Chromatin Remodeling by SWI/SNF Results in Nucleosome Mobilization to Preferential Positions in the Rat Osteocalcin Gene Promoter
2007; Elsevier BV; Volume: 282; Issue: 13 Linguagem: Inglês
10.1074/jbc.m609847200
ISSN1083-351X
AutoresJosé L. Gutiérrez, Roberto Paredes, Fernando Cruzat, David A. Hill, André J. van Wijnen, Jane B. Lian, Gary S. Stein, Janet L. Stein, Anthony N. Imbalzano, Martı́n Montecino,
Tópico(s)Cancer Mechanisms and Therapy
ResumoChanges in local chromatin structure accompany transcriptional activation of eukaryotic genes. In vivo these changes in chromatin organization can be catalyzed by ATP-dependent chromatin-remodeling complexes, such as SWI/SNF. These complexes alter the tight wrapping of DNA in the nucleosomes and can facilitate the mobilization of the histone octamer to adjacent DNA segments, leaving promoter regulatory elements exposed for transcription factor binding. To gain understanding of how the activity of SWI/SNF complexes may be modulated by the different DNA sequences within a natural promoter, we have reconstituted nucleosomes containing promoter segments of the transcriptionally active cell type-specific osteocalcin (OC) gene and determined how they affect the directional movements of the nucleosomes. Our results indicate that SWI/SNF complexes induce octamer sliding to preferential positions in the OC promoter, leading to a nucleosomal organization that resembles that described in intact cells expressing the OC gene. Our studies demonstrate that the position of the histone octamer is primarily determined by sequences within the OC promoter that include or exclude nucleosomes. We propose that these sequences are critical components of the regulatory mechanisms that mediate expression of this tissue-specific gene. Changes in local chromatin structure accompany transcriptional activation of eukaryotic genes. In vivo these changes in chromatin organization can be catalyzed by ATP-dependent chromatin-remodeling complexes, such as SWI/SNF. These complexes alter the tight wrapping of DNA in the nucleosomes and can facilitate the mobilization of the histone octamer to adjacent DNA segments, leaving promoter regulatory elements exposed for transcription factor binding. To gain understanding of how the activity of SWI/SNF complexes may be modulated by the different DNA sequences within a natural promoter, we have reconstituted nucleosomes containing promoter segments of the transcriptionally active cell type-specific osteocalcin (OC) gene and determined how they affect the directional movements of the nucleosomes. Our results indicate that SWI/SNF complexes induce octamer sliding to preferential positions in the OC promoter, leading to a nucleosomal organization that resembles that described in intact cells expressing the OC gene. Our studies demonstrate that the position of the histone octamer is primarily determined by sequences within the OC promoter that include or exclude nucleosomes. We propose that these sequences are critical components of the regulatory mechanisms that mediate expression of this tissue-specific gene. Within the eukaryotic nucleus the DNA is organized as a highly structured nucleoprotein complex named chromatin. The nucleosome, the fundamental unit of chromatin, is formed by the association of a DNA segment of 147 bp around a histone octamer composed of two of each histone H2A, H2B, H3, and H4 (1Richmond T. Dadey L. Nature. 2003; 423: 145-150Crossref PubMed Scopus (921) Google Scholar, 2Ramakrishman V. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 83-112Crossref PubMed Scopus (138) Google Scholar). The packaging of DNA sequences in nucleosomes and higher order chromatin structures has been implicated in the regulation of key events in eukaryotic cells such as replication and transcription (3Narlikar G. Fan H. Kingston R. Cell. 2002; 108: 475-487Abstract Full Text Full Text PDF PubMed Scopus (1228) Google Scholar, 4Neely K. Workman J. Biochim. Biophys. Acta. 2002; 1603: 19-29PubMed Google Scholar). The presence of nucleosomes is generally considered to block accessibility of most transcription factors to their cognate binding sequences. Moreover, gene activity is often accompanied by perturbations in the nucleosomal array, as evidenced by increased nuclease hypersensitivity of specific promoter and enhancer elements (3Narlikar G. Fan H. Kingston R. Cell. 2002; 108: 475-487Abstract Full Text Full Text PDF PubMed Scopus (1228) Google Scholar, 4Neely K. Workman J. Biochim. Biophys. Acta. 2002; 1603: 19-29PubMed Google Scholar). Multiple studies have established that there are intrinsic differences in the nucleosome binding capacity of different transcription factors. Although many factors cannot bind when their sites are assembled into nucleosomes, others can specifically recognize and interact with nucleosome-engaged binding sequences (3Narlikar G. Fan H. Kingston R. Cell. 2002; 108: 475-487Abstract Full Text Full Text PDF PubMed Scopus (1228) Google Scholar, 4Neely K. Workman J. Biochim. Biophys. Acta. 2002; 1603: 19-29PubMed Google Scholar), although with different degrees of affinity. Studies based on nucleosome reconstitution assays in vitro have established the existence of DNA sequences that possess higher capacity to be organized as nucleosomal particles. The first segment reported to have this property was a region from the 5 S rRNA gene of sea urchin (5Simpson R. Stafford D. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 51-55Crossref PubMed Scopus (276) Google Scholar), which has become the standard for “nucleosome positioning” sequences. To date, no specific pattern can be associated with the ability to position nucleosomes. However, it appears that these sequences may exhibit certain parameters, such as the presence of AA duplets or AAA triplets, or CTG triplets every 10 or 11 bp (6Thastrom A. Lowary P. Widlund H. Cao H. Kubista M. Widom J. J. Mol. Biol. 1999; 288: 213-229Crossref PubMed Scopus (297) Google Scholar, 7Kiyama R. Trifonov E. FEBS Lett. 2002; 523: 7-11Crossref PubMed Scopus (66) Google Scholar). Interestingly, there have also been reports suggesting the existence of “nucleosome-excluding” sequences, which include short repeats of adenine (A16) (8Anderson J. Widom J. Mol. Cell Biol. 2001; 21: 3830-3839Crossref PubMed Scopus (122) Google Scholar), long CCG triplet repeats (9Wang Y. Gellibolian R. Shimizu M. Wells R. Griffith J. J. Mol. Biol. 1996; 263: 511-516Crossref PubMed Scopus (106) Google Scholar), TGGA repeats (10Cao H. Windlund H. Simonsson T. Kubista M. J. Mol. Biol. 1998; 281: 253-260Crossref PubMed Scopus (69) Google Scholar), or the [(G/C)3NN]n motif (11Wang Y. Griffith J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8863-8867Crossref PubMed Scopus (45) Google Scholar). These nucleosome-excluding sequences may lead to nucleosome-free promoter domains that could allow preferential access for transcription factors to their cognate elements. During the last decade, a large family of protein complexes that function to promote transcription by altering chromatin structure have been described (3Narlikar G. Fan H. Kingston R. Cell. 2002; 108: 475-487Abstract Full Text Full Text PDF PubMed Scopus (1228) Google Scholar, 4Neely K. Workman J. Biochim. Biophys. Acta. 2002; 1603: 19-29PubMed Google Scholar, 12Becker P. Horz W. Annu. Rev. Biochem. 