Context-dependent GATA Factor Function
2007; Elsevier BV; Volume: 282; Issue: 19 Linguagem: Inglês
10.1074/jbc.m700792200
ISSN1083-351X
AutoresRyan J. Wozniak, Meghan E. Boyer, Jeffrey A. Grass, Youngsook Lee, Emery H. Bresnick,
Tópico(s)Hemoglobinopathies and Related Disorders
ResumoGATA factors are fundamental components of developmentally important transcriptional networks. By contrast to common mechanisms in which transacting factors function directly at promoters, the hematopoietic GATA factors GATA-1 and GATA-2 often assemble dispersed complexes over broad chromosomal regions. For example, GATA-1 and GATA-2 occupy five conserved regions over ∼100 kb of the Gata2 locus in the transcriptionally repressed and active states, respectively, in erythroid cells. Since it is unknown whether the individual complexes exert qualitatively distinct or identical functions to regulate Gata2 transcription in vivo, we compared the activity of the -3.9 and +9.5 kb sites of the Gata2 locus in transgenic mice. The +9.5 site functioned as an autonomous enhancer in the endothelium and fetal liver of embryonic day 11 embryos, whereas the -3.9 site lacked such activity. Mechanistic studies demonstrated critical requirements for a GATA motif and a neighboring E-box within the +9.5 site for enhancer activity in endothelial and hematopoietic cells. Surprisingly, whereas this GATA-E-box composite motif was sufficient for enhancer activity in an erythroid precursor cell line, its enhancer function in primary human endothelial cells required additional regulatory modules. These results identify the first molecular determinant of Gata2 transcription in vascular endothelium, composed of a core enhancer module active in both endothelial and hematopoietic cells and regulatory modules preferentially required in endothelial cells. GATA factors are fundamental components of developmentally important transcriptional networks. By contrast to common mechanisms in which transacting factors function directly at promoters, the hematopoietic GATA factors GATA-1 and GATA-2 often assemble dispersed complexes over broad chromosomal regions. For example, GATA-1 and GATA-2 occupy five conserved regions over ∼100 kb of the Gata2 locus in the transcriptionally repressed and active states, respectively, in erythroid cells. Since it is unknown whether the individual complexes exert qualitatively distinct or identical functions to regulate Gata2 transcription in vivo, we compared the activity of the -3.9 and +9.5 kb sites of the Gata2 locus in transgenic mice. The +9.5 site functioned as an autonomous enhancer in the endothelium and fetal liver of embryonic day 11 embryos, whereas the -3.9 site lacked such activity. Mechanistic studies demonstrated critical requirements for a GATA motif and a neighboring E-box within the +9.5 site for enhancer activity in endothelial and hematopoietic cells. Surprisingly, whereas this GATA-E-box composite motif was sufficient for enhancer activity in an erythroid precursor cell line, its enhancer function in primary human endothelial cells required additional regulatory modules. These results identify the first molecular determinant of Gata2 transcription in vascular endothelium, composed of a core enhancer module active in both endothelial and hematopoietic cells and regulatory modules preferentially required in endothelial cells. Tissue-specific gene regulation is a tightly controlled process directed and altered by a plethora of cellular cues. Although numerous cell type-specific transcription factors have been identified, and common biochemical mechanisms have emerged (1Kadonaga J.T. Cell. 2004; 116: 247-257Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 2Orphanides G. Reinberg D. Cell. 2002; 108: 439-451Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar), many questions remain unanswered regarding how ensembles of such factors function at complex mammalian loci. Considerable progress has been made in analyzing how members of the GATA transcription factor family (GATA-1 to -6) establish transcriptional networks that orchestrate developmental processes, including hematopoietic stem cell differentiation into diverse blood cell types (3Bresnick E.H. Martowicz M.L. Pal S. Johnson K.D. J. Cell. Physiol. 