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Tyrosine Phosphorylation of HoxA10 Decreases DNA Binding and Transcriptional Repression during Interferon γ-induced Differentiation of Myeloid Leukemia Cell Lines

2000; Elsevier BV; Volume: 275; Issue: 26 Linguagem: Inglês

10.1074/jbc.m907915199

1083-351X

Elizabeth A. Eklund, Annika Jalava, Renu Kakar,

Cancer-related gene regulation

The DNA binding affinity of HoxA10 is increased by partnering with Pbx proteins. A consensus sequence for Pbx1-HoxA10 DNA binding has been derived, but genuine target genes have not been identified. We noted that the derived Pbx-HoxA10 DNA-binding consensus is similar to a repressor element in the CYBB promoter. TheCYBB gene, which encodes the respiratory burst oxidase component gp91 phox, is expressed only in myeloid cells that have differentiated beyond the promyelocyte stage. In these studies, we demonstrate that interferon γ (IFN-γ)-induced differentiation of myeloid cell lines abolishes in vitro Pbx-HoxA10 binding to either the derived consensus or the similar CYBB sequence. We also demonstrate that HoxA10, overexpressed in myeloid cell lines, represses reporter gene expression from artificial promoter constructs with Pbx-HoxA10 binding sites. We determine that HoxA10 has endogenous repression domains that are not functionally altered by IFN-γ treatment. However, IFN-γ-induced differentiation of myeloid cell lines leads to HoxA10 tyrosine phosphorylation, which decreasesin vitro DNA binding to Pbx-HoxA10 binding sites. Therefore, these investigations identify the CYBB gene as a potential target for HoxA10 and define repression of genes expressed in mature myeloid cells as a novel role for HoxA10 during myeloid differentiation. The DNA binding affinity of HoxA10 is increased by partnering with Pbx proteins. A consensus sequence for Pbx1-HoxA10 DNA binding has been derived, but genuine target genes have not been identified. We noted that the derived Pbx-HoxA10 DNA-binding consensus is similar to a repressor element in the CYBB promoter. TheCYBB gene, which encodes the respiratory burst oxidase component gp91 phox, is expressed only in myeloid cells that have differentiated beyond the promyelocyte stage. In these studies, we demonstrate that interferon γ (IFN-γ)-induced differentiation of myeloid cell lines abolishes in vitro Pbx-HoxA10 binding to either the derived consensus or the similar CYBB sequence. We also demonstrate that HoxA10, overexpressed in myeloid cell lines, represses reporter gene expression from artificial promoter constructs with Pbx-HoxA10 binding sites. We determine that HoxA10 has endogenous repression domains that are not functionally altered by IFN-γ treatment. However, IFN-γ-induced differentiation of myeloid cell lines leads to HoxA10 tyrosine phosphorylation, which decreasesin vitro DNA binding to Pbx-HoxA10 binding sites. Therefore, these investigations identify the CYBB gene as a potential target for HoxA10 and define repression of genes expressed in mature myeloid cells as a novel role for HoxA10 during myeloid differentiation. CCAAT displacement protein interferon γ base pair electrophoretic mobility shift assay chloramphenicol acetyltransferase polyacrylamide gel electrophoresis The 39 human and murine HOX genes encode homeodomain transcription factors necessary for embryogenesis (1.Izpisua-Belmonte J. Falkenstein H. Dolle P. Renucci A. Duboule D. EMBO J. 1991; 10: 2279-2289Crossref PubMed Scopus (281) Google Scholar, 2.Dolle P. Izpisua-Belmonte J.-C. Falkenstein H. Renucci A. Duboule D. Nature. 