. Pillus L. Berger S.L. EMBO J. 2005; 24: 997-1008Crossref PubMed Scopus (79) Google Scholar) Open table in a new tab Growth Conditions—For glucose concentration shift experiments, cells were grown in SC medium (0.67% (w/v) yeast nitrogen base without amino acids (Difco) supplemented with complete supplement mixture (Qbiogene)) containing 4% glucose. When cells had reached mid log (A600 = 0.55–0.6), 300 ml of the cell culture was cross-linked by addition of 1% formaldehyde (corresponding to time t = 0) for 20 min at room temperature. The remaining cells were harvested by centrifugation for 6 min at 1700 × g in a Sorvall SLA3000 rotor, resuspended in a small volume of SC with 4% glucose, and diluted 40-fold in SC without glucose to adjust the glucose concentration to 0.1%. Cells were incubated at 30 °C and subjected to cross-linking at the indicated time points. For RNA analysis cells were grown in SC medium containing 4% glucose. When cells had reached mid log (A600 = 0.55–0.6), 10 ml of the culture was directly added to 25 ml of liquid N2 (t = 0). After shifting to low glucose, 10 ml of the culture was removed at the indicated time points and was frozen directly in liquid N2. After thawing, cells were collected by centrifugation, refrozen in liquid N2, and stored at -80 °C upon further processing. Northern Blotting—RNA isolation and hybridization conditions were described previously (46.Andrau J.-C. van Oevelen C.J.C. van Teeffelen H.A.A.M. Weil P.A. Holstege F.C.P. Timmers H.T.M. EMBO J. 2002; 21: 5173-5183Crossref PubMed Scopus (47) Google Scholar). Oligonucleotide sequences and the labeling procedure used for HXT genes have been described previously (47.Diderich J.A. Schepper M. van Hoek P. Luttik M.A. van Dijken J.P. Pronk J.T. Klaassen P. Boelens H.F. de Mattos M.J. van Dam K. Kruckeberg A.L. J. Biol. Chem. 1999; 274: 15350-15359Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). A 1-kb fragment of the ACT1 coding region (spanning the region +324 to +1347) was used to analyze variations in mRNA loading. The ACT1 DNA fragment was labeled with the Rediprime™II system according to the manufacturer's protocol (Amersham Biosciences). PhosphorImager quantification of HXT2 and HXT4 mRNA signals has been described previously (46.Andrau J.-C. van Oevelen C.J.C. van Teeffelen H.A.A.M. Weil P.A. Holstege F.C.P. Timmers H.T.M. EMBO J. 2002; 21: 5173-5183Crossref PubMed Scopus (47) Google Scholar). Data from triplicate experiments were used for quantification. Chromatin Immunoprecipitation Assay—Chromatin extracts were prepared as previously described (46.Andrau J.-C. van Oevelen C.J.C. van Teeffelen H.A.A.M. Weil P.A. Holstege F.C.P. Timmers H.T.M. EMBO J. 2002; 21: 5173-5183Crossref PubMed Scopus (47) Google Scholar). Chromatin extract (200 μl) was used for immunoprecipitation, and 10μl of chromatin extract was used for input control preparation. Specific protein-DNA complexes were recovered as described previously (46.Andrau J.-C. van Oevelen C.J.C. van Teeffelen H.A.A.M. Weil P.A. Holstege F.C.P. Timmers H.T.M. EMBO J. 2002; 21: 5173-5183Crossref PubMed Scopus (47) Google Scholar) with 25 μl of Protein G-agarose beads (Roche Applied Science) pre-bound to 10 μg of anti-yTBP, 30 μg of anti-HA (12CA5), 10 μg of anti-Pol II CTD (8WG16), 1 μl of anti-H3 (Abcam ab1791), 2 μl of anti-acetylated H3K9 (Upstate 07-352), 2 μl of anti-acetylated H3K14 (Upstate 07–353), and 2 μl of anti-acetylated H3K18 (Abcam ab1191) antibodies. To recover tandem affnity purification-tagged proteins, 30 μl of IgG-Sepharose™ 6 fast flow beads (Amersham Biosciences) was used. After elution cross-links were reversed by incubating the eluates with proteinase K to a final concentration of 1 μg/μl for 2 h at 37°C and overnight at 65 °C. DNA from input samples was prepared similarly. After DNA purification from the eluates using QIAquick columns (Qiagen) DNA was diluted (1:10) and analyzed by real-time PCR with an MJ PTC200 thermal cycler accommodated with a Chromo4™ real-time detector (MJ Research/Bio-Rad). Data were analyzed using Opticon 3 Monitor™ software and Microsoft Excel 2003. Activation and Repression of HXT2 and HXT4 mRNA Expression—Transcription of the HXT2 and HXT4 genes is regulated by glucose levels (23.Ozcan