FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2014279477> ?p ?o ?g. }

• W2014279477 endingPage "32243" @default.
• W2014279477 startingPage "32236" @default.
• W2014279477 abstract "The binding of all known linker histones, named H1a through H1e, including H10 and H1t, to a model chromatin complex based on a DNA fragment containing the mouse mammary tumor virus long terminal repeat promotor was systematically studied. As for the histone subtype H1b, we found a dissociation constant of 8–16 nm to a single mononucleosome (210 base pairs), whereas the binding constant of all other subtypes varied between 2 and 4 nm. Most of the H1 histones, namely H1a, H1c, H1d/e, and H10, completely aggregate polynucleosomes (1.3 kilobase pairs, 6 nucleosomes) at 270–360 nm, corresponding to a molar ratio of six to eight H1 molecules per reconstituted nucleosome. To form aggregates with the histones H1t and H1b, however, greater amounts of protein were required. Furthermore, our results show that specific types of in vivo phosphorylation of the linker histone tails influence both the binding to mononucleosomes and the aggregation of polynucleosomes. S phase-specific phosphorylation with one to three phosphate groups at specific sites in the C terminus influences neither the binding to a mononucleosome nor the aggregation of polynucleosomes. In contrast, highly phosphorylated H1 histones with four to five phosphate groups in the C and N termini reveal a very high binding affinity to a mononucleosome but a low chromatin aggregation capability. These findings suggest that specific S phase or mitotic phosphorylation sites act independently and have distinct functional roles. The binding of all known linker histones, named H1a through H1e, including H10 and H1t, to a model chromatin complex based on a DNA fragment containing the mouse mammary tumor virus long terminal repeat promotor was systematically studied. As for the histone subtype H1b, we found a dissociation constant of 8–16 nm to a single mononucleosome (210 base pairs), whereas the binding constant of all other subtypes varied between 2 and 4 nm. Most of the H1 histones, namely H1a, H1c, H1d/e, and H10, completely aggregate polynucleosomes (1.3 kilobase pairs, 6 nucleosomes) at 270–360 nm, corresponding to a molar ratio of six to eight H1 molecules per reconstituted nucleosome. To form aggregates with the histones H1t and H1b, however, greater amounts of protein were required. Furthermore, our results show that specific types of in vivo phosphorylation of the linker histone tails influence both the binding to mononucleosomes and the aggregation of polynucleosomes. S phase-specific phosphorylation with one to three phosphate groups at specific sites in the C terminus influences neither the binding to a mononucleosome nor the aggregation of polynucleosomes. In contrast, highly phosphorylated H1 histones with four to five phosphate groups in the C and N termini reveal a very high binding affinity to a mononucleosome but a low chromatin aggregation capability. These findings suggest that specific S phase or mitotic phosphorylation sites act independently and have distinct functional roles. mouse mammary tumor virus long terminal repeat high performance liquid chromatography high performance capillary electrophoresis base pair(s) kilobase pair(s). H1 histones are a heterogeneous group of at least five subtypes with closely related but nonetheless different primary structures (1Kinkade Jr., J.M. Cole R.D. J. Biol. Chem. 1966; 241: 5798-5805Abstract Full Text PDF PubMed Google Scholar,2Rall S.C. Cole R.D. J. Biol. Chem. 1971; 246: 7175-7190Abstract Full Text PDF PubMed Google Scholar). Two further H1 subtypes are known: the histone H10, which is found in nonreplicative tissues (3Panyim S. Chalkley R. Biochem. Biophys. Res. Commun. 1969; 37: 1042-1049Crossref PubMed Scopus (239) Google Scholar, 4Yasuda H. Mueller R.D. Logan K.A. Bradbury E.M. J. Biol. Chem. 1986; 261: 2349-2354Abstract Full Text PDF PubMed Google Scholar) and in rapidly proliferating cells (5D’Anna J.A. Gurley L.R. Becker R.R. Biochemistry. 