2002; 71: 247-273Crossref PubMed Scopus (619) Google Scholar, 13Peterson C. EMBO J. 2002; 3: 319-322Crossref Scopus (68) Google Scholar). Among them is the SWI/SNF complex subfamily, which remodels chromatin in an ATP-dependent manner (3Narlikar G. Fan H. Kingston R. Cell. 2002; 108: 475-487Abstract Full Text Full Text PDF PubMed Scopus (1228) Google Scholar, 4Neely K. Workman J. Biochim. Biophys. Acta. 2002; 1603: 19-29PubMed Google Scholar, 12Becker P. Horz W. Annu. Rev. Biochem. 2002; 71: 247-273Crossref PubMed Scopus (619) Google Scholar, 13Peterson C. EMBO J. 2002; 3: 319-322Crossref Scopus (68) Google Scholar). One feature of ATP-dependent chromatin remodelers is their ability to mobilize nucleosomes. Repositioning of histone octamers may occur along the same DNA segment by sliding (14Hamiche A. Sandaltzopoulos R. Gdula G. Wu C. Cell. 1999; 97: 833-842Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 15Langst G. Bonte E. Corona D. Becker P. Cell. 1999; 97: 843-852Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 16Whitehouse I. Flaus A. Cairns B. White M. Workman J. Owen-Hughes T. Nature. 1999; 400: 784-787Crossref PubMed Scopus (283) Google Scholar) or to a nucleosome-free DNA segment by octamer transfer (17Phelan M. Schwitzler G. Kingston R. Mol. Cell Biol. 2000; 20: 6380-6389Crossref PubMed Scopus (91) Google Scholar). The molecular mechanism by which the SWI/SNF complexes alter chromatin structure has not been definitively established; however, it has been proposed that they can bind to the linker region and push the DNA toward the nucleosome to slide the histone octamer (3Narlikar G. Fan H. Kingston R. Cell. 2002; 108: 475-487Abstract Full Text Full Text PDF PubMed Scopus (1228) Google Scholar, 12Becker P. Horz W. Annu. Rev. Biochem. 2002; 71: 247-273Crossref PubMed Scopus (619) Google Scholar). Osteoblast-specific transcription of the rat osteocalcin (OC) 2The abbreviations used are: OC, osteocalcin; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; nt, nucleotide(s); TPCK, l-1-tosylamido-2-phenylethyl chloromethyl ketone; ssDNA, single-stranded DNA; ROSF, partially purified SWI/SNF complex from ROS 17/2.8 cells. 2The abbreviations used are: OC, osteocalcin; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; nt, nucleotide(s); TPCK, l-1-tosylamido-2-phenylethyl chloromethyl ketone; ssDNA, single-stranded DNA; ROSF, partially purified SWI/SNF complex from ROS 17/2.8 cells. gene is controlled by a series of modularly distributed basal and hormone-responsive promoter regulatory elements located within two DNase I-hypersensitive sites (–600 to –400 and –170 to –70) (18Montecino M. Lian J. Stein G. Stein J. Biochemistry. 1996; 35: 5093-5102Crossref PubMed Scopus (63) Google Scholar). The DNA segment between these two hypersensitive sites is organized as a nucleosome. The translational position of this nucleosome might reflect specific protein-DNA interactions occurring in the proximal promoter region that account for both formation of the proximal hypersensitive site and OC gene transcriptional activity (18Montecino M. Lian J. Stein G. Stein J. Biochemistry. 1996; 35: 5093-5102Crossref PubMed Scopus (63) Google Scholar, 19Montecino M. Frenkel B. Lian J. Stein J. Stein G. J. Cell Biochem. 1996; 63: 221-228Crossref PubMed Google Scholar, 20Gutierrez S. Javed A. Tennant D. van Rees M. Montecino M. Stein G. Stein J. Lian J. J. Biol. Chem. 2002; 277: 1316-1323Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Alternatively, the specific location of this nucleosome might be the result of nucleosome-positioning sequences at this region of the OC promoter (21Gutierrez J. Sierra J. Medina R. Puchi M. Imschenetzky M. van Wijnen A. Lian J. Stein G. Stein J. Montecino M. Biochemistry. 