YY1 Binds Five cis-Elements and Trans-activates the Myeloid Cell-restricted gp91 Promoter
1999; Elsevier BV; Volume: 274; Issue: 42 Linguagem: Inglês
10.1074/jbc.274.42.29984
ISSN1083-351X
AutoresBritta M. Jacobsen, David G. Skalnik,
Tópico(s)Peptidase Inhibition and Analysis
ResumoFour transcriptional activatingcis-elements within the gp91phox promoter bind a protein complex of similar mobility and binding specificity, denoted BID (binding increased during differentiation). The intensity of BID complexes increases upon myeloid cell differentiation, coincident with induction of gp91phox expression, and BID competes with the transcriptional repressor CDP for binding to each of these promoter elements. To determine the identity of BID, an expression library was ligand screened with the BID-binding site that surrounds the −145-base pair (bp) region of the gp91phox promoter. One recovered factor that exhibits the expected binding specificity is YY1, a ubiquitous multifunctional transcription factor. BID complexes that form with the four binding sites within the gp91phox promoter are disrupted by YY1 antiserum, and a fifth YY1-binding site was detected in the −412-bp promoter region. Overexpression of YY1 in transient co-transfection assays trans-activates a minimal promoter containing two copies of the −145-bp binding site from the gp91phox promoter. Neither the level of YY1 protein nor DNA binding activity increases during myeloid cell differentiation. These studies identify a target gene of YY1 function in mature myeloid cells, and demonstrate that YY1 function can be controlled during myeloid development by the modulation of a competing DNA-binding factor. Four transcriptional activatingcis-elements within the gp91phox promoter bind a protein complex of similar mobility and binding specificity, denoted BID (binding increased during differentiation). The intensity of BID complexes increases upon myeloid cell differentiation, coincident with induction of gp91phox expression, and BID competes with the transcriptional repressor CDP for binding to each of these promoter elements. To determine the identity of BID, an expression library was ligand screened with the BID-binding site that surrounds the −145-base pair (bp) region of the gp91phox promoter. One recovered factor that exhibits the expected binding specificity is YY1, a ubiquitous multifunctional transcription factor. BID complexes that form with the four binding sites within the gp91phox promoter are disrupted by YY1 antiserum, and a fifth YY1-binding site was detected in the −412-bp promoter region. Overexpression of YY1 in transient co-transfection assays trans-activates a minimal promoter containing two copies of the −145-bp binding site from the gp91phox promoter. Neither the level of YY1 protein nor DNA binding activity increases during myeloid cell differentiation. These studies identify a target gene of YY1 function in mature myeloid cells, and demonstrate that YY1 function can be controlled during myeloid development by the modulation of a competing DNA-binding factor. Phagocytic blood cells such as monocyte/macrophages and neutrophils use the NADPH oxidase to produce a respiratory burst to kill microbes. Several subunits comprise the NADPH oxidase, including p67phox, p47phox, p22phox, and gp91phox(1Orkin S.H. Annu. Rev. Immunol. 1989; 7: 277-307Crossref PubMed Google Scholar). The gp91phox gene is transcriptionally controlled and is expressed nearly exclusively in mature myeloid cells (2Royer-Pokora B. Kunkel L.M. Monaco A.P. Goff S.C. Newburger P.E. Baehner R.L. Cole F.S. Curnutte J.T. Orkin S.H. Nature. 1986; 322: 32-38Crossref PubMed Scopus (594) Google Scholar). Previously we demonstrated that the −450 to +12 base pair (bp) 1The abbreviations used are: bpbase pairsBIDbinding increased during differentiationCDPCCAAT displacement proteinEMSAelectrophoretic mobility shift assayIFNinterferonPMAphorbol 12-myristate 13-acetatePBSphosphate-buffered salineIRFIFN regulatory factor region of the gp91phox promoter is capable of directing reporter gene expression in a subset of monocyte/macrophages in transgenic mice (3Skalnik D.G. Dorfman D.M. Perkins A.S. Jenkins N.A. Copeland N.G. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8505-8509Crossref PubMed Scopus (55) Google Scholar), and responds to interferon (IFN)-γ stimulation in transfected myeloid cell lines (4Eklund E.A. Skalnik D.G. J. Biol. Chem. 1995; 270: 8267-8273Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). An enhancer region located 50 kilobases upstream of the proximal promoter is additionally required to direct appropriate expression of gp91phox in the full spectrum of mature myeloid cells (5Lien L.L. Lee Y. Orkin S.H. Mol. Cell. Biol. 1997; 17: 2279-2290Crossref PubMed Scopus (23) Google Scholar). base pairs binding increased during differentiation CCAAT displacement protein electrophoretic mobility shift assay interferon phorbol 12-myristate 13-acetate phosphate-buffered saline IFN regulatory factor Several DNA-binding proteins interact with the −450 to +12-bp region of the gp91phox promoter (Fig. 1). These include the transcriptional repressor CCAAT displacement protein (CDP) (6Skalnik D.G. Strauss E.C. Orkin S.H. J. Biol. Chem. 1991; 266: 16736-16744Abstract Full Text PDF PubMed Google Scholar, 7Luo W. Skalnik D.G. J. Biol. Chem. 1996; 271: 18203-18210Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 8Luo W. Skalnik D.G. J. Biol. Chem. 1996; 271: 23445-23451Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), as well as several transcriptional activators including the CCAAT-binding factor CP1 (4Eklund E.A. Skalnik D.G. J. Biol. Chem. 1995; 270: 8267-8273Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 6Skalnik D.G. Strauss E.C. Orkin S.H. J. Biol. Chem. 1991; 266: 16736-16744Abstract Full Text PDF PubMed Google Scholar, 7Luo W. Skalnik D.G. J. Biol. Chem. 1996; 271: 18203-18210Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), IFN regulatory factor (IRF)-1, IRF-2 (8Luo W. Skalnik D.G. J. Biol. Chem. 1996; 271: 23445-23451Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 9Eklund E.A. Luo W. Skalnik D.G. J. Immunol. 1996; 157: 2418-2429PubMed Google Scholar), PU.1 (10Suzuki S. Kumatori A. Haagen I.-A. Fujii Y. Sadat M.A. Jun H.L. Tsuji Y. Roos D. Nakamura M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6085-6090Crossref PubMed Scopus (93) Google Scholar, 11Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 12Voo K.S. Skalnik D.G. Blood. 1999; 93: 3512-3520Crossref PubMed Google Scholar), IFN consensus sequence-binding protein (11Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), Elf-1 (12Voo K.S. Skalnik D.G. Blood. 1999; 93: 3512-3520Crossref PubMed Google Scholar), and an unidentified factor denoted BID (binding increased during differentiation) (8Luo W. Skalnik D.G. J. Biol. Chem. 1996; 271: 23445-23451Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 9Eklund E.A. Luo W. Skalnik D.G. J. Immunol. 1996; 157: 2418-2429PubMed Google Scholar). In immature myeloid cells, the transcriptional repressor CDP binds to at least five sites in the promoter, preventing binding of transcriptional activators to overlapping binding sites, and the gp91phox protein is not expressed. However, CDP DNA binding activity is post-translationally down-regulated in mature myeloid cells (6Skalnik D.G. Strauss E.C. Orkin S.H. J. Biol. Chem. 1991; 266: 16736-16744Abstract Full Text PDF PubMed Google Scholar, 7Luo W. Skalnik D.G. J. Biol. Chem. 1996; 271: 18203-18210Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), allowing transcriptional activators to bind to the promoter and induce gp91phox expression. Previously we reported that BID binds to four sites within the −450 to +12 bp gp91phox promoter (8Luo W. Skalnik D.G. J. Biol. Chem. 1996; 271: 23445-23451Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 9Eklund E.A. Luo W. Skalnik D.G. J. Immunol. 1996; 157: 2418-2429PubMed Google Scholar). These conclusions were based on eletrophoretic mobility shift assay (EMSA) analysis that demonstrated complexes of similar mobility and binding specificity with each of four promoter probes (8Luo W. Skalnik D.G. J. Biol. Chem. 1996; 271: 23445-23451Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 9Eklund E.A. Luo W. Skalnik D.G. J. Immunol. 