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• W1998433561 abstract "The precise positioning of nucleosomes plays a critical role in the regulation of gene expression by modulating the DNA binding activity of trans-acting factors. However, molecular determinants responsible for positioning are not well understood. We examined whether the removal of the core histone tail domains from nucleosomes reconstituted with specific DNA fragments led to alteration of translational positions. Remarkably, we find that removal of tail domains from a nucleosome assembled on a DNA fragment containing a Xenopus borealis somatic-type 5S RNA gene results in repositioning of nucleosomes along the DNA, including two related major translational positions that move about 20 bp further upstream with respect to the 5S gene. In a nucleosome reconstituted with a DNA fragment containing the promoter of a Drosophila alcohol dehydrogenase gene, several translational positions shifted by about 10 bp along the DNA upon tail removal. However, the positions of nucleosomes assembled with a DNA fragment known to have one of the highest binding affinities for core histone proteins in the mouse genome were not altered by removal of core histone tail domains. Our data support the notion that the basic tail domains bind to nucleosomal DNA and influence the selection of the translational position of nucleosomes and that once tails are removed movement between translational positions occurs in a facile manner on some sequences. However, the effect of the N-terminal tails on the positioning and movement of a nucleosome appears to be dependent on the DNA sequence such that the contribution of the tails can be masked by very high affinity DNA sequences. Our results suggest a mechanism whereby sequence-dependent nucleosome positioning can be specifically altered by regulated changes in histone tail-DNA interactions in chromatin. The precise positioning of nucleosomes plays a critical role in the regulation of gene expression by modulating the DNA binding activity of trans-acting factors. However, molecular determinants responsible for positioning are not well understood. We examined whether the removal of the core histone tail domains from nucleosomes reconstituted with specific DNA fragments led to alteration of translational positions. Remarkably, we find that removal of tail domains from a nucleosome assembled on a DNA fragment containing a Xenopus borealis somatic-type 5S RNA gene results in repositioning of nucleosomes along the DNA, including two related major translational positions that move about 20 bp further upstream with respect to the 5S gene. In a nucleosome reconstituted with a DNA fragment containing the promoter of a Drosophila alcohol dehydrogenase gene, several translational positions shifted by about 10 bp along the DNA upon tail removal. However, the positions of nucleosomes assembled with a DNA fragment known to have one of the highest binding affinities for core histone proteins in the mouse genome were not altered by removal of core histone tail domains. Our data support the notion that the basic tail domains bind to nucleosomal DNA and influence the selection of the translational position of nucleosomes and that once tails are removed movement between translational positions occurs in a facile manner on some sequences. However, the effect of the N-terminal tails on the positioning and movement of a nucleosome appears to be dependent on the DNA sequence such that the contribution of the tails can be masked by very high affinity DNA sequences. Our results suggest a mechanism whereby sequence-dependent nucleosome positioning can be specifically altered by regulated changes in histone tail-DNA interactions in chromatin. In the eukaryotic cell, assembly of DNA into chromatin serves to organize and compact several meters worth of DNA several thousand-fold into a nucleus about 10 μm in diameter (1van Holde K.E. Chromatin, Springer Verlag, New York. 1989; Google Scholar). This condensation is accomplished in sequential steps. First, 147-bp stretches of DNA are wrapped around an octamer of core histone proteins to form a nucleosome core, which are spaced at ∼200-base pair intervals along the genomic DNA. In higher eukaryotes, approximately one linker histone binds each nucleosome and the “linker” DNA between the cores. Under physiological conditions, strings of nucleosomes spontaneously condense into secondary structures such as the 30-nm chromatin fiber and higher order tertiary structures and beyond (2Hansen J.C. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 361-392Crossref PubMed Scopus (420) Google Scholar, 3Belmont A.S. Bruce K. J. Cell Biol. 1994; 127: 287-302Crossref PubMed Scopus (277) Google Scholar). The assembly of DNA into chromatin plays an integral role in the regulation of gene expression, primarily by regulating access of genomic DNA to trans-acting factors. For example, the activity of sequence-specific DNA-binding proteins is greatly reduced for DNA targets located within the 147-bp nucleosome core region compared with linker DNA (4Polach K.J. Widom J. J. Mol. Biol. 1995; 254: 130-149Crossref PubMed Scopus (532) Google Scholar) and initiation of transcription typically involves multiple processes that result in exposure or occlusion of cognate DNA sites within promoter regions (5Grunstein M. Sci. Am. 1992; 267: 68-74Crossref PubMed Scopus (69) Google Scholar, 6Boeger H. Griesenbeck J. Strattan J.S. Kornberg R.D. Mol. Cell. 2003; 11: 1587-1598Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 7Korber P. Luckenbach T. Blaschke D. Horz W. Mol. Cell. Biol. 2004; 24: 10965-10974Crossref PubMed Scopus (77) Google Scholar, 8Whitehouse I. Tsukiyama T. Nat. Struct. Mol. Biol. 2006; 13: 633-640Crossref PubMed Scopus (107) Google Scholar). Classic experiments by the Grunstein (10Han M. Grunstein M. Cell. 1988; 55: 1137-1145Abstract Full Text PDF PubMed Scopus (314) Google Scholar) and Horz (9Almer A. Rudolph H. Hinnen A. Horz W. EMBO J. 1986; 5: 2689-2696Crossref PubMed Scopus (349) Google Scholar) laboratories showed that nucleosomes are intimately involved in gene regulation and can act as specific repressors of transcription. Indeed recent genome-wide determinations of nucleosome positions indicate specific positioning of nucleosomes in the vicinity of many promoters in yeast (11Segal E. Fondufe-Mittendorf Y. Chen L. Thastrom A. Field Y. Moore I.K. Wang J.P. Widom J. Nature. 2006; 442: 772-778Crossref PubMed Scopus (1187) Google Scholar, 12Ioshikhes I.P. Albert I. Zanton S.J. Pugh B.F. Nat. Genet. 2006; 38: 1210-1215Crossref PubMed Scopus (262) Google Scholar), requiring nucleosome eviction or repositioning (sliding) to allow transcription (13Fazzio T.G. Tsukiyama T. Mol. Cell. 2003; 12: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 14Becker P.B. EMBO J. 2002; 21: 4749-4753Crossref PubMed Scopus (94) Google Scholar, 15Badenhorst P. Xiao H. Cherbas L. Kwon S.Y. Voas M. Rebay I. Cherbas P. Wu C. Genes Dev. 2005; 19: 2540-2545Crossref PubMed Scopus (113) Google Scholar). Generally nucleosomes are mobilized by ATP-dependent remodeling complexes such as the SWI/SNF, MI-2, NURF, or ISWI complexes to effect gene activation (16Whitehouse I. Flaus A. Cairns B.R. White M.F. Workman J.L. Owen-Hughes T. Nature. 1999; 400: 784-787Crossref PubMed Scopus (290) Google Scholar, 17Guschin D. Wade P.A. Kikyo N. Wolffe A.P. Biochemistry. 2000; 39: 5238-5245Crossref PubMed Scopus (74) Google Scholar, 18Hamiche A. Sandaltzopoulos R. Gdula D.A. Wu C. Cell. 1999; 97: 833-842Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 19Becker P.B. Horz W. Annu. Rev. Biochem. 2002; 71: 247-273Crossref PubMed Scopus (624) Google Scholar, 20Laöngst G. Bonte E.J. Corona D.F. Becker P.B. Cell. 1999; 97: 843-852Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar) or repression (8Whitehouse I. Tsukiyama T. Nat. Struct. Mol. Biol. 2006; 13: 633-640Crossref PubMed Scopus (107) Google Scholar). In some cases, specific post-translational modifications of core histone tails are involved in the recruitment of remodeling complexes to target sites and thus may facilitate nucleosome sliding by these complexes (21Agalioti T. Lomvardas S. Parekh B. Yie J. Maniatis T. Thanos D. Cell. 2000; 103: 667-678Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar, 22Lomvardas S. Thanos D. Cell. 2001; 106: 685-696Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). For example, acetylation is required for transcription of the interferon-γ gene in mammalian cells or the HO gene in yeast cells (22Lomvardas S. Thanos D. Cell. 2001; 106: 685-696Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 23Cosma M.P. Mol. Cell. 2002; 10: 227-236Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). However, the actual mechanism of nucleosome mobilization or sliding remains the subject of much debate (24Flaus A. Owen-Hughes T. Biopolymers. 