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• W2078199021 abstract "The Rpd3 histone deacetylase (HDAC) functions in a large complex containing many proteins including Sin3 and Sap30. Previous evidence indicates that the pho23,rpd3, sin3, and sap30 mutants exhibit similar defects in PHO5 regulation. We report thatpho23 mutants like rpd3, sin3, andsap30 are hypersensitive to cycloheximide and heat shock and exhibit enhanced silencing of rDNA, telomeric, andHMR loci, suggesting that these genes are functionally related. Based on these observations, we explored whether Pho23 is a component of the Rpd3 HDAC complex. Our results demonstrate that Myc-Pho23 co-immunoprecipitates with HA-Rpd3 and HA-Sap30. Furthermore, similar levels of HDAC activity were detected in immunoprecipitates of HA-Pho23, HA-Rpd3, or HA-Sap30. In contrast, HDAC activity was not detected in immunoprecipitates of HA-Pho23 or HA-Sap30 from strains lacking Rpd3, suggesting that Rpd3 is the HDAC associated with these proteins. However, HDAC activity was detected in immunoprecipitates of HA-Sap30 or HA-Rpd3 from cells lacking Pho23, although levels were significantly lower than those detected in wild-type cells, indicating that Rpd3 activity is compromised in the absence of Pho23. Together, our genetic and biochemical studies provide strong evidence that Pho23 is a component of the Rpd3 HDAC complex, and is required for the normal function of this complex. The Rpd3 histone deacetylase (HDAC) functions in a large complex containing many proteins including Sin3 and Sap30. Previous evidence indicates that the pho23,rpd3, sin3, and sap30 mutants exhibit similar defects in PHO5 regulation. We report thatpho23 mutants like rpd3, sin3, andsap30 are hypersensitive to cycloheximide and heat shock and exhibit enhanced silencing of rDNA, telomeric, andHMR loci, suggesting that these genes are functionally related. Based on these observations, we explored whether Pho23 is a component of the Rpd3 HDAC complex. Our results demonstrate that Myc-Pho23 co-immunoprecipitates with HA-Rpd3 and HA-Sap30. Furthermore, similar levels of HDAC activity were detected in immunoprecipitates of HA-Pho23, HA-Rpd3, or HA-Sap30. In contrast, HDAC activity was not detected in immunoprecipitates of HA-Pho23 or HA-Sap30 from strains lacking Rpd3, suggesting that Rpd3 is the HDAC associated with these proteins. However, HDAC activity was detected in immunoprecipitates of HA-Sap30 or HA-Rpd3 from cells lacking Pho23, although levels were significantly lower than those detected in wild-type cells, indicating that Rpd3 activity is compromised in the absence of Pho23. Together, our genetic and biochemical studies provide strong evidence that Pho23 is a component of the Rpd3 HDAC complex, and is required for the normal function of this complex. histone acetyltransferase histone deacetylase plant homeodomain polymerase chain reaction green fluorescent protein phosphate-buffered saline Modifications of chromatin by histone acetyltransferases (HATs)1 and histone deacetylases (HDACs) play important roles in transcriptional regulation (1Struhl K. Genes Dev. 1998; 12: 599-606Crossref PubMed Scopus (1546) Google Scholar, 2Sterner D.E. Berger S.L. Microbiol. Mol. Biol. Rev. 2000; 64: 435-459Crossref PubMed Scopus (1398) Google Scholar, 3Cheung W.L. Briggs S.D. Allis C.D. Curr. Opin. Cell Biol. 2000; 12: 326-333Crossref PubMed Scopus (260) Google Scholar, 4Brown C.E. Lechner T. Howe L. Workman J.L. Trends Biochem. Sci. 2000; 25: 15-19Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). Many proteins possessing intrinsic HAT activity have been identified from various organisms, and many of these proteins have been shown to be transcriptional coactivators or have other