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• W2104687412 abstract "Drosophila nucleosome remodeling factor (NURF) is an ISWI-containing protein complex that facilitates nucleosome mobility and transcriptional activation in an ATP-dependent manner. Numerous studies have implicated histone acetylation in transcriptional activation. We investigated the relative contributions of these two chromatin modifications to transcription in vitro of a chromatinized adenovirus E4 minimal promoter that contains binding sites for the GAL4-VP16 activator. We found that NURF could remodel chromatin and stimulate transcription irrespective of the acetylation status of histones. In contrast, hyperacetylation of histones in the absence of NURF was unable to stimulate transcription, suggesting that NURF-dependent chromatin remodeling is an obligatory step in E4 promoter activation. When chromatin templates were first hyperacetylated and then incubated with NURF, significantly greater transcription stimulation was observed. The results suggest that changes in chromatin induced by acetylation of histones and the mobilization of nucleosomes by NURF combine synergistically to facilitate transcription. Experiments using single and multiple rounds of transcription indicate that these chromatin modifications stimulate transcription preinitiation as well as reinitiation. Drosophila nucleosome remodeling factor (NURF) is an ISWI-containing protein complex that facilitates nucleosome mobility and transcriptional activation in an ATP-dependent manner. Numerous studies have implicated histone acetylation in transcriptional activation. We investigated the relative contributions of these two chromatin modifications to transcription in vitro of a chromatinized adenovirus E4 minimal promoter that contains binding sites for the GAL4-VP16 activator. We found that NURF could remodel chromatin and stimulate transcription irrespective of the acetylation status of histones. In contrast, hyperacetylation of histones in the absence of NURF was unable to stimulate transcription, suggesting that NURF-dependent chromatin remodeling is an obligatory step in E4 promoter activation. When chromatin templates were first hyperacetylated and then incubated with NURF, significantly greater transcription stimulation was observed. The results suggest that changes in chromatin induced by acetylation of histones and the mobilization of nucleosomes by NURF combine synergistically to facilitate transcription. Experiments using single and multiple rounds of transcription indicate that these chromatin modifications stimulate transcription preinitiation as well as reinitiation. base pair(s) histone acetyltransferase nucleosome remodeling factor polyacrylamide gel electrophoresis micrococcal nuclease The compaction of the eukaryotic genome in nucleosomes and the higher order folding of nucleosome arrays present barriers to regulatory proteins and the multisubunit enzymes that process genetic information (reviewed in Refs. 1Grunstein M. Annu. Rev. Cell Biol. 1990; 6: 643-678Crossref PubMed Google Scholar, 2Kornberg R.D. Lorch Y. Annu. Rev. Cell Biol. 1992; 8: 563-587Crossref PubMed Google Scholar, 3Van Holde K. Zlatanova J. Arents G. Moudrianakis E. Elgin S.C.R. Chromatin Structure and Gene Expression. Oxford University Press, Oxford1995: 1-26Google Scholar, 4Fletcher T.M. Hansen J.C. Crit. Rev. Eukaryotic Gene. Expression. 1996; 6: 149-188Crossref PubMed Google Scholar, 5Ramakrishnan V. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 83-112Crossref PubMed Scopus (132) Google Scholar). In the nucleosome core particle, winding of 147 bp1 of DNA over an octamer of histones creates severe distortion of the DNA helix and obscures roughly half of the helix surface (reviewed in Refs. 6Luger K. Richmond T.J. Curr. Opin. Genet. Dev. 1998; 8: 140-146Crossref PubMed Scopus (384) Google Scholar and7Kornberg R.D. Lorch Y. Curr. Opin. Genet. Dev. 1999; 9: 148-151Crossref PubMed Scopus (189) Google Scholar). To counteract the constraints imposed by chromatin architecture, cells employ several distinct mechanisms. Strategies to destabilize chromatin include the use of homopolymeric stretches of DNA that resist bending; architectural, high mobility group-type proteins that unfold nucleosome arrays; histone-modifying enzymes that covalently alter specific residues of the histone tails; and ATP-dependent chromatin remodeling complexes that facilitate nucleosome mobility (reviewed in Refs. 8Wu C. J. Biol. Chem. 1997; 272: 28171-28174Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 9Armstrong J.A. Emerson B.M. Curr. Opin. Genet. Dev. 1998; 8: 165-172Crossref PubMed Scopus (76) Google Scholar, 10Kadonaga J.T. Cell. 1998; 92: 307-313Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar, 11Workman J.L. Kingston R.E. Annu. Rev. Biochem. 1998; 67: 545-579Crossref PubMed Scopus (925) Google Scholar, 12Bustin M. Mol. Cell. Biol. 1999; 19: 5237-5246Crossref PubMed Google Scholar, 13Kingston R.E. Narlikar G.J. 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SWI2/SNF2-containing complexes are large assemblies in the megadalton size range and composed of 11–15 distinct polypeptides. ISWI-containing complexes are smaller and are composed of 2–5 subunits. Both types of chromatin remodelers use the free energy of ATP hydrolysis to increase nucleosome mobility by changing nucleosome conformation (19Lorch Y. Zhang M. Kornberg R.D. Cell. 1999; 96: 389-392Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 20Hamiche A. Sandaltzopoulos R. Gdula D.A. Wu C. Cell. 1999; 97: 833-842Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 21Langst G. Bonte E.J. Corona D.F. Becker P.B. Cell. 1999; 97: 843-852Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 22Whitehouse I. Flaus A. Cairns B.R. White M.F. Workman J.L. Owen-Hughes T. Nature. 1999; 400: 784-787Crossref PubMed Scopus (274) Google Scholar). A large body of evidence implicates the SWI2/SNF2-containing complexes in transcription regulation. Theswi2/snf2 gene was originally identified genetically as a transcriptional regulator in yeast (reviewed in Refs. 23Winston F. Carlson M. Trends Genet. 1992; 8: 387-391Abstract Full Text PDF PubMed Google Scholar and 24Peterson C.L. Tamkun J.W. Trends Biochem. Sci. 1995; 20: 143-146Abstract Full Text PDF PubMed Scopus (338) Google Scholar). Recent genetic studies show that Drosophila iswiis required for engrailed and Ultrabithoraxexpression in vivo (25Deuring R. Fanti L. Armstrong J.A. Sarte M. Papoulas O. Prestel M. Daubresse G. Verardo M. Moseley S.L. Berloco M. Tsukiyama T. Wu C. Pimpinelli S. Tamkun J.W. Mol. Cell. 2000; 5: 355-365Abstract Full Text Full Text PDF PubMed Google Scholar). In addition, SWI2/SNF2- and ISWI-containing complexes isolated from yeast, flies, and mammals can assist the transcriptional activation of model chromatin templatesin vitro (26Ito T. Bulger M. Pazin M.J. Kobayashi R. Kadonaga J.T. Cell. 1997; 90: 145-155Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar, 27Mizuguchi G. Tsukiyama T. Wisniewski J. Wu C. Mol. Cell. 1997; 1: 141-150Abstract Full Text Full Text PDF PubMed Google Scholar, 28Cote J. Peterson C.L. Workman J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4947-4952Crossref PubMed Scopus (163) Google Scholar, 29Armstrong J.A. Bieker J.J. Emerson B.M. Cell. 1998; 95: 93-104Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 30LeRoy G. Orphanides G. Lane W.S. Reinberg D. Science. 