Nucleosome Disruption by Human SWI/SNF Is Maintained in the Absence of Continued ATP Hydrolysis
1996; Elsevier BV; Volume: 271; Issue: 34 Linguagem: Inglês
10.1074/jbc.271.34.20726
ISSN1083-351X
AutoresAnthony N. Imbalzano, Gavin R. Schnitzler, Robert E. Kingston,
Tópico(s)interferon and immune responses
ResumoWe have examined the requirement for ATP in human (h) SWI/SNF-mediated alteration of nucleosome structure and facilitation of transcription factor binding to nucleosomal DNA. hSWI/SNF-mediated nucleosome alteration requires hydrolysis of ATP or dATP. The alteration is stable upon removal of ATP from the reaction or upon inhibition of activity by excess ATPγS, indicating that continued ATP hydrolysis is not required to maintain the altered nucleosome structure. This stable alteration is sufficient to facilitate binding of a transcriptional activator protein; concurrent ATP hydrolysis was not required to facilitate binding. These data suggest sequential steps that can occur in the process by which transcription factors gain access to nucleosomal DNA. We have examined the requirement for ATP in human (h) SWI/SNF-mediated alteration of nucleosome structure and facilitation of transcription factor binding to nucleosomal DNA. hSWI/SNF-mediated nucleosome alteration requires hydrolysis of ATP or dATP. The alteration is stable upon removal of ATP from the reaction or upon inhibition of activity by excess ATPγS, indicating that continued ATP hydrolysis is not required to maintain the altered nucleosome structure. This stable alteration is sufficient to facilitate binding of a transcriptional activator protein; concurrent ATP hydrolysis was not required to facilitate binding. These data suggest sequential steps that can occur in the process by which transcription factors gain access to nucleosomal DNA. INTRODUCTIONIn vivo, DNA is compacted via association with histones and nonhistone proteins to form chromatin, which, in general, inhibits the interaction of the transcriptional machinery with promoter sequences and is therefore refractory to gene expression. While inactive promoters are generally incorporated in nucleosomal arrays, regulatory sequences controlling the expression of genes being actively transcribed have been shown to exist in a more open conformational state, as shown by their increased sensitivity to cleavage by nucleases. Thus, there must be mechanisms utilized by the cell to disrupt chromatin and render relevant DNA sequences accessible to the transcriptional machinery.A number of different mechanisms may exist to explain how chromatin structure is altered on promoter/enhancer sequences. Many activators, such as Sp1 (Chen et al., 5Chen H. Li B. Workman J.L. EMBO J. 1994; 13: 380-390Crossref PubMed Scopus (137) Google Scholar; Li et al., 28Li B. Adams C.C. Workman J.L. J. Biol. Chem. 1994; 269: 7756-7763Abstract Full Text PDF PubMed Google Scholar), synthetic derivatives of the yeast GAL4 transcriptional activator (Taylor et al., 41Taylor I.C.A. Workman J.L. Schuetz T.J. Kingston R.E. Genes Dev. 1991; 5: 1285-1298Crossref PubMed Scopus (202) Google Scholar; Workman and Kingston, 51Workman J.L. Kingston R.E. Science. 1992; 258: 1780-1784Crossref PubMed Scopus (149) Google Scholar), progesterone receptor (Pham et al., 37Pham T.A. McDonnell D.P. Tsai M.-J. O'Malley B.W. Biochemistry. 