Genome-wide Transcriptional Dependence on TAF1 Functional Domains
2006; Elsevier BV; Volume: 281; Issue: 10 Linguagem: Inglês
10.1074/jbc.m513776200
ISSN1083-351X
AutoresJordan D. Irvin, B. Franklin Pugh,
Tópico(s)RNA Research and Splicing
ResumoTranscription factor IID (TFIID) plays a central role in regulating the expression of most eukaryotic genes. Of the 14 TBP-associated factor (TAF) subunits that compose TFIID, TAF1 is one of the largest and most functionally diverse. Yeast TAF1 can be divided into four regions including a putative histone acetyltransferase domain and TBP, TAF, and promoter binding domains. Establishing the importance of each region in gene expression through deletion analysis has been hampered by the cellular requirement of TAF1 for viability. To circumvent this limitation we introduced galactose-inducible deletion derivatives of previously defined functional regions of TAF1 into a temperature-sensitive taf1ts2 yeast strain. After galactose induction of the TAF1 mutants and temperature-induced elimination of the resident Taf1ts2 protein, we examined the properties and phenotypes of the mutants, including their impact on genome-wide transcription. Virtually all TAF1-dependent genes, which comprise ∼90% of the yeast genome, displayed a strong dependence upon all regions of TAF1 that were tested. This finding might reflect the need for each region of TAF1 to stabilize TAF1 against degradation or may indicate that all TAF1-dependent genes require the many activities of TAF1. Paradoxically, deletion of the region of TAF1 that is important for promoter binding interfered with the expression of many genes that are normally TFIID-independent/SAGA (Spt-Ada-Gcn5-acetyltransferase)-dominated, suggesting that this region normally prevents TAF1 (TFIID) from interfering with the expression of SAGA-regulated genes. Transcription factor IID (TFIID) plays a central role in regulating the expression of most eukaryotic genes. Of the 14 TBP-associated factor (TAF) subunits that compose TFIID, TAF1 is one of the largest and most functionally diverse. Yeast TAF1 can be divided into four regions including a putative histone acetyltransferase domain and TBP, TAF, and promoter binding domains. Establishing the importance of each region in gene expression through deletion analysis has been hampered by the cellular requirement of TAF1 for viability. To circumvent this limitation we introduced galactose-inducible deletion derivatives of previously defined functional regions of TAF1 into a temperature-sensitive taf1ts2 yeast strain. After galactose induction of the TAF1 mutants and temperature-induced elimination of the resident Taf1ts2 protein, we examined the properties and phenotypes of the mutants, including their impact on genome-wide transcription. Virtually all TAF1-dependent genes, which comprise ∼90% of the yeast genome, displayed a strong dependence upon all regions of TAF1 that were tested. This finding might reflect the need for each region of TAF1 to stabilize TAF1 against degradation or may indicate that all TAF1-dependent genes require the many activities of TAF1. Paradoxically, deletion of the region of TAF1 that is important for promoter binding interfered with the expression of many genes that are normally TFIID-independent/SAGA (Spt-Ada-Gcn5-acetyltransferase)-dominated, suggesting that this region normally prevents TAF1 (TFIID) from interfering with the expression of SAGA-regulated genes. DNA binding sequence-specific activators regulate eukaryotic genes at many stages including the recruitment of chromatin remodeling factors that increase the accessibility of promoters to the transcription machinery. Activators also assist in the loading of the general