1981; 20: 4501-4505Crossref PubMed Scopus (23) Google Scholar), and the testis-specific histone variant H1t (6Meistrich M.L. Bucci L.R. Trostle-Weige P.K. Brock W.A. Dev. Biol. 1985; 112: 230-240Crossref PubMed Scopus (127) Google Scholar). The various linker histones containing a globular central region flanked by highly basic and hydrophilic C- and N-terminal tails (7Bradbury E.M. Cary P.D. Chapman G.E. Crane-Robinson C. Damby S.E. Rattle H.W.E. Boublik M. Palau J. Avilés F.X. Eur. J. Biochem. 1975; 52: 605-613Crossref PubMed Scopus (108) Google Scholar,8Isenberg I. Annu. Rev. Biochem. 1979; 48: 159-191Crossref PubMed Scopus (463) Google Scholar) bind to the nucleosome and promote the organization of nucleosomes to a higher order structure (9Finch J.T. Klug A. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 1897-1901Crossref PubMed Scopus (958) Google Scholar, 10Thoma F. Koller T. Klug A. J. Cell Biol. 1979; 83: 403-427Crossref PubMed Scopus (1180) Google Scholar). There is evidence that histone H1 may interact differently with transcriptionally active and inactive regions of chromatin (11Nacheva G.A. Guschin D.Y. Preobrazhenskaya O.V. Karpov V.L. Ebralidse K.K. Mirzabekov A.D. Cell. 1989; 58: 27-36Abstract Full Text PDF PubMed Scopus (222) Google Scholar). Linker histones are also thought to modulate nucleosome position (12Wolffe A., P. Cell. 1994; 77: 13-16Abstract Full Text PDF PubMed Scopus (227) Google Scholar, 13Clark K.L. Halay E.D. Lai E., D. Burley S.K. Nature. 1993; 364: 412-417Crossref PubMed Scopus (1098) Google Scholar) and to influence replication efficiency in vitro (14Halmer L. Gruss C. Nucleic Acids Res. 1996; 24: 1420-1427Crossref PubMed Scopus (46) Google Scholar). The presence of this large number of various H1 histone subtypes and their possible posttranslational modifications, such as phosphorylation (15Talasz H. Helliger W. Puschendorf B. Lindner H. Biochemistry. 1996; 35: 1761-1767Crossref PubMed Scopus (81) Google Scholar), make it very probable that H1 histones play numerous structural and functional roles in chromatin. Until now, no specific role for the various variants has been established although Kaludov et al. (16Kaludov N.K. Pabón-Peña L. Seavy M. Robinson G. Hurt M.M. J. Biol. Chem. 1997; 272: 15120-15127Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar) showed that the mouse histone H1b binds preferentially to a regulatory sequence within a mouse H3.2 replication-dependent histone gene. Previous analysis of the structural role of H1 histones demonstrated that three subfractions of H1 histones differ in their effectiveness in condensing DNA fibers into ordered aggregates (17Liao L.W. Cole K.D. J. Biol. Chem. 1981; 256: 10124-10128Abstract Full Text PDF PubMed Google Scholar) and that histone subtype H1t, compared with other subtypes, differs in its ability to condense chromatin (18Delucia F. Faraone-Menella M.R. D’Erme M. Quesada P. Caiafa P. Farina B. Biochem. Biophys. Res. Commun. 1994; 198: 32-39Crossref PubMed Scopus (70) Google Scholar,19Khadake J.R. Rao M.R.S. Biochemistry. 1997; 36: 1041-1051Crossref PubMed Scopus (46) Google Scholar). Furthermore, differences in the binding of H1 variants to DNA or phosphorylated H1 histones to DNA have been shown (20Clark D.J. Thomas J.O. Eur. J. Biochem. 1988; 178: 225-233Crossref PubMed Scopus (57) Google Scholar, 21Hill C.S. Rimmer J.M. Green B.N. Finch J.T. Thomas J.O. EMBO J. 1991; 10: 1939-1948Crossref PubMed Scopus (90) Google Scholar). The present work was undertaken to extend these earlier binding studies using an in vitro assay developed by Wolffe and Hayes (22Wolffe A.P. Hayes J.J. Methods Mol. Genet. 1993; 2: 314-330Google Scholar), enabling us to systematically study the binding of all known linker histones to a model chromatin complex. The complex is based on a DNA fragment containing the mouse mammary tumor virus long terminal repeat (MMTV LTR)1 promotor, the chromatin structure of which has been well characterized (23Richard-Foy H. Hager G.L. EMBO J. 1987; 6: 2321-2328Crossref PubMed Scopus (450) Google Scholar). The MMTV LTR reproducibly acquires a series of six positioned nucleosomes (24Archer T.K. Cordingly M.G. Wolford R.G. Hager G.L. Mol. Cell. Biol. 1991; 11: 688-698Crossref PubMed Scopus (294) Google Scholar). We have extended our systematic study of the histone-chromatin complexes formed by all known various H1 subtypes, H1a to H1e, H10, and H1t, to differently phosphorylated H1 histones according to the various phases of the cell cycle. The in vitro results