Characterization of a Functional ZBP-89 Binding Site That Mediates Gata1 Gene Expression during Hematopoietic Development
2009; Elsevier BV; Volume: 284; Issue: 44 Linguagem: Inglês
10.1074/jbc.m109.026948
ISSN1083-351X
AutoresKinuko Ohneda, Shin’ya Ohmori, Yasushi Ishijima, Mayu Nakano, Masayuki Yamamoto,
Tópico(s)Acute Myeloid Leukemia Research
ResumoGATA-1 is a lineage-restricted transcription factor that plays essential roles in hematopoietic development. The Gata1 gene hematopoietic enhancer allowed Gata1 reporter expression in erythroid cells and megakaryocytes of transgenic mice. The Gata1 hematopoietic enhancer activity is strictly dependent on a GATA site located in the 5′ region of the enhancer. However, the importance of the GC-rich region adjacent to the 3′-end of this GATA site has been also suggested. In this study, we show that this GC-rich region contains five contiguous deoxyguanosine residues (G5 string) that are bound by multiple nuclear proteins. Interestingly, deletion of one deoxyguanosine residue from the G5 string (G4 mutant) specifically eliminates binding to ZBP-89, a Krüppel-like transcription factor, but not to Sp3 and other binding factors. We demonstrate that GATA-1 and ZBP-89 occupy chromatin regions of the Gata1 enhancer and physically associate in vitro through zinc finger domains. Gel mobility shift assays and DNA affinity precipitation assays suggest that binding of ZBP-89 to this region is reduced in the absence of GATA-1 binding to the G1HE. Luciferase reporter assays demonstrate that ZBP-89 activates the Gata1 enhancer depending on the G5 string sequence. Finally, transgenic mouse studies reveal that the G4 mutation significantly reduced the reporter activity of the Gata1 hematopoietic regulatory domain encompassing an 8.5-kbp region of the Gata1 gene. These data provide compelling evidence that the G5 string is necessary for Gata1 gene expression in vivo and ZBP-89 is the functional trans-acting factor for this cis-acting region. GATA-1 is a lineage-restricted transcription factor that plays essential roles in hematopoietic development. The Gata1 gene hematopoietic enhancer allowed Gata1 reporter expression in erythroid cells and megakaryocytes of transgenic mice. The Gata1 hematopoietic enhancer activity is strictly dependent on a GATA site located in the 5′ region of the enhancer. However, the importance of the GC-rich region adjacent to the 3′-end of this GATA site has been also suggested. In this study, we show that this GC-rich region contains five contiguous deoxyguanosine residues (G5 string) that are bound by multiple nuclear proteins. Interestingly, deletion of one deoxyguanosine residue from the G5 string (G4 mutant) specifically eliminates binding to ZBP-89, a Krüppel-like transcription factor, but not to Sp3 and other binding factors. We demonstrate that GATA-1 and ZBP-89 occupy chromatin regions of the Gata1 enhancer and physically associate in vitro through zinc finger domains. Gel mobility shift assays and DNA affinity precipitation assays suggest that binding of ZBP-89 to this region is reduced in the absence of GATA-1 binding to the G1HE. Luciferase reporter assays demonstrate that ZBP-89 activates the Gata1 enhancer depending on the G5 string sequence. Finally, transgenic mouse studies reveal that the G4 mutation significantly reduced the reporter activity of the Gata1 hematopoietic regulatory domain encompassing an 8.5-kbp region of the Gata1 gene. These data provide compelling evidence that the G5 string is necessary for Gata1 gene expression in vivo and ZBP-89 is the functional trans-acting factor for this cis-acting region. The differentiation and lineage-specification of hematopoietic cells are coordinated by the combinatorial functions of lineage-restricted