transcription factors and RNA polymerase II at promoters to form a preinitiation complex that is capable of transcribing the gene. The transcription machinery assembles at promoters via two major pathways in yeast, one that involves TFIID 3The abbreviations used are: TFIID, transcription factor IID; TBP, TATA box-binding protein; FHT, Flu-His6-TEV; HA, hemagglutinin; TEV, tobacco etch virus; 5-FOA, 5-fluoroo-rotic acid; WT, wild type; SAGA, Spt-Ada-Gcn5-acetyltransferase; TAF, TBP-associated factor; TAND, TAF1 N-terminal domain; HAT, histone acetyltransferase; gal, galactose; YPR, yeast extract, peptone, raffinose; CSM, complete synthetic medium; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; ts, temperature-sensitive; HA, hemagglutinin. and the other involving a compositionally related complex called SAGA (1Naar A.M. Lemon B.D. Tjian R. Annu. Rev. Biochem. 2001; 70: 475-501Crossref PubMed Scopus (441) Google Scholar, 2Green M.R. Trends Biochem. Sci. 2000; 25: 59-63Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 3Huisinga K.L. Pugh B.F. Mol. Cell. 2004; 13: 573-585Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). TFIID is composed of the TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs), 4In this manuscript, we have followed the systematic TAF naming rules proposed by Tora (39Tora L. Genes Dev. 2002; 16: 673-675Crossref PubMed Scopus (194) Google Scholar). of which all but one are essential for cell viability. Several of these TAFs, along with TBP, are also found in SAGA (4Grant P.A. Schieltz D. Pray-Grant M.G. Steger D.J. Reese J.C. Yates III, J.R. Workman J.L. Cell. 1998; 94: 45-53Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). Although TBP is widely regarded as responsible for delivering TFIID to promoters through interactions of TBP with the TATA box, TFIID largely functions at TATA-less promoters (3Huisinga K.L. Pugh B.F. Mol. Cell. 2004; 13: 573-585Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 5Basehoar A.D. Zanton S.J. Pugh B.F. Cell. 2004; 116: 699-709Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar, 6Zanton S.J. Pugh B.F. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16843-16848Crossref PubMed Scopus (64) Google Scholar, 7Kim T.H. Barrera L.O. Zheng M. Qu C. Singer M.A. Richmond T.A. Wu Y. Green R.D. Ren B. Nature. 2005; 436: 876-880Crossref PubMed Scopus (761) Google Scholar). TATA-containing promoters tend to be TFIID-independent and instead prefer to load TBP via the SAGA assembly pathway (3Huisinga K.L. Pugh B.F. Mol. Cell. 2004; 13: 573-585Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). The vast majority of all yeast genes (80–90%) are regulated through a TFIID/TATA-less arrangement, whereas a smaller minority depend primarily on a SAGA/TATA arrangement. Strikingly, the latter class largely includes stress-induced genes. Thus transcription complex assembly via the SAGA pathway might provide a greater level of inducibility, which is characteristic of stress-induced responses (3Huisinga K.L. Pugh B.F. Mol. Cell. 2004; 13: 573-585Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 6Zanton S.J. Pugh B.F. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16843-16848Crossref PubMed Scopus (64) Google Scholar, 8Gasch A.P. Spellman P.T. Kao C.M. Carmel-Harel O. Eisen M.B. Storz G. Botstein D. Brown P.O. Mol. Biol. Cell. 2000; 11: 4241-4257Crossref PubMed Scopus (3763) Google Scholar). Under normal growth conditions, SAGA is not essential for cell viability (4Grant P.A. Schieltz D. Pray-Grant M.G. Steger D.J. Reese J.C. Yates III, J.R. Workman J.L. Cell. 1998; 94: 45-53Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 9Lee T.I. Causton H.C. Hostege F.C. Shen W.C. Hannett N. Jennings E.G. Winston F. Green M.R. Young R.A. Nature. 