Contribution of the Histone H3 and H4 Amino Termini to Gcn4p- and Gcn5p-mediated Transcription in Yeast
2006; Elsevier BV; Volume: 281; Issue: 14 Linguagem: Inglês
10.1074/jbc.m513178200
ISSN1083-351X
AutoresCailin Yu, Michael J. Palumbo, Charles E. Lawrence, Randall H. Morse,
Tópico(s)RNA Research and Splicing
ResumoHistone amino termini are post-translationally modified by both transcriptional coactivators and corepressors, but the extent to which the relevant histone modifications contribute to gene expression, and the mechanisms by which they do so, are incompletely understood. To address this issue, we have examined the contributions of the histone H3 and H4 amino termini, and of the coactivator and histone acetyltransferase Gcn5p, to activation of a small group of Gcn4p-activated genes. The histone H3 tail exerts a modest (about 2-fold) but significant effect on activation that correlates with a requirement for Gcn5p and is distributed over multiple lysine residues. The H4 tail also plays a positive role in activation of some of those genes tested, but this does not correlate as closely with Gcn5p coactivation. Microarray experiments did not reveal a close correspondence between those genes activated by Gcn4p and genes requiring the H3 or H4 tail, and analysis of published microarray data indicates that Gcn4p-regulated genes are not in general strongly dependent on Gcn5p. However, a large fraction of genes activated by Gcn4p were found to be repressed by the H3 and H4 amino termini under non-inducing conditions, indicating that one role for Gcn4p is to overcome repression mediated by the histone tails. Histone amino termini are post-translationally modified by both transcriptional coactivators and corepressors, but the extent to which the relevant histone modifications contribute to gene expression, and the mechanisms by which they do so, are incompletely understood. To address this issue, we have examined the contributions of the histone H3 and H4 amino termini, and of the coactivator and histone acetyltransferase Gcn5p, to activation of a small group of Gcn4p-activated genes. The histone H3 tail exerts a modest (about 2-fold) but significant effect on activation that correlates with a requirement for Gcn5p and is distributed over multiple lysine residues. The H4 tail also plays a positive role in activation of some of those genes tested, but this does not correlate as closely with Gcn5p coactivation. Microarray experiments did not reveal a close correspondence between those genes activated by Gcn4p and genes requiring the H3 or H4 tail, and analysis of published microarray data indicates that Gcn4p-regulated genes are not in general strongly dependent on Gcn5p. However, a large fraction of genes activated by Gcn4p were found to be repressed by the H3 and H4 amino termini under non-inducing conditions, indicating that one role for Gcn4p is to overcome repression mediated by the histone tails. Investigations into chromatin structure and function performed over the past decade have revealed that the association of DNA with histones in eukaryotes confers an added layer of regulatory complexity that has dwarfed expectations. Chromatin transactions are involved in transcription, replication, repair, and recombination, and a variety of cellular machinery contributes to these transactions by remodeling chromatin structure and/or post-translationally modifying the histones (1Peterson C.L. Laniel M.A. Curr. Biol. 2004; 14: R546-R551Abstract Full Text Full Text PDF PubMed Scopus (988) Google Scholar). Some post-translational modifications of the histones occur in the central, structured domains, but histone modifications are particularly concentrated in the highly conserved, unstructured amino termini, or "tails" (2Jaskelioff M. Peterson C.L. Nat. Cell. Biol. 2003; 5: 395-399Crossref PubMed Scopus (69) Google Scholar, 3Cosgrove M.S. Boeke J.D. Wolberger C. Nat. Struct. Mol. 