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HOXA9 Activates Transcription of the Gene Encoding gp91 during Myeloid Differentiation

2005; Elsevier BV; Volume: 280; Issue: 13 Linguagem: Inglês

10.1074/jbc.m408138200

ISSN

1083-351X

Autores

Ling Bei, YuFeng Lu, Elizabeth A. Eklund,

Tópico(s)

S100 Proteins and Annexins

Resumo

The CYBB gene encodes gp91Phox; a component of the phagocyte respiratory burst oxidase. CYBB transcription is restricted to myeloid cells differentiated beyond the promyelocyte stage. In undifferentiated myeloid cells, the homeodomain (HD) transcription factor HoxA10 represses CYBB transcription via a cis element in the proximal promoter. During myelopoiesis, phosphorylation of conserved tyrosine residues in the HD decreases HoxA10 binding to this CYBB cis element. In the current studies, we found HoxA9 activates CYBB transcription in differentiated myeloid cells via the same cis element. We find HoxA9-mediated CYBB-transcription requires Pbx1 but is inhibited by Meis1. Additionally, phosphorylation of the conserved HD tyrosines increases HoxA9 binding to the CYBB promoter. The HOXA9 gene is involved in leukemia-associated translocations with the gene encoding Nup98, a nucleopore protein. We find expression of a Nup98-hoxA9 fusion protein blocks HoxA9-induced CYBB transcription in differentiating myeloid cells. In comparison to HoxA9, Nup98-hoxA9 has greater binding affinity for the CYBB cis element, but binding is not altered by HD tyrosine phosphorylation. Therefore, these studies identify CYBB as a common target gene repressed by HoxA10 and activated by HoxA9. These studies also suggest overexpression of Meis1 or Nup98-hoxA9 represses myeloid-specific gene transcription, thereby contributing to differentiation block in leukemogenesis. The CYBB gene encodes gp91Phox; a component of the phagocyte respiratory burst oxidase. CYBB transcription is restricted to myeloid cells differentiated beyond the promyelocyte stage. In undifferentiated myeloid cells, the homeodomain (HD) transcription factor HoxA10 represses CYBB transcription via a cis element in the proximal promoter. During myelopoiesis, phosphorylation of conserved tyrosine residues in the HD decreases HoxA10 binding to this CYBB cis element. In the current studies, we found HoxA9 activates CYBB transcription in differentiated myeloid cells via the same cis element. We find HoxA9-mediated CYBB-transcription requires Pbx1 but is inhibited by Meis1. Additionally, phosphorylation of the conserved HD tyrosines increases HoxA9 binding to the CYBB promoter. The HOXA9 gene is involved in leukemia-associated translocations with the gene encoding Nup98, a nucleopore protein. We find expression of a Nup98-hoxA9 fusion protein blocks HoxA9-induced CYBB transcription in differentiating myeloid cells. In comparison to HoxA9, Nup98-hoxA9 has greater binding affinity for the CYBB cis element, but binding is not altered by HD tyrosine phosphorylation. Therefore, these studies identify CYBB as a common target gene repressed by HoxA10 and activated by HoxA9. These studies also suggest overexpression of Meis1 or Nup98-hoxA9 represses myeloid-specific gene transcription, thereby contributing to differentiation block in leukemogenesis. Hox proteins are homeodomain transcription factors that are highly conserved from Drosophila to human (1.Acampora D. D'Esposito M. Faiella A. Pannese M. Migliaccio E. Morelli F. Stornaiuolo A. Nitro V. Simeone A. Boncinelli A. Nucleic Acids Res. 1989; 17: 10385-10400Crossref PubMed Scopus (269) Google Scholar). Human HOX genes are arranged in four paralog groups (HoxA–D) on four chromosomes. During definitive hematopoiesis, HOX gene transcription proceeds 3′ to 5′ through each paralog group (1.Acampora D. D'Esposito M. Faiella A. Pannese M. Migliaccio E. Morelli F. Stornaiuolo A. Nitro V. Simeone A. Boncinelli A. Nucleic Acids Res. 1989; 17: 10385-10400Crossref PubMed Scopus (269) Google Scholar). Therefore, HOX1–4 genes are maximally transcribed in hematopoietic stem cells and HOX7–13 in committed progenitors (2.