2000; 405: 701-704Crossref PubMed Scopus (302) Google Scholar). In the absence of SAGA, expression of virtually the entire measurable yeast genome becomes TFIID-dependent. Thus, TFIID may be capable of setting up transcription complexes at all polymerase II-transcribed genes. TAFs perform a variety of functions including interactions with transcriptional activators, other general transcription factors, and promoter DNA (10Shen W.C. Bhaumik S.R. Causton H.C. Simon I. Zhu X. Jennings E.G. Wang T.H. Young R.A. Green M.R. EMBO J. 2003; 22: 3395-3402Crossref PubMed Scopus (81) Google Scholar, 11Kirschner D.B. vom Baur E. Thibault C. Sanders S.L. Gangloff Y.G. Davidson I. Weil P.A. Tora L. Mol. Cell. Biol. 2002; 22: 3178-3193Crossref PubMed Scopus (27) Google Scholar, 12Mencia M. Struhl K. Mol. Cell. Biol. 2001; 21: 1145-1154Crossref PubMed Scopus (14) Google Scholar, 13Kirchner J. Sanders S.L. Klebanow E. Weil P.A. Mol. Cell. Biol. 2001; 21: 6668-6680Crossref PubMed Scopus (15) Google Scholar, 14Durso R.J. Fisher A.K. Albright-Frey T.J. Reese J.C. Mol. Cell. Biol. 2001; 21: 7331-7344Crossref PubMed Scopus (36) Google Scholar, 15Kokubo T. Swanson M.J. Nishikawa J.I. Hinnebusch A.G. Nakatani Y. Mol. Cell. Biol. 1998; 18: 1003-1012Crossref PubMed Scopus (102) Google Scholar). Genome-wide studies using temperature-sensitive alleles of various TAFs indicate that some TAFs may be selective in the genes they activate (9Lee T.I. Causton H.C. Hostege F.C. Shen W.C. Hannett N. Jennings E.G. Winston F. Green M.R. Young R.A. Nature. 2000; 405: 701-704Crossref PubMed Scopus (302) Google Scholar, 10Shen W.C. Bhaumik S.R. Causton H.C. Simon I. Zhu X. Jennings E.G. Wang T.H. Young R.A. Green M.R. EMBO J. 2003; 22: 3395-3402Crossref PubMed Scopus (81) Google Scholar, 16Holstege F.C.P. Jennings E.G. Wyrick J.J. Lee T.I. Hengartner C.J. Green M.R. Golub T.R. Lander E.S. Young R.A. Cell. 1998; 95: 717-728Abstract Full Text Full Text PDF PubMed Scopus (1598) Google Scholar). This suggests that distinct parts of TFIID might play important promoter-specific roles. Similarly, a variety of temperature-sensitive alleles located throughout TAF10 reveal potential promoter-selective roles for distinct regions of a single TAF (11Kirschner D.B. vom Baur E. Thibault C. Sanders S.L. Gangloff Y.G. Davidson I. Weil P.A. Tora L. Mol. Cell. Biol. 2002; 22: 3178-3193Crossref PubMed Scopus (27) Google Scholar). TAF1 is considered to be a "hallmark" of TFIID in that it resides only in TFIID and not in SAGA, and it may serve as a scaffold upon which TBP and TAFs assemble, although other TAFs might also play a scaffolding role (12Mencia M. Struhl K. Mol. Cell. Biol. 2001; 21: 1145-1154Crossref PubMed Scopus (14) Google Scholar, 17Chen J.L. Attardi L.D. Verrijzer C.P. Yokomori K. Tjian R. Cell. 1994; 79: 93-105Abstract Full Text PDF PubMed Scopus (328) Google Scholar, 18Singh M.V. Bland C.E. Weil P.A. Mol. Cell. Biol. 2004; 24: 4929-4942Crossref PubMed Scopus (12) Google Scholar, 19Bai Y. Perez G.M. Beechem J.M. Weil P.A. Mol. Cell. Biol. 1997; 17: 3081-3093Crossref PubMed Scopus (61) Google Scholar). When the studies reported here were initiated, TAF1 had been systematically dissected into four functional domains: an N-terminal TBP-binding domain termed TAND, a TAF-TAF interaction domain, a putative histone acetyltransferase (HAT) domain, and a promoter recognition domain (12Mencia M. Struhl K. Mol. Cell. Biol. 2001; 21: 1145-1154Crossref PubMed Scopus (14) Google Scholar, 15Kokubo T. Swanson M.J. Nishikawa J.I. Hinnebusch A.G. Nakatani Y. Mol. Cell. Biol. 1998; 18: 1003-1012Crossref PubMed Scopus (102) Google Scholar, 18Singh M.V. Bland C.E. Weil P.A. Mol. Cell. Biol. 