S. Johnston M. Microbiol. Mol. Biol. Rev. 1999; 63: 554-569Crossref PubMed Google Scholar). Previously, it was shown that deletion of components of the repression complex (i.e. MIG1, RGT1, SSN6, and TUP1) leads to (partial) derepression of the HXT2 and HXT4 promoters depending on the growth conditions (18.Ozcan S. Johnston M. Mol. Cell. Biol. 1995; 15: 1564-1572Crossref PubMed Google Scholar, 19.Ozcan S. Johnston M. Mol. Cell. Biol. 1996; 16: 5536-5545Crossref PubMed Scopus (84) Google Scholar). In contrast, deletion of the SNF1 gene completely abolishes HXT2 and HXT4 induction under conditions of low glucose (18.Ozcan S. Johnston M. Mol. Cell. Biol. 1995; 15: 1564-1572Crossref PubMed Google Scholar). In these experiments HXT2 and HXT4 promoter activity was measured in a plasmid-based system using the LacZ reporter gene. To study the early kinetics of HXT2 and HXT4 mRNA induction we have developed a rapid cell harvesting protocol (40.van Oevelen C.J. van Teeffelen H.A. Timmers H.T. Mol. Cell. Biol. 2005; 25: 4863-4872Crossref PubMed Scopus (20) Google Scholar). Using this method, we studied the kinetics of mRNA induction from the endogenous HXT2 and HXT4 genes in repression and derepression mutant strains (Table 1). To analyze variations in mRNA loading, HXT2 and HXT4 mRNA were quantified relative to ACT1 mRNA levels (Fig. 1, C and D), which is invariant during the glucose concentration shift (data not shown). As shown in Fig. 1 and as previously described (40.van Oevelen C.J. van Teeffelen H.A. Timmers H.T. Mol. Cell. Biol. 2005; 25: 4863-4872Crossref PubMed Scopus (20) Google Scholar), a 20- to 25-fold induction of HXT2 and HXT4 mRNA was observed, which occurred already 5 min after a shift to low glucose. Deletion of SNF1 completely abolished induction of HXT2 and HXT4 mRNA, which is in agreement with previous results (18.Ozcan S. Johnston M. Mol. Cell. Biol. 1995; 15: 1564-1572Crossref PubMed Google Scholar). Mig1p and Rgt1p are repressors recruiting the Ssn6p-Tup1p complex to glucose-repressed genes (1.Carlson M. Curr. Opin. Microbiol. 1999; 2: 202-207Crossref PubMed Scopus (458) Google Scholar). Mutant cells deleted for either MIG1 or RGT1 displayed a 3- to 5-fold increase of basal levels compared with wild-type cells (Fig. 1). Nevertheless, shifting to low glucose still resulted in mRNA induction of HXT2 and HXT4 to levels, which slightly exceeded wild-type levels. In contrast, deletion of TUP1 resulted in (an almost complete) derepression and deletion of SSN6 completely derepressed HXT2 and HXT4 transcription under non-inducing conditions. Thus, the Ssn6p-Tup1p complex represses mRNA expression of the HXT2 and HXT4 genes under non-inducing conditions and relief of repression requires Snf1p as has been described previously (18.Ozcan S. Johnston M. Mol. Cell. Biol. 1995; 15: 1564-1572Crossref PubMed Google Scholar, 19.Ozcan S. Johnston M. Mol. Cell. Biol. 1996; 16: 5536-5545Crossref PubMed Scopus (84) Google Scholar). The results also suggest that both Mig1p and Rgt1p can recruit the Ssn6p-Tup1p complex to the HXT2 and HXT4 promoters. A Decrease in Tup1p Binding Precedes Decreased Histone H3 Binding and Preinitiation Complex Formation—The Ssn6p-Tup1p repressor complex is recruited by Mig1p to several glucose-repressed genes such as CYC1 and GAL1 (3.Treitel M.A. Carlson M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3132-3136Crossref PubMed Scopus (337) Google Scholar, 24.Papamichos-Chronakis M. Gligoris T. Tzamarias D. EMBO Rep. 2004; 5: 368-372Crossref PubMed Scopus (95) Google Scholar, 48.Tzamarias D. Struhl K. Nature. 1994; 369: 758-761Crossref PubMed Scopus (279) Google Scholar). Furthermore, deletion of either component of the Ssn6p-Tup1p complex resulted in a constitutive expression of the HXT2 and HXT4 genes (Fig. 1). Therefore, to investigate the direct association of the Ssn6p-Tup1p complex to the HXT2 and HXT4 promoters, we determined the binding kinetics of Tup1p during a shift to low glucose. This was investigated by chromatin immunoprecipitation assays employing yeast cells expressing a hemagglutinin (HA)-tagged version of Tup1p (see Table 1). Cells were grown in SC medium containing 4% glucose and shifted to 0.1% glucose. Subsequently, formaldehyde-cross-linked chromatin was isolated at various times after the glucose shift. Immunopurified DNA was analyzed by real-time PCR using primer sets, which amplify the TATA or upstream regions of the HXT2 and HXT4 genes (Fig. 2A). An intragenic fragment of the POL1 gene was used as a normalization control (49.Kuras L. Struhl K. Nature. 