1996; 157: 2418-2429PubMed Google Scholar). Mutations of the putative BID-binding sites surrounding the −355, −225, and −145 bp regions of the gp91phox promoter result in decreased promoter activity in PLB-985 myeloid cells in response to IFN-γ stimulation (9Eklund E.A. Luo W. Skalnik D.G. J. Immunol. 1996; 157: 2418-2429PubMed Google Scholar). Truncation of the gp91phox promoter to −102 to +12 bp removes four CDP-binding sites and reveals a promiscuous promoter that is active in some cells not expressing the endogenous gp91phox gene (7Luo W. Skalnik D.G. J. Biol. Chem. 1996; 271: 18203-18210Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Specific ablation of a BID-binding site at −90 bp decreases this promiscuous promoter activity by 50% in HEL cells (8Luo W. Skalnik D.G. J. Biol. Chem. 1996; 271: 23445-23451Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). These data indicate that BID functions as a transcriptional activator. Data base searching with the four putative BID-binding sites revealed no common consensus binding sites for known transcription factors. Eklund and Kakar (13Eklund E.A. Kakar R. J. Biol. Chem. 1997; 272: 9344-9355Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) reported the cloning of a novel component of the BID complex (denoted TF1phox in that report). However, data from Yamit-Heziet al. (14Yamit-Hezi A. Levy Z. Neuman S. Nudel U. Gene ( Amst. ). 1997; 185: 99-103Crossref PubMed Scopus (6) Google Scholar) demonstrate that this clone is a bacterial contaminant present in some commercially available libraries. Hence, the identity of the BID complex remained to be identified. We undertook a molecular cloning approach to identify BID. Ligand screening of a λgt11 HeLa cell cDNA expression library was performed using as a probe the −145-bp BID-binding site derived from the gp91phox promoter. One sequence specific DNA-binding factor obtained is YY1, a ubiquitously expressed multifunctional member of the GLI Krüppel-related family of zinc finger transcription factors. Further examination of the −450 to +12 bp gp91phox promoter revealed a fifth potential YY1-binding site. YY1 is present in all five identified BID complexes within the gp91phox promoter, and transient transfection studies confirm that YY1 functions as a transcriptional activator of the gp91phox promoter. A β-globin TATA box minimal promoter fragment was excised from a human growth hormone gene reporter vector (gift of Ellis Neufeld, Harvard) with HindIII andBamHI and cloned into the luciferase vector, pXP2 (15Nordeen S.K. BioTechniques. 1988; 6: 454-456PubMed Google Scholar), that was digested with HindIII and BglII. This construct was digested with BamHI, de-phosphorylated with shrimp alkaline phosphatase (Amersham Pharmacia Biotech/U. S. Biochemical, Cleveland, OH), and ligated to phosphorylated −145 Core or −145 mut oligonucleotides (see below). The nucleotide sequence was determined for constructs containing a dimer of each oligonucleotide, and those with a dimer in the forward orientation were prepared by cesium chloride ultracentrifugation to be used in transient transfections (see below). HeLa human cervical choriocarcinoma cells, K562 human chronic myelogenous leukemia cells, and HEL human erythroleukemia cells were obtained from the American Type Culture Collection (Rockville, MD). The PLB-985 human myelomonoblastic cell line (16Tucker K.A. Lilly M.B. Heck Jr., L. Rado T.A. Blood. 1987; 70: 372-378Crossref PubMed Google Scholar) was a gift of Thomas Rado (Birmingham, AL). Suspension cells (HEL, K562, and PLB-985) were grown in RPMI 1640 medium with 10% fetal bovine serum or Fetal Clone III (Bovine Serum Product, HyClone, Logan, UT) and 0.2 mm glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin at 37 °C and 5% CO2. Adherent cells (HeLa) were grown in similarly supplemented Dulbecco's modified Eagle's media. Cells were diluted in media to a concentration of 107 cells/300 μl and placed into electroporation cuvettes (0.4-cm gap). One microgram of luciferase test plasmid and 5 μg of CB6+ CMV driven expression plasmid or CB6+-YY1 (17Gualberto A. LePage D. Pons G. Mader S.L. Park K. Atchison M.L. Walsh K. Mol. Cell. Biol. 1992; 12: 4209-4214Crossref PubMed Scopus (146) Google Scholar) (gifts of Kenneth Walsh, Tufts) were added to each cuvette. Each sample also contained 0.25 μg of cytomegalovirus