FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2002191009> ?p ?o ?g. }

• W2002191009 endingPage "9296" @default.
• W2002191009 startingPage "9287" @default.
• W2002191009 abstract "Histone posttranslational modifications that accompany DNA replication, nucleosome assembly, and H2A/H2B exchange were examined in human tissue culture cells. Through microsequencing analysis and chromatin immunoprecipitation, it was found that a subset of newly synthesized H3.2/H3.3 is modified by acetylation and methylation at sites that correlate with transcriptional competence. Immunoprecipitation experiments suggest that cytosolic predeposition complexes purified from cells expressing FLAG-H4 contain H3/H4 dimers, not tetramers. Studies of the deposition of newly synthesized H2A/H2B onto replicating and nonreplicating chromatin demonstrated that H2A/H2B exchange takes place in chromatin regions that contain acetylated H4; however, there is no single pattern of H4 acetylation that accompanies exchange. H2A/H2B exchange is also largely independent of the deposition of replacement histone variant, H3.3. Finally, immunoprecipitation of nucleosomes replicated in the absence of de novo nucleosome assembly showed that histone modifications do not prevent the transfer of parental histones to newly replicated DNA and thus have the potential to serve as means of epigenetic inheritance. Our experiments provide an in-depth analysis of the “histone code” associated with chromatin replication and dynamic histone exchange in human cells. Histone posttranslational modifications that accompany DNA replication, nucleosome assembly, and H2A/H2B exchange were examined in human tissue culture cells. Through microsequencing analysis and chromatin immunoprecipitation, it was found that a subset of newly synthesized H3.2/H3.3 is modified by acetylation and methylation at sites that correlate with transcriptional competence. Immunoprecipitation experiments suggest that cytosolic predeposition complexes purified from cells expressing FLAG-H4 contain H3/H4 dimers, not tetramers. Studies of the deposition of newly synthesized H2A/H2B onto replicating and nonreplicating chromatin demonstrated that H2A/H2B exchange takes place in chromatin regions that contain acetylated H4; however, there is no single pattern of H4 acetylation that accompanies exchange. H2A/H2B exchange is also largely independent of the deposition of replacement histone variant, H3.3. Finally, immunoprecipitation of nucleosomes replicated in the absence of de novo nucleosome assembly showed that histone modifications do not prevent the transfer of parental histones to newly replicated DNA and thus have the potential to serve as means of epigenetic inheritance. Our experiments provide an in-depth analysis of the “histone code” associated with chromatin replication and dynamic histone exchange in human cells. The nuclear DNA of eukaryotic cells is complexed with histone proteins to form the nucleoprotein complex termed chromatin. An octamer of the core histones (two each of histones H2A, H2B, H3, and H4) is encircled by approximately two turns of DNA to produce the fundamental repeating unit of chromatin, the nucleosome (1van Holde K.E. Chromatin.in: Rich A. Springer-Verlag, New York1988Google Scholar). Although once widely thought to be primarily involved with DNA packaging, it is now known that histones are fundamental participants in the regulation of dynamic chromatin processes. Transcription, replication, repair, recombination, and silencing can be controlled by the precise alignment of nucleosomes on DNA and by the various posttranslational modifications that histones undergo (including acetylation, phosphorylation, methylation, ubiquitylation, and poly(ADP-ribosylation)) (2Wolffe A.P. Chromatin: Structure and Function. 3rd Ed. Academic Press, Inc., San Diego, CA1999Google Scholar). Most of the posttranslational modifications are clustered on the histone N-terminal (and, in the case of H2A, C-terminal) “tail” domains, which project beyond the surrounding DNA (1van Holde K.E. Chromatin.in: Rich A. Springer-Verlag, New York1988Google Scholar). The modifications constitute a network of interdependent signals, providing controllable marks for the specific targeting of trans-acting factors to chromatin. It has been suggested that histone modifications can provide an epigenetic language, or “histone code,” that modulates the genetic information transmitted in DNA (3Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6130) Google Scholar, 4Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7149) Google Scholar, 5Turner B.M. BioEssays. 