1992; 31: 1570-1578Crossref PubMed Scopus (16) Google Scholar), glucocorticoid receptor (Perlmann and Wrange, 33Perlmann T. Wrange Ö EMBO J. 1988; 7: 3073-3079Crossref PubMed Scopus (234) Google Scholar; Pina et al., 38Pina B. Bruggemeier U. Beato M. Cell. 1990; 60: 719-731Abstract Full Text PDF PubMed Scopus (334) Google Scholar; Archer et al., 2Archer T.K. Cordingley M.G. Wolford R.G. Hager G.L. Mol. Cell. Biol. 1991; 11: 688-698Crossref PubMed Scopus (293) Google Scholar; Li and Wrange, 1993; Li and Wrange, 1995), TFIIIA 1The abbreviations used are: TFtranscription factorhhumanbpbase pair(s)ATPγSadenosine 5′-O-(thiotriphosphate)AMP-PNPadenosine 5′-(β,γ-imino)triphosphateNURFnucleosome remodeling factorBSAbovine serum albumin. (Rhodes, 40Rhodes D. EMBO J. 1985; 4: 3473-3482Crossref PubMed Google Scholar; Lee et al., 25Lee D.Y. Hayes J.J. Pruss D. Wolffe A.P. Cell. 1993; 72: 73-84Abstract Full Text PDF PubMed Scopus (955) Google Scholar), upstream stimulatory factor (Chen et al., 5Chen H. Li B. Workman J.L. EMBO J. 1994; 13: 380-390Crossref PubMed Scopus (137) Google Scholar; Adams and Workman, 1Adams C.C. Workman J.L. Mol. Cell. Biol. 1995; 15: 1405-1421Crossref PubMed Google Scholar), and Max and c-Myc-Max heterodimers (Wechsler et al., 48Wechsler D.S. Papoulas O. Dang C.V. Kingston R.E. Mol. Cell. Biol. 1994; 14: 4097-4107Crossref PubMed Scopus (45) Google Scholar) have been shown to bind to nucleosome particles in vitro. In some cases, the binding of activators can destablize the nucleosome, as shown by the observation that the binding of GAL4 derivatives to mononucleosome particles containing five GAL4 sites facilitates the displacement of histones to histone acceptors (Workman and Kingston, 51Workman J.L. Kingston R.E. Science. 1992; 258: 1780-1784Crossref PubMed Scopus (149) Google Scholar; Chen et al., 5Chen H. Li B. Workman J.L. EMBO J. 1994; 13: 380-390Crossref PubMed Scopus (137) Google Scholar; Walter et al., 47Walter P.P. Owen-Hughes T.A. Côté J. Workman J.L. Mol. Cell. Biol. 1995; 15: 6178-6187Crossref PubMed Scopus (113) Google Scholar). In other cases, direct modification of the histone proteins comprising the nucleosome can alter the accessibility of the nucleosome to transcription factors. For example, TFIIIA binding to mononucleosomes can be facilitated by acetylation of the N-terminal tails of the core histones (Lee et al., 25Lee D.Y. Hayes J.J. Pruss D. Wolffe A.P. Cell. 1993; 72: 73-84Abstract Full Text PDF PubMed Scopus (955) Google Scholar).Other proposed mechanisms involve activities that mediate energy-dependent chromatin alteration (Cote et al., 7Cote J. Quinn J. Workman J.L. Peterson C.L. Science. 1994; 265: 53-60Crossref PubMed Scopus (718) Google Scholar; Kwon et al., 21Kwon H. Imbalzano A.N. Khavari P.A. Kingston R.E. Green M.R. Nature. 1994; 370: 477-481Crossref PubMed Scopus (640) Google Scholar; Imbalzano et al., 1994a14Imbalzano A.N. Kwon H. Green M.R. Kingston R.E. Nature. 1994; 370 (a): 481-485Crossref PubMed Scopus (520) Google Scholar; Tsukiyama et al., 43Tsukiyama T. Becker P.B. Wu C. Nature. 1994; 367: 525-531Crossref PubMed Scopus (558) Google Scholar; Tsukiyama and Wu, 42Tsukiyama T. Wu C. Cell. 1995; 83: 1011-1020Abstract Full Text PDF PubMed Scopus (511) Google Scholar; Wall et al., 46Wall G. Varga-Weisz P.D. Sandaltzopoulos R. Becker P.B. EMBO J. 1995; 14: 1727-1736Crossref PubMed Scopus (99) Google Scholar; Varga-Weisz et al., 45Varga-Weisz P.D. Blank T.A. Becker P.B. EMBO J. 1995; 14: 2209-2216Crossref PubMed Scopus (144) Google Scholar; Pazin et al., 32Pazin M.J. Kamakaka R.T. Kadonaga J.T. Science. 1994; 266: 2007-2011Crossref PubMed Scopus (136) Google Scholar). Some of these activities are due to large protein complexes (e.g. SWI/SNF, NURF) that hydrolyze ATP and structurally alter nucleosome particles (Cote et al., 7Cote J. Quinn J. Workman J.L. Peterson C.L. Science. 1994; 265: 53-60Crossref PubMed Scopus (718) Google Scholar; Kwon et al., 21Kwon H. Imbalzano A.N. Khavari P.A. Kingston R.E. Green M.R. Nature. 1994; 370: 477-481Crossref PubMed Scopus (640) Google Scholar; Imbalzano et al., 1994a14Imbalzano A.N. Kwon H. Green M.R. Kingston R.E. Nature. 