presented here suggest that (i) differences in binding to a single mononucleosome (210 bp) and (ii) differences in aggregation of polynucleosomes (1.3 kbp, 6 nucleosomes) exist among the various linker histones. Furthermore, we found that (iii) specific types of in vivo phosphorylation of the linker histone tails do not influence binding to mononucleosomes, whereas (iv) aggregation of polynucleosomes was decreased in the case of highly phosphorylated mitotic H1 histones. NIH 3T3 fibroblasts were grown in monolayer cultures and cultivated in DMEM (Life Technologies, Inc.) supplemented with 10% fetal calf serum, penicillin (60 μg/ml), and streptomycin (100 μg/ml) in the presence of 5% CO2. To obtain cells from the G1 phase, S phase, and mitosis, we used a cell synchronization method described by Talasz et al. (15Talasz H. Helliger W. Puschendorf B. Lindner H. Biochemistry. 1996; 35: 1761-1767Crossref PubMed Scopus (81) Google Scholar). Routine microscopic examination and FACS analysis showed that more than 90% of the cells were in the various cell cycle phases. For isolation of H1 histones, nuclei from mouse liver or NIH fibroblasts were prepared as described by Zubay and Doty (25Zubay G. Doty P. J. Mol. Biol. 1959; 1: 1-20Crossref Scopus (275) Google Scholar) and Talasz et al. (15Talasz H. Helliger W. Puschendorf B. Lindner H. Biochemistry. 1996; 35: 1761-1767Crossref PubMed Scopus (81) Google Scholar). H1 histones were isolated from the resulting nuclear preparations by extracting with 5% perchloric acid at 4 °C for 1 h. The mixture was centrifuged at 10,000 × g for 20 min. The supernatant was mixed with trichloroacetic acid to the final concentration of 20% and allowed to stand for 1 h at 4 °C. The precipitate was washed once with acetone-HCl and thereafter with acetone, resuspended in water containing 10 mm 2-mercaptoethanol, and freeze-dried. The equipment used consisted of two Beckman model 114M pumps, a 421A system controller, and a model 165 variable-wavelength UV-visible detector. The effluent was monitored at 210 nm and the peaks were recorded using Beckman System Gold software. Protein separation was performed on a Nucleosil 300–5 C4 column (12.5 or 25 cm × 0.8 cm, 5-μm beads, 300 Å). The freeze-dried proteins were dissolved in 0.04 m 2-mercaptoethanol/water containing 0.1% trifluoroacetic acid, and samples of 100–300 μg of histones were loaded onto the column. At a constant flow rate of 1 and 1.5 ml/min, the H1 histones were eluted using a 45-min linear gradient from 34 to 54% solvent B (solvent A, water containing 15% ethylene glycolmonomethylether and 0.1% trifluoroacetic acid; solvent B, ethylene glycolmonomethylether (15%)/70% acetonitrile (85%) with 0.1% trifluoroacetic acid) (26Lindner H. Helliger W. Puschendorf B. J. Chromatogr. 1988; 450: 309-316Crossref PubMed Scopus (20) Google Scholar, 27Lindner H. Helliger W. Chromatographia. 1990; 30: 518-522Crossref Scopus (20) Google Scholar, 28Talasz H. Helliger W. Puschendorf B. Lindner H. Biochemistry. 1993; 32: 1188-1193Crossref PubMed Scopus (11) Google Scholar). High performance capillary electrophoresis of histone H1 proteins was performed with slight modifications according to Lindner et al. (29Lindner H. Helliger W. Dirschlmayer A. Talasz H. Wurm M. Sarg B. Jaquemar M. Puschendorf B. J. Chromatogr. 1992; 608: 211-216Crossref PubMed Scopus (39) Google Scholar, 30Lindner H. Helliger W. Sarg B. Meraner C. Electrophoresis. 1995; 16: 604-610Crossref PubMed Scopus (45) Google Scholar). In short, an untreated capillary (57/50 cm) was used, and separation was performed in a 10 mmtriethylamine/H3PO4 buffer system (pH 2.0) containing 90 mm perchloric acid and 0.02% hydroxypropylmethyl cellulose. The plasmid pMMTV-CAT was prepared from Escherichia coli cells harboring this plasmid using the alkali lysis method and the PC-500 Nucleobond kit (Macherey-Nagel). Plasmid DNA was digested either with restriction endonucleases SacI and BamHI (Boehringer Mannheim) at 37 °C (2 units/μg of DNA overnight), which deliberated a 210-bp insert containing part of the MMTV LTR promotor region, or with restriction endonucleases BamHI and HindIII (Boehringer Mannheim) at 37 °C (2 units/μg of DNA overnight), which liberated a 1.3-kbp insert containing the whole MMTV LTR promotor region. The inserts were purified from