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W1977208266 abstract "The MgCl2-induced folding of defined 12-mer nucleosomal arrays, in which ubiquitinated histone H2A (uH2A) replaced H2A, was analyzed by quantitative agarose gel electrophoresis and analytical centrifugation. Both types of analysis showed that uH2A arrays attained a degree of compaction similar to that of control arrays in 2 mm MgCl2. These results indicate that attachment of ubiquitin to H2A has little effect on the ability of nucleosomal arrays to form higher order folded structures in the ionic conditions tested. In contrast, uH2A arrays were found to oligomerize at lower MgCl2 concentrations than control nucleosomal arrays, suggesting that histone ubiquitination may play a role in nucleosomal fiber association. The MgCl2-induced folding of defined 12-mer nucleosomal arrays, in which ubiquitinated histone H2A (uH2A) replaced H2A, was analyzed by quantitative agarose gel electrophoresis and analytical centrifugation. Both types of analysis showed that uH2A arrays attained a degree of compaction similar to that of control arrays in 2 mm MgCl2. These results indicate that attachment of ubiquitin to H2A has little effect on the ability of nucleosomal arrays to form higher order folded structures in the ionic conditions tested. In contrast, uH2A arrays were found to oligomerize at lower MgCl2 concentrations than control nucleosomal arrays, suggesting that histone ubiquitination may play a role in nucleosomal fiber association. ubiquitinated histones H2A and H2B base pair(s) gel-free electrophoretic mobility effective macromolecular radius effective gel pore size Although for many years histones were thought to be merely structural components of nucleosomes, the primary level of DNA organization required to compact the genome in the nucleus, they are now recognized as important players in the mechanisms underlying gene expression. One of the keys to chromatin's dynamic nature is post-translational modification of the flexible histone tails. These modifications include acetylation, phosphorylation, methylation, and ubiquitination (1van Holde K.E. Chromatin. Spinger-Verlag, New York1989Crossref Google Scholar, 2Davie J.R. Curr. Opin. Genet. Dev. 1998; 8: 173-178Crossref PubMed Scopus (178) Google Scholar, 3Spencer V.A. Davie J.R. Gene. 1999; 240: 1-12Crossref PubMed Scopus (265) Google Scholar). Ubiquitin is a small, mainly globular and highly conserved protein consisting of 76 amino acids found, as its name implies, in most living organisms. Ubiquitin has been found to be conjugated in vivo to histones H2A, H2B, H3, and H1 (4Goldknopf I.L. French M.F. Musso R. Busch H. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5492-5495Crossref PubMed Scopus (93) Google Scholar, 5West M.H.P. Bonner W.M. Nucleic Acids Res. 1980; 8: 4671-4680Crossref PubMed Scopus (163) Google Scholar, 6Chen H.Y. Sun J.M. Zhang Y. Davie J.R. Meistrich M.L. J. Biol. Chem. 1998; 273: 13165-13169Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 7Pham A.-D. Sauer F. Science. 2000; 289: 2357-2360Crossref PubMed Scopus (203) Google Scholar). Ubiquitin is reversibly attached to bovine H2A by means of an isopeptide bond between its terminal glycine and the ε amino group of H2A lysine 119 (8Goldknopf I.L. Busch H. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 864-868Crossref PubMed Scopus (306) Google Scholar), which lies in the trypsin-accessible region of the carboxyl-terminal tail (9Böhm L. Crane-Robinson C. Sautiére P. Eur. J. Biochem. 1980; 106: 525-530Crossref PubMed Scopus (58) Google Scholar). Histones are among the most abundant ubiquitin-protein conjugates in higher eukaryotes, where 5–15% of the total H2A is ubiquitinated (10Rechsteiner M. Ubiquitin. Plenum Press, New York1988Crossref Google Scholar). The function of histone ubiquitination remains unclear. Although ubiquitin has been shown to play an important role in the degradation of many short-lived proteins (for reviews see Refs. 11Finley D. Chau V. Ann. Rev. Cell Biol. 1991; 7: 25-69Crossref PubMed Scopus (421) Google Scholar, 12Jennissen H.P. Eur. J. Biochem. 