2000; 39: 13565-13574Crossref PubMed Scopus (31) Google Scholar). To gain understanding about the basis for specific nucleosome positioning at the active OC gene promoter, we have characterized nucleosomes reconstituted using segments of this promoter. We find that SWI/SNF activity produces preferential histone octamer sliding to positions that resemble those found in intact osteoblastic cells expressing the OC gene. This preferential octamer location is determined by specific sequences within the proximal OC promoter region that include or exclude nucleosomes. DNA Segments From the Rat OC Gene Promoter—DNA fragments from the rat OC gene promoter containing the sequences –500 to –270, –500 to –87, –500 to –355, –451 to –306, –441 to –206, –380 to –145, –351 to –206, –351 to –116, –287 to –57, –257 to –106, –207 to –56, and –161 to –10 were generated by PCR using specific primers, one of which had been previously end-labeled with polynucleotide kinase (New England Biolabs, Beverly, MA) and [γ-32P]ATP (PerkinElmer Life Sciences), as previously described (21Gutierrez J. Sierra J. Medina R. Puchi M. Imschenetzky M. van Wijnen A. Lian J. Stein G. Stein J. Montecino M. Biochemistry. 2000; 39: 13565-13574Crossref PubMed Scopus (31) Google Scholar). To map nucleosome positions, labeled DNA fragments were obtained by PCR using specific primers and including [α-32P]dATP in the reaction mix as described before (21Gutierrez J. Sierra J. Medina R. Puchi M. Imschenetzky M. van Wijnen A. Lian J. Stein G. Stein J. Montecino M. Biochemistry. 2000; 39: 13565-13574Crossref PubMed Scopus (31) Google Scholar). Nucleosome Reconstitution—Nucleosome reconstitutions were carried out by the histone octamer transfer method as previously described (21Gutierrez J. Sierra J. Medina R. Puchi M. Imschenetzky M. van Wijnen A. Lian J. Stein G. Stein J. Montecino M. Biochemistry. 2000; 39: 13565-13574Crossref PubMed Scopus (31) Google Scholar). Donor oligonucleosomes were obtained as described previously (21Gutierrez J. Sierra J. Medina R. Puchi M. Imschenetzky M. van Wijnen A. Lian J. Stein G. Stein J. Montecino M. Biochemistry. 2000; 39: 13565-13574Crossref PubMed Scopus (31) Google Scholar). To map nucleosome positions, 2 pmol of labeled DNA fragments and 6 μg of oligonucleosomes isolated from chicken red blood cells were used. In all the other reconstitution experiments, 1 pmol of acceptor DNA and 3 μg of oligonucleosomes were used. After reconstitution, the final composition of the nucleosome-containing buffer was 10 mm Tris-Cl (pH 7.4), 1 mm EDTA (pH 8.0), 5 mm DTT, 0.5 mm PMSF, 100 mm NaCl, 100 μg/ml bovine serum albumin, 10% glycerol, and 0.05% Nonidet P-40. Electrophoretic Analysis and Isolation of Nucleosome Populations—Following reconstitution, the nucleosomes were analyzed by electrophoresis in non-denaturing 5% polyacrylamide (40:1 acrylamide:bisacrylamide ratio) gels and 0.5× TBE (45 mm Tris-Cl (pH 8.0), 45 mm Boric Acid, 1 mm EDTA), at 4 °C and 200 V. Gels were dried, and the nucleosome pattern as well as the efficiency of reconstitution were monitored using a Molecular Imager FX phosphorimaging device (Bio-Rad). Alternatively, the dried gels were exposed to Kodak X-OMAT films at –80 °C. The nucleosome populations obtained after the reconstitution were isolated and purified following protocols described by Studitsky et al. (22Studitsky V. Clark D. Felsenfeld G. Methods Enzymol. 1996; 274: 246-256Crossref PubMed Scopus (7) Google Scholar). Briefly, the different samples were concentrated up to 5-fold using centricon-10 tubes (Amicon) and fractionated in non-denaturing polyacrylamide gels. Bands corresponding to naked DNA, lateral, and central nucleosome populations were cut from the wet gels and extracted with elution buffer (10 mm Tris-Cl (pH 7.4), 1 mm EDTA (pH 8.0), 5 mm DTT, 0.5 mm PMSF, 100 mm NaCl, and 100 μg/ml bovine serum albumin) by agitation overnight. Tubes were centrifuged 1 min at maximum speed in a microcentrifuge to decant gel pieces. The supernatant containing the nucleosomes was transferred to