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• W2146780090 abstract "Locus control regions (LCRs) are capable of activating target genes over substantial distances and establishing autonomously regulated chromatin domains. The basis for this action is poorly defined. Human growth hormone gene (hGH-N) expression is activated by an LCR marked by a series of DNase I-hypersensitive sites (HSI–III and HSV) in pituitary chromatin. These HSs are located between −15 and −32 kilobases (kb) relative to thehGH transcription start site. To establish a mechanistic basis for hGH LCR function, we carried out acetylation mapping of core histones H3 and H4 in chromatin encompassing thehGH cluster. These studies revealed that the entire LCR was selectively enriched for acetylation in chromatin isolated from a human pituitary somatotrope adenoma and in pituitaries of mice transgenic for the hGH locus, but not in hepatic or erythroid cells. Quantification of histone modification in the pituitary revealed a dramatic peak at HSI/II, the major pituitary-specific hGHLCR determinant (−15 kb), with gradually decreasing levels of modification extending from this site in both 5′- and 3′-directions. The 5′-border of the acetylated domain coincided with the 5′ mosthGH LCR element, HSV (−34 kb); and the 3′-border included the expressed hGH-N gene, but did not extend farther 3′ into the placenta-specific region of the gene cluster. These data support a model of LCR function involving targeted recruitment and subsequent spreading of histone acetyltransferase activity to encompass and activate a remote target gene. Locus control regions (LCRs) are capable of activating target genes over substantial distances and establishing autonomously regulated chromatin domains. The basis for this action is poorly defined. Human growth hormone gene (hGH-N) expression is activated by an LCR marked by a series of DNase I-hypersensitive sites (HSI–III and HSV) in pituitary chromatin. These HSs are located between −15 and −32 kilobases (kb) relative to thehGH transcription start site. To establish a mechanistic basis for hGH LCR function, we carried out acetylation mapping of core histones H3 and H4 in chromatin encompassing thehGH cluster. These studies revealed that the entire LCR was selectively enriched for acetylation in chromatin isolated from a human pituitary somatotrope adenoma and in pituitaries of mice transgenic for the hGH locus, but not in hepatic or erythroid cells. Quantification of histone modification in the pituitary revealed a dramatic peak at HSI/II, the major pituitary-specific hGHLCR determinant (−15 kb), with gradually decreasing levels of modification extending from this site in both 5′- and 3′-directions. The 5′-border of the acetylated domain coincided with the 5′ mosthGH LCR element, HSV (−34 kb); and the 3′-border included the expressed hGH-N gene, but did not extend farther 3′ into the placenta-specific region of the gene cluster. These data support a model of LCR function involving targeted recruitment and subsequent spreading of histone acetyltransferase activity to encompass and activate a remote target gene. locus control regions DNase I-hypersensitive site cAMP-responsive element-binding protein-binding protein human growth hormone growth hormone kilobase(s) base pair(s) growth hormone-releasing factor chromatin immunoprecipitation adrenocorticotropic hormone The majority of DNA in the eukaryotic nucleus is packaged into a compact chromatin conformation. For a gene to be expressed, this chromatin structure must be disrupted to accommodate transcription factor binding and RNA polymerase assembly and passage (1.Felsenfeld G. Cell. 1996; 86: 13-19Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 2.Workman J.L. Kingston R.E. Annu. Rev. Biochem. 