1989; 342: 767-772Crossref PubMed Scopus (474) Google Scholar) and definitive hematopoiesis (3.Sauvageau G. Lansdorp P.M. Eaves C.J. Hogge D.E. Dragowska W.H. Reid D.S. Largman C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12223-12227Crossref PubMed Scopus (441) Google Scholar). Genes of the HOX A andB paralog groups are preferentially expressed in CD34+ bone marrow progenitor cells and are activated 3′ to 5′ during hematopoiesis (3.Sauvageau G. Lansdorp P.M. Eaves C.J. Hogge D.E. Dragowska W.H. Reid D.S. Largman C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12223-12227Crossref PubMed Scopus (441) Google Scholar). Expression of 3′ HOX A and B genes increases in early CD34+ cells and decreases in CD34+ committed progenitors. In contrast, transcription of the 5′ genes (i.e. HOX9-13) is invariant in CD34+ cells, and decreases in mature phagocytes (3.Sauvageau G. Lansdorp P.M. Eaves C.J. Hogge D.E. Dragowska W.H. Reid D.S. Largman C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12223-12227Crossref PubMed Scopus (441) Google Scholar). In comparison with normal, mature myeloid cells, expression ofHOXA10 is increased in acute myeloid leukemia, chronic myeloid leukemia, or myelodysplasia (4.Lawrence H.J. Sauvageau G. Ahmadi N. Lopez A.R. Le Beau M.M. Link M. Humphries K. Largman C. Exp. Hematol. 1995; 23: 1160-1166PubMed Google Scholar). Consistent with this, overexpression of HoxA10 in murine bone marrow induces a myeloproliferative disorder, which evolves to acute leukemia (5.Thorsteinsdottir U. Sauvageau G. Hough M.R. Dragowska W. Lansdorp P.M. Lawrence H.J. Largman C. Humphries R.K. Mol. Cell. Biol. 1995; 17: 495-505Crossref Scopus (296) Google Scholar). These results suggest that HoxA10 is involved in progression of myeloid differentiation. Although the most abundant HoxA10 transcript in human myeloid cells encodes a 406-amino acid protein (predicted molecular mass of 50 kDa) (6.Lowney P. Corral J. Detmer K. Le Beau M.M. Deaven L. Lawrence H.J. Largman C. Nucleic Acids Res. 1991; 19: 3443-3449Crossref PubMed Scopus (67) Google Scholar), alternatively spliced transcripts have been described at various stages of murine embryogenesis (7.Benson G.V. Nguyen T.-H.E. Maas R.L. Mol. Cell. Biol. 1995; 15: 1591-1601Crossref PubMed Google Scholar) and in transformed cell lines (4.Lawrence H.J. Sauvageau G. Ahmadi N. Lopez A.R. Le Beau M.M. Link M. Humphries K. Largman C. Exp. Hematol. 1995; 23: 1160-1166PubMed Google Scholar). In myeloid leukemia cell lines, a HoxA10 transcript is present encoding a protein that initiates 20 amino acids N-terminal to the homeodomain (6.Lowney P. Corral J. Detmer K. Le Beau M.M. Deaven L. Lawrence H.J. Largman C. Nucleic Acids Res. 1991; 19: 3443-3449Crossref PubMed Scopus (67) Google Scholar). It is hypothesized that the 80-amino acid (15-kDa) "short A10" may contribute to immortalization of myeloid cell lines, although the role of short A10 in normal myelopoiesis is unknown. Similar to the other Abd-like Hox proteins (Hox9–13), DNA binding affinity of HoxA10 is increased by partnering with Pbx proteins (8.Chang C.-P. Brocchieri L Shen W.-F. Largman C. Cleary M.L. Mol. Cell. Biol. 1996; 16: 1734-1745Crossref PubMed Scopus (250) Google Scholar). Although consensus sequences for Pbx-HoxA10 binding have been derived (8.Chang C.-P. Brocchieri L Shen W.-F. Largman C. Cleary M.L. Mol. Cell. Biol. 1996; 16: 1734-1745Crossref PubMed Scopus (250) Google Scholar, 9.Shen W.