promoter/β-galactosidase (CMV/β-gal) plasmid that serves as an internal control for transfection efficiency. Samples were electroporated using a Bio-Rad Gene Pulser at 960 microfarads and 220 V, re-suspended in 10 ml of media and incubated at 37 °C for 24 h. Cells were harvested, washed with PBS, resuspended in 100 μl of lysis buffer (Promega Inc., Madison, WI), and incubated at room temperature for 15 min. Twenty microliters of cell lysate was assayed for luciferase activity as described by the manufacturer (Promega, Inc.) using a Lumat LB 9501 (Berthold, Gaithersburg, MD) luminometer. β-Gal activity was detected as described (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and used to adjust luciferase values to correct for differences in transfection efficiency. Each sample was transfected in duplicate, and two independent plasmid preparations of each construct were used in five independent experiments. Nuclear extracts were prepared by the method of Dignam et al. (19Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar). PLB-985 cells were treated with agents that induce gp91phox expression as described previously (9Eklund E.A. Luo W. Skalnik D.G. J. Immunol. 1996; 157: 2418-2429PubMed Google Scholar). To prepare fractionated extract, six liters of K562 cells were grown to log phase and nuclear extract was prepared as described above. Nuclear extract was then fractionated by heparin-agarose (Sigma) chromatography using a stepwise gradient of KCl in Dignam Buffer D supplemented with protease inhibitors. Fractions exhibiting abundant BID DNA binding activity (0.2–0.3 mKCl) were pooled and dialyzed to 0.1 m KCl. For mini-nuclear extracts, 107 HEL cells were transiently transfected with 20 μg of CB6+ or CB6-YY1 expression plasmids as described above, incubated for 12 h, and mini-nuclear extracts prepared as described (20Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2211) Google Scholar). Complimentary oligonucleotides were annealed and radiolabeled using T4 polynucleotide kinase and [γ-32 P]ATP. Radiolabeled probes were purified as described previously (8Luo W. Skalnik D.G. J. Biol. Chem. 1996; 271: 23445-23451Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). EMSA was performed as described previously (6Skalnik D.G. Strauss E.C. Orkin S.H. J. Biol. Chem. 1991; 266: 16736-16744Abstract Full Text PDF PubMed Google Scholar), using 4–9 μg of nuclear extract and 0.1–0.5 μg of poly(dI-dC). Non-radioactive competitor oligonucleotides were added to designated samples and incubated on ice for 12 min. One to two microliters of YY1 antiserum (catalog number sc-1703x, Santa Cruz Biotech, Santa Cruz, CA) or Ets-2 antiserum (catalog number sc-351x, Santa Cruz Biotech) was added to designated samples and incubated on ice for 40 min. Radiolabeled oligonucleotide probe (20,000 cpm) was added (except where otherwise indicated) and samples were incubated on ice for an additional 30 min. The reactions were loaded onto native 6% polyacrylamide gels (except where otherwise indicated) and electrophoresis was performed at 25 mA for 1.25 h at 4 °C in 0.5 × TBE. Gels were then dried and exposed to x-ray film at −70 °C. Complimentary oligonucleotides were synthesized on an Applied Biosystems model 394 synthesizer. Sequences correspond to the upper strand of the human gp91phox promoter (6Skalnik D.G. Strauss E.C. Orkin S.H. J. Biol. Chem. 1991; 266: 16736-16744Abstract Full Text PDF PubMed Google Scholar). Mutated bases are underlined, and all oligonucleotides contain BamHI linkers (not shown): −90 (−102 to −65 bp), 5′-ctgataaaagaaaaggaaaccgattgccccagggctgc-3′; -145 Core (−155 to −130 bp), 5′-aagtttgttatggatgcaagcttttc-3′; −145 mut, 5′-aagtttgttataagtacaagctttt-3′; −225 Core (−240 to −215 bp), 5′-gaaattggtttcattttccactatgt-3′; −355 Core (−369 to −344 bp), 5′-tacccagcacgaagtcatgtctagtt-3′; −412 (−424 to −399 bp), 5′-gcaaggctatgaatgctgttccagcc-3′; BID-mut (−107 to −65 bp), 5′-aatttctgataaaagaaacttcaaccgattgccccagggctgc-3′; −182 to −112 bp, 5′-tttgtagttgttgaggtttaaagatttaagtttgttatggatgcaagcttttcagttgaccaatgattat-3′. The sequence of a high affinity CDP-binding site, E36 (21Andres V. Chiara M.D. Mahdavi V. Genes Dev. 1994; 8: 245-257Crossref PubMed Scopus (96) Google Scholar), is as follows: 