1998; 67: 545-579Crossref PubMed Scopus (956) Google Scholar). A specific set of distal regulatory elements termed locus control regions (LCRs)1 are postulated to function in promoting changes in chromatin structure conducive to gene activation. Critical determinants that constitute an LCR co-map to one or more DNase I-hypersensitive sites (HSs) flanking LCR-dependent genes in the chromatin of expressing cells (3.Boyes J. Felsenfeld G. EMBO J. 1996; 15: 2496-2507Crossref PubMed Scopus (145) Google Scholar). The functions of such LCR determinants are operationally defined by their ability to establish autonomously functioning transgene chromatin domains that are independent of the site of integration within the host genome (4.Grosveld F. Blom van Assendelft G. Greaves D.R. Kollias G. Cell. 1987; 51: 975-985Abstract Full Text PDF PubMed Scopus (1429) Google Scholar, 5.Kioussis D. Festenstein R. Curr. Opin. Genet. Dev. 1997; 7: 614-619Crossref PubMed Scopus (125) Google Scholar). The biochemical mechanism(s) by which LCRs activate their target genes has not been determined. Histone-modifying enzymes are directly involved in modulating chromatin structures relevant to gene transcription (6.Wolffe A.P. Cell. 1994; 77: 13-16Abstract Full Text PDF PubMed Scopus (225) Google Scholar, 7.Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Edmondson D.G. Roth S.Y. Allis C.D. Cell. 1996; 84: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1273) Google Scholar, 8.Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2351) Google Scholar, 9.Hampsey M. Trends Genet. 1997; 13: 427-429Abstract Full Text PDF PubMed Scopus (33) Google Scholar, 10.Wade P.A. Pruss D. Wolffe A.P. Trends Biochem. Sci. 1997; 22: 128-132Abstract Full Text PDF PubMed Scopus (400) Google Scholar, 11.Struhl K. Genes Dev. 1998; 12: 599-606Crossref PubMed Scopus (1530) Google Scholar). The acetylation of nucleosomal histones at the promoters of certain genes promotes chromatin disruption (12.Wolffe A.P. Pruss D. Cell. 1996; 84: 817-819Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 13.Gregory P.D. Schmid A. Zavari M. Lui L. Berger S.L. Horz W. Mol. Cell. 1998; 1: 495-505Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 14.Nightingale K.P. Wellinger R.E. Sogo J.M. Becker P.B. EMBO J. 1998; 17: 2865-2876Crossref PubMed Scopus (121) Google Scholar, 15.Steger D.J. Eberharter A. John S. Grant P.A. Workman J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12924-12929Crossref PubMed Scopus (111) Google Scholar) and facilitates transcriptional activation (16.Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (456) Google Scholar, 17.Kuo M.-H. Zhou J. Jambeck P. Churchill M.E.A. Allis C.D. Genes Dev. 1998; 12: 627-639Crossref PubMed Scopus (394) Google Scholar, 18.Wang L. Liu L. Berger S.L. Genes Dev. 1998; 12: 640-653Crossref PubMed Scopus (218) Google Scholar). Histone deacetylase activity has the opposite effect, resulting in gene silencing (19.Nagy L. Kao H.-Y. Chakravarti D. Lin R.J. Hassig C.A. Ayer D.E. Schreiber S.L. Evans R.M. Cell. 1997; 89: 373-380Abstract Full Text Full Text PDF PubMed Scopus (1098) Google Scholar, 20.Kadosh D. Struhl K. Mol. Cell. Biol. 1998; 18: 5121-5127Crossref PubMed Scopus (265) Google Scholar). The linkage between histone acetylation and transcriptional activation was strongly supported by the discovery that a number of transcriptional coactivators (GCN5, CBP/p300, SRC1, TAF11250, and PCAF) possess histone acetyltransferase activity (7.Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Edmondson D.G. Roth S.Y. Allis C.D. Cell. 1996; 84: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1273) Google Scholar, 21.Mizzen C.A. Yang X.