1998; 282: 1900-1904Crossref PubMed Google Scholar, 31Okada M. Hirose S. Mol. Cell. Biol. 1998; 18: 2455-2461Crossref PubMed Google Scholar, 32Kal A.J. Mahmoudi T. Zak N.B. Verrijzer C.P. Genes Dev. 2000; 14: 1058-1071PubMed Google Scholar). The histone acetyltransferases (HATs) and histone deacetylases, which catalyze the reversible modification of specific lysines on the N-terminal histone tails, are the most extensively studied of the histone-modifying enzymes. The similarity of Tetrahymena p55 HAT and mammalian HDAC1 to genetically defined transcriptional regulators provided the first link between these histone-modifying enzymes and transcription (33Brownell 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 (1210) Google Scholar, 34Taunton J. Hassig C.A. Schreiber S.L. Science. 1996; 272: 408-411Crossref PubMed Google Scholar). Subsequent analysis of the growing family of HATs and HDACs has shown that they can form large, multimeric complexes that can be recruited to gene promoters (reviewed in Refs.35Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2233) Google Scholar, 36Wade P.A. Wolffe P.A. Curr. Biol. 1997; 7: 82-84Abstract Full Text Full Text PDF PubMed Google Scholar, 37Davie J.R. Curr. Opin. Genet. Dev. 1998; 8: 173-178Crossref PubMed Scopus (176) Google Scholar, 38Mizzen C.A. Allis C.D. Cell. Mol. Life. Sci. 1998; 54: 6-20Crossref PubMed Scopus (177) Google Scholar, 39Struhl K. Genes Dev. 1998; 12: 599-606Crossref PubMed Google Scholar, 40Howe L. Brown C.E. Lechner T. Workman J.L. Crit. Rev. Eukaryot. Gene. Expr. 1999; 9: 231-243Crossref PubMed Google Scholar, 41Tyler J.K. Kadonaga J.T. Cell. 1999; 99: 443-446Abstract Full Text Full Text PDF PubMed Google Scholar, 42Brown C.E. Lechner T. Howe L. Workman J.L. Trends. Biochem. Sci. 2000; 25: 15-19Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 43Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6068) Google Scholar, 44Kouzarides T. EMBO J. 2000; 19: 1176-1179Crossref PubMed Google Scholar). Experiments in vitro on reconstituted chromatin templates have shown that histone acetylation can facilitate transcription (45Sheridan P.L. Mayall T.P. Verdin E. Jones K.A. Genes Dev. 1997; 11: 3327-3340Crossref PubMed Google Scholar, 46Steger D.J. Eberharter A. John S. Grant P.A. Workman J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12924-12929Crossref PubMed Scopus (111) Google Scholar, 47Nightingale K.P. Wellinger R.E. Sogo J.M. Becker P.B. EMBO J. 1998; 17: 2865-2876Crossref PubMed Scopus (118) Google Scholar, 48Utley R.T. Ikeda K. Grant P.A. Cote J. Steger D.J. Eberharter A. John S. Workman J.L. Nature. 1998; 394: 498-502Crossref PubMed Scopus (430) Google Scholar, 49Ikeda K. Steger D.J. Eberharter D.A. Workman J.L. Mol. Cell. Biol. 1999; 19: 855-863Crossref PubMed Google Scholar, 50Wallberg A.E. Neely K.E. Gustafsson J.A. Workman J.L. Wright A.P. Grant P.A. Mol. Cell. Biol. 1999; 19: 5952-5959Crossref PubMed Scopus (52) Google Scholar, 51Kraus W.L. Manning E.T. Kadonaga J.T. Mol. Cell. Biol. 1999; 19: 8123-8135Crossref PubMed Google Scholar, 52Akhtar A. Becker P.B. Mol. Cell. 2000; 5: 367-375Abstract Full Text Full Text PDF PubMed Google Scholar, 53Vignali M. Steger D.J. Neely K.E. Workman J.L. EMBO J. 2000; 19: 2629-2640Crossref PubMed Google Scholar, 54Kundu T.K. Palham V.B. Wang Z. An W. Cole P.A. Roeder R. Mol. Cell. 2000; 6: 551-561Abstract Full Text Full Text PDF PubMed Google Scholar). Although the separate contributions of histone