5′-cggatccgaattcatcgataatcgattat-3′. Oligonucleotides containing a high affinity consensus binding site or mutated consensus binding site for YY1 as listed in the Santa Cruz Biotechnology, Inc. catalog were synthesized by Life Technologies, Inc. (Gaithersburg, MD) and contained BamHI linkers (not shown): YY1, 5′-cgctccgcggccatcttggcggctggt-3′; mut YY1 (mutated bases are underlined), 5′-cgctccgcgattatcttggcggctggt-3′. A λgt11 cDNA expression library derived from HeLa cells (CLONTECH, Palo Alto, CA) was a generous gift of Dr. Saw Yin Oh (Indianapolis, IN). Y1090 cells were incubated with bacteriophage for 15 min at 37 °C and plated at a density of 50,000 plaques/150-mm plate, incubated at 42 °C for 5 h, and nitrocellulose filters impregnated with 10 mmisopropyl-β-d-thiogalactoside were placed on the plates overnight at 37 °C. Denaturation/renaturation has been shown to increase the DNA binding affinity of some factors (22Vinson C.R. LaMarco K.L. Johnson P.E. Landschultz W.H. McKnight S.L. Genes Dev. 1988; 2: 901-906Crossref PubMed Scopus (346) Google Scholar). Filters were twice immersed in 6 m guanidine hydrochloride (Roche Molecular Biochemicals, Indianapolis, IN) for 5 min at 4 °C, then in successive 2-fold dilutions of denaturant to slowly renature the fusion proteins. The filters were then blocked overnight at 4 °C in blocking buffer (2.5% dried milk, 25 mm HEPES (pH 8.0), 1 mm dithiothreitol, 10% glycerol, 50 mm NaCl, 0.05% lauryldimethylamine oxide (Calbiochem), 1 mm EGTA). Filters were rinsed briefly in TNE-50 (10 mm Tris (pH 7.5), 50 mm NaCl, 1 mm EGTA, 1 mmdithiothreitol). The oligonucleotide containing a high affinity BID-binding site (−145 Core) was phosphorylated, concatenated using T4 DNA ligase, and radiolabeled with [α-32 P]dCTP using random priming. Probe was added to 106 cpm/ml of 1 × binding buffer (25 mm HEPES (pH 7.9), 3 mm MgCl2, and 40 mm KCl). Herring sperm DNA was added as a nonspecific competitor to a final concentration of 4 μg/ml. Filters were incubated with probe at 4 °C overnight, washed briefly three times in TNE-50 at room temperature, then exposed to x-ray film overnight at −70 °C. A total of 2.5 × 106 plaques were analyzed. Positive plaques were picked and eluted for a second round of ligand screening to test for binding specificity, for which filters were cut in half and probed with either the wild type −145 Core or −145 mut-concatenated probes. Clones exhibiting an appropriate binding specificity were purified by four successive rounds of ligand screening. DNA was prepared from purified plaques using the Wizard λ preparation (Promega, Inc., Madison, WI). Isolated DNA was digested with EcoRI and electrophoresis was performed on a 1% agarose gel. Insert fragments were recovered and cloned into Bluescript (Stratagene, La Jolla, CA). The nucleotide sequence of subcloned fragments was determined by the dideoxy chain termination method using M13 and T3 primers. Obtained sequences were used to search the GenBank data base. Nuclear extracts were quantitated using the method of Bradford (23Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216357) Google Scholar). SDS loading dye was added to 40 μg of nuclear extract and the samples were boiled for 10 min then loaded onto a 4–20% Tris glycine gel (Novex, San Diego, CA) and electrophoresis was performed at 180 V for 1.5 h at 4 °C. The proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad) for 2 h at 80 V, and the membrane was blocked in PBS + 0.1% Tween 20 (PBS-T) containing 5% low fat milk. Following blocking, the membranes were washed three times with PBS-T and incubated with an antiserum raised against the full-length YY1 protein (Santa Cruz Biotech., Inc) diluted 1:20,000 in blocking buffer for 1 h at 25 °C. The membrane was washed three times with PBS-T and secondary antibody conjugated to horseradish peroxidase was diluted 1:20,000 in PBS-T containing 5% low fat milk and incubated with the membrane for 1 h at room temperature. The membrane was washed five times in PBS-T, and chemiluminescent detection was performed according to the manufacturer's instructions (Amersham Pharmacia Biotech). Numerous DNA-binding proteins interact with the proximal gp91phox promoter to direct myeloid