1995; 231: 1-30Crossref PubMed Scopus (112) Google Scholar), two independent studies have shown that ubiquitination does not tag histones for degradation (13Seale R.L. Nucleic Acids Res. 1981; 9: 3151-3158Crossref PubMed Scopus (44) Google Scholar, 14Wu R.S. Kohn K.W. Bonner W.M. J. Biol. Chem. 1981; 256: 5916-5920Abstract Full Text PDF PubMed Google Scholar). Nucleosomes can be reconstituted with two molecules of uH2A or uH2B1 without obvious perturbation of the nucleosomal structure (15Kleinschmidt A.M. Martinson H.G. Nucleic Acids Res. 1981; 9: 2423-2431Crossref PubMed Scopus (30) Google Scholar, 16Davies N. Lindsey G. Biochim. Biophys. Acta. 1994; 1218: 187-193Crossref PubMed Scopus (40) Google Scholar). Although some studies have reported an enrichment of uH2A in transcriptionally poised or active chromatin (17Barsoum J. Varshavsky A. J. Biol. Chem. 1985; 260: 7688-7697Abstract Full Text PDF PubMed Google Scholar, 18Levinger L. Varshavsky A. Cell. 1982; 28: 375-385Abstract Full Text PDF PubMed Scopus (236) Google Scholar) others do not find this correlation (19Huang S.Y. Barnard M.B. Xu M. Matsui S. Rose S.M. Garrard W.T. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3738-3742Crossref PubMed Scopus (46) Google Scholar, 20Parlow M.H. Haas A.L. Lough J. J. Biol. Chem. 1990; 265: 7507-7512Abstract Full Text PDF PubMed Google Scholar, 21Dawson B.A. Herman T. Haas A.L. Lough J. J. Cell. Biochem. 1991; 46: 166-173Crossref PubMed Scopus (24) Google Scholar). Moreover, inhibition of transcription does not alter the levels of uH2A in a variety of cell lines (22Ericsson C. Goldknopf I.L. Daneholt B. Exp. Cell Res. 1986; 167: 127-134Crossref PubMed Scopus (17) Google Scholar, 23Davie J.R. Murphy L.C. Biochemistry. 1990; 29: 4752-4757Crossref PubMed Scopus (121) Google Scholar, 24Davie J.R. Murphy L.C. Biochem. Biophys. Res. Commun. 1994; 203: 344-350Crossref PubMed Scopus (40) Google Scholar), whereas inhibitors of hnRNA synthesis were found to cause a decrease in uH2B levels (23Davie J.R. Murphy L.C. Biochemistry. 1990; 29: 4752-4757Crossref PubMed Scopus (121) Google Scholar,24Davie J.R. Murphy L.C. Biochem. Biophys. Res. Commun. 1994; 203: 344-350Crossref PubMed Scopus (40) Google Scholar). Cell cycle studies have shown that, in cells undergoing mitosis, uH2A levels decrease progressively to non-detectable levels at metaphase but increase again in late anaphase (14Wu R.S. Kohn K.W. Bonner W.M. J. Biol. Chem. 1981; 256: 5916-5920Abstract Full Text PDF PubMed Google Scholar, 25Matsui S. Seon B.K. Sandberg A.A. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6386-6390Crossref PubMed Scopus (147) Google Scholar, 26Mueller R.D. Yasuda H. Hatch C.L. Bonner W.M. Bradbury E.M. J. Biol. Chem. 1985; 260: 5147-5153Abstract Full Text PDF PubMed Google Scholar). Based on these and other observations, several authors have proposed that H2A ubiquitination could perturb chromatin structure (e.g. Refs.6Chen H.Y. Sun J.M. Zhang Y. Davie J.R. Meistrich M.L. J. Biol. Chem. 1998; 273: 13165-13169Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 17Barsoum J. Varshavsky A. J. Biol. Chem. 1985; 260: 7688-7697Abstract Full Text PDF PubMed Google Scholar, 18Levinger L. Varshavsky A. Cell. 1982; 28: 375-385Abstract Full Text PDF PubMed Scopus (236) Google Scholar), but until now this model has not been tested. In this report we analyze the folding of defined nucleosomal arrays containing uH2A in response to MgCl2 using quantitative agarose gel electrophoresis and analytical centrifugation.DISCUSSIONHistone modifications such as acetylation and phosphorylation mediate changes in chromatin largely through alteration of the charge of amino acid residues in the amino-terminal histone tails. Ubiquitination is, by comparison, a bulky modification that has led researchers to postulate its function to lie in hindering chromatin folding (e.g. Refs. 6Chen H.Y. Sun J.M. Zhang Y. Davie J.R. Meistrich M.L. J. Biol. Chem. 1998; 273: 13165-13169Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 17Barsoum J. Varshavsky A. J. Biol. Chem. 1985; 260: 7688-7697Abstract Full Text PDF PubMed Google Scholar, 18Levinger L. Varshavsky A. Cell. 1982; 28: 375-385Abstract Full Text PDF PubMed Scopus (236) Google Scholar). This postulate has been difficult to confirm in vivo, because the enzymes involved in conjugating ubiquitin to histones are also required for the ubiquitination of many other proteins that may directly or indirectly affect chromatin folding. We have therefore used an in vitromodel system to assay the impact of histone H2A ubiquitination on the Mg2+-induced folding and oligomerization of nucleosomal arrays. Moreover, the extent of H2A ubiquitination used in this study was far greater than that in vivo where it is more common for only one H2A molecule to be ubiquitinated per nucleosome (51Levinger L. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3244-3248Crossref PubMed Scopus (45) Google Scholar). In the absence of linker histones, nucleosomal arrays equilibrate between moderately folded and extensively folded structures in buffers containing 2 mm MgCl2 (31Hansen J.C. Ausió J. Stanik V.H. van Holde K.E. Biochemistry. 1989; 28: 9129-9136Crossref PubMed Scopus (195) Google Scholar, 37Garcia-Ramirez M. Dong F. Ausió J. J. Biol. Chem. 1992; 267: 19587-19595Abstract Full Text PDF PubMed Google Scholar, 47Schwarz P.M. Hansen J.C. J. Biol. Chem. 1994; 269: 16284-16289Abstract Full Text PDF PubMed Google Scholar). The data obtained from quantitative agarose gel electrophoresis (Tables I andII) and analytical ultracentrifugation (Fig. 4) show that uH2A and control arrays attained a similar extent of compaction in 2 mm MgCl2 relative to low salt conditions. This indicates that uH2A does not affect this degree of nucleosomal array folding. Thus, although the tail domains of histones are crucial for the salt-induced folding of nucleosomal arrays (37Garcia-Ramirez M. Dong F. Ausió J. J. Biol. Chem. 1992; 267: 19587-19595Abstract Full Text PDF PubMed Google Scholar, 48Moore S.C. Ausió J. Biochem. Biophys. Res. Commun. 1997; 230: 136-139Crossref PubMed Scopus (67) Google Scholar, 49Tse C. Hansen J.C. Biochemistry. 1997; 36: 11381-11388Crossref PubMed Scopus (127) Google Scholar, 52Allan J. Cowling G.J. Harborne N. Cattini P. Craigie R. Gould H. J. Cell Biol. 1981; 90: 279-288Crossref PubMed Scopus (107) Google Scholar), the carboxyl-terminal tails of H2A can be ubiquitinated without much impact on the folding process. Furthermore, the results shown in Fig.4 A in 2 mm MgCl2 are almost identical to those previously reported for unmodified arrays (see Fig. 3A of Ref. 47Schwarz P.M. Hansen J.C. J. Biol. Chem. 1994; 269: 16284-16289Abstract Full Text PDF PubMed Google Scholar). Therefore, it is possible to conclude that histone H2A ubiquitination neither affects the 28–40 S folding transition, which is characteristic of the histone H1-depleted chromatin in either the presence of monovalent (31Hansen J.C. Ausió J. Stanik V.H. van Holde K.E. Biochemistry. 1989; 28: 9129-9136Crossref PubMed Scopus (195) Google Scholar, 37Garcia-Ramirez M. Dong F. Ausió J. J. Biol. Chem. 1992; 267: 19587-19595Abstract Full Text PDF PubMed Google Scholar) or low concentrations of divalent ions (47Schwarz P.M. Hansen J.C. J. Biol. Chem. 1994; 269: 16284-16289Abstract Full Text PDF PubMed Google Scholar), nor the maximum folding (40–55 S transition), which occurs at higher levels of histone saturation in the presence of MgCl2 (43Tse C. Sera T. Wolffe A.P. Hansen J.C. Mol. Cell. Biol. 1998; 18: 4629-4638Crossref PubMed Scopus (478) Google Scholar, 44Carruthers L.M. Bednar J. Woodcock C.L. Hansen J.C. Biochemistry. 1998; 37: 14776-14787Crossref PubMed Scopus (207) Google Scholar, 47Schwarz P.M. Hansen J.C. J. Biol. Chem. 1994; 269: 16284-16289Abstract Full Text PDF PubMed Google Scholar, 49Tse C. Hansen J.C. Biochemistry. 1997; 36: 11381-11388Crossref PubMed Scopus (127) Google Scholar).Although support for uH2A playing a role in hindering the final stages of chromatin compaction has been provided by reports of the loss of the uH2A ubiquitin moiety at metaphase (25Matsui S. Seon B.K. Sandberg A.A. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6386-6390Crossref PubMed Scopus (147) Google Scholar, 26Mueller R.D. Yasuda H. Hatch C.L. Bonner W.M. Bradbury E.M. J. Biol. Chem. 1985; 260: 5147-5153Abstract Full Text PDF PubMed Google Scholar), not all compact chromatin structures are devoid of ubiquitin. In mice spermatocytes, uH2A has been associated with the inactive sex body that contains the heterochromatic X and Y chromosomes (53Baarends W.M. Hoogerbrugge J.W. Roest H.P. Ooms M. Vreeburg J. Hoeijmakers J.H. Grootegoed J.A. Dev. Biol. 1999; 207: 322-333Crossref PubMed Scopus (240) Google Scholar), and in Drosophilaubiquitin has been shown to be mainly associated with the band domains of polytene chromosomes (54Izquierdo M. Chromosoma. 