hyperacetylation and ATP-driven chromatin remodeling to transcription are well known, the interrelationships between these two major types of chromatin modifications and their relative contributions to the activation process are only beginning to be explored. Genetic studies indicate that the yeast SWI2/SNF2 and GCN5 genes perform independent but overlapping functions during transcriptional activation (55Pollard K.J. Peterson C.L. Mol. Cell. Biol. 1997; 17: 6212-6222Crossref PubMed Scopus (190) Google Scholar, 56Roberts S.M. Winston F. Genetics. 1997; 147: 451-465Crossref PubMed Google Scholar, 57Sudarsanam P. Cao Y. Wu L. Laurent B.C. Winston F. EMBO J. 1999; 18: 3101-3106Crossref PubMed Scopus (90) Google Scholar, 58Biggar S.R. Crabtree G.R. EMBO J. 1999; 18: 2254-2264Crossref PubMed Google Scholar, 59Cosma M.P. Tanaka T. Nasmyth K. Cell. 1999; 97: 299-311Abstract Full Text Full Text PDF PubMed Scopus (586) Google Scholar, 60Krebs J.E. Kuo M.H. Allis C.D. Peterson C.L. Genes Dev. 1999; 13: 1412-1421Crossref PubMed Google Scholar, 61Syntichaki P. Topalidou I. Thireos G. Nature. 2000; 404: 414-417Crossref PubMed Scopus (165) Google Scholar). To date, however, no biochemical studies have examined the mechanistic relationship between histone hyperacetylation and ATP-dependent chromatin remodeling. To address this question, we analyzed the relative contributions ofDrosophila nucleosome remodeling factor (NURF) and histone acetylation to the activation of a model chromatin template in vitro. Drosophila NURF was identified as a four-subunit, ISWI-containing complex that has been implicated in transcriptional activation of chromatin (62Tsukiyama T. Becker P.B. Wu C. Nature. 1994; 367: 525-532Crossref PubMed Scopus (542) Google Scholar, 63Tsukiyama T. Daniel C. Tamkun J. Wu C. Cell. 1995; 83: 1021-1026Abstract Full Text PDF PubMed Scopus (297) Google Scholar, 64Tsukiyama T. Wu C. Cell. 1995; 83: 1011-1020Abstract Full Text PDF PubMed Scopus (489) Google Scholar, 65Georgel P.T. Tsukiyama T. Wu C. EMBO J. 1997; 16: 4717-4726Crossref PubMed Scopus (113) Google Scholar, 66Martinez-Balbas M.A. Tsukiyama T. Gdula D. Wu C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 132-137Crossref PubMed Scopus (141) Google Scholar, 67Gdula D.A. Sandaltzopoulos R. Tsukiyama Ossipow V. Wu C. Genes Dev. 1998; 12: 3206-3216Crossref PubMed Google Scholar). Other ISWI-containing complexes related to NURF have subsequently been characterized fromDrosophila (ACF (68Varga-Weisz P.D. Wilm M. Bonte E. Dumas K. Mann M. Becker P.B. Nature. 1997; 388: 598-602Crossref PubMed Scopus (413) Google Scholar) and CHRAC (21Langst G. Bonte E.J. Corona D.F. Becker P.B. Cell. 1999; 97: 843-852Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar)) and human cells (RSF (30LeRoy G. Orphanides G. Lane W.S. Reinberg D. Science. 1998; 282: 1900-1904Crossref PubMed Google Scholar) and WCRF/hACF (69Bochar D.A. Savard J. Wang W. Lafleur D.W. Moore P. Cote J. Shiekhattar R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1038-1043Crossref PubMed Scopus (124) Google Scholar, 70LeRoy G. Loyola A. Lane W.S. Reinberg D. J. Biol. Chem. 2000; 275: 14787-14790Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar)). In a previous report, we demonstrated that purified NURF enables activation of a chromatin template by GAL4-HSF at an early step in the process of transcription (27Mizuguchi G. Tsukiyama T. Wisniewski J. Wu C. Mol. Cell. 1997; 1: 141-150Abstract Full Text Full Text PDF PubMed Google Scholar). Here, we demonstrate synergism between the hyperacetylation of histones and nucleosome remodeling by NURF. Histone hyperacetylation alone did not