Disruption of an SP2/KLF6 Repression Complex by SHP Is Required for Farnesoid X Receptor-induced Endothelial Cell Migration
2006; Elsevier BV; Volume: 281; Issue: 51 Linguagem: Inglês
10.1074/jbc.m607720200
ISSN1083-351X
AutoresAmitava Das, Martín E. Fernández-Zapico, Sheng Cao, Janet Yao, Stefano Fiorucci, Robert P. Hebbel, Raúl Urrutia, Vijay H. Shah,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoThe farnesoid X receptor (FXR) signaling pathway regulates bile acid and cholesterol homeostasis. Here, we demonstrate, using a variety of gain- and loss-of-function approaches, a role of FXR in the process of cell motility, which involves the small heterodimeric partner (SHP)-dependent up-regulation of matrix metalloproteinase-9. We use this observation to reveal a transcriptional regulatory mechanism involving the SP/KLF transcription factors, SP2 and KLF6. Small interference RNA-based silencing studies in combination with promoter, gel shift, and chromatin immunoprecipitation assays indicate that SP2 and KLF6 bind to the matrix metalloproteinase-9 promoter and together function to maintain this gene in a silenced state. However, upon activation of FXR, SHP interacts with SP2 and KLF6, disrupting the SP2/KLF6 repressor complex. Thus, together, these studies identify a mechanism for antagonizing Sp/KLF protein repression function via SHP, with this process regulating endothelial cell motility. The farnesoid X receptor (FXR) signaling pathway regulates bile acid and cholesterol homeostasis. Here, we demonstrate, using a variety of gain- and loss-of-function approaches, a role of FXR in the process of cell motility, which involves the small heterodimeric partner (SHP)-dependent up-regulation of matrix metalloproteinase-9. We use this observation to reveal a transcriptional regulatory mechanism involving the SP/KLF transcription factors, SP2 and KLF6. Small interference RNA-based silencing studies in combination with promoter, gel shift, and chromatin immunoprecipitation assays indicate that SP2 and KLF6 bind to the matrix metalloproteinase-9 promoter and together function to maintain this gene in a silenced state. However, upon activation of FXR, SHP interacts with SP2 and KLF6, disrupting the SP2/KLF6 repressor complex. Thus, together, these studies identify a mechanism for antagonizing Sp/KLF protein repression function via SHP, with this process regulating endothelial cell motility. Farnesoid X Receptor (FXR) 3The abbreviations used are: FXR, farnesoid X receptor; BOEC, blood outgrowth endothelial cell; CDCA, chenodeoxycholic acid; 6-ECDCA, 6-ethylchenodeoxycholic acid; SHP, small heterodimeric partner; MMP-9, matrix metalloproteinase-9; Sp, stimulatory protein; KLF, Kruppel-like factor; siRNA, short interfering RNA; ChIP, chromatin immunoprecipitation; RLA, relative luciferase activity; RT, reverse transcription; qRT-PCR, quantitative real-time PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; VEGF, vascular epidermal growth factor; rFXR, retroviral FXR; rSHP, retroviral SHP; EMSA, electrophoretic mobility shift assay. is a member of the nuclear receptor superfamily of transcription factors. In response to ligand binding, FXR regulates expression of genes involved in bile acid, cholesterol, and triglyceride metabolism (1Davis R.A. Miyake J.H. Hui T.Y. Spann N.J. J. 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Thus, whereas SP2 and KLF6 repress MMP-9 promoter activity under basal conditions, activation of FXR results in a process by which SHP displaces SP2/KLF6 from the MMP-9 promoter, thereby up-regulating MMP-9 expression and function. Together, these studies identify SHP as a key disruptor of SP2/KLF6-mediated gene silencing to allow transcriptional activation by FXR. The functional importance of this pathway is supported by its biological role in FXR-induced endothelial cell motility. Cell Culture—Blood outgrowth endothelial cells (BOECs) are a blood-derived endothelial cell commonly utilized for investigations of vascular cell motility and remodeling (29Lin Y. Weisdorf D.J. Solovey A. Hebbel R.P. J. Clin. Invest. 2000; 105: 71-77Crossref PubMed Scopus (1338) Google Scholar). BOECs (P4–P6) were prepared from human blood using primary cell isolation conditions that have been previously described (29Lin Y. Weisdorf D.J. Solovey A. Hebbel R.P. J. Clin. Invest. 2000; 105: 71-77Crossref PubMed Scopus (1338) Google Scholar). Cells were grown in EBM-2 medium supplemented with EGM-2, 10% fetal bovine serum, and 1% streptomycin/penicillin. Cells were incubated with CDCA, 6-ECDCA, or equivalent volume of vehicle (Me2SO) at concentrations and durations that were based on prior work (27Fiorucci S. Antonelli E. Rizzo G. Renga B. Mencarelli A. Riccardi L. Orlandi S. Pellicciari R. Morelli A. Gastroenterology. 