1994; 370 (a): 481-485Crossref PubMed Scopus (520) Google Scholar; Tsukiyama and Wu, 42Tsukiyama T. Wu C. Cell. 1995; 83: 1011-1020Abstract Full Text PDF PubMed Scopus (511) Google Scholar; Tsukiyama et al., 44Tsukiyama T. Daniel C. Tamkun J. Wu C. Cell. 1995; 83: 1021-1026Abstract Full Text PDF PubMed Scopus (312) Google Scholar). The yeast SWI/SNF complex is comprised of the products of several yeast SWI and SNF genes that function together in a large, multisubunit complex (Laurent and Carlson, 22Laurent B.C. Carlson M. Genes Dev. 1992; 6: 1707-1715Crossref PubMed Scopus (122) Google Scholar; Peterson and Herskowitz, 34Peterson L. Herskowitz I. Cell. 1992; 68: 573-583Abstract Full Text PDF PubMed Scopus (462) Google Scholar; Peterson et al., 36Peterson C.L. Dingwall A. Scott M.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2905-2908Crossref PubMed Scopus (338) Google Scholar; Cairns et al., 3Cairns B.R. Kim Y.-J. Sayre M.H. Laurent B.C. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1950-1954Crossref PubMed Scopus (343) Google Scholar) that is required for the transcription of a large number of inducible genes and has been shown to enhance the function of many yeast, fly, and human transcriptional activators in yeast cells (Laurent and Carlson, 22Laurent B.C. Carlson M. Genes Dev. 1992; 6: 1707-1715Crossref PubMed Scopus (122) Google Scholar; Peterson and Herskowitz, 34Peterson L. Herskowitz I. Cell. 1992; 68: 573-583Abstract Full Text PDF PubMed Scopus (462) Google Scholar; Yoshinaga et al., 54Yoshinaga S.K. Peterson C.L. Herskowitz I. Yamamoto K.R. Science. 1992; 258: 1598-1604Crossref PubMed Scopus (412) Google Scholar; reviewed in Winston and Carlson, 50Winston F. Carlson M. Trends Genetics. 1992; 8: 387-391Abstract Full Text PDF PubMed Scopus (481) Google Scholar). Similarly, two human homologs of the helicase-related ATPase SNF2/SWI2 have been shown to enhance nuclear hormone receptor function in mammalian cells (Khavari et al., 17Khavari P.A. Peterson C.L. Tamkun J.W. Crabtree G.R. Nature. 1993; 366: 170-174Crossref PubMed Scopus (533) Google Scholar; Muchardt and Yaniv, 31Muchardt C. Yaniv M. EMBO J. 1993; 12: 4279-4290Crossref PubMed Scopus (522) Google Scholar; Chiba et al., 6Chiba H. Muramatsu M. Nomoto A. Kato H. Nucleic Acids Res. 1994; 22: 1815-1820Crossref PubMed Scopus (287) Google Scholar) and to purify as part of a large molecular weight complex (Khavari et al., 17Khavari P.A. Peterson C.L. Tamkun J.W. Crabtree G.R. Nature. 1993; 366: 170-174Crossref PubMed Scopus (533) Google Scholar; Kwon et al., 21Kwon H. Imbalzano A.N. Khavari P.A. Kingston R.E. Green M.R. Nature. 1994; 370: 477-481Crossref PubMed Scopus (640) Google Scholar).Although individual yeast SNF genes have been shown to function as activators when fused to DNA binding domains (Laurent et al., 23Laurent B.C. Treitel M.A. Carlson M. Mol. Cell. Biol. 1990; 10: 5616-5625Crossref PubMed Scopus (103) Google Scholar; Laurent et al., 24Laurent B.C. Treitel M.A. Carlson M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2687-2691Crossref PubMed Scopus (209) Google Scholar; Laurent and Carlson, 22Laurent B.C. Carlson M. Genes Dev. 1992; 6: 1707-1715Crossref PubMed Scopus (122) Google Scholar), the SWI/SNF complex appears not to function as an activator or bridging coactivator. Instead, many lines of evidence point to a role in chromatin disruption. First, phenotypes caused by mutations in yeast SWI and SNF genes can be suppressed by mutations in histone and nonhistone chromatin proteins and by mutations that alter histone expression levels (Kruger and Herskowitz, 19Kruger W. Herskowitz I. Mol. Cell. Biol. 1991; 11: 4135-4146Crossref PubMed Scopus (128) Google