2000; 22: 836-845Crossref PubMed Scopus (907) Google Scholar). One of the first demonstrated assignments of a defined histone modification to a specific cellular process involved the acetylation of newly synthesized H4 (6Ruiz-Carrillo A. Wangh L.J. Allfrey V.G. Science. 1975; 190: 117-128Crossref PubMed Google Scholar, 7Jackson V. Shires A. Tanphaichitr N. Chalkley R. J. Mol. Biol. 1976; 104: 471-483Crossref PubMed Scopus (138) Google Scholar, 8Allis C.D. Chicoine L.G. Richman R. Schulman I.G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8048-8052Crossref PubMed Google Scholar, 9Waterborg J.H. Matthews J.H. Eur. J. Biochem. 1984; 142: 329-335Crossref PubMed Google Scholar). During chromatin replication and assembly, new H4 is acetylated at lysines 5 and 12 prior to deposition onto nascent DNA (10Chicoine L.G. Schulman I.G. Richman R. Cook R.G. Allis C.D. J. Biol. Chem. 1986; 261: 1071-1076Abstract Full Text PDF PubMed Google Scholar, 11Sobel R.E. Cook R.G. Perry C.A. Annunziato A.T. Allis C.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1237-1241Crossref PubMed Scopus (402) Google Scholar). Deacetylation of new H4 occurs over the next 30-60 min (7Jackson V. Shires A. Tanphaichitr N. Chalkley R. J. Mol. Biol. 1976; 104: 471-483Crossref PubMed Scopus (138) Google Scholar) and is required for proper chromatin maturation (12Annunziato A.T. Seale R.L. J. Biol. Chem. 1983; 258: 12675-12684Abstract Full Text PDF PubMed Google Scholar). The “Lys5/Lys12” acetylation pattern of newly synthesized H4 is highly conserved and has been found in organisms as diverse as protozoa (Tetrahymena), Drosophila, and humans (11Sobel R.E. Cook R.G. Perry C.A. Annunziato A.T. Allis C.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1237-1241Crossref PubMed Scopus (402) Google Scholar). (Note that in Tetrahymena, the acetylation sites are at lysines 4 and 11, due to a deletion of the arginine at position 3; also, new H4 in Physarum is predominantly monoacetylated (9Waterborg J.H. Matthews J.H. Eur. J. Biochem. 1984; 142: 329-335Crossref PubMed Google Scholar).) Despite this widespread occurrence, the function of H4 acetylation during chromatin biosynthesis remains undefined. Notably, in the yeast Saccharomyces cerevisiae, the lysines at positions 5 and 12 of H4 are inessential for nucleosome assembly, and Lys5, Lys8, and Lys12 act redundantly during histone deposition (13Ma X.J. Wu J.S. Altheim B.A. Schultz M.C. Grunstein M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6693-6698Crossref PubMed Scopus (108) Google Scholar). There is also evidence that the acetylatable lysines of H4 may be required for efficient nuclear import in yeast (14Glowczewski L. Waterborg J.H. Berman J.G. Mol. Cell. Biol. 2004; 24: 10180-10192Crossref PubMed Scopus (35) Google Scholar). In contrast to the conserved acetylation pattern of new H4, the acetylation of nascent H3 varies among species. For example, in Tetrahymena, Lys9 and Lys14 are the predominant sites, whereas in Drosophila, lysines 14 and 23 are preferred (11Sobel R.E. Cook R.G. Perry C.A. Annunziato A.T. Allis C.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1237-1241Crossref PubMed Scopus (402) Google Scholar). In budding yeast, the lysines at positions 9, 14, 23, and 27 all show some acetylation (with Lys9 and Lys27 preferred), but most new H3 is monoacetylated (15Kuo M.H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Nature. 1996; 383: 269-272Crossref PubMed Scopus (486) Google Scholar). There is also evidence that new H3 in S. cerevisiae is acetylated at lysine 56 (16Masumoto H. Hawke D. Kobayashi R. Verreault A. Nature. 2005; 436: 294-298Crossref PubMed Scopus (446) Google Scholar). The variability of nascent H3 modifications may indicate a nonuniform requirement and/or function for the acetylation of H3 during chromatin assembly. Human cells contain four distinct histone H3 variants, termed H3.1, H3.2, H3.3, and H3.4, which differ only slightly in amino acid sequence (17Albig W. Bramlage B. Gruber K. Klobeck H.G. Kunz J. Doenecke D. Genomics. 