2004; 24: 4929-4942Crossref PubMed Scopus (12) Google Scholar, 20Sanders S.L. Garbett K.A. Weil P.A. Mol. Cell. Biol. 2002; 22: 6000-6013Crossref PubMed Scopus (89) Google Scholar, 21Mizzen C.A. Yang X.J. Kokubo T. Brownell J.E. Bannister A.J. Owen-Hughes T. Workman J. Wang L. Berger S.L. Kouzarides T. Nakatani Y. Allis C.D. Cell. 1996; 87: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar, 22Kotani T. Banno K. Ikura M. Hinnebusch A.G. Nakatani Y. Kawaichi M. Kokubo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7178-7183Crossref PubMed Scopus (40) Google Scholar). More recently, a fifth domain that interacts with TAF7 has been identified (18Singh M.V. Bland C.E. Weil P.A. Mol. Cell. Biol. 2004; 24: 4929-4942Crossref PubMed Scopus (12) Google Scholar). In addition, the physiological significance of the yeast TAF1 HAT activity has come into question (23Durant M. Pugh B.F. Mol. Cell. Biol. 2006; (in press)Google Scholar). Collectively, the potentially gene-specific roles of TAFs and the potential modularity of TAF1 and other TAFs led us to consider whether the various functional domains of TAF1 play gene-specific roles in vivo. Because TFIID contributes to the expression of nearly the entire yeast genome, a greater understanding of the yeast gene regulatory network might be achieved by assessing the contribution of each of the TFIID activities on genome-wide transcription. Any investigation into the genome-wide function of TAF1 or any other essential factor is hampered by the fact that deleterious mutations block cell growth. Creation of temperature-sensitive alleles has been a powerful and productive means of dissecting essential functional regions. However, this approach can be biased and restrictive in that any mutation must knock out an essential function at the nonpermissive temperature but renders the protein functional at the permissive temperature. Because a large fraction of yeast genes are not essential for cell growth, this strategy could miss TAF1 mutations that are specifically defective in expression of many nonessential genes. As an alternative strategy, we employed a systematic targeted approach by disrupting known functional domains of TAF1. To minimize potential indirect effects caused by constitutive expression of the TAF1 mutants, we chose to express each mutant under an inducible promoter. Because TAF1 is essential, this approach necessitated the use of a functional copy of TAF1 to promote cell growth. However, when assaying for mutant function it was desirable to eliminate the functional TAF1 copy so that it would not obscure or suppress the deletion construct. This was achieved by using a temperature-sensitive TAF1 allele (3Huisinga K.L. Pugh B.F. Mol. Cell. 2004; 13: 573-585Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 24Walker S.S. Shen W.C. Reese J.C. Apone L.M. Green M.R. Cell. 1997; 90: 607-614Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Using this approach we first assayed for the ability of an induced version of wild type or mutant TAF1 to functionally complement the growth defect of a temperature-sensitive TAF1 allele. Next we characterized the expression, subcellular localization, TFIID integrity, and stability of the TAF1 mutants in order to better assess its potential to impact gene expression. Lastly, genome-wide expression studies were performed to evaluate the contribution of each functional domain to transcription. YCp50 (TAF1 WT, URA3), and pRS313 taf1ts2 HIS3 (25Reese J.C. Apone L. Walker S.S. Griffin L.A. Green M.R. Nature. 