1994; 103: 193-197Crossref PubMed Scopus (10) Google Scholar). Further investigations are required to determine if uH2A affects the higher degree of folding attained by nucleosomal arrays containing linker histones in response to elevated salt concentrations (44Carruthers L.M. Bednar J. Woodcock C.L. Hansen J.C. Biochemistry. 1998; 37: 14776-14787Crossref PubMed Scopus (207) Google Scholar). It also remains to be investigated if ubiquitination of H2A could affect the binding of other proteins involved in the formation of mitotic chromosomes. Finally, it has been suggested that histone ubiquitination could label specific chromatin regions (26Mueller R.D. Yasuda H. Hatch C.L. Bonner W.M. Bradbury E.M. J. Biol. Chem. 1985; 260: 5147-5153Abstract Full Text PDF PubMed Google Scholar, 55Goldknopf I.L. Wilson G. Ballal N.R. Busch H. J. Biol. Chem. 1980; 255: 10555-10558Abstract Full Text PDF PubMed Google Scholar) and as such could be part of the “histone code” (56Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6507) Google Scholar). This ubiquitin tag could direct as yet unidentified or known cellular machinery such as chromatin remodeling complexes (57Muchardt C. Yaniv M. J. Mol. Biol. 1999; 293: 187-198Crossref PubMed Scopus (159) Google Scholar) to uH2A-enriched chromatin regions such as the 5′-end of the mouse dihydrofolate reductase gene (17Barsoum J. Varshavsky A. J. Biol. Chem. 1985; 260: 7688-7697Abstract Full Text PDF PubMed Google Scholar) or the copia and hsp 70 genes in Drosophila (18Levinger L. Varshavsky A. Cell. 1982; 28: 375-385Abstract Full Text PDF PubMed Scopus (236) Google Scholar). Although for many years histones were thought to be merely structural components of nucleosomes, the primary level of DNA organization required to compact the genome in the nucleus, they are now recognized as important players in the mechanisms underlying gene expression. One of the keys to chromatin's dynamic nature is post-translational modification of the flexible histone tails. These modifications include acetylation, phosphorylation, methylation, and ubiquitination (1van Holde K.E. Chromatin. Spinger-Verlag, New York1989Crossref Google Scholar, 2Davie J.R. Curr. Opin. Genet. Dev. 1998; 8: 173-178Crossref PubMed Scopus (178) Google Scholar, 3Spencer V.A. Davie J.R. Gene. 1999; 240: 1-12Crossref PubMed Scopus (265) Google Scholar). Ubiquitin is a small, mainly globular and highly conserved protein consisting of 76 amino acids found, as its name implies, in most living organisms. Ubiquitin has been found to be conjugated in vivo to histones H2A, H2B, H3, and H1 (4Goldknopf I.L. French M.F. Musso R. Busch H. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5492-5495Crossref PubMed Scopus (93) Google Scholar, 5West M.H.P. Bonner W.M. Nucleic Acids Res. 1980; 8: 4671-4680Crossref PubMed Scopus (163) Google Scholar, 6Chen H.Y. Sun J.M. Zhang Y. Davie J.R. Meistrich M.L. J. Biol. Chem. 1998; 273: 13165-13169Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 7Pham A.-D. Sauer F. Science. 2000; 289: 2357-2360Crossref PubMed Scopus (203) Google Scholar). Ubiquitin is reversibly attached to bovine H2A by means of an isopeptide bond between its terminal glycine and the ε amino group of H2A lysine 119 (8Goldknopf I.L. Busch H. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 864-868Crossref PubMed Scopus (306) Google Scholar), which lies in the trypsin-accessible region of the carboxyl-terminal tail (9Böhm L. Crane-Robinson C. Sautiére P. Eur. J. Biochem. 1980; 106: 525-530Crossref PubMed Scopus (58) Google Scholar). Histones are among the most abundant ubiquitin-protein conjugates in higher eukaryotes, where 5–15% of the total H2A is ubiquitinated (10Rechsteiner M. Ubiquitin. Plenum Press, New York1988Crossref Google Scholar). The function of histone ubiquitination remains unclear. Although ubiquitin has been shown to play an important role in the degradation of many short-lived proteins (for reviews see Refs. 11Finley D. Chau V. Ann. Rev. Cell Biol. 1991; 7: 25-69Crossref PubMed Scopus (421) Google Scholar, 12Jennissen H.P. Eur. J. Biochem. 