2005; 205: 1-9Crossref PubMed Scopus (90) Google Scholar, 4Cantor A.B. Orkin S.H. Semin. Cell Dev. Biol. 2005; 16: 117-128Crossref PubMed Scopus (116) Google Scholar). In addition to conferring developmental control, GATA factors can function in certain differentiated cell types, including endothelial cells. GATA-2 was initially identified as an activator of endothelin-1 expression in endothelial cells (5Lee M.E. Temizer D.H. Clifford J.A. Quertermous T. J. Biol. Chem. 1991; 266: 16188-16192Abstract Full Text PDF PubMed Google Scholar, 6Dorfman D.M. Wilson D.B. Bruns G.A. Orkin S.H. J. Biol. 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GATA-2 occupies these sites at the transcriptionally active locus, and GATA-1 displaces GATA-2 concomitant with repression (26Grass J.A. Boyer M.E. Paul S. Wu J. Weiss M.J. Bresnick E.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8811-8816Crossref PubMed Scopus (298) Google Scholar, 27Martowicz M.L. Grass J.A. Boyer M.E. Guend H. Bresnick E.H. J. Biol. Chem. 2005; 280: 1724-1732Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 29Grass J.A. Jing H. Kim S-I. Martowicz M.L. Pal S. Blobel G.A. Bresnick E.H. Mol. Cell. Biol. 2006; 26: 7056-7067Crossref PubMed Scopus (118) Google Scholar). Although it is unknown whether the GATA switch sites function similarly or distinctly to regulate Gata2 transcription in vivo, they exhibit certain structural and functional differences. 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In principle, context-dependent functions can arise at the level of chromatin occupancy or postchromatin occupancy. Although GATA motifs are abundant throughout the genome, only a small subset are occupied in cells (29Grass J.A. Jing H. Kim S-I. Martowicz M.L. Pal S. Blobel G.A. Bresnick E.H. Mol. Cell. Biol. 2006; 26: 7056-7067Crossref PubMed Scopus (118) Google Scholar, 37Johnson K.D. Grass J.D. Boyer M.E. Kiekhaefer C.M. Blobel G.A. Weiss M.J. Bresnick E.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11760-11765Crossref PubMed Scopus (110) Google Scholar, 38Pal S. Nemeth M.J. Bodine D.M. Miller J.L. Svaren J. Thein S.L. Lowry P.J. Bresnick E.H. J. Biol. Chem. 2004; 279: 31348-31356Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 39Bresnick E.H. Johnson K.D. Kim S-I. Im H. Prog. Nucleic Acids Res. Mol. Biol. 2006; 81: 435-471Crossref PubMed Scopus (24) Google Scholar). 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Context-dependent post-chromatin occupancy activities can involve the combinatorial arrangement of GATA motifs with neighboring cis-elements; a particularly instructive example involves an Ets motif adjacent to a GATA motif. This configuration allows a GATA-1-FOG-1 complex to activate the megakaryocytic αIIB promoter in a transient transfection assay; without the Ets motif, the GATA-1-FOG-1 complex represses the reporter (40Wang X. Crispino J.D. Letting D.L. Nakazawa M. Poncz M. Blobel G.A. EMBO J. 2002; 21: 5225-5234Crossref PubMed Scopus (142) Google Scholar). In addition, a GATA-E-box composite element, which mediates assembly of a multiprotein complex containing GATA-1, LMO2, LDB1, SCL/TAL1 and E2A in erythroid cells (41Wadman I.A. Osada H. Grutz G.G. Agulnick A.D. Westphal H. Forster A. Rabbitts T.H. EMBO J. 1997; 16: 3145-3157Crossref PubMed Scopus (732) Google Scholar, 42Xu Z. Huang S. Chang L.S. Agulnick A.D. Brandt S.J. Mol. Cell. 