1994; 371: 523-527Crossref PubMed Scopus (148) Google Scholar) were gifts from J. Reese of this department (Department of Biochemistry and Molecular Biology, Pennsylvania State University). pYN2 (TAF145 WT, TRP1) (15Kokubo T. Swanson M.J. Nishikawa J.I. Hinnebusch A.G. Nakatani Y. Mol. Cell. Biol. 1998; 18: 1003-1012Crossref PubMed Scopus (102) Google Scholar) was a gift from T. Kokubo (Yokohama City University, Japan). pJI11 (pRS315 taf1ts2 LEU2) and pJI12 (pRS315 TAF1 WT LEU2) were created by amplifying the TAF1 gene from pRS313 taf1ts2 or pYN2, respectively, with taf1ts2 oligonucleotides (Integrated DNA Technologies, supplemental Table S2) containing NotI and SalI restriction sites. The 4,456-bp PCR products were digested with NotI and SalI (New England Biolabs) (4,442 bp) and ligated into digested pRS315 (5,953 bp) to create 10,396-bp plasmids pJI11 and pJI12. The PCR product contained 494 bp upstream of TAF1 open reading frame and 741 bp downstream. To confirm the taf1ts2 temperature-sensitive (ts) phenotype, pJI11 and pJI12 were transformed into Y13.2 (15Kokubo T. Swanson M.J. Nishikawa J.I. Hinnebusch A.G. Nakatani Y. Mol. Cell. Biol. 1998; 18: 1003-1012Crossref PubMed Scopus (102) Google Scholar) and plated on CSM-LEU. pYN1 (TAF1 WT URA3) was shuffled out of Y13.2 by plating cells on CSM-LEU + 5-FOA (Zymo Research). Cells were then grown at 23 or 37 °C for 3 days on CSM-LEU (dextrose) plates to confirm the ts phenotype. The temperature-sensitive phenotype was confirmed independently in the taf1::KanMX null strain (yjdi381) on CSM-LEU (dextrose). pCALF-T(PGK) (26Kou H. Irvin J.D. Huisinga K.L. Mitra M. Pugh B.F. Mol. Cell. Biol. 2003; 23: 3186-3201Crossref PubMed Scopus (14) Google Scholar) was converted to pCALF-FHT-T(PGK) 2.2 by inserting a 66-bp HIS-TEV oligo into the NdeI site. pUG6-FHT-P (4,170 bp) was made by PCR-amplifying a 259-bp fragment containing the FHT sequence from pCALFHT-T(PGK) 2.2 plasmid. PCR product was digested with SalI, and 161 bp was ligated into SalI-digested pUG6 plasmid (4,009 bp) such that the orientation was FHT-loxP-KanMX-loxP. Saccharomyces cerevisiae strain BY4743 (27Brachmann C.B. Davies A. Cost G.J. Caputo E. Li J. Hieter P. Boeke J.D. Yeast. 1998; 14: 115-132Crossref PubMed Scopus (2646) Google Scholar) (Invitrogen) was used as the parental strain. Initially, the stain was transformed with pSH47 (URA3) (28Guldener U. Heck S. Fielder T. Beinhauer J. Hegemann J.H. Nucleic Acids Res. 1996; 24: 2519-2524Crossref PubMed Scopus (1372) Google Scholar) encoding galactose inducible Cre recombinase. 70-mer oligonucleotides F4 and R2 (supplemental Table S2; regions of homology to TAF1 are in bold) were used to PCR-amplify 1991 bp of pFA6a-His3MX6-PGAL1 containing the HIS3 gene and GAL1 promoter (29Longtine M.S. McKenzie III, A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4193) Google Scholar). The PCR product was transformed into BY4743 using a high efficiency lithium acetate method (28Guldener U. Heck S. Fielder T. Beinhauer J. Hegemann J.H. Nucleic Acids Res. 1996; 24: 2519-2524Crossref PubMed Scopus (1372) Google Scholar) to replace 550 bp of the endogenous TAF1 promoter with the GAL1 promoter, creating strain yLAC3. HIS+ homologous recombination transformants were selected on CSM-HIS-URA media and verified with colony PCR. Regions of TAF1 were deleted by replacing coding sequences with an FHT tag. The FHT tag encodes three HA (Flu) repeats, a hexahistidine (H) sequence, and the TEV protease sequence (T). The kanamycin resistance region of pUG6-FHT-p was PCR-amplified with 68-mer oligonucleotides with 50-bp homology to distinct regions of TAF1 (supplemental Table S2, Fig. 1). 