Interaction between Hex and GATA Transcription Factors in Vascular Endothelial Cells Inhibits flk-1/KDR-mediated Vascular Endothelial Growth Factor Signaling
2004; Elsevier BV; Volume: 279; Issue: 20 Linguagem: Inglês
10.1074/jbc.m308730200
ISSN1083-351X
AutoresTakashi Minami, Takeshi Murakami, Keiko Horiuchi, Mai Miura, Tamio Noguchi, Jun–ichi Miyazaki, Takao Hamakubo, William C. Aird, Tatsuhiko Kodama,
Tópico(s)Single-cell and spatial transcriptomics
ResumoRecent evidence supports a role for GATA transcription factors as important signal intermediates in differentiated endothelial cells. The goal of this study was to identify proteins that interact with endothelial-derived GATA transcription factors. Using yeast two-hybrid screening, we identified hematopoietically expressed homeobox (Hex) as a GATA-binding partner in endothelial cells. The physical association between Hex and GATA was confirmed with immunoprecipitation in cultured cells. Hex overexpression resulted in decreased flk-1/KDR expression, both at the level of the promoter and the endogenous gene, and attenuated vascular endothelial growth factor-mediated tube formation in primary endothelial cell cultures. In electrophoretic mobility shift assays, Hex inhibited the binding of GATA-2 to the flk-1/KDR 5′-untranslated region GATA motif. Finally, in RNase protection assays, transforming growth factor β1, which has been previously shown to decrease flk-1 expression by interfering with GATA binding activity, was shown to increase Hex expression in endothelial cells. Taken together, the present study provides evidence for a novel association between Hex and GATA and suggests that transforming growth factor β-mediated repression of flk-1/KDR and vascular endothelial growth factor signaling involves the inducible formation of inhibitory Hex-GATA complexes. Recent evidence supports a role for GATA transcription factors as important signal intermediates in differentiated endothelial cells. The goal of this study was to identify proteins that interact with endothelial-derived GATA transcription factors. Using yeast two-hybrid screening, we identified hematopoietically expressed homeobox (Hex) as a GATA-binding partner in endothelial cells. The physical association between Hex and GATA was confirmed with immunoprecipitation in cultured cells. Hex overexpression resulted in decreased flk-1/KDR expression, both at the level of the promoter and the endogenous gene, and attenuated vascular endothelial growth factor-mediated tube formation in primary endothelial cell cultures. In electrophoretic mobility shift assays, Hex inhibited the binding of GATA-2 to the flk-1/KDR 5′-untranslated region GATA motif. Finally, in RNase protection assays, transforming growth factor β1, which has been previously shown to decrease flk-1 expression by interfering with GATA binding activity, was shown to increase Hex expression in endothelial cells. Taken together, the present study provides evidence for a novel association between Hex and GATA and suggests that transforming growth factor β-mediated repression of flk-1/KDR and vascular endothelial growth factor signaling involves the inducible formation of inhibitory Hex-GATA complexes. 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Taken together, these data suggest that GATA transcription factors are involved in highly complex regulatory pathways and that the dissection of these networks may provide valuable insight into the transcriptional control of endothelial phenotypes. To that end, the goal of the present study was to identify partner proteins that interact with GATA factors in endothelial cells. Using a yeast two-hybrid system, we describe a physical interaction between GATA family of transcription factors and the homeobox protein Hex. The interaction between Hex-GATA is associated with reduced GATA binding activity and transcriptional activity, decreased expression of the GATA-2 target gene, flk-1/KDR, and secondary attenuation of VEGF-mediated 1The abbreviations used are: VEGF, vascular endothelial growth factor; TGF, transforming growth factor; HUVEC, human