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Patient R.K. Green A.R. EMBO J. 2002; 21: 3039-3050Crossref PubMed Scopus (194) Google Scholar). Enhancer activities in transfection assays often do not recapitulate transcriptional mechanisms in vivo, and therefore we tested whether GATA switch sites at the Gata2 locus have distinct enhancer activities in transgenic mouse embryos. We demonstrate that the +9.5 GATA switch site functions autonomously as a strong enhancer in endothelial cells and the fetal liver, a major site of erythropoiesis during embryogenesis. By contrast, the -3.9 GATA switch site, which binds GATA-1 and GATA-2 in erythroid precursor cells (27Martowicz M.L. Grass J.A. Boyer M.E. Guend H. Bresnick E.H. J. Biol. Chem. 2005; 280: 1724-1732Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 29Grass J.A. Jing H. Kim S-I. Martowicz M.L. Pal S. Blobel G.A. Bresnick E.H. Mol. Cell. Biol. 2006; 26: 7056-7067Crossref PubMed Scopus (118) Google Scholar), lacks autonomous activity in vivo and has little to no activity in cultured endothelial cells. Mechanistic analyses revealed that the +9.5 enhancer critically requires a core module, consisting of a GATA motif and a neighboring E-box, in both endothelial cells and erythroid precursor cells. Surprisingly, although the core module was sufficient for enhancer activity in the erythroid precursor cell line, its enhancer function in primary human endothelial cells required additional regulatory modules. These studies provide evidence that the combinatorial usage of enhancer modules can establish context-dependent GATA factor functions. Cell Culture—HUVECs and HAECs (Cascade Biologics) were maintained in Medium 200 (Cascade Biologics) containing 1% penicillin/streptomycin (Invitrogen) and Low Serum Growth Supplement (Cascade Biologics). G1E cells (31Weiss M.J. Yu C. Orkin S.H. Mol. Cell. Biol. 1997; 17: 1642-1651Crossref PubMed Scopus (295) Google Scholar) were maintained in Iscove’s modified Dulbecco’s medium (Gibco/Invitrogen) containing 1% penicillin/streptomycin (Gibco/Invitrogen), 2 units/ml erythropoietin, 120 nm monothioglycerol (Sigma), 0.6% conditioned medium from a Kit ligand-producing Chinese hamster ovary cell line, and 15% fetal bovine serum (Gibco/Invitrogen). Plasmid Constructs—GATA-2 sequences were cloned from a murine 129SV bacterial artificial chromosome DNA isolated by Research Genetics/Invitrogen. Primers used to amplify genomic regions of Gata2 for the creation of the plasmid constructs used herein are available upon request. The integrity of cloned sequences was confirmed by DNA sequence analysis. The pGL3basic luciferase reporter plasmid was obtained from Promega. For LacZ reporter constructs, sequences identical to the respective transient construct were cloned into the pSVβ vector (Clontech). Generation of the 1SLuc, (-3.9)1SLuc, (-1.8)1SLuc, (+9.5)1SLuc, and (+9.5 mtG-1,2,3)1SLuc constructs was described previously (27Martowicz M.L. Grass J.A. Boyer M.E. Guend H. Bresnick E.H. J. Biol. Chem. 2005; 280: 1724-1732Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 29Grass J.A. Jing H. Kim S-I. Martowicz M.L. Pal S. Blobel G.A. Bresnick E.H. Mol. Cell. Biol. 2006; 26: 7056-7067Crossref PubMed Scopus (118) Google Scholar). The (+9.5 mtG-2)1SLuc and (+9.5 mtE)1SLuc constructs were generated by replacing the central GATA motif (AGATAA) with an EcoRI site (GAATTC), whereas the E-box (CATCTG) was replaced by a SalI site (GTCGAC). The individual 5′ and 3′ arm deletions, as well as the 5′ arm truncations (1Kadonaga J.T. Cell. 2004; 116: 247-257Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 2Orphanides G. Reinberg D. Cell. 2002; 108: 439-451Abstract Full Text Full Text PDF PubMed Scopus (714) Google Scholar, 3Bresnick E.H. Martowicz M.L. Pal S. Johnson K.D. J. Cell. Physiol. 