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). Recent investigations indicate "Abd" HoxA proteins (HoxA7–11) are of particular interest to myelopoiesis and myeloid leukemogenesis. One HoxA protein that has received particular attention is HoxA9. Although few genuine HoxA9 target genes have been identified, much is known about the impact of this protein on myelopoiesis and leukemogenesis. For example, HoxA9 overexpression immortalizes cultured murine bone marrow myeloid cells, but does not block ex vivo differentiation (4.Calvo K.R. Sykes D.B. Pasillas M. Kapms M.P. Mol. Cell. Biol. 2000; 20: 3274-3285Crossref PubMed Scopus (117) Google Scholar). Similarly, HoxA9 overexpression expands the progenitor pool but does not block differentiation in murine bone marrow transplantation experiments (5.Thorsteinsdottir U. Mamo A. Kroon E. Jerome L. Bijl J. Lawrence H.J. Humphries K. Sauvageau G. Blood. 2002; 99: 121-129Crossref PubMed Scopus (292) Google Scholar). In such studies, HoxA9 overexpression induces leukemia only inconsistently, after a long latency (5.Thorsteinsdottir U. Mamo A. Kroon E. Jerome L. Bijl J. Lawrence H.J. Humphries K. Sauvageau G. Blood. 2002; 99: 121-129Crossref PubMed Scopus (292) Google Scholar, 6.Kroon E. Krosl J. Thorsteindottir U. Banban S. Buchberg A.M. Sauvageau G. EMBO J. 1998; 17: 3714-3725Crossref PubMed Scopus (549) Google Scholar). Of interest to the current studies, HoxA9 overexpression in murine myeloid cells does not impair granulocyte-colony stimulating factor- or macrophage-colony stimulating factor-induced expression of gp91Phox, a phagocyte respiratory burst oxidase protein (4.Calvo K.R. Sykes D.B. Pasillas M. Kapms M.P. Mol. Cell. Biol. 2000; 20: 3274-3285Crossref PubMed Scopus (117) Google Scholar). Gp91Phox is encoded by the CYBB gene, and transcription of this gene is restricted to myeloid cells differentiated beyond the promyelocyte stage (7.Royer-Pokora B. Kunkel L.M. Monaco A.P. Goff S.C. Newburger P.E. Baihner L. Cole F.S. Curnutte J.T. Orkin S.H. Nature. 1986; 322: 32-38Crossref PubMed Scopus (596) Google Scholar, 8.Newburger P.E. Dai Q. Whitney C. J. Biol. Chem. 1991; 266: 16171-16177Abstract Full Text PDF PubMed Google Scholar). Other investigations associate HoxA9 with myeloid leukemia. For example, HoxA9 overexpression is highly correlated with myeloid phenotype in pediatric leukemia (9.Golub T.R. Slonim D.K. Tamoy P. Huard C. Gassenbeek M. Mesirov J.P. Coller H. Loh M.L. Downing J.R. Caligiuri M.A. Bloomfield C.D. Landr E.S. Science. 1999; 286: 531-537Crossref PubMed Scopus (9261) Google Scholar). Additionally, expression of leukemia-associated MLL (mixed lineage leukemia) fusion proteins increases expression of HoxA7, -9, -10, and Meis1 (also a homeodomain (HD) 1The abbreviations used are: HD, homeodomain; HA, hemagglutinin; IFN, interferon; EMSA, electrophoretic mobility shift assay; CMV, cytomegalovirus; CAT, chloramphenicol acetyl transferase. protein) in human leukemia and murine models (10.Armstrong S.A. Staunton J.E. Silverman L.B. Nat. Genet. 2002; 30: 41-47Crossref PubMed Scopus (1617) Google Scholar, 11.Corral J. Lavenir I. Impey H. Cell. 1996; 85: 853-861Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). However, MLL fusion proteins also induce leukemia in HoxA9-deficient mice, although the phenotype is "less myeloid" (12.Kumar A.R. Hudson W.A. Chen W. Nishiuchi R. Yao Q. Kersey J.H. Blood. 2004; 103: 1823-1828Crossref PubMed Scopus (110) Google Scholar). The HOXA9 gene is itself involved in leukemia-associated translocations. For example, translocations involving HOXA9 and the gene encoding the nucleopore protein Nup98 occur in some myeloid leukemias (13.Borrow J. Shearman A.M. Stanton V.P. Becher R. Collins T. Williams A.J. Dube I. Katz F. Kwong Y.L. Morris C. Ohuashiki K. Toyama K. Rowley J. Housman D.E. Nat. Genet. 