umbilical vein endothelial cell(s); HEK, human embryonic kidney; GBD, Gal4 DNA-binding domain; GAD, Gal4 activation domain; UTR, untranslated region; EGFP, enhanced green fluorescence protein; IRES, internal ribosome entry sites; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 1The abbreviations used are: VEGF, vascular endothelial growth factor; TGF, transforming growth factor; HUVEC, human umbilical vein endothelial cell(s); HEK, human embryonic kidney; GBD, Gal4 DNA-binding domain; GAD, Gal4 activation domain; UTR, untranslated region; EGFP, enhanced green fluorescence protein; IRES, internal ribosome entry sites; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. signaling. Finally, we present data supporting the notion that TGF-β exerts its anti-angiogenesis effect by a Hex-GATA-flk-1/KDR-dependent mechanism. Cell Culture—Human umbilical vein endothelial cells (HUVEC) (Clonetics, La Jolla, CA) were cultured in EGM-2 MV medium (Clonetics). Human embryonic kidney (HEK)-293 cells (ATCC CRL-1573) and COS-7 cells (ATCC CRL-1651) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum. HUVEC were used within the first eight passages. Plasmids—The construction of the flk-1/KDR-luc, flk-1/KDR (GATA mut), and flk-1/KDR (SP1 mut) plasmids were previously described (30Minami T. Aird W.C. J. Biol. Chem. 2001; 276: 47632-47641Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Human GATA-2 expression plasmid (pMT2-GATA2) was a kind gift from Dr. Stuart H. Orkin (Harvard Medical School, Boston, MA). Human GATA-3 and -6 and Hex cDNA fragments were amplified using PCR from reverse-transcribed HUVEC total RNA. To generate the Hex expression vector, Hex cDNA was subcloned into the pcDNA3 vector (Invitrogen). To generate the plasmids expressing Gal4 DNA-binding domain (GBD) fused with GATA-2, -3, and -6, each of the three GATA cDNA fragments was inserted into pGBKT7 (Clontech, Palo Alto, CA). To construct the plasmid expressing Gal4 activation domain (GAD) fused to Hex, the Hex cDNA fragment was subcloned into pGAD424 (Clontech). To generate a FLAG-tagged Hex (pFLAG-Hex) and FLAG-tagged GATA-2 (pFLAG-GATA2), Hex and GATA-2 cDNA fragments were subcloned into pFLAG (Sigma), respectively. For construction of pGEM-hflk, a 266-bp human flk-1/KDR cDNA fragment was amplified from reverse-transcribed HUVEC total RNA and subcloned into pGEM-T-easy (Promega). Similarly, pGEM-hHex and pGEM-hGAPDH were derived by ligating a 296-bp human Hex cDNA fragment and a 283-bp human GAPDH fragment into pGEM-T-easy. Orientation was confirmed by automated DNA sequencing. Yeast Two-hybrid Screening and β-Galactosidase Assays—A HUVEC cDNA library was constructed using a two-hybrid cDNA library construction kit (Clontech). AH109 yeast were transfected sequentially with the GATA-6 bait vector and cDNA library and then spread on synthesized dropout medium plate in the absence of tryptophan, leucine, and histidine. After 5 days of incubation at 30 °C, colonies complemented by histidine autotrophy were isolated and confirmed to be positive by β-galactosidase assay according to the manufacturer's instruction (Clontech; yeast protocols handbook). The plasmids from the positive colonies were purified, and the inserts were subsequently sequenced. To analyze the protein-protein interaction, Y190 yeast were co-transfected with pGAD-Hex and a plasmid in which GBD was fused either with GATA-2, -3, and -6. Similarly, Y190 yeast were co-transfected with pGBD-GATA2 and a plasmid containing GAD fused with Hex. The cells were spread and incubated on the synthesized dropout medium plate without tryptophan and leucine. Transfection of COS-7 Cells and Immunoprecipitation Assays— COS-7 cells were co-transfected with either pFLAG-Hex and pMT2-GATA2, or pFLAG-GATA2 and pcDNA3-Hex expression plasmids using the FuGENE 6 reagent (Roche Applied Science) as instructed by the manufacturer. Two days later, the transfected cells were freeze-thawed three times and incubated for 30 min on ice with cell lysis buffer (0.1% IGEPAL CA-630, 50 mm Tris-HCl, 150 mm NaCl, 5 mm EDTA, 0.5 μg/ml pepstatin A, 10 μg/ml