and general transcription factors. The precise expression profile of lineage-restricted transcription factors is critical for promoting the differentiation program. GATA-1 is a lineage-restricted transcription factor required for normal erythropoiesis and megakaryopoiesis. The expression of Gata1 is restricted to erythroid cells, megakaryocytes, eosinophils, mast cells, and dendritic cells within the hematopoietic system (1Ferreira R. Ohneda K. Yamamoto M. Philipsen S. Mol. Cell Biol. 2005; 25: 1215-1227Crossref PubMed Scopus (312) Google Scholar, 2Gutiérrez L. Nikolic T. van Dijk T.B. Hammad H. Vos N. Willart M. Grosveld F. Philipsen S. Lambrecht B.N. Blood. 2007; 110: 1933-1941Crossref PubMed Scopus (52) Google Scholar). We previously generated transgenic mice expressing a β-galactosidase (lacZ) reporter gene driven by the Gata1 hematopoietic regulatory domain (Gata1-HRD) 2The abbreviations used are: HRDhematopoietic regulatory domainG1HEGata1 gene hematopoietic enhancerKLFKrüppel-like transcription factorsX-gal5-bromo-4-chloro-3-indolyl-β-d-galactosideDBDDNA binding domainChIPchromatin immunoprecipitationMELmouse erythroleukemiaIPimmunoprecipitationDAPADNA affinity precipitation assayGSTglutathione S-transferaseFOG-1friend of GATA-1MBPmaltose-binding proteinEMSAelectrophoretic mobility shift assayPBSphosphate-buffered salinesiRNAsmall interfering RNAGFPgreen fluorescent protein. (3Onodera K. Takahashi S. Nishimura S. Ohta J. Motohashi H. Yomogida K. Hayashi N. Engel J.D. Yamamoto M. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 4487-4492Crossref PubMed Scopus (143) Google Scholar, 4Nishimura S. Takahashi S. Kuroha T. Suwabe N. Nagasawa T. Trainor C. Yamamoto M. Mol. Cell. Biol. 2000; 20: 713-723Crossref PubMed Scopus (100) Google Scholar). The Gata1-HRD (5Motohashi H. Katsuoka F. Shavit J.A. Engel J.D. Yamamoto M. Cell. 2000; 103: 865-875Abstract Full Text Full Text PDF PubMed Google Scholar) is an 8.5-kbp region of the murine Gata1 gene and comprises a 3.9-kbp region 5′ of the proximal first exon (IE) and the entire first intron. Expression of the Gata1-HRD-driven reporter recapitulated endogenous GATA-1 expression in erythroid cells and megakaryocytes (3Onodera K. Takahashi S. Nishimura S. Ohta J. Motohashi H. Yomogida K. Hayashi N. Engel J.D. Yamamoto M. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 4487-4492Crossref PubMed Scopus (143) Google Scholar, 4Nishimura S. Takahashi S. Kuroha T. Suwabe N. Nagasawa T. Trainor C. Yamamoto M. Mol. Cell. Biol. 2000; 20: 713-723Crossref PubMed Scopus (100) Google Scholar). Furthermore, transgenic expression of GATA-1 cDNA driven by the Gata1-HRD could abolish lethal anemia in Gata1 germline mutant mice, demonstrating that the Gata1-HRD can drive sufficient transcriptional activity in vivo (6Takahashi S. Shimizu R. Suwabe N. Kuroha T. Yoh K. Ohta J. Nishimura S. Lim K.C. Engel J.D. Yamamoto M. Blood. 2000; 96: 910-916Crossref PubMed Google Scholar, 7Ferreira R. Wai A. Shimizu R. Gillemans N. Rottier R. von Lindern M. Ohneda K. Grosveld F. Yamamoto M. Philipsen S. Blood. 2007; 109: 5481-5490Crossref PubMed Scopus (37) Google Scholar). hematopoietic regulatory domain Gata1 gene hematopoietic enhancer Krüppel-like transcription factors 5-bromo-4-chloro-3-indolyl-β-d-galactoside DNA binding domain chromatin immunoprecipitation mouse erythroleukemia immunoprecipitation DNA affinity precipitation assay glutathione S-transferase friend of GATA-1 maltose-binding protein electrophoretic mobility shift assay phosphate-buffered saline small interfering RNA green fluorescent protein. Four discrete cis-acting regions have been identified as playing essential roles in the activity of the Gata1-HRD. These regions are: the Gata1 gene hematopoietic enhancer (G1HE, also referred to as hypersensitive site I, mouse hypersensitive site-3.5); the two upstream promoter elements containing a double GATA site and a CACCC box, referred to as hypersensitive site II; and a region containing multiple GATA repeats located in the first intron (3Onodera K. Takahashi S. Nishimura S. Ohta J. Motohashi H. Yomogida K. Hayashi N. Engel J.D. Yamamoto M. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 4487-4492Crossref PubMed Scopus (143) Google Scholar, 4Nishimura S. Takahashi S. Kuroha T. Suwabe N. Nagasawa T. Trainor C. Yamamoto M. Mol. Cell. Biol. 2000; 20: 713-723Crossref PubMed Scopus (100) Google Scholar, 8Nicolis S. Bertini C. Ronchi A. Crotta S. Lanfranco L. Moroni E. Giglioni B. Ottolenghi S. Nucleic Acids Res. 1991; 19: 5285-5291Crossref PubMed Scopus (69) Google Scholar, 9Ohneda K. Shimizu R. Nishimura S. Muraosa Y. Takahashi S. Engel J.D. Yamamoto M. Genes Cells. 2002; 7: 1243-1254Crossref PubMed Scopus (47) Google Scholar, 10Tsai S.F. Strauss E. Orkin S.H. Genes Dev. 1991; 5: 919-931Crossref PubMed Scopus (261) Google Scholar, 11Vyas P. McDevitt M.A. Cantor A.B. Katz S.G. Fujiwara Y. Orkin S.H. Development. 1999; 126: 2799-2811Crossref PubMed Google Scholar). The G1HE is located 3.9-2.6 kbp 5′ to the hematopoietic cell-specific first exon (IE). This region allows high expression levels of reporter genes in erythroid cells and megakaryocytes, as demonstrated by transgenic mouse reporter analysis (4Nishimura S. Takahashi S. Kuroha T. Suwabe N. Nagasawa T. Trainor C. Yamamoto M. Mol. Cell. Biol. 2000; 20: 713-723Crossref PubMed Scopus (100) Google Scholar, 11Vyas P. McDevitt M.A. Cantor A.B. Katz S.G. Fujiwara Y. Orkin S.H. Development. 1999; 126: 2799-2811Crossref PubMed Google Scholar, 12Suzuki M. Moriguchi T. Ohneda K. Yamamoto M. Mol. Cell. Biol. 2009; 29: 1163-1175Crossref PubMed Scopus (44) Google Scholar). G1HE activity is strictly reliant on a GATA site located in the 5′ region of the G1HE, because disruption of this GATA site completely abolished reporter expression in both lineages. Interestingly, however, we and others demonstrated that sequential deletion in the 3′ region of the G1HE reduced reporter expression in fetal liver, even in the presence of the critical GATA site (4Nishimura S. Takahashi S. Kuroha T. Suwabe N. Nagasawa T. Trainor C. Yamamoto M. Mol. Cell. Biol. 2000; 20: 713-723Crossref PubMed Scopus (100) Google Scholar, 11Vyas P. McDevitt M.A. Cantor A.B. Katz S.G. Fujiwara Y. Orkin S.H. Development. 1999; 126: 2799-2811Crossref PubMed Google Scholar). These results suggest that an additional cis-acting element 3′ of the GATA site is required for full activity of the G1HE. However, the properties of this additional region have not been fully characterized. A Krüppel-type zinc finger transcription factor ZBP-89 (also referred to as zfp148, BERF-1, and BFCOL1 (13Antona V. Cammarata G. De Gregorio L. Dragani T.A. Giallongo A. Feo S. Cytogenet. Cell Genet. 1998; 83: 90-92Crossref PubMed Scopus (9) Google Scholar, 14Hasegawa T. Takeuchi A. Miyaishi O. Isobe K. de Crombrugghe B. J. Biol. Chem. 1997; 272: 4915-4923Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 15Merchant J.L. Iyer G.R. Taylor B.R. Kitchen J.R. Mortensen E.R. Wang Z. Flintoft R.J. Michel J.B. Bassel-Duby R. Mol. Cell. Biol. 1996; 16: 6644-6653Crossref PubMed Scopus (117) Google Scholar)) was recently identified as a novel GATA-1-associated transcription factor in both erythroid cells and megakaryocytes (16Woo A.J. Moran T.B. Schindler Y.L. Choe S.K. Langer N.B. Sullivan M.R. Fujiwara Y. Paw B.H. Cantor A.B. Mol. Cell. Biol. 2008; 28: 2675-2689Crossref PubMed Scopus (56) Google Scholar). A high level of ZBP-89 chromatin occupancy at the G1HE was found in both erythroid and megakaryocytic cell lines (16Woo