-F. Rosenfeld S. Lawrence H.J. Largman C. J. Biol. Chem. 1997; 272: 8198-8296Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 10.Neuteboon S.T.C. Murre C. Mol. Cell. Biol. 1997; 17: 4696-4706Crossref PubMed Scopus (62) Google Scholar), genuine Pbx-HoxA10 target genes have not been identified. It has been hypothesized that HoxA10 regulates myeloid differentiation by activating transcription of genes that are necessary for progression of myelopoiesis. Conversely, HoxA10 might repress transcription of genes characteristic of differentiated myeloid cells, or HoxA10 might activate transcription at one stage of myelopoiesis and repress transcription at another, as has been described for homologousDrosophila proteins, during embryogenesis (11.Pinsonneault J. Florence B. Vaessin H. McGinnis W. EMBO J. 1997; 16: 2032-2042Crossref PubMed Scopus (125) Google Scholar). We have been studying regulation of genes encoding the respiratory burst oxidase proteins, gp91 phox (the CYBB gene) (12.Royer-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 (596) Google Scholar) and p67 phox (the NCF2 gene) (13.Leto T.L. Lomax K.J. Volpp B.D. Nunio H. Sechler J.M.G. Nauseef W.M. Clark R. Gallin J.I. Malech H.L. Science. 1990; 248: 727-735Crossref PubMed Scopus (299) Google Scholar). These genes are transcribed in cells differentiated beyond the promyelocyte stage and therefore provide a model for gene regulation during late myelopoiesis. Both the CYBB and NCF2 genes contain sequences similar to the Pbx-HoxA10 binding consensus sequence. One of these CYBB sequences is within a previously described repressor element (14.Skalnik D.G. Strauss E.C. Orkin S.H. J. Biol. Chem. 1991; 266: 16736-16744Abstract Full Text PDF PubMed Google Scholar), which binds the CCAAT displacement protein (CDP)1 in electrophoretic mobility shift assays (EMSA) with HeLa or K562 nuclear proteins (14.Skalnik D.G. Strauss E.C. Orkin S.H. J. Biol. Chem. 1991; 266: 16736-16744Abstract Full Text PDF PubMed Google Scholar,15.Neufeld E.J. Skalnik D.G. Lievens P.M.-J. Orkin S.H. Nat. Genet. 1992; 1: 50-55Crossref PubMed Scopus (186) Google Scholar). In NIH 3T3 cells, overexpression of CDP represses an artificial promoter construct containing the CYBB element (16.Lievens P.M.J. Donady J.J. Tufarelli C. Neufeld E.J. J. Biol. Chem. 1995; 270: 12745-12750Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). In contrast, our previous investigations demonstrated that CDP is not a component of the complex binding to the CYBB repressor element in EMSA with nuclear proteins from the myeloid lines PLB985 and U937 (17.Eklund E.A. Kakar R. J. Biol. Chem. 1997; 272: 9344-9355Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Since HoxA10 mRNA is present in these myeloid cell lines, but not in HeLa or K562 cells (4.Lawrence H.J. Sauvageau G. Ahmadi N. Lopez A.R. Le Beau M.M. Link M. Humphries K. Largman C. Exp. Hematol. 1995; 23: 1160-1166PubMed Google Scholar), our current studies investigate the hypothesis that, in committed myeloid progenitors, HoxA10 interacts with repressor elements and suppresses transcription of some myeloid-specific genes until later stages of myelopoiesis. Previously, we demonstrated that IFN-γ-induced myeloid differentiation decreases in vitro protein binding to theCYBB repressor element, coincident with increasedCYBB transcription (17.Eklund E.A. Kakar R. J. Biol. Chem. 1997; 272: 9344-9355Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Therefore, we also investigate the effect of IFN-γ-induced differentiation on HoxA10 DNA binding and functional activity in myeloid cells. Artificial promoter/reporter constructs were generated as described previously (18.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13967Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), in the minimal promoter/reporter vector, p-TATACAT (19.Scholer H.R. Balling R. Hazopoulos A.K. Suzuki N. Gruss P. EMBO J. 1989; 8: 2551-2558Crossref PubMed Scopus (271) Google Scholar) (obtained from Dr. A. Kraft, University of Colorado, Denver). Constructs were generated with three copies (in the forward orientation) of the consensus sequence for HoxA10-Pbx binding (p-a10TATACAT) (8.Chang C.