2003; 68: 563-578Crossref PubMed Scopus (71) Google Scholar). The core histones are each comprised of a histone-fold domain that oligomerizes to form the central protein spool onto which nucleosomal DNA is wrapped and an N-terminal tail domain, which represents about 20–25% of the mass of each core histone and projects out from the main body of the nucleosome core (25Bohm L. Crane-Robinson C. Biosci. Rep. 1984; 4: 365-386Crossref PubMed Scopus (130) Google Scholar, 26Luger K. Mader A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-260Crossref PubMed Scopus (6973) Google Scholar, 27Arents G. Moudrianakis E.N. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10489-10493Crossref PubMed Scopus (313) Google Scholar). The N-terminal tail domains play a central role in epigenetic regulation of gene expression presumably due to their direct role in defining the structural and functional state of chromatin. The tails are essential for folding of arrays of nucleosomes into condensed secondary and tertiary chromatin structures and are also the location of most of the transcription-related post-translational modifications within the core histones. Post-translational modifications such as acetylation can facilitate transcription by directly affecting the stability of higher order chromatin structures (28Garcia-Ramirez M. Dong F. Ausio J. J. Biol. Chem. 1992; 267: 19587-19595Abstract Full Text PDF PubMed Google Scholar, 29Garcia-Ramirez M. Rocchini C. Ausio J. J. Biol. Chem. 1995; 270: 17923-17928Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 30Allan J. Harborne N. Rau D.C. Gould H. J. Cell Biol. 1982; 93: 285-297Crossref PubMed Scopus (201) Google Scholar, 31Tse C. Sera T. Wolffe A.P. Hansen J.C. Mol. Cell. Biol. 1998; 18: 4629-4638Crossref PubMed Scopus (482) Google Scholar) and/or by directing the binding of ancillary proteins, which in turn alter chromatin structure (2Hansen J.C. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 361-392Crossref PubMed Scopus (420) Google Scholar, 32Mellor J. Cell. 2006; 126: 22-24Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Interestingly, recent evidence suggests that a major determinant of nucleosome positioning in vivo can be ascribed to DNA sequence-dependent effects such as anisotropic flexibility and inherent curvature within DNA (11Segal E. Fondufe-Mittendorf Y. Chen L. Thastrom A. Field Y. Moore I.K. Wang J.P. Widom J. Nature. 2006; 442: 772-778Crossref PubMed Scopus (1187) Google Scholar). Using an algorithm based on a set of selected DNA sequences that exhibit high affinity for binding to the core histone octamer, Widom and colleagues (11Segal E. Fondufe-Mittendorf Y. Chen L. Thastrom A. Field Y. Moore I.K. Wang J.P. Widom J. Nature. 2006; 442: 772-778Crossref PubMed Scopus (1187) Google Scholar) accurately predicted about 50% the nucleosome positions observed in vivo in yeast cells. Likewise, using a similar consensus nucleosome positioning element, Ioshikhes et al. (12Ioshikhes I.P. Albert I. Zanton S.J. Pugh B.F. Nat. Genet. 2006; 38: 1210-1215Crossref PubMed Scopus (262) Google Scholar) demonstrated that DNA sequence is likely the dominant influence directing the location of nucleosomes in the vicinity of promoters in yeast. They find a distinct class of promoters in which the TATA box is buried within a well defined nucleosome positioning element that is typically associated with genes known to be highly dependent on chromatin remodeling and histone modification enzymes. Thus, it is becoming clear that understanding molecular determinants of sequence-directed nucleosome positioning is critical to a complete understanding of the regulation of gene expression. Older experiments suggested that the tail domains do not contribute to the “choice” of translational position adopted by nucleosomes along a DNA sequence. The translational positioning of nucleosomes assembled with DNA fragments containing 5S genes from both Xenopus and sea urchin did not appear to be altered by the acetylation or removal of core histone tail domains (33Dong F. Hansen J.C. van Holde K.