the digest on 1% agarose gels in 1× TBE buffer (90 mm Tris, 90 mm boric acid, 2.5 mm Na2EDTA, pH 8.3) using the GenElute Agarose Spin Columns (Supelco, Bellefonte). The 210-bp and 1.3-kbp DNA fragments were 5′-end labeled with T4 polynucleotide kinase and used for nucleosome reconstitution experiments. The naked nonspecific DNA (146 bp) was recovered from mouse liver mononucleosome particles with phenol-chloroform extraction after proteinase K (50 μg/1 mg of DNA) digestion in the presence of 1% SDS at 37 °C for 2 h. Mononucleosomes were recovered from mouse liver nuclei as described in Ref. 31Zalenskaya I.A. Pospelov V.A. Zalensky A.O. Vorob’ev V.I. Nucleic Acids Res. 1981; 9: 473-487Crossref PubMed Scopus (50) Google Scholar. Nuclei from mouse liver were prepared (25Zubay G. Doty P. J. Mol. Biol. 1959; 1: 1-20Crossref Scopus (275) Google Scholar), and the crude nuclear extract was centrifuged in a Beckman SW28 rotor through a 2.2 m sucrose (50 mm Tris-HCl, pH 7.5, 1 mm MgCl2, 0.1 mm phenylmethanesulfonyl fluoride, 0.1 mmbenzamidine) cushion at 25,000 rpm for 1 h at 4 °C to remove residual cytoplasmic components. Soluble chromatin was obtained by brief micrococcal nuclease (Sigma) digestion of the nuclei (31Zalenskaya I.A. Pospelov V.A. Zalensky A.O. Vorob’ev V.I. Nucleic Acids Res. 1981; 9: 473-487Crossref PubMed Scopus (50) Google Scholar). Digestion of nuclei with 10 units of micrococcal nuclease per 1 mg of DNA for 30 s at 37 °C in 5 mm Tris-HCl, pH 7.5, 0.3m sucrose, 1 mm CaCl2 (Buffer 1) produces chromatin lengths of 10–30 nucleosomes. To remove linker histone H1 this soluble chromatin was treated with Dowex 50 W-X2 (Sigma) in 0.4 m NaCl, 0.05 m sodium phosphate buffer (pH 7.4). Soluble chromatin without H1 histones was transferred to a dialysis bag (with a molecular size limit of 3 kDa) and dialyzed against Buffer 1 for 4 h. Thereafter, chromatin was redigested with 60 units of micrococcal nuclease per 1 mg of DNA for 10 min. Core particles were separated on 5–20% (w/w) sucrose gradients containing 5 mm Tris-HCl, pH 7.5, 1 mm Na2EDTA by centrifuging in a Beckman SW28 rotor at 25,000 rpm for 17 h at 4 °C. Nucleosomes were reconstituted onto radiolabeled DNA fragments by exchange with mouse liver core particles (donor chromatin) according to Drew and Travers (32Drew H.R. Travers A.A. J. Mol. Biol. 1985; 186: 773-790Crossref PubMed Scopus (554) Google Scholar). In the case of reconstitution onto a 1.3-kbp DNA fragment, the original 6 μl of exchange reaction containing ∼3200 ng of donor chromatin, 520 ng of naked nonspecific DNA (146 bp), and 200 ng of labeled 1.3-kbp fragment of the MMTV promotor was incubated in 1 m NaCl for 1 h (all incubations at room temperature). By adding 2.75 μl of TE 1 m NaCl was then diluted to 0.8 m NaCl followed by addition of 4.55 μl of TE to 0.6 m NaCl, for 30 min each (TE consisted of 10 mm Tris-HCl, pH 8.0, 1 mm EDTA). Thereafter, it was diluted to 0.4 m NaCl by addition of 9.1 μl of TE and to 0.1 m NaCl by addition of 82 μl TE, for 30 min each. The salt concentration was finally diluted to 0.05 m NaCl with 109 μl of TE. In this condition, 100% of the labeled 1.3-kbp MMTV promotor fragment was assembled to a saturated oligonucleosome chain. In the case of reconstitution onto 210-bp DNA fragments, the original 6 μl of exchange reaction containing ∼1300 ng of donor chromatin, ∼260 ng of naked nonspecific DNA (146 bp), and 80–100 ng of labeled 210-bp fragment of the MMTV promotor was incubated for 1 h (all incubations at room temperature) in 1 m NaCl. This was then diluted from 1 m NaCl to 0.05 mNaCl with TE as described for the exchange reaction onto 1.3-kbp. About 50–60% of the labeled MMTV LTR promotor 210-bp fragment was assembled to mononucleosome cores as monitored with electrophoresis. To ensure accurate protein concentration determination BCA protein assay (Pierce), spectrophotometry at 230 nm and reversed-phase HPLC with peak integration were used for concentration measurement of H1 histones. Additionally, we used the dotMETRIC protein assay (Geno Technology, Inc.), because the results obtained with this assay are not dependent on the amino acid composition of the protein, making the assay independent of protein-to-protein variation. Nucleosomes reconstituted with approximately 7 ng of radioactively end-labeled 210-bp