-J. Kokubo T. Brownell J.E. Bannister A.J. Owen-Hughes T. Workman J. Wang L. Berger S.L. Kouzarides T. Nakatani Y. Allis C.D. Cell. 1996; 87: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar, 22.Shikama N. Lyon J. La Thangue N.B. Trends Cell Biol. 1997; 7: 230-236Abstract Full Text PDF PubMed Scopus (424) Google Scholar, 23.Spencer T.E. Jenster G. Burcin M.M. Allis C.D. Zhou J. Mizzen C.A. McKenna N.J. Onate S.A. Tsai S.Y. Tsai M.-J. O'Malley B.W. Nature. 1997; 389: 194-198Crossref PubMed Scopus (1049) Google Scholar, 24.Ogryzko V.V. Kotani T. Zhang X. Schlita R.L. Howard T. Yang X.-J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 25.Korzus E. Torchia J. Rose D.W. Xu L. Kurokawa R. McInerney E.M. Mullen T.M. Glass C.K. Rosenfeld M.G. Science. 1998; 279: 703-706Crossref PubMed Scopus (556) Google Scholar), whereas a number of transcriptional repressors (Rpd3, HDAC1, and HDAC2) associate with histone deacetylases (26.Alland L. Muhle R. Hou H.J. Potes J. Chin L. Schriber-Agus N. DePinho R.A. Nature. 1997; 387: 49-55Crossref PubMed Scopus (730) Google Scholar, 27.Brehm A. Miska E.A. McCance D.J. Reid J.L. Bannister A.J. Kouzarides T. Nature. 1998; 391: 597-601Crossref PubMed Scopus (1062) Google Scholar, 28.Kadosh D. Struhl K. Genes Dev. 1998; 12: 797-805Crossref PubMed Scopus (203) Google Scholar, 29.Kao H.-Y. Ordentlich P. Koyano-Nakagawa N. Tang Z. Downes M. Kintner C.R. Evans R.M. Kadesch T. Genes Dev. 1998; 12: 2269-2277Crossref PubMed Scopus (486) Google Scholar, 30.Magnaghi-Jaulin L. Groisman R. Naguibneva I. Robin P. Lorain S. LeVillain J.P. Troalen F. Trouche D. Harel-Bellan A. Nature. 1998; 391: 601-605Crossref PubMed Scopus (797) Google Scholar, 31.Rundlett S.E. Carmen A.A. Suka N. Turner B.M. Grunstein M. Nature. 1998; 392: 831-835Crossref PubMed Scopus (360) Google Scholar). Collectively, these data provide strong evidence that acetylases and deacetylases activate or repress gene expression by being recruited to specific promoters and/or proximal enhancer elements. The role of such histone acetylation and deacetylation in LCR function remains to be explored. The human growth hormone gene (hGH) cluster comprises five closely linked genes: 5′-hGH-N/hCS-L/hCS-A/hGH-V/hCS-B-3′. Expression of hGH-N is limited to the somatotrope and somatolactotrope cells of the anterior pituitary, whereas expression of the remaining four genes is restricted to the syncytiotrophoblast layer of the placental villi (32.Liebhaber S.A. Urbanek M. Ray J. Tuan R.S. Cooke N.E. J. Clin. Invest. 1989; 83: 1985-1991Crossref PubMed Scopus (80) Google Scholar, 33.McWilliams D. Boime I. Endocrinology. 1980; 107: 761-765Crossref PubMed Scopus (33) Google Scholar). A set of tissue-specific DNase I-hypersensitive sites located between −15 and −32 kb upstream of thehGH gene have been identified and shown to be required for appropriate tissue-specific expression of the hGH gene cluster in transgenic mice. Pituitary chromatin contains a subset of four HSs (HSI–III and HSV), and chromatin isolated from placental syncytiotrophoblasts contains a partially overlapping set of three HSs (HSIII–V). The full set of HSs renders expression of hGH-Ntransgenes reproducibly copy number-dependent and site of integration-independent in the mouse pituitary (34.Jones B.K. Monks B.R. Liebhaber S.A. Cooke N.E. Mol. Cell. Biol. 1995; 15: 7010-7021Crossref PubMed Scopus (150) Google Scholar). HSI and HSII, which are closely linked and thus considered as a single determinant, are located 15 kb 5′ to the hGH gene and are unique to the pituitary (34.Jones B.K. Monks B.R. Liebhaber S.A. Cooke N.E. Mol. Cell. Biol. 1995; 15: 7010-7021Crossref PubMed Scopus (150) Google Scholar). They are fully sufficient to confer high level, developmentally appropriate, somatotrope-specific and position-independent expression on a linked hGH-N transgene (35.Bennani-Baiti I.M. Asa S.L. Song D. Iratni R. Liebhaber S.A. Cooke N.