1,826-bp PCR products were transformed into yLAC3 and selected on CSM-HIS-URA (dextrose) plates containing 500 μg/ml G418 (Invitrogen). The kanamycin resistance cassette flanked by loxP sites was removed by induction of Cre recombinase with 2% galactose for 4 h, leaving the FHT tag N-terminal to the mutation in TAF1. Kanamycin-sensitive colonies were identified by replica plating on media containing and lacking G418. Additionally, mutations were verified by colony PCR with primers specific to each mutation. Kanamycin-sensitive FHT-TAF1 strains (Table 1) were plated on CSM-HIS + 5-FOA to select cells having lost pSH47 (30Adams A. Gottschling D.E. Kaiser C.A. Stearns T. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997Google Scholar) and verified by replica plating on CSM-HIS and CSM-HIS-URA. Strains were then transformed with YCp50 (TAF1 WT, URA3) and transformants selected on CSM-HIS-URA media. Strains were plated on pre-sporulation medium (1% yeast extract, 2% peptone, 10% dextrose) for 2 days at 30 °C. Cells were cultured in sporulation medium (0.3% potassium acetate, 0.02% raffinose) for 3 days at 30 °C. 200 μl of the culture was pelleted, resuspended in 1.2 m sorbitol, 10 mm Tris, pH 7.4, and treated with 20 units of (1 mg/ml) zymolyase (MP Biomedicals) at room temperature for 20 min. Tetrads were dissected according to standard yeast techniques on YPD plates (yeast extract, peptone, dextrose). Spores were replica plated onto CSM-HIS-URA medium to select for the HIS3 gene (and therefore the GAL1 promoter). Mating types of the taf1 strains were confirmed with MATa and MATα sex tester strains. MATα leu- HIS+ LYS+ tetrads were selected. Strains were then transformed with pJI11 (taf1ts2, LEU2) or pJI12 (TAF1 WT, LEU2) and selected on CSM-LEU media. Cells that lost YCp50 (TAF1 WT, URA3) were selected by plating on CSM-LEU + 5-FOA.TABLE 1Yeast strainsStrainPromoterTAF1 allelePlasmidMATRef.BY4743TAF1WTDiploid27Brachmann C.B. Davies A. Cost G.J. Caputo E. Li J. Hieter P. Boeke J.D. Yeast. 1998; 14: 115-132Crossref PubMed Scopus (2646) Google ScholaryLAC3GAL1WTpSH47DiploidThis studyyjdi307TAF1WTYCp50 TAF1 WT URA3αThis studyyjdi302GAL1WTYCp50 TAF1 WT URA3αThis studyyjdi270GAL1FHT-WT1YCp50 TAF1 WT URA3αThis studyyjdi275GAL1FHT-DT1 (Δ10–88)YCp50 TAF1 WT URA3αThis studyyjdi280GAL1FHT-TF1 (Δ208–303)YCp50 TAF1 WT URA3αThis studyyjdi288GAL1FHT-HT4 (Δ645–768)YCp50 TAF1 WT URA3αThis studyyjdi295GAL1FHT-PB1 (Δ912–992)YCp50 TAF1 WT URA3αThis studyyjdi352GAL1FHT-WT1pJI12 TAF1 WT LEU2αThis studyyjdi353GAL1FHT-WT1pJI11 taf1ts2 LEU2αThis studyyjdi354GAL1FHT-DT1 (Δ10–88)pJI12 TAF1 WT LEU2αThis studyyjdi355GAL1FHT-DT1 (Δ10–88)pJI11 taf1ts2 LEU2αThis studyyjdi356GAL1FHT-TF1 (Δ208–303)pJI12 TAF1 WT LEU2αThis studyyjdi357GAL1FHT-TF1 (Δ208–303)pJI11 taf1ts2 LEU2αThis studyyjdi358GAL1FHT-HT4 (Δ645–768)pJI12 TAF1 WT LEU2αThis studyyjdi359GAL1FHT-HT4 (Δ645–768)pJI11 taf1ts2 LEU2αThis studyyjdi360GAL1FHT-PB1 (Δ912–992)pJI12 TAF1 WT LEU2αThis studyyjdi361GAL1FHT-PB1 (Δ912–992)pJI11 taf1ts2 LEU2αThis studyyjdi362GAL1WTpJI12 TAF1 WT LEU2αThis studyyjdi363GAL1WTpJI11 taf1ts2 LEU2αThis studyyjdi366TAF1WTpJI12 TAF1 WT LEU2αThis studyyjdi367TAF1WTpJI11 taf1ts2 LEU2αThis studyyjdi375GAL1taf1::KanMXpJI12 TAF1 WT LEU2αThis studyyjdi379GAL1taf1::KanMXYCp50 TAF1 WT URA3αThis studyyjdi381GAL1taf1::KanMXpJI11 taf1ts2 LEU2αThis studyY13.2TAF1ΔTAF1pYN1 TAF1 WT, URA3α15Kokubo T. Swanson M.J. Nishikawa J.I. Hinnebusch A.G. Nakatani Y. Mol. Cell. Biol. 1998; 18: 1003-1012Crossref PubMed Scopus (102) Google Scholar Open table in a new tab 1× 25 mm MgCl2 buffer (Gene Choice), 2.5 units of Taq polymerase (Gene Choice), 0.0002 units of Pfu polymerase (Stratagene), 0.4 mm dNTPs, and each primer at 0.2 μm was used per 50-μl reaction for 32 cycles. Viability—MATα haploid strains carrying YCp50 (TAF1 WT, URA3) were grown at 25 °C in YPD medium (yeast extract, peptone, dextrose). Cells were diluted into YPR + 0.2% galactose and grown to mid-log phase. 