MMTV LTR DNA in the presence of 110 ng of unlabeled nonspecific DNA were incubated with various amounts of histone H1 subtypes (see legend to Fig. 2) in 17 μl of binding buffer (10 mm Tris-HCl, pH 8.0, 50 mm NaCl, 1 mm EDTA, 5% (v/v) glycerol). Because about 40% of labeled DNA is free DNA, 4.2 ng (60%) of labeled DNA is reconstituted in mononucleosomes. Because the DNA is 210 base pairs, the concentration of the labeled mononucleosomes is about 2 nm. In the case of a 1.3-kbp fragment, approximately 10 ng of labeled fragment in reconstituted nucleosomes was incubated with various amounts of histone H1 subtypes (see legend to Fig. 7) in 10 μl of binding buffer in the presence of 120 ng of unlabeled nonspecific DNA (146 bp). For the 1.3-kbp fragment we used conditions where no free labeled DNA exists, this means that 10 ng of labeled DNA was reconstituted in polynucleosomes. Because the DNA was 1.3 kbp, the concentration of the polynucleosomes was about 1 nm.Figure 7Chromatin aggregation properties of the various H1 histone subtypes. A, nucleoprotein gel shift assay of the polynucleosome aggregation behavior of H1a, H1b, H1c, H1d/e, H10, and H1t. The position of free DNA, 6-mer polynucleosomes (PolyNuc), H1-polynucleosome complex (PolyNuc-H1), and aggregates is indicated. Polynucleosomes reconstituted with radioactively end-labeled 1.3-kbp MMTV LTR DNA were incubated with 0, 12, 24, 36, 48, 72, and 96 ng of H1 subtypes, corresponding to 0, 45, 90, 135, 180, 270, and 360 nm(lanes 2–8, respectively). In the case of H1b, we used higher concentrations and titrated with 0, 48, 72, 96, 120, 144, and 192 ng (H1b high conc.). Lane 1, free 1.3-kbp DNA. Complexes were separated on 0.7% agarose gels, and the gels were autoradiographed. B, quantitative analysis of the polynucleosome H1 aggregates formed by titration with various H1 subtypes. The quantity of H1 subtype-bound polynucleosome aggregates observed on autoradiographs of gel mobility shift experiments like those shown in A was determined and plotted against histone concentration (the mean of two experiments is shown).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the case of naked DNA linker histone reconstitution experiments, we used 7 ng of end-labeled 210-bp MMTV LTR DNA (2 nm) in the presence of 110 ng of unlabeled unspecific DNA (146 bp) without core particles. DNA was incubated with various amounts of histone H1 subtypes (see legend to Fig. 3) in 17 μl of binding buffer (10 mm Tris-HCl, pH 8.0, 50 mm NaCl, 1 mm EDTA, 5% (v/v) glycerol). Samples from reconstitution of nucleosomes or DNA with linker histones were incubated at room temperature for 15–20 min and loaded directly onto running 0.7% agarose gels in 0.1× TB (1× TB is 90 mm Tris, 90 mm boric acid, pH 8.3) or in 0.5× TBE. Running buffer was circulated throughout the experiment at a rate sufficient to prevent formation of either pH or ion gradients. After electrophoresis, the gels were dried and autoradiographed. The quality and quantity of the dried gels were determined by PhosphorImager analysis (Molecular Dynamics Inc.). For amplification and internal labeling of a 210-bp DNA fragment corresponding to the first A nucleosome of the MMTV LTR promotor (24Archer T.K. Cordingly M.G. Wolford R.G. Hager G.L. Mol. Cell. Biol. 1991; 11: 688-698Crossref PubMed Scopus (294) Google Scholar), we used the plasmid pMMTV-CAT and the primers 5′-CTT AGT GTT CTA TTT TCC TAT GTT CT-3′ and 5′-GAT GTG CGG GGG GAC-3′. Polymerase chain reaction radiolabeling reactions (50 μl) contained 1 ng of supercoiled plasmid template; 2 μm each primer; 10 mm Tris/HCl, pH 8.85; 25 mm KCl; 5 mm (NH4)2SO4; 2 mm MgSO4; 200 μm each of the three nucleotides dATP, dGTP, and dTTP; 20 μm dCTP; and 2.5 units of Pwo DNA polymerase (Boehringer Mannheim). In addition, 5 μl of [32P]dCTP (6000 Ci/mmol, 3.3 μm) was added to a final concentration of 0.33 μm. Thirty cycles of polymerase chain reaction were performed (denaturation, 30 s at 94 °C; annealing, 1 min at 51 °C; elongation, 1 min at 72 °C) after an initial 5 min of denaturation at 95 °C. The samples were precipitated and washed with ethanol and dried. Mononucleosomes (100 ng of specific internally labeled 210-bp DNA and 1600 ng of nonspecific 146-bp DNA) in the presence or absence of 200 ng of histone H1 (w/w ratio of histone to DNA