leupeptin, 2 μg/ml aprotinin, 200 μm phenylmethylsulfonyl fluoride, pH 7.5), followed by centrifugation at 20,000 × g for 5 min. The supernatant was incubated with anti-FLAG polyclonal antibody (Sigma) overnight at 4 °C. The resulting mixture was then mixed and incubated with protein G-Sepharose (Amersham Biosciences) for 1 h at 4 °C. The immobilized beads were washed five times with 1 ml of cell lysis buffer containing 1.5% IGEPAL CA-630. Each sample was separated on 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Amersham Biosciences). The membrane was incubated either with anti-FLAG M5 monoclonal antibody (Sigma), anti-Myc monoclonal antibody (Invitrogen) or anti-GATA-2 antibody (Santa Cruz, CA). Alternatively, subconfluent HUVEC (2 × 107 cells) were harvested for nuclear extracts according to the mild nitrogen cavitation method (31Brock T.G. Paine 3rd, R. Peters-Golden M. J. Biol. Chem. 1994; 269: 22059-22066Abstract Full Text PDF PubMed Google Scholar) to keep intact the protein-protein associations. 1 μg of nuclear extracts was precleaned by centrifugation with 4 μg of control IgG (Santa Cruz). The resulting supernatant was incubated with 30 μg of agarose-conjugated anti-GATA-2 monoclonal antibody (Santa Cruz, sc-267 AC) or an identical amount of isotype-matched mouse control IgG (Santa Cruz, sc-2343) overnight at 4 °C. The immobilized samples were separated and transferred to a polyvinylidene difluoride membrane. The membrane was then probed for Hex and GATA-2 by Western blot analysis, using anti-Hex antibody (generated by Dr. Tamio Noguchi, Japan) and anti-GATA-2 antibody (Santa Cruz, sc-16044, and generated by Dr. Stuart H. Orkin, Harvard Medical School, Boston), respectively. The complexes were visualized with an ECL advance Western blotting detection kit (Amersham Biosciences). RNA Isolation and RNase Protection Assays—HUVEC were serum-starved in EBM-2 medium containing 0.5% fetal bovine serum. 18 h later, HUVEC were treated with 2.5–10 ng/ml TGF-β1 (Peprotec, Rocky Hill, NJ) for 18 h (for Hex) or 24h (for flk-1/KDR). Alternatively, HUVEC were infected with adenoviruses encoding the IRES-mediated green fluorescence protein (EGFP) (Adeno-Blank) or IRES coupled to Hex and EGFP (Adeno-Hex). Adeno-Blank and Adeno-Hex were generated with a ligation into adenovirus cosmid vector and a co-transfection into HEK-293 (32Chiba T. Kogishi K. Wang J. Xia C. Matsushita T. Miyazaki J. Saito I. Hosokawa M. Higuchi K. Am. J. Pathol. 1999; 155: 1319-1326Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Infections were carried out at a multiplicity of infection of 20 for 24 h. HUVEC were harvested for total RNA at the times indicated, using the Isogen reagent (Nippon Gene). For in vitro transcription, flk-1/KDR-, Hex-, and GAPDH-specific 32P-labeled riboprobes were synthesized from pGEM-hflk, pGEM-hHex, and pGEM-hGAPDH, respectively. Riboprobes were synthesized using SP6 (for flk-1/KDR and GAPDH) or T7 (for Hex) RNA polymerase (Ambion, Austin, TX) and purified with a G-50 spun column (Amersham Biosciences). RNase protection assays were performed with a RPA III kit (Ambion) according to the manufacturer's instructions. Transfection of HEK-293 Cells or HUVEC and Analysis of Luciferase Activity—HEK-293 cells and HUVEC were transfected as described previously (30Minami T. Aird W.C. J. Biol. Chem. 2001; 276: 47632-47641Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Briefly, either 2 × 105 cells/well of HEK-293 cells or 1 × 105 cells/well of HUVEC were seeded onto 12-well plates 18–24 h before transfection. 0.05 pmol of the luciferase reporter plasmid (either KDR-, KDR (GATA mut)-, or KDR (SP1 mut)-luc), 50 ng of pRL-CMV (Promega), 0.075 pmol of the GATA expression vector and either 0.0375 or 0.075 pmol of Hex expression vector were incubated with 2 μl of FuGENE 6. As a negative control, empty vector (pMT2 and pcDNA3) was transfected instead of GATA-2 and Hex expression vector, respectively. 