A.J. Moran T.B. Schindler Y.L. Choe S.K. Langer N.B. Sullivan M.R. Fujiwara Y. Paw B.H. Cantor A.B. Mol. Cell. Biol. 2008; 28: 2675-2689Crossref PubMed Scopus (56) Google Scholar, 17Guyot B. Murai K. Fujiwara Y. Valverde-Garduno V. Hammett M. Wells S. Dear N. Orkin S.H. Porcher C. Vyas P. J. Biol. Chem. 2006; 281: 13733-13742Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Moreover, erythroid and megakaryocyte differentiation is severely attenuated in the absence of ZBP-89 in zebrafish and mouse embryos (16Woo A.J. Moran T.B. Schindler Y.L. Choe S.K. Langer N.B. Sullivan M.R. Fujiwara Y. Paw B.H. Cantor A.B. Mol. Cell. Biol. 2008; 28: 2675-2689Crossref PubMed Scopus (56) Google Scholar, 18Li X. Xiong J.W. Shelley C.S. Park H. Arnaout M.A. Development. 2006; 133: 3641-3650Crossref PubMed Scopus (26) Google Scholar), overlapping with the phenotype of Gata1-deficient animals (19Fujiwara Y. Browne C.P. Cunniff K. Goff S.C. Orkin S.H. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 12355-12358Crossref PubMed Scopus (619) Google Scholar, 20Lyons S.E. Lawson N.D. Lei L. Bennett P.E. Weinstein B.M. Liu P.P. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 5454-5459Crossref PubMed Scopus (125) Google Scholar, 21Takahashi S. Onodera K. Motohashi H. Suwabe N. Hayashi N. Yanai N. Nabesima Y. Yamamoto M. J. Biol. Chem. 1997; 272: 12611-12615Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). These findings confirmed that ZBP-89 plays essential roles for Gata1 gene regulation during hematopoietic development. ZBP-89 binds to a GC-rich sequence that is also bound by numerous Krüppel-like transcription factors (KLFs), such as Sp1, Sp3, and EKLF/KLF1. Sp1 and EKLF interacted physically with GATA-1 and synergistically activated reporter transcription in transient transfection assays (22Gregory R.C. Taxman D.J. Seshasayee D. Kensinger M.H. Bieker J.J. Wojchowski D.M. Blood. 1996; 87: 1793-1801Crossref PubMed Google Scholar, 23Merika M. Orkin S.H. Mol. Cell. Biol. 1995; 15: 2437-2447Crossref PubMed Scopus (437) Google Scholar). Thus, a functional redundancy might exist among KLFs in the regulation of Gata1 gene expression. In addition, because ZBP-89, Sp1, and EKLF associate with GATA-1, these factors might be able to act as cofactors and direct DNA binding of these factors might not be necessary. To test these possibilities, we examined the GC-rich region in the G1HE by transgenic mouse reporter assays and gel mobility shift assays (EMSAs). We found that five contiguous deoxyguanosine residues (G5 string) within the GC-rich sequence are essential for Gata1 gene reporter expression in vivo. Interestingly, a series of EMSAs demonstrated that deletion of one deoxyguanosine residue from the G5 string (G4 mutant) specifically abolished binding to ZBP-89, but not to Sp3 and other binding factors. Furthermore, we show that the G4 mutation significantly reduced the Gata1-HRD reporter activity in fetal liver of transgenic mice. These data indicate that the G5 string is necessary for Gata1 gene expression and binding of ZBP-89 to this region is critical for the G1HE activity in vivo. IE3.9LacZ and IE3.9intLacZ reporter genes were constructed as described previously (3Onodera K. Takahashi S. Nishimura S. Ohta J. Motohashi H. Yomogida K. Hayashi N. Engel J.D. Yamamoto M. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 4487-4492Crossref PubMed Scopus (143) Google Scholar). For assembling the mutated and deleted reporter plasmids, a 1.3-kb G1HE fragment was excised from IE3.9intLacZ and cloned into pBluescript II KS+ plasmid between the XhoI and EcoRI sites (pBSG1HE). Fragments 1–235 and 1–207 of the G1HE were amplified by PCR using pBSG1HE as a template and inserted into the 5′-end of IE2.6LacZ that was constructed as described previously (3Onodera K. Takahashi S. Nishimura S. Ohta J. Motohashi H. Yomogida K. Hayashi N. Engel J.D. Yamamoto M. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 4487-4492Crossref PubMed Scopus (143) Google Scholar). Mutations were created in pBSG1HE using a QuikChangeTM site-directed mutagenesis kit according to the manufacturer's directions. For IE3.9intG4LacZ, the mutated G1HE fragment was removed from pBSG1HE and replaced by the G1HE in IE3.9intLacZ. For 1–235mutIE2.6LacZ and 1–235IE2.6G4LacZ constructs, the G1HE-(1–235) fragments were amplified using mutated pBSG1HE as a template and cloned into the 5′-end of IE2.6LacZ. The GATA-1 plasmids for maltose-binding protein (MBP) were constructed by means of a pMAL-c2 vector as described previously (24Nishikawa K. Kobayashi M. Masumi A. Lyons S.E. Weinstein B.M. Liu P.P. Yamamoto M. Mol. Cell. Biol. 2003; 23: 8295-8305Crossref PubMed Scopus (36) Google Scholar). The prokaryotic expression plasmid pGEXZBP-89 for generating the glutathione S-transferase (GST)-ZBP-89 protein was a generous gift from Dr. Juanita L. Merchant, University of Michigan. The cDNAs for selected domains of ZBP-89 were generated by PCR and cloned into a pGEX vector with modified restriction enzyme sites. The eukaryotic expression plasmid pCDNA3.1BFCOL1 was a generous gift from Dr. Ken-ichi Isobe, University of Nagoya. The expression plasmid for the C-terminal Myc-tagged BFCOL-1 (ZBP-89-Myc) was made from pCDNA3.1BFCOL1. The cDNAs for GATA-1 and its deletion mutants were cloned into a pEF-BOS eukaryotic expression vector (25Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5322Crossref PubMed Scopus (1501) Google Scholar) as described previously (26Shimizu R. Takahashi S. Ohneda K. Engel J.D. Yamamoto M. EMBO J. 2001; 20: 5250-5260Crossref PubMed Scopus (111) Google Scholar). For the luciferase reporter constructs, the G1HE-(124–235) fragment was amplified by PCR and cloned into the thymidine kinase minimal promoter reporter plasmid pT81-luc (27Nordeen S.K. BioTechniques. 1988; 6: 454-458PubMed Google Scholar). Transgenic reporter mice were generated by microinjection into fertilized BDF-1 eggs using standard procedures (28Hogan B. Beddington R. Constantini F. Lacy E. Manipulating the Mouse Embryo. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994Google Scholar). Transgenic founders were dissected on embryonic day 14.5 and transgene integration was verified by PCR amplification of a lacZ gene fragment (4Nishimura S. Takahashi S. Kuroha T. Suwabe N. Nagasawa T. Trainor C. Yamamoto M. Mol. Cell. Biol. 2000; 20: 713-723Crossref PubMed Scopus (100) Google Scholar). Reporter expression was examined by 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal) staining using cryosections of fetal liver as described previously (4Nishimura S. Takahashi S. Kuroha T. Suwabe N. Nagasawa T. Trainor C. Yamamoto M. Mol. Cell. Biol. 2000; 20: 713-723Crossref PubMed Scopus (100) Google Scholar). At least two separate fetal liver sections stained with X-gal were prepared for each embryo. At least 200 erythroid cells and 50 megakaryocytes were counted in each fetal liver section. Fetal liver with more than 10% of the total cells counted expressing β-galactosidase was scored as positive. We did not observe a founder with an X-gal staining that was positive only for erythroid cells, but not for megakaryocytes, or vice versa. Micrograph images were taken with a BX40F4 research microscope (Olympus) with an UplanF1 objective lens (20 × 0.40 or 40 × 0.75). Data acquisition was carried out with a DP70 digital camera and DP-controller software (Olympus). Nuclear extract preparation was performed as previously reported (4Nishimura S. Takahashi S. Kuroha T. Suwabe N. Nagasawa T. Trainor C. Yamamoto M. Mol. Cell. Biol. 