2005; 205: 1-9Crossref PubMed Scopus (90) Google Scholar, 4Cantor A.B. Orkin S.H. Semin. Cell Dev. Biol. 2005; 16: 117-128Crossref PubMed Scopus (116) Google Scholar, 5Lee M.E. Temizer D.H. Clifford J.A. Quertermous T. J. Biol. Chem. 1991; 266: 16188-16192Abstract Full Text PDF PubMed Google Scholar) of the +9.5 site, were created by amplifying the relevant region with the appropriate primers (primers available upon request), digesting with KpnI and XhoI, and then ligating the digested, purified product into 1SLuc. For concurrent deletion of both the 5′ and 3′ arms of the +9.5 site, oligonucleotides were annealed and inserted into the 1SLuc plasmid digested with KpnI and XhoI. Similarly, insertion of the +9.5 GATA motifs/E-box in place of the -3.9 GATA sites was accomplished by ligating two sets of annealed oligonucleotides into the (-3.9)1SLuc plasmid digested with HinfI and NcoI. Finally, the -3.9 kb GATA motifs were inserted in place of the +9.5 GATA motifs/E-box by using chimeric primers partially homologous to the (+9.5)1SLuc template and partially nonhomologous (the -3.9 GATA motifs). The purified amplicon was digested with MboI and RsrII and ligated into an identically digested (+9.5)1SLuc plasmid. The 5′ arm was cloned upstream of pGL3pro (Promega), containing the SV40 promoter, to generate (+9.5 5′arm)SV40Luc. Transient Transfection Assay—HUVECs and HAECs were plated 1 day prior to transfection and were ∼60-70% confluent at the time of transfection. An equal amount of each plasmid (2 μg) was added to 100 μl of Opti-MEM (Invitrogen) reduced serum medium, incubated with Lipofectin reagent (6 μl/1 μg of DNA; Invitrogen) for 15 min at room temperature, and then added to the cells. The cells were incubated with the transfection mixture for 3 h before the readdition of Medium 200. Cell lysates were harvested 48 h post-transfection and were assayed for luciferase activity using the Luciferase Assay System (Promega). G1E cell transfections were conducted as described previously (29Grass J.A. Jing H. Kim S-I. Martowicz M.L. Pal S. Blobel G.A. Bresnick E.H. Mol. Cell. Biol. 2006; 26: 7056-7067Crossref PubMed Scopus (118) Google Scholar). The luciferase activity for each sample was normalized to the protein concentration of the lysate, as determined by a Bradford assay (Bio-Rad) using γ-globulin as a standard. At least two independent preparations of each plasmid were analyzed. Transgenic Mice—Transgenic mice harboring LacZ reporter constructs were generated by standard procedures by the University of Wisconsin Transgenic Animal Facility. Briefly, DNA constructs for F0 transgenic analysis were linearized, purified with an Elutip-d column (Schleicher & Schuell), and microinjected into fertilized mouse oocytes. To identify embryos containing LacZ transgenes, genomic DNA from yolk sac was subjected to PCR analysis with the following primers: LacZ Forward (5′-GATCTTCCTGAGGCCGATACTGTCGTCGTCCCCTCA-3′) and LacZ Reverse (5′-GTAGTCGGTTTATGCAGCAACGAGACGTCACGG-3′). For whole mount analysis, 5-bromo-4-chloro-3-indolyl β-galactoside (X-gal; Sigma) staining was performed with E11 embryos as described previously (62Onodera 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). Embryos were fixed with 2% formaldehyde, 0.2% glutaraldehyde, and 0.02% Nonidet P-40 (Sigma) in PBS for 2 h at 4 °C. Embryos were washed twice with PBS and then incubated overnight at 37 °C in 2 mm MgCl2, 5 mm K3Fe(CN)6, 5 mm K4Fe(CN)6, and X-gal (0.5 mg/ml) in PBS. After X-gal staining, embryos were washed twice with PBS and postfixed with 4% formaldehyde overnight at 4 °C. For tissue sections, the post-fixed embryos were dehydrated through progressive washes in 50, 70, 85, 95, and 100% ethanol. Samples were embedded in paraffin and dried overnight at room temperature before being sectioned. The sectioned embryos (10 μm) were counter-stained with 0.1% Nuclear Fast Red staining solution in 5% aluminum sulfate. Distinct Functions of Gata2 Locus GATA Switch Sites in Vivo—GATA-1 and GATA-2 occupy five highly conserved regions (Fig. 1) of the Gata2 locus in erythroid precursor cells, and these GATA switch sites confer qualitatively and quantitatively distinct enhancer activities in GATA-1- and GATA-2-expressing hematopoietic cells in vitro (26Grass J.A. Boyer M.E. Paul S. Wu J. Weiss M.J. Bresnick E.