1995; 30: 264-272Crossref PubMed Scopus (50) Google Scholar, 18Albig W. Kioschis P. Poustka A. Meergans K. Doenecke D. Genomics. 1997; 40: 314-322Crossref PubMed Scopus (65) Google Scholar, 19Koessler H. Doenecke D. Albig W. DNA Cell Biol. 2003; 22: 233-241Crossref PubMed Scopus (11) Google Scholar, 20Marzluff W.F. Gongidi P. Woods K.R. Jin J.P. Maltais L.J. Genomics. 2002; 80: 487-498Crossref PubMed Google Scholar). The major variant (H3.1) is referred to as replication-dependent, because its synthesis rises sharply in S phase and is linked to DNA replication (21Wu R.S. Bonner W.M. Cell. 1981; 27: 321-330Abstract Full Text PDF PubMed Scopus (243) Google Scholar, 22Wu R.S. Tsai S. Bonner W.M. Cell. 1982; 31: 367-374Abstract Full Text PDF PubMed Scopus (162) Google Scholar); H3.1 is encoded by 10 genes (19Koessler H. Doenecke D. Albig W. DNA Cell Biol. 2003; 22: 233-241Crossref PubMed Scopus (11) Google Scholar). H3.2 is also replication-dependent but is encoded by only one gene copy (19Koessler H. Doenecke D. Albig W. DNA Cell Biol. 2003; 22: 233-241Crossref PubMed Scopus (11) Google Scholar, 20Marzluff W.F. Gongidi P. Woods K.R. Jin J.P. Maltais L.J. Genomics. 2002; 80: 487-498Crossref PubMed Google Scholar); it is distinguished from H3.1 by a single amino acid change at position 96 (cysteine in H3.1, serine in H3.2) (20Marzluff W.F. Gongidi P. Woods K.R. Jin J.P. Maltais L.J. Genomics. 2002; 80: 487-498Crossref PubMed Google Scholar, 23Franklin S.G. Zweidler A. Nature. 1977; 266: 273-275Crossref PubMed Google Scholar). Synthesis of the replication-independent variant H3.3 is not coupled to DNA replication but continues at a basal level throughout the cell cycle (21Wu R.S. Bonner W.M. Cell. 1981; 27: 321-330Abstract Full Text PDF PubMed Scopus (243) Google Scholar, 22Wu R.S. Tsai S. Bonner W.M. Cell. 1982; 31: 367-374Abstract Full Text PDF PubMed Scopus (162) Google Scholar). H3.3 can be deposited in a replication-independent manner, often in association with transcription (24Ray-Gallet D. Quivy J.P. Scamps C. Martini E.M.D. Lipinski M. Almouzni G. Mol. Cell. 2002; 9: 1091-1100Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 25Ahmad K. Henikoff S. Mol. Cell. 2002; 9: 1191-1200Abstract Full Text Full Text PDF PubMed Scopus (836) Google Scholar, 26Janicki S.M. Tsukamoto T. Salghetti S.E. Tansey W.P. Sachidanandam R. Prasanth K.V. Ried T. Shav-Tal Y. Bertrand E. Singer R.H. Spector D.L. Cell. 2004; 116: 683-698Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 27Schwartz B.E. Ahmad K. Gene Dev. 2005; 19: 804-814Crossref PubMed Scopus (0) Google Scholar, 28Chow C.M. Georgiou A. Szutorisz H. Silva A.M.E. Pombo A. Barahona I. Dargelos E. Canzonetta C. Dillon N. EMBO Rep. 2005; 6: 354-360Crossref PubMed Scopus (117) Google Scholar, 29Wirbelauer C. Bell O. Schubeler D. Gene Dev. 2005; 19: 1761-1766Crossref PubMed Scopus (0) Google Scholar, 30Mito Y. Henikoff J.G. Henikoff S. Nat. Genet. 2005; 37: 1090-1097Crossref PubMed Scopus (408) Google Scholar, 31Choi E.S. Shin J.A. Kim H.S. Jang Y.K. Nucleic Acids Res. 2005; 33: 7102-7110Crossref PubMed Scopus (31) Google Scholar). Human H3.4 appears to be expressed solely in testis (32Witt O. Albig W. Doenecke D. Exp. Cell Res. 1996; 229: 301-306Crossref PubMed Scopus (82) Google Scholar). In a previous report, we showed that newly synthesized H3.1 from human (HeLa) cells was essentially unmodified, in contrast to nascent H3 from Tetrahymena, Drosophila, and S. cerevisiae (11Sobel R.E. Cook R.G. Perry C.A. Annunziato A.T. Allis C.