1995; 231: 1-30Crossref PubMed Scopus (112) Google Scholar), two independent studies have shown that ubiquitination does not tag histones for degradation (13Seale R.L. Nucleic Acids Res. 1981; 9: 3151-3158Crossref PubMed Scopus (44) Google Scholar, 14Wu R.S. Kohn K.W. Bonner W.M. J. Biol. Chem. 1981; 256: 5916-5920Abstract Full Text PDF PubMed Google Scholar). Nucleosomes can be reconstituted with two molecules of uH2A or uH2B1 without obvious perturbation of the nucleosomal structure (15Kleinschmidt A.M. Martinson H.G. Nucleic Acids Res. 1981; 9: 2423-2431Crossref PubMed Scopus (30) Google Scholar, 16Davies N. Lindsey G. Biochim. Biophys. Acta. 1994; 1218: 187-193Crossref PubMed Scopus (40) Google Scholar). Although some studies have reported an enrichment of uH2A in transcriptionally poised or active chromatin (17Barsoum J. Varshavsky A. J. Biol. Chem. 1985; 260: 7688-7697Abstract Full Text PDF PubMed Google Scholar, 18Levinger L. Varshavsky A. Cell. 1982; 28: 375-385Abstract Full Text PDF PubMed Scopus (236) Google Scholar) others do not find this correlation (19Huang S.Y. Barnard M.B. Xu M. Matsui S. Rose S.M. Garrard W.T. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3738-3742Crossref PubMed Scopus (46) Google Scholar, 20Parlow M.H. Haas A.L. Lough J. J. Biol. Chem. 1990; 265: 7507-7512Abstract Full Text PDF PubMed Google Scholar, 21Dawson B.A. Herman T. Haas A.L. Lough J. J. Cell. Biochem. 1991; 46: 166-173Crossref PubMed Scopus (24) Google Scholar). Moreover, inhibition of transcription does not alter the levels of uH2A in a variety of cell lines (22Ericsson C. Goldknopf I.L. Daneholt B. Exp. Cell Res. 1986; 167: 127-134Crossref PubMed Scopus (17) Google Scholar, 23Davie J.R. Murphy L.C. Biochemistry. 1990; 29: 4752-4757Crossref PubMed Scopus (121) Google Scholar, 24Davie J.R. Murphy L.C. Biochem. Biophys. Res. Commun. 1994; 203: 344-350Crossref PubMed Scopus (40) Google Scholar), whereas inhibitors of hnRNA synthesis were found to cause a decrease in uH2B levels (23Davie J.R. Murphy L.C. Biochemistry. 1990; 29: 4752-4757Crossref PubMed Scopus (121) Google Scholar,24Davie J.R. Murphy L.C. Biochem. Biophys. Res. Commun. 1994; 203: 344-350Crossref PubMed Scopus (40) Google Scholar). Cell cycle studies have shown that, in cells undergoing mitosis, uH2A levels decrease progressively to non-detectable levels at metaphase but increase again in late anaphase (14Wu R.S. Kohn K.W. Bonner W.M. J. Biol. Chem. 1981; 256: 5916-5920Abstract Full Text PDF PubMed Google Scholar, 25Matsui S. Seon B.K. Sandberg A.A. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6386-6390Crossref PubMed Scopus (147) Google Scholar, 26Mueller R.D. Yasuda H. Hatch C.L. Bonner W.M. Bradbury E.M. J. Biol. Chem. 1985; 260: 5147-5153Abstract Full Text PDF PubMed Google Scholar). Based on these and other observations, several authors have proposed that H2A ubiquitination could perturb chromatin structure (e.g. Refs.6Chen H.Y. Sun J.M. Zhang Y. Davie J.R. Meistrich M.L. J. Biol. Chem. 1998; 273: 13165-13169Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 17Barsoum J. Varshavsky A. J. Biol. Chem. 1985; 260: 7688-7697Abstract Full Text PDF PubMed Google Scholar, 18Levinger L. Varshavsky A. Cell. 1982; 28: 375-385Abstract Full Text PDF PubMed Scopus (236) Google Scholar), but until now this model has not been tested. In this report we analyze the folding of defined nucleosomal arrays containing uH2A in response to MgCl2 using quantitative agarose gel electrophoresis and analytical centrifugation. DISCUSSIONHistone modifications such as acetylation and phosphorylation mediate changes in chromatin largely through alteration of the charge of amino acid residues in the amino-terminal histone tails. Ubiquitination is, by comparison, a bulky modification that has led researchers to postulate its function to lie in hindering chromatin folding (e.g. Refs. 6Chen H.Y. Sun J.M. Zhang Y. Davie J.R. Meistrich M.L. J. Biol. Chem. 1998; 273: 13165-13169Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 17Barsoum J. Varshavsky A. J. Biol. Chem. 1985; 260: 7688-7697Abstract Full Text PDF PubMed Google Scholar, 18Levinger L. Varshavsky A. Cell. 1982; 28: 375-385Abstract Full Text PDF PubMed Scopus (236) Google Scholar). This postulate has been difficult to confirm in vivo, because the enzymes involved in conjugating ubiquitin to histones are also required for the ubiquitination of many other proteins that may directly or indirectly affect chromatin folding. We have therefore used an in vitromodel system to assay the impact of histone H2A ubiquitination on the Mg2+-induced folding and oligomerization of nucleosomal arrays. Moreover, the extent of H2A ubiquitination used in this study was far greater than that in vivo where it is more common for only one H2A molecule to be ubiquitinated per nucleosome (51Levinger L. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3244-3248Crossref PubMed Scopus (45) Google Scholar). In the absence of linker histones, nucleosomal arrays equilibrate between moderately folded and extensively folded structures in buffers containing 2 mm MgCl2 (31Hansen J.C. Ausió J. Stanik V.H. van Holde K.E. Biochemistry. 1989; 28: 9129-9136Crossref PubMed Scopus (195) Google Scholar, 37Garcia-Ramirez M. Dong F. Ausió J. J. Biol. Chem. 1992; 267: 19587-19595Abstract Full Text PDF PubMed Google Scholar, 47Schwarz P.M. Hansen J.C. J. Biol. Chem. 1994; 269: 16284-16289Abstract Full Text PDF PubMed Google Scholar). The data obtained from quantitative agarose gel electrophoresis (Tables I andII) and analytical ultracentrifugation (Fig. 4) show that uH2A and control arrays attained a similar extent of compaction in 2 mm MgCl2 relative to low salt conditions. This indicates that uH2A does not affect this degree of nucleosomal array folding. Thus, although the tail domains of histones are crucial for the salt-induced folding of nucleosomal arrays (37Garcia-Ramirez M. Dong F. Ausió J. J. Biol. Chem. 1992; 267: 19587-19595Abstract Full Text PDF PubMed Google Scholar, 48Moore S.C. Ausió J. Biochem. Biophys. Res. Commun. 1997; 230: 136-139Crossref PubMed Scopus (67) Google Scholar, 49Tse C. Hansen J.C. Biochemistry. 1997; 36: 11381-11388Crossref PubMed Scopus (127) Google Scholar, 52Allan J. Cowling G.J. Harborne N. Cattini P. Craigie R. Gould H. J. Cell Biol. 1981; 90: 279-288Crossref PubMed Scopus (107) Google Scholar), the carboxyl-terminal tails of H2A can be ubiquitinated without much impact on the folding process. Furthermore, the results shown in Fig.4 A in 2 mm MgCl2 are almost identical to those previously reported for unmodified arrays (see Fig. 3A of Ref. 47Schwarz P.M. Hansen J.C. J. Biol. Chem. 1994; 269: 16284-16289Abstract Full Text PDF PubMed Google Scholar). Therefore, it is possible to conclude that histone H2A ubiquitination neither affects the 28–40 S folding transition, which is characteristic of the histone H1-depleted chromatin in either the presence of monovalent (31Hansen J.C. Ausió J. Stanik V.H. van Holde K.E. Biochemistry. 1989; 28: 9129-9136Crossref PubMed Scopus (195) Google Scholar, 37Garcia-Ramirez M. Dong F. Ausió J. J. Biol. Chem. 1992; 267: 19587-19595Abstract Full Text PDF PubMed Google Scholar) or low concentrations of divalent ions (47Schwarz P.M. Hansen J.C. J. Biol. Chem. 1994; 269: 16284-16289Abstract Full Text PDF PubMed Google Scholar), nor the maximum folding (40–55 S transition), which occurs at higher levels of histone saturation in the presence of MgCl2 (43Tse C. Sera T. Wolffe A.P. Hansen J.C. Mol. Cell. Biol. 1998; 18: 4629-4638Crossref PubMed Scopus (478) Google Scholar, 44Carruthers L.M. Bednar J. Woodcock C.L. Hansen J.C. Biochemistry. 1998; 37: 14776-14787Crossref PubMed Scopus (207) Google Scholar, 47Schwarz P.M. Hansen J.C. J. Biol. Chem. 1994; 269: 16284-16289Abstract Full Text PDF PubMed Google Scholar, 49Tse C. Hansen J.C. Biochemistry. 1997; 36: 11381-11388Crossref PubMed Scopus (127) Google Scholar).Although support for uH2A playing a role in hindering the final stages of chromatin compaction has been provided by reports of the loss of the uH2A ubiquitin moiety at metaphase (25Matsui S. Seon B.K. Sandberg A.A. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6386-6390Crossref PubMed Scopus (147) Google Scholar, 26Mueller R.D. Yasuda H. Hatch C.L. Bonner W.M. Bradbury E.M. J. Biol. Chem. 1985; 260: 5147-5153Abstract Full Text PDF PubMed Google Scholar), not all compact chromatin structures are devoid of ubiquitin. In mice spermatocytes, uH2A has been associated with the inactive sex body that contains the heterochromatic X and Y chromosomes (53Baarends W.M. Hoogerbrugge J.W. Roest H.P. Ooms M. Vreeburg J. Hoeijmakers J.H. Grootegoed J.A. Dev. Biol. 1999; 207: 322-333Crossref PubMed Scopus (240) Google Scholar), and in Drosophilaubiquitin has been shown to be mainly associated with the band domains of polytene chromosomes (54Izquierdo M. Chromosoma. 