24 h later for HEK-293 cells and 48 h later for HUVEC, the cells were washed with phosphate-buffered saline, lysed, and assayed for luciferase activity using the dual luciferase reporter assay system (Promega) and a Lumat LB 9507 luminometer (Berthold, Gaithersburg, MD). Sandwich Tube Formation Assays—400-μl aliquots of type-I collagen gel (Koken) containing EGM-2 MV medium (Clonetics) without basic fibroblast growth factor were added to 24-well plates and allowed to gel for 1 h at37 °C. HUVEC infected with Adeno-Blank or Adeno-Hex were seeded at 1 × 105 cells/well and incubated for 24 h in 5% CO2. The medium was removed, and the HUVEC were covered with 400 μlofthe gel. The plate was incubated for 30 min at 37 °C. The cells were incubated with 1 ml of EGM-2 MV media in the absence of basic fibroblast growth factor, in the presence or absence of SU1498 (Calbiochem, San Diego, CA). Two days later, a branched capillary network was visualized under a microscope. Images from at least three different areas in each well were captured by a digital camera under a microscope. Nuclear Extracts and Electrophoretic Mobility Shift Assays—Nuclear extracts were prepared as previously described (30Minami T. Aird W.C. J. Biol. Chem. 2001; 276: 47632-47641Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 33Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9131) Google Scholar). Double-stranded oligonucleotides were labeled with [α-32P]dCTP and Klenow fragment and purified by spun column (Amersham Biosciences). 10 μg of HUVEC nuclear extracts were incubated with 10 fmol of 32P-labeled probe, 1 μg of poly(dI-dC), and 3 μl of 10× binding buffer (100 mm Tris HCl, pH 7.5, 50% glycerol, 10 mm dithiothreitol, 10 mm EDTA) for 20 min at the room temperature, followed by 30 min at 4 °C. The following oligonucleotides sequences were used for probes: 5′-UTR GATA motifs, 5′-GGCAGCCTGGATATCCTCTCCTA-3′; GATA-mut motifs, 5′-GGCAGCCTGTTTAAGCTCTCCTA-3′; flk-1/KDR SP1 motifs, 5′-GGTGAGGGGCGGGGCTGGCCGC-3′; and flk-1/KDR SP1-mut motifs, 5′-GGTGAGGTTCGGTTCTGGTTGC-3′. To test the effect of antibodies on DNA-protein binding, the nuclear extracts were preincubated with monoclonal antibody to GATA-2 (we prepared from the antigen GATA-2 (amino acids 9–258)) or polyclonal antibody to p65 (Santa Cruz) for 30 min at the room temperature. DNA-protein complexes were resolved on a 5% nondenaturing polyacrylamide gel containing 5% glycerol in 0.5× TBE (50 mm Tris, 50 mm boric acid, and 1 mm EDTA). The loaded gel was fixed with 10% methanol and 10% acetic acid and then imaged by BAS-1800 (Fuji Film, Japan). The signals were quantified with NIH Image. Identification of Hematopoietically Expressed Hex as a GATA-interacting Protein by Yeast Two-hybrid Screening—The GATA family of transcription factors has been implicated not only in the early differentiation of endothelial cells but also in the transduction of extracellular signals in the adult endothelium. Several members of the GATA family have been identified in endothelial cells, including GATA-2, -3, and -6. Previous studies have shown that GATA-1 and -2 interact with partner proteins in erythroid and megakaryocyte cells (34Cantor A.B. Orkin S.H. Oncogene. 2002; 21: 3368-3376Crossref PubMed Scopus (479) Google Scholar). Our goal was to identify the proteins that interact with GATA transcription factors in endothelial cells. To that end, we employed a yeast two-hybrid system in which full-length human GATA-6 served as bait. From a screen of 8.6 × 106 clones, a total of 58 clones demonstrated histidine auxotroph. After elimination of false positives, 16 clones were selected, one of which encoded a fragment (amino acids 75–270) of Hex (Fig. 1A). To confirm the specificity of interaction between Hex and GATA-6, constructs containing the Gal4-activating domain fused with full-length Hex (pGAD-Hex) and either the Gal4-binding domain fused with GATA-6 (pGBD-GATA6) or the Gal4-binding domain alone (pGBD) were co-expressed in yeast AH109. As shown in Fig. 1B, co-expression of pGAD-Hex and pGBD-GATA6 resulted in a 4.2-fold increase in β-galactosidase activity compared with co-expression of pGBD and pGAD-Hex. We next wished to determine whether Hex interacts with human GATA-2 and -3. To that end, pGAD-Hex