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1237-1241Crossref PubMed Scopus (402) Google Scholar). However, in that report, we also presented evidence that newly synthesized human H3.2 and/or H3.3 showed detectable acetylation at lysines 14 and 18. In light of the observation that bulk H3.3 is enriched in modifications that are indicative of transcriptional competence (33McKittrick E. Gaften P.R. Ahmad K. Henikoff S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1525-1530Crossref PubMed Scopus (389) Google Scholar, 34Hake S.B. Garcia B.A. Duncan E.M. Kauer M. Dellaire G. Shabanowitz J. Bazett-Jones D.P. Allis C.D. Hunt D.F. J. Biol. Chem. 2005; 281: 559-568Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar), we have more closely examined the modifications of newly synthesized H3.2 and H3.3 in human cells, in this case including the analysis of histone methylation. We have also initiated a characterization of the histone modifications that are present in newly replicated nucleosomes and on parental histones that are segregated to newly replicated DNA (35Annunziato A.T. J. Biol. Chem. 2005; 280: 12065-12068Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). In conjunction with these studies, we have investigated the exchange of newly synthesized H2A and H2B into replicating and nonreplicating chromatin regions, through the use of antibodies that recognize specific acetylated H4 isoforms. Our results demonstrate that a subset of new H3 carries modifications that are characteristic of transcriptionally active chromatin immediately following their deposition onto DNA. We also show that specific modifications of parental H3 and H4 can persist through the replication process without preventing the transfer of old histones to new DNA and thus may act as effectors of epigenetic inheritance. It is further demonstrated that the exchange of new H2A/H2B dimers into chromatin is concomitant with the acetylation of H4 and can occur independently of the deposition of new H3.3. Finally, we provide evidence that the human cytosolic H3/H4 predeposition complex contains an H3/H4 dimer (not a tetramer) and that cytosolic H2A can be acetylated at lysine 5. Taken together, our results provide a detailed description of histone modifications during chromatin replication, nucleosome assembly, and H2A/H2B exchange in human cells. Cell Culture and Labeling—HeLa S-3 cells were maintained in spinner culture at 37 °C in minimal essential medium, Joklik modification (MEM), 3The abbreviations used are: MEM, Eagle's minimal essential medium (Joklik modification); HU, hydroxyurea; TSA, trichostatin A; PIPES, Piperazine-1,4-bis(2-ethanesulfonic acid); HPLC, high pressure liquid chromatography; TAU, Triton-acid-urea; GFP, green fluorescent protein; acH4, acetylated histone 4. supplemented with 5-10% calf serum. Newly replicated DNA was labeled with [3H]thymidine for 5 min in vivo as described previously (36Perry C.A. Dadd C.A. Allis C.D. Annunziato A.T. Biochemistry. 1993; 32: 13605-13614Crossref PubMed Google Scholar). For microsequencing newly synthesized histones, cells were synchronized using the double thymidine block procedure as described previously (37Annunziato A.T. Eason M.B. Perry C.A. Biochemistry. 1995; 34: 2916-2924Crossref PubMed Scopus (64) Google Scholar) and released into S phase for 2-4 h prior to radiolabeling. Cells were then pulse-labeled with l-[4,5-3H]lysine (85 Ci/mol; PerkinElmer Life Sciences) for 8-10 min at 100 μCi/ml, as described (38Chang L. Ryan C.A. Schneider C.A. Annunziato A.T. Wassarman P.M. Wolffe A.P. Methods in Enzymology. 304. Academic Press, Inc., San Diego, CA1999: 76-99Google Scholar). To inhibit histone deacetylation, all labeling of DNA and histones was performed in the presence of 50 mm sodium butyrate (39Cousens L.S. Alberts B.M. J. Biol. Chem. 1982; 257: 3945-3949Abstract Full Text PDF PubMed Google Scholar, 40Perry C.A. Annunziato A.T. Nucleic Acids Res. 1989; 17: 4275-4291Crossref PubMed Google Scholar) and 1 μm trichostatin A (TSA). Separation of histones by reverse-phase HPLC and microsequencing analysis of newly synthesized H3 was performed as described previously (11Sobel R.E. Cook R.G. Perry C.A. Annunziato A.T. Allis C.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1237-1241Crossref PubMed Scopus (402) Google Scholar); for cycles 4, 9, 14, 18, 23, and 27, 1-min fractions from the sequencer in-line phenylthiohydantoin analyzer HPLC were collected and analyzed by scintillation counting; the gradient was run under conditions that separate acetylated, monomethylated, di-/trimethylated, and unmodified lysine (11Sobel R.E. Cook R.G. Perry C.A. Annunziato A.T. Allis C.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1237-1241Crossref PubMed Scopus (402) Google Scholar). To inhibit DNA replication, cells were concentrated 10-fold in warm MEM, preincubated with 10 mm hydroxyurea (HU) for 10 min, and labeled for 15 min in the continuous presence of hydroxyurea (plus sodium butyrate and TSA) (36Perry C.A. Dadd C.A. Allis C.D. Annunziato A.T. Biochemistry. 