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10655-10660Crossref PubMed Scopus (53) Google Scholar). As such, HSI/II constitutes the major element of thehGH LCR in the pituitary. Critical cis-acting determinants that bind the pituitary-specific POU homeodomaintrans-factor Pit-1 have recently been identified within HSI/II (36.Shewchuk B.M. Asa S.L. Cooke N.E. Liebhaber S.A. J. Biol. Chem. 1999; 274: 35725-35733Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). These sites are necessary for HSI/II function in vivo, but the mechanistic basis for their action is not defined. A central question raised by studies of LCR function concerns the mechanism by which LCRs selectively alter chromatin structures and establish transcriptionally productive chromatin environments in specific tissues or cell types. Given the effects of histone modification on modulating chromatin structure, we have investigated the potential association of histone acetylation with LCR function. The data support a role for LCR-mediated histone acetylation in the establishment of a pituitary-specific, transcriptionally active chromatin environment required for hGH-N gene activation. Mouse GHFT1 presomatotrope cells (37.Lew D. Brady H. Klausing K. Yaginuma K. Theill L.E. Stauber C. Karin M. Mellon P.L. Genes Dev. 1993; 7: 683-693Crossref PubMed Scopus (115) Google Scholar) and human K562 erythroid cells were maintained in Dulbecco's modified Eagle's medium and RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. The GHFT1/HSIII cell line was generated by transfection of GHFT1 cells with the HSIII/hGHneo r construct. 2I. Bennani-Baiti, A. Abu-Daya, N. E. Cooke, and S. A. Liebhaber, unpublished data. A portion of a surgically removed GH-secreting human pituitary adenoma was utilized. Mouse pituitaries and livers were isolated from the indicated transgenic lines. Pituitary and liver samples were dissociated in cell dissociation buffer (Life Technologies, Inc.). Nuclei were isolated from the dissociated pituitary and liver cells and cultured GHFT1/HSIII and K562 cells by hypotonic lysis in the presence of mild detergent as described previously (38.Ginder G. Methods Hematol. 1989; 20: 111-123Google Scholar). Mice carrying the hGH/P1transgene were generated by microinjection of a linearized P1 plasmid carrying the hGH/P1 clone (encompassing the entirehGH LCR and the first four genes of the cluster) into fertilized mouse oocytes to establish founder lines (39.Su Y. Liebhaber S.A. Cooke N.E. J. Biol. Chem. 2000; 275: 7902-7909Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Frozen embryos carrying the human growth hormone-releasing factor (GRF) transgene were a kind gift from R. Brinster (University of Pennsylvania). To generate hGH/P1× GRFcompound transgenic mice, an hGH/P1 transgenic line was crossed to the GRF line. Doubly positive transgenic mice were identified by dot blot analysis of tail DNA using the probes described below. Nuclei were resuspended in 1.0 ml of digestion buffer (50 mm NaCl, 20 mmTris-HCl (pH 7.5), 3.0 mm MgCl2, 1.0 mm CaCl2, 10 mm sodium butyrate, and 0.1 mm phenylmethylsulfonyl fluoride) at a concentration of 0.3 mg/ml. They were digested with 25 units of micrococcal nuclease (Amersham Pharmacia Biotech) for 6 min at 37 °C. The digestion was terminated by the addition of EDTA to 0.5 mm, and the sample was centrifuged at 12,000 ×g in a microcentrifuge for 10 min at 4 °C to generate supernatant S1. The pellet was resuspended in 300 μl of low salt lysis buffer (10 mm Tris-HCl (pH 7.5), 10 mmsodium butyrate, 0.25 mm EDTA, and 0.1 mmphenylmethylsulfonyl fluoride), incubated on ice for 2 min, and then centrifuged as before. The resulting supernatant, S2, was combined with S1. The soluble chromatin was concentrated using Microcon centrifugal filters (Amicon, Inc., Bedford, MA). The chromatin immunoprecipitation (ChIP) procedure was carried out according to previously reported methods (40.O'Neill L. Turner B.M. Methods Enzymol. 