2000; 20: 713-723Crossref PubMed Scopus (100) Google Scholar). The plasmid pGEXZBP-89 DNA binding domain (DBD) was generated by PCR. GST fusion proteins were expressed in the Escherichia coli strain BL21(DE3)pLysS (Invitrogen) and purified as previously reported (15Merchant J.L. Iyer G.R. Taylor B.R. Kitchen J.R. Mortensen E.R. Wang Z. Flintoft R.J. Michel J.B. Bassel-Duby R. Mol. Cell. Biol. 1996; 16: 6644-6653Crossref PubMed Scopus (117) Google Scholar). Probe labeling and the DNA-binding assay were performed as previously described (26Shimizu R. Takahashi S. Ohneda K. Engel J.D. Yamamoto M. EMBO J. 2001; 20: 5250-5260Crossref PubMed Scopus (111) Google Scholar). For supershift assays, a mouse monoclonal antibody for Sp1, a rabbit polyclonal antibody for Sp3 (sc-420 and sc-644, respectively, Santa Cruz), and a rabbit polyclonal antibody for ZBP-89 (code number 100-401-685, Rockland) were used. The p21 and p21 mut sequences shown in Fig. 3, A and B, are as follows: p21 (forward), 5′-ggttggtcctgcctctgagggggcggggcctgggccgag-3′; p21 (reverse), 5′-ctcggcccaggccccgccccctcagaggcaggaccaacc-3′; p21 mut (forward), 5′-ggttggtcctgcctctgagggttcggggcctgggccgag-3′; and p21 mut (reverse), 5′-ctcggcccaggccccgaaccctcagaggcaggaccaacc-3′. The ChIP assay was performed using mouse erythroleukemia (MEL) cells. A total of 106 cells per immunoprecipitation (IP) were fixed with 0.4% formaldehyde for 10 min at room temperature. Cross-links were quenched with 125 mm glycine. Cells were washed with phosphate-buffered saline (PBS), resuspended in SDS cell lysis buffer (10 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% SDS, 1 mm EDTA and protease inhibitor mixture) and sonicated for four 15-s pulses using a TOMY UD-201 sonicator. The lysates were diluted 5 times with ChIP dilution buffer (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100 and protease inhibitor mixture). The diluted samples were used for IP with a rat monoclonal antibody for GATA-1 (N6, sc-265, Santa Cruz) or a rabbit polyclonal antibody for ZBP-89 (number 100-401-685, Rockland). To prepare antibody-bead complexes, 15 μl of Dynabeads-Protein A (Invitrogen) was equilibrated with PBS for 30 min at 4 °C. The beads were resuspended with 675 μl of PBS containing 50 μg/ml bovine serum albumin, and 5 μg of anti-rat IgG rabbit antibody (Jackson ImmunoResearch) as a secondary antibody and incubated on a rotator for 2 h at 4 °C. Then the beads were washed twice with PBS and resuspended with 675 μl of PBS containing 50 μg/ml bovine serum albumin and 1 μg of anti-GATA-1 antibody as a primary antibody, incubated on a rotator for 2 h at 4 °C. The antibody-bead complexes were then washed twice with ChIP dilution buffer. For ZBP-89 ChIP, 3 μg of anti-ZBP-89 antibody was conjugated directly to Dynabeads-Protein A without a secondary antibody. The prepared antibody-bead complexes were resuspended with the diluted sample and incubated on a rotator overnight at 4 °C. The immune complexes were washed twice with ChIP dilution buffer, once with Wash buffer I (20 mm Tris-HCl, pH 8.0, 500 mm NaCl, 2 mm EDTA, 1% Triton X-100, 0.1% SDS and protease inhibitor mixture), once with Wash buffer II (10 mm Tris-HCl, pH 8.0, 500 mm NaCl, 1 mm EDTA, 0.25 m LiCl, 0.5% deoxycholate, 0.5% Nonidet P-40 and protease inhibitor mixture), and twice with TE buffer. The TE buffer was replaced with 300 μl of elution buffer (50 mm Tris-HCl, pH 8.0, 10 mm EDTA, and 1% SDS). After incubation for 2 h at 65 °C, Proteinase K was added and incubated overnight at 65 °C. The DNA fragments were extracted with phenol-chloroform, precipitated with ethanol, and suspended with 50 μl of TE buffer. Purified DNA from IP was analyzed in triplicates by a Mx3000P real time PCR system (Stratagene) with 2 μl of DNA solution