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Cell. 1988; 55: 27-39Abstract Full Text PDF PubMed Scopus (313) Google Scholar, 10Durrin L.K. Mann R.K. Kayne P.S. Grunstein M. Cell. 1991; 65: 1023-1031Abstract Full Text PDF PubMed Scopus (256) Google Scholar, 11Thompson J.S. Ling X. Grunstein M. Nature. 1994; 369: 245-247Crossref PubMed Scopus (207) Google Scholar, 12Park E.C. Szostak J.W. Mol. Cell. Biol. 1990; 10: 4932-4934Crossref PubMed Scopus (147) Google Scholar), using the budding yeast Saccharomyces cerevisiae, established that the H3 and H4 tails are not essential for viability, but are important for growth and mating-type regulation. Those studies also revealed redundancy with regard to viability and GAL gene regulation among the modifiable lysine residues in the H3 and H4 amino termini, and more recent investigations have indicated that these lysines function redundantly in transcription on a genome-wide level in yeast (13Dion M.F. Altschuler S.J. Wu L.F. Rando O.J. Proc. Natl. Acad. Sci. U. S. 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Biol. 2004; 14: R546-R551Abstract Full Text Full Text PDF PubMed Scopus (988) Google Scholar). More recent reports have arrived at conflicting conclusions regarding the extent to which patterns of histone modifications specify transcriptional output versus the extent to which modifications function redundantly (18Kurdistani S.K. Tavazoie S. Grunstein M. Cell. 2004; 117: 721-733Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 19Liu C.L. Kaplan T. Kim M. Buratowski S. Schreiber S.L. Friedman N. Rando O.J. PLoS Biol. 2005; 3: e328Crossref PubMed Scopus (401) Google Scholar). The first coactivator identified as a histone acetyltransferase, Gcn5p, when tested as a recombinant protein was found to target Lys-14 of histone H3 and Lys-8 and Lys-16 of histone H4 for acetylation when presented with purified histones, but to have little activity toward nucleosomal histones (20Kuo M.H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Nature. 1996; 383: 269-272Crossref PubMed Scopus (508) Google Scholar) (although under optimal buffer conditions, recombinant Gcn5p can acetylate histone H3 in reconstituted nucleosome arrays (21Tse C. Georgieva E.I. Ruiz-Garcia A.B. Sendra R. Hansen J.C. J. Biol. Chem. 1998; 273: 32388-32392Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar)). In vivo, however, Gcn5p exists predominantly as a component of the SAGA and ADA complexes (22Grant 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 (887) Google Scholar). In vitro assays indicate that SAGA principally targets Lys-9, -14, and -18 of histone H3 for acetylation (23Grant P.A. Eberharter A. John S. Cook R.G. Turner B.M. Workman J.L. J. Biol. Chem. 1999; 274: 5895-5900Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar), whereas in vivo investigations revealed Lys-9 and -18 of H3 as important targets of Gcn5p but indicated that additional targets exist (24Zhang W. Bone J.R. Edmondson D.G. Turner B.M. Roth S.Y. EMBO J. 1998; 17: 3155-3167Crossref PubMed Scopus (277) Google Scholar). These results suggest that for genes requiring Gcn5p as a coactivator, specific lysine residues in histone H3, and possibly H4, could be important for transcriptional activation. However, Gcn5p-dependent activation of a reporter gene by the chimeric activator Gal4-VP16 was increased