1996; 274: 189-203Crossref PubMed Scopus (55) Google Scholar, 41.Crane-Robinson C. Hebbes T.R. Clayton A.L. Thorne A.W. Methods. 1997; 12: 48-56Crossref PubMed Scopus (28) Google Scholar) with minor modifications. Antisera specific to the acetylated lysine residues of histone H3 or H4 were diluted 1:100 and used in a 1:1 mixture. Alternatively, 15 μl of antiserum to unacetylated histone H3 was used. Each antiserum was kindly provided by C. D. Allis (University of Virginia). Immunoprecipitations contained 250-μg aliquots of chromatin DNA in a total volume of 500 μl. Protein A-Sepharose (Amersham Pharmacia Biotech) precipitates were generated and washed, and DNA and proteins were harvested from pellets and supernatants according to published protocols (40.O'Neill L. Turner B.M. Methods Enzymol. 1996; 274: 189-203Crossref PubMed Scopus (55) Google Scholar). Input, unbound, and bound DNA samples were each analyzed by electrophoresis on 1% agarose gels stained with ethidium bromide prior to use to ensure the quality of oligonucleosome preparations. Southern hybridization of micrococcal nuclease-digested input DNA using sheared 32P-labeled total genomic DNA as a probe demonstrated that the majority of DNA ranged in size from ∼0.16 to 1 kb. Proteins from bound and unbound fractions were obtained from the first phenol/chloroform phase to which 8 μg of bovine serum albumin was added as carrier. HCl was then added to 0.1 m, followed by precipitation with 12 volumes of acetone as described previously (40.O'Neill L. Turner B.M. Methods Enzymol. 1996; 274: 189-203Crossref PubMed Scopus (55) Google Scholar,41.Crane-Robinson C. Hebbes T.R. Clayton A.L. Thorne A.W. Methods. 1997; 12: 48-56Crossref PubMed Scopus (28) Google Scholar). Proteins were analyzed by electrophoresis on 15% SDS-polyacrylamide gels and then transferred to nitrocellulose filters and separately incubated with anti-acetylated H4 and anti-acetylated H3 (both at 1:1000 dilution) and anti-unacetylated H3 (1:250 dilution) antibodies. The blots were developed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (used at 1:3000 dilution; Roche Molecular Biochemicals), and immune complexes were visualized by the Lumi-Light Western blotting substrate system (Roche Molecular Biochemicals). Equal masses of DNA (1.0 μg) from input (unfractionated chromatin), antibody-bound, and unbound fractions were loaded onto Zetabind membranes (Cuno, Inc., Meriden, CT) using a slot-blot manifold. The blots were incubated overnight at 65 °C with hybridization solution containing 1–2 × 106 cpm/ml random primer-labeled probe. Subsequent washes were at 60 °C in 0.1% SDS and 0.5× SSC. Signals were quantified by PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) using ImageQuant software, and the ratios between bound and unbound DNA fractions were calculated for each probe used. To correct for potentially unequal slot-blot loadings, each blot was rehybridized with random primer-labeled sheared genomic DNA (see below). All ratios are reported after normalization to this loading control. The majority of DNA fragments used as hybridization probes were generated by polymerase chain reaction using AmpliTaq DNA polymerase (Perkin-Elmer). The template for the polymerase chain reaction was a P1 clone encompassing thehGH LCR and the first four of the five clusteredhGH genes (39.Su Y. Liebhaber S.A. Cooke N.E. J. Biol. Chem. 2000; 275: 7902-7909Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The primer sets are as shown in TableI. Probe p9 was a subcloned 263-bpEcoRI fragment of a repeated element (“P-element”) (42.Nachtigal M.W. Nickel B.E. Cattini P.A. J. Biol. Chem. 1993; 268: 8473-8479Abstract Full Text PDF PubMed Google Scholar) located 2 kb 5′ to hCS-L, hCS-A,hGH-V, and hCS-B. Probe p10 was an amplified fragment of a repeated element (“enhancer element”) (43.Jiang S.