and Platinum SYBR Green qPCR SuperMix (Invitrogen). The results were normalized with those from input DNA without IP. The PCR primers were as follows: 5′-tccaggaatgaagaaatggg-3′ (forward) and 5′-gtatgggggagtctcattgg-3′ (reverse) for G1HE; 5′-ggtccaggaaaaggcataag-3′ (forward) and 5′-tactgcccacctctatcagg-3′ (reverse) for a Gata1 coding region (exon 6) as a negative control. These primer pairs amplified a single product confirmed by 2% agarose gel electrophoresis and melting-curve analysis. For reChIP assays, the first immunoprecipitated complexes were eluted from Dynabeads-Protein A with reChIP elution buffer (20 mm dithiothreitol and 0.01% Triton X-100 in PBS) at 37 °C for 30 min. The eluted complex was diluted 50 times with ChIP dilution buffer and the ChIP procedure was repeated again. MBPs for GATA-1 were prepared and the binding assay with GST-ZBP-89 was performed as previously described (29Shimizu R. Trainor C.D. Nishikawa K. Kobayashi M. Ohneda K. Yamamoto M. J. Biol. Chem. 2007; 282: 15862-15871Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Immunoblotting was performed with anti-MBP antibody (sc-808, Santa Cruz). The siRNA for human GATA-1 (D-009610), ZBP-89 (D-012658), Sp3 (D-23096), and control (D-001210) were purchased from Thermo Scientific. One hundred pmol of siRNA was transfected into K562 cells (2 × 106 cells per transfection) by using the Cell Line Nucleofector Kit V (Lonza) according to the manufacturer's protocol. Cells were harvested 24 h after the transfection and nuclear extracts or total RNA were prepared for analyses. Western blotting was performed as described previously (26Shimizu R. Takahashi S. Ohneda K. Engel J.D. Yamamoto M. EMBO J. 2001; 20: 5250-5260Crossref PubMed Scopus (111) Google Scholar). GATA-1 (C20) and Lamin B (M20) antibodies were purchased from Santa Cruz Biotechnology. For densitometry analysis, x-ray films were scanned by LAS-3000 image analyzer and density measurements were done by using MultiGauge version 3.0 software (FUJI FILM). Total RNA was isolated by using ISOGEN (NIPPON GENE). Reverse transcription reactions were performed by using Superscript II (Invitrogen) according to the manufacturer's protocol. The quantitative real time PCRs were performed as described in ChIP assays. Primer sequences are as follows: GATA-1 (forward), 5′-gctcaactgtatggagggga-3′; GATA-1 (reverse), 5′-cagttgaggcagggtagagc-3′; ZBP-89 (forward), 5′-cgcatttagaagatgcgtca-3′; ZBP-89 (reverse), 5′-attttgctccagtggctgtt-3′; and glyceraldehyde-3-phosphate dehydrogenase (forward), 5′-agatcatcagcaatgcctcc-3′; glyceraldehyde-3-phosphate dehydrogenase (reverse), 5′-tgtggtcatgagtccttcca-3′. DAPA was performed according to the methods of Billon et al. (30Billon N. Carlisi D. Datto M.B. van Grunsven L.A. Watt A. Wang X.F. Rudkin B.B. Oncogene. 1999; 18: 2872-2882Crossref PubMed Scopus (122) Google Scholar) with minor modifications. K562 cells were transfected with the expression plasmid for Myc-tagged ZBP-89 by using the Cell Line Nucleofector Kit V. Nuclear extracts were collected 24 h after the transfection. To prepare DNA probes, the G1HE-(131–230) oligonucleotides (wild type, 5′-gattcccttatctatgccttcccagctgcctccctgctggctgaactgtggccacagacttctgggccttgcaccccctccacagggatgggggagggaa-3′; GATA box mutant, 5′-gattccctggcttatgccttcccagctgcctccctgctggctgaactgtggccacagacttctgggccttgcaccccctccacagggatgggggagggaa-3′; and G5 strings mutant, 5′-gattcccttatctatgccttcccagctgcctccctgctggctgaactgtggccacagacttctgggccttgcacgcactccacagggatgcacgagggaa-3′) were biotinylated at their 5′ termini (Operon Biotechnologies) and annealed with the corresponding antisense-strand oligonucleotides. The DAPA was performed by mixing 1 μg of the biotinylated probe, 20 μg of salmon testes DNA, and 200 μg of K562 nuclear extracts in binding