1996; 12: 159-167Crossref PubMed Scopus (405) Google Scholar). These leukemias express a fusion protein with N-terminal Nup98 domains and the HoxA9 HD. Nup98-HoxA9 overexpression rapidly induces leukemia in murine bone marrow transplantation experiments (14.Kroon E. Thorsteinsdottir U. Mayotte N. Nakamura T. Sauvageau G. EMBO J. 2001; 20: 350-361Crossref PubMed Scopus (184) Google Scholar). In microarray experiments, differences in gene expression were identified in myeloid leukemia cell lines overexpressing HoxA9 versus Nup98-A9 (3.Ghannam G. Takeda A. Camarata T. Maoore M.A. Viale A. Yaseen N.R. J. Biol. Chem. 2004; 279: 866-875Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). These results suggest HoxA9 and Nup98-hoxA9 either interact with different target genes, exhibit differential impact on myelopoiesis (and therefore secondarily on gene expression), or both. HoxA9 binds DNA as a multiprotein complex with Pbx and/or Meis HD proteins. A DNA-binding site consensus sequence has been derived for HoxA9/Pbx, but few genuine target genes have been identified (15.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). Function of DNA-bound HoxA9/Pbx may vary during differentiation, because Pbx proteins interact with either co-repressors or co-activators in a signal-dependant manner (16.Saleh M. Rambaldi I. Yang X.-J. Featherstone M.S. Mol. Cell. Biol. 2000; 20: 8623-8633Crossref PubMed Scopus (168) Google Scholar). HoxA9 also partners with Meis1 to bind DNA, with and without Pbx1 (17.Shen W.F. Rozenfeld S. Kwong A. Komuves L.G. Lawrence H.J. Largman D. Mol. Cell. Biol. 1999; 19: 3051-3061Crossref PubMed Scopus (215) Google Scholar). Overexpression of HoxA9 plus Meis1 (but not HoxA9 plus Pbx1) rapidly induces myeloid leukemia in murine bone marrow transplantation experiments (6.Kroon E. Krosl J. Thorsteindottir U. Banban S. Buchberg A.M. Sauvageau G. EMBO J. 1998; 17: 3714-3725Crossref PubMed Scopus (549) Google Scholar). Overexpression of HoxA9 plus Meis1 also blocks ex vivo cytokine-induced myeloid differentiation (18.Calvo K.R. Koepfler P.S. Sykes D.B. Pasillas M.P. Kamps M.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13120-13125Crossref PubMed Scopus (82) Google Scholar). Of interest to the current studies, this includes blocked induction of gp91Phox expression in differentiating cells (18.Calvo K.R. Koepfler P.S. Sykes D.B. Pasillas M.P. Kamps M.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13120-13125Crossref PubMed Scopus (82) Google Scholar). HoxA9 and -10 are similarly expressed during myeloid differentiation and in myeloid leukemia (2.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). Additionally, both interact with Pbx proteins and recognize similar DNA-binding consensus sequences (15.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). We previously found HoxA10 represses transcription of the CYBB gene via a cis element homologous to the Hox/Pbx-DNA binding consensus (19.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). HoxA10 repression of the CYBB gene is Pbx1-independent and occurs in undifferentiated myeloid cells (20.Lu Y. Goldenberg I. Bei L. Andrijec J. Eklund E.A. J. Biol. Chem. 2003; 278: 47792-47802Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). During differentiation, phosphorylation of conserved tyrosine residues in the HoxA10 HD decreases HoxA10 interaction with the CYBB cis element, decreasing repression (21.Eklund E.A. Goldenberg I. Lu Y. Andrejic J. Kakar R. J. Biol. Chem. 2002; 277: 36878-36888Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Although conserved through the HD and Pbx interaction domains, HoxA9 and -10 proteins are otherwise divergent. Perhaps consistent with this, overexpressed HoxA10 induces myeloid leukemia in a Meis1-independent manner in murine bone marrow transplantation experiments (22.Thorsteinsdottir U. Sauvageau G. Hough M.R. Dragowska W. Lansdorp P.M. Lawrence H.J. Largman E. Humphries R.K. Mol. Cell. Biol. 1997; 17: 495-505Crossref PubMed Scopus (296) Google Scholar). One mechanism contributing to leukemogenesis is the HoxA10 differentiation block due to repression of myeloid specific genes (i.e. the CYBB gene). However, repression activity is abrogated by cytokine-induced HoxA10 tyrosine phosphorylation. This suggests that differentiation block by overexpressed HoxA10 would require additional mutations in cytokine signaling pathways. Consistent with