-P. Brocchieri L Shen W.-F. Largman C. Cleary M.L. Mol. Cell. Biol. 1996; 16: 1734-1745Crossref PubMed Scopus (250) Google Scholar) or four copies (in the forward direction) the −94 to −134 bp sequence from the CYBBpromoter (p-cybba10TATACAT). This CYBB promoter sequence has previously been demonstrated to function as a repressor element in myeloid cell lines (16.Lievens P.M.J. Donady J.J. Tufarelli C. Neufeld E.J. J. Biol. Chem. 1995; 270: 12745-12750Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). An artificial promoter construct with five copies of the Gal4 DNA binding site and a minimal promoter from the thymidine kinase gene linked to a chloramphenicol acetyltransferase (CAT) reporter (p-gal4TKCAT) was obtained from T. Gabig (Indiana University, Indianapolis). The cDNAs for human HoxA10 and "short A10" were obtained from C. Largman (University of California, San Francisco) and subcloned in to the mammalian expression vector pSRα (20.Takebe, Y., Seki, M., Fujisajwa, J.-I. Hoy, P., Yokota, K., Arai, K.-I., Yoshida, M., and Arai, N. (1988) Mol. Cell. Biol.Google Scholar). The human Pbx1 cDNA and a FLAG epitope-tagged HoxA10 cDNA were obtained from M. Cleary (Stanford University, Stanford, CA) (8.Chang C.-P. Brocchieri L Shen W.-F. Largman C. Cleary M.L. Mol. Cell. Biol. 1996; 16: 1734-1745Crossref PubMed Scopus (250) Google Scholar) and also subcloned into the pSRα vector. The cDNAs for HoxA10 and "short A10" were also subcloned into the vector pM2(GAL4), for expression as a fusion protein with the DNA binding domain of GAL4 (21.Sadowski I. Ma J. Trieznberg S. Ptashne M. Nature. 1988; 335: 563-564Crossref PubMed Scopus (991) Google Scholar). Oligonucleotides were synthesized by the Core Facility of the Comprehensive Cancer Center (University of Alabama, Birmingham) or the Riley Pediatric Research Center (Indiana University, Indianapolis). Oligonucleotides used were as follows: derived consensus sequence for Pbx-HoxA10 binding (dsA10), 5′-tgcgatgat ttatgaccgc-3′; the similar sequence from the CYBB promoter (−94 to −134 bp) (dscybbA10) (14.Skalnik D.G. Strauss E.C. Orkin S.H. J. Biol. Chem. 1991; 266: 16736-16744Abstract Full Text PDF PubMed Google Scholar), 5′-ttcagttgaccaat gat tattagccaattttctgataaaa-3′; a mutant sequence from the CYBB promoter (−94 to −134 bp) (dscybbmut), 5′-ttcagttgaccaat gattcggcgccaatttctgataaaa-3′; the similar sequence from the NCF2 gene (−600 to −637 bp) (dsncf2A10), 5′-aaaaggcattagtcaagagataat taattgggaaagag-3′; a mutant sequence from the NCF2 gene (−600 to −637 bp) (dsncf2A10mut), 5′-aaaaggcattagtcaagagataat gccgtgggaaagag-3′; an irrelevant sequence from the NCF2 gene (−535 to −575 bp) (dsncf2irf), 5′-cactctaggtcacgggtttcatttgggaccactagcctagt-3′; anotherCYBB promoter sequence similar to the Pbx-HoxA10 binding consensus sequence (−194 to −242 bp) (dscybb5′A10), 5′-agaaattggtttcattttccact atgtt taattgtgactggatcatta-3′; the CCAAT box from the α-globin gene (urccaat) (22.Dorn A. Bollenkens J. Staub A. Benoist C. Mathis D. Cell. 1987; 50: 863-875Abstract Full Text PDF PubMed Scopus (474) Google Scholar), 5′-ccgggctccgcgccagccaatgagcgccgcgg-3′. In these oligonucleotides, the HoxA10 core (or mutated core) is in boldface type, the Pbx core is in italics, and ccaat boxes are underlined. All cell lines were of human origin. The promyelocytic leukemia cell line PLB985 was obtained from Thomas Rado (University of Alabama, Birmingham). The myelomonocytic cell line U937 (23.Larrick J.W. Anderson S.J. Koren H.S. J. Immunol. 1980; 125: 6-14Crossref PubMed Google Scholar) was obtained from Andrew Kraft (University of Colorado, Denver). Cell lines were maintained and differentiated as described (17.Eklund E.A. Kakar R. J. Biol. Chem. 1997; 272: 9344-9355Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 18.