was co-expressed in yeast AH109 with constructs containing the Gal4-binding domain fused either with GATA-2 or -3 (pGBD-GATA2 or GATA3, respectively) As shown in Fig. 1C, co-expression with human GATA-2 and -3 resulted in 5.8- and 4.7-fold induction of the β-galactosidase activity, respectively, compared with co-expression with Gal4-binding domain alone (pGBD). Taken together, these results suggest that Hex interacts with GATA-2, -3, and -6. Physical Interaction between Hex and GATA-2 in Mammalian Cells—Having identified the interaction between GATA and Hex in the yeast two-hybrid system, we wished to determine whether this interaction occurs in mammalian cells. Of the various members of the GATA family of transcription factors, GATA-2 is expressed most abundantly in cultured endothelial cells (data not shown) and is believed to play a predominant role in endothelial cell biology. Therefore, we chose to focus on the interaction between Hex and GATA-2. To that end, COS-7 cells were transiently transfected with expression plasmids for human GATA-2 (pMT2-GATA2), FLAG-tagged human GATA-2 (pFLAG-GATA2), FLAG- or Myc-tagged human Hex (pFLAG-Hex or pMyc-Hex), or vector alone (pMT2, pMyc or pFLAG) and then processed for immunoprecipitation. The transfected cells expressed high levels of GATA-2 and Hex (Fig. 2, A and B, lanes 1–3). In co-transfections (pMT2-GATA2 and pFLAG-Hex), GATA-2 co-precipitated with the anti-FLAG antibody (Fig. 2A, lane 6, closed arrowhead) but not with the nonimmune control (Fig. 2A, lane 7). In contrast, in experiments in which either (pMT2-GATA2 and pFLAG) or (pMT2 and pFLAG-tagged Hex) were transfected, GATA-2 did not co-precipitate (Fig. 2A, lanes 4 and 5). Furthermore, in cotransfections (pFLAG-GATA2 and pMyc-Hex), Myc-tagged Hex co-precipitated with the anti-FLAG antibody (Fig. 2B, lane 6, closed arrowhead). Although in experiments in which either (pFLAG and pMyc-Hex) or (pFLAG-GATA2 and pMyc) were transfected, Hex did not co-precipitate (Fig. 2B, lanes 4 and 5). Next, we wished to identify the physical interaction between endogenous GATA-2 and Hex in endothelial cells. To that end, nuclear extracts were prepared and processed for immunoblotting and co-immunoprecipitations. As shown in Fig. 2C, endogenous GATA-2 and Hex were detected in nuclear extracts from HUVEC but not COS-7. Endogenous Hex was co-precipitated with anti-GATA2 antibody but not with isotype-matched control IgG (Fig. 2D, lanes 2 and 3). In semi-quantitative calculations from two independent experiments, anti-GATA-2 antibody immunoprecipitated 75.2% of total GATA-2 in HUVEC. Moreover, 8.29% of total cellular Hex was physically associated with GATA-2. Collectively, these findings suggest that GATA-2 and Hex specifically interact with one another in cultured endothelial cells. Hex Inhibits GATA-2-mediated flk-1/KDR Promoter Activity—We next wished to study the functional relevance of the interaction between GATA-2 and Hex. We have previously shown that GATA-2 binds to a GATA motif in the 5′-UTR region of the flk-1/KDR promoter and that this effect is necessary for full expression (35Minami T. Rosenberg R.D. Aird W.C. J. Biol. Chem. 2001; 276: 5395-5402Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). To determine the effect of Hex on GATA-2-mediated activation of flk-1/KDR, transactivation assays were carried out in HEK-293 cells (nonendothelial cells) and HUVEC (endothelial cells) co-transfected with KDR-luc (–115 and +296 flk-1/KDR coupled to luciferase) and an expression plasmid containing either human GATA-2 (pMT2-GATA2) or Hex (pcDNA3-HEX). As a negative control, the cells were co-transfected with vector alone (pMT2 or pcDNA3). Consistent with our previous findings, the basal level of flk-1/KDR promoter activity in HEK-293 cells was significantly transactivated by 4.2-fold with overexpression of GATA-2 (Fig. 3A). GATA-2-mediated stimulation of promoter activity was completely abrogated by co-expression of Hex in a dose-dependent manner. Moreover, overexpression of Hex did not change the basal level of the promoter activity (Fig. 3A). In HUVEC, a high