cell-restricted expression (Fig. 1). The transcriptional repressor CDP binds to multiple sites in the proximal promoter in undifferentiated myeloid cells, thus excluding the binding of transcriptional activators to overlapping binding sites. Myeloid precursor cells treated with agents that induce gp91phox expression, such as phorbol ester (PMA), dimethylformamide, or IFN-γ exhibit decreased CDP DNA binding activity and a concomitant increase in the intensity of BID complexes (6Skalnik D.G. Strauss E.C. Orkin S.H. J. Biol. Chem. 1991; 266: 16736-16744Abstract Full Text PDF PubMed Google Scholar, 7Luo W. Skalnik D.G. J. Biol. Chem. 1996; 271: 18203-18210Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 24Newburger P.E. Ezekowitz R.A.B. Whitney C. Wright J. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5215-5219Crossref PubMed Scopus (109) Google Scholar, 25Barker K.A. Orkin S.H. Newburger P.E. Mol. Cell. Biol. 1988; 8: 2804-2810Crossref PubMed Scopus (23) Google Scholar). CDP and BID bind to the gp91phox promoter in a mutually exclusive manner (7Luo W. Skalnik D.G. J. Biol. Chem. 1996; 271: 18203-18210Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). To facilitate study of BID, a truncated oligonucleotide was created which lacks several of the bases necessary for CDP binding to the −145 bp region of the gp91phox promoter. This oligonucleotide (−145 Core) was designed to include the binding site of BID, as deduced from methylation interference analysis (9Eklund E.A. Luo W. Skalnik D.G. J. Immunol. 1996; 157: 2418-2429PubMed Google Scholar). A similar strategy was used to design the −225 Core and −355 Core oligonucleotides (see below). The −145 Core oligonucleotide is capable of binding the BID protein, but does not bind CDP (Fig. 2, and data not shown). The −145 mutated oligonucleotide (−145 mut) contains a 4-bp mutation that disrupts BID contact sites and no longer binds the BID protein, as it no longer disrupts the BID EMSA complex formed with the wild type −145 Core probe (Fig. 2). The −145 Core oligonucleotide probe was used to assess the distribution of the BID binding activity in the absence of competing CDP complexes (Fig. 2 A). The BID complex is disrupted upon addition of homologous oligonucleotide competitor (lanes 3, 6, 9, and 12) but not by the −145 mut competitor which no longer binds the BID complex (lanes 4, 7, 10, and13). The BID complex is present in nuclear extracts derived from PLB-985 (lanes 2–4), HeLa (lanes 5–7), K562 (lanes 8–10), and HEL (lanes 11–13) cells. Thus, expression of BID is not lineage specific. There are also faster migrating complexes in both the HeLa extract (lanes 5–7) and the K562 extract (lanes 8–10) which behave with the same binding specificity as the previously identified slow mobility BID complex. We speculate that these may correspond to products of partial proteolysis of BID. EMSA was performed using the −145 Core binding site probe and nuclear extracts derived from PLB-985 cells treated with agents that induce gp91phox expression and were previously found to induce increased BID complex intensity (9Eklund E.A. Luo W. Skalnik D.G. J. Immunol. 1996; 157: 2418-2429PubMed Google Scholar, 25Barker K.A. Orkin S.H. Newburger P.E. Mol. Cell. Biol. 1988; 8: 2804-2810Crossref PubMed Scopus (23) Google Scholar). The BID complex, which is disrupted with the −145 Core oligonucleotide competitor (Fig.2 B, lanes 3, 6, and 9) but not with the −145 mut oligonucleotide (lanes 4, 7, and 10), is as intense in nuclear extract derived from untreated PLB-985 cells (lanes 2–4) as nuclear extracts derived from PLB-985 cells treated with PMA (lanes 5–7) or IFN-γ (lanes 8–10). Effective induction of the PLB-985 cell cultures was confirmed by demonstrating the down-regulation of CDP DNA binding activity by EMSA analysis (data not shown). Hence, increased intensity of BID complexes with composite BID/CDP probes upon induction of gp91phox expression is the result of decreased binding of CDP to overlapping binding sites. Searches of the Transcription Factor Sites data base (Genetics Computer Group Inc., Madison, WI) (26Ghosh D. Nucleic Acids Res. 1993; 21: 3117-3118Crossref PubMed Scopus (119) Google Scholar) with the −145 Core BID-binding site sequence produced no matches with consensus binding sites for known transcription factors. Hence, a molecular approach was taken to determine the identity of the BID protein. Because BID is ubiquitously expressed (Fig. 2 A), ligand screening was performed with a λgt11 HeLa cDNA expression library and the concatenated −145 Core BID-binding site probe. The −145 Core site demonstrates the cleanest EMSA complex and the highest affinity for the BID complex. 