this, leukemia induction due to HoxA10 overexpression has a latency of several months in murine bone marrow transplantation experiments (22.Thorsteinsdottir U. Sauvageau G. Hough M.R. Dragowska W. Lansdorp P.M. Lawrence H.J. Largman E. Humphries R.K. Mol. Cell. Biol. 1997; 17: 495-505Crossref PubMed Scopus (296) Google Scholar). Although this latency is longer than in experiments with Nup98-A9 or Meis1 plus HoxA9 overexpression, it is rapid and reproducible in comparison to HoxA9-induced leukemia (6.Kroon E. Krosl J. Thorsteindottir U. Banban S. Buchberg A.M. Sauvageau G. EMBO J. 1998; 17: 3714-3725Crossref PubMed Scopus (549) Google Scholar). Based on these results, the current studies investigate whether CYBB is a HoxA9 target gene. The first goal of these investigations is to determine whether HoxA9 and HoxA10 have redundant function with respect to regulation of myeloid-specific gene transcription during differentiation. Because of specific structural similarities and differences between HoxA9 and HoxA10, the second goal of these studies is to determine whether HD tyrosine phosphorylation similarly regulates DNA-binding affinity of the two proteins. Also, any model of HoxA9 function must address differences between HoxA9 and Nup98-A9, and the role of Meis1 in modulating HoxA9 activity. Our third goal is to address these issues in the context of CYBB gene regulation. Plasmids and PCR Mutagenesis—An HA-tagged HoxA9 cDNA was obtained from Dr. Corey Largman (University of California Veterans Affairs Medical Center, San Francisco, CA), and subcloned into the pcDNAamp vector (HA-HoxA9/pcDNAamp). HoxA9 (not tagged) was obtained by PCR, and also subcloned into the pcDNAamp vector (HoxA9/pcDNAamp). The cDNA for Nup98 and Nup98a-A9 were obtained from Dr. Nabel Yaseen (Northwestern University, Chicago, IL). These cDNAs were subcloned into the pcDNAamp vector with and without HA epitope tags. The cDNA for Pbx1a was obtained from M. Cleary (University of California, San Francisco, CA) and subcloned into the pSRα vector, as previously described (19.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The cDNA for Meis1b was obtained from Dr. Jeffrey Lawrence (University of California, San Francisco, CA) and subcloned into the pSRα vector. A HoxA9 cDNA sequence with point mutation of two residues in the Pbx interaction domain (residues 198 and 199 mutated to alanine, referred to as 198/199 HoxA9) or the two tyrosine residues in the homeodomain (tyrosines 212 and 225 to phenylalanine, referred to as Y212F/Y225F HoxA9) were generated by site-directed mutagenesis using the Clontech QuikChange protocol. These mutant cDNAs were subcloned into the pcDNAamp vector for in vitro translation and transfection experiments. Mutant cDNAs were sequenced to verify that no unintended mutations had been introduced. Reporter gene assays were performed with a promoter/reporter construct with 450 bp of the promoter from the gene encoding gp91Phox (the CYBB gene) in the pCATE vector (Promega). This construct has differentiation-inducible reporter activity, as previously described (20.Lu Y. Goldenberg I. Bei L. Andrijec J. Eklund E.A. J. Biol. Chem. 2003; 278: 47792-47802Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 21.Eklund E.A. Goldenberg I. Lu Y. Andrejic J. Kakar R. J. Biol. Chem. 2002; 277: 36878-36888Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). For other assays, an artificial promoter construct was generated with four copies of a CYBB cis element homologous to the Hox/Pbx binding consensus linked to a minimal promoter and a CAT reporter was used (the pTATACAT vector) (23.Scholer H.R. Balling R. Hazopoulos A.K. Suzuki N. Gruss P. EMBO J. 1989; 8: 2551-2558Crossref PubMed Scopus (271) Google Scholar). This construct is referred to as cybbTATACAT and has been previously described (19.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 20.Lu Y. Goldenberg I. Bei L. Andrijec J. Eklund E.A. J. Biol. Chem. 2003; 278: 47792-47802Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 24.Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). Oligonucleotides—Oligonucleotides were synthesized by the Core Facility of the University of Alabama, Birmingham Comprehensive Cancer Center, or the Core Facility of the Robert H. Lurie Comprehensive Cancer Center at Northwestern University. Oligonucleotides: derived consensus sequence for HoxA10/Pbx binding (dsA10); 5′-tgcgatgatttatgaccgc-3′, the Hox/Pbx-binding sequence from the CYBB promoter (–106 to –125 bp) (dscybb) (25.Skalnik D.G. Strauss E.C. Orkin S.H. J. Biol. Chem. 1991; 266: 16736-16744Abstract Full Text PDF PubMed Google Scholar); 5′-ccaatgattattagccaatt-3′. In these oligonucleotides, the Hox core is in boldface, the Pbx core is in italics, and ccaat boxes are underlined. Oligonucleotide nucleotides used in PCR for chromatin immunoprecipitation experiments were designed to flank the Hox/Pbx binding CYBB cis element: 5′ PCR oligonucleotide (–133 to –106); 5′-tcagttgaccaatgattattagccaatt-3′,3′ PCR oligonucleotide (–6 to –32); and 5′-ctatgcttcttcttccaatgaccaaat-3′. Myeloid Cell Line Culture—The human myelomonocytic cell line U937 (26.Larrick J.W. Anderson S.J. Koren H.S. J. Immunol. 1980; 125: 6-14Crossref PubMed Google Scholar) was obtained from Andrew Kraft (Medical University of South Carolina, Charleston, SC). Cells were maintained and differentiated as described previously (19.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). U937 cells were differentiated for 48 h with 500 units/ml human recombinant IFNγ (Roche Applied Science). Electrophoretic Mobility Shift Assays—Nuclear extract proteins were prepared by the method of Dignam (27.Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1993; 11: 1475-1479Crossref Scopus (9164) Google Scholar) with protease inhibitors (as described) (28.Kautz B. Kakar R. David E. Eklund E.A. J. Biol. Chem. 2001; 276: 37868-37878Abstract Full Text Full Text PDF PubMed Google Scholar). Oligonucleotides probes were prepared, and EMSA and antibody supershift assays were performed as described (19.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Antibody to HoxA9, Pbx1a, Meis1, and HA epitope tag were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The HoxA9 antibody recognizes a peptide present in Nup98-hoxA9 but is not cross-reactive with other Hox proteins. In Vitro Translated Proteins—In vitro transcribed HoxA9, Y212F/Y225F HoxA9, and Nup98-A9 mRNA were generated from linearized template DNA using the Riboprobe System, according to manufacture's instructions (Promega, Madison, WI). In vitro translated proteins were generated in rabbit reticulocyte lysate, according to manufacturer's instructions (Promega). Control lysates were generated in similar reactions in the absence of input RNA. In vitro translated proteins were tyrosine de-phosphorylated with Yop protein tyrosine phosphatase (New England Biolabs). Proteins (10 μl of in vitro translated protein) were incubated 30 min at 30 °C, in a 20-μl reaction volume with 50 units of Yop and 1× reaction buffer. Control proteins were incubated similarly in 1× reaction buffer without Yop. De-phosphorylation of [35S]methionine-labeled, in vitro translated proteins was verified by SDS-PAGE and autoradiography of anti-phospho tyrosine immunoprecipitated proteins, as previously described (21.Eklund E.A. Goldenberg I. Lu Y. Andrejic J. Kakar R. J. Biol. Chem. 2002; 277: 36878-36888Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). EMSA with in vitro translated proteins was performed as described (21.Eklund E.A. Goldenberg I. Lu Y. Andrejic J. Kakar R. J. Biol. Chem. 2002; 277: 36878-36888Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The amounts of in vitro translated proteins in DNA binding reactions were equalized by SDS-PAGE of [35S]methionine-labeled proteins. Transfection and Reporter Gene Assays—Cells were transfected by electroporation as described (21.Eklund E.A. Goldenberg I. Lu Y. Andrejic J. Kakar R. J. Biol. Chem. 2002; 277: 36878-36888Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). U937 cells (32 × 106 per sample) were transfected with 50 μg of 450pCATE or pCATE (see above); 60 μg of pcDNAamp, wild type or mutant HoxA9/pcDNAmp, or Nup98-A9/pcDNAamp; 30 μg of