1994; 103: 193-197Crossref PubMed Scopus (10) Google Scholar). Further investigations are required to determine if uH2A affects the higher degree of folding attained by nucleosomal arrays containing linker histones in response to elevated salt concentrations (44Carruthers L.M. Bednar J. Woodcock C.L. Hansen J.C. Biochemistry. 1998; 37: 14776-14787Crossref PubMed Scopus (207) Google Scholar). It also remains to be investigated if ubiquitination of H2A could affect the binding of other proteins involved in the formation of mitotic chromosomes. Finally, it has been suggested that histone ubiquitination could label specific chromatin regions (26Mueller R.D. Yasuda H. Hatch C.L. Bonner W.M. Bradbury E.M. J. Biol. Chem. 1985; 260: 5147-5153Abstract Full Text PDF PubMed Google Scholar, 55Goldknopf I.L. Wilson G. Ballal N.R. Busch H. J. Biol. Chem. 1980; 255: 10555-10558Abstract Full Text PDF PubMed Google Scholar) and as such could be part of the “histone code” (56Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6507) Google Scholar). This ubiquitin tag could direct as yet unidentified or known cellular machinery such as chromatin remodeling complexes (57Muchardt C. Yaniv M. J. Mol. Biol. 1999; 293: 187-198Crossref PubMed Scopus (159) Google Scholar) to uH2A-enriched chromatin regions such as the 5′-end of the mouse dihydrofolate reductase gene (17Barsoum J. Varshavsky A. J. Biol. Chem. 1985; 260: 7688-7697Abstract Full Text PDF PubMed Google Scholar) or the copia and hsp 70 genes in Drosophila (18Levinger L. Varshavsky A. Cell. 1982; 28: 375-385Abstract Full Text PDF PubMed Scopus (236) Google Scholar). Histone modifications such as acetylation and phosphorylation mediate changes in chromatin largely through alteration of the charge of amino acid residues in the amino-terminal histone tails. Ubiquitination is, by comparison, a bulky modification that has led researchers to postulate its function to lie in hindering chromatin folding (e.g. Refs. 6Chen H.Y. Sun J.M. Zhang Y. Davie J.R. Meistrich M.L. J. Biol. Chem. 1998; 273: 13165-13169Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 17Barsoum J. Varshavsky A. J. Biol. Chem. 1985; 260: 7688-7697Abstract Full Text PDF PubMed Google Scholar, 18Levinger L. Varshavsky A. Cell. 1982; 28: 375-385Abstract Full Text PDF PubMed Scopus (236) Google Scholar). This postulate has been difficult to confirm in vivo, because the enzymes involved in conjugating ubiquitin to histones are also required for the ubiquitination of many other proteins that may directly or indirectly affect chromatin folding. We have therefore used an in vitromodel system to assay the impact of histone H2A ubiquitination on the Mg2+-induced folding and oligomerization of nucleosomal arrays. Moreover, the extent of H2A ubiquitination used in this study was far greater than that in vivo where it is more common for only one H2A molecule to be ubiquitinated per nucleosome (51Levinger L. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3244-3248Crossref PubMed Scopus (45) Google Scholar). In the absence of linker histones, nucleosomal arrays equilibrate between moderately folded and extensively folded structures in buffers containing 2 mm MgCl2 (31Hansen J.C. Ausió J. Stanik V.H. van Holde K.E. Biochemistry. 1989; 28: 9129-9136Crossref PubMed Scopus (195) Google Scholar, 37Garcia-Ramirez M. Dong F. Ausió J. J. Biol. Chem. 1992; 267: 19587-19595Abstract Full Text PDF PubMed Google Scholar, 47Schwarz P.M. Hansen J.C. J. Biol. Chem. 1994; 269: 16284-16289Abstract Full Text PDF PubMed Google Scholar). The data obtained from quantitative agarose gel electrophoresis (Tables I andII) and analytical ultracentrifugation (Fig. 4) show that uH2A and control arrays attained a similar extent of compaction in 2 mm MgCl2 relative to low salt conditions. This indicates that uH2A does not affect this degree of nucleosomal array folding. Thus, although the tail domains of histones are crucial for the salt-induced folding of nucleosomal arrays (37Garcia-Ramirez M. Dong F. Ausió J. J. Biol. Chem. 1992; 267: 19587-19595Abstract Full Text PDF PubMed Google Scholar, 48Moore S.C. Ausió J. Biochem. Biophys. Res. Commun. 1997; 230: 136-139Crossref PubMed Scopus (67) Google Scholar, 49Tse C. Hansen J.C. Biochemistry. 