1993; 32: 13605-13614Crossref PubMed Google Scholar); this concentration of HU effectively inhibits DNA replication in HeLa cells (41Nadeau P. Oliver D.R. Chalkley R. Biochemistry. 1978; 17: 4885-4893Crossref PubMed Google Scholar). 4K. Tong and A. T. Annunziato, unpublished results. To inhibit protein synthesis, cells were concentrated 10-fold in warm MEM, pretreated with 200 μg/ml cycloheximide for 8-10 min, and then labeled with [3H]thymidine at 75 μCi/ml in the presence of cycloheximide for 20 min (37Annunziato A.T. Eason M.B. Perry C.A. Biochemistry. 1995; 34: 2916-2924Crossref PubMed Scopus (64) Google Scholar, 42Annunziato A.T. Seale R.L. Biochemistry. 1982; 21: 5431-5438Crossref PubMed Scopus (32) Google Scholar). Labeling of newly replicated DNA with [3H]TTP in isolated nuclei was performed according to the method of Seale (43Seale R.L. Biochemistry. 1977; 16: 2847-2853Crossref PubMed Scopus (9) Google Scholar), modified as previously described (44Perry C.A. Allis C.D. Annunziato A.T. Biochemistry. 1993; 32: 13615-13623Crossref PubMed Google Scholar). Prior to isolating nuclei for in vitro replication, cells were treated with 200 μg/ml cycloheximide for 5 min, to interpose a stretch of unassembled DNA between the last nucleosomes assembled in vivo and the DNA to be labeled in vitro (44Perry C.A. Allis C.D. Annunziato A.T. Biochemistry. 1993; 32: 13615-13623Crossref PubMed Google Scholar). Plasmid Construction—An (amino-terminal) FLAG-H4 construct was obtained by PCR using N-FLAG H4/pET3a plasmid as the template (N-FLAG H4/pET3a was a gift from Drs. Christophe Thiriet and Jeffrey J. Hayes), PCR Master Mix (Promega), and the following primers: forward primer, 5′-CCCGGGGATATAGCCATGGAC-3′; reverse primer, 5′-CAGCCGGATCCTTAACCACCGAAACCGTAC-3′. The forward primer introduced a XmaI restriction site (underlined) and a Kozak sequence (boldface type); the reverse primer introduced a BamHI restriction site (underlined). Purified PCR products were ligated into pGEM-T vector (Promega) and then excised using XmaI and BamHI. The enzyme digestion products were purified and ligated into pIRES-hrGFPII (Stratagene), which had been cut using XmaI and BamHI. The constructed plasmid was amplified following transformation into Escherichia coli strain JM109, confirmed by sequencing, and purified using the EndoFree plasmid kit (Qiagen). Transfection of 293-H Cells—293-H cells were obtained from Invitrogen and maintained according to the supplier's instructions. Lipofectamine 2000 (Invitrogen) was used for transfection; stable cell lines were obtained by G418 selection after transfection with N-FLAG-H4/pIRES-hrGFPII. Green fluorescence was observed ∼30 h after transfection. Stable cell lines were grown in minimal essential medium supplemented with 10% fetal bovine serum, 4 mm l-glutamine, 50 units/ml penicillin, 50 μg/ml streptomycin sulfate, and 400 μg/ml G418. FLAG-H4 expression was confirmed by immunofluorescence using an anti-FLAG M2 antibody (Sigma) and immunoblotting as described previously (45Lu M.J. Dadd C.A. Mizzen C.A. Perry C.A. McLachlan D.R. Annunziato A.T. Allis C.D. Chromosoma. 1994; 103: 111-121PubMed Google Scholar); at least 95% of the stably transfected cells expressed the FLAG epitope. FLAG-H4 represented <10% of total H4 in the transfected cells, as determined by Western analysis using antibodies recognizing unmodified H4 (the generous gift of Dr. Judith Berman; see Fig. 8). Nuclear Isolation, Histone, and Chromatin Preparation—Isolated nuclei, S100 cytosolic extracts, and acid-soluble nuclear proteins were prepared as described previously (36Perry C.A. Dadd C.A. Allis C.D. Annunziato A.T. Biochemistry. 1993; 32: 13605-13614Crossref PubMed Google Scholar); for immunoprecipitation studies, acid-extracted histones were dialyzed overnight in 1 liter of distilled water at 4 °C. Under these experimental conditions, H3 and H4 can associate and co-immunoprecipitate; however, the formation of H3/H4 complexes is less efficient than that observed using salt-extracted histones (46Isenberg I. Annu. Rev. Biochem. 