buffer containing 20 mm HEPES, pH 7.9, 10% glycerol, 50 mm KCl, 0.2 mm EDTA, 1.5 mm MgCl2, 10 μm ZnCl2, 1 mm dithiothreitol, and 0.25% Triton X-100. The sample was incubated on ice for 50 min and 25 μl of NeutrAvidin-agarose beads (Thermo Scientific) were added to the reactions. After a 2-h incubation at 4 °C, the agarose beads were washed 4 times in binding buffer and then once in PBS. The DNA-bound protein was eluted by adding 25 μl of 2× SDS sample buffer and heating at 95 °C for 5 min. The eluted protein was resolved on SDS-protein gel and Western blot analysis was performed using anti-GATA-1 antibody (C20) and anti-c-Myc antibody (Sigma). Quail fibrosarcoma QT6 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. A total of 5 × 104 cells per transfection were seeded on 12-well plates 24 h before transfection. Cells were co-transfected with various combinations of a firefly luciferase reporter (either wild type or mutant), expression plasmids, and a Renilla luciferase control plasmid (pRLTK luc). Cells were harvested 48 h after transfection. Cell lysis and luciferase activity measurements were performed with a Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. Data were shown as average ± S.E. Statistical analysis was performed using Student's t test. Previously, we and other groups found that 3′-deletion of the G1HE reduced Gata1 reporter expression in the fetal livers of transgenic mice, even in the presence of the critical GATA box located in the 5′ region of the G1HE (4Nishimura S. Takahashi S. Kuroha T. Suwabe N. Nagasawa T. Trainor C. Yamamoto M. Mol. Cell. Biol. 2000; 20: 713-723Crossref PubMed Scopus (100) Google Scholar, 11Vyas P. McDevitt M.A. Cantor A.B. Katz S.G. Fujiwara Y. Orkin S.H. Development. 1999; 126: 2799-2811Crossref PubMed Google Scholar). In this study, we sought to identify the cis-acting element required for reporter expression in fetal livers of transgenic mice. We generated several reporter constructs (Fig. 1A) and examined their expressions in the fetal livers of transgenic founders on embryonic day 14.5 (E14.5). When the lacZ reporter gene was linked to the IE exon and its 3.9-kbp upstream region (IE3.9LacZ), both erythroid cells and megakaryocytes in fetal livers were stained with X-gal in 9 of 17 transgenic embryos (Table 1). Similar results were obtained when the 3′ 236–1174-bp portion of the G1HE was deleted (1–235IE2.6LacZ, see Table 1 and Fig. 1B). In contrast, no X-gal positive embryos were obtained when base pairs 208–1174 of the G1HE was deleted (1–207IE2.6LacZ, Table 1 and Fig. 1B). These results indicate that a critical cis-acting element for reporter expression in the fetal liver resides within the 208–235-bp region of the G1HE.TABLE 1Expression of LacZ in the E14.5 fetal livers of transgenic foundersConstructsNumber of foundersNumber of LacZ-positive embryosIE3.9LacZ1791–235IE2.6LacZ1571–207IE2.6LacZ1601–235mutIE2.6LacZ170 Open table in a new tab To define the sequences obligatory for transcription factor binding within this region of the G1HE, we performed an EMSA using nuclear extracts prepared from K562 human leukemia cells that express GATA-1 abundantly. We employed oligonucleotides based on the G1HE-(201–235) bp sequence as a probe. This region contains a high GC content (69%) and is conserved in the corresponding region of the human GATA1 gene, except that 209A is replaced by G in human. Multiple retarded bands were observed using nuclear extracts from K562 cells (Fig. 1C). To determine the sequence critical for the formation of complexes, we generated six unlabeled competitors (C1–C6) based on serial mutations of the G1HE-(201–235) sequence and examined their abilities to compete with probe binding (Fig. 1
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