transcription-related functions. Similarly, several HDACs have been identified in different organisms as multiprotein complexes that are associated with transcriptional repressors and co-repressors (5Ayer D.E. Trends Cell Biol. 1999; 9: 193-198Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 6Davie J.R. Curr. Opin. Genet. Dev. 1998; 8: 173-178Crossref PubMed Scopus (178) Google Scholar, 7Kornberg R.D. Lorch Y. Curr. Opin. Genet. Dev. 1999; 9: 148-151Crossref PubMed Scopus (198) Google Scholar). In many cases, HATs and HDACs are targeted to specific promoters through their interaction with DNA-binding transcription factors, suggesting that they regulate transcriptional activity by modifying the local chromatin structure at target promoters (8Kuo M.H. Zhou J. Jambeck P. Churchill M.E. Allis C.D. Genes Dev. 1998; 12: 627-639Crossref PubMed Scopus (398) Google Scholar, 9Kadosh D. Struhl K. Mol. Cell. Biol. 1998; 18: 5121-5127Crossref PubMed Scopus (268) Google Scholar, 10Rundlett S.E. Carmen A.A. Suka N. Turner B.M. Grunstein M. Nature. 1998; 392: 831-835Crossref PubMed Scopus (363) Google Scholar). However, recent reports suggest that HATs also function in an untargeted manner to acetylate histones on a genome-wide scale (11Reid J.L. Iyer V.R. Brown P.O. Struhl K. Mol. Cell. 2000; 6: 1297-1307Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 12Kuo M. vom, B. E. Struhl K. Allis C.D. Mol. Cell. 2000; 6: 1309-1320Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Packaging of DNA into chromatin is thought to affect transcription by impeding the access of transcription factors to DNA regulatory sequences. HATs acetylate lysine residues on core histones, thereby neutralizing the positive charge of the histone tails and decreasing their affinity for DNA and/or adjacent nucleosomes in higher order chromosomal structures (7Kornberg R.D. Lorch Y. Curr. Opin. Genet. Dev. 1999; 9: 148-151Crossref PubMed Scopus (198) Google Scholar, 13Hong L. Schroth G.P. Matthews H.R. Yau P. Bradbury E.M. J. Biol. Chem. 1993; 268: 305-314Abstract Full Text PDF PubMed Google Scholar). Such a modification of chromatin is thought to increase the accessibility of DNA to transcription regulatory complexes (14Lee D.Y. Hayes J.J. Pruss D. Wolffe A.P. Cell. 1993; 72: 73-84Abstract Full Text PDF PubMed Scopus (964) Google Scholar, 15Vettese-Dadey M. Grant P.A. Hebbes T.R., C Allis C.D. Workman J.L. EMBO J. 1996; 15: 2508-2518Crossref PubMed Scopus (376) Google Scholar). Thus, in general, hyperacetylation of histones correlates with activation of gene expression, whereas deacetylation represses transcription (16Hebbes T.R. Thorne A.W. Crane-Robinson C. EMBO J. 1988; 7: 1395-1402Crossref PubMed Scopus (707) Google Scholar, 17Braunstein M. Rose A.B. Holmes S.G. Allis C.D. Broach J.R. Genes Dev. 1993; 7: 592-604Crossref PubMed Scopus (711) Google Scholar). Consistent with this model, the targeted recruitment of the Gcn5 HAT to specific promoters correlates with both transcriptional activation and acetylation of core histones in the vicinity of the promoters, and the HAT activity of Gcn5 is required for transcriptional activation of such target genes (8Kuo M.H. Zhou J. Jambeck P. Churchill M.E. Allis C.D. Genes Dev. 1998; 12: 627-639Crossref PubMed Scopus (398) Google Scholar,12Kuo M. vom, B. E. Struhl K. Allis C.D. Mol. Cell. 2000; 6: 1309-1320Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Also consistent with this model, targeted recruitment of the Rpd3 HDAC complex to specific promoters correlates with both decreased acetylation and transcriptional repression, and Rpd3 catalytic activity is important for transcriptional repression (9Kadosh D. Struhl K. Mol. Cell. Biol. 1998; 18: 5121-5127Crossref PubMed Scopus (268) Google Scholar, 10Rundlett S.E. Carmen A.A. Suka N. Turner B.M. Grunstein M. Nature. 