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13967Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). U937 cells were treated with 200 units/ml human recombinant IFN-γ (Roche Molecular Biochemicals). Nuclear extract proteins were prepared by the method of Dignam et al. (24.Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1993; 11: 1475-1479Crossref Scopus (9164) Google Scholar), with protease and phosphatase inhibitors, as described (18.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13967Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Oligonucleotides probes were prepared, and EMSA and antibody supershift assays were performed as described (18.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13967Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Antiserum to HoxA10 (not cross-reactive with other Hox proteins) was obtained from Covance Research Products (Richmond, CA). Pbx antibodies (C-20 and P-20), blocking peptides, and an antibody to CDP were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal antiserum raised to whole CDP protein (purified from HeLa cells) was a generous gift of Ellis Neufeld (Boston Children's Hospital, Boston, MA). In vitro transcribed HoxA10, short A10, and Pbx1 mRNA were generated from linearized template DNA, using the Riboprobe System, according to the manufacturer's instructions (Promega, Madison, WI). In vitro translated proteins were generated in rabbit reticulocyte lysate, according to the manufacturer's instructions (Promega). Control (unprogrammed) lysates were generated in similar reactions in the absence of input RNA.In vitro translated proteins and nuclear proteins were tyrosine-dephosphorylated with Yop protein-tyrosine phosphatase (New England Biolabs, Beverly, MA). Proteins (either 10 μl of in vitro translated protein or 2 μg of nuclear proteins) were incubated 30 min at 30 °C, in a 20-μl reaction volume with 50 units of Yop and 1× reaction buffer, according to the manufacturer's instructions. Control proteins were incubated, similarly, in 1× reaction buffer withoutYop. EMSA with the in vitro translated proteins was performed as described (18.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13967Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Binding assays with in vitro translated proteins and the dscybbA10 oligonucleotide were performed in the presence of a 200-fold molar excess of the urccaat oligonucleotide. The urccaat oligonucleotide competes for binding of CP1, found in reticulocyte lysate, to the dscybbA10 probe. Cells were transfected by electroporation as described (18.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13967Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). U937 cells (32 × 106/sample) were transfected with 70 μg of p-TATACAT, p-a10TATACAT, or p-cybba10TATACAT; 30 μg of pSRα, HoxA10/pSRα, shortA10/pSRα, or Pbx1/pSRα or 15 μg each of HoxA10/pSRα plus Pbx1/pSRα; and 15 μg of p-CMVβ-gal (to normalize for transfection efficiency). In other experiments, U937 cells were transfected with 20 or 2 μg of p-gal4TKCAT; 20 μg of HoxA10/pm2(GAL4), shortA10/pm2GAL4, or control pM2GAL4; and 15 μg of p-CMVβ-gal. Transfectants were incubated for 24 h at 37 °C, 5% CO2, followed by 24 h with or without IFN-γ (200 units/ml). Preparation of cell extracts, β-galactosidase, and CAT assays were performed as described (25.Seed B. Sheen J.-Y. Gene (Amst.). 1988; 67: 271-275Crossref PubMed Scopus (830) Google Scholar, 26.Sambrook H. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). In other experiments, U937 cells were transfected with 30 μg of pSRα, HoxA10/pSRα, or HoxA10(FLAG)/pSRα. The cells were incubated for 48 h at 37 °C, 5% CO2 and harvested for extraction of nuclear proteins or total cellular RNA. Western blots were performed with 30 μg of nuclear proteins extracted from U937 cells, with or without 48 h IFN-γ incubation. Proteins were separated on 12% SDS-PAGE, transferred to nitrocellulose, and probed with HoxA10 antiserum (Covance Research Products) or control rabbit pre-immune serum, and proteins were detected by chemiluminescence, according to the manufacturer's instructions (Amersham Pharmacia Biotech). In other experiments, Western blots of nuclear proteins from U937 cells transiently transfected with pSRα or FLAG epitope-tagged HoxA10/pSRα were performed with anti-FLAG antibody. To determine HoxA10 phosphorylation state, U937 cells, with or without 48 h of IFN-γ differentiation, were incubated for 4 h at 37 °C with 32P-orthophosphate, as described (27.Brugge J.S. Erickson R.L. Nature. 1977; 269: 346-348Crossref PubMed Scopus (479) Google Scholar). Cells were lysed, under denaturing conditions, and proteins were immunoprecipitated for 4 h at 4 °C, with 2 μl of HoxA10 antiserum or 2 μl of control rabbit preimmune serum, followed by a 1-h incubation with 30 μl of 50% staph protein A-Sepharose bead slurry, as described (27.Brugge J.S. Erickson R.L. Nature. 1977; 269: 346-348Crossref PubMed Scopus (479) Google Scholar). Immunoprecipitated proteins were washed with radioimmune precipitation buffer, eluted in SDS sample buffer, and identified by autoradiography of 12% SDS-PAGE. Immunoprecipitation experiments were also performed with 100 μg of nuclear proteins extracted from U937 cells, with or without 48-h IFN-γ incubation. Nuclear proteins were diluted into radioimmune precipitation buffer, with protease and phosphatase inhibitors, as described (28.Cuatrecasas P. J. Biol. Chem. 1970; 245: 3059-3065Abstract Full Text PDF PubMed Google Scholar), and incubated with either 1 μl of anti-phosphotyrosine antibody (4G10; Upstate Biotechnology, Inc., Lake Placid, NY) or irrelevant antibody (mouse anti-rabbit IgG), for 4 h at 4 °C, followed by a 1-h incubation with 15 μl of 50% staph protein A-Sepharose bead slurry. Beads were washed with radioimmune precipitation buffer, and proteins were eluted in SDS sample buffer, separated on 12% SDS-PAGE, and transferred to nitrocellulose. Blots were probed with HoxA10 antibody (Covance Research Products) or control rabbit preimmune serum. Similar experiments were performed to determine tyrosine phosphorylation of in vitro translated HoxA10. In vitro translated proteins (10 μl), with or withoutYop treatment, and control lysate were diluted into radioimmune precipitation buffer and immunoprecipitated as described above. The proteins were detected by autoradiography of SDS-PAGE. Total cellular RNA was extracted from U937 cells, as described (17.Eklund E.A. Kakar R. J. Biol. Chem. 1997; 272: 9344-9355Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), 48 h after transfection with HoxA10/pSRα, FLAG epitope-tagged HoxA10/pSRα, or control pSRα. Northern blots were performed with 20 μg of total cellular RNA, as described (17.Eklund E.A. Kakar R. J. Biol. Chem. 1997; 272: 9344-9355Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Probes for Northern blots were generated by random primer labeling of cDNAs encoding human gp91 phox, p67 phox and chicken γ-actin, as described (17.Eklund E.A. Kakar R. J. Biol. Chem. 1997; 272: 9344-9355Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The consensus sequence for HoxA10 binding to DNA as a heterodimer with Pbx1 (8.Chang C.-P. Brocchieri L Shen W.