HoxA10 Activates Transcription of the Gene Encoding Mitogen-activated Protein Kinase Phosphatase 2 (Mkp2) in Myeloid Cells
2007; Elsevier BV; Volume: 282; Issue: 22 Linguagem: Inglês
10.1074/jbc.m610556200
ISSN1083-351X
AutoresHao Wang, YuFeng Lu, Weiqi Huang, Eleftherios T. Papoutsakis, Peter G. Fuhrken, Elizabeth A. Eklund,
Tópico(s)Ubiquitin and proteasome pathways
ResumoHoxA10 is a homeodomain transcription factor that is frequently overexpressed in human acute myeloid leukemia. In murine bone marrow transplantation studies, HoxA10 overexpression induces a myeloproliferative disorder with accumulation of mature phagocytes in the peripheral blood and tissues. Over time, differentiation block develops in these animals, resulting in acute myeloid leukemia. In immature myeloid cells, HoxA10 represses transcription of some genes that confer the mature phagocyte phenotype. Therefore, overexpressed HoxA10 blocks differentiation by repressing myeloid-specific gene transcription in differentiating myeloid cells. In contrast, target genes involved in myeloproliferation due to HoxA10 overexpression have not been identified. To identify such genes, we screened a CpG island microarray with HoxA10 co-immunoprecipitating chromatin. We identified the DUSP4 gene, which encodes mitogen-activated protein kinase phosphatase 2 (Mkp2), as a HoxA10 target gene. We analyzed the DUSP4 5′-flank and identified two proximal-promoter cis elements that are activated by HoxA10. We find that DUSP4 transcription and Mkp2 expression decrease during normal myelopoiesis. However, this down-regulation is impaired in myeloid cells overexpressing HoxA10. In hematopoietic cells, c-Jun N-terminal kinases (Jnk) are the preferred substrates for Mkp2. Therefore, Mkp2 inhibits apoptosis by dephosphorylating (inactivating) Jnk. Consistent with this, HoxA10 overexpression decreases apoptosis in differentiating myeloid cells. Therefore, our studies identify a mechanism by which overexpressed HoxA10 contributes to inappropriate cell survival during myelopoiesis. HoxA10 is a homeodomain transcription factor that is frequently overexpressed in human acute myeloid leukemia. In murine bone marrow transplantation studies, HoxA10 overexpression induces a myeloproliferative disorder with accumulation of mature phagocytes in the peripheral blood and tissues. Over time, differentiation block develops in these animals, resulting in acute myeloid leukemia. In immature myeloid cells, HoxA10 represses transcription of some genes that confer the mature phagocyte phenotype. Therefore, overexpressed HoxA10 blocks differentiation by repressing myeloid-specific gene transcription in differentiating myeloid cells. In contrast, target genes involved in myeloproliferation due to HoxA10 overexpression have not been identified. To identify such genes, we screened a CpG island microarray with HoxA10 co-immunoprecipitating chromatin. We identified the DUSP4 gene, which encodes mitogen-activated protein kinase phosphatase 2 (Mkp2), as a HoxA10 target gene. We analyzed the DUSP4 5′-flank and identified two proximal-promoter cis elements that are activated by HoxA10. We find that DUSP4 transcription and Mkp2 expression decrease during normal myelopoiesis. However, this down-regulation is impaired in myeloid cells overexpressing HoxA10. In hematopoietic cells, c-Jun N-terminal kinases (Jnk) are the preferred substrates for Mkp2. Therefore, Mkp2 inhibits apoptosis by dephosphorylating (inactivating) Jnk. Consistent with this, HoxA10 overexpression decreases apoptosis in differentiating myeloid cells. Therefore, our studies identify a mechanism by which overexpressed HoxA10 contributes to inappropriate cell survival during myelopoiesis. The 39 human and murine HOX genes are arranged in four paralog groups on four different chromosomes These genes encode homeodomain transcription factors that are highly conserved from Drosophila to humans. During embryogenesis, HOX gene transcription is activated 3′ to 5′ with the 3′-most genes regulating cephlad development and the 5′-most genes regulating caudal development (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 (268) Google