pSRα, Pbx1a/pSRα, or Meis1b/pSRα; and 15 μg of p-CMVβ-gal (to normalize for transfection efficiency). In other experiments, cells were transfected with the artificial promoter constructs 70 μg of cybbTATACAT, or control TATACAT (see above); 60 μg of pcDNAamp, wild type or mutant HoxA9/pcDNAmp, Nup98-A9/pcDNAamp, or Nup98 fragment/pcDNAamp; 30 μg of pSRα, Pbx1a/pSRα, or Meis1b/pSRα; and 15 μg of p-CMVβ-gal (to normalize for transfection efficiency). Transfectants were incubated for 24 h at 37 °C, 5% CO2 followed by 48 h with or without IFNγ (500 units/ml). Preparation of cell extracts, β-galactosidase and chloramphenicol acetyl transferase (CATs) assays were performed as described (29.Seed B. Sheen J.-Y. Gene (Amst.). 1988; 67: 271-275Crossref PubMed Scopus (830) Google Scholar, 30.Sambrook H. Fritch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). To generate lines stably expressing HoxA9 or Nup98-hoxA9, U937 cells were transfected with the mammalian expression vector pcDNAamp with HoxA9 or Nup98-hoxA9 with and without an HA-epitope tag or empty vector control plus the pSRα vector (for antibiotic selection). Cells were transfected and selected in G418 as previously described (28.Kautz B. Kakar R. David E. Eklund E.A. J. Biol. Chem. 2001; 276: 37868-37878Abstract Full Text Full Text PDF PubMed Google Scholar). Stable transfectants were analyzed for HoxA9 or Nup98-hoxA9 overexpression by Western blot. Each experiment was repeated with two or three different transfectant pools and representative results are shown. Immunoprecipitation and Western Blotting—Immunoprecipitation was performed with 300 μg of nuclear proteins from U937 cells. Proteins were immunoprecipitated with anti-HoxA9 antibody (Santa Cruz Biotechnology) or irrelevant rabbit anti-mouse antibody. Proteins were immunoprecipitated for 4 h at 4 °C with 2 μl of HoxA9 antibody or 2 μl of control rabbit antibody followed with 1-h incubation with 30 μlof50% Staphylococcus protein A-Sepharose bead slurry, as described (21.Eklund E.A. Goldenberg I. Lu Y. Andrejic J. Kakar R. J. Biol. Chem. 2002; 277: 36878-36888Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Immunoprecipitated proteins were washed with radioimmune precipitation assay buffer, eluted in SDS sample buffer, and separated on 12% SDS-PAGE. Immunoprecipitated proteins were transferred to nitrocellulose, and blots were probed with anti-HoxA9 or anti-phospho-tyrosine antibody (Santa Cruz Biotechnology). Immunoreactive proteins were detected by chemiluminescence, according to manufacturer's instructions (Amersham Biosciences). Chromatin Immunoprecipitation—Stably transfected U937 cells were cultured with or without IFNγ for 48 h. Cells for chromatin immunoprecipitation were incubated with formaldehyde prior to lysis, and lysates were sonicated to generate chromatin fragments with an average size of 2.0 kb, as described (31.Wells J. Farnham P.J. Methods. 2002; 26: 37-47Crossref PubMed Scopus (208) Google Scholar). Lysates underwent one round of immunoprecipitation with antibody to HoxA9, Meis1, or HA-epitope tag (Santa Cruz Biotechnology), as indicated under "Results." Control immunoprecipitation was performed with an irrelevant glutathione S-transferase antibody, as described (31.Wells J. Farnham P.J. Methods. 2002; 26: 37-47Crossref PubMed Scopus (208) Google Scholar). Immunoprecipitated chromatin was analyzed by PCR for antibody-specific co-precipitation of the Hox/Pbx binding cis element in the CYBB gene. Input chromatin was a positive control and chromatin precipitated by glutathione S-transferase antibody was a negative control. PCR products were analyzed by acrylamide gel electrophoresis. The identity of the PCR product was verified by subcloning into a plasmid vector followed by dideoxy sequencing. HoxA9 Overexpression Increases CYBB Transcription in Differentiating U937 Myeloid Cells—Previously, we found HoxA10 represses CYBB transcription in undifferentiated U937 myeloid cells (19.