1998; 392: 831-835Crossref PubMed Scopus (363) Google Scholar, 18Kadosh D. Struhl K. Genes Dev. 1998; 12: 797-805Crossref PubMed Scopus (205) Google Scholar). However, in contradiction to this model, transcriptional silencing of centromeric heterochromatin of Drosophila melanogaster and the yeast mating-type loci require acetylation of histone H4 (19Turner B.M. Birley A.J. Lavender J. Cell. 1992; 69: 375-384Abstract Full Text PDF PubMed Scopus (466) Google Scholar, 20Braunstein M. Sobel R.E. Allis C.D. Turner B.M. Broach J.R. Mol. Cell. Biol. 1996; 16: 4349-4356Crossref PubMed Scopus (328) Google Scholar) and mutation of Rpd3 enhances silencing at rDNA, telomeric, and mating-type loci (21De Rubertis F. Kadosh D. Henchoz S. Pauli D. Reuter G. Struhl K. Spierer P. Nature. 1996; 384: 589-591Crossref PubMed Scopus (194) Google Scholar, 22Rundlett S.E. Carmen A.A. Kobayashi R. Bavykin S. Turner B.M. Grunstein M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14503-14508Crossref PubMed Scopus (522) Google Scholar, 23Vannier D. Balderes D. Shore D. Genetics. 1996; 144: 1343-1353Crossref PubMed Google Scholar, 24Smith J.S. Caputo E. Boeke J.D. Mol. Cell. Biol. 1999; 19: 3184-3197Crossref PubMed Scopus (185) Google Scholar, 25Sun Z.W. Hampsey M. Genetics. 1999; 152: 921-932Crossref PubMed Google Scholar), implying that Rpd3 functions to counteract rather than to establish or maintain silencing. One possible explanation for how acetylation is required for both activation and silencing of genes is that the acetylated states of different regions of chromatin may provide distinct recognition signals for transcriptional activator and repressor protein complexes (25Sun Z.W. Hampsey M. Genetics. 1999; 152: 921-932Crossref PubMed Google Scholar, 26Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6578) Google Scholar). Loss of Rpd3 may also indirectly enhance silencing of telomeric genes sensitive to histone depletion through an increase in histone gene expression (27Bernstein B.E. Tong J.K. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13708-13713Crossref PubMed Scopus (368) Google Scholar). Genetic and biochemical studies of yeast Rpd3 and the human homologs HDAC1 and HDAC2 first established the role of HDACs in gene regulation. Rpd3 is the catalytic component of a large multiprotein complex that contains Sin3, Sap30, Sds3, and many other proteins (22Rundlett S.E. Carmen A.A. Kobayashi R. Bavykin S. Turner B.M. Grunstein M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14503-14508Crossref PubMed Scopus (522) Google Scholar, 28Kasten M.M. Dorland S. Stillman D.J. Mol. Cell. Biol. 1997; 17: 4852-4858Crossref PubMed Scopus (114) Google Scholar, 29Kadosh D. Struhl K. Cell. 1997; 89: 365-371Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 30Zhang Y. Sun Z.W. Iratni R. Erdjument-Bromage H. Tempst P. Hampsey M. Reinberg D. Mol. Cell. 1998; 1: 1021-1031Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 31Laherty C.D. Billin A.N. Lavinsky R.M. Yochum G.S. Bush A.C. Sun J.M. Mullen T.M. Davie J.R. Rose D.W. Glass C.K. Rosenfeld M.G. Ayer D.E. Eisenman R.N. Mol. Cell. 1998; 2: 33-42Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 32Lechner T. Carrozza M.J., Yu, Y. Grant P.A. Eberharter A. Vannier D. Brosch G. Stillman D.J. Shore D. Workman J.L. J. Biol. Chem. 2000; 275: 40961-40966Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Some of these papers report the identification of a smaller (∼0.6 MDa) HDAC complex containing Rpd3 (22Rundlett S.E. Carmen A.A. Kobayashi R. Bavykin S. Turner B.M. Grunstein M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14503-14508Crossref PubMed Scopus (522) Google Scholar, 32Lechner