stimulate transcription from our model chromatin template. However, in combination, the addition of NURF to hyperacetylated chromatin leads to synergistic activation of transcription. We suggest that a hierarchy exists in which NURF-dependent chromatin remodeling is an obligatory step during promoter activation. By the use of single and multiple round transcription experiments we demonstrate that the two types of chromatin modification stimulate transcription of chromatin both at the preinitiation and reinitiation stages. FLAG epitope-tagged human p300 was expressed in Sf9 cells after infection with recombinant baculovirus, and protein was purified as described elsewhere (71Yang X.J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1283) Google Scholar). Human P/CAF complex was purified from nuclear extracts of a HeLa cell line expressing FLAG epitope-tagged P/CAF as described (72Ogryzko V.V. Kotani T. Zhang X. Schlitz R.L. Howard T. Yang X.J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). Plasmid pGM2a expressing the GAL4-VP16 fusion protein was constructed by inserting the XhoI–BamHI fragment of pJL2 (73Chasman D.I. Leatherwood J. Carey M. Ptashne M. Kornberg R.D. Mol. Cell. Biol. 1989; 9: 4746-4749Crossref PubMed Scopus (136) Google Scholar), which encodes amino acids 95–147 of GAL4 and the carboxyl-terminal 291 amino acids of the VP16 transactivation domain, of plasmid pJL2 into plasmid pGM1 digested with XhoI and BamHI (27Mizuguchi G. Tsukiyama T. Wisniewski J. Wu C. Mol. Cell. 1997; 1: 141-150Abstract Full Text Full Text PDF PubMed Google Scholar). Expression of GAL4-VP16 was induced in Escherichia coli BL21 (DE3) pLysE (Novagen) and purified as described (27Mizuguchi G. Tsukiyama T. Wisniewski J. Wu C. Mol. Cell. 1997; 1: 141-150Abstract Full Text Full Text PDF PubMed Google Scholar, 74Mizuguchi G. Wu C. Methods Mol. Biol. 1999; 119: 333-342PubMed Google Scholar). NURF was purified from nuclear extracts of 0–12 h Drosophila embryos up to the glycerol gradient step as described (64Tsukiyama T. Wu C. Cell. 1995; 83: 1011-1020Abstract Full Text PDF PubMed Scopus (489) Google Scholar). Recombinant Drosophila TFIIA, TFIIB, TFIIE, and TFIIF were expressed and purified as described (75Hansen S.K. Tjian R. Cell. 1995; 82: 565-575Abstract Full Text PDF PubMed Scopus (98) Google Scholar).Drosophila TFIID was immunoprecipitated with mAb 2B2 againstDrosophila TAF250 (76Weinzierl R.O. Dynlacht B.D. Tjian R. Nature. 1993; 362: 511-517Crossref PubMed Scopus (140) Google Scholar) from the MonoQ-TFIID fraction prepared as described (75Hansen S.K. Tjian R. Cell. 1995; 82: 565-575Abstract Full Text PDF PubMed Scopus (98) Google Scholar). The TFIID complex was washed in 0.1m KCl-HEMGND buffer (25 mm HEPES-KOH (pH 7.6), 0.1 mm EDTA, 12.5 mm MgCl2, 10% (v/v) glycerol, 0.1% (v/v) Nonidet P-40, 1 mmdithiothreitol) and eluted with epitope-containing peptide in 0.1m KCl-HEMGND containing 0.5 m guanidine hydrochloride. The eluted TFIID was dialyzed against 0.1 mKCl-HEMGND and used for in vitro transcription.Drosophila TFIIH and RNA polymerase II were purified fromDrosophila embryo nuclear extract as described (75Hansen S.K. Tjian R. Cell. 1995; 82: 565-575Abstract Full Text PDF PubMed Scopus (98) Google Scholar, 77Austin R.J. Biggin M.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5788-5792Crossref PubMed Scopus (23) Google Scholar). Recombinant Drosophila TFIIS was synthesized in E. coli BL21 (DE3) (Novagen) and purified as described (78Guo H. Price D.H. J. Biol. Chem. 1993; 268: 18762-18770Abstract Full Text PDF PubMed Google Scholar). Chromatin was assembled using aDrosophila embryo S-190 extract (79Kamakaka R.T. Bulger M. Kadonaga J.T. Genes Dev. 1993; 7: 1779-1795Crossref PubMed Google Scholar) and cosmid pWEGIE-0 as a template. Cosmid pWEGIE-0 was constructed by insertion of the 1208-bp AatII–EcoRI fragment of pGIE-0 (see Ref. 80Pazin M.J. Kamakaka R.T. Kadonaga J.T. Science. 