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• W2006943966 abstract "Previously, we characterized a DNA-binding protein, HS2NF5, that bound tightly to a conserved region within hypersensitive site 2 (HS2) of the human β-globin locus control region (LCR) (Lam, L. T., and Bresnick, E. H. (1996) J. Biol. Chem. 271, 32421–32429). The β-globin LCR controls the chromatin structure, transcription, and replication of the β-globin genes. We have now purified HS2NF5 to near-homogeneity from fetal bovine thymus. Two polypeptides of 56 and 61 kDa copurified with the DNA binding activity. The two proteins bound to the LCR recognition site with an affinity (3.1 nm) and specificity similar to mouse erythroleukemia cell HS2NF5. The amino acid sequences of tryptic peptides of purified HS2NF5 revealed it to be identical to the murine homolog of the suppressor of hairless transcription factor, also known as recombination signal binding protein Jκ or C promoter binding factor 1 (CBF1). The CBF1 site within HS2 resides near sites for hematopoietic regulators such as GATA-1, NF-E2, and TAL1. An additional conserved, high affinity CBF1 site was localized within HS4 of the LCR. As CBF1 is a downstream target of the Notch signaling pathway, we propose that Notch may modulate LCR activity during hematopoiesis. Previously, we characterized a DNA-binding protein, HS2NF5, that bound tightly to a conserved region within hypersensitive site 2 (HS2) of the human β-globin locus control region (LCR) (Lam, L. T., and Bresnick, E. H. (1996) J. Biol. Chem. 271, 32421–32429). The β-globin LCR controls the chromatin structure, transcription, and replication of the β-globin genes. We have now purified HS2NF5 to near-homogeneity from fetal bovine thymus. Two polypeptides of 56 and 61 kDa copurified with the DNA binding activity. The two proteins bound to the LCR recognition site with an affinity (3.1 nm) and specificity similar to mouse erythroleukemia cell HS2NF5. The amino acid sequences of tryptic peptides of purified HS2NF5 revealed it to be identical to the murine homolog of the suppressor of hairless transcription factor, also known as recombination signal binding protein Jκ or C promoter binding factor 1 (CBF1). The CBF1 site within HS2 resides near sites for hematopoietic regulators such as GATA-1, NF-E2, and TAL1. An additional conserved, high affinity CBF1 site was localized within HS4 of the LCR. As CBF1 is a downstream target of the Notch signaling pathway, we propose that Notch may modulate LCR activity during hematopoiesis. locus control region C promoter from Epstein-Barr virus C promoter binding factor 1 electrophoretic mobility shift assay Epstein-Barr virus Epstein-Barr virus nuclear antigen 2 hairy enhancer of split high pressure liquid chromatography hypersensitive site recombination signal binding protein immunoglobulin Jκ suppressor of hairless polyacrylamide gel electrophoresis base pair(s) mass spectroscopy mutant. Transcription of the β-globin genes is controlled by a powerful genetic element called the β-globin locus control region (LCR).1 The human LCR consists of four erythroid-specific DNase I HSs, 10–50-kilobase pairs upstream of the β-globin genes (1Tuan D. London I.M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 2718-2722Crossref PubMed Scopus (83) Google Scholar, 2Forrester W.C. Thompson C. Elder J.T. Groudine M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1359-1363Crossref PubMed Scopus (270) Google Scholar). The LCR controls the chromatin structure, transcriptional activity, and replication timing of the β-globin locus (3Grosveld F. van Assendelft G.B. Greaves D.R. Kollias G. Cell. 1987; 51: 975-985Abstract Full Text PDF PubMed Scopus (1436) Google Scholar, 4Forrester W.C. Takegawa