Scholar; Peterson et al., 35Peterson C.L. Kruger W. Herskowitz I. Cell. 1991; 64: 1135-1143Abstract Full Text PDF PubMed Scopus (138) Google Scholar; Hirschhorn et al., 13Hirschhorn J.N. Brown S.A. Clark C.D. Winston F. Genes Dev. 1992; 6: 2288-2298Crossref PubMed Scopus (436) Google Scholar; Kruger et al., 20Kruger W. Peterson C.L. Sil A. Coburn C. Arents G. Moudrianakis E.N. Herskowitz I. Genes Dev. 1995; 9: 2770-2779Crossref PubMed Scopus (217) Google Scholar). SWI/SNF mutations have also been shown to mediate structural changes in chromatin in vivo (Hirschhorn et al., 13Hirschhorn J.N. Brown S.A. Clark C.D. Winston F. Genes Dev. 1992; 6: 2288-2298Crossref PubMed Scopus (436) Google Scholar; Matallana et al., 30Matallana E. Franco L. Perez-Ortin J.E. Mol. Gen. Genetics. 1992; 231: 395-400Crossref PubMed Scopus (51) Google Scholar). More recently, both the yeast and human SWI/SNF complexes have been purified and shown to directly alter nucleosome structure as well as to facilitate transcription factor binding to nucleosomal DNA in an ATP-dependent manner (Cote et al., 7Cote J. Quinn J. Workman J.L. Peterson C.L. Science. 1994; 265: 53-60Crossref PubMed Scopus (718) Google Scholar; Kwon et al., 21Kwon H. Imbalzano A.N. Khavari P.A. Kingston R.E. Green M.R. Nature. 1994; 370: 477-481Crossref PubMed Scopus (640) Google Scholar; Imbalzano et al., 1994a14Imbalzano A.N. Kwon H. Green M.R. Kingston R.E. Nature. 1994; 370 (a): 481-485Crossref PubMed Scopus (520) Google Scholar). Another clue to the mechanism of SWI/SNF complex function is provided by the observation that active SWI/SNF complex is a component of the yeast RNA polymerase II holoenzyme (Wilson et al., 49Wilson C.J. Chao D.M. Imbalzano A.N. Schnitzler G.R. Kingston R.E. Young R.A. Cell. 1996; 84: 235-244Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar), which is thought to be the form of polymerase responsible for mRNA synthesis in vivo (reviewed in Carey, 4Carey M.F. Current Biol. 1995; 9: 1003-1005Abstract Full Text Full Text PDF Scopus (15) Google Scholar; Emili and Ingles, 10Emili A. Ingles C.J. Curr. Opin. Genet. & Dev. 1995; 5: 204-209Crossref PubMed Scopus (19) Google Scholar; Koleske and Young, 18Koleske A.J. Young R.A. Trends Biochem. Sci. 1995; 20: 113-116Abstract Full Text PDF PubMed Scopus (266) Google Scholar). This suggests that holoenzyme, perhaps targetted to an inactive promoter by activators capable of weakly binding chromatin, carries with it an activity capable of disrupting repressive chromatin structure and allowing preinitiation complex formation.Thus, understanding the mechanism by which SWI/SNF complexes alter chromatin structure is likely to be important in determining how transcriptionally inert genes become activated and may be applicable as well to other processes involving utilization of the DNA in chromatin, such as replication and viral integration (Dunaief et al., 8Dunaief J.L. Strober B.E. Guha S. Khavari P.A. Ålin K. Luban J. Begemann M. Crabtree G.R. Goff S.P. Cell. 1994; 79: 119-130Abstract Full Text PDF PubMed Scopus (551) Google Scholar; Kalpana et al., 16Kalpana G.V. Marmon S. Wang W. Crabtree G.R. Goff S.P. Science. 1994; 266: 2002-2006Crossref PubMed Scopus (456) Google Scholar). To further characterize the mechanism by which the human SWI/SNF (hSWI/SNF) complexes alter chromatin structure, we have investigated the ATP requirement for hSWI/SNF activity as well as the nature of the structural change induced by hSWI/SNF. We report that the change in chromatin structure induced by hSWI/SNF is stable, even in the absence of continued ATP hydrolysis, on both mononucleosome and nucleosomal plasmid templates. In addition, hSWI/SNF-facilitated transcription factor binding to nucleosomal