1979; 48: 159-191Crossref PubMed Google Scholar). For the analysis of chromatin-bound radiolabeled histones, soluble chromatin was prepared with micrococcal nuclease (Sigma) at 5 units/ml at 4 °C, in 10 mm PIPES, 20 mm sodium butyrate, and 80 mm NaCl, 0.5 mm CaCl2, pH 7.0 (36Perry C.A. Dadd C.A. Allis C.D. Annunziato A.T. Biochemistry. 1993; 32: 13605-13614Crossref PubMed Google Scholar). To generate mononucleosomes, nuclei were isolated in Buffer A (10 mm Tris-HCl, 10 mm sodium butyrate, 3 mm MgCl2, 2 mm 2-mercaptoethanol, pH 7.6 (43Seale R.L. Biochemistry. 1977; 16: 2847-2853Crossref PubMed Scopus (9) Google Scholar)), resuspended in Buffer A at 40 A260/ml, adjusted to 0.5 mm CaCl2, and digested with 1.2 units/ml micrococcal nuclease for 1.5-2.0 min at 37 °C; the reaction was stopped by adding EGTA to a final concentration of 5 mm. Digested nuclei were incubated on ice for 15-30 min and then centrifuged for 10 min at ∼10,000 × g; the supernatant, containing mononucleosomes, was termed S1 (47Annunziato A.T. Schindler R.K. Riggs M.G. Seale R.L. J. Biol. Chem. 1982; 257: 8507-8515Abstract Full Text PDF PubMed Google Scholar). The resulting pellet was resuspended in 2 mm EDTA, pH 7.2, incubated on ice for 20 min, and again centrifuged; this supernatant, containing mono- and polynucleosomes, was designated S2. The residual pellet was designated P (see supplemental Fig. S1) (47Annunziato A.T. Schindler R.K. Riggs M.G. Seale R.L. J. Biol. Chem. 1982; 257: 8507-8515Abstract Full Text PDF PubMed Google Scholar). Scintillation counting of trichloroacetic acid-precipitable radioactivity was measured as described previously, using a biodegradable fluor (Ecoscint A; National Diagnostics) (36Perry C.A. Dadd C.A. Allis C.D. Annunziato A.T. Biochemistry. 1993; 32: 13605-13614Crossref PubMed Google Scholar, 44Perry C.A. Allis C.D. Annunziato A.T. Biochemistry. 1993; 32: 13615-13623Crossref PubMed Google Scholar). Immunoprecipitation—In this study, several different antibodies were used for immunoprecipitation experiments. For immunoprecipitations of newly replicated and newly assembled chromatin, acetylated H4-specific antibodies (generated using a peptide representing the H4 N-terminal domain acetylated at lysines 5 and 12; i.e. the sites acetylated in newly synthesized HeLa H4) were routinely used; these antibodies have been described previously (38Chang L. Ryan C.A. Schneider C.A. Annunziato A.T. Wassarman P.M. Wolffe A.P. Methods in Enzymology. 304. Academic Press, Inc., San Diego, CA1999: 76-99Google Scholar, 48Chang L. Loranger S.S. Mizzen C. Ernst S.G. Allis C.D. Annunziato A.T. Biochemistry. 1997; 36: 469-480Crossref PubMed Scopus (143) Google Scholar). During the course of our experiments, it was necessary to produce additional antibodies to the same Lys5/Lys12-acetylated H4 peptide. These antibodies (identified in the figure legends) have a minor reaction with denatured acetylated H2A in Western blots (supplemental Fig. S2A); however, they do not immunoprecipitate either acetylated (cytosolic) or newly synthesized H2A (data not presented; also see Fig. 6). Antibodies were also produced using an H4 N-terminal peptide acetylated at lysines 8 and 16, which recognize acetylated H4 primarily at lysine 8 (supplemental Fig. S2B). All other antibodies were purchased from Upstate (Charlottesville, VA). Chromatin immunoprecipitations were performed essentially as described, with the exception that high salt wash buffer contained 400 mm NaCl (36Perry C.A. Dadd C.A. Allis C.D. Annunziato A.T. Biochemistry. 1993; 32: 13605-13614Crossref PubMed Google Scholar, 48Chang L. Loranger S.S. Mizzen C. Ernst S.G. Allis C.D. Annunziato A.T. Biochemistry. 1997; 36: 469-480Crossref PubMed Scopus (143) Google Scholar). All immunoprecipitations used 50-100 μl of antiserum or antibody, and 40-80 μl of packed Protein A-Sepharose beads (GE Healthcare). Antibody/chromatin excess was monitored by sequential immunoprecipitations of the unbound fraction or by doubling the amount of antibody used, as described previously (44Perry C.A. Allis C.D. Annunziato A.T. Biochemistry. 