S. Papayannopoulou T. Stamatoyannopoulos G. Groudine M. Nucleic Acids Res. 1987; 15: 10159-10177Crossref PubMed Scopus (292) Google Scholar, 5Forrester W.C. Epner E. Driscoll M.C. Enver T. Brice M. Papayannopoulou T. Groudine M. Genes Dev. 1990; 4: 1637-1649Crossref PubMed Scopus (388) Google Scholar). A defining feature of the LCR is its ability to confer copy number-dependent and position-independent expression to a linked gene that is stably integrated into chromosomal DNA (3Grosveld F. van Assendelft G.B. Greaves D.R. Kollias G. Cell. 1987; 51: 975-985Abstract Full Text PDF PubMed Scopus (1436) Google Scholar). The physiological importance of the LCR is highlighted by a chromosomal deletion associated with Hispanic β-thalassemia, which removes part of the LCR and correlates with repression of the β-globin genes (5Forrester W.C. Epner E. Driscoll M.C. Enver T. Brice M. Papayannopoulou T. Groudine M. Genes Dev. 1990; 4: 1637-1649Crossref PubMed Scopus (388) Google Scholar). An activity of the LCR distinct from traditional enhancers is that the activation property can be shared by multiple genes over long distances on a chromosome (6Bresnick E.H. Felsenfeld G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1314-1317Crossref PubMed Scopus (32) Google Scholar, 7Furukawa T. Zitnik G. Leppig K. Papayannopoulou T. Stamatoyannopoulos G. Blood. 1994; 83: 1412-1419Crossref PubMed Google Scholar, 8Wijgerde M. Grosveld F. Fraser P. Nature. 1995; 377: 209-213Crossref PubMed Scopus (429) Google Scholar). We have proposed a mechanism of coordinate promoter activation involving the recruitment of chromatin remodeling enzymes, which mediate the decondensation of chromatin throughout the β-globin domain (9Bresnick E.H. Tze L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4566-4571Crossref PubMed Scopus (41) Google Scholar, 10Bresnick E.H. Chemtracts Biochem. Mol. Biol. 1997; 10: 1001-1009Google Scholar). The increase in DNA accessibility is manifested as general DNase I sensitivity (11Forrester W.C. Thompson C. Elder J.T. Groudine M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1359-1363Crossref PubMed Google Scholar). We hypothesize that this chromatin transition is necessary for the subsequent protein-DNA interactions occurring through promoters, enhancers, and silencers that determine the developmental expression pattern of the β-globin genes. An alternative mechanism favors looping interactions between the LCR and individual β-globin gene promoters (12Hanscombe O. Whyatt D. Fraser P. Yannoutsos N. Greaves D. Dillon N. Grosveld F. Genes Dev. 1991; 5: 1387-1394Crossref PubMed Scopus (225) Google Scholar, 13Engel J.D. Trends Genet. 1993; 9: 304-309Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 14Tuan D.Y. Solomon W.B. London I.M. Lee D.P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2554-2558Crossref PubMed Scopus (205) Google Scholar), controlling the assembly of preinitiation complexes on the promoters. The chromatin disruption and looping models are not mutually exclusive, as the disruption may be necessary for subsequent promoter interactions. Both mechanisms share the requirement for transactivating proteins that function through the LCR. Multiple hematopoietic and ubiquitous transcription factors interact with conserved sequences within the HSs of the LCR. HS2 is a strong erythroid-specific enhancer, which has been studied extensively (15Sorrentino B. Ney P. Nienhuis A.W. Nucleic Acids Res. 1990; 18: 2721-2731Crossref PubMed Scopus (55) Google Scholar, 16Ryan T.M. Behringer R.R. Martin N.C. Townes T.M. Palmiter R.D. Brinster R.L. Genes Dev. 1989; 3: 314-323Crossref PubMed Scopus (179) Google Scholar, 17Talbot D. Grosveld F. EMBO J. 1991; 10: 1391-1398Crossref PubMed Scopus (180) Google Scholar, 18Moi P. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9000-9004Crossref PubMed Scopus (78) Google Scholar, 19Lloyd J.A. Krakowsky J.M. Crable S.C. Lingrel J.B. Mol. Cell. Biol. 1992; 12: 1561-1567Crossref PubMed Google Scholar, 20Ellis J. Talbot D. Dillon N. Grosveld F. EMBO J. 1993; 12: 127-134Crossref PubMed Scopus (104) Google Scholar, 21Hardison R. Xu J. Jackson J. Mansberger J. Selifonova O. Grotch B. Biesecker J. Petrykowska H. Miller W. Nucleic Acids Res. 1993; 21: 1265-1272Crossref PubMed Scopus (43) Google Scholar, 22Caterina J.J. Ciavatta D.J. Donze D. Behringer R.R. Townes T.M. Nucleic Acids Res. 1994; 22: 1006-1011Crossref PubMed Scopus (80) Google Scholar, 23Enver T. Li Q. Gale K.B. Hu M. May G.E. Karlinsey J.E. Jimenez G. Papayannopoulou T. Costantini F. Dev. Biol. 1994; 165: 574-584Crossref PubMed Scopus (10) Google Scholar, 24Reddy P.M. Stamatoyannopoulos G. Papayannopoulou T. Shen C.K. J. Biol. Chem. 1994; 269: 8287-8295Abstract Full Text PDF PubMed Google Scholar, 25Fiering S. Epner E. Robinson K. Zhuang Y. Telling A. Hu M. Martin D.I. Enver T. Ley T.J. Groudine M. Genes Dev. 1995; 9: 2203-2213Crossref PubMed Scopus (295) Google Scholar). Factors interacting with HS2 include NF-E2 (26Andrews N.C. Erdjument-Bromage H. Davidson M.B. Tempst P. Orkin S.H. Nature. 1993; 362: 722-728Crossref PubMed Scopus (566) Google Scholar, 27Ney P.A. Andrews N.C. Jane S.M. Safer B. Purucker M.E. Weremowicz S. Morton C.C. Goff S.C. Orkin S.H. Nienhuis A.W. Mol. Cell. Biol. 1993; 13: 5604-5612Crossref PubMed Scopus (162) Google Scholar), GATA-1 (28Tsai S.F. Martin D.I. Zon L.I. D'Andrea A.D. Wong G.G. Orkin S.H. Nature. 1989; 339: 446-451Crossref PubMed Scopus (664) Google Scholar, 29Evans T. Felsenfeld G. Cell. 1989; 58: 877-885Abstract Full Text PDF PubMed Scopus (451) Google Scholar), TAL-1 (30Elnitski L. Miller W. Hardison R. J. Biol. Chem. 1997; 272: 369-378Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), USF (20Ellis J. Talbot D. Dillon N. Grosveld F. EMBO J. 1993; 12: 127-134Crossref PubMed Scopus (104) Google Scholar, 31Bresnick E.H. Felsenfeld G. J. Biol. Chem. 1993; 268: 18824-18834Abstract Full Text PDF PubMed Google Scholar), SP1 (32Talbot D. Philipsen S. Fraser P. Grosveld F. EMBO J. 1990; 9: 2169-2177Crossref PubMed Scopus (234) Google Scholar), YY1 (33Yant S.R. Zhu W. Millinoff D. Slightom J.L. Goodman M. Gumucio D.L. Nucleic Acids Res. 1995; 23: 4353-4362Crossref PubMed Scopus (132) Google Scholar), and HS2NF5 (34Lam L.T. Bresnick E.H. J. Biol. Chem. 1996; 271: 32421-32429Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Mutational analysis suggests that many of these factors are required for optimal HS2-mediated transactivation in transgenic mice and stably transfected cell lines (20Ellis J. Talbot D. Dillon N. Grosveld F. EMBO J. 1993; 12: 127-134Crossref PubMed Scopus (104) Google Scholar, 22Caterina J.J. Ciavatta D.J. Donze D. Behringer R.R. Townes T.M. Nucleic Acids Res. 1994; 22: 1006-1011Crossref PubMed Scopus (80) Google Scholar, 34Lam L.T. Bresnick E.H. J. Biol. Chem. 1996; 271: 32421-32429Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Previously, we had characterized protein-DNA interactions within a conserved region of HS2 containing an E box (34Lam L.T. Bresnick E.H. J. Biol. Chem. 1996; 271: 32421-32429Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), which was a putative TAL1-binding site. TAL1 is a hematopoietic basic helix-loop-helix transcription factor that regulates hematopoiesis (35Porcher C. Swat W. Rockwell K. Fujiwara Y. Alt F.W. Orkin S.H. Cell. 1997; 86: 47-57Abstract Full Text Full Text PDF Scopus (612) Google Scholar, 36Robb L. Elwood N.J. Elefanty A.G. Kontgen F. Li R. Barnett L.D. Begley C.G. EMBO J. 1996; 15: 4123-4129Crossref