1997; 36: 11381-11388Crossref PubMed Scopus (127) Google Scholar, 52Allan J. Cowling G.J. Harborne N. Cattini P. Craigie R. Gould H. J. Cell Biol. 1981; 90: 279-288Crossref PubMed Scopus (107) Google Scholar), the carboxyl-terminal tails of H2A can be ubiquitinated without much impact on the folding process. Furthermore, the results shown in Fig.4 A in 2 mm MgCl2 are almost identical to those previously reported for unmodified arrays (see Fig. 3A of Ref. 47Schwarz P.M. Hansen J.C. J. Biol. Chem. 1994; 269: 16284-16289Abstract Full Text PDF PubMed Google Scholar). Therefore, it is possible to conclude that histone H2A ubiquitination neither affects the 28–40 S folding transition, which is characteristic of the histone H1-depleted chromatin in either the presence of monovalent (31Hansen J.C. Ausió J. Stanik V.H. van Holde K.E. Biochemistry. 1989; 28: 9129-9136Crossref PubMed Scopus (195) Google Scholar, 37Garcia-Ramirez M. Dong F. Ausió J. J. Biol. Chem. 1992; 267: 19587-19595Abstract Full Text PDF PubMed Google Scholar) or low concentrations of divalent ions (47Schwarz P.M. Hansen J.C. J. Biol. Chem. 1994; 269: 16284-16289Abstract Full Text PDF PubMed Google Scholar), nor the maximum folding (40–55 S transition), which occurs at higher levels of histone saturation in the presence of MgCl2 (43Tse C. Sera T. Wolffe A.P. Hansen J.C. Mol. Cell. Biol. 1998; 18: 4629-4638Crossref PubMed Scopus (478) Google Scholar, 44Carruthers L.M. Bednar J. Woodcock C.L. Hansen J.C. Biochemistry. 1998; 37: 14776-14787Crossref PubMed Scopus (207) Google Scholar, 47Schwarz P.M. Hansen J.C. J. Biol. Chem. 1994; 269: 16284-16289Abstract Full Text PDF PubMed Google Scholar, 49Tse C. Hansen J.C. Biochemistry. 1997; 36: 11381-11388Crossref PubMed Scopus (127) Google Scholar). Although support for uH2A playing a role in hindering the final stages of chromatin compaction has been provided by reports of the loss of the uH2A ubiquitin moiety at metaphase (25Matsui S. Seon B.K. Sandberg A.A. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6386-6390Crossref PubMed Scopus (147) Google Scholar, 26Mueller R.D. Yasuda H. Hatch C.L. Bonner W.M. Bradbury E.M. J. Biol. Chem. 1985; 260: 5147-5153Abstract Full Text PDF PubMed Google Scholar), not all compact chromatin structures are devoid of ubiquitin. In mice spermatocytes, uH2A has been associated with the inactive sex body that contains the heterochromatic X and Y chromosomes (53Baarends W.M. Hoogerbrugge J.W. Roest H.P. Ooms M. Vreeburg J. Hoeijmakers J.H. Grootegoed J.A. Dev. Biol. 1999; 207: 322-333Crossref PubMed Scopus (240) Google Scholar), and in Drosophilaubiquitin has been shown to be mainly associated with the band domains of polytene chromosomes (54Izquierdo M. Chromosoma. 1994; 103: 193-197Crossref PubMed Scopus (10) Google Scholar). Further investigations are required to determine if uH2A affects the higher degree of folding attained by nucleosomal arrays containing linker histones in response to elevated salt concentrations (44Carruthers L.M. Bednar J. Woodcock C.L. Hansen J.C. Biochemistry. 1998; 37: 14776-14787Crossref PubMed Scopus (207) Google Scholar). It also remains to be investigated if ubiquitination of H2A could affect the binding of other proteins involved in the formation of mitotic chromosomes. Finally, it has been suggested that histone ubiquitination could label specific chromatin regions (26Mueller R.D. Yasuda H. Hatch C.L. Bonner W.M. Bradbury E.M. J. Biol. Chem. 1985; 260: 5147-5153Abstract Full Text PDF PubMed Google Scholar, 55Goldknopf I.L. Wilson G. Ballal N.R. Busch H. J. Biol. Chem. 1980; 255: 10555-10558Abstract Full Text PDF PubMed Google Scholar) and as such could be part of the “histone code” (56Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6507) Google Scholar). This ubiquitin tag could direct as yet unidentified or known cellular machinery such as chromatin remodeling complexes (57Muchardt C. Yaniv M. J. Mol. Biol. 1999; 293: 187-198Crossref PubMed Scopus (159) Google Scholar) to uH2A-enriched chromatin regions such as the 5′-end of the mouse dihydrofolate reductase gene (17Barsoum J. Varshavsky A. J. Biol. Chem. 1985; 260: 7688-7697Abstract Full Text PDF PubMed Google Scholar) or the copia and hsp 70 genes in Drosophila (18Levinger L. Varshavsky A. Cell. 1982; 28: 375-385Abstract Full Text PDF PubMed Scopus (236) Google Scholar). We are very grateful to Dr. R. T. Simpson for the p5S 208-12 plasmid construct and Dr. J. C. Hansen for providing details of the quantitative agarose gel electrophoresis apparatus." @default.
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