Scholar). Transcription of the HOX genes is also tightly regulated during definitive hematopoiesis. Genes 3′ in the locus (HOX1 to -4) are actively transcribed in hematopoietic stem cells, and more 5′ genes (HOX5 to -11) are actively transcribed in lineage-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 (440) Google Scholar). Therefore, HOX1 to -4 genes are transcribed in CD34+CD38– cells. In contrast, HOX5 to -11 genes (also referred to as ABD HOX genes) are transcribed throughout the CD34+ compartment and down-regulated in CD34– cells. In poor prognosis AML, the normal decrease in HoxA7, A9, and A10 expression in CD34– cells does not occur (3.Kawagoe H. Humphries R.K. Blair A. Sutherland H.J. Hogge D.E. Leukemia. 1999; 13: 687-698Crossref PubMed Scopus (181) Google Scholar, 4.Drabkin H.A. Parsy C. Gerguson K. Guilhot F. Lacotte L. Roy R. Zeng C. Baron A. Hunger S.P. Varella-Garcia M. Gemmill R. Brizard F. Brizard A. Roche J. Leukemia. 2002; 16: 186-195Crossref PubMed Scopus (145) Google Scholar). Consistent with this, mice transplanted with bone marrow overexpressing either HoxA9 or HoxA10 rapidly develop a myeloproliferative disorder, which evolves to acute myeloid leukemia over time. This myeloproliferative disorder is characterized by an increase in mature phagocytes in the peripheral blood and tissues. Murine studies also indicate that forced overexpression of HoxA9 or 10 has a common effect of expanding the bone marrow myeloid progenitor pool (5.Calvo K.R. Sykes D.B. Pasillas M. Kamps M.P. Mol. Cell. Biol. 2000; 20: 3274-3285Crossref PubMed Scopus (117) Google Scholar, 6.Lawrence H.J. Helgason C.D. Sauvageau G. Fong S. Izon D.J. Humphries R.K. Largman C. Blood. 1997; 89: 1922-1930Crossref PubMed Google Scholar, 7.Buske C. Feuring-Buske M. Antonchuk J. Rosten P. Hogge D.E. Eaves C.J. Humphries R.K. Blood. 2001; 97: 2286-2292Crossref PubMed Scopus (89) Google Scholar). In contrast, the effect of HoxA9 and HoxA10 on myeloid differentiation is nonredundant. HoxA9 expression appears to be necessary for acquisition of the myeloid phenotype in normal and leukemic cells (5.Calvo K.R. Sykes D.B. Pasillas M. Kamps M.P. Mol. Cell. Biol. 2000; 20: 3274-3285Crossref PubMed Scopus (117) Google Scholar). In contrast, HoxA10 overexpression is associated with differentiation block in animal studies and in vitro (7.Buske C. Feuring-Buske M. Antonchuk J. Rosten P. Hogge D.E. Eaves C.J. Humphries R.K. Blood. 2001; 97: 2286-2292Crossref PubMed Scopus (89) Google Scholar). Despite the many elegant studies of the role of Abd HoxA proteins in myelopoiesis and leukemogenesis, relatively few genuine target genes have been identified for these homeodomain transcription factors. To address this issue, initial studies were performed to derive consensus sequences for HoxA protein-DNA binding. These studies identified consensus sequences for DNA binding of Hox-Pbx heterodimers (8.Chang C.P. Brocchieri L. Shen W.F. Largman C. Cleary M.L. Mol. Cell. Biol. 1996; 16: 1734-1745Crossref PubMed Scopus (247) Google Scholar). Pbx proteins are homeodomain transcription factors that increase the affinity of Hox proteins for DNA-binding sites. Based on the results of these studies, HoxA9 and HoxA10 target genes were identified that encode proteins involved in phagocyte functions. For example, a HoxA9/Pbx1 heterodimer activates homologous cis elements in the genes encoding the respiratory burst oxidase proteins gp91phox and p67phox in differentiating myeloid cells (9.Bei L. Lu Y.F. Eklund E.A. J. Biol. Chem. 2005; 280: 12359-12370Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Additional studies determined that transcription of these two oxidase genes is repressed by interaction of a HoxA10/Pbx1 heterodimer with the same cis elements in undifferentiated myeloid cells (10.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 11.Eklund E.A. Goldenberg I. Lu Y. Andrejic J. Kakar R. J. Biol. Chem. 2002; 39: 36878-36888Abstract Full Text Full Text PDF Scopus (49) Google Scholar, 12.Lindsey S. Zhu C. Lu Y.F. Eklund E.A. J. Immunol. 