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). This repression requires HoxA10 interaction with a CYBB cis element homologous to the derived Hox/Pbx DNA-binding consensus sequence (referred to as the Hox/Pbx consensus-like CYBB cis element). U937 myeloid leukemia cells represent a pro-monocyte stage of differentiation and express very low levels of gp91Phox. IFNγ differentiation increases CYBB transcription and gp91Phox expression in these cells (19.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 24.Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar). Also, HoxA10 becomes tyrosine-phosphorylated during U937 differentiation (19.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Therefore, because tyrosine phosphorylation decreases HoxA10 binding affinity for the CYBB cis element, IFNγ treatment decreases HoxA10 repression activity. Because the DNA-binding consensus sequences for HoxA9 and HoxA10 are similar, the current studies investigated the impact of HoxA9 on endogenous gp91Phox mRNA abundance and CYBB promoter activity. For these studies, U937 cells were stably transfected with a vector to express HoxA9 or empty vector control. Transfectant pools were selected (instead of clones) to compensate for potential integration site effects and three independent pools were studied. In initial experiments, we analyzed HoxA9 protein expression in these transfectants. Consistent with previous reports (32.Pinsonneault J. Florence B. Vaessin H. McGinnis W. EMBO J. 1997; 16: 2032-2042Crossref PubMed Scopus (125) Google Scholar), we found little HoxA9 expression in control U937 transfectants. In contrast, HoxA9 protein abundance was increased in stable transfectants with HoxA9 expression vector (Fig. 1A). Therefore, we investigated the impact of HoxA9 overexpression on gp91Phox mRNA abundance in U937 cells, with and without IFNγ differentiation. Total cellular RNA was isolated and analyzed by Northern blot for gp91Phox message (Fig. 1B). We found HoxA9 overexpression does not alter gp91Phox expression in undifferentiated U937 cell in comparison to control transfectants. In contrast, overexpression of HoxA9 in IFNγ-treated cells increases gp91Phox expression in comparison to similarly treated control U937 transfectants. To determine whether this increase in gp91Phox mRNA represents increased CYBB transcription, the impact of HoxA9 on CYBB promoter activity was determined. U937 cells were transfected with a CAT reporter construct with 450 bp of the proximal CYBB promoter (previously described) (20.Lu Y. Goldenberg I. Bei L. Andrijec J. Eklund E.A. J. Biol. Chem. 2003; 278: 47792-47802Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 21.Eklund E.A. Goldenberg I. Lu Y. Andrejic J. Kakar R. J. Biol. Chem. 2002; 277: 36878-36888Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) or reporter vector control. This 450 bp has IFNγ-inducible promoter activity and includes the Hox/Pbx consensus-like cis element, discussed above. U937 cells were co-transfected with vectors to overexpress various combinations of HoxA9 and Pbx1a. Transfectants were analyzed for reporter expression with and without IFNγ differentiation. In undifferentiated U937 transfectants, overexpression of HoxA9 did not alter CYBB promoter activity, with or without Pbx1a co-overexpression (Fig. 1C). Consistent with our previous results (24.Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105Crossref PubMed Google Scholar), IFNγ increased CYBB promoter activity in U937 cells (Fig. 1C). In addition, HoxA9 overexpression induced a significant increase in CYBB promoter activity in differentiated transfectants (n = 7, p < 0.001). This HoxA9-transcriptional effect was significantly augmented by co-overexpression of Pbx1a in IFNγ-treated transfectants (n = 7, p < 0.001). Nup98-HoxA9 Blocks Induction of CYBB Transcription during U937 Differentiation—Overexpression of the Nup98-hoxA9 leukemia-associated fusion protein induces acute myeloid leukemia in murine bone marrow transplantation experiments (19.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Any model of HoxA9 function must address differences between overexpressed HoxA9 and this leukemia-associated protein. Since

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