2B. M. Jacobsen, unpublished observations. The −145 mut oligonucleotide, which contains a 4-bp mutation and no longer binds the BID complex, was used as a probe to examine the sequence specificity of recovered DNA binding activities. Approximately 2.5 × 106 plaques were screened, and 43 reproducibly positive DNA-binding clones were examined for binding specificity. Three clones bind to the wild type −145 Core probe, but not to the −145 mut probe (Fig. 3). The other 40 clones encode nonspecific DNA-binding factors as they bind both the −145 Core and −145 mut probes, and were not pursued further. A search of the GenBank data base revealed that the determined nucleotide sequence of a sequence specific clone matches the cDNA sequence of YY1 (data not shown), a ubiquitous multifunctional transcription factor that is a member of the GLI Krüppel zinc finger factor family (27Park K. Atchinson M.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9804-9808Crossref PubMed Scopus (296) Google Scholar). The recovered cDNA encompasses amino acid 85 to the stop codon and a portion of the 3′-untranslated region, and includes the region of YY1 previously reported to contain the DNA-binding domain (28Hariharan N. Kelley D.E. Perry R.P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9799-9803Crossref PubMed Scopus (246) Google Scholar). Antiserum directed against YY1 was tested in EMSA analysis using the −145 Core probe (Fig. 4 A). The BID complex that forms upon addition of nuclear extract derived from HEL cells (lane 2) is disrupted by homologous oligonucleotide competition (lane 3), but not by the −145 mut oligonucleotide competitor (lane 4), and is also abolished upon addition of YY1 antiserum (lane 5). The other complexes visible in the lanes do not exhibit the appropriate binding site specificity for BID and are not affected by the YY1 antiserum. Also, the BID complex is not affected by an antiserum directed against Ets-2 (lane 6), thus demonstrating the specificity of the disruption by YY1 antiserum. Additional EMSA studies were performed using a previously described YY1-binding site oligonucleotide to compare the behavior of BID and YY1. The −145 Core site probe (Fig. 4 B, lanes 1–6) produces the BID complex (lane 2) which is disrupted by homologous competitor oligonucleotide (lane 3) and not by the −145 mut competitor (lane 4). The BID complex is also disrupted by the YY1 consensus binding site competitor (lane 5), but is not disrupted by a mutated YY1-binding site competitor (lane 6). Additionally, EMSA using the YY1 consensus binding site as a probe (Fig. 4 B, lanes 7–10) demonstrates a complex of similar mobility (lane 8) and binding specificity (lanes 9 and 10) as the BID complex formed with the −145 Core probe. EMSA studies with nuclear extract derived from HeLa cells and the −145 Core site probe demonstrate that the previously detected faster migrating complexes (Fig. 2 A) are also disrupted by the YY1 specific antibody (data not shown). A complex of slower mobility also binds to the −145 Core site (lane 2). However, this complex does not contain YY1 because it is disrupted by both homologous and mutant competitors (lanes 3and 4), and is not disrupted by either the YY1 consensus oligonucleotide (lane 5) or YY1 antiserum (Fig. 4 A, lane 5). Nuclear extracts prepared from HEL cells transiently transfected with the CB6-YY1 expression vector (or empty parental expression vector) were analyzed by EMSA using the −145 Core site probe (Fig.4 C). The BID complex is greatly enhanced in cells overexpressing YY1 (lane 3) compared with those transfected with the parental CB6+ expression vector (lane 2). The intensified complex is disrupted by competition with homologous oligon
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