1994; 266: 2007-2011Crossref PubMed Google Scholar; the DNA fragment contains five GAL4-binding sites and a TATA box from the adenovirus type 5 E4 promoter) into the AatII andEcoRI sites of cosmid pWE15 (Promega) DNA. The S-190 extract (275 μl; ∼7.0 mg of protein) was incubated with purifiedDrosophila core histones (4.5–5.0 μg) (81Simon R.H. Felsenfeld G. Nucleic Acids Res. 1979; 6: 689-696Crossref PubMed Scopus (287) Google Scholar, 20Hamiche A. Sandaltzopoulos R. Gdula D.A. Wu C. Cell. 1999; 97: 833-842Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar) and buffer R (10 mm HEPES-KOH (pH7.6), 0.5 mm EGTA, 1.5 mm MgCl, 10% (v/v) glycerol, 10 mm KCl, 10 mm β-glycerophosphate, 1 mm dithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride) in a total volume of 630 μl at 4 °C for 30 min. Cosmid pWEGIE-0 DNA (5 μg), ATP (3 mm), creatine phosphate (30 mm), creatine phosphokinase (1 μg/ml), and MgCl2 (7 mm) were added in a final volume of 700 μl, and assembly was carried out at 26 °C for 6 h. Sucrose gradient purification of preassembled chromatin was performed essentially as described (79Kamakaka R.T. Bulger M. Kadonaga J.T. Genes Dev. 1993; 7: 1779-1795Crossref PubMed Google Scholar). Briefly, 2.8 ml of preassembled chromatin mixture was applied onto a Beckman SW41 tube and centrifuged in a Beckman SW41 rotor at 26,000 rpm for 16 h at 4 °C. The sucrose gradient buffer was 10 mm HEPES-KOH (pH 7.6), 1 mm EDTA, 35 mm NaCl containing from 30 to 50% (w/v) sucrose. 1.0-ml gradient fractions were collected, and DNA concentration was determined after 1% agarose gel electrophoresis of an aliquot (40 μl) by ethidium bromide staining. The sucrose gradient fraction was directly subjected to treatment of the histone acetyltransferases. Typically, 100 μl (1.4 μg of DNA equivalent) of the gradient fraction was incubated at 26 °C for 30 min with 1 mm dithiothreitol, 1 mm sodium butylate, 1 mm acetyl-CoA (lithium salt; Amersham Pharmacia Biotech), and ∼5 pmol of p300 or P/CAF complex. Hyperacetylated chromatin was further purified and subjected to chromatin remodeling and transcription. For conventional purification of hyperacetylated chromatin, Sepharose CL4B (Amersham Pharmacia Biotech) was used in a SizeSep-400 spin column (Amersham Pharmacia Biotech) preequilibrated with elution buffer (10 mm HEPES-KOH (pH 7.6), 0.5 mm EGTA, 5 mm MgCl, 10% (v/v) glycerol, 50 mm KCl, 10 mm β-glycerophosphate, 1 mmdithiothreitol). Bovine serum albumin (Roche Molecular Biochemicals) was added to chromatin fractions after purification to a final concentration of 0.5 mg/ml. DNA content was estimated by agarose gel electrophoresis and ethidium bromide staining. For analysis of histone acetylation by SDS-PAGE and fluorography, 100 μl (1.4 μg of DNA equivalent) of the sucrose gradient purified chromatin was processed as described above, except that 1 nmol of [3H]acetyl-CoA was introduced in place of 1 mm acetyl-CoA (lithium salt). The reaction was quenched by the addition of SDS-PAGE sample buffer and analyzed on 15% SDS-PAGE. The gels were then stained by Coomassie Brilliant Blue and exposed for fluorography. For Triton-acid-urea gel analysis, samples were processed as described above, using 1 mm acetyl-CoA (lithium salt). The acetylated chromatin was then precipitated with trichloroacetic acid and analyzed on a TAU gel, essentially as described (82Krajewski W.A. Becker P.