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5724-5728Crossref PubMed Scopus (169) Google Scholar, 34Hayes J.J. Clark D.J. Wolffe A.P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6829-6833Crossref PubMed Scopus (228) Google Scholar, 35Bauer W.R. Hayes J.J. White J.H. Wolffe A.P. J. Mol. Biol. 1994; 236: 685-690Crossref PubMed Scopus (114) Google Scholar). However, these experiments employed relatively low-resolution techniques that might be insensitive to small changes in translational positioning. Indeed, acetylation of the core histone tail domains was later shown to be required for the movement of nucleosomes already remodeled by SWI/SNF (22Lomvardas S. Thanos D. Cell. 2001; 106: 685-696Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar) and reconstitution of nucleosomes onto a 359-bp DNA fragment containing the Drosophila hsp70 promoter have shown that nucleosomes lacking the H2B N-terminal tail domain exhibited a different distribution of translational positions compared with nucleosomes containing wild type histones (36Hamiche A. Kang J.G. Dennis C. Xiao H. Wu C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14316-14321Crossref PubMed Scopus (138) Google Scholar). Moreover these studies (33Dong F. Hansen J.C. van Holde K.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5724-5728Crossref PubMed Scopus (169) Google Scholar, 34Hayes J.J. Clark D.J. Wolffe A.P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6829-6833Crossref PubMed Scopus (228) Google Scholar, 35Bauer W.R. Hayes J.J. White J.H. Wolffe A.P. J. Mol. Biol. 1994; 236: 685-690Crossref PubMed Scopus (114) Google Scholar, 36Hamiche A. Kang J.G. Dennis C. Xiao H. Wu C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14316-14321Crossref PubMed Scopus (138) Google Scholar) compared translational positions in samples of nucleosomes reconstituted with either native or tailless histones, whereas changes occurring upon alteration of tail-DNA interactions of existing nucleosomes may be more relevant to gene regulation in vivo. Here we investigate the effect of removal of the core histone tails on translational positions of nucleosomes reconstituted with specific DNA sequences. We demonstrate that the tail domains do indeed contribute to sequence-directed translational positioning as removal of tail domains from existing nucleosomes can lead to a re-distribution of translational positions along a DNA fragment. Preparation of the Radiolabeled DNA Fragments—A 215-bp DNA fragment containing nucleotides –78 to +137 of a Xenopus borealis somatic-type 5S RNA gene was obtained from plasmid pXP-10 (37Wolffe A.P. Jordan E. Brown D.D. Cell. 1986; 44: 381-389Abstract Full Text PDF PubMed Scopus (105) Google Scholar) by digestion with EcoRI (New England Biolabs). The digested plasmid was treated with alkaline phosphatase (Roche Applied Science), phosphorylated with T4 polynucleotide kinase (New England Biolabs) using [γ-32P]ATP, then cut with DdeI (New England Biolabs). All DNA fragments were purified from 8% polyacrylamide gels in 1× TBE buffer. A 238-bp DNA fragment containing the 5S RNA gene, nucleotides –102 to +135, were prepared by end labeling after digestion with XbaI then digestion with HpaII by the same method. A 182-bp DNA fragment containing 12 (TATAAACGCC) repeats was prepared from plasmid pHCn41 by first cleaving with EcoRI, radiolabeling, then cutting with BamHI (38Widlund H.R. Kuduvalli P.N. Bengtsson M. Cao H. Tullius T.D. Kubista M. J. Biol. Chem. 1999; 274: 31847-31852Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). A 227-bp DNA fragment containing the promoter of an alcohol dehydrogenase (Adh) 3The abbreviations used are: Adh, alcohol dehydrogenase; APB, 4-azidophenacyl bromide; ExoIII, exonuclease III. gene from Drosophila melanogaster was obtained from plasmid pX5′r-168 by digesting with XbaI, end labeling, then cleaving with ScaI. Reconstitution of Nucleosomes Containing Native, H2A-A12C-APB, H2A-A45C-APB, and H2B-S56C-APB Core Histones—Core histones were purified from chicken blood as H2A/H2B dimers and (H3/H4)2 tetramers according to standard procedures (39Simon R.H. Felsenfeld G. Nucleic Acids Res. 1979; 6: 689-696Crossref PubMed Scopus (293) Google Scholar). Mutant histone proteins H2A-A12C, H2A-A45C, and H2B-S56C were expressed in Escherichia coli, purified, then modified with the cross-linking agent, 4-azidophenacyl bromide (APB), as described (40Yang Z. Zheng C. Thiriet C. Hayes J.J. Mol. Cell. Biol. 2005; 25: 241-249Crossref PubMed Scopus (27) Google Scholar). Nucleosomes were reconstituted with chicken H3/H4 and recombinant H2A/H2B except where indicated via standard salt dialysis (41Hayes J.J. Lee K.M. Methods. 