-F. Largman C. Cleary M.L. Mol. Cell. Biol. 1996; 16: 1734-1745Crossref PubMed Scopus (250) Google Scholar) consists of a Pbx1 site adjacent to a HoxA10 site: 5′-ATGAT TTATGA-3′ (HoxA10 site in boldface type, the Pbx site in italics) (9.Shen W.-F. Rosenfeld S. Lawrence H.J. Largman C. J. Biol. Chem. 1997; 272: 8198-8296Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Inspection of the promoter regions of the genes encoding gp91 phox (the CYBBgene) and p67 phox (the NCF2 gene) identified sequences similar to the Pbx-HoxA10 consensus (Fig.1 A). The CYBBsequence includes the core sequence preferred in HoxA10 binding site selection experiments (TTAT) and the Pbx consensus (7.Benson G.V. Nguyen T.-H.E. Maas R.L. Mol. Cell. Biol. 1995; 15: 1591-1601Crossref PubMed Google Scholar). However, unlike sequences identified by binding site selection, there was overlap of the Pbx and Hox binding cores. The NCF2 sequence includes an alternative HoxA10 core, identified by binding site selection (TAAT) (7.Benson G.V. Nguyen T.-H.E. Maas R.L. Mol. Cell. Biol. 1995; 15: 1591-1601Crossref PubMed Google Scholar), and a Pbx consensus, altered at position 3. The Pbx-HoxA10-like sequence in the CYBB promoter is within a 30-bp region previously demonstrated to function as a repressor element in undifferentiated cells (16.Lievens P.M.J. Donady J.J. Tufarelli C. Neufeld E.J. J. Biol. Chem. 1995; 270: 12745-12750Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). In EMSA with nuclear proteins from the promyelocytic leukemia cell line PLB985, an unidentified, specific protein complex interacts with this CYBB repressor element (referred to as complex A) (14.Skalnik D.G. Strauss E.C. Orkin S.H. J. Biol. Chem. 1991; 266: 16736-16744Abstract Full Text PDF PubMed Google Scholar, 15.Neufeld E.J. Skalnik D.G. Lievens P.M.-J. Orkin S.H. Nat. Genet. 1992; 1: 50-55Crossref PubMed Scopus (186) Google Scholar). We previously demonstrated that IFN-γ-induced differentiation of PLB985 cells abolishes in vitro binding of complex A to the repressor element, coincident with an increase in CYBB transcription (17.Eklund E.A. Kakar R. J. Biol. Chem. 1997; 272: 9344-9355Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). We hypothesized that HoxA10 is a component of complex A binding theCYBB repressor element and that myeloid differentiation decreases HoxA10 DNA binding. To pursue this hypothesis, we investigated whether in vitro protein binding to the derived Pbx-HoxA10 consensus sequence decreases during IFN-γ-induced myeloid differentiation. In these experiments, we used U937 cells, a monocyte-committed cell line that expresses HoxA10 mRNA (4.Lawrence H.J. Sauvageau G. Ahmadi N. Lopez A.R. Le Beau M.M. Link M. Humphries K. Largman C. Exp. Hematol. 1995; 23: 1160-1166PubMed Google Scholar). IFN-γ treatment of U937 cells results in monocyte differentiation and increased CYBB and NCF2 transcription (17.Eklund E.A. Kakar R. J. Biol. Chem. 1997; 272: 9344-9355Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). EMSAs were performed, using nuclear proteins from U937 cells and a radiolabeled probe with the derived consensus sequence for Pbx-HoxA10 binding (referred to as dsA10 oligonucleotide) (Fig. 1 B). A complex binds to dsA10 that is of similar mobility to complex A generated by U937 nuclear proteins and the homologous CYBBsequence (the dscybbA10 oligonucleotide). Binding of this complex to the dsA10 probe (referred to as complex A1) is competed for by excess, unlabeled, homologous oligonucleotide and by oligonucleotides with the similar sequences from the CYBBand NCF2 genes (the dsncf2A10 oligonucleotide) but not by several dissimilar oligonucleotides (Fig. 1 B). Binding of both complex A1 to the dsA10 probe and of A to the dscybbA10 probe is decreased in EMSA