rather than diminished by point mutations examined in Lys-9 or Lys-14 of histone H3 or in the lysines of the H4 tail (24Zhang W. Bone J.R. Edmondson D.G. Turner B.M. Roth S.Y. EMBO J. 1998; 17: 3155-3167Crossref PubMed Scopus (277) Google Scholar). Here, we have sought to obtain new insight into the roles of the H3 and H4 amino termini in transcriptional activation, with particular focus on the H3 tail, by focusing on a few genes that are activated by the general transcriptional activator Gcn4p and/or that require Gcn5p for full activation. We have tested how expression of these genes is affected by successively larger truncations of the H3 amino terminus, or by point mutation of specific lysine residues, to determine whether their activation depends on a specific region of the H3 tail, as the "histone code" hypothesis would suggest, or whether more delocalized effects might regulate gene expression. Gcn4p interacts with the SAGA complex in vitro (25Drysdale C.M. Jackson B.M. McVeigh R. Klebanow E.R. Bai Y. Kokubo T. Swanson M. Nakatani Y. Weil P.A. Hinnebusch A.G. Mol. Cell. Biol. 1998; 18: 1711-1724Crossref PubMed Scopus (85) Google Scholar, 26Natarajan K. Jackson B.M. Rhee E. Hinnebusch A.G. Mol. Cell. 1998; 2: 683-692Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and is capable of recruiting Gcn5p during gene activation in vivo (27Kuo M.-H. Vom Baur E. Struhl K. Allis C.D. Mol. Cell. 2000; 6: 1309-1320Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 28Syntichaki P. Topalidou I. Thireos G. Nature. 2000; 404: 414-417Crossref PubMed Scopus (169) Google Scholar). This suggests that many of the large number of genes activated by Gcn4p (29Natarajan K. Meyer M.R. Jackson B.M. Slade D. Roberts C. Hinnebusch A.G. Marton M.J. Mol. Cell. Biol. 2001; 21: 4347-4368Crossref PubMed Scopus (569) Google Scholar) could depend on Gcn5p (26Natarajan K. Jackson B.M. Rhee E. Hinnebusch A.G. Mol. Cell. 1998; 2: 683-692Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and therefore could be expected to show significant dependence for their activation on the H3 and/or H4 tail. To test this idea, we examined genome-wide effects of the loss of the H3 or H4 amino terminus on transcription under conditions of amino acid starvation, in which most Gcn4p-regulated genes are active. In addition, we have used our own and other publicly available genome-wide expression data to examine the dependence of genes that bind Gcn5p on the histone H3 and H4 amino termini for expression. Our results suggest a modest dependence of Gcn5p-dependent genes on the H3 and H4 amino termini, but very little dependence of most Gcn4p-activated genes on the H3 and H4 tails for activation. However, we show that a large fraction of genes activated by Gcn4p are repressed by H3 and H4 amino termini under non-inducing conditions, indicating that one function of Gcn4p is to overcome chromatin-mediated repression that depends on the histone tails. Plasmids—Plasmid pMS308 was generated by cloning the HHT1-HHF1 fragment from pMS329, which contains a URA3 marker, into pMS358, which contains a LEU2 marker (8Morgan B.A. Mittman B.A. Smith M.M. Mol. Cell. Biol. 1991; 11: 4111-4120Crossref PubMed Scopus (89) Google Scholar). To generate plasmids pCY318, pCY328, pCY338, and pCY348, which harbor the genes encoding histone H3(Δ1-20), H3(Δ1-15), H3(Δ1-10), and H3(Δ1-5) respectively, fragments encoding the corresponding histone H3 deletions were generated by PCR and cloned into pMS358 as SmaI-EcoRI fragments in place of the hht1-2 gene. All mutant hht1 genes were verified by sequencing. Plasmids pCY101, pCY102, pCY103, pCY201, pCY202, and