1997; 12: 2-9Crossref PubMed Scopus (91) Google Scholar). Preparation of Tailless Nucleosomes Containing H2A-A12C-APB, H2A-A45C-APB, and H2B-S56C-APB—Five ml of nucleosomes (400 μg) reconstituted with wild type H2A, H2A-A12C-APB, H2A-A45C-APB, and H2B-S56C-APB were concentrated to 0.5 ml by spin filtration (Millipore, YM-50), then treated with 0.04 ml of trypsin cross-linked agarose beads (Sigma) for 15 min at room temperature (42Yang Z. Hayes J.J. Methods. 2004; 33: 25-32Crossref PubMed Scopus (9) Google Scholar). The digest was then centrifuged to remove the beads, the supernatant was transferred to a fresh tube and then the extent of trypsinization examined by 18% SDS-PAGE. Restriction Enzyme Selection for Major Translational Positions—Five ml of nucleosomes containing H2A-A45C-APB reconstituted with the 215-bp 5S DNA fragment were concentrated to 1 ml using centrifugal-based filter (Millipore) as described above, then treated with 50 units of BamHI (New England Biolabs) for 15 min at 37 °C. BamHI cleaves this DNA fragment at position –61 (Fig. 1). BamHI-resistant nucleosomes were then purified on sucrose gradients (10 ml, 5–20%), and fractions (∼1 ml) were subjected to a buffer-exchange procedure with 10 mm Tris-Cl (pH 8.0) to remove sucrose and the sample concentrated to a volume of 0.5 ml. The nucleosomes were then incubated with trypsin-agarose beads to remove core histone tails by the method above (42Yang Z. Hayes J.J. Methods. 2004; 33: 25-32Crossref PubMed Scopus (9) Google Scholar). The major translational positions within the H2A-A45C-APB-containing 5S nucleosomes reconstituted onto the 238-bp DNA fragment were enriched by AluI digestion by the above protocol because AluI cleaves the DNA template at nucleotide –54 (Fig. 1). To check efficiency of selection, samples were loaded into 5% translational polyacrylamide gel (20 mm HEPES, pH 7.5), electrophoresed at 106 V for 2 h at room temperature, dried, and analyzed by phosphorimager (43Aoyagi S. Wade P.A. Hayes J.J. J. Biol. Chem. 2003; 278: 30562-30568Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Determination of Restriction Enzyme Accessibility—The 5S nucleosomes were reconstituted, purified by sucrose gradient, then incubated with restriction enzymes (New England Biolabs), BamHI (1,000 units/ml), BbvI (500 units/ml), EcoRV (1,000 units/ml), RsaI (500 units/ml), and FokI (500 units/ml), respectively, for the times indicated in the figures at 37 °C. Samples were removed at each reaction time, quenched by adding SDS-EDTA stop solution (40 mm EDTA, 0.2% SDS), loaded into 6% SDS-PAGE, then quantitated by phosphorimager. Determination of Cross-linking Positions—5S nucleosomes containing H2A-A45C-APB or H2B-S56C-APB core histones were purified by sucrose gradient, loaded into 0.7% agarose nucleoprotein gels. The nucleosomes were irradiated at 365 nm for 30 s before or after the removal of core histone tail domains. DNA was purified from each radioactive band in the preparative nucleoprotein gel, then cross-linked DNA was separated from uncross-linked on 6% SDS-polyacrylamide gels as described (44Lee K.M. Hayes J.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8959-8964Crossref PubMed Scopus (52) Google Scholar). DNA purified from the polyacrylamide gel was treated with NaOH, precipitated, then equivalent amounts of radioactivity were loaded onto 6% sequencing gels. The gels were dried and analyzed by phosphorimager (45Lee K.M. Chafin D.R. Hayes J.J. Methods Enzymol. 