DNA requires nucleosome alteration but does not require concurrent ATP hydrolysis.RESULTSTo investigate the requirements for and to assess changes in nucleosome structure due to hSWI/SNF activity, rotationally phased nucleosome particles were assembled in vitro from 32P-end-labeled, gel-purified 150-bp DNA fragments and purified HeLa cell histone octamers. Mononucleosome particles were separated from unassembled DNA by glycerol gradient centrifugation. The purified mononucleosome particles showed decreased mobility relative to naked DNA on native polyacrylamide gels (see below), were resistant to micrococcal nuclease digestion (Imbalzano et al., 1994a14Imbalzano A.N. Kwon H. Green M.R. Kingston R.E. Nature. 1994; 370 (a): 481-485Crossref PubMed Scopus (520) Google Scholar), and exhibited a 10-bp cleavage ladder upon digestion with DNase I (Fig. 1), which is typical of a mononucleosome population that is rotationally phased.Nucleotide Requirements for hSWI/SNF FunctionPrevious work has demonstrated that hSWI/SNF-mediated nucleosome disruption requires ATP and is not promoted by nonhydrolyzable ATP analogs (Kwon et al., 21Kwon H. Imbalzano A.N. Khavari P.A. Kingston R.E. Green M.R. Nature. 1994; 370: 477-481Crossref PubMed Scopus (640) Google Scholar). To further investigate the nucleotide requirements for nucleosome disruption, ATP was replaced by different nucleoside di- or triphosphates, and concentrations ranging from 20 µM to 2 mM of each nucleotide were tested (Fig. 1). hSWI/SNF activity resulted in a decrease in the intensity of the cleavage products comprising the 10-bp repeat pattern and the appearance of novel cleavages throughout the length of the template. 20 µM ATP was almost as effective as 2 mM ATP at eliciting maximal activity (lanes 4-6; similar results were seen at lower amounts of hSWI/SNF, data not shown). This change in accessibility to DNase I indicates that hSWI/SNF mediates an ATP-dependent alteration in the structure of the nucleosomal DNA (compare lanes 3 and 4). Lanes 22-27 confirm that ATP hydrolysis is required for hSWI/SNF function as neither ATPγS nor AMP-PNP, both nonhydrolyzable ATP analogs, supported nucleosome disruption. Of the analogs tested, only dATP could substitute for ATP, although approximately 10-fold more dATP than ATP was required to see disruption (lanes 7-9). UTP, GTP, CTP, and ADP did not promote disruption (lanes 10-21).Prior work has demonstrated that two chromatographically separable fractions that contain hSWI/SNF nucleosome disruption activity can be obtained from fractionation of HeLa cell nuclear extract. These fractions were termed A and B (Kwon et al., 21Kwon H. Imbalzano A.N. Khavari P.A. Kingston R.E. Green M.R. Nature. 1994; 370: 477-481Crossref PubMed Scopus (640) Google Scholar). Both contained immunoreactivity to BRG1, a SWI2/SNF2 homolog (Khavari et al., 17Khavari P.A. Peterson C.L. Tamkun J.W. Crabtree G.R. Nature. 1993; 366: 170-174Crossref PubMed Scopus (533) Google Scholar), and they showed no functional differences in all previous studies (Kwon et al., 21Kwon H. Imbalzano A.N. Khavari P.A. Kingston R.E. Green M.R. Nature. 1994; 370: 477-481Crossref PubMed Scopus (640) Google Scholar; Imbalzano et al., 1994a14Imbalzano A.N. Kwon H. Green M.R. Kingston R.E. Nature. 