T. Carrozza M.J., Yu, Y. Grant P.A. Eberharter A. Vannier D. Brosch G. Stillman D.J. Shore D. Workman J.L. J. Biol. Chem. 2000; 275: 40961-40966Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar); however, it is not clear whether these reports reflect the existence of structurally and functionally distinct Rpd3 complexes in vivo or whether they represent different forms of the same complex. Several components of the Rpd3 HDAC complex, including Rpd3, Sin3, and Sap30, have been highly conserved both structurally and functionally in eucaryotes (30Zhang Y. Sun Z.W. Iratni R. Erdjument-Bromage H. Tempst P. Hampsey M. Reinberg D. Mol. Cell. 1998; 1: 1021-1031Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 31Laherty C.D. Billin A.N. Lavinsky R.M. Yochum G.S. Bush A.C. Sun J.M. Mullen T.M. Davie J.R. Rose D.W. Glass C.K. Rosenfeld M.G. Ayer D.E. Eisenman R.N. Mol. Cell. 1998; 2: 33-42Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar,33Ayer D.E. Lawrence Q.A. Eisenman R.N. Cell. 1995; 80: 767-776Abstract Full Text PDF PubMed Scopus (524) Google Scholar, 34Taunton J. Hassig C.A. Schreiber S.L. Science. 1996; 272: 408-411Crossref PubMed Scopus (1530) Google Scholar, 35Yang W.M. Inouye C. Zeng Y. Bearss D. Seto E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12845-12850Crossref PubMed Scopus (482) Google Scholar). In mammals, the HDAC1,2·Sin3·Sap30 complex mediates transcriptional repression by interacting with the Mad family of DNA-binding transcriptional repressors (33Ayer D.E. Lawrence Q.A. Eisenman R.N. Cell. 1995; 80: 767-776Abstract Full Text PDF PubMed Scopus (524) Google Scholar, 36Schreiber-Agus N. Chin L. Chen K. Torres R. Rao G. Guida P. Skoultchi A.I. DePinho R.A. Cell. 1995; 80: 777-786Abstract Full Text PDF PubMed Scopus (335) Google Scholar, 37Laherty C.D. Yang W.M. Sun J.M. Davie J.R. Seto E. Eisenman R.N. Cell. 1997; 89: 349-356Abstract Full Text Full Text PDF PubMed Scopus (842) Google Scholar) and the nuclear hormone receptors N-CoR and SMRT (31Laherty C.D. Billin A.N. Lavinsky R.M. Yochum G.S. Bush A.C. Sun J.M. Mullen T.M. Davie J.R. Rose D.W. Glass C.K. Rosenfeld M.G. Ayer D.E. Eisenman R.N. Mol. Cell. 1998; 2: 33-42Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 38Alland L. Muhle R. Hou H. Potes J. Chin L. Schreiber-Agus N. DePinho R.A. Nature. 1997; 387: 49-55Crossref PubMed Scopus (735) Google Scholar, 39Heinzel T. Lavinsky R.M. Mullen T.M. Soderstrom M. Laherty C.D. Torchia J. Yang W.M. Brard G. Ngo S.D. Davie J.R. Seto E. Eisenman R.N. Rose D.W. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 43-48Crossref PubMed Scopus (1081) Google Scholar, 40Nagy L. Kao H.Y. Chakravarti D. Lin R.J. Hassig C.A. Ayer D.E. Schreiber S.L. Evans R.M. Cell. 1997; 89: 373-380Abstract Full Text Full Text PDF PubMed Scopus (1103) Google Scholar). Similarly, in yeast, the Rpd3 complex is required for transcriptional repression by Ume6, a zinc finger protein that binds URS1 elements and regulates genes involved in meiosis and arginine catabolism (29Kadosh D. Struhl K. Cell. 1997; 89: 365-371Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). In yeast, mutants of Rpd3, Sin3, and Sds3 are associated with similar defects in the regulation of many target genes, including PHO5 (27Bernstein B.E. Tong J.K. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13708-13713Crossref PubMed Scopus (368) Google Scholar,41Vidal M. Gaber R.F. Mol. Cell. Biol. 1991; 11: 6317-6327Crossref PubMed Scopus (262) Google Scholar, 42Vidal M. Strich R. Esposito R.E. Gaber R.F. Mol. Cell. Biol. 1991; 11: 6306-6316Crossref PubMed Scopus (142) Google Scholar, 43Dorland S. Deegenaars M.L. Stillman D.J. Genetics. 