1999; 304: 231-251Crossref PubMed Scopus (9) Google Scholar). Cross-linking within nucleosomes containing the (TATAAACGCC)12 repeats or the alcohol dehydrogenase gene promoter were determined by the same method. ExoIII Nuclease Assay—Intact or tailless nucleosomes purified by sucrose gradient were incubated with 0.5 μl of ExoIII nuclease (100 kilounits/ml, New England Biolabs) for 2, 5, or 10 min, then the reaction was quenched with an EDTA-SDS stop solution (43Aoyagi S. Wade P.A. Hayes J.J. J. Biol. Chem. 2003; 278: 30562-30568Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). DNA from each reaction was precipitated, then the cleavage products analyzed after separation on 6% sequencing gels as above. We wished to determine whether the core histone tail domains contribute to the selection of sequence-dependent nucleosome translational positions and specifically whether alteration of tail-DNA interactions would lead to mobilization and redistribution of nucleosome positions. In previous work we used a site-directed histone → DNA cross-linking method in which a APB was attached to the 12th residue in H2A, a position located adjacent to the DNA gyre, about 40 bp from the nucleosome dyad (44Lee K.M. Hayes J.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8959-8964Crossref PubMed Scopus (52) Google Scholar). We observed that the pattern of cross-links within nucleosomes assembled on a 215-bp DNA fragment containing a Xenopus 5S RNA gene was drastically altered upon proteolytic removal of the tail domains, suggesting that the tails contribute to choice of translational positions on this DNA (40Yang Z. Zheng C. Thiriet C. Hayes J.J. Mol. Cell. Biol. 2005; 25: 241-249Crossref PubMed Scopus (27) Google Scholar). To further investigate this possibility, we repeated the cross-linking approach with APB attached to residue 45 within the histone-fold of H2A as APB attachment at this position has been used to precisely map nucleosome translational positions (46Kassabov S.R. Henry N.M. Zofall M. Tsukiyama T. Bartholomew B. Mol. Cell. Biol. 2002; 22: 7524-7534Crossref PubMed Scopus (92) Google Scholar). Nucleosomes were reconstituted with H2A-A45C-APB, native core histones H2B, H3 and H4, and the 215-bp 5S DNA fragment. Previous characterization of translational positions of nucleosomes reconstituted on this DNA fragment (40Yang Z. Zheng C. Thiriet C. Hayes J.J. Mol. Cell. Biol. 2005; 25: 241-249Crossref PubMed Scopus (27) Google Scholar, 43Aoyagi S. Wade P.A. Hayes J.J. J. Biol. Chem. 2003; 278: 30562-30568Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 47Rhodes D. EMBO J. 1985; 4: 3473-3482Crossref PubMed Google Scholar, 48Hayes J.J. Tullius T.D. Wolffe A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7405-7409Crossref PubMed Scopus (289) Google Scholar, 49Panetta G. Buttinelli M. Flaus A. Richmond T.J. Rhodes D. J. Mol. Biol. 1998; 282: 683-697Crossref PubMed Scopus (54) Google Scholar) showed two main translational positions with dyad axes at approximately positions –3 and +8 and a set of minor positions at the downstream end of the fragment with dyad axes at approximately +45, +55, and +65 nucleotides (Fig. 1A). Importantly, we found that the APB modification did not significantly alter the overall distribution of translational positions as determined by translational gel analysis and restriction enzyme accessibility assays (results not shown, but see below) or the ability of trypsin to specifically remove the tail domains (Fig. 1, B and C). To determine whether the core histone tail domains influence selection of translational positions, 5S nucleosomes containing H2A-A45C-APB were irradiated to induce cross-linking before or after careful proteolytic removal of core histone tails (see “Experimental Procedures”). When these nucleosomes were irradiated before treatment with trypsin, we detected cross-links at positions +93, +83, +70, +45, +16, +6, and –8 (Fig. 2, lanes 5 and 6). Because H2A-A45C forms cross-links about 39 bp to either side of the dyad axis (46Kassabov S.R. Henry N.M. Zofall M. Tsukiyama T. Bartholomew B. Mol. Cell. Biol. 2002; 22: 7524-7534Crossref PubMed Scopus (92) Google Scholar), cross-links at +93 and +16 correspond to a nucleosome position with a dyad axis at nucleotides +55, whereas cross-links at +83/+6 and +70/–8 correspond to nucleosomes with dyads at +45 and +31. These positions correspond to two of the minor downstream positions mentioned above. The cross-link at position +45 corresponds to a previously identified “major” position having a dyad axis at nucleotide +8 (50Pruss D. Bartholomew B. Persinger J. Hayes J. Arents G. Moudrianakis E.N. Wolffe A.P. Science. 