0.5 A600 units of cells were removed, and 5 μl of 10-fold serial dilutions were plated. Cells having lost the YCp50 plasmid were selected by growing on CSM + 5-FOA with 2% dextrose or 2% galactose at 25 or 37 °C. Toxicity—MATα haploid strains carrying pJI12 (TAF1 WT LEU2) plasmid were grow in CSM-LEU raffinose to mid-log phase. 0.5 A600 of cells were serially diluted 10-fold, and 5 μl was plated on CSM-LEU + 2% dextrose or 2% galactose at 19, 25, and 37 °C. Photographs were taken after 96 h for 25 and 37 °C plates and after 120 h for 19 °C plates. FHT-TAF1 strains were grown in YPR at 25 °C until the A600 was ∼0.8. Galactose was added to 2%, and strains were incubated at 25 °C for 45 min. Dextrose was then added to 2%, and the cultures were placed in a 37 °C water bath. Equal-volume aliquots were removed at 15, 30, 45, 60, 120, and 180 min after the addition of dextrose. After Western blotting, quantitation was performed on four independent replicates using a densitometer and ImageQuant software from Amersham Biosciences. Exposure times were chosen so that all signals were in the linear range of detection. Local background was subtracted from the signal band at each time point. Intensity peaked at 15 min in dextrose (at 37 °C) for all strains. This value was set as 100%, and the percent of TAF1 remaining over time was plotted. Whole cells containing constitutively expressed HA-Bdf1 were loaded in each lane of the immunoblots as an internal control for protein extraction, recovery, transfer, and immunodetection but were not included in the quantitation. Cultures were grown in YPR overnight at 30 °C, diluted to A600, an A600 of 0.2, and grown for 3.5 h in YPR + 2% galactose at 30 °C. 2.0 A600 units were fixed in 3.7% formaldehyde, treated with zymolyase, and bound to polylysine-coated slides. FHT-tagged TAF1 proteins were visualized by incubating with anti-HA.11 monoclonal antibodies (1:1000, Babco) and then with goat anti-mouse IgG-Alexa Fluor 488 (Molecular Probes). 4′,6-Diamidino-2-phenylindole was used to visualize nucleic acids (31Manzini G. Barcellona M.L. Avitabile M. Quadrifoglio F. Nucleic Acids Res. 1983; 11: 8861-8876Crossref PubMed Scopus (144) Google Scholar). The method was adapted from Ref. 30Adams A. Gottschling D.E. Kaiser C.A. Stearns T. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997Google Scholar. Samples were viewed on an Axioplan epifluorescence microscope (Carl Zeiss, Inc.). TIFF images were collected using a Spot2 cooled charge-coupled device digital camera (Diagnostic Instruments). Cells were washed in 0.5 ml of 0.1 m sodium hydroxide for 5 min at room temperature, spun, resuspended in 2× protein sample buffer, and heated to 95 °C (32Kushnirov V.V. Yeast. 2000; 16: 857-860Crossref PubMed Scopus (679) Google Scholar). HA-TAF1 mutants were electrophoresed in 7.8% Bis-acrylamide gels (PAGE) and transferred to nitrocellulose in 80% Tris-glycine-SDS/20% methanol for 120 min at 1.0 A. FHT-TAF1 mutants were detected with anti-HA (HA.11, Babco) and anti-mouse horseradish peroxidase antibodies (Amersham Biosciences) and exposed to Hyperfilm (Amersham Biosciences) with ECL (Amersham Biosciences). TAF1 strains were induced with 2% galactose in CSM-LEU media for 75 min at 25 °C. ∼10 A600 equivalents of cellular lysate were immunoprecipitated with HA antibodies (Babco) and protein A-Sepharose (Amersham Biosciences) as described previously (19Bai Y. Perez G.M. Beechem J.M. Weil P.A. Mol. Cell. Biol. 1997; 17: 3081-3093Crossref PubMed Scopus (61) Google Scholar). Approximately 2.5 A600 of cells were loaded onto 4–12% Bis-Tris NuPAGE Novex gradient gels (Invitrogen) and transferred to polyvinylidene difluoride membrane in 80% Tris-glycine-SDS/20% methanol for 120 min at 1.0 A. Polyclonal TAF antibodies (gifts of P. A. Weil, Vanderbilt University), polyclonal HA antibodies (Rockland), polyclonal yTBP antibodies, and anti-rabbit horseradish peroxidase antibodies (Amersham Biosciences) were used to detect co-immunoprecipitated TAFs and TBP. For TAF1 production after galactose induction, cultures were grown in CSM-LEU raffinose at room temperature and induced with 2% galactose. 