2000; 154: 573-586Crossref PubMed Google Scholar), and exhibit enhanced silencing of rDNA, telomeric, and mating-type loci (22Rundlett S.E. Carmen A.A. Kobayashi R. Bavykin S. Turner B.M. Grunstein M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14503-14508Crossref PubMed Scopus (522) Google Scholar, 23Vannier D. Balderes D. Shore D. Genetics. 1996; 144: 1343-1353Crossref PubMed Google Scholar, 24Smith J.S. Caputo E. Boeke J.D. Mol. Cell. Biol. 1999; 19: 3184-3197Crossref PubMed Scopus (185) Google Scholar, 25Sun Z.W. Hampsey M. Genetics. 1999; 152: 921-932Crossref PubMed Google Scholar, 43Dorland S. Deegenaars M.L. Stillman D.J. Genetics. 2000; 154: 573-586Crossref PubMed Google Scholar). rpd3, sin3, andsap30 mutants also exhibit other similar phenotypes, including hypersensitivity to cycloheximide, mating defects, and an inability to sporulate as homozygous diploids (30Zhang Y. Sun Z.W. Iratni R. Erdjument-Bromage H. Tempst P. Hampsey M. Reinberg D. Mol. Cell. 1998; 1: 1021-1031Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 41Vidal M. Gaber R.F. Mol. Cell. Biol. 1991; 11: 6317-6327Crossref PubMed Scopus (262) Google Scholar). Deletion ofRPD3 has also been shown to extend the life-span of yeast (44Kim S. Benguria A. Lai C.Y. Jazwinski S.M. Mol. Biol. Cell. 1999; 10: 3125-3136Crossref PubMed Scopus (181) Google Scholar). In addition to Rpd3, their are five other yeast HDACs, including Hda1, Hos1, Hos2, Hos3, and Sir2 (7Kornberg R.D. Lorch Y. Curr. Opin. Genet. Dev. 1999; 9: 148-151Crossref PubMed Scopus (198) Google Scholar, 22Rundlett S.E. Carmen A.A. Kobayashi R. Bavykin S. Turner B.M. Grunstein M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14503-14508Crossref PubMed Scopus (522) Google Scholar), and HDAC complexes containing Hda1 and Hos3 have been identified (22Rundlett S.E. Carmen A.A. Kobayashi R. Bavykin S. Turner B.M. Grunstein M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14503-14508Crossref PubMed Scopus (522) Google Scholar, 45Carmen A.A. Griffin P.R. Calaycay J.R. Rundlett S.E. Suka Y. Grunstein M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12356-12361Crossref PubMed Scopus (73) Google Scholar). hda1 mutants, likerpd3 mutants, exhibit decreased acetylation of histones H3 and H4 and increased silencing at telomeric loci (22Rundlett S.E. Carmen A.A. Kobayashi R. Bavykin S. Turner B.M. Grunstein M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14503-14508Crossref PubMed Scopus (522) Google Scholar). Sir2 has also been defined genetically as being required for silencing in addition to DNA recombination, repair, and longevity (46Kaeberlein M. McVey M. Guarente L. Genes Dev. 1999; 13: 2570-2580Crossref PubMed Scopus (1758) Google Scholar) (reviewed in Refs. 47Grunstein M. Cell. 1998; 93: 325-328Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar and48Haber J.E. Cell. 1999; 97: 829-832Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Pho23 was identified by a screen for yeast mutants that constitutively express PHO5 (49Lau W.W. Schneider K.R. O'Shea E.K. Genetics. 1998; 150: 1349-1359PubMed Google Scholar), suggesting that Pho23 is linked genetically and functionally to the Rpd3 HDAC complex. Pho23 is closely related to two other yeast proteins, Yng1 and Yng2, and human Ing1, a candidate tumor suppressor (50Loewith R. Meijer M. Lees-Miller S.P. Riabowol K. Young D. Mol. Cell. Biol. 2000; 20: 3807-3816Crossref PubMed Scopus (141) Google Scholar). The carboxyl-terminal regions of these proteins contain PHD domains, which are found in several proteins implicated in chromatin-mediated gene regulation (51Aasland R. Gibson T.J. Stewart A.F. Trends Biochem. Sci. 1995; 20: 56-59Abstract Full Text PDF PubMed Scopus (754) Google Scholar, 52Jacobson S. Pillus L. Curr. Opin. Genet. Dev. 1999; 9: 175-184Crossref PubMed Scopus (111) Google Scholar). We have previously shown that Yng1 and Yng2 are associated with specific HAT complexes in yeast (50Loewith R. Meijer M. Lees-Miller S.P. Riabowol K. Young D. Mol. Cell. Biol. 2000; 20: 3807-3816Crossref PubMed Scopus (141) Google Scholar). In this paper we report evidence that Pho23 is associated with an Rpd3 HDAC complex and that it is required for the normal function of Rpd3 in the silencing of rDNA, telomeric, and mating-type loci. The genotypes of yeast strains used in this study are listed in TableI . Saccharomyces cerevisiaeculture, transformation, mating, tetrad analysis, and other genetic manipulations were performed as described previously (53Adams A. Gottschling D.E. Kaiser C.A. Stearns T. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Plainview, NY1998Google Scholar,54Elble R. BioTechniques. 1992; 13: 18-20PubMed Google Scholar).Table IS. cerevisiae strainsStrainGenotypeSourceBY4742Matα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0Research Geneticsrpd3ΔBY4742 rpd3Δ∷kanMX4Research Geneticssap30ΔBY4742 sap30Δ∷kanMX4Research Geneticspho23ΔMatα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pho23Δ∷kanMX4Research GeneticsJC1Matα ade8 his3 leu2 lys2 trp1 ura3 can1J. Colicelli, UCLAJS311Matα his3Δ200 leu2Δ1 met15Δ0 trp1Δ63 ura3–167 RDN1∷Ty1-MET15, mURA3/HIS3Ref. 24Smith J.S. Caputo E. Boeke J.D. Mol. Cell. Biol. 1999; 19: 3184-3197Crossref PubMed Scopus (185) Google ScholarJS493JS311 sin3Δ∷kanMX4Ref. 24Smith J.S. Caputo E. Boeke J.D. Mol. Cell. Biol. 1999; 19: 3184-3197Crossref PubMed Scopus (185) Google ScholarJS694YNB9rpd3Δ∷kanMX4This studyJS753YCB647sin3Δ∷kanMX4This studyJS754YCB647rpd3Δ∷kanMX4This studyJS755YCB647rpd3Δ∷kanMX4This studyJS756YNB9sin3Δ∷kanMX4This studyJS758YNB9pho23Δ∷kanMX4This studyJS767JS311pho23Δ∷kanMX4This studyJS768JS311pho23Δ∷kanMX4 rpd3Δ∷kanMX4This studyM475JS311sap30∷mTn3∷LEU2∷lacZRef. 24Smith J.S. Caputo E. Boeke J.D. Mol. Cell. Biol. 1999; 19: 3184-3197Crossref PubMed Scopus (185) Google ScholarM480JS311rpd3∷mTn3∷LEU2∷lacZRef. 24Smith J.S. Caputo E. Boeke J.D. Mol. Cell. Biol. 1999; 19: 3184-3197Crossref PubMed Scopus (185) Google ScholarTWY7YCB647pho23Δ∷kanMX4This studyTWY9YCB647pho23Δ∷kanMX4This studyTWY17YLS59pho23Δ∷kanMX4This studyYCB647MATa his3Δ200 leu2Δ1∷TRP1 lys2Δ202 trp1Δ63 ura3–52 ADH4∷TEL∷URA3Ref. 62Smith J.S. Brachmann C.B. Celic I. Kenna M.A. Muhammad S. Starai V.J. Avalos J.L. Escalante-Semerena J.C. Grubmeyer C. Wolberger C. Boeke J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6658-6663Crossref PubMed Scopus (614) Google ScholarYLS59MATα ade2–1 can1–100 his3–11,15 leu2–3,112 trp1–1 ura3–1 hmrΔA∷TRP1Ref. 63Sussel L. Shore D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7749-7753Crossref PubMed Scopus (180) Google ScholarYNB9MATα ade2Δ∷hisG his3Δ200 leu2Δ1 lys2Δ0 met15Δ0 ura3–167 RDN1∷Ty1-MET15, mURA3/HIS3 TELV∷ADE2This studyYSK661Matα ura3–52 lys2–801 ade2–101 trp1-Δ63 his3-Δ200 leu2-Δ1 rpd3Δ∷URA3Ref. 44Kim S. Benguria A. Lai C.Y. Jazwinski S.M. Mol. Biol. Cell. 1999; 10: 3125-3136Crossref PubMed Scopus (181) Google ScholarYSK663Matα ura3–52 lys2–801 ade2–101 trp1-Δ63 his3-Δ200 leu2-Δ1Ref. 44Kim S. Benguria A. Lai C.Y. Jazwinski S.M. Mol. Biol. Cell. 1999; 10: 3125-3136Crossref PubMed Scopus (181) Google ScholarYSK664Matα ura3–52 lys2–801 ade2–101 trp1-Δ63 his3-Δ200 leu2-Δ1 hda1Δ∷HIS3Ref. 44Kim S. Benguria A. Lai C.Y. Jazwinski S.M. Mol. Biol. Cell. 1999; 10: 3125-3136Crossref PubMed Scopus (181) Google Scholar Open table in a new tab Procedures used for DNA manipulation and analysis (purification, cloning, electrophoresis, transformation, etc.) were described previously (55Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory Press, Plainview, NY1989Google Scholar). PCR was performed as described previously (56Matviw H., Yu, G. Young D. Mol. Cell. Biol. 1992; 12: 5033-5040Crossref PubMed Scopus (62) Google Scholar). pAD4H and pUAD6 contain the 2-μm origin of replication and ADH1 promoter and encode the HA-epitope and Myc epitope, respectively (57Hubberstey A., Yu, G. Loewith R. Lakusta C. Young D. J. Cell. Biochem. 1996; 61: 459-466Crossref PubMed Scopus (45) Google Scholar). pADGFPHA was derived by cloning the PCR-derived coding sequence of the enhanced green fluorescent protein (eGFP, CLONTECH) into pAD4H as described previously (50Loewith R. Meijer M. Lees-Miller S.P. Riabowol K. Young D. Mol. Cell. Biol. 2000; 20: 3807-3816Crossref PubMed Scopus (141) Google Scholar). pADHA-Pho23, pADHA-Rpd3, and pADHA-Sap30 were generated by cloning the PCR-derived open reading frames of Pho23, Rpd3, and Sap30 into pAD4H. pADMyc-Pho23 was generated by cloning the PCR-derived coding region of Pho23 in pUAD6. Proteins were isolated from yeast cultures grown in synthetic media to anA600 of ∼1.0. Cells from 150 ml of culture were collected by centrifugation, washed once with lysis buffer (20 mm Tris, pH 7.4, 150 mm NaCl, 2 mm EDTA, 0.2% Triton X-100), and resuspended in 2 ml of lysis buffer with protease inhibitors (1 μg/ml pepstatin A, 200 μm phenylmethylsulfonyl fluoride, 500 μmbenzamidine HCl, 10 μg/ml aprotinin, 1 μg/ml leupeptin). Cell suspensions were aliquoted into two tubes with 1.5 g of glass beads (425–600 nm, Sigma) and shaken for 5 min in a Mini-BeadBeater (Biospec Products) at 4 °C. Cell debris was removed by centrifugation for 2 min at 2500 rpm and 2 × 5 min at 10,000 rpm. Protein concentrations (typically 10–15 mg/ml) were determined at this point using a Bio-Rad Protein assay. 2 mg of protein was used in 1-ml immunoprecipitation reactions in lysis buffer with protease inhibitors at 4 °C with gentle rotation. Immunoprecipitation reactions were precleared with 40 μl of protein A-Sepharose beads for 20 min. After removal of the beads, 60 μl of protein A-Sepharose beads cross-linked to 12CA5 (anti-HA) was added, and reactions were incubated overnight. Beads were collected by a 1-min centrifugation at 2,200 rpm and washed 5–10 × with 1 ml of lysis buffer. For some experiments, one-third of the beads were removed at this point for HDAC assays. For Western analyses, 20 μl of protein sample buffer was added to the beads, boiled, and separated by SDS-PAGE. Proteins were electroblotted onto nitrocellulose membranes (1.5 h, 100 V), blocked for 1–12 h in 5% milk in TBS (137 mm NaCl, 2.7 mm KCl, 25 mm Tris, pH 7.4), and incubated 1 h with primary antibody (1:2000 12CA5, 1:30 9E10) in TBS + 0.05% Tween 20. After washing, tagged proteins were detected with horseradish peroxidase-conjugated anti-mouse secondary antibodies and ECL reagents (Amersham Pharmacia Biotech). HDAC assays were performed as described previously (58Hendzel M.J. Delcuve G.P. Davie J.R. J. Biol. Chem. 1991; 266: 21936-21942Abstract Full Text PDF PubMed Google Scholar, 59Kolle D. Brosch G. Lechner T. Lusser A. Loidl P. Methods ( San Diego ). 1998; 15: 323-331Crossref PubMed Scopus (95) Google Scholar), with minor modifications. Immunoprecipitates were rotated at 23 °C for 1 h in a total volume of 200 μl containing 5.5 μg of [3H]acetyl-labeled HeLa histones, 40 μl of 5× HDAC buffer (50 mm Tris-HCL, pH 8.0, 0.75 m NaCl, 50% glycerol), and ±50 μl of 1 m sodium butyrate. The reaction was stopped with the addition of 50 μl of Quench solution (259 μl of HCl, 28 μl of acetic acid, 2713 μl of H2O) and 600 μl of ethyl acetate. Samples were vortexed and spun for 1 min in a microcentrifuge, and 200 μl of the organic phase was counted to detect the released [3H]acetate. [3H]Acetyllysine-labeled histones were prepared from HeLa cells as described previously (60Carmen A.A. Rundlett S.E. Grunstein M. J. Biol. Chem. 1996; 271: 15837-15844Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar) with minor modifications. 1 liter of HeLa cells were grown to a density of 2 × 105 cells/ml, pelleted (1500 × g), resuspended in 25 ml of PBS (140 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, and 1.8 mm KH2PO4, pH 7.4) containing 100 μg/ml cycloheximide, 10 mm sodium butyrate, and 0.2 mCi/ml [3H]acetic acid, and incubated for 1 h at 37 °C. Cells were chilled on ice, washed three times in 10 ml of PBS plus 10 mm sodium butyrate, and then lysed in 8 ml of NIB (1% Nonidet P-40 in IB buffer (10 mmTris-HCl, pH 7.4, 2 mm MgCl2, 3 mmCaCl2, 10 mm sodium butyrate, and 1 mm phenylmethylsulfonyl fluoride)). Nuclei were harvested, washed twice in 8 ml of NIB, washed once in NIB with 100 mmNaCl, and washed once in 8 ml of IB with 100 mm NaCl. Nuclei were high salt-extracted twice in 8 ml of IB with 400 mm NaCl followed by centrifugation, and the nuclear pellet was extracted twice in 10 volumes of 0.2 mH2SO4 for 90 min on ice and centrifuged (30,000 × g, 25 min). Pooled supernatants were dialyzed extensively against 100 mm acetic acid at 4 °C, and the extracted histones were lyophilized and resuspended in H2O (4 mg/ml). 4 ml of histones were dialyzed against PBS; 1 ml of Quench solution and 12 ml of ethyl acetate were added; and the aqueous phase was recovered and dialyzed against 0.5× PBS, yielding prequenched [3H]acetyllysine-labeled histones (1.1 mg/ml, 2 × 106 cpm/mg). Mutants for RPD3, SIN3, andSAP30, which encode components of the Rpd3 HDAC complex, have previously been shown to exhibit derepressed silencing of rDNA, telomeric, and mating-type loci (22Rundlett S.E. Carmen A.A. Kobayashi R. Bavykin S. Turner B.M. Grunstein M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14503-14508Crossref PubMed Scopus (522) Google Scholar, 23Vannier D. Balderes D. Shore D. Genetics. 1996; 144: 1343-1353Crossref PubMed Google Scholar, 24Smith J.S. Caputo E. Boeke J.D. Mol. Cell. Biol. 1999; 19: 3184-3197Crossref PubMed Scopus (185) Google Scholar, 25Sun Z.W. Hampsey M. Genetics. 1999; 152: 921-932Crossref PubMed Google Scholar). We previously identified mutants in a genetic screen that either enhanced or suppressed silencing of rDNA (24Smith J.S. Caputo E. Boeke J.D. Mol. Cell. Biol. 1999; 19: 3184-3197Crossref PubMed Scopus (185) Google Scholar). Random Tn3 transposon insertions were generated in a strain that harbored three different polymerase II-transcribed reporter genes in the rDNA: MET15,HIS3, and mURA3 (24Smith J.S. Caputo E. Boeke J.D. Mol. Cell. Biol. 1999; 19: 3184-3197Crossref PubMed Scopus (185) Google Scholar, 61Smith J.S. Boeke J.D. Genes Dev. 1997; 11: 241-254Crossref PubMed Scopus (501) Google Scholar). Mutants that strengthened silencing of all three reporters were isolated, and the disrupted genes were named IRS (increasedrDNA silencing) (24Smith J.S. Caputo E. Boeke J.D. Mol. Cell. Biol. 1999; 19: 3184-3197Crossref PubMed Scopus (185) Google Scholar). Therefore, one of the normal functions of the IRS genes is to antagonize rDNA silencing. RPD3 (IRS2) and SAP30(IRS8) were both isolated from this screen (24Smith J.S. Caputo E. Boeke J.D. Mol. Cell. Biol. 1999; 19: 3184-3197Crossref PubMed Scopus (185) Google Scholar). Also, as predicted, deletion of the SIN3 gene caused anirs phenotype (24Smith J.S. Caputo E. Boeke J.D. Mol. Cell. Biol. 1999; 19: 3184-3197Crossref PubMe" @default.
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• W2078199021 title "Pho23 Is Associated with the Rpd3 Histone Deacetylase and Is Required for Its Normal Function in Regulation of Gene Expression and Silencing in Saccharomyces cerevisiae" @default.
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