1993; 32: 13615-13623Crossref PubMed Google Scholar). For immunoprecipitations of extracted histones, Protein A-Sepharose beads were blocked with 1 mg/ml acetylated bovine serum albumin (Promega) and 0.1 mg/ml ubiquitin (Sigma). For the analysis of immunoprecipitated histones by gel electrophoresis, washes contained 0.1% SDS; this did not affect the immunoprecipitation of intact newly assembled nucleosomes (see Fig. 4). In preparation for electrophoresis, soluble chromatin in the unbound fraction was adjusted to 10 mm MgCl2 and precipitated with two volumes of ethanol; free histones were precipitated with 25% trichloroacetic acid (12Annunziato A.T. Seale R.L. J. Biol. Chem. 1983; 258: 12675-12684Abstract Full Text PDF PubMed Google Scholar). To separate immunoprecipitated histones in gels containing SDS, immunopellets were resuspended in sample buffer and placed in boiling water for 5 min. To resolve immunoprecipitated histones in Triton-acid-urea gels, proteins were extracted from the immunopellets using the method of Crane-Robinson et al. (49Crane-Robinson C. Myers F.A. Hebbes T.R. Clayton A.L. Thorne A.W. Wassarman P.M. Wolffe A.P. Methods in Enzymology. 304. Academic Press, Inc., San Diego, CA1999: 533-547Google Scholar). Cytoplasmic S100 extracts from 293-H cells were prepared as described previously (36Perry C.A. Dadd C.A. Allis C.D. Annunziato A.T. Biochemistry. 1993; 32: 13605-13614Crossref PubMed Google Scholar); S1 chromatin fractions were prepared as described above for HeLa cells. Prior to immunoprecipitation, ∼20% of each sample was saved as an “input” fraction. For immunoprecipitation, samples were adjusted to 1 mm phenylmethylsulfonyl fluoride, 1 mm EGTA, 0.25% Triton X-100, 0.6 μg/ml leupeptin, 0.8 μg/ml pepstatin, and 0.5 μg/ml mycrocystin and then incubated with EZview™ Red Anti-FLAG M2 affinity gel (Sigma), which had been equilibrated with 50 mm Tris-HCl, 150 mm NaCl, pH 7.4. As a control, an equivalent volume of sample was incubated with mouse IgG-agarose affinity gel (Sigma). Immunoprecipitations were performed at 4 °C overnight. Bound and unbound fractions were separated by centrifugation for 2 min at 1200 × g. Input and unbound S100 fractions were precipitated with 25% trichloroacetic acid, washed with acetone, and dried; input and unbound S1 chromatin fractions were adjusted to 10 mm MgCl2 and precipitated with 2 volumes of ethanol. Bound, immunoprecipitated fractions were washed five times with high salt wash buffer (1% Triton X-100, 0.4 m NaCl, 2 mm EDTA, 20 mm Tris-HCl, pH 8.1) and once with 10 mm Tris-HCl, pH 8.1 (36Perry C.A. Dadd C.A. Allis C.D. Annunziato A.T. Biochemistry. 1993; 32: 13605-13614Crossref PubMed Google Scholar). In some cases, S100 preparations from HeLa or 293-H cells were treated with 5-10 mm MgCl2 and centrifuged at 12,000 × g for 10 min, to remove any possible contaminating chromatin. Gel Electrophoresis, Fluorography, and Immunoblotting—Radiolabeled DNA was separated in 4% polyacrylamide gels prior to analysis by fluorography (50Annunziato A.T. Schindler R.K. Thomas Jr., C.A. Seale R.L. J. Biol. Chem. 1981; 256: 11880-11886Abstract Full Text PDF PubMed Google Scholar). Proteins were subjected to SDS-PAGE in 18% polyacrylamide gels (51Thomas J.O. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 2626-2630Crossref PubMed Google Scholar), as described previously (40Perry C.A. Annunziato A.T. Nucleic Acids Res. 1989; 17: 4275-4291Crossref PubMed Google Scholar). To resolve histone H3 variants, acid-soluble proteins were separated in 0.4% Triton X-100, 0.9 m acetic acid, 6 m urea, 15% polyacrylamide (TAU) gels (52Zweidler A. Methods Cell Biol. 1978; 17: 223-233Crossref PubMed Scopus (307) Google Scholar, 53Ryan C.A. Annunziato A.T. Curr. Protocols Mol. Biol. 1999; 21: 1-10Google Scholar). Fluorography was performed according to published methods (54Bonner W.M. Laskey R.A. Eur. J. Biochem. 1974; 46: 83-88Crossref PubMed Google Scholar, 55Laskey R.A. Mills A.D. FEBS Lett. 1977; 82: 314-316Crossref PubMed Scopus (594) Google Scholar). For quantitation, films were scanned using a Bio-Rad GS-800 densitometer; the intensity of the bands was determined using Quantity One software. For immunoblotting, proteins were transferred to Immobilon-P membrane according to the methods of Towbin et al. (56Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Google Scholar) and analyzed as previously described (36Perry C.A. Dadd C.A. Allis C.D. Annunziato A.T. Biochemistry. 19" @default.
• W2002191009 created "2016-06-24" @default.