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1540-1545Crossref PubMed Scopus (79) Google Scholar). In vitro transcription and primer extension analysis were performed as described previously (27Mizuguchi G. Tsukiyama T. Wisniewski J. Wu C. Mol. Cell. 1997; 1: 141-150Abstract Full Text Full Text PDF PubMed Google Scholar, 83Becker P.B. Rabindran S.K. Wu C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4109-4113Crossref PubMed Google Scholar, 84Orphanides G. LeRoy G. Chang C.H. Luse D.S. Reinberg D. Cell. 1998; 92: 105-116Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). For the purified transcription system, typically 40 ng of chromatin (40 μl of spin column-purified chromatin) was preincubated in 80 μl (final volume) containing 25 ng of TFIIA, 15 ng of TFIIB, ∼120 ng of immunopurified TFIID, 50 ng of TFIIE, 50 ng of TFIIF, ∼40 ng of purified TFIIH, 10 ng of TFIIS, ∼30 ng of RNA polymerase II, 20 mm HEPES-KOH (pH 7.6), 5 mm MgCl, 40 mm KCl, 2.6% (v/v) polyethylene glycol 8000 (final concentration), 3.75 mm(NH4)2SO4, 1 mmdithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride for 30 min at 26 °C to form preinitiation complexes and then transcribed for 10 min at 26 °C with the addition of ribonucleotide triphosphates (0.55 mm final concentration). Transcripts were detected by primer extension using a 32P-labeled AdE4 primer corresponding to positions 72–99 of the transcribed strand of pWEGIE-0. cDNA products were analyzed on a 6% denaturing polyacrylamide gel. Quantitation of transcripts was performed on a Fuji Bio-Image Analyzer. For transcription in a crude system, soluble nuclear fraction (nuclear extract) was prepared from 0–12-h Drosophila embryos as described (85Kamakaka R.T. Tyree C.M. Kadonaga J.T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1024-1028Crossref PubMed Scopus (73) Google Scholar, 86Kamakaka R.T. Kadonaga J.T. Methods Cell Biol. 1994; 44: 225-235Crossref PubMed Scopus (39) Google Scholar). Reactions were performed as described above, except that 70 μg of the soluble nuclear fraction was used instead of purified general transcription factors and RNA polymerase II. For the heparin challenge protocol, reactions were performed essentially as described for transcription with the purified system. A synthetic RNA polymerase II pause at +5 site was created by the addition of 0.55 mm ATP, 0.55 mm CTP, and 0.55 mm UTP. Following incubation at 26 °C for 5 min, 400 ng of heparin and 0.55 mm GTP were added to block reinitiation of RNA polymerase II, and the reactions were incubated for a further 30 min at 30 °C (87Zheng X.M. Moncollin V. Egly J.M. Chambon P. Cell. 1987; 50: 361-368Abstract Full Text PDF PubMed Google Scholar, 88Conaway R.C. Conaway J.W. J. Biol. Chem. 1990; 265: 7559-7563Abstract Full Text PDF PubMed Google Scholar). Transcribed RNAs were analyzed by primer extension as above. Micrococcal nuclease (MNase) digestion analysis and sequential Southern blot hybridization were performed as described (62Tsukiyama T. Becker P.B. Wu C. Nature. 1994; 367: 525-532Crossref PubMed Scopus (542) Google Scholar,80Pazin M.J. Kamakaka R.T. Kadonaga J.T. Science. 