clean microtubes, and 0.25 volumes of elution buffer, including 50% glycerol and 0.25% Nonidet P-40, were immediately added. The samples were stored at 4 °C for up to 2 weeks until use. Mapping of Nucleosome Positions—The purified nucleosome populations were subjected to micrococcal nuclease digestion for 5 min at 37 °C. The concentration of micrococcal nuclease used ranged from 0.1 to 1 unit/μl, depending on the particular nucleosome population and was determined by previous titration. The digested DNA products were purified and fractionated by electrophoresis in a 12% polyacrylamide gel (0.5× TBE) at 200 V. The ∼146-bp micrococcal nuclease-resistant band was detected by direct autoradiography, cut from the gel, and purified. The mapping of the nuclease-resistant fragment was then assessed by digestion with restriction enzymes. The enzymes chosen in each analysis varied according to the DNA segment and are explained in the corresponding Fig. legend. The digestion products were visualized by electrophoresis in a sequencing gel and autoradiography. Isolation of Nucleosome Remodeling Activity from Osteoblastic Cells—A partially purified nucleosome remodeling complex analogous to hSWI/SNF and free of ISWI-containing complexes (see results shown in Fig. 5), was obtained according to a method described previously (23Imbalzano A. Kwon H. Green M. Kingston R. Nature. 1994; 370: 481-485Crossref PubMed Scopus (519) Google Scholar, 24Kwon H. Imbalzano A. Khavari P. Kingston R. Green M. Nature. 1994; 370: 477-481Crossref PubMed Scopus (636) Google Scholar, 25Wang W. Cote J. Xue Y. Zhou S. Kahvari P. Biggar S. Muchardt C. Kalpana G. Goff S. Yaniv M. Workman J. Crabtree G. EMBO J. 1996; 15: 5370-5372Crossref PubMed Scopus (668) Google Scholar). Nuclear extracts from ROS 17/2.8 (rat osteosarcoma) cells were obtained as described (26Dignam J. Lebovitz R. Roeder R. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9131) Google Scholar), except that buffer C contained 0.6 m KCl instead of 0.42 m. Nuclear extracts were dialyzed against 20 mm HEPES (pH 7.9), 0.5 mm PMSF, and 0.5 mm β-mercaptoethanol, until KCl concentration reached 125 mm. Glycerol was added to the samples up to a final concentration of 20% (leaving KCl at a final concentration of 100 mm). The nuclear extract samples were then frozen in liquid nitrogen and kept at –80 °C until use. Prior to the first chromatographic step, nuclear extracts were centrifuged for 5 min at 5000 rpm. The supernatant was loaded on a phosphocellulose column and eluted with buffer A (20 mm HEPES (pH 7.9), 0.2 mm EDTA, 2 mm DTT, 0.5 mm PMSF, 7 μg/ml TPCK, 10 μg/ml trypsin inhibitor, 1 μg/ml leupeptin, 1 μg/ml pepstatin) and increasing concentrations of KCl (0.1, 0.3, 0.5, and 0.8 m). The protein concentration in each chromatographic fraction was determined by Bradford's method, and the peak at 0.5 m KCl was collected, dialyzed against buffer B (20 mm HEPES (pH 7.9), 0.1 m KCl, 2.0 mm MgCl2, 2 mm DTT, 0.1 mm EDTA, 0.5 mm PMSF, and 10% glycerol), and loaded onto a ssDNA column (Sigma). The sample was eluted using buffer B (also including a proteinase inhibitors mixture) with increasing concentrations of KCl (0.1, 0.3, and 0.5 m). The fractions were frozen in liquid nitrogen and stored at –80 °C. Enrichment in SWI/SNF complexes was monitored by analyzing aliquots from the different fractions by Western blot using antibodies against BRG-1 and by evaluating the presence of nucleosome remodeling activity. Nucleosome Remodeling Reactions—The nucleosome remodeling assays were performed in 15 μl of remodeling buffer (60 mm KCl, 20 mm HEPES (pH 7.9), 0.2 mm EDTA, 0.5 mm PMSF, 2.5 mm DTT, 0.05% Nonidet P-40, 10% glycerol, 7 mm MgCl2, 4 mm ATP, and 100 μg/ml bovine serum albumin), including 0.73 nm of purified human SWI/SNF