pCY203 were generated by point mutagenesis using the QuikChange II XL site-directed mutagenesis kit (Stratagene). All mutations were verified by sequencing. Plasmid pRS416-DED1pr-GCN4 was created by cloning the SacI-PstI fragment of pAB71 2A. Bortvin, unpublished data. (30Yu L. Morse R.H. Mol. Cell. Biol. 1999; 19: 5279-5288Crossref PubMed Scopus (91) Google Scholar) into pRS416. This single-copy plasmid constitutively expresses GCN4 from the DED1 promoter. Strains and Media—Strains used in this study are listed in Table 1. Strain LYY256 was constructed beginning with strain MX15-3B, generously provided by Prof. Mitch Smith (University of Virginia). MX15-3B is a meiotic segregant constructed by crossing MX4-22A (MATa ura3-52 lys2-Δ201 leu2-3,-112 Δ(HHT1 HHF1) Δ(HHT2 HHF2) pMS329(CEN4 ARS1 HHT1 HHF1 URA3)) (7Megee P.C. Morgan B.A. Mittman B.A. Smith M.M. Science. 1990; 247: 841-845Crossref PubMed Scopus (271) Google Scholar) with a congenic strain derived from L3110 (gcn4-2 bas1-2 bas2-2) (31Arndt K.T. Styles C. Fink G.R. Science. 1987; 237: 874-880Crossref PubMed Scopus (176) Google Scholar) and dissecting tetrads. The histone deletion loci were confirmed by dependence on pMS329 and Southern blot analysis, and the gcn4, bas1-2, and bas2-2 alleles were confirmed by tetrad analysis, growth requirements, and complementation analysis (31Arndt K.T. Styles C. Fink G.R. Science. 1987; 237: 874-880Crossref PubMed Scopus (176) Google Scholar). The plasmid pMS358, a LEU2 marked plasmid that encodes histone H3 lacking the amino-terminal 28 amino acids, was transformed into MX15-3B. Leu+ transformants were selected, and the URA3-marked plasmid expressing wild-type histone H3 was shuttled out by selection on complete synthetic medium (CSM) 3The abbreviations used are: CSM, complete synthetic dropout medium; FDR, false discovery rates; 3-AT, 3-aminotriazole. -Leu plates containing 5-fluoroorotic acid. The loss of the plasmid was confirmed by PCR using primers specific for H3(Δ1-28). The new strain (hht1, hht2, bas1, bas2, gcn4, HIS4, [pHHF1-hht1(Δ1-28) LEU2]) was mated with LYY599 (30Yu L. Morse R.H. Mol. Cell. Biol. 1999; 19: 5279-5288Crossref PubMed Scopus (91) Google Scholar). Diploids were selected on CSM-Ura-Leu plates and tested for inability to mate with haploid tester strains. After random sporulation, haploids carrying only the hht1-2 (H3(Δ1-28)) gene and containing the wild-type HIS4 promoter were identified by PCR. LYY256 was then constructed by introducing wild-type histone H3 expression plasmid pMS329 into this strain and shuttling out pMS358. Finally, pAB71 or pRS416-DED1pr-GCN4 was introduced into these strains to express Gcn4p.TABLE 1Yeast strainsStrainGenotype or descriptionRef.LYY256MA Ta ura3-52 leu2-3, 112 gcn4-2 bas1-2 bas2-2 Δ(HHT2-HHF2) pMS329 [CEN ARS URA3 HHT1-HHF1] pAB71 [LEU2 DED 1pr-GCN4]This workCYY356MA Ta ura3-52 leu2-3, 112 gcn4-2 bas1-2 bas2-2 Δ(HHT2-HHF2) pCY318 [CEN ARS LEU2 hht1 (Δ1-20)-HHF1] pRS416-DED1pr-GCN4This workCYY456MA Ta ura3-52 leu2-3, 112 gcn4-2 bas1-2 bas2-2 Δ(HHT2-HHF2) pCY328 [CEN ARS LEU2 hht1 (Δ1-15)-HHF1] pRS416-DED1pr-GCN4This workCYY556MA Ta ura3-52 leu2-3, 112 gcn4-2 bas1-2 bas2-2 Δ(HHT2-HHF2) pCY338 [CEN ARS LEU2 hht1 (Δ1-10)-HHF1] pRS416-DED1pr-GCN4This workCYY656MA Ta ura3-52 leu2-3, 112 gcn4-2 bas1-2 bas2-2 Δ(HHT2-HHF2) pCY348 [CEN ARS LEU2 hht1 (Δ1-5)-HHF1] pRS416-DED1pr-GCN4This workCYY756MA Ta ura3-52 leu2-3, 112 gcn4-2 bas1-2 bas2-2 Δ(HHT2-HHF2) pCY358 [CEN ARS LEU2 hht1H3 (K4,9,14,18,23,27Q)-HHF1] pRS416-DED1pr-GCN4This workNSY429MA Tαura3-52 lys2Δ201 leu2-3, 112 trp1-289 his3Δ1 Δ(hht1-hhf1)(hht2-hhf2) pNS329 [CEN ARS TRP1 HHT1-HHF1]36NSY438MA