-W. Trujillo M.A. Eberhardt N.L. Mol. Endocrinol. 1997; 11: 1233-1244Crossref PubMed Scopus (8) Google Scholar) located 2 kb 3′ to hCS-L, hCS-A, and hCS-B. Due to the high sequence homology between each of the hGHpromoters, primers specific for the hGH-N promoter were designed to amplify a 200-bp region −0.8 kb to −1 kb upstream of thehGH-N transcription start site. The mouse ζ-globin probe was a 1.3-kb BamHI fragment encompassing the mouse ζ-globin coding region. The human ζ-globin probe was a 1.8-kbBglII fragment encompassing the human ζ-globin coding region. The probe used as a loading control was generated by random primer labeling of sonicated mouse or human total genomic DNA. Transgenic genotypes were determined by tail blots using two probes: the GRF probe was a 1.7-kbBglII/HindIII fragment encompassing the humanGRF coding region, and the hGH probe was a 500-bpEcoRI/BamHI fragment encompassing thehGH-N promoter.Table IAmplification primer setsProbeSizePrimer setbpHSI/II4005′-GGAATTCCCAAGCCTTTCCCAGTTATAC-3′5′-GGAATTCGATCTTGGCCTAGGCCTCGGA-3′HSIII3635′-GCGAATTCGAGGAGAGACTAGAGAAGCACCCAG-3′5′-AAGGATCCACTCATAACCCACCCATAAACACC-3′HSIV4605′-TGCCTCTACGTGGACATCTC-3′5′-TATCAGCAGAGAGTGCACAA-3′HSV5005′-CGAGTGGACCACCTTAACTT-3′5′-TAGAGGATAAGTGTGAGGAC-3′p13405′-GATTACAAGCGCCCACTACC-3′5′-GGGAGAGAATAAGCCAGGAGGTG-3′p24055′-TGCTCAGACCAGCCTATGCA-3′5′-TCAACAGGAAGTGGAGCACA-3′p32205′-GCTCCCCCAGAAGACTGAGAG-3′5′-GGAGGAAAACGTGRAACTGC-3′p44705′-GCTGTATTCTTCCAGACAAG-3′5′-GAGCTAAGCTATGAGGATGC-3′p53205′-GCCTCAAAACCTGATTGG-3′5′-GGAGATCTCTGAGGCTGG-3′p62605′-CCTGGGTGGCGTAGAGATG-3′5′-GACCCACGTTGTCGTAGTTG-3′p74165′-GCTCATCAAGATCATTGGCA-3′5′-AGTAAATTGAGAACTACGGG-3′p82405′-TGCCAGGAGGTGGGAGTT-3′5′-TGGCCCTGGGCTTTTGTGTAC-3′p102405′-GTCTACATTTCAGCTCATCA-3′5′-CCTTGTTTACATGTTAGAATC-3′hGH-N-specific probe2005′-GCCTCGCCACCCCTGT-3′5′-CTTCCATGTTCCTCC-3′ Open table in a new tab A set of studies were designed to establish the specificity and accuracy of the ChIP approach used in this report. Salt-soluble, unfixed oligonucleosome preparations from each cell or tissue type studied (K562 erythroid cells, GHFT1/HSIII presomatotrope cells, human pituitary tumor tissue, and P1/GRF transgenic mouse pituitary and liver tissue) were generated by micrococcal nuclease digestion of purified nuclei. To ensure the quality of the chromatin preparations and to determine the size distribution of the resulting oligonucleosomes for each sample, the DNA from the digested chromatin preparation was analyzed by gel electrophoresis. In each case, this analysis revealed a typical oligonucleosome ladder; the majority of DNA ranged from ∼160 bp (mononucleosomes) to 1 kb, and minimal DNA could be visualized above 2 kb (Fig. 1 A). To establish the specificity of the antibodies for acetylated H3 and H4 under the experimental ChIP conditions used for our studies, soluble oligonucleosomes prepared from the K562 erythroleukemia cell line (Fig.1 A) were immunoprecipitated with a 1:1 mixture of antibodies specific to acetylated histones H3 and H4. DNA and proteins were separately isolated from the total input chromatin, the unacetylated (unbound) chromatin, and the acetylated (bound) chromatin. DNA from each fraction was applied via a slot-blot manifold to nylon membranes for hybridization analysis (see below). In parallel, equal amounts