complex or 12.3 ng/μl(<6.2 nm SWI/SNF complex) of partially purified SWI/SNF complex from ROS 17/2.8 cells (ROSF), and 15.4 nm total nucleosome mixture (0.7 nm nucleosome probe, approximately). Samples were incubated for 30 min at 30 °C. Then, 750 ng of salmon sperm DNA (Sigma) and 500 ng of cold oligonucleosomes were added to the reaction mix and incubated for 30 min at 30 °C. The remodeled nucleosome samples were subsequently analyzed by electrophoresis in non-denaturant 5% polyacrylamide gels (0.5× TBE) or by direct restriction enzyme digestion (see below). Restriction Endonuclease Enzyme Accessibility Analysis—Reconstituted nucleosomes or mock reconstituted DNA segments were digested with restriction enzymes for 20 min at 30 °C. The amount of each restriction enzyme used in the assays was determined previously as that sufficient to obtain a partial digestion of the naked DNA (close to 90%). Digestions were stopped by the addition of EDTA (20 mm final concentration). The nucleosome samples that had been previously subjected to remodeling reactions were incubated with 1 unit of apyrase (1 unit/μl) for 20 min at 30 °C, prior to the addition of restriction enzyme. Incubation with the restriction enzyme was carried out for 20 min at 30 °C, followed by the addition of 750 ng of salmon sperm DNA (Sigma), 500 ng of non-labeled oligonucleosomes and EDTA (20 mm final concentration). This reaction mix was incubated for 30 min at 30 °C. All samples were analyzed by electrophoresis in a 5% polyacrylamide gel as described above. The percentage of digestion was determined using a Molecular Imager FX (Bio-Rad). Chromatin Immunoprecipitation Analysis—Chromatin immunoprecipitation studies were performed as described earlier (27Villagra A. Cruzat F. Carvallo L. Olate J. van Wijnen A. Stein G. Lian J. Stein J. Imbalzano A.N. Montecino M. J. Biol. Chem. 2006; 281: 22695-22706Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) with modifications. All the steps were performed at 4 °C. ROS 17/2.8 cell cultures (100-mm-diameter plates) were washed with 10 ml of phosphate-buffered saline, scraped off in the same volume of phosphate-buffered saline, and collected by centrifugation at 1,000 × g for 5 min. The cell pellet was resuspended in 3 ml of lysis buffer (50 mm Hepes (pH 7.8), 20 mm KCl, 3 mm MgCl2, 0.1% Nonidet P-40, and a mixture of proteinase inhibitors) and homogenized in a Dounce homogenizer using the loose pestle. The nuclear fraction was collected by centrifugation at 1,000 × g for 5 min and then incubated with the restriction endonuclease StuI (500 units/ml for 30 min at 37 °C) according to previous reports (18Montecino M. Lian J. Stein G. Stein J. Biochemistry. 1996; 35: 5093-5102Crossref PubMed Scopus (63) Google Scholar). This enzyme cleaves between the proximal OC promoter region and the nucleosome positioned immediately upstream, therefore dividing the promoter and preventing that chromatin fragments containing sequences recognized by more than one specific set of primers are amplified. The reaction was stopped by the addition of 25 mm EDTA (final concentration), and the digested nuclear fraction was collected by centrifugation at 1,000 × g for 5 min and resuspended in 3.0 ml of sonication buffer (50 mm Hepes (pH 7.9), 140 mm NaCl, 1 mm EDTA, 1% Triton X-100, 0.1% deoxycholate acid, 0.1% SDS, and a mixture of proteinase inhibitors). To reduce the length of the chromatin fragments to ∼300 bp or smaller (confirmed by electrophoretic analysis and PCR amplification (not shown), the extract was sonicated with a Misonix sonicator (model 3000), using ten 15-s pulses at 30% power. After centrifugation at 16,000 × g, the supernatant was collected, frozen in liquid nitrogen, and kept at –80 °C. An aliquot was used for A260 measurements. Extracts (6 units of A260) were resuspended in sonication buffer to a final volume of 500 μl. The samples were ple-cleared by incubation with 30 μl of protein A/G-agarose beads pre-blocked with bovine serum albumin (Santa Cruz Biotechnology, Santa Cruz, CA) for 15 min at 4 °C with agitation. After centrifugation at 1,000 × g for 5 min, the supernatant was collected and immunoprecipitated with either an anti-histone H3 polyclonal antibody (Santa Cruz Biotechnology, FL-136), anti-histone H4 polyclonal antibody (Santa Cruz Biotechnology, H-97), anti-acetylated histone H3 polyclonal antibody (Upstate Biotechnology, 06-599), or anti-acetylated histone H4 polyclonal antibody (Upstate Biotechnology, 06-866). As a control for nonspecific precipitation, normal rabbit-purified IgG fraction (Santa Cruz Biotechnology, sc-2027) was used. The immunocomplexes were recovered with the addition of 30 μl of protein A-agarose beads and subsequent incubation for 1 h at 4°C with agitation. The complexes were washed twice with sonication buffer, twice with sonication buffer plus 500 mm NaCl, twice with LiCl buffer (100 mm Tris-HCl (pH 8.0), 500 mm LiCl, 0.1% Nonidet P-40, and 0.1% deoxycholic acid), and twice with dialysis buffer (2 mm EDTA and 50 mm Tris-HCl, pH 8.0), with the solution incubated at each washing for 5 min at 4 °C. The histone-DNA complexes were then eluted by incubation with 100 μl of elution buffer (50 mm NaHCO3 and 1% SDS) for 15 min at 65 °C. After centrifugation at 1,000 × g for 5 min, the supernatant was collected and incubated with 10 μg of RNase A per ml for 1 h at 42 °C. The proteins were then digested with 200 μg/ml proteinase K for 2 h at 50°C. The DNA was recovered by phenolchloroform extraction and ethanol precipitation using tRNA (5 μg/ml) as a precipitation carrier. The PCR conditions used to evaluate the OC proximal and distal promoter regions were reported previously (27Villagra A. Cruzat F. Carvallo L. Olate J. van Wijnen A. Stein G. Lian J. Stein J. Imbalzano A.N. Montecino M. J. Biol. Chem. 2006; 281: 22695-22706Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). To amplify the precipitated OC promoter sequences the following primers were used: forward (–315) 5′-CGATTTTAGATCTCTGTACCCTCTCTAGG-3′; reverse (–206) 5′-GACATGGCCCCAGACCTCTTCC-3′ or reverse (–28) 5′-TATATCCACTGCCTGAGCCC-3′; forward (–148) 5′-AGCTGCAGTCACCAACCAC-3′; and reverse (–57) 5′-GCAGGAGGGGCAGGAGATGCT-3′. The Proximal Region of the Rat Osteocalcin Gene Promoter Contains a Nucleosome-positioning Sequence—Multiple studies have established that following nucleosomal reconstitution in vitro, DNA segments larger than 150 bp (e.g. 230 bp) tend to position the histone octamer at their ends (5′-or3′-end) either immediately after reconstitution or after thermal redistribution, because these are the thermodynamically most stable conformations (28Meersseman G. Pennings S. Bradbury M. EMBO J. 1992; 11: 2951-2959Crossref PubMed Scopus (258) Google Scholar, 29Flaus A. Richmond T. J. Mol. Biol. 1998; 275: 427-441Crossref PubMed Scopus (113) Google Scholar, 30Flaus A. Owen-Hughes T. Mol. Cell Biol. 2003; 23: 7767-7779Crossref PubMed Scopus (83) Google Scholar). This result is valid for any given DNA fragment unless it possesses a nucleotide sequence that favors a preferential position for the histone octamer different from the ends. We have previously reported that nucleosome reconstitution of segments from the proximal rat OC gene promoter results in a non-random positioning of the histone octamer (21Gutierrez J. Sierra J. Medina R. Puchi M. Imschenetzky M. van Wijnen A. Lian J. Stein G. Stein J. Montecino M. Biochemistry. 