was 0.12) were digested with 0.15, 0.3, or 0.6 units of micrococcal nuclease (Sigma) for 5 min at 37 °C. Incubation with histone H1 was performed as described above. Ca2+ was adjusted to 2 mm concomitantly with the addition of micrococcal nuclease. Micrococcal nuclease digestions were terminated by adding of EDTA (5 mm), SDS (0.25%, w/v), and proteinase K (0.5 μg/ml). Digestion with proteinase K lasted 2 h at 37 °C. DNA was recovered by phenol extraction. The labeled DNA fragments were separated by electrophoresis in nondenaturing 9% polyacrylamide gels with 1× TBE as running buffer. After electrophoresis, the gels were dried and autoradiographed. H1 histones from mitotic cells were digested with alkaline phosphatase basically as described (33Sherod D. Johnson G. Chalkley R. Biochemistry. 1970; 9: 4611-4616Crossref PubMed Scopus (60) Google Scholar, 34Harisanova N.T. Ralchev K.H. Mol. Biol. Rep. 1986; 11: 199-203Crossref PubMed Scopus (3) Google Scholar). About 50 μg of H1 histones in 140 μl of 10 mm Tris-HCl, pH 8.0, and 1 mmphenylmethanesulfonyl fluoride was mixed with 2 units of E. coli alkaline phosphatase (Sigma), corresponding to 40 units/mg histone. Incubation at 22 °C was terminated after 16 h by adjusting the incubation mixture to 5% perchloric acid. The histone H1 was resolved as described above. To test mononucleosome cores for their ability to bind the various linker histones we first fractionated mouse liver H1 histones using reversed-phase HPLC. The resulting subtypes and subfractions obtained were designated according to the nomenclature of Lennox et al. (35Lennox R.W. Oshima R.G. Cohen L.H. J. Biol. Chem. 1982; 257: 5183-5189Abstract Full Text PDF PubMed Google Scholar). In mouse liver we found H1a, H1b, H1c, and H10 as pure subtypes, whereas one subfraction was a mixture containing the variants H1d and H1e (Fig. 1 A) (28Talasz H. Helliger W. Puschendorf B. Lindner H. Biochemistry. 1993; 32: 1188-1193Crossref PubMed Scopus (11) Google Scholar). Histone subtype H1t was obtained by means of reversed-phase HPLC fractionation of mouse testis (Fig. 1 B) (36Lindner H. Helliger W. Parvez H. Caudy P. Parvez S. Roland-Gosselin P. Progress in HPLC-HPCE. 5. VSP, Utrecht, The Netherlands1997: 97-113Google Scholar). Mononucleosome cores were prepared as described under “Experimental Procedures” and reconstituted with a DNA fragment of the MMTV LTR promotor. We used a 210-bp fragment corresponding to the first A nucleosome described by Archer et al. (24Archer T.K. Cordingly M.G. Wolford R.G. Hager G.L. Mol. Cell. Biol. 1991; 11: 688-698Crossref PubMed Scopus (294) Google Scholar). We assembled about 50–60% of the labeled MMTV LTR DNA with a single octamer, whereas the rest remained unbound and served as a naked DNA control (Fig. 2 A, lane 2). Fig. 2 shows the binding pattern of the mouse H1 histones H1a to H1e, H10, H1t, and total H1 histones from mouse liver and testis. Similar to reconstitution experiments performed by other groups (37Hayes J.J. Wolffe A.P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6415-6419Crossref PubMed Scopus (134) Google Scholar), we found that the reconstitutes were preferentially bound by the various linker histone subtypes over the naked DNA (Fig. 2 A, lanes 3–8). After the octamer DNA complex shifted completely to the H1 octamer DNA complex, the H1 histone started to bind to naked DNA (disappearance of free DNA) and an H1 DNA complex appeared (Fig. 2 A, lanes 7 and 8). This phenomenon is visible at titration with H1a, H1c, H1d/e, H10, and total liver H1, but not clearly pronounced with H1b, H1t, and total testis H1 histones. Nucleoprotein gels, such as those shown in Fig. 2 A, were quantitatively analyzed to determine the relative nucleosome binding affinity of the various linker histone subtypes (Fig. 2 B). This analysis revealed that none of the subtypes has significantly different affinity, with the exception of histone H1b. For this H1b subtype we found a dissociation constant of 8–16 nm, whereas the dissociation constant of all other subtypes varied between 1 and 3 nm. In the cases of H1b and H1t, the binding to free DNA appears to be less strong than in the case of H1a. To test whether the various H1 subtypes have different affinities for free DNA we directly measured the binding of H1 subtypes to not nucleosomal organized DNA. These results (Fig. 3) further confirmed our finding that H1b and H1t have less stronger affinities not only to nucleosomal organized DNA but also to free DNA. As shown by Ura et al. (38Ura K. Wolffe A.P. Hayes J.J. J. Biol. Chem. 1994; 269: 27171-27174Abstract Full Text PDF PubMed Google Scholar), micrococcal nuclease digestion of H1 histone-bound chromatin leads to an accumulation of a kinetic intermediate containing all the histones and 166–168 bp of DNA, known as the chromatosome (39Simpson R.T. Biochemistry. 