1994; 370 (a): 481-485Crossref PubMed Scopus (520) Google Scholar). Fig. 2A demonstrates that the alteration in the DNase I digestion pattern of mononucleosomes mixed with either hSWI/SNF A or hSWI/SNF B is the same. Both hSWI/SNF A and hSWI/SNF B were used separately in all of the experiments presented in this paper, and essentially identical results were obtained for each experiment (data not shown). The fact that all results are observed with both SWI/SNF complexes argues that they are unlikely to be caused by a fortuitously co-purifying activity because the hSWI/SNF A and B fractions are both highly enriched and they chromatograph differently on both phosphocellulose and single strand DNA-cellulose columns.Fig. 2A, nucleosome disruption by hSWI/SNF A and B is identical. Reaction conditions were as described for Fig. 1, except that, where indicated, reactions contained 4 mM ATP and 200 ng hSWI/SNF B fraction or 400 ng of hSWI/SNF A fraction. B, nucleosome disruption by hSWI/SNF is stable upon removal of ATP by apyrase. Reaction conditions were as described for Fig. 1, except that, where indicated, reactions contained 0.02 mM ATP and 300 ng of hSWI/SNF A fraction. Nucleosome disruption as assessed by DNase digestion 10 min after addition of hSWI/SNF is seen in lane 4; disruption after 40 min is seen in lane 11. Addition of 1 unit of apyrase prior to hSWI/SNF addition prevented nucleosome disruption (lane 5). A titration of apyrase concentration indicated that 0.1 unit was sufficient to prevent nucleosome disruption (data not shown). Apyrase was added to identical reactions 10 min after addition of hSWI/SNF (lanes 6-10), and disruption was assessed by DNase I digestion 2 min (lane 6), 5 min (lane 7), 10 min (lane 8), 20 min (lane 9), or 30 min (lane 10) after addition of apyrase. Reaction start times were staggered such that reactions presented in lanes 6-11 were started 5 min after the reactions presented in lanes 1-5. Solid dots were placed at some of the bands or groups of bands where the frequency of DNase I cleavage was altered by hSWI/SNF. N represents naked DNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)ATP Is Not Required to Maintain Disruption of MononucleosomesTo characterize the alteration of nucleosome structure by hSWI/SNF, we sought to determine whether ATP was continuously required in order to maintain a disrupted pattern in the presence of hSWI/SNF. If ATP were continuously required for activity, that is, if the structural change induced by hSWI/SNF and ATP were transient, and the nucleosome reverted to its original form after ATP mediated disruption, then removal of ATP from the reaction prior to DNase I cleavage should generate the same 10-bp ladder of cleavage products seen when untreated nucleosomes are digested with DNase I. Alternatively, if the change in structure induced by hSWI/SNF is stable, then the altered DNase I digestion pattern should be maintained, even after ATP is removed from the reaction. To facilitate this experiment, the ATP concentration in the reactions was decreased to 20 µM, which is sufficient for disruption (Fig. 1, lane 4).When apyrase, which cleaves ATP, was added to a reaction containing nucleosomes and ATP before the addition of hSWI/SNF, ATP-dependent nucleosome disruption was inhibited (Fig. 2B, compare lanes 4 and 5). In the reactions presented in lanes 6-10, identical samples containing nucleosomes, ATP, and hSWI/SNF were incubated for 10 min, were subsequently exposed to apyrase, and then were digested with DNase I at times ranging from 2 to 30 min following apyrase addition to determine whether the altered nucleosome would revert to its original structure. Lanes 6-10 of Fig. 2B indicate that the altered DNase I digestion pattern was maintained for up to 30 min past the addition of apyrase. Other experiments indicate that the altered digestion pattern was maintained for up to 2.5 h after apyrase addition (data not shown). This result indicates that the alteration in nucleosome structure induced by hSWI/SNF is stable, even in the absence of ATP.To confirm that hSWI/SNF could stably alter nucleosome structure, the nonhydrolyzable ATP analog, ATPγS, was used to