Tαura3-52 lys2Δ201 leu2-3, 112 trp1-289 his3Δ1 Δ(hht1-hhf1)(hht2-hhf2) pNS338 [CEN ARS TRP1 HHT1-hhf1-8(H4Δ2-26)]36MX1-4CMA Tαura3-52 lys2Δ201 leu2-3, 112 trp1-289 his3Δ1 Δ(hht1-hhf1)(hht2-hhf2) pMS329 [CEN ARS URA3 HHT1-HHF1]8CY1-4CMA Tαura3-52 lys2Δ201 leu2-3, 112 trp1-289 his3Δ1 Δ(hht1-hhf1)(hht2-hhf2) pMS308 [CEN ARS LEU2 HHT1-HHF1]This workCY2-4CMA Tαura3-52 lys2Δ201 leu2-3, 112 trp1-289 his3Δ1 Δ(hht1-hhf1)(hht2-hhf2) pMS358 [CEN ARS LEU2 hht1-2(Δ1-28)-HHF1]This workCY3-4CMA Tαura3-52 lys2Δ201 leu2-3, 112 trp1-289 his3Δ1 Δ(hht1-hhf1)(hht2-hhf2) pCY318 [CEN ARS LEU2 hht1(Δ1-20)-HHF1]This workCY4-4CMA Tαura3-52 lys2Δ201 leu2-3, 112 trp1-289 his3Δ1 Δ(hht1-hhf1)(hht2-hhf2) pCY328 [CEN ARS LEU2 hht1(Δ1-15)-HHF1]This workCY5-4CMA Tαura3-52 lys2Δ201 leu2-3, 112 trp1-289 his3Δ1 Δ(hht1-hhf1)(hht2-hhf2) pCY338 [CEN ARS LEU2 hht1(Δ1-10)-HHF1]This workCY6-4CMA Tαura3-52 lys2Δ201 leu2-3, 112 trp1-289 his3Δ1 Δ(hht1-hhf1)(hht2-hhf2) pCY348 [CEN ARS LEU2 hht1(Δ1-5)-HHF1]This workCLY460MA Tαura3-52 lys2Δ201 leu2-3, 112 trp1-289 his3Δ1 Δ(hht1-hhf1)(hht2-hhf2) pCL460 [CEN ARS LEU2 hht1-3(K4,9,14,18,23,27Q)-HHF1]44CYY101MA Ta ura3-52 leu2-3, 112 gcn4-2 bas1-2 bas2-2 Δ(HHT2-HHF2) pCY101 [CEN ARS LEU2 hht1H3(K4Q)-HHF1] pRS416-DED1pr-GCN4This workCYY201MA Ta ura3-52 leu2-3, 112 gcn4-2 bas1-2 bas2-2 Δ(HHT2-HHF2) pCY201 [CEN ARS LEU2 hht1H3(K4R)-HHF1] pRS416-DED1pr-GCN4This workCYY102MA Ta ura3-52 leu2-3, 112 gcn4-2 bas1-2 bas2-2 Δ(HHT2-HHF2) pCY102 [CEN ARS LEU2 hht1H3(K14Q)-HHF1] pRS416-DED1pr-GCN4This workCYY202MA Ta ura3-52 leu2-3, 112 gcn4-2 bas1-2 bas2-2 Δ(HHT2-HHF2) pCY202 [CEN ARS LEU2 hht1H3(K14R)-HHF1] pRS416-DED1pr-GCN4This workCYY103MA Ta ura3-52 leu2-3, 112 gcn4-2 bas1-2 bas2-2 Δ(HHT2-HHF2) pCY103 [CEN ARS LEU2 hht1H3(K18Q)-HHF1] pRS416-DED1pr-GCN4This workCYY203MA Ta ura3-52 leu2-3, 112 gcn4-2 bas1-2 bas2-2 Δ(HHT2-HHF2) pCY203 [CEN ARS LEU2 hht1H3(K18R)-HHF1] pRS416-DED1pr-GCN4This work Open table in a new tab The LYY256 and MX1-4C derivative strains were generated by plasmid shuffling. Genes encoding histone mutants were amplified by PCR from the resulting yeast strains and verified by sequencing. The gcn5Δ strain CY8-4C was constructed by transforming CY1-4C with a PCR fragment obtained by amplifying gcn5::KanMx and flanking sequences from the corresponding yeast deletion collection strain (32Giaever G. Chu A.M. Ni L. Connelly C. Riles L. Veronneau S. Dow S. Lucau-Danila A. Anderson K. Andre B. Arkin A.P. Astromoff A. El-Bakkoury M. Bangham R. Benito R. Brachat S. Campanaro S. Curtiss M. Davis K. Deutschbauer A. Entian K.D. Flaherty P. Foury F. Garfinkel D.J. Gerstein M. Gotte D. Guldener U. Hegemann J.H. Hempel S. Herman Z. Jaramillo D.F. Kelly D.E. Kelly S.L. Kotter P. LaBonte D. Lamb D.C. Lan N. Liang H. Liao H. Liu L. Luo C. Lussier M. Mao R. Menard P. Ooi S.L. Revuelta J.L. Roberts C.J. Rose M. Ross-Macdonald P. Scherens B. Schimmack G. Shafer B. Shoemaker D.D. Sookhai-Mahadeo S. Storms R.K. Strathern J.N. Valle G. Voet M. Volckaert G. Wang C.Y. Ward T.R. Wilhelmy J. Winzeler E.A. Yang Y. Yen G. Youngman E. Yu K. Bussey H. Boeke J.D. Snyder M. Philippsen P. Davis R.W. Johnston M. Nature. 2002; 418: 387-391Crossref PubMed Scopus (3267) Google Scholar) and selecting on geneticin-containing plates. Yeast cells were grown at 30 °C in complete synthetic dropout medium (CSM-) (6.7 g/liter yeast nitrogen base without amino acids, 2% glucose, and CSM dropout mixture (Bio101)). 