2004; 127: 1497-1512Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar) and that are indicated in individual experiments. HepG2 cells were also used in some experiments in which the molecular intervention was not feasible in BOECs. Retroviral Overexpression—FXR and SHP plasmid constructs (from Benjamin Shneider and David Mangelsdorf) and lacZ were subcloned into the retroviral vector pMMP using standard molecular approaches to generate pMMP-rFXR, pMMP-rSHP, and pMMP-rLacZ (30Zeng H. Sanyal S. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 32714-32719Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). All constructs were sequenced for confirmation. In brief, to generate retrovirus, 5 × 106 293T/17 cells in 100-mm dishes were co-transfected with three plasmids, 1.5 μg of pMD.MLV gag.pol, 0.5 μg of pMD.G, and 2 μg of relevant retroviral vector using Effectene™ transfection reagent (Qiagen). Cell culture medium containing retrovirus was collected 48 h after transfection. For cell transduction, 2 ml of viral stock and 8 ml of fresh Dulbecco's modified Eagle's medium were mixed and added in a 100-mm dish of 0.5 × 106 BOECs with 8 μg/ml Polybrene. Transduction efficiency using this approach was uniformly >90%. Cells were used for experiments after 18–24 h. Microarray Analysis—Cells were incubated with vehicle or CDCA (100 μm) for 48 h. Total RNA was isolated using an RNeasy kit according to the manufacturer's instruction (Qiagen), and 3 μg was used for the probe preparation using the GEArray AmpoLabeling-LPR kit and biotin-16,2′-deoxyuridine-5′-triphosphate. GEArray Q Series Human Endothelial Cell Biology Gene Array (HS-036) membrane was used for hybridization with the synthesized probe and detected by using the Chemiluminescent detection kit (Super Array Bioscience Corp., Frederick, MD) according to the manufacturer's protocol. Changes in expression were assessed by the software provided by the manufacturer. siRNA Gene Silencing—siRNA targeting human FXR, SHP, MMP-9, SP2, KLF6, and a scrambled control were obtained from Ambion (Austin, TX). Cells were transfected with siRNA using Oligofectamine (Invitrogen) as we described previously (16Cao S. Fernandez-Zapico M.E. Jin D. Puri V. Cook T.A. Lerman L.O. Zhu X.Y. Urrutia R. Shah V. J. Biol. Chem. 2005; 280: 1901-1910Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Conditions and concentrations required for specificity of knockdown with high transfection efficiency were individually established for each of the siRNA (see supplemental materials). Semiquantitative and Quantitative RT-PCR—Total RNA was extracted from cells as described above. 1.0 μg of total RNA was used for the cDNA synthesis using oligo(dT) primer of SuperScript III First-Strand Synthesis System (Invitrogen) and appropriate forward and reverse primers of FXR and GAPDH for the semiquantitative PCR. Thermal cycling conditions were: 2 min at 94 °C, followed by 40 cycles of 94 °C for 15 s, 55 °C for 30 s, and 72 °C for 60 s and a final extension by 1 cycle of 72 °C for 10 min in a Hybaid PCR express instrument. For TaqMan-based quantitative real-time PCR analysis (qRT-PCR) 25 ng of each cDNA was added to the TaqMan Universal PCR Master Mix along with 900 nm of each primer and 200 nm of probe according to the manufacturer's instruction (Applied Biosystems, Foster City, CA) (7Sirvent A. Claudel T. Martin G. Brozek J. Kosykh V. Darteil R. Hum D.W. Fruchart J.C. Staels B. FEBS Lett. 2004; 566: 173-177Crossref PubMed Scopus (86) Google Scholar, 12Zhao A. Lew J.L. Huang L. Yu J. Zhang T. Hrywna Y. Thompson J.R. de Pedro N. Blevins R.A. Pelaez F. Wright S.D. Cui J. J. Biol. Chem. 2003; 278: 28765-28770Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Real-time fluorescence monitoring was performed with the Applied Biosystems 7500 Real Time PCR System instrument. Amplification of human GAPDH and eukaryotic 18 S rRNA (Applied Biosystems) was used in the same reaction of all samples as an internal control. FXR, SHP, MMP-9, SP2, and KLF6 mRNA was normalized to GAPDH mRNA and shown as the -fold change. Gelatin Zymography—Conditioned media samples from cells incubated with vehicle, CDCA, or 6-ECDCA for 48 h were mixed with electrophoresis loading buffer and subjected to 8% SDS-PAGE co-polymerized with gelatin type B (2 mg/ml). After washing with 2.5% Triton X-100 and incