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• W2122911345 abstract "H2A.Z and H2A.1 nucleosome core particles and oligonucleosome arrays were obtained using recombinant versions of these histones and a native histone H2B/H3/H4 complement reconstituted onto appropriate DNA templates. Analysis of the reconstituted nucleosome core particles using native polyacrylamide gel electrophoresis and DNase I footprinting showed that H2A.Z nucleosome core particles were almost structurally indistinguishable from its H2A.1 or native chicken erythrocyte counterparts. While this result is in good agreement with the recently published crystallographic structure of the H2A.Z nucleosome core particle (Suto, R. K., Clarkson, M J., Tremethick, D. J., and Luger, K. (2000) Nat. Struct. Biol. 7, 1121–1124), the ionic strength dependence of the sedimentation coefficient of these particles exhibits a substantial destabilization, which is most likely the result of the histone H2A.Z-H2B dimer binding less tightly to the nucleosome. Analytical ultracentrifuge analysis of the H2A.Z 208-12, a DNA template consisting of 12 tandem repeats of a 208-base pair sequence derived from the sea urchin Lytechinus variegatus 5 S rRNA gene, reconstituted oligonucleosome complexes in the absence of histone H1 shows that their NaCl-dependent folding ability is significantly reduced. These results support the notion that the histone H2A.Z variant may play a chromatin-destabilizing role, which may be important for transcriptional activation. H2A.Z and H2A.1 nucleosome core particles and oligonucleosome arrays were obtained using recombinant versions of these histones and a native histone H2B/H3/H4 complement reconstituted onto appropriate DNA templates. Analysis of the reconstituted nucleosome core particles using native polyacrylamide gel electrophoresis and DNase I footprinting showed that H2A.Z nucleosome core particles were almost structurally indistinguishable from its H2A.1 or native chicken erythrocyte counterparts. While this result is in good agreement with the recently published crystallographic structure of the H2A.Z nucleosome core particle (Suto, R. K., Clarkson, M J., Tremethick, D. J., and Luger, K. (2000) Nat. Struct. Biol. 7, 1121–1124), the ionic strength dependence of the sedimentation coefficient of these particles exhibits a substantial destabilization, which is most likely the result of the histone H2A.Z-H2B dimer binding less tightly to the nucleosome. Analytical ultracentrifuge analysis of the H2A.Z 208-12, a DNA template consisting of 12 tandem repeats of a 208-base pair sequence derived from the sea urchin Lytechinus variegatus 5 S rRNA gene, reconstituted oligonucleosome complexes in the absence of histone H1 shows that their NaCl-dependent folding ability is significantly reduced. These results support the notion that the histone H2A.Z variant may play a chromatin-destabilizing role, which may be important for transcriptional activation. base pair The packaging of DNA around histone octamers creates a thermodynamic obstacle to processive enzyme complexes such as RNA polymerase. To lower the energy of activation and display a template more amenable for expression, the nucleus uses several mechanisms to biochemically alter the nature of histone-DNA and histone-histone interactions. These mechanisms include post-translational modifications, nucleosome remodeling complexes, and the introduction of histone variants into the octameric core (for current reviews, see Refs. 1Wolffe A.P. Guschin D. J. Struct. Biol. 2000; 129: 102-122Crossref PubMed Scopus (290) Google Scholar, 2Flaus A. Owen-Hughes T. Curr. Opin. Genet. Dev. 2001; 11: 148-154Crossref PubMed Scopus (161) Google Scholar, 3Aalfs J.D. Kingston R.E. Trends Biochem. Sci. 2000; 25: 548-555Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Histone variants are nonallelic isoforms of the major H2A, H2B, H3, and H4 proteins that interact through inherent histone fold domains in nucleosomes throughout the genome (for current reviews, see Refs. 4Kornberg R.D. Lorch Y. Cell. 1999; 98: 285-294Abstract Full Text Full Text PDF PubMed Scopus (1421) Google Scholar and 5Hayes J.J. Hansen J.C. Curr. Opin. Genet. Dev. 2001; 11: 124-129Crossref PubMed Scopus (100) Google Scholar). By inserting variant histones into the octamer noncovalent interactions between the players are altered, possibly creating particles with modified stability or functional novelty. This epigenetic feature may be utilized to silence nonessential genes in differentiated tissues or to lower the binding constants of replication, transcription, and repair machinery in active chromatin.H2A.Z is an H2A subtype that has been identified in organisms as diverse as Saccharomyces cerevisiae (6Jackson J.D. Falciano V.T. Gorovsky M.A. Trends Biochem. Sci. 1996; 21: 466-467Abstract Full Text PDF PubMed Scopus (45) Google Scholar),Tetrahymena (7Allis C.D. Glover C.V. Bowen J.K. Gorovsky M.A. Cell. 1980; 20: 609-617Abstract Full Text PDF PubMed Scopus (139) Google Scholar), Drosophila (8van Daal A. White E.M. Gorovsky M.A. Elgin S.C. Nucleic Acids Res. 1988; 16: 7487-7497Crossref PubMed Scopus (64) Google Scholar), and Homo sapiens (9Hatch C.L. Bonner W.M. Nucleic Acids Res. 1988; 16: 1113-1124Crossref PubMed Scopus (52) Google Scholar). The protein displays 60% homology with H2A and 90% homology between species. Mutagenic assays have demonstrated that H2A.Z is essential for development in yeast (10Jackson J.D. Gorovsky M.A. Nucleic Acids Res. 2000; 28: 3811-3816Crossref PubMed Scopus (103) Google Scholar) and for viability in Tetrahymena (11Liu X. Li B. Gorovsky M.A. Mol. Cell. Biol. 1996; 16: 4305-4311Crossref PubMed Scopus (102) Google Scholar) and Drosophila (12van Daal A. Elgin S.C. Mol. Biol. Cell. 1992; 3: 593-602Crossref PubMed Scopus (175) Google Scholar, 13Clarkson M.J. Wells J.R.E. Gibson F. Saint R. Tremethick D.J. Nature. 1999; 399: 694-697Crossref PubMed Scopus (168) Google Scholar). Initial immunochemical characterization of this protein uncovered that H2A.Z is exclusive to transcriptionally active domains in Tetrahymena(14Allis C.D. Ziegler Y.S. Gorovsky M.A. Olmsted J.B. Cell. 1982; 31: 131-136Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 15Wenkert D. Allis C.D. J. Cell Biol. 1984; 98: 2107-2117Crossref PubMed Scopus (71) Google Scholar, 16Stargell L.A. Bowen J. Dadd C.A. Dedon P.C. Davis M. Cook R.G. Allis C.D. Gorovsky M.A. Genes Dev. 1993; 7: 2641-2651Crossref PubMed Scopus (108) Google Scholar). Recently H2A.Z has been observed to be located at yeast promoters and to display a redundant role with ATP-dependent nucleosome remodeling complexes (17Santisteban M.S. Kalashnikova T. Smith M.M. Cell. 2000; 103: 411-422Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar) and interact directly with transcriptional machinery during gene expression (18Adam M. Robert F. Larochelle M. Gaudreau L. Mol. Cell. Biol. 2001; 21: 6270-6279Crossref PubMed Scopus (181) Google Scholar). However, the functional dynamics of H2A.Z enrichment in active chromatin remains enigmatic as other studies describe H2A.Z deposition to have a nonspecific (19Leach T.J. Mazzeo M. Chotkowski H.L. Madigan J.P. Wotring M.G. Glaser R.L. J. Biol. Chem. 2000; 275: 23267-23272Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) and repressive effect on expression (20Dhillon N. Kamakaka R.T. Mol. Cell. 2000; 6: 769-780Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar).The characterization of the H2A.Z nucleosome crystal structure by Suto and colleagues (21Suto R.K. Clarkson M.J. Tremethick D.J. Luger K. Nat. Struct. Biol. 2000; 7: 1121-1124Crossref PubMed Scopus (435) Google Scholar) provides a snapshot of histone-DNA and histone-histone interactions within the nucleosome core particle containing this histone variant. Divergent amino acid residues in H2A.Z octamers conform to similar nucleic acid-binding sites as native octamers (22Luger K. Mader A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-260Crossref PubMed Scopus (6786) Google Scholar) and do not distort the superhelical path of DNA around the nucleosome perimeter (21Suto R.K. Clarkson M.J. Tremethick D.J. Luger K. Nat. Struct. Biol. 2000; 7: 1121-1124Crossref PubMed Scopus (435) Google Scholar). Despite this lack of effect on the DNA trajectory, it appears that internal protein-protein interactions are affected. Substitution of H2A Gln104 by Gly104in H2A.Z destabilizes the 2(H2A.Z-H2B)-(H3-H4)2association and presents an opportune particle for DNA activation (13Clarkson M.J. Wells J.R.E. Gibson F. Saint R. Tremethick D.J. Nature. 1999; 399: 694-697Crossref PubMed Scopus (168) Google Scholar, 17Santisteban M.S. Kalashnikova T. Smith M.M. Cell. 2000; 103: 411-422Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Also the molecular surface of the variant nucleosome displays a novel acidic patch and a divalent cation-binding site, which may facilitate the rearrangement of higher order structure through internucleosomal electrostatic interactions or the recruitment of remodeling factors (21Suto R.K. Clarkson M.J. Tremethick D.J. Luger K. Nat. Struct. Biol. 2000; 7: 1121-1124Crossref PubMed Scopus (435) Google Scholar). However, these observations are only speculative as it is difficult to determine the direct implications of H2A.Z for chromatin from static crystallographic images.To address this problem we have reconstituted nucleosome core particles and oligonucleosome arrays containing major histone H2A.1 variant and variant histone H2A.Z and have characterized the ionic strength dependence of these complexes by analytical ultracentrifugation. These experiments provide the first clues into the folding dynamics of H2A.Z mononucleosome and chromatin complexes.RESULTS AND DISCUSSIONWe have used recombinant human H2A.Z and H2A.1 histone variants to reconstitute nucleosome core particles and 208-12 nucleosome arrays. Fig. 1, A and B, display the electrophoretic nature of the histone component of these particles.H2A.Z octamers were reconstituted onto random sequence 146-bp DNA fragments obtained from chicken erythrocyte nucleosomes. The generated particles are shown in Fig. 2 A. As it can be seen in this figure, histone octamers consisting of H2A.1 or H2A.Z are equally able to produce mononucleosome particles with identical electrophoretic mobility. The slightly lower electrophoretic mobility of these complexes when compared with native (purified) chicken erythrocyte mononucleosomes (Fig. 2 A, lane 4) can be ascribed to differences in the ionic strength of the sample buffer. Indeed the fraction of free DNA that is present in lane 2 of the Fig. 2 A (see white arrow) also exhibits a similar extent of mobility retardation when compared with the same DNA template used for the reconstitution of these particles (see Fig. 2 A,lane 5). The structural similarity between reconstituted H2A.1 and H2A.Z nucleosome core particles and to native chicken erythrocyte particles can be further depicted from the DNase I footprints which are shown in Fig. 2 B.Figure 2A, native (4%) polyacrylamide gel of H2A.Z-containing (lane 2) and H2A.1-containing (lane 3) reconstituted nucleosome core particles. Lane 4 is native chicken erythrocyte nucleosome core particles, and lane 5 corresponds to the 146-bp DNA obtained from these native particles and which was used in the reconstitution of the complexes shown in lanes 2 and 3. Lane 1 is a DNA marker obtained by digesting pBr 322 with HhaI. The white arrow points to the presence of a small fraction of free DNA. B, DNase I footprinting analysis of H2A.1-containing (lane 2) and H2A.Z-containing (lane 3) reconstituted nucleosome core particles in comparison to native chicken erythrocyte nucleosome core particles (lane 1).View Large Image Figure ViewerDownload Hi-res image Download (PPT)These results clearly demonstrate that recombinant histone H2A.Z variants can be equally reconstituted into nucleosome core particles that are otherwise structurally very similar to native nucleosome core particles. This result is not surprising, and it was to be anticipated from the recently determined crystal structure of H2A.Z nucleosomes (21Suto R.K. Clarkson M.J. Tremethick D.J. Luger K. Nat. Struct. Biol. 2000; 7: 1121-1124Crossref PubMed Scopus (435) Google Scholar), which showed that the overall structure of this particle was similar to that of a nucleosome core particle that did not contain this unique histone H2A variant (22Luger K. Mader A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-260Crossref PubMed Scopus (6786) Google Scholar).The apparent lack of a significant structural difference between H2A.Z and native nucleosome core particles consisting of the two major histone H2A isoforms (H2A.1 and H2A.2) is intriguing. Indeed, in contrast to H2A.1/H2A.2 variants, histone H2A.Z has been shown to be essential for survival in organisms phylogenetically as diverse as Tetrahymena (11Liu X. Li B. Gorovsky M.A. Mol. Cell. Biol. 1996; 16: 4305-4311Crossref PubMed Scopus (102) Google Scholar) and Drosophila (12van Daal A. Elgin S.C. Mol. Biol. Cell. 1992; 3: 593-602Crossref PubMed Scopus (175) Google Scholar, 13Clarkson M.J. Wells J.R.E. Gibson F. Saint R. Tremethick D.J. Nature. 1999; 399: 694-697Crossref PubMed Scopus (168) Google Scholar). It has been determined by substitution experiments with H2A.1 homologous regions that the indispensable portion of histone H2A.Z maps to the carboxyl-terminal of the molecule (13Clarkson M.J. Wells J.R.E. Gibson F. Saint R. Tremethick D.J. Nature. 1999; 399: 694-697Crossref PubMed Scopus (168) Google Scholar). Interestingly enough, of all core histones (H2A, H2B, H3, and H4), H2A is the only histone with a prominent carboxyl-terminal “tail” extending beyond the histone fold (39Arents G. Moudrianakis E.N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11170-11174Crossref PubMed Scopus (286) Google Scholar). The COOH domain also introduces inherent functional novelty in the case of three other histone H2A variants. H2A-Bbd is a recently identified protein that displays a truncated carboxyl-terminal tail and is enriched in active chromatin (40Chadwick B.P. Willard H.F. J. Cell Biol. 2001; 152: 375-384Crossref PubMed Scopus (163) Google Scholar), H2AX contains a carboxyl-terminal DNA-dependent protein kinase/ataxia telangiectasia mutated consensus recognition sequence (SQE) and is a phosphorylation substrate during double strand break DNA repair (41Downs J.A. Lowndes N.F. Jackson S.P. Nature. 2000; 408: 1001-1004Crossref PubMed Scopus (532) Google Scholar) and meiosis (42Mahadevaiah S.K. Turner J.M.A. Baudat F. Rogakou E.P. de Boer P. Blanco-Rodriguez J. Jasin M. Keeney S. Bonner W.M. Burgoyne P.S. Nat. Genet. 2001; 27: 271-276Crossref PubMed Scopus (687) Google Scholar), and macroH2A has a large COOH nonhistone region, which specializes the protein for transcriptionally silenced chromatin (43Pehrson J.R. Fried V.A. Science. 1992; 257: 1398-1400Crossref PubMed Scopus (278) Google Scholar).In the case of the major H2A.1 and -2 subtypes, the carboxyl terminus has been shown to play an important role in the stability of the nucleosome core particle. Cleavage of the last 15 COOH-terminal amino acids of this region by an endogenous chromatin-bound protease (44Eickbush T.H. Watson D.K. Moudrianakis E.N. Cell. 1976; 9: 785-792Abstract Full Text PDF PubMed Scopus (57) Google Scholar) has been shown to substantially lower the affinity of the histone H2A-H2B dimer for the H3-H4 tetramer (45Eickbush T.H. Godfrey J.E. Elia M.C. Moudrianakis E.N. J. Biol. Chem. 1988; 263: 18972-18978Abstract Full Text PDF PubMed Google Scholar). Based upon these observations, it is reasonable to assume that H2A isoform substitution within the nucleosome may have functional and/or structural implications for chromatin nucleoprotein folding dynamics in vivo.Alterations of nucleosome core particle stability in solution can be monitored by changes in their conformation resulting from variations of the ionic strength within the range of salt concentrations where histones still remain bound to DNA (≤0.6 m NaCl) (32Ausió J. Seger D. Eisenberg H. J. Mol. Biol. 1984; 176: 77-104Crossref PubMed Scopus (107) Google Scholar, 46Greulich K.O. Ausió J. Eisenberg H. J. Mol. Biol. 1985; 186: 167-173Crossref PubMed Scopus (25) Google Scholar). Changes in the ionic environment of the nucleosome under these conditions are physiologically relevant as they can mimic, to a large extent, the changes in the ionic environment resulting from interactions with other protein complexes such as RNA polymerase or chromatin-remodeling complexes (3Aalfs J.D. Kingston R.E. Trends Biochem. Sci. 2000; 25: 548-555Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) among others.We (25Ausió J. Dong F. van Holde K.E. J. Mol. Biol. 1989; 206: 451-463Crossref PubMed Scopus (282) Google Scholar, 32Ausió J. Seger D. Eisenberg H. J. Mol. Biol. 1984; 176: 77-104Crossref PubMed Scopus (107) Google Scholar, 33Ausió J. Biophys. Chem. 2000; 86: 141-153Crossref PubMed Scopus (38) Google Scholar, 47Ausió J.A. van Holde K.E. Biochemistry. 