PubMed Scopus (285) Google Scholar). Although TAL1 binding was not detected using MEL and K562 cell nuclear extracts, a novel protein was identified, HS2NF5, that bound tightly to a site overlapping the E box. Recently, we performed a quantitative analysis of recombinant TAL1/E12 heterodimer DNA binding specificity (37Gould K.A. Bresnick E.H. Gene Expr. 1998; 7: 87-101PubMed Google Scholar). TAL1/E12 bound to oligonucleotides containing the HS2 E box with very low affinity, in contrast to the high affinity interaction with an optimal TAL1 site. As protein-protein interactions between TAL1 and LIM domain proteins alter the DNA binding specificity of TAL1 (38Wadman I.A. Osada H. Grutz G.G. Agulnick A.D. Westphal H. Forster A. Rabbitts T.H. EMBO J. 1997; 16: 3145-3157Crossref PubMed Scopus (728) Google Scholar), we postulated that additional factors may allow TAL1 to associate stably with the E box. In contrast to our results with nuclear extracts, Etlinski et al. (30Elnitski L. Miller W. Hardison R. J. Biol. Chem. 1997; 272: 369-378Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) detected TAL1 binding to the E box using K562 extracts. The importance of HS2NF5 in LCR function was suggested by a mutational analysis of the HS2NF5-binding site of HS2 (34Lam L.T. Bresnick E.H. J. Biol. Chem. 1996; 271: 32421-32429Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). This site was necessary for optimal enhancer activity in stable and transient transfection assays. Since HS2NF5 had an apparently unique DNA binding specificity, a variable distribution in cell lines, and was the major activity that bound with high affinity to a functionally important region of HS2, we chose to purify HS2NF5. Here, we describe the identification of two forms of HS2NF5 as the previously cloned transcription factor known as Su(H) in Drosophila (39Furukawa T. Maruyama S. Kawaichi M. Honjo T. Cell. 1992; 69: 1191-1197Abstract Full Text PDF PubMed Scopus (101) Google Scholar, 40Schweisguth F. Posakony J.W. Cell. 1992; 69: 1199-1212Abstract Full Text PDF PubMed Scopus (229) Google Scholar), and RBPJκ or CBF1 2CBF1 and RBP-Jκ refer to the same protein, which will be indicated as CBF1 throughout.2CBF1 and RBP-Jκ refer to the same protein, which will be indicated as CBF1 throughout. in mammals (41Matsunami N. Hamaguchi Y. Yamamoto Y. Kuze K. Kangawa K. Matsuo H. Kawaichi M. Honjo T. Nature. 1989; 342: 934-937Crossref PubMed Scopus (131) Google Scholar, 42Henkel T. Ling P.D. Hayward S.D. Peterson M.G. Science. 1994; 265: 92-95Crossref PubMed Scopus (364) Google Scholar). As Su(H) and CBF1 are critical developmental regulators and downstream targets of the Notch signaling pathway, we discuss a potential role for Notch signaling in LCR activity and hematopoiesis. Buffer A contained 20 mm HEPES (pH 7.2), 0.1 mm EDTA, 5% glycerol, 50 mm NaCl. Buffer B contained 20 mm Tris (pH 7.8), 0.1 mm EDTA, 5% glycerol, 50 mm NaCl. Buffer C contained 25 mmHEPES (pH 7.6), 20% glycerol. Buffer D contained 20 mmTris (pH 7.5), 150 mm NaCl, 0.2 mm EDTA. Buffer E contained 20 mm HEPES (pH 7.5), 250 mmsucrose, 500 mm NaCl, 0.5 mmphenylmethylsulfonyl fluoride, 0.1 mm benzamide. Buffer H contained 20 mm HEPES (pH 7.5), 250 mm sucrose, 25 mm KCl, 5 mm MgCl2, 0.5 mm phenylmethylsulfonyl fluoride, 0.1 mmbenzamide. All buffers contained 5 mm dithiothreitol. The mouse erythroleukemia cell line MEL was propagated in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 4 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. The cell line was grown in a humidified incubator at 37 °C, in the presence of 5% carbon dioxide. MEL cells were treated with 1.5% dimethyl sulfoxide for 72 h prior to isolating nuclear extract. Fetal calf thymus (from approximately 