0.5 A600 aliquots were removed at 10-min intervals after the addition of galactose and immunoblotted. For TAF1 induction for use in microarray analysis, cultures were grown in CSM-LEU raffinose at room temperate until A600 ∼ 0.8. Galactose was added to 2% at 0, 15, 30 or 60 min before shifting culture to 37 °C with warm CSM-LEU + 2% galactose. After shift to 37 °C, the cultures were incubated for 45 min before harvesting and use in expression profiling. In generating histograms of log2 expression as a function of time in galactose prior to temperature shift, strain yjdi375 (PTAF1- TAF1 WT + taf1ts2) was used as reference. Test strains were yjdi363 (PGAL1-TAF1 WT + taf1ts2) and yjdi381 (PGAL1-taf1::kanMX + taf1ts2). Microarrays were performed essentially as described (3Huisinga K.L. Pugh B.F. Mol. Cell. 2004; 13: 573-585Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 33Chitikila C. Huisinga K.L. Irvin J.D. Basehoar A.D. Pugh B.F. Mol. Cell. 2002; 10: 871-882Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Briefly, cultures were grown at ∼24 °C in CSM-LEU + 3% raffinose to an A600 of ∼0.8. FHT-TAF1 mutants were induced by adding galactose to 2% at 30 min prior to temperature shift. Cultures were shifted to 37 °C by adding an equal volume of warm CSM-LEU + 2% galactose and placed in a 37 °C incubator for 45 min to inactivate taf1ts2. Cells were harvested by centrifugation at room temperature, washed in RNase-free (diethyl pyrocarbonate-treated) double distilled H2O, and frozen in liquid nitrogen. Total RNA and poly(A+) mRNA purification, reverse transcription, and labeling with fluorescent dyes (Cy3 and Cy5 (Amersham Biosciences)), hybridization, and scanning were all performed as described (3Huisinga K.L. Pugh B.F. Mol. Cell. 2004; 13: 573-585Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 33Chitikila C. Huisinga K.L. Irvin J.D. Basehoar A.D. Pugh B.F. Mol. Cell. 2002; 10: 871-882Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). 4 μg of mRNA was used for hybridizations instead of the conventional 2 μg. Slides were treated with Dye Saver2 (Genisphere) according to manufacturer's instructions to preserve signal intensity. Raw data are accessible at GeneExpression Omnibus (www.ncbi.nlm.nih.gov/geo/; GenBank™ accession numbers GSM65302 to GSM65315). Processed data are presented in supplemental Table S3. R software was used to mode-center replicates (dye swaps) (34Ihaka R.G., R. J. Comp. Graph. Stat. 1996; 5: 299-314Google Scholar). Bacillus subtilis transcripts (Phe, Lys, Dap, Thr, Trp) were added to each culture prior to total RNA isolation based on A600 units. These hybridize to cognate spots on each of the 16 grids per microarray slide. R output (mode-centered) data were normalized by the spiking controls. Genes were filtered by several criteria to minimize false positives. 1) Genes were eliminated if their signal on the array was greater than 25% saturated. 2) The mean foreground signal minus the median background signal had to be greater than standard deviation of background signal. 3) Quality data were needed from both replicates of the dye swap. 4) The directional change of signal of the mutant (relative to reference) had to be equivalent in the replicates. K-M
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