level of flk-1/KDR promoter activity (8.7-fold higher than SV40 promoter plus enhancer construct (pGL2-control), not shown) occurred, and overexpression of GATA-2 resulted in 2.8-fold transactivation of the promoter (Fig. 3B). More importantly, overexpression of Hex resulted in huge reduction of the flk-1/KDR promoter activity (15.9-fold compared without expression of GATA-2 and Hex). Co-expression of GATA-2 failed to recover the Hex-mediated attenuation of the flk-1/KDR promoter activity (2.3-fold induction compared Hex expression alone) (Fig. 3B). To confirm that the Hex-mediated down-regulation of the promoter activity was mediated by the 5′-UTR GATA motif on the flk-1/KDR gene, either the GATA element or the SP1 element (as a control) point-mutated plasmid was transfected into HUVEC. KDR promoter activity from SP1 point-mutated plasmid was down to 63.1% and markedly reduced to 3.4% by the co-expression with Hex. In contrast, the promoter activity from GATA point-mutated plasmid was down to 33.1%, whereas there was no significant reduction in the presence of Hex (Fig. 3B). Previous studies have shown that Hex directly binds to a consensus element (5′-CAATTAAA-3′) in the promoter region of its target genes, resulting in transcriptional activation (36Crompton M.R. Bartlett T.J. MacGregor A.D. Manfioletti G. Buratti E. Giancotti V. Goodwin G.H. Nucleic Acids Res. 1992; 20: 5661-5667Crossref PubMed Scopus (155) Google Scholar). A search of the 4-kb 5′-flanking region and 3-kb first intron of the flk-1/KDR failed to reveal such a motif. Taken together, these results suggest that Hex represses the GATA-mediated flk-1/KDR promoter activation. Hex Suppresses flk-1/KDR mRNA Expression in Primary Human Endothelial Cells—To determine whether Hex modulates the expression of the endogenous flk-1/KDR gene, HUVEC were infected with adenovirus expressing either IRES-mediated-EGFP (Blank) or IRES-mediated-rat Hex and EGFP (Hex) at a multiplicity of infection of 20. Using this approach, over 80% of the cells were infected as determined by EGFP expression. Western blot assays of infected cells demonstrated high levels of Hex protein (Fig. 4A). More importantly, overexpression of Hex in HUVEC resulted in a 85% reduction (mean of three independent experiments) in flk-1/KDR mRNA by RNase protection assay (Fig. 4B, compare lanes 4 and 5). These findings indicate that Hex suppresses flk-1/KDR activity not only at the level of the promoter but also at the level of the endogenous gene. Hex Inhibits Binding of GATA-2 to the flk-1/KDR 5′-UTR GATA Motif—Based on the above findings, we hypothesized that Hex inhibits flk-1/KDR expression by interfering with GATA binding to the 5′-UTR. To test this hypothesis, we performed electrophoretic mobility shift assays in which nuclear extracts derived from HUVEC either expressing IRES-mediated EGFP (Adeno-Blank) or IRES-mediated Hex and EGFP (Adeno-Hex) were incubated with a radiolabeled probe spanning the 5′-UTR GATA motif (+98 to +122). As shown in Fig. 5 (A and B), incubation of nuclear extract from Adeno-Blank infected HUVEC with the 32P-labeled probe resulted in the appearance of two specific DNA-protein complexes (closed and open arrows). These DNA-protein complexes were inhibited by the addition of 10-, 100-, and 500-fold molar excess unlabeled self-competitor (Fig. 5A, lanes 3–5) but not by 500-fold molar excess GATA mutant competitor (Fig. 5A, lane 6). The mobility shift pattern was identical in Hex-overexpressing cells (the more slowly migrated complexes designated with the open arrows appeared with longer exposure time). However, Hex overexpression resulted in a significant reduction (54.2% reduction by densitometry) in the intensity of the GATA-binding complexes (Fig. 5, A, compare lanes 3–5 and 9–11, and B, compare lanes 1 and 2). As previously reported, the DNA-protein complexes were inhibited by preincubation with the anti-GATA-2 antibody (34Cantor A.B. Orkin S.H. Oncogene. 2002; 21: 3368-3376Crossref PubMed Scopus (479) Google Scholar). In contrast, the complex was
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