5-Fluoroorotic acid was used at 1 g/liter with addition of 50 mg/liter uracil. Rich medium (YPD) contained 1% Bacto-yeast extract, 2% Bacto-peptone, and 2% glucose. Yeast transformations were performed using a standard lithium acetate method (33Hill J. Donald K.A. Griffiths D.E. Donald G. Nucleic Acids Res. 1991; 19: 5791Crossref PubMed Scopus (460) Google Scholar). Northern Analysis—RNA was prepared from log phase cultures by the hot phenol method (34Schmitt M.E. Brown T.A. Trumpower B.L. Nucleic Acids Res. 1990; 18: 3091-3092Crossref PubMed Scopus (1155) Google Scholar). Northern blotting was performed as described previously (35Tsang S.S. Yin X. Guzzo-Arkuran C. Jones V.S. Davison A.J. Bio-Techniques. 1993; 14: 380-381Google Scholar) and blots were hybridized using probes produced by random primer labeling. Prior to reprobing, Northern membranes were stripped by rinsing 2-4 min in near boiling solution with 15 mm NaCl, 0.1× SSC, 1% SDS. Northern blots were scanned on a Amersham Biosciences Phosphorimager and quantitated using ImageQuant. The XhoI-XbaI fragment of pDN42 (a gift of Dr. D. Nag) containing most of the HIS4 sequence was used as the HIS4 Northern probe. The BglII fragment of pGEM-PYK1 was used as the PYK1 Northern probe (a gift from Dr. J. Curcio). The Northern probes for HIS3, HOM2, LYS1, ARG1, TRP2, and SAM2 were generated by PCR (primer sequences available upon request). Transcript levels were normalized to PYK1 mRNA. For normalization across a set of samples (e.g. from the series of histone H3 mutants in Fig. 1, A and B), the transcript levels (normalized to PYK1) were summed for each experiment (e.g. HIS3/PYK1 values for wild-type, H3Δ1-5, etc. were summed for experiment 1, where transcript levels from each yeast strain were measured on the same Northern blot) and the sums were set to the same arbitrary value for each experiment. The renormalized values thus obtained for each individual transcript were then used to obtain averages and standard deviations for each transcript as shown in the figures. Microarray Analysis and Computational Methods—RNA was prepared from exponentially growing yeast cultured in CSM dropout medium using the Masterpure Yeast RNA purification kit (Epicenter Technology, Madison, WI). RNA was further purified using the RNeasy purification kit (Qiagen). Processing and hybridization to Affymetrix (Santa Clara, CA) S98 microarrays were done according to the manufacturer's protocol as described previously (36Sabet N. Tong F. Madigan J.P. Volo S. Smith M.M. Morse R.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4084-4089Crossref PubMed Scopus (39) Google Scholar). Changes in gene expression were derived by averaging log2 expression changes. False discovery rates (FDRs) were derived according to Storey (37Storey J.D. J. R. Stat. Soc. B. 2002; 64: 479-498Crossref Scopus (3910) Google Scholar). p Values for overlaps were derived using a hypergeometric distribution. Comparative analysis and clustering were done using Excel (Microsoft), Genetraffic (Iobion Informatics), and Genespring (Affymetrix). Enrichment of gene sets in specific functional categories, as defined by the MIPS data base (38Mewes H.W. Frishman D. Guldener U. Mannhaupt G. Mayer K. Mokrejs M. Morgenstern B. Munsterkotter M. Rudd S. Weil B. 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Biosci. 