1978; 17: 5524-5531Crossref PubMed Scopus (447) Google Scholar). We used this chromatosomal stop (40Allan J. Hartman P.G. Crane-Robinson C. Aviles F.X. Nature. 1980; 288: 675-679Crossref PubMed Scopus (526) Google Scholar) as a diagnostic tool for correct incorporation of the various linker histone subtypes into chromatin. Chromatosome-length DNA was observed regardless of the histone subtype used to reconstitute the MMTV LTR promotor mononucleosomal cores. Fig. 4compares total H1 histones from mouse liver and H1b subtype (other subtypes not shown). With an H1/DNA ratio (w/w) of 0.12, corresponding to a calculated molar ratio of about one MMTV LTR promotor DNA H1 histone molecule per reconstituted nucleosome core, both total H1 from mouse liver and pure mouse H1b subtype induced the appearance of chromatosome-length DNA. To test the influence of phosphorylation of the histone tails on binding to the MMTV LTR promotor DNA octamer complex, we prepared linker histones in various phosphorylation states (see under “Experimental Procedures”). Total H1 histones from mouse NIH 3T3 fibroblasts were prepared from synchronized cells passing through G1 phase, S phase, or mitosis. Capillary electrophoresis was used to measure the phosphorylation state of total H1 histones (Fig. 5). In growth-arrested cells at G1 phase, linker histones are unphosphorylated, whereas in late S phase, the H1 variants exist as a combination of molecules containing one to three phosphate groups, according to the particular subtype (15Talasz H. Helliger W. Puschendorf B. Lindner H. Biochemistry. 1996; 35: 1761-1767Crossref PubMed Scopus (81) Google Scholar). The fastest migrating components in capillary electrophoresis are the unphosphorylated H1 histones (29Lindner H. Helliger W. Dirschlmayer A. Talasz H. Wurm M. Sarg B. Jaquemar M. Puschendorf B. J. Chromatogr. 1992; 608: 211-216Crossref PubMed Scopus (39) Google Scholar, 36Lindner H. Helliger W. Parvez H. Caudy P. Parvez S. Roland-Gosselin P. Progress in HPLC-HPCE. 5. VSP, Utrecht, The Netherlands1997: 97-113Google Scholar) (Fig. 5 A, peaks between 18 and 20.5 min). Histones from S phase cells (Fig. 5 B) and, to a great extent, highly phosphorylated H1 histone subtypes from cells in mitosis containing four to five phosphate groups migrate at lower speed. In the case of the most highly phosphorylated H1 histones, the main peaks are visible at 20.5–21.5 min (Fig. 5 C). When we digested the mitotic H1 histones with alkaline phosphatase, only peaks between 18 and 20.5 min were visible, corresponding to unphosphorylated H1 histones (Fig. 5 D). Fig. 6 A shows the association of cell cycle-specific linker histones with the MMTV LTR nucleosome core, whereas Fig. 6 B presents the quantitative analysis of these nucleoprotein gels. Both the unphosphorylated linker histones prepared from G1 cells and the medium phosphorylated H1 histones from S phase seem to have a binding constant of about 3–5 nm, with a total shift to the chromatosome at 32 nm. Interestingly, the very highly phosphorylated H1 histones from mitotic cells also have a binding constant of about 3–5 nm and shifted completely to the chromatosome with 32 nm (Fig. 6 A, mitosis). Khadake and Rao (19Khadake J.R. Rao M.R.S. Biochemistry. 1997; 36: 1041-1051Crossref PubMed Scopus (46) Google Scholar) showed that a mixture of histone subtypes H1b, H1c, H1d, and H1e retarded the mobility of a polynucleosome in a conc" @default.
• W2014279477 created "2016-06-24" @default.
• W2014279477 creator A5030274834 @default.
• W2014279477 creator A5055193163 @default.
• W2014279477 creator A5056989229 @default.
• W2014279477 creator A5060320016 @default.
• W2014279477 creator A5069412131 @default.
• W2014279477 date "1998-11-01" @default.
• W2014279477 modified "2023-10-16" @default.