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8811-8816Crossref PubMed Scopus (298) Google Scholar, 27Martowicz M.L. Grass J.A. Boyer M.E. Guend H. Bresnick E.H. J. Biol. Chem. 2005; 280: 1724-1732Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 29Grass J.A. Jing H. Kim S-I. Martowicz M.L. Pal S. Blobel G.A. Bresnick E.H. Mol. Cell. Biol. 2006; 26: 7056-7067Crossref PubMed Scopus (118) Google Scholar). To determine if the differential activities in vitro reflect unique activities in vivo, we analyzed the +9.5 and -3.9 sites in F0 transgenic mouse embryos. These elements, located at +9.5 and -3.9 kb relative to the Gata2 1S promoter, were cloned upstream of the 1S promoter fused to LacZ, injected into mouse oocytes, and implanted into recipient females. Embryos were harvested at E11 and stained with X-gal to reveal LacZ expression. The (+9.5)1SLacZ transgene displayed a reproducible expression pattern in 7 of the 31 embryos containing the LacZ transgene (Fig. 2A). The remaining embryos showed no detectable transgene expression. Transverse sections of all seven expressing embryos revealed expression throughout vascular endothelium, in endocardial cells lining the interior of the heart, and in a subset of cells in the fetal liver (Fig. 2A and supplemental Fig. 1), which is heavily colonized with GATA-1- and GATA-2-expressing hematopoietic precursor cells at E11 (53McGrath K.E. Palis J. Exp. Hematol. 2005; 33: 1021-1028Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 54Mikkola H.K.A. Orkin S.H. Development. 2006; 133: 3733-3744Crossref PubMed Scopus (392) Google Scholar). Although quantitative differences in expression were apparent (compare whole mount images of two representative (+9.5)1SLacZ embryos in Fig. 2A), no ectopic staining was detected. Thus, the +9.5 element confers enhancer activity in multiple sites of endogenous Gata2 expression.FIGURE 2Endothelial enhancer activity of Gata2 +9. 5 site in vivo. A, the photographs illustrate representative whole mount and transverse sections of two (left and right columns) E11 transgenic embryos expressing LacZ (blue cells) under control of the Gata2 +9.5 site fused to the Gata2 1S promoter. Histological sections reveal expression of the (+9.5)1SLacZ construct in vascular endothelium, including the dorsal aorta (DA) and endocardium (EC), and also in the fetal liver (FL), which is heavily colonized by hematopoietic precursors at this stage of development. The arrowheads highlight LacZ-positive cells. B, the photographs illustrate representative whole mount and transverse sections of two (left and right columns) representative E11 transgenic embryos containing the Gata2 -3.9 site fused to the Gata2 1S promoter. Endothelial and hematopoietic (FL) staining was absent in (-3.9)1SLacZ transgenic embryos. C, enhancer activities of Gata2 switch site regions (-3.9, -1.8, and +9.5) in human endothelial cells. HUVECs and HAECs were transiently transfected with reporter plasmids derived from the pGL3 luciferase vector containing the Gata2 1S promoter cloned upstream of luciferase (1SLuc). The plots depict luciferase activities of the cell lysates normalized by the protein concentrations of the lysates. The activity of the 1SLuc construct was designated 1.0 (means ± S.E.). In HUVECs, the 1SLuc, (+9.5)1SLuc, (-3.9)1SLuc, and (-1.8)1SLuc constructs were analyzed in 8, 8, 5, and 4 independent experiments, respectively; in HAEC, all constructs were analyzed in two independent experiments. In each experiment, transfections were performed in triplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT) By contrast to the +9.5 site, analysis of (-3.9)1SLacZ in 15 E11 embryos containing the transgene revealed no e
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