1989; 5: 89-96PubMed Google Scholar) to report sites that match the model at a chosen level of statistical significance. Microarray Accession Number—Microarray gene expression data are available at the Gene Expression Omnibus under accession number GSE4135. Effects of Histone H3 Amino-terminal Deletions and Lys to Gln Mutation on Transcriptional Activation of Selected Genes—To examine the role of the histone H3 amino terminus in transcriptional activation, we constructed a series of yeast strains having deletions of the first 5, 10, 15, or 20 amino acids of histone H3. We then used these strains, together with previously described strains (44Sabet N. Volo S. Yu C. Madigan J.P. Morse R.H. Mol. Cell. Biol. 2004; 24: 8823-8833Crossref PubMed Scopus (45) Google Scholar) lacking the first 28 amino acids of histone H3 or having the 6 lysine residues replaced by glutamines (Lys to Gln mutant), and the corresponding wild-type strains (Table 1), to analyze the levels of six Gcn4p-dependent transcripts (HIS4, HOM2, LYS1, HIS3, ARG1, and TRP2) and one control transcript (SAM2) that does not show Gcn4p dependence (29Natarajan K. Meyer M.R. Jackson B.M. Slade D. Roberts C. Hinnebusch A.G. Marton M.J. Mol. Cell. Biol. 2001; 21: 4347-4368Crossref PubMed Scopus (569) Google Scholar). Based on microarray data from the Hinnebusch laboratory (29Natarajan K. Meyer M.R. Jackson B.M. Slade D. Roberts C. Hinnebusch A.G. Marton M.J. Mol. Cell. Biol. 2001; 21: 4347-4368Crossref PubMed Scopus (569) Google Scholar), these transcripts show from 3- to 15-fold induction after treatment with 100 mm 3-aminotriazole (3-AT), and their induced levels of transcripts are reduced 6-19-fold in gcn4Δ yeast (Table 2). A lower (10 mm) concentration of 3-AT results in 4.5-20-fold induction, with the exception of TRP2, which is not induced. We confirmed the Gcn4p dependence of these genes in one of the two strains (both derived from strain S288C) used here, and found results generally consistent with those obtained by the Hinnebusch laboratory (29Natarajan K. Meyer M.R. Jackson B.M. Slade D. Roberts C. Hinnebusch A.G. Marton M.J. Mol. Cell. Biol. 2001; 21: 4347-4368Crossref PubMed Scopus (569) Google Scholar), although we did observe a modest dependence of SAM2 expression on Gcn4p not found previously (Table 2). After this work was underway, a genome-wide location analysis study yielded data indicating that five of the GCN4-dependent transcripts examined here (HIS4, LYS1, HIS3, ARG1, and TRP2) are indeed direct targets of Gcn4p (having p values for Gcn4p association of <2 × 10-4, where 1 × 10-3 is judged to be significant), whereas binding of Gcn4p to the HOM2 (and SAM2) promoter was not observed (29Natarajan K. Meyer M.R. Jackson B.M. Slade D. Roberts C. Hinnebusch A.G. Marton M.J. Mol. Cell. Biol. 2001; 21: 4347-4368Crossref PubMed Scopus (569) Google Scholar). HOM2 may therefore be indirectly regulated by Gcn4p.TABLE 2Dependence on GCN4 and induction by 3AT of genes examined in this workGeneGCN4+/gcn4-aFrom Natarajan et al. (29).10 mm 3-ATaFrom Natarajan et al. (29).100 mm 3-ATaFrom Natarajan et al. (29).GCN4+/gcn4-bAverage from two independent determinations (strain LYY256 with and without pRS416-DED1-GCN4).HIS418.689.416.2HIS39.410.28.1NDLYS113.520.415.58.4ARG19311.612.342TRP25.80.822.82.8HOM211.54.53.14.4SAM20.730.230.182.7a From Natarajan et al. (29).b Average from two independent determinations (strain LYY256 with and without pRS416-DED1-GCN4). Open table in a new tab We chose to examine Gcn4p-dependent transcripts because a large number of genes are regulated by this well studied transcriptional activator, and considerable knowledge exists regarding Gcn4p-mediated transcriptional activation (29Natarajan K. Meyer M.R. Jackson B.M. Slade D. Roberts C. Hinnebusch A.G. Marton M.J. Mol. Cell. Biol. 2001; 21: 4347-4368Crossref PubMed Scopus (569) Google Scholar, 45Harbison C.T. Gordon D.B. Lee T.I. Rinaldi N.J. Macisaac K.D. Danford T.W. Hannett N.M. Tagne J.B. Reynolds D.B. Yoo J. Jennings E.G. Zeitlinger J. Pokholok D.K. Kellis M. Rolfe P.A. Takusagawa K.T. Lander E.S. 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