• W2014279477 title "In Vitro Binding of H1 Histone Subtypes to Nucleosomal Organized Mouse Mammary Tumor Virus Long Terminal Repeat Promotor" @default.
• W2014279477 cites W1481236667 @default.
• W2014279477 cites W1493507586 @default.
• W2014279477 cites W1511253669 @default.
• W2014279477 cites W1520024145 @default.
• W2014279477 cites W1542748993 @default.
• W2014279477 cites W1548407034 @default.
• W2014279477 cites W1555044868 @default.
• W2014279477 cites W1556841387 @default.
• W2014279477 cites W1589555651 @default.
• W2014279477 cites W1596717967 @default.
• W2014279477 cites W1897686051 @default.
• W2014279477 cites W1960503626 @default.
• W2014279477 cites W1963882904 @default.
• W2014279477 cites W1968802467 @default.
• W2014279477 cites W1970020592 @default.
• W2014279477 cites W1970764247 @default.
• W2014279477 cites W1979204453 @default.
• W2014279477 cites W1980550935 @default.
• W2014279477 cites W1981790067 @default.
• W2014279477 cites W1989538824 @default.
• W2014279477 cites W2004360936 @default.
• W2014279477 cites W2005144583 @default.
• W2014279477 cites W2012119941 @default.
• W2014279477 cites W2013059397 @default.
• W2014279477 cites W2013179009 @default.
• W2014279477 cites W2018356630 @default.
• W2014279477 cites W2018626566 @default.
• W2014279477 cites W2024438743 @default.
• W2014279477 cites W2026371011 @default.
• W2014279477 cites W2031236107 @default.
• W2014279477 cites W2031882083 @default.
• W2014279477 cites W2039979009 @default.
• W2014279477 cites W2040034427 @default.
• W2014279477 cites W2040126142 @default.
• W2014279477 cites W2042214898 @default.
• W2014279477 cites W2046001002 @default.
• W2014279477 cites W2046609493 @default.
• W2014279477 cites W2047979686 @default.
• W2014279477 cites W2049920155 @default.
• W2014279477 cites W2058914439 @default.
• W2014279477 cites W2061014609 @default.
• W2014279477 cites W2066492784 @default.
• W2014279477 cites W2067623697 @default.
• W2014279477 cites W2070922804 @default.
• W2014279477 cites W2071478793 @default.
• W2014279477 cites W2084413077 @default.
• W2014279477 cites W2095791513 @default.
• W2014279477 cites W2099114692 @default.
• W2014279477 cites W2102136241 @default.
• W2014279477 cites W2805946960 @default.
• W2014279477 cites W4242845182 @default.
• W2014279477 doi "https://doi.org/10.1074/jbc.273.48.32236" @default.
• W2014279477 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9822702" @default.
• W2014279477 hasPublicationYear "1998" @default.
• W2014279477 type Work @default.
• W2014279477 sameAs 2014279477 @default.
• W2014279477 citedByCount "64" @default.
• W2014279477 countsByYear W20142794772012 @default.
• W2014279477 countsByYear W20142794772013 @default.
• W2014279477 countsByYear W20142794772014 @default.
• W2014279477 countsByYear W20142794772015 @default.
• W2014279477 countsByYear W20142794772016 @default.
• W2014279477 countsByYear W20142794772017 @default.
• W2014279477 countsByYear W20142794772018 @default.
• W2014279477 countsByYear W20142794772019 @default.
• W2014279477 countsByYear W20142794772020 @default.
• W2014279477 countsByYear W20142794772021 @default.
• W2014279477 countsByYear W20142794772023 @default.
• W2014279477 crossrefType "journal-article" @default.
• W2014279477 hasAuthorship W2014279477A5030274834 @default.
• W2014279477 hasAuthorship W2014279477A5055193163 @default.
• W2014279477 hasAuthorship W2014279477A5056989229 @default.
• W2014279477 hasAuthorship W2014279477A5060320016 @default.
• W2014279477 hasAuthorship W2014279477A5069412131 @default.
• W2014279477 hasBestOaLocation W20142794771 @default.
• W2014279477 hasConcept C101762097 @default.
• W2014279477 hasConcept C104317684 @default.
• W2014279477 hasConcept C121608353 @default.
• W2014279477 hasConcept C141231307 @default.
• W2014279477 hasConcept C150194340 @default.
• W2014279477 hasConcept C153911025 @default.
• W2014279477 hasConcept C199491388 @default.
• W2014279477 hasConcept C202751555 @default.
• W2014279477 hasConcept C2522874641 @default.
• W2014279477 hasConcept C2776187077 @default.
• W2014279477 hasConcept C2779777497 @default.
• W2014279477 hasConcept C502942594 @default.
• W2014279477 hasConcept C530470458 @default.

• next