4-month-old calves) was obtained from Peck Slaughterhouse (Milwaukee, WI). The fresh thymus was washed with cold phosphate-buffered saline and then mixed with 2 volumes of buffer H at 4 °C. The mixture was homogenized in a Waring blender with two pulses of 40 s each. The blended material was filtered through four sheets of cheesecloth and then centrifuged at 545 × g for 15 min. The pellet was washed with 2 volumes of buffer H by resuspension and centrifugation at 545 ×g for 5 min. The pellet was then resuspended by vigorous shaking in 2 volumes of buffer E, and the mixture was centrifuged at 142,413 × g for 40 min. The supernatant was adjusted to 25% (NH4)2SO4 by gradual addition of solid (NH4)2SO4 with stirring. The mixture was stirred for 30 min, and the precipitate was collected by centrifugation at 21,000 × g for 15 min. The supernatant was adjusted to 50% (NH4)2SO4 as described above, and the precipitated material was collected by centrifugation at 21,000 × g for 15 min. The pellet was dissolved in a minimal volume of buffer A for Resource-S chromatography. The solution was dialyzed against the same buffer overnight. The insoluble material was removed by centrifugation for 15 min at 21,000 ×g, and the resulting nuclear extract was used for the chromatography. All procedures were performed at 4 °C. DNA binding reactions were performed as described previously (34Lam L.T. Bresnick E.H. J. Biol. Chem. 1996; 271: 32421-32429Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). DNA binding activity was quantitated by analyzing gels with a PhosphorImager (Molecular Dynamics). All chromatographic steps were carried out with an Amersham Pharmacia Biotech fast pressure liquid chromatography system at 4 °C. The crude extract was chromatographed in three consecutive runs on a 20-ml Resource-S cation exchange column (Amersham Pharmacia Biotech) equilibrated in buffer A. Proteins were resolved with a 50–450 mm NaCl gradient in buffer A at a flow rate of 2.5 ml/min. HS2NF5 DNA binding was measured by EMSA in the presence of 1 μg of poly(dI-dC) (Amersham Pharmacia Biotech). HS2NF5 activity eluted as a homogeneous peak between 200 and 380 mm NaCl. The fractions containing maximal HS2NF5 activity were pooled and dialyzed overnight against 2 liters of buffer B. The dialyzed material from the Resource-S column was centrifuged for 15 min at 21,000 ×g at 4 °C. The supernatant was applied to a 12-ml Q-Sepharose anion-exchange column (Amersham Pharmacia Biotech) equilibrated in buffer B. Proteins were resolved with a 100–400 mm NaCl gradient in buffer B at a flow rate of 2.5 ml/min. HS2NF5 DNA binding was measured by EMSA in the presence of 0.5 μg of poly(dI-dC). HS2NF5 activity eluted as a major peak between 160 and 260 mm NaCl. A minor peak eluted at a slightly lower salt concentration but was not analyzed further, as it was present in fractions containing a high concentration of protein, which would have reduced considerably the fold purification. The fractions containing maximal HS2NF5 activity were pooled. Ammonium sulfate was added to the pooled material from the Q-Sepharose column to a final concentration of 1 m. The mixture was applied to a 10-ml phenyl-Sepharose hydrophobic column (Amersham Pharmacia Biotech) equilibrated in buffer B containing 1 m ammonium sulfate. Proteins were resolved with a 1 to 0 m ammonium sulfate gradient in buffer B at a flow rate of 2 ml/min. HS2NF5 DNA binding was measured in the presence of 0.25 μg of poly(dI-dC). HS2NF5 activity eluted as a homogeneous peak between 150 and 300 mmammonium sulfate. The fractions containing maximal HS2NF5 activity were pooled and dialyzed overnight against 1 liter of buffer C. The pooled material from the phenyl-Sepharose column was applied to a 5-ml