1996; 274: 614-617Crossref PubMed Scopus (165) Google Scholar), whereas very faint bands near +36 correspond to the major position with a dyad at –3. The positions and relative strengths of cross-links were not affected when the core histone tails were removed after irradiation, as expected (Fig. 2, lane 6). However, when H2A-A45C-APB 5S nucleosomes were irradiated after the removal of core histone tails, both the positions and intensities of cross-link bands were changed, with cross-links now found at +23, +64, +80, +102, +105, +108, and approximately +115, indicating that the distribution of translational positions was altered upon tail removal (Fig. 2, lane 7). These results were not dependent on the position of the cross-linker within the nucleosome as identical results were obtained with nucleosomes containing either H2A-A12C-APB, as mentioned above (40Yang Z. Zheng C. Thiriet C. Hayes J.J. Mol. Cell. Biol. 2005; 25: 241-249Crossref PubMed Scopus (27) Google Scholar), or H2B-S56C-APB (46Kassabov S.R. Henry N.M. Zofall M. Tsukiyama T. Bartholomew B. Mol. Cell. Biol. 2002; 22: 7524-7534Crossref PubMed Scopus (92) Google Scholar) (results not shown). These data indicate that the core histone tail domains play a role in selection of translational positions and that removal of these domains on pre-formed 5S nucleosomes induces a re-distribution of nucleosomes along the 5S DNA fragment. As mentioned above, previous results have shown that about 70% of the nucleosomes on the 5S DNA fragment occupy either of two related translational positions with dyads at approximately –3 and +8, near the start site of transcription of the 5S gene (43Aoyagi S. Wade P.A. Hayes J.J. J. Biol. Chem. 2003; 278: 30562-30568Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 48Hayes J.J. Tullius T.D. Wolffe A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7405-7409Crossref PubMed Scopus (289) Google Scholar, 50Pruss D. Bartholomew B. Persinger J. Hayes J. Arents G. Moudrianakis E.N. Wolffe A.P. Science. 1996; 274: 614-617Crossref PubMed Scopus (165) Google Scholar, 51Thiriet C. Hayes J.J. J. Biol. Chem. 1998; 273: 21352-21358Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). However, cross-links corresponding to these major positions were much less prominent compared with signals corresponding to the minor downstream translational positions in the above experiments. To investigate potential reasons for the non-correspondence between cross-link signal intensity and nucleosome population, and to more accurately assess the effect of core histone tail removal on the main translational positions by the cross-linking methods, we prepared samples in which the population of nucleosomes was highly enriched for the major translational positions. Cleavage of the reconstituted nucleosomes with the restriction enzyme BamHI at position –61 (Fig. 1A) effectively removes the radiolabel from all nucleosomes with translational positions downstream from the major positions (43Aoyagi S. Wade P.A. Hayes J.J. J. Biol. Chem. 2003; 278: 30562-30568Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Analysis of the 5S nucleosome sample before BamHI digestion on a polyacrylamide nucleoprotein gel, which resolves translational positions, shows a set of 5–6 bands (Fig. 3A, lane 1). The upper bands on the gel correspond to the more centrally located downstream minor translational positions, whereas the lower bands correspond to the major positions near the 5′ end of the fragment (43Aoyagi S. Wade P.A. Hayes J.J. J. Biol. Chem. 2003; 278: 30562-30568Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Upon digestion with BamHI, the upper bands were largely absent from the sample, whereas the lower bands were resistant to BamHI digestion, as expected (43Aoyagi S. Wade P.A. Hayes J.J. J. Biol. Chem. 2003; 278: 30562-30568Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Interestingly, th" @default.
• W1998433561 created "2016-06-24" @default.
• W1998433561 creator A5005262749 @default.
• W1998433561 creator A5018233941 @default.
• W1998433561 creator A5080879554 @default.
• W1998433561 date "2007-03-01" @default.
• W1998433561 modified "2023-10-10" @default.
• W1998433561 title "The Core Histone Tail Domains Contribute to Sequence-dependent Nucleosome Positioning" @default.
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