2005; 175: 5269-5279Crossref PubMed Scopus (29) Google Scholar). Interestingly, a HoxA10/Pbx2 heterodimer activates transcription of the gene encoding β3 integrin in endometrial cells (13.Daftary G.S. Troy P.J. Bagot C.N. Young S.L. Taylor H.S. Mol. Endocrinol. 2002; 16: 571-579Crossref PubMed Scopus (160) Google Scholar). Since β3 integrin is expressed in myeloid cells, these results suggest the possibility that HoxA10 is a multifunction transcription factor during myelopoiesis. In contrast, relatively few target genes that encode proteins involved in regulating cell proliferation or survival have been identified for any Abd HoxA protein. In hematopoietic stem cells and myeloid progenitor cells, these functions are regulated by a variety of different mechanisms. To identify HoxA10 target genes involved in myeloproliferation in differentiating myeloid cells, we coupled chromatin co-immunoprecipitation with high throughput screening of a CpG island microarray (14.Oberley M.J. Farnham P.J. Methods Enzymol. 2003; 371: 577-596Crossref PubMed Scopus (56) Google Scholar). By this approach, we identified DUSP4 as a potential HoxA10 target gene in undifferentiated myeloid cells. This gene encodes Dusp4 (dual specific phosphatase 4), also known as Mkp2 (mitogen-activated protein kinase phosphatase 2). Mkp1 and -2 are structurally related proteins that are expressed in hematopoietic cells but have different substrate specificities. In vivo, Mkp2 preferentially dephosphorylates (and therefore inactivates) c-Jun N-terminal kinases 1 and 2 (Jnk1 and -2) but not p38 or extracellular signal-regulated kinase mitogen-activated protein kinases (15.Cadalbert L. Sloss C.M. Cameron P. Plevin R. Cell. Signal. 2005; 17: 1254-1264Crossref PubMed Scopus (44) Google Scholar). Jnk1 is activated (phosphorylated) in response to genotoxic stress or hematopoietic cytokines and mediates apoptosis in differentiating myeloid cells (16.Yoshizumi M. Abe J. Haendlerer J. Huang Q. Berk B.C. J. Biol. Chem. 2000; 275: 11706-11712Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 17.Terada K. Kaziro Y. Satoh T. J. Biol. Chem. 1997; 272: 4544-4548Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Therefore, if HoxA10 activates DUSP4 transcription, HoxA10 overexpression in myeloid malignancy might increase cell survival during cytokine-induced myelopoiesis. In this study, we investigate the role of HoxA10 in DUSP4 transcription and in regulation of apoptosis. DUSP4 5′-Flank Reporter Constructs−Genomic clones for the DUSP4 5′-flank were obtained by PCR using genomic DNA isolated from U937 cells. The 3′-primer for each of the reactions encompassed +30 to 0 relative to the ATG start codon. A series of 5′-primers were generated to amplify 5′-flank sequences, including –2933 to +30 bp, –1772 to +30 bp, –1722 to +30 bp, –1205 to +30 bp, and –1142 to +30 bp. These genomic sequences were subcloned into the pCATE reporter vector (Stratagene, Cedar Creek, TX). Clones were completely sequenced on both strands, and the sequence was compared with the NCBI Human Genome Data Base to ensure that no mutations had been introduced. Artificial Promoter Constructs−Artificial promoter/reporter constructs were generated as previously described (10.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 12.Lindsey S. Zhu C. Lu Y.F. Eklund E.A. J. Immunol. 2005; 175: 5269-5279Crossref PubMed Scopus (29) Google Scholar) in the minimal promoter/reporter vector, p-TATACAT (18.Scholer H.R. Balling R. Hazopoulos A.K. Suzuki N. Gruss P. EMBO J. 1989; 8: 2551-2558Crossref PubMed Scopus (264) Google Scholar) (obtained from Dr. A. Kraft (Hollings Cancer Center at the Medical University of South Carolina, Charleston, SC)). Constructs were generated with four copies (in the forward direction) of the –1755 to –1717 bp sequence (referred to as "A") or the –1174 to –1136 bp sequence (referred to as "B") from the DUSP4 promoter (p-ADUSP4-TATACAT and BDUSP4-TATACAT, respectively). cDNA Sequences−The cDNA for human HoxA10 was obtained from C. Largman (University of California, San Francisco, CA) (19.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). This cDNA sequence represents the major transcript in mammalian hematopoietic cells, encoding a 393-amino acid, 55-kDa protein (20.Takebe Y. Seiki M. Fujisajwa J.