1986; 25: 1421-1428Crossref PubMed Scopus (197) Google Scholar, 48Dong F. Nelson C. Ausió J. Biochemistry. 1990; 29: 10710-10716Crossref PubMed Scopus (14) Google Scholar) and others (35Yager T.D. van Holde K.E. J. Biol. Chem. 1984; 259: 4212-4222Abstract Full Text PDF PubMed Google Scholar, 49Stein A. J. Mol. Biol. 1979; 130: 103-134Crossref PubMed Scopus (125) Google Scholar, 50Cotton R.W. Hamkalo B.A. Nucleic Acids Res. 1981; 9: 445-457Crossref PubMed Scopus (56) Google Scholar, 51Widom J. Methods Mol. Biol. 1999; 119: 61-77PubMed Google Scholar) have shown that nucleosomes in solution are not static entities but rather they are highly transient structures. A dynamic equilibrium exists between the constitutive histone octamer and the nucleosomal DNA (32Ausió J. Seger D. Eisenberg H. J. Mol. Biol. 1984; 176: 77-104Crossref PubMed Scopus (107) Google Scholar), which besides the ionic strength is also dependent on many other physical parameters such as temperature and sample concentration.With this in mind, we decided to characterize the ionic strength dependence of the sedimentation coefficient of reconstituted H2A.1 and H2A.Z nucleosomes. The results of such analysis are shown in Fig. 3 A. They indicate that H2A.1 nucleosomes behave in a way that is almost indistinguishable from native nucleosome core particles. In contrast, although H2A.Z reconstituted nucleosome core particles exhibit a very similar sedimentation coefficient at low salt (≤0.1 m NaCl), this parameter displays a characteristic declining trend to a sedimentation coefficient value of 8.3 S at 600 mm NaCl. This drop in the S value is clearly indicative of a conformational change of the nucleosome core particle. Integral distribution analysis (38van Holde K.E. Weischet W.O. Biopolymers. 1978; 17: 1387-1403Crossref Scopus (318) Google Scholar) shows that at 0.6 m NaCl, about 30–40% of the H2A.Z nucleosome core particles sediment at 8.3 S, 30% sediment at 5.4 S, and the remaining 30% sediment with intermediate values (see Fig. 3 B). This is in contrast to reconstituted H2A.1 nucleosomes, which under the same conditions exhibit a 9.4 S (70%), 5.4 S (15%), and 15% component of intermediate sedimenting particles (see Fig. 3 B) in what is almost indistinguishable from native nucleosome core particles (32Ausió J. Seger D. Eisenberg H. J. Mol. Biol. 1984; 176: 77-104Crossref PubMed Scopus (107) Google Scholar, 33Ausió J. Biophys. Chem. 2000; 86: 141-153Crossref PubMed Scopus (38) Google Scholar). The 5.4 S value corresponds to free nucleosomal DNA, which is reversibly dissociating from the nucleosome core particle (32Ausió J. Seger D. Eisenberg H. J. Mol. Biol. 1984; 176: 77-104Crossref PubMed Scopus (107) Google Scholar). While the conformational change in the native and H2A.1 nucleosome core particles corresponding to 9.4 S at 0.6m NaCl is not yet clearly understood (52Bauer W.R. Hayes J.J. White J.H. Wolffe A.P. J. Mol. Biol. 1994; 236: 685-690Crossref PubMed Scopus (113) Google Scholar), the value of 8.3 S observed with H2A.Z nucleosome core particles under the same conditions could be accounted for by partial H2A-H2B depletion. We have experimentally determined the sedimentation velocity coefficient of a nucleosome core particle deficient in one H2A-H2B dimer to be 8.6 S and that of the nucleosome core particle lacking both dimers to be 6.9 S, whereas the sedimentation coefficient of free 146-bp DNA was determined to be 5.2 S. 2J. Ausió, unpublished results. These values are in good agreement with similar experimental values reported by other groups (53Read C.M. Baldwin J.P. Crane-Robinson C. Biochemistry. 1985; 24: 4435-4450Crossref PubMed Scopus (30) Google Scholar). Thus it is possible that the decrease in sedimentation observed in the case of the H2A.Z nucleosome core particle corresponds to the progressive loss of particle integrity (H2A.Z-H2B dissociation).Figure 3A, ionic strength-dependent variation of the sedimentation coefficient s20,w of H2A.Z-containing (●) and H2A.1-containing (○) reconstituted nucleosome core particles in comparison to native chicken erythrocyte nucleosome core particles (▵). The dashed line corresponds to previously published data (25Ausió J. Dong F. van Holde K.E. J. Mol. Biol. 1989; 206: 451-463Crossref PubMed Scopus (282) Google Scholar, 47Ausió J.A. van Holde K.E. Biochemistry. 1986; 25: 1421-1428Crossref PubMed Scopus (197) Google Scholar). B, integral distribution of the sedimentation coefficient (in Svedberg units (S)) of H2A.Z-containing (●) and H2A.1-containing (○) reconstituted nucleosome core particles in comparison to native chicken erythrocyte nucleosome core particles (▵) in 0.6 m NaCl. These integral distributions were obtained after analysis of the sedimentation boundaries using the method of van Holde and Weischet (38van Holde K.E. Weischet W.O. Biopolymers. 1978; 17: 1387-1403Crossref Scopus (318) Google Scholar). In these plots the ordinate represents the fraction of nucleosome arrays sedimenting with an s20,wgiven in the abscissa. All experiments were carried out at 20 °C and 40,000 rpm. The buffer used was 10 mm Tris-HCl (pH 7.5), 0.1 mm EDTA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)H2A.Z particle lability shown in Fig. 3 was also observed elsewhere during sample manipulation. H2A.Z particles displayed an increased electrophoretic mobility compared with the unmodified patterns of its major H2A and native nucleosome counterparts following concentration (see Fig. 4). This observation corroborates the ultracentrifuge analysis and indicates that the H2A.Z nucleosome core particles have a reduced stability. The results are in very good agreement and support the crystallographic data that pointed to the existence of a “subtle destabilization of the interaction between the (H2A.Z-H2B) dimer and the (H3-H4)2 tetramer” in the crystal structure (21Suto R.K. Clarkson M.J. Tremethick D.J. Luger K. Nat. Struct. Biol. 2000; 7: 1121-1124Crossref PubMed Scopus (435) Google Scholar).Figure 4Native (4%) polyacrylamide gel of nucleosomes that have been concentrated 5-fold with Centricon YM-10 (Amicon, Millipore) before being loaded on the gel. Lane 2, native chicken erythrocyte nucleosome core particles; lane 3, H2A.1-containing reconstituted nucleosome core particles; lane 4, H2A.Z-containing reconstituted nucleosome core particles; lane 5, 146-bp random sequence DNA. Lane 1 consists of a DNA marker obtained by cutting pBr 322 with HhaI.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We decided next to look at the effects of the H2A.Z histone variant in the modulation of internucleosomal interactions by analyzing reconstituted oligonucleosome complexes (28Ausió J. Moore S.C. Methods. 1998; 14: 333-342Crossref Scopus (36) Google Scholar). The results of this analysis are shown in Fig. 5.Figure 5A, integral distribution of the sedimentation coefficient (in Svedberg units (S)) of H2A.Z reconstituted 208-12 nucleosome arrays at different NaCl concentrations (0, 75, and 150 mm as indicated) in 10 mmTris-HCl (pH 7.5), 0.1 mm EDTA buffer. B, ionic strength (NaCl concentration) dependence of the average sedimentation coefficient s20,w of 208-12 oligonucleosome complexes containing histone H2A.1 (○) and 208-12 oligonucleosome arrays containing H2A.Z (●). The dotted line represents the ionic strength dependence of 208-12 oligonucleosome complexes reconstituted with native chicken erythrocyte histones (28Ausió J. Moore S.C. Methods. 1998; 14: 333-342Crossref Scopus (36) Google Scholar). C, integral distribution of the sedimentation coefficient (in Svedberg units (S)) at 150 mm NaCl of H2A.Z-containing (●) and H2A.1-containing (○) reconstituted 208-12 nucleosome arrays and 208-12 oligonucleosome complexes reconstituted with native chicken erythrocyte histones. The analytical ultracentrifuge runs were carried out at 20 °C and 20,000 rpm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Saturated H2A.Z 208-12 oligonucleosome complexes (30Simpson R.T. Thoma F. Brubaker J.M. Cell. 1985; 42: 799-808Abstract Full Text PDF PubMed Scopus (371) Google Scholar) sedimented in low salt (10 mm Tris-HCl (pH 7.5), 0.1 mm EDTA (TE buffer)) as a very homogeneous population with a sedimentation coefficient of 29.5 S (see Fig. 5 A), similar to its H2A.1 208-12 counterpart (results not shown). However, as the salt was titrated to 150 mm, the increase in the sedimentation coefficient of the 208-12 H2A.Z reconstituted complexes was consistently lower than that of the 208-12 complexes reconstituted with either H2A.1 octamers or native histone octamers (28Ausió J. Moore S.C. Methods. 1998; 14: 333-342Crossref Scopus (36) Google Scholar, 31Garcia-Ramirez M. Dong F. Ausió J. J. Biol. Chem. 1992; 267: 19587-19595Abstract Full Text PDF PubMed Google Scholar). As in H2A.1 or in native histone 208-12 complexes, a plateau was reached at 100–150 mm NaCl but at a (10%) lower average sedimentation coefficient value (34 S) (see Fig. 5 B). The increase in sedimentation coefficient of the 208-12 reconstituted complexes under these conditions reflects an increase in the folding of the complexes (31Garcia-Ramirez M. Dong F. Ausió J. J. Biol. Chem. 1992; 267: 19587-19595Abstract Full Text PDF PubMed Google Scholar, 33Ausió J. Biophys. Chem. 2000; 86: 141-153Crossref PubMed Scopus (38) Google Scholar, 54Hansen J.C. Ausió J. Stanik V.H. van Holde K.E. Biochemistry. 1989; 28: 9129-9136Crossref PubMed Scopus (195) Google Scholar). The very similar values of the sedimentation coefficients of H2A.1 and H2A.Z reconstituted 208-12 complexes at low salt was to be expected from the similarity of the sedimentation coefficient values of the nucleosome core particles under the same conditions (see Fig. 3 A), which suggest that both species of nucleosome core particles have a very similar conformation at this low salt. The reason for the inability of the 208-12 H2A.Z nucleosome arrays to fold to the same extent as the H2A.1 counterpart (as indicated by the lower sedimentation coefficient values observed in this later instance) is not clear. However, it could possibly be attributed to novel internucleosomal electrostatic interactions resulting from the H2A.Z-H2B dimer acidic patch, which was observed in the crystal structure of the H2A.Z nucleosome core particles (21Suto R.K. Clarkson M.J. Tremethick D.J. Luger K. Nat. Struct. Biol. 2000; 7: 1121-1124Crossref PubMed Scopus (435) Google Scholar). It is important to note, however, that this does not preclude the formation of the 40–50 S higher folding structures (see Fig. 5 C), which are observed for 208-12-mers reconstituted with native core histones under physiological ionic strength conditions of 100–150 mm NaCl and which have also been well characterized in the presence of divalent ions such as magnesium (55Fletcher T.M. Hansen J.C. Crit. Rev. Eukaryot. Gene Expr. 1996; 6: 149-188Crossref PubMed Scopus (130) Google Scholar).When the results described above are considered together, the data suggest that histone variant H2A.Z has a destabilizing effect on both intranucleosomal histone-histone interactions and at the internucleosomal level. Such destabilization is consistent with the physiological roles attributed to this variant, especially its participation in the regulation of transcription through its enrichment at promoter sites and redundancy with nucleosome remodeling complexes (17Santisteban M.S. Kalashnikova T. Smith M.M. Cell. 2000; 103: 411-422Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar) and in recruitment of RNA polymerase II (20Dhillon N. Kamakaka R.T. Mol. Cell. 2000; 6: 769-780Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Such instability of the nucleosome core particle complexes possibly resulting from the loss of H2A.Z-H2B dimers could facilitate the movement of the RNA polymerase complex through the nucleosomal DNA during transcriptional elongation (56van Holde K.E. Lohr D.E. Robert C. J. Biol. Chem. 1992; 267: 2837-2840Abstract Full Text PDF PubMed Google Scholar).In the future, it will be" @default.
• W2122911345 created "2016-06-24" @default.
• W2122911345 creator A5001104135 @default.
• W2122911345 creator A5021636704 @default.
• W2122911345 creator A5029363724 @default.
• W2122911345 creator A5060968786 @default.
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• W2122911345 date "2001-11-01" @default.
• W2122911345 modified "2023-10-09" @default.
• W2122911345 title "Characterization of the Stability and Folding of H2A.Z Chromatin Particles" @default.
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