double-stranded DNA-cellulose (Sigma) column equilibrated in buffer C. Proteins were resolved with a 150–450 mm KCl gradient in buffer C at a flow rate of 1 ml/min. HS2NF5 DNA binding was assayed in the presence of 0.1 μg of poly(dI-dC). HS2NF5 activity eluted as a homogenous peak between 270 and 390 mm KCl. Protein concentrations of the pooled samples from each of the first three columns were determined by the Bradford assay. Proteins were analyzed by resolving on 9% SDS-PAGE, followed by silver staining. In place of the DNA-cellulose step, we also used sequence-specific DNA affinity chromatography with the concatamerized HS2NF5 site coupled to Sepharose. Although HS2NF5 could also be isolated with this column, the yield of HS2NF5 was lower than with the DNA-cellulose column (data not shown). The pooled material from the DNA-cellulose column was dialyzed against buffer D and concentrated by lyophilization. The mixture was applied to a Superdex 200 HR 10/30 column (Amersham Pharmacia Biotech) equilibrated in buffer D, and proteins were eluted at a flow rate of 0.5 ml/min. HS2NF5 DNA binding was measured by EMSA in the presence of 0.1 μg of poly(dI-dC). After dialysis against Milli-Q filtered water and lyophilization, proteins were analyzed by 9% SDS-PAGE and silver staining. Fractions of DNA-cellulose-purified material were dialyzed against Milli-Q water and concentrated by lyophilization. The proteins were resolved by 9% SDS-PAGE and detected by Coomassie Blue staining. After thorough destaining, gel pieces containing the 56- and 61-kDa forms of HS2NF5 were excised and subjected to carboxyamidomethylation proteolysis with trypsin as described previously (43Nash H.M. Bruner S.D. Scharer O.D. Kawate T. Addona T.A. Spooner E. Lane W.S. Verdine G.L. Curr. Biol. 1996; 6: 968-980Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar). Mass spectroscopy sequencing was performed by Bill Lane of the Harvard Microchemistry Facility. The eluted peptides were separated by microcapillary reverse-phase HPLC coupled to the electrospray ionization source of a Finnigan LCQ quadrupole ion trap mass spectrometer. Peptides were eluted from the column with a linear gradient of 0–50% acetonitrile in 0.05% acetic acid at a flow rate of 700 nl/min. Ionization was assisted with a coaxial sheath liquid of 70% methanol, 0.05% acetic acid. Spectra were acquired as successive sets of three scan modes as follows: full scan MS over them/z range 395–1118 amu, followed by two data-dependent scans on the most abundant ion in that full scan. These data-dependent scans allowed the automatic acquisition of a high resolution (zoom) scan to determine charge state and exact mass and MS/MS spectra for peptide sequence information. Base peak relative ion abundance corresponded to a load of 5–35 fmol by comparison with a standard peptide mixture analyzed under identical conditions. Interpretation of the resulting MS/MS spectra of the peptides was facilitated by searching NCBI non-redundant protein and EST data bases with the algorithm Sequest (44Eng J.K. McCormick A.L. Yates III., J.R. J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Crossref PubMed Scopus (5411) Google Scholar). Quantitative DNA binding studies suggested that the amount of HS2NF5 in nuclear extracts of MEL and K562 cells was low, which would make purification of ample amounts for sequencing difficult. Instead, we found that HS2NF5 DNA binding activity was present in nuclear extracts of fetal calf thymus, and it resembled HS2NF5 from MEL and K562 cells. To compare the DNA sequence specificity of MEL and thymus HS2NF5, we performed EMSAs using nuclear extracts fractionated on a Resource-S column, followed by a Q-Sepharose column. As shown in Fig. 1, titrations were