competitively inhibit ATP hydrolysis by hSWI/SNF. Fig. 3 shows that concurrent addition of ATP and a 200-fold excess of ATPγS before addition of hSWI/SNF prevented nucleosome disruption for up to 60 min, presumably because ATPγS acts as a competitive inhibitor (lanes 6 and 13). When a 200-fold excess of ATPγS was added subsequent to hSWI/SNF addition, the altered DNase I digestion pattern was maintained (lanes 7-11), confirming that hSWI/SNF induced a stable change in nucleosome structure that was maintained in the absence of further hydrolysis. Addition of a 200-fold excess of AMP-PNP to the reaction did not inhibit hSWI/SNF activity (data not shown), probably reflecting a lower affinity of this analog for the ATP binding site.Fig. 3Nucleosome disruption by hSWI/SNF is stable when hSWI/SNF activity is competitively inhibited by excess ATPγS. The experiment is similar to that presented in Fig. 2B. PH MLT nucleosomes were labeled at the BamHI end. Where indicated, reactions contained 600 ng of hSWI/SNF B fraction, 0.02 mM ATP, and 4 mM ATPγS (200-fold excess). ATP-dependent nucleosome disruption 30 or 60 min after hSWI/SNF addition, as assayed by DNase I digestion, is seen in lanes 4 and 12. Substitution of excess ATPγS for ATP did not support nucleosome disruption (lane 5 and Fig. 1). Addition of excess ATPγS to reactions containing ATP prior to addition of hSWI/SNF prevented nucleosome disruption when assayed 30 min (lane 6) or 60 min (lane 13) later. Excess ATPγS was added to identical reactions 30 min after addition of hSWI/SNF (lanes 7-11), and disruption was assessed by DNase I digestion 2 min (lane 7), 5 min (lane 8), 10 min (lane 9), 20 min (lane 10), or 30 min (lane 11) after addition of ATPγS. A time course of hSWI/SNF B activity indicated that the maximal change in the DNase I digestion pattern required 30 min (data not shown). Therefore, the reactions containing hSWI/SNF B, nucleosomes, and ATP were incubated for 30 min prior to ATPγS addition. This difference in time required for maximal disruption is not thought to represent a functional difference between the A and B fractions but instead reflects differences in hSWI/SNF concentration and the age of the preparations (data not shown). Solid dots and lines were placed at some of the bands or groups of bands where the frequency of DNase I cleavage was altered by hSWI/SNF. N represents naked DNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)ATP Is Not Required to Maintain Altered Supercoiling of Plasmid TemplatesPreviously, we demonstrated that the hSWI/SNF fractions can reduce the linking number of closed circular DNA that was assembled into a nucleosomal template (Kwon et al., 21Kwon H. Imbalzano A.N. Khavari P.A. Kingston R.E. Green M.R. Nature. 1994; 370: 477-481Crossref PubMed Scopus (640) Google Scholar). This result is consistent with the observation that hSWI/SNF fractions can increase the accessibility of mononucleosomes to nucleases such as DNase I and indicates that hSWI/SNF can alter chromatin structure on nucleosomal plasmid templates as well as on mononucleosome particles. In the experiment presented in Fig. 4A, 32P-labeled, closed circular DNA was reconstituted into nucleosomes using octamers purified from HeLa cells and a Xenopus egg heat-treated extract that is competent for nucleosome assembly (Workman et al., 1991b53Workman J.L. Taylor I.C.A. Kingston R.E. Roeder R.G. Methods Cell Biol. 1991; 35 (b): 419-447Crossref PubMed Scopus (50) Google Scholar). The reconstituted template was purified by glycerol gradient centrifugation and was mixed with hSWI/SNF, ATP, and topoisomerase I. Following a 90-min incubation, the DNA was purified, and topoisomers were resolved by agarose gel electrophoresis. In an