1994; 266: 2007-2011Crossref PubMed Google Scholar, 89Becker P.B. Wu C. Mol. Cell. Biol. 1992; 12: 2241-2249Crossref PubMed Google Scholar). A restriction enzyme accessibility assay was performed essentially as described (90Tsukiyama T. Palmer J. Landel C.C. Shiloach J. Wu C. Genes Dev. 1999; 13: 686-697Crossref PubMed Google Scholar). Typically, 50 μl (∼50 ng of DNA equivalent) of spin column-purified chromatin, 0.5 μl of NURF fraction, ATP (0.5 mm final concentration), and 0.5 pmol of GAL4 derivative (GAL4-VP16) were mixed and adjusted to 65 μl with elution buffer. The reaction was incubated at 26 °C for 30 min and subjected to micrococcal nuclease or restriction enzyme digestion. For MNase digestion, the reaction mixture was directly treated with enzyme and analyzed as described (27Mizuguchi G. Tsukiyama T. Wisniewski J. Wu C. Mol. Cell. 1997; 1: 141-150Abstract Full Text Full Text PDF PubMed Google Scholar). For restriction enzyme digestion, the reaction mixture was incubated with either 0.5 unit of BamHI (digested at position −46) or 0.5 unit of HaeII (digested at position +98), at 26 °C for 30 min. Digested DNA samples were deproteinized, precipitated with ethanol, and redigested withPstI and ClaI. Samples were loaded on a 1.3% agarose gel and analyzed by Southern blot hybridization as for the MNase assay. To analyze the relative contributions of histone acetylation and ATP-dependent chromatin remodeling to transcription, we used a 9-kilobase pair cosmid (pWEGIE-0) containing five tandemly repeated GAL4 binding sites upstream of the adenovirus E4 core promoter (80Pazin M.J. Kamakaka R.T. Kadonaga J.T. Science. 1994; 266: 2007-2011Crossref PubMed Google Scholar). The experimental procedure for reconstitution and acetylation of pWEGIE-0 chromatin is outlined in Fig.1 A. After assembly with theDrosophila embryo S-190 extract and purifiedDrosophila core histones, chromatin is purified by sucrose gradient sedimentation. This procedure removes the bulk of nonhistone proteins, leaving histones as the predominant proteins in the chromatin preparation, as judged by SDS-PAGE and silver staining (79Kamakaka R.T. Bulger M. Kadonaga J.T. Genes Dev. 1993; 7: 1779-1795Crossref PubMed Google Scholar). The purified chromatin is then modified with a HAT and acetyl-CoA, followed by repurification on a Sepharose CL4B spin column, to preclude acetylation of remodeling proteins and transcription factors that are introduced subsequently. Chromatin is next incubated with saturating amounts of the transcription activator GAL4-VP16 and saturating amounts of purified NURF and ATP to allow the mobilization of nucleosomes. This is followed by the assembly of preinitiation complexes with either aDrosophila soluble nuclear fraction that has little ATP-dependent chromatin remodeling activity (27Mizuguchi G. Tsukiyama T. Wisniewski J. Wu C. Mol. Cell. 1997; 1: 141-150Abstract Full Text Full Text PDF PubMed Google Scholar, 80Pazin M.J. Kamakaka R.T. Kadonaga J.T. Science. 1994; 266: 2007-2011Crossref PubMed Google Scholar) or a purified transcription system consisting of native and recombinantDrosophila general transcription factors and RNA polymerase II. Transcription is then initiated upon the addition of ribonucleotide triphosphates (NTPs), and the RNA products after 10 min of transcription are analyzed by primer extension. We note thatDrosophila embryo extracts contain histone acetyltransferases and deacetylases that can modify histones during chromatin assembly (45Sheridan P.L. Mayall T.P. Verdin E. Jones K.A. Genes Dev. 1997; 11: 3327-3340Crossref PubMed Google Scholar, 47Nightingale K.P. Wellinger R.E. Sogo J.M. Becker P.B. EMBO J. 1998; 17: 2865-2876Crossref PubMed Scopus (" @default.
• W2104687412 created "2016-06-24" @default.
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• W2104687412 date "2001-01-01" @default.
• W2104687412 modified "2023-10-16" @default.
• W2104687412 title "ATP-dependent Nucleosome Remodeling and Histone Hyperacetylation Synergistically Facilitate Transcription of Chromatin" @default.
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