with nuclear proteins from IFN-γ-treated U937 cells, in comparison with nuclear proteins from undifferentiated cells (Fig. 1, B and C). These results are consistent with our previous data with nuclear proteins from PLB985 cells and the dscybbA10 probe (17.Eklund E.A. Kakar R. J. Biol. Chem. 1997; 272: 9344-9355Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). In EMSA with U937 nuclear proteins, binding of the A complex to the dscybbA10 probe was competed for by homologous oligonucleotide and the dsncf2A10 and dsA10 oligonucleotides, but not by unrelated oligonucleotides or by dscybbA10 or dsncf2A10 oligonucleotides with mutations in the TTAT or TAAT sequences (Fig. 1 D). However, there are differences in the complexes shifted by the dsA10 and dscybbA10 probes. Binding of complex A1 to the dsA10 probe is of lower affinity than complex A binding to the dscybbA10 probe (in terms of pmol of shifted probe/μg of nuclear proteins; Fig.1, compare B and C). Also, the dsA10 probe binds several higher mobility protein complexes, in addition to A1, which are competed for by homologous oligonucleotide or dscybbA10 but not dsncf2A10 (Fig. 1 B). These higher mobility complexes may represent proteins encoded by alternatively spliced HoxA10 transcripts (6.Lowney P. Corral J. Detmer K. Le Beau M.M. Deaven L. Lawrence H.J. Largman C. Nucleic Acids Res. 1991; 19: 3443-3449Crossref PubMed Scopus (67) Google Scholar, 7.Benson G.V. Nguyen T.-H.E. Maas R.L. Mol. Cell. Biol. 1995; 15: 1591-1601Crossref PubMed Google Scholar). Alternatively, these complexes may represent proteolytic fragments of HoxA10, although U937 nuclear proteins used in these experiments were tested for integrity in other EMSAs with characterized binding sites. To determine if HoxA10 is a component of either complex A1binding to the dsA10 probe or complex A binding to the dscybbA10 probe, we used an antibody raised to the HoxA10 C terminus that does not recognize other Hox proteins. In preliminary experiments, we determined that this antibody does not recognize recombinant CDP (data not shown). In EMSA with the dsA10 probe and nuclear proteins from U937 cells, binding of complex A1 is disrupted by HoxA10 antibody but not preimmune serum (Fig. 2 A). Antibody to HoxA10 also disrupts binding of complex A to the dscybbA10 probe (Fig. 2 B). Identical results were obtained with nuclear proteins from PLB985 cells (not shown). In addition, HoxA10 antibody disrupts two high mobility complexes binding the dsA10 probe, suggesting that they contain the HoxA10 C terminus, consistent with previously described, alternatively spliced messages (6.Lowney P. Corral J. Detmer K. Le Beau M.M. Deaven L. Lawrence H.J. Largman C. Nucleic Acids Res. 1991; 19: 3443-3449Crossref PubMed Scopus (67) Google Scholar, 7.Benson G.V. Nguyen T.-H.E. Maas R.L. Mol. Cell. Biol. 1995; 15: 1591-1601Crossref PubMed Google Scholar). Interestingly, the dscybbA10 probe does not bind high mobility species, cross-immunoreactive with HoxA10. EMSAs were also performed to determine if Pbx1 is a component of either complex A1 (binding to dsA10) or complex A (binding to dscybbA10). EMSAs were performed with U937 nuclear proteins and Pbx antibodies, with or without blocking peptides. In EMSA with an antibody to a peptide in the N terminus of Pbx1, inconsistent disruption of both complexes A and A1 is demonstrated (not shown). However, in EMSA with an antibody to a peptide in the Pbx C terminus, binding of complex A1 to the dsA10 probe and of complex A to the dscybbA10 probe is disrupted (Fig. 1, C and D). However, these results do not exclude the possibility that additional, unidentified proteins bind to dsA10 or dscybbA10 and participate in these complexes. C