ATP-dependent manner, hSWI/SNF caused the appearance of a number of DNA topoisomers that have reduced mobility in the gel, indicating a loss of superhelical density (compare lanes 2, 5, and 7). Addition of an 18-fold excess of ATPγS prior to the addition of hSWI/SNF prevented alteration of the template (lane 6). When an 18-fold excess of ATPγS was added to the reaction subsequent to addition of hSWI/SNF and ATP, DNA species with reduced superhelical density were still present (lanes 9 and 10), indicating that the structural alteration in the nucleosomal plasmid template caused by hSWI/SNF was stable, even upon inhibition by excess ATPγS. Similarly, addition of apyrase at the start of the reaction but prior to the addition of hSWI/SNF prevented alteration of the template (lane 13). When apyrase was added after the addition of hSWI/SNF and ATP, topoisomers with reduced superhelical density were present (lanes 14-15), indicating that removal of ATP from the reaction did not reverse the alteration in nucleosomal DNA structure. We therefore conclude that the alteration in structure of a nucleosomal plasmid template by hSWI/SNF is also maintained in the absence of ATP hydrolysis.To increase the resolution of the topoisomers showing a hSWI/SNF-induced reduction in supercoiling, similar reactions were resolved on agarose gels containing chloroquine (Fig. 4B). The change in superhelical density in the presence of hSWI/SNF and ATP is shown in lane 9 (compare to lane 1). Addition of apyrase after the addition of hSWI/SNF did not change the distribution of topoisomers, again indicating that removal of ATP from the reaction did not reverse the alteration in nucleosomal DNA structure (lanes 7-8). The increase in resolution provided by the presence of chloroquine also indicated that hSWI/SNF has a small effect on nucleosome structure in the absence of ATP (lanes 3, 5, and 6). ATP-independent effects of yeast SWI/SNF on DNA topology have previously been noted (Quinn et al., 39Quinn J. Fyrberg A.M. Ganster R.W. Schmidt M.C. Peterson C.L. Nature. 1996; 379: 844-847Crossref PubMed Scopus (171) Google Scholar).Estimations of the size of the hSWI/SNF complex (1 MD by gel filtration (Kwon et al., 21Kwon H. Imbalzano A.N. Khavari P.A. Kingston R.E. Green M.R. Nature. 1994; 370: 477-481Crossref PubMed Scopus (640) Google Scholar)) and of the purity of the fractions used (3-10%, as estimated by visual examination of silver-stained SDS-polyacrylamide gels, see "Materials and Methods") allowed a crude estimate of stoichiometry to be made in these experiments. We calculate that hSWI/SNF is approximately equimolar with mononucleosome particles at concentrations of hSWI/SNF where nucleosome disruption is maximal. For nucleosomal plasmid templates, we calculated that a ∼20-fold excess of hSWI/SNF to template was sufficient to see maximal changes in supercoiling. This template contains 16 nucleosomes (on average), thereby resulting in an approximate equimolar ratio between hSWI/SNF and nucleosomes.ATP Is Required to Initiate SWI/SNF Activity on NucleosomesThe above results could be explained by a model in which ATP was necessary to produce an "active" version of hSWI/SNF, which would then be capable of altering nucleosome structure in the absence of further ATP hydrolysis. Alternatively, ATP hydrolysis could be required during hSWI/SNF disruption of the nucleosome. To distinguish between these possibilities, an order of addition experiment was performed in which hSWI/SNF was mixed with ATP, was subsequently treated with apyrase, and then was mixed with the nucleosomes (Fig. 5, lane 10). The results show that the unaltered 10-bp ladder of DNase I cleavage products
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