performed with increasing concentrations of radiolabeled oligonucleotides containing a wild-type or mutated HS2NF5 site (Fig. 1 A) and a constant amount of fractionated extract. The HS2NF5-DNA complexes formed with MEL cell and thymus extracts had an identical mobility on the nondenaturing gel. A mutant oligonucleotide, Mut-1, was tested, which had been shown previously not to bind HS2NF5 (34Lam L.T. Bresnick E.H. J. Biol. Chem. 1996; 271: 32421-32429Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). No HS2NF5 binding to Mut-1 was detected with either extract. In contrast, another mutated oligonucleotide, Mut-2, which had been shown previously not to affect HS2NF5 binding (34Lam L.T. Bresnick E.H. J. Biol. Chem. 1996; 271: 32421-32429Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) was bound by both MEL and thymus HS2NF5. Thus, the DNA binding activity of MEL and thymus HS2NF5 appeared to be indistinguishable, justifying the use of thymus extract for purifying HS2NF5. The HS2NF5 purification scheme is outlined in Fig. 2. Fetal calf thymus nuclear extract was fractionated by ammonium sulfate precipitation, followed by chromatography on Resource-S, Q-Sepharose, phenyl-Sepharose, and double-stranded DNA-cellulose columns (data not shown). Protein samples from each stage of the purification were resolved by SDS-PAGE and analyzed by silver staining to evaluate the purity of HS2NF5. As shown in the extensively silver-stained gel of Fig. 3 A, after four chromatographic steps, two major protein bands of 56 and 61 kDa were present in fractions 22–28. A smaller amount of protein from another purification is shown on the right to illustrate the copurification and purity of the 56- and 61-kDa proteins (Fig. 3 B). Both the 56- and 61-kDa bands were characterized by microheterogeneity.Figure 3SDS-PAGE analysis of purified HS2NF5. A, fetal calf thymus nuclear extract (10 μg), the Resource-S pool (10 μg), the Q-Sepharose pool (10 μg), the phenyl-Sepharose pool (10 μg), and fractions from the DNA-cellulose column were analyzed by SDS-PAGE and visualized by silver staining. The positions of the two forms of HS2NF5 are indicated. B, purified HS2NF5 from another purification (fraction 24) was analyzed by SDS-PAGE and silver staining. C, comparison of the relative amount of 56- and 61-kDa polypeptides detected by silver staining and the DNA binding activity of HS2NF5 quantitated from EMSA (data not shown). D, densitometric scan of stained proteins in fraction 24 of panel A.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To show further that the 56- and 61-kDa bands represent HS2NF5, we compared the relative amount of DNA binding activity measured by EMSA (data not shown) with the relative amount of the 56- and 61-kDa polypeptides detected by densitometric analysis of the silver-stained bands (Fig. 3 A). As the binding activity and protein correlated well, this supports the identity of the 56- and 61-kDa bands as HS2NF5 (Fig. 3 C). The purity of HS2NF5 in fraction 24 from the DNA-cellulose column was estimated to be 94% by densitometric analysis (Fig. 3 D) and reverse-phase chromatography on a microcapillary C18 HPLC column (data not shown). The purification results are summarized in Table I. A 52,450-fold purification of HS2NF5 was achieved, with a yield of 27%. These results were representative of 10 independent purifications with different samples of fetal calf thymus.Table IPurification of HS2NF5 from fetal calf thymusFractionTotal proteinTotal activitySpecific activityTotal yieldPurificationmgpmolpmol/mg%-foldI. Nuclear extract11250.0II. Ammonium sulfate6030.0807.10.1100.01.0III. Resource-S191.4608.63.275.423.7IV. Q-Sepharose22.6467.220.757.9154.4V. Phenyl-Sepharose3.6380.4105.447.0786.9VI. DNA-celluloseFraction 