of core histones isolated from each fraction were analyzed by SDS-polyacrylamide gel electrophoresis (Fig. 1 B )and then Western-blotted and immunostained with antibodies specific to acetylated H3 or H4 (Fig. 1 C). These results demonstrated that the immunoprecipitates were enriched for acetylated histones H3 and H4 compared with the unbound fractions. The specificity of the immunoprecipitation was further validated by demonstrating a reciprocal enrichment of unacetylated histone H3 in the unbound fraction (Fig.1 C, lower panel). As expected, prolonged exposure of the blot revealed a small amount of residual unacetylated H3 in the bound fraction as well (not shown). These protein studies demonstrated that this ChIP procedure enriched chromatin fractions for acetylated histones H3 and H4. K562 DNA isolated by the ChIP procedure was analyzed by hybridization using probes corresponding to promoter sequences of actively transcribed ζ-globin and unexpressed hGH (Fig.1 D). Equal amounts of intact DNA extracted from input, unacetylated (unbound), and acetylated (bound) chromatin were applied to membranes via a slot-blot manifold and sequentially probed for specific DNA sequence content. Hybridization signal intensities in each of these three fractions were normalized for minor differences in DNA loading by directly quantifying the DNA in each slot using a labeled total genomic DNA probe. As expected from previous acetylation studies of active human globin genes (44.Clayton A.L. Hebbes T.R. Thorne A.W. Crane-Robinson C. FEBS Lett. 1993; 336: 23-26Crossref PubMed Scopus (43) Google Scholar), the chromatin encompassing the expressed ζ-globin promoter was enriched for acetylation (boundversus unbound ratio of 3.7). The acetylation of the chromatin encompassing the silent hGH promoter was at background levels (ratio of 1.0). The difference between these two ratios was highly significant (p < 0.001). In five independent ChIP experiments using K562 chromatin, the ratios of the ζ-globin promoter ranged between 3.5 and 3.8. The specificity of the immunoprecipitation was further validated by demonstrating that K562 nuclear chromatin immunoprecipitated with antibodies to unacetylated histone H3 was not enriched for ζ-globin promoter sequences. These DNA hybridization studies demonstrated the specificity and reproducibility of the ChIP procedure that was used in the studies that follow. The association of HS formation with localized histone acetylation was initially tested in a cell culture setting. HSIII of the hGH LCR (Fig.2 A) was used for these studies because the DNase I-hypersensitive structure of this chromatin element could be reproduced in stably transfected pituitary cells.2A 3150-kb fragment encompassing HSIII was linked to 500 bp of thehGH-N promoter region driving a neomycin resistance (neor) cassette (HSIII/hGHneo r) (Fig.2 B, bottom). Following stable transfection into GHFT1 presomatotrope cells, a neor cell line (GHFT1/HSIII) was obtained. A DNase I-hypersensitive site formed in the integrated 3150-bp HSIII segment at a position corresponding precisely to that of native HSIII in primary pituitary and placental tissue (data not shown). Cell nuclei isolated from these neor cells were digested with micrococcal nuclease, and the resulting soluble oligonucleosomes were immunoprecipitated with a 1:1 mixture of antibodies specific to acetylated histones H3 and H4 using the ChIP procedure (40.O'Neill L. Turner B.M. Methods Enzymol. 1996; 274: 189-203Crossref PubMed Scopus (55) Google Scholar, 41.Crane-Robinson C. Hebbes T.R. Clayton A.L. Thorne A.W. Methods. 