• W2002191009 creator A5000012529 @default.
• W2002191009 creator A5001139865 @default.
• W2002191009 creator A5017547067 @default.
• W2002191009 creator A5033939754 @default.
• W2002191009 creator A5037221487 @default.
• W2002191009 creator A5044786646 @default.
• W2002191009 creator A5066253842 @default.
• W2002191009 creator A5081952589 @default.
• W2002191009 creator A5082367235 @default.
• W2002191009 date "2006-04-01" @default.
• W2002191009 modified "2023-10-06" @default.
• W2002191009 title "Modifications of H3 and H4 during Chromatin Replication, Nucleosome Assembly, and Histone Exchange" @default.
• W2002191009 cites W1501349652 @default.
• W2002191009 cites W1519860621 @default.
• W2002191009 cites W1526700183 @default.
• W2002191009 cites W1535387945 @default.
• W2002191009 cites W1554740131 @default.
• W2002191009 cites W1571366008 @default.
• W2002191009 cites W158157254 @default.
• W2002191009 cites W1599764563 @default.
• W2002191009 cites W1752502457 @default.
• W2002191009 cites W1876643910 @default.
• W2002191009 cites W1885556624 @default.
• W2002191009 cites W1964275197 @default.
• W2002191009 cites W1964480889 @default.
• W2002191009 cites W1964541915 @default.
• W2002191009 cites W1965469784 @default.
• W2002191009 cites W1968177936 @default.
• W2002191009 cites W1968264633 @default.
• W2002191009 cites W1969691479 @default.
• W2002191009 cites W1971229961 @default.
• W2002191009 cites W1973096737 @default.
• W2002191009 cites W1973798439 @default.
• W2002191009 cites W1974590591 @default.
• W2002191009 cites W1975859486 @default.
• W2002191009 cites W1976040047 @default.
• W2002191009 cites W1977987411 @default.
• W2002191009 cites W1978684090 @default.
• W2002191009 cites W1982604241 @default.
• W2002191009 cites W1983866602 @default.
• W2002191009 cites W1984751522 @default.
• W2002191009 cites W1985634754 @default.
• W2002191009 cites W1987440057 @default.
• W2002191009 cites W1988927816 @default.
• W2002191009 cites W1992555811 @default.
• W2002191009 cites W1994158526 @default.
• W2002191009 cites W1996504826 @default.
• W2002191009 cites W1998478452 @default.
• W2002191009 cites W1998555020 @default.
• W2002191009 cites W1999284470 @default.
• W2002191009 cites W1999529939 @default.
• W2002191009 cites W2000939708 @default.
• W2002191009 cites W2002686161 @default.
• W2002191009 cites W2002698073 @default.
• W2002191009 cites W2003917529 @default.
• W2002191009 cites W2007538089 @default.
• W2002191009 cites W2008409661 @default.
• W2002191009 cites W2008543992 @default.
• W2002191009 cites W2008890975 @default.
• W2002191009 cites W2010817679 @default.
• W2002191009 cites W2012751802 @default.
• W2002191009 cites W2013516320 @default.
• W2002191009 cites W2018307111 @default.
• W2002191009 cites W2029399086 @default.
• W2002191009 cites W2031505662 @default.
• W2002191009 cites W2032733245 @default.
• W2002191009 cites W2034277598 @default.
• W2002191009 cites W2035915355 @default.
• W2002191009 cites W2039478057 @default.
• W2002191009 cites W2040415752 @default.
• W2002191009 cites W2041383836 @default.
• W2002191009 cites W2042198281 @default.
• W2002191009 cites W2044007120 @default.
• W2002191009 cites W2046306550 @default.
• W2002191009 cites W2046663888 @default.
• W2002191009 cites W2048988091 @default.
• W2002191009 cites W2049968571 @default.
• W2002191009 cites W2056373980 @default.
• W2002191009 cites W2060624618 @default.
• W2002191009 cites W2060863780 @default.
• W2002191009 cites W2062147146 @default.
• W2002191009 cites W2063516501 @default.
• W2002191009 cites W2066332561 @default.
• W2002191009 cites W2067487392 @default.
• W2002191009 cites W2067490392 @default.
• W2002191009 cites W2067651011 @default.
• W2002191009 cites W2069681995 @default.
• W2002191009 cites W2069877725 @default.
• W2002191009 cites W2070312499 @default.
• W2002191009 cites W2071955851 @default.
• W2002191009 cites W2082623015 @default.
• W2002191009 cites W2083118156 @default.
• W2002191009 cites W2085971700 @default.
• W2002191009 cites W2088468607 @default.
• W2002191009 cites W2089805605 @default.
• W2002191009 cites W2093497792 @default.

• next