220.01779.54676.09.846760.0Fraction 240.01481.45814.010.158140.0Fraction 2636.84.6Fraction 2817.02.1Three hundred and fifteen grams of fetal calf thymus was used in this purification. The amount of 32P-oligonucleotide bound was measured with a PhosphorImager. The binding activity of HS2NF5 in the nuclear extract could not be accurately determined, since the crude extract contained substances that inhibited binding. The protein concentrations of fractions 22 and 24 of the DNA-cellulose column were estimated by SDS-PAGE and silver staining relative to standard proteins. The protein concentrations of fractions 26 and 28 were too low to be measured. Open table in a new tab Three hundred and fifteen grams of fetal calf thymus was used in this purification. The amount of 32P-oligonucleotide bound was measured with a PhosphorImager. The binding activity of HS2NF5 in the nuclear extract could not be accurately determined, since the crude extract contained substances that inhibited binding. The protein concentrations of fractions 22 and 24 of the DNA-cellulose column were estimated by SDS-PAGE and silver staining relative to standard proteins. The protein concentrations of fractions 26 and 28 were too low to be measured. The DNA binding specificity of purified HS2NF5 was assessed by EMSA with a labeled wild-type HS2NF5 oligonucleotide and unlabeled wild-type and mutant competitors (Fig. 4 A). Two mutations were shown previously (34Lam L.T. Bresnick E.H. J. Biol. Chem. 1996; 271: 32421-32429Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) to reduce HS2 enhancer activity (E box mut, CAGATG changed to GTCGAC; HS2NF5 mut, TTCTCA changed to CTGCAG). In addition, as a control, we tested a third mutant (TAA mut, GCC changed to TAA), which we had shown previously to have no effect on HS2NF5 DNA binding (34Lam L.T. Bresnick E.H. J. Biol. Chem. 1996; 271: 32421-32429Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The wild-type and TAA mut oligonucleotides strongly reduced binding (wild type, 94.8 and 97.9% decrease at 50- and 200-fold excess, respectively; TAA mut, 79 and 93.2% decrease at 50- and 200-fold excess, respectively) (Fig. 4 C). In contrast, the E box mut and HS2NF5 mut oligonucleotides only weakly competed for binding (E box mut, 48 and 61% decrease at 50- and 200-fold excess, respectively; HS2NF5 mut, 8.0 and 43% decrease at 50- and 200-fold excess, respectively), which agrees with our previous analysis of HS2NF5 DNA binding specificity in MEL cell nuclear extracts (34Lam L.T. Bresnick E.H. J. Biol. Chem. 1996; 271: 32421-32429Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Since the mutations of the E box mut and HS2NF5 mut oligonucleotides prevent HS2NF5 DNA binding and were shown previously to decrease HS2 enhancer activity (34Lam L.T. Bresnick E.H. J. Biol. Chem. 1996; 271: 32421-32429Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), these results suggest that impaired HS2NF5 binding may be responsible for the inhibition. To determine whether the 56- and 61-kDa forms of HS2NF5 have distinct DNA binding properties or bind to DNA as a heteromer, we resolved the purified material on an analytical gel filtration column. As shown in Fig. 5, A and B, the 56- and 61-kDa proteins were partially separated by the" @default.
• W2006943966 created "2016-06-24" @default.
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• W2006943966 title "Identity of the β-Globin Locus Control Region Binding Protein HS2NF5 as the Mammalian Homolog of the Notch-regulated Transcription Factor Suppressor of Hairless" @default.
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• W2006943966 doi "https://doi.org/10.1074/jbc.273.37.24223" @default.
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