2000; 39: 13565-13574Crossref PubMed Scopus (31) Google Scholar). This finding suggests that there may be sequences within this region of the promoter that preferentially organize nucleosomes. To address this hypothesis we initially tested the –287/–57 OC proximal promoter segment, which encompasses all the basic tissue-specific regulatory elements (19Montecino M. Frenkel B. Lian J. Stein J. Stein G. J. Cell Biochem. 1996; 63: 221-228Crossref PubMed Google Scholar, 20Gutierrez S. Javed A. Tennant D. van Rees M. Montecino M. Stein G. Stein J. Lian J. J. Biol. Chem. 2002; 277: 1316-1323Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Fig. 1 shows that nucleosome reconstitution of this fragment generates two main bands as determined by native PAGE (Fig. 1A, lane 2). These bands reflect at least two principal histone octamer positions at either end of the –287/–57 segment (the faster migration band) and at the center of this segment (retarded migration band) (28Meersseman G. Pennings S. Bradbury M. EMBO J. 1992; 11: 2951-2959Crossref PubMed Scopus (258) Google Scholar, 31Duband-Goulet I. Carot V. Ulyanov A. Douc-Rasy S. Prunell A. J. Mol. Biol. 1992; 224: 981-1001Crossref PubMed Scopus (34) Google Scholar). Both nucleosome forms were isolated and purified as described under “Experimental Procedures” (Fig. 1A, lanes 4 and 5); thus indicating that they represent bona fide nucleosome particles that are stable at 4 °C for several days (data not shown). The precise position of these nucleosomes along the –287/–57 DNA segment was determined (21Gutierrez J. Sierra J. Medina R. Puchi M. Imschenetzky M. van Wijnen A. Lian J. Stein G. Stein J. Montecino M. Biochemistry. 2000; 39: 13565-13574Crossref PubMed Scopus (31) Google Scholar). This approach involves digesting the isolated nucleosomes with micrococcal nuclease and mapping the position of the ∼146-bp micrococcal nuclease-resistant fragment by digesting with three different restriction enzymes. Mapping of the faster migrating nucleosome band confirmed that the histone octamer is positioned at both ends of the –287/–57 segment (Fig. 1, B and C). However, the significant differences in intensity of the bands generated by cleavage with the restriction enzymes indicate that the principal nucleosomal location (which accounts for ∼70% of the nucleosomal population) is at the 5′-end of the segment (Fig. 1B). For example, the bands of 126 nt and 20 nt corresponding to cleavage products generated after digestion of the micrococcal nuclease-resistant –287/–142 fragment with StuI are reproducibly and significantly more intense than the bands of 105 nt and 41 nt generated by cleavage of the micrococcal nuclease-resistant –202/–57 fragment with this same restriction endonuclease (Fig. 1B, lane 4). Mapping of the lower migration nucleosome band (Fig. 1B, lanes 7–10) confirmed that some of the histone octamers are located at the center of the –287/–57 segment, mainly positioned between –250 and –105 (Fig. 1D). Based on the restriction enzyme cleavage pattern (Fig. 1B, lanes 7–10) we determined that after isolation this central nucleosome population also exhibits a fraction of nucleosomes positioned at the ends of the –287/–57 segment (see also Fig. 1A, lane 5). This indicates that soon after purification (which proceeds at 4 °C), a fraction of the histone octamers are spontaneously mobilized toward the ends (28Meersseman G. Pennings S. Bradbury M. EMBO J. 1992; 11: 2951-2959Crossref PubMed Scopus (258) Google Scholar, 29Flaus A. Richmond T. J. Mol. Biol. 1998; 275: 427-441Crossref PubMed Scopus (113) Google Scholar, 30Flaus A. Owen-Hughes T. Mol. Cell Biol. 2003; 23: 7767-7779Crossref PubMed Scopus (83) Google Scholar). Our previous studies had established that in osteoblastic cells expressing OC, the promoter region of this gene exhibits a nucleosome positioned within the sequence –350 to –170 (18Mon
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