1999; 399: 609-613Crossref PubMed Scopus (399) Google Scholar). When cells were grown in 4% glucose we observed strong and specific binding of Tup1p to the putative UAS regions of the HXT2 and HXT4 promoters, which was 35- to 40-fold above the POL1 background signal (Fig. 2B). Directly after cells were shifted to low glucose, Tup1p binding was slightly decreased (less than 2-fold) at the putative UAS of HXT2 and HXT4. Association of Tup1 with the flanking regions was lower, and activation did not reduce this association. Tup1p has been reported to bind to the N-terminal tails of histone H3 and H4, and Tup1p binding correlates with hypoacetylated histone H3 on the STE2 and STE6 promoters in vivo (15.Edmondson D.G. Smith M.M. Roth S.Y. Genes Dev. 1996; 10: 1247-1259Crossref PubMed Scopus (405) Google Scholar, 16.Davie J.K. Trumbly R.J. Dent S.Y. Mol. Cell. Biol. 2002; 22: 693-703Crossref PubMed Scopus (63) Google Scholar). Therefore we analyzed the binding of histone H3 to the HXT2 and HXT4 promoters by using an antibody directed against the C-terminal 12 residues of H3. Under levels of high glucose, we observed histone H3 binding to the UAS and TATA regions of the HXT2 and HXT4 promoters. Shifting cells to low glucose resulted in a 3- to 8-fold decrease in histone H3 binding to both UAS and TATA regions with minimal binding after 2–5 min (Fig. 2C). Interestingly, H3 binding inversely correlated with TBP and polymerase II binding, which is at maximum levels 2–5 min after the glucose shift (Fig. 2D) as we observed previously (40.van Oevelen C.J. van Teeffelen H.A. Timmers H.T. Mol. Cell. Biol. 2005; 25: 4863-4872Crossref PubMed Scopus (20) Google Scholar, 46.Andrau J.-C. van Oevelen C.J.C. van Teeffelen H.A.A.M. Weil P.A. Holstege F.C.P. Timmers H.T.M. EMBO J. 2002; 21: 5173-5183Crossref PubMed Scopus (47) Google Scholar). Thus, after a shift to low glucose the small decrease in Tup1p association precedes a strong decrease in histone H3 binding and increases in TBP and polymerase II association. HXT2 and HXT4 Promoters Are Preferentially Acetylated at Histone H3K18 after Activation—A correlation between Tup1p association and histone H3 acetylation was reported for the STE2, RNR3, and SUC2 genes (16.Davie J.K. Trumbly R.J. Dent S.Y. Mol. Cell. Biol. 2002; 22: 693-703Crossref PubMed Scopus (63) Google Scholar, 17.Bone J.R. Roth S.Y. J. Biol. Chem. 2001; 276: 1808-1813Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). In these cases, deletion of TUP1 leads to an increase in acetylated histone H3 on these promoters. Therefore, we tested whether a decrease in Tup1p binding correlated with an increase in acetylated histone H3 on the HXT genes. Cross-linked chromatin was subjected to immunoprecipitations using specific antibodies directed against individual acetylated lysine residues of histone H3. We observed a strong increase in H3K18Ac relative to a control region of the POL1 open reading frame (46.Andrau J.-C. van Oevelen C.J.C. van Teeffelen H.A.A.M. Weil P.A. Holstege F.C.P. Timmers H.T.M. EMBO J. 2002; 21: 5173-5183Crossref PubMed Scopus (47) Google Scholar, 50.Kuras L. Kosa P. Mencia M. Struhl K. Science. 2000; 288: 1244-1248Crossref PubMed Scopus (145) Google Scholar) on both HXT2 and HXT4 promoters after a shift to low glucose (Fig. 3A). In contrast, we did not detect changes in H3K14Ac levels (Fig. 3B) and only a 2-fold increase in H3K9Ac levels (supplementary Fig. S1). However, when compared with the total amount of histone H3 on the HXT2 and HXT4 promoters, H3K14Ac (and H3K9Ac) increased after the glucose shift (Fig. 3, C and D). In addition, normalization to total histone H3 levels further confirms that the HXT2 and HXT4 promoters are preferentially acetylated on H3K18 (Fig. 3, C and D). Thus, analysis of the modification status of histone H3 reveals a specific histone H3 acetylation pattern on the HXT2 and HXT4 prom

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