1997; 12: 48-56Crossref PubMed Scopus (28) Google Scholar). Equal amounts of intact DNA extracted from the input, unbound, and bound chromatin fractions were sequentially probed for specific DNA sequence content as described above. The normalized ratio of HSIII sequences in the bound versus unbound chromatin fractions was 3.3, and that for the hGH-N promoter was 2.8. In contrast, the ratios for the inactive α-fetoprotein and ζ-globin genes in the same chromatin samples were 1.1 and 1.2, respectively (Fig. 2 B). In four independent experiments using GHFT1/HSIII chromatin, the ratios of HSIII ranged between 3.2 and 3.5. Specificity was validated by demonstrating that antibodies to unacetylated H3 failed to enrich for HSIII sequences (Fig.2 B). These data further established the specificity and reproducibility of this ChIP procedure and demonstrated the enrichment of HSIII in the acetylated chromatin fraction of a stably transfected, pituitary-derived cell line. Analysis ofhGH LCR chromatin acetylation was next expanded to anin vivo setting in which the entire hGH cluster and adjacent sequences could be studied in their native setting. The chromatin of a primary GH-secreting human pituitary adenoma was analyzed using a set of probes corresponding to the hGH-Npromoter, each of the hGH LCR HSs, and two sites located 5′ to the LCR. Tissue specificity of the acetylation map was established by comparing these results with those obtained from analysis of chromatin isolated from the human K562 erythroid cell line. The positions of the probes in relation to the hGH cluster, the closely linked CD79b (the B lymphocyte-specific immunoglobulin receptor subunit gene encoding Igβ) (45.Bennani-Baiti I.M. Cooke N.E. Liebhaber S.A. Genomics. 1998; 48: 258-264Crossref PubMed Scopus (33) Google Scholar), andSCN4A (the striated muscle-specific sodium channel gene) (46.Bennani-Baiti I.M. Jones B.K. Liebhaber S.A. Cooke N.E. Genomics. 1995; 29: 647-652Crossref PubMed Scopus (32) Google Scholar) are shown in Fig. 3 A. ChIP assays of the human pituitary adenoma chromatin revealed that the segments encompassing each of the five HSs as well as the adjacenthGH-N promoter were all enriched for acetylation (all ratios = 2.0) (Fig. 3 B). The most highly modified region coincided with HSI/II (3.4-fold enrichment). Acetylation at the two sites upstream of the LCR (probes p7 and p8) (Fig. 3 B) was insignificant (ratios of 1.4). There was virtually no acetylation enrichment at any of these sites in erythroid (K562) cell chromatin. These data demonstrated tissue-specific enrichment of acetylation at all HSs in the chromatin of this human pituitary cell line enriched for somatotropes. Furthermore, they identified a 5′-boundary to the LCR modification in the pituitary just upstream of HSV and suggested that HSI/II was the most highly modified among the HSs. Of additional note was the acetylation of HSIV. Because HSIV does not form in the pituitary, its modification in this tissue suggested that acetylation of the hGH locus in pituitary chromatin might not be limited to the immediate locale of the active HSs, but might instead be generally distributed throughout the LCR domain. The pituitary is made up of six differentiated hormone-secreting cell types: the somatotropes (secreting GH), somatolactotropes (GH and prolactin), lactotropes (prolactin), thyrotropes (thyrotropin-releasing hormone), gonadotropes (luteinizing hormone and follicle-stimulating hormone), and corticotropes (AC" @default.
• W2146780090 created "2016-06-24" @default.
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• W2146780090 creator A5080563290 @default.
• W2146780090 date "2000-05-01" @default.
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• W2146780090 title "Targeted Recruitment of Histone Acetyltransferase Activity to a Locus Control Region" @default.
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