-I. Hoy P. Yokota K. Arai K.-I. Yoshida M. Arai N. Mol. Cell. Biol. 1988; 8: 466-472Crossref PubMed Google Scholar). Wild type HoxA10 cDNA sequence was subcloned into the pSRα vector for expression in mammalian cells (per the manufacturer's instructions (Stratagene)). The human Mkp2 cDNA was obtained from Clontech (Mountain View, CA) and subcloned into the pSRα vector for expression in mammalian cells. Plasmids for Short Hairpin RNA (shRNA) 2The abbreviations used are: shRNA, short hairpin RNA; IFNγ, interferon γ; EMSA, electrophoretic mobility shift assay(s); CAT, chloramphenicol acetyltransferase; Jnk, c-Jun N-terminal kinase. Expression−Plasmids were generated to express shRNA to human Mkp2 using the pLKO.1 vector (kindly provided by Dr. K. Rundell, Northwestern University, Chicago, IL). Oligonucleotide sequences for Mkp2-specific shRNA or scrambled control shRNA were designed with the assistance of the software on the Promega Web site (Promega, Madison, WI). Oligonucleotides were synthesized by the Core Facility of the Robert H. Lurie Comprehensive Cancer Center at Northwestern University. Double-stranded, synthetic oligonucleotides were generated representing the –1755 to –1717 bp "A" sequence from the DUSP4 promoter (5′-TAACTCCCTTGCTCTGTGATTAATTCTCACTAACAAGA-3′), the –1174 to –1136 bp "B" sequence from the DUSP4 promoter (5′-CTATAAAACTGATTTAATGGCTTTAGATGAAAATCGATC-3′), or the –94 to –134 bp sequence of the CYBB promoter (5′-ttcagttgaccaatgattattagccaattttctgataaaa-3′). In these oligonucleotides, the HoxA10 core is in boldface type, and the Pbx core is in italic type. The human myelomonocytic cell line U937 (22.Larrick J.W. Anderson S.J. Koren H.S. J. Immunol. 1980; 125: 6-14PubMed Google Scholar) was obtained from Andrew Kraft. Cells were maintained and differentiated as described (10.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 12.Lindsey S. Zhu C. Lu Y.F. Eklund E.A. J. Immunol. 2005; 175: 5269-5279Crossref PubMed Scopus (29) Google Scholar, 23.Lu Y. Goldenberg I. Bei L. Andrejic J. Eklund E.A. J. Biol. Chem. 2003; 278: 47792-47802Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). U937 cells were treated with 500 units/ml human recombinant IFNγ for 24 or 48 h, as indicated (Hoffman-LaRoche). U937 cells were cultured with or without IFNγ for 48 h, as described (10.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 12.Lindsey S. Zhu C. Lu Y.F. Eklund E.A. J. Immunol. 2005; 175: 5269-5279Crossref PubMed Scopus (29) Google Scholar, 23.Lu Y. Goldenberg I. Bei L. Andrejic J. Eklund E.A. J. Biol. Chem. 2003; 278: 47792-47802Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Chromatin immunoprecipitation and CpG island microarray hybridization were performed as described (14.Oberley M.J. Farnham P.J. Methods Enzymol. 2003; 371: 577-596Crossref PubMed Scopus (56) Google Scholar). Briefly, cells were incubated with formaldehyde and lysed, and lysates were sonicated to generate chromatin fragments with an average size of 2.0 kb. Lysates were precipitated with HoxA10 antiserum (Covance) or control preimmune serum. Chromatin was recovered by precipitation, proteins were stripped from the chromatin, and the chromatin was PCR-amplified. Several batches of immunoprecipitated, amplified chromatin were combined for each experiment. Aliquots of HoxA10-specific and preimmune serum control-precipitated, amplified chromatin were labeled with Cy3 or Cy5 by the random primer method. Labeled DNA was used to probe a CpG island microarray, as described (14.Oberley M.J. Farnham P.J. Methods Enzymol. 2003; 371: 577-596Crossref PubMed Scopus (56) Google Scholar). CpG island microarrays were obtained from the Microarray Center, University Health Network (Ontario Cancer Institute, Ontario, Canada). "Spots" with 3-fold enhancement in HoxA10-specific versus control preimmune serum-precipitated chromatin in three independent hybridization experiments were further considered. Dye swapping experiments were performed as controls for differences in efficiency in incorporation of Cy3 versus Cy5 into DNA. Arrays were scanned using an Agilent microarray scanner (G2565BA, Wilmington, DE), and feature intensity statistics were extracted using GenePix (Molecular Devices, Union City, CA). The GenBank™ accession number from the array was used to search the NCBI human genome data base for adjacent genes. Specificity of chromatin immunoprecipitation was confirmed by independent chromatin immunoprecipitation with HoxA10-specific antiserum or preimmune serum control. Precipitated chromatin was PCR-amplified with overlapping primer sets that were designed to encompass the proximal 4 kb of 5′-flank sequences, based on location of the CpG island (–1734 to –2025 bp relative to the ATG start codon). Primer sets were designed to amplify –2025 to +30 bp or –2025 to –3613 bp. Nuclear extract proteins were prepared by the method of Dignam (24.Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1993; 11: 1475-1479Crossref Scopus (9150) Google Scholar) with protease inhibitors (as described) (10.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Oligonucleotides probes were prepared, and EMSA and antibody supershift assays were performed, as described (10.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 12.Lindsey S. Zhu C. Lu Y.F. Eklund E.A. J. Immunol. 2005; 175: 5269-5279Crossref PubMed Scopus (29) Google Scholar). For all experiments, at least three independent batches of nuclear proteins were tested in at least two independent experiments. Integrity of the nuclear proteins and equality of protein loading was determined in control EMSA with a classical CCAAT box from the α globin gene promoter. Antiserum to HoxA10 (not cross-reactive with other Hox proteins) was obtained from Covance Research Products (Richmond, CA) and from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies to Pbx1 and Pbx2 (not cross-reactive with each other or other Pbx proteins) were obtained from Santa Cruz Biotechnology. All EMSA were performed several times with at least two different batches of nuclear proteins, and representative data are shown. Reporter Gene Assays−Cells were transfected by electroporation as described (10.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 12.Lindsey S. Zhu C. Lu Y.F. Eklund E.A. J. Immunol. 2005; 175: 5269-5279Crossref PubMed Scopus (29) Google Scholar, 23.Lu Y. Goldenberg I. Bei L. Andrejic J. Eklund E.A. J. Biol. Chem. 2003; 278: 47792-47802Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). U937 cells (32 × 106 cells/sample) were transfected with 70 μg of reporter plasmid with various truncations of the DUSP4 5′-flank (or empty vector control), 50 μg of HoxA10/pSRα or control pSRα, and 15 μg of p-CMVβ-gal (to normalize for transfection efficiency). In other experiments, cells were transfected with p-TATACAT, ADUSP4-TATACAT, or BDUSP4-TATACAT minimal promoter reporter vectors, 50 μg of HoxA10/pSRα or control pSRα, and 15 μg of p-CMVβ-gal (to normalize for transfection efficiency). Transfectants were incubated for 48 h at 37 °C, 5% CO2, with and without IFNγ (500 units/ml). Preparation of cell extracts, β-galactosidase, and chloramphenicol acetyltransferase (CAT) assays were performed as described (10.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 12.Lindsey S. Zhu C. Lu Y.F. Eklund E.A. J. Immunol. 2005; 175: 5269-5279Crossref PubMed Scopus (29) Google Scholar, 23.Lu Y. Goldenberg I. Bei L. Andrejic J. Eklund E.A. J. Biol. Chem. 2003; 278: 47792-47802Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Stable Transfectants−Stable U937 transfectants were generated with pSRα vectors to overexpress HoxA10, Mkp2, or empty vector control. Stable transfectants were selected in G418, as previously described (10.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). At least three transfectant pools were tested for each construct. In some experiments, U937 cells were transfected with PLKO.1 vectors to express shRNA for Mkp2 or scrambled shRNA control and selected with puromycin. Total cell lysate proteins from U937 cells or U937 stable transfectants (50 μg) were separated by SDS-PAGE and transferred to nitrocellulose. Western blots were serially probed with antibodies to various proteins. Antibodies to tubulin, HoxA10, Jnk1, and phospho-Jnk were obtained from Santa Cruz Biotechnology. Each experiment was performed several times, and representative blots are shown. In some experiments, RNA was isolated from U937 cells using the Triazol reagent, according to manufacturer's instructions (Invitrogen). RNA was tested by denaturing gel electrophoresis to determine the integrity of the 18 and 28 S ribosomal bands. Primers were designed with the software from Integrated DNA Technologies, and real time PCR was performed using SYBR green according to the "standard curve" method. Results were normalized to 18 S and actin to control for RNA abundance in various samples. In other experiments, chromatin co-immunoprecipitating from U937 cells with antibody to HoxA10, anti-acetyl-histone 3, or control preimmune serum was analyzed by real time PCR. Primers were designed to amplify ∼80 bp centered around the A and B HoxA10 DNA-binding consensus sequences using the Integrated DNA Technologies software, as above. Results were normalized to PCR product abundance in nonprecipitated, total chromatin samples. Experiments were performed in triplicate for each of three different immunoprecipitation experiments. Annexin V/propidium iodide (Beckman) double staining was used according to the manufacturer's instructions. The cells were washed with ice-cold culture medium, adjusted to a concentration of 1 × 106 cells/ml, and incubated with annexin V-fluorescein isothiocyanate solution (2.5 μg/ml) and propidium iodide (12.5 μg/ml) on ice for 15 min, and cell preparations were analyzed on a BD Biosciences FACScan flow cytometer. Statistical significance of comparisons between two groups was determined by Student's t test. Statistical significance of comparisons between larger groups was determined using analysis of variance. Statistical calculations were performed using the SigmaPlot and SigmaStat software. HoxA10 Interacts with the DUSP4 Promoter in Vitro and in Vivo−HoxA10 target genes were identified by chromatin co-immunoprecipitation from U937 myeloid leukemia cells. This myeloid cell line undergoes differentiation in response to various cytokines, including IFNγ (22.Larrick J.W. Anderson S.J. Koren H.S. J. Immunol. 1980; 125: 6-14PubMed Google Scholar). U937 differentiation is characterized by acquisition of mature phagocyte characteristics, including respiratory burst activity and phagocytosis (10.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 2000; 275: 20117-20126Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 12.Lindsey S. Zhu C. Lu Y.F. Eklund E.A. J. Immunol. 2005; 175: 5269-5279Crossref PubMed Scopus (29) Google Scholar, 22.Larrick J.W. Anderson S.J. Koren H.S. J. Immunol. 1980; 125: 6-14PubMed Google Scholar, 25.Levy R. Rotrosen D. Nagauker O. Leto T.L. Malech H.L. J. Immunol. 1990; 145: 2595-2601PubMed Google Scholar). Differentiation is also characterized by cell cycle arrest within 24 h and programmed cell death over 72 h. Therefore, this cell line provides a reasonable model of the events of myelopoiesis. For these studies, U937 cells were treated with formaldehyde to generate in vivo DNA-protein cross-links, and cell lysates were immunoprecipitated with antiserum to HoxA10 or control preimmune serum. Prior to immunoprecipitation, the lysates were sonicated to generate 2.0-kb chromatin fragments (14.Oberley M.J. Farnham P.J. Methods Enzymol. 2003; 371: 577-596Crossref PubMed Scopus (56) Google Scholar). Precipitated chromatin was labeled with Cy3 or Cy5 and used to hybridize a human CpG island microarray, as described (14.Oberley M.J. Farnham P.J. Methods Enzymol. 2003; 371: 577-596Crossref PubMed Scopus (56) Google Scholar). Triplicate array experiments were performed (with independent co-precipitations), and only "spots" with at least 3-fold enhancement in all three were further considered. One of the identified CpG islands localized to chromosome 8 in the 5′-flank of the DUSP4 gene (Fig. 1A; location of the CpG island is –2025 to –1734 bp relative to the ATG start codon). This gene encodes dual specific phosphatase 4, also known as Mkp2 (mitogen-activated protein kinase phosphatase 2). This result was of considerable interest, since Mkp2 has been implicated in regulating apoptosis in myeloid cells via inactivation (dephosphorylation) of proapoptotic Jnk proteins (14.Oberley M.J. Farnham P.J. Methods Enzymol. 2003; 371: 577-596Crossref PubMed Scopus (56) Google Scholar). Since co-precipitating DNA was sheared to generate 2.0-kb fragments, the location of the CpG island (∼–2.0 kb) indicated that the HoxA10 binding site(s) should be within the proximal 4.0 kb of the DUSP4 5′-flank. Therefore, we performed initial experiments to verify interaction of HoxA10 with the DUSP4 5′-flank and to locate the binding site within either the proximal –2.0 kb of the 5′-flank or between –2.0 and –4.0 kb. To determine the impact of differentiation stage on HoxA10-binding, chromatin was co-immunoprecipitated from U937 cells with and without 48 h of IFNγ differentiation. Precipitating chromatin was amplified by PCR, separated by acrylamide gel electrophoresis, and visualized by ethidium bromide staining. Two primer sets were used to amplify fragments from –2025 bp to +30 bp (relative to the ATG) or from –2025 to –3613 bp. For these experiments, nonprecipitated chromatin was a positive control, and chromatin co-precipitating with preimmune serum was a negative control. We found specific amplification of HoxA10 co-precipitating chromatin with the proximal primer set (Fig. 1B). Although not quantitative, these studies also suggested that HoxA10 interaction with the DUSP4 5′-flank decreased during U937 differentiation. In contrast, HoxA10 antibody did not co-precipitate chromatin with the more distal 5′-flank sequence (not shown). The results of these chromatin immunoprecipitation studies suggest that HoxA10 interacts with a binding site within the proximal 2.0 kb of DUSP4 5′-flank in vivo. Therefore, we analyzed this 2.0-kb sequence to determine if it contained any HoxA10 DNA-binding consensus sequences. This analysis resulted in identification of two sequences that conform to the derived consensus for DNA binding of HoxA10-Pbx heterodimers. These sequences were found at –1731 to –1738 bp (referred to as "A") and –1158 to –1166 bp (referred to as "B") relative to the ATG start codon in the DUSP4 5′-flank (Fig. 1C). These results provided initial evidence in support of our hypothesis that DUSP4 is a HoxA10 target gene. Therefore, we performed additional experiments to determine the impact on Mkp2 expression of myeloid differentiation and HoxA10 overexpression. Mkp2 Expression Is Decreased by Differentiation and Increased by HoxA10 Overexpression in Myeloid Cells−Mkp2 is expressed in myeloid cells, but differentiation stage-specific regulation of Mkp2 expression has not been previously investigated (14.Oberley M.J. Farnham P.J. Methods Enzymol. 2003; 371: 577-596Crossref PubMed Scopus (56) Google Scholar). Differentiation of U937 cells is associated with increased Jnk activation (phosphorylation) and Jnk-induced apoptosis. Since Mkp2 dephosphorylates Jnk, one might anticipate a decrease in Mkp2 expression as differentiation proceeds. Therefore, we initially investigated the impact of IFNγ differentiation on Mkp2 mRNA abundance in U937 cells. For these studies, expression was analyzed by real time PCR using RNA isolated from U937 cells over a 72-h period of differentiation. We found that Mkp2 mRNA abundance in U937 cells decreased significantly after 24 h of IFNγ treatment but did not decrease further between 24 and 72 h post-IFNγ (F = 0.40, p = 0.68, n = 4) (Fig. 2A). Based on these results, we investigated the impact of U937 differentiation and HoxA10 overexpression on Mkp2 protein abundance. For these studies, stable U937 transfectant pools were generated with a vector to overexpress HoxA10 or empty control vector. Transfectants were analyzed with and without IFNγ differentiation. Three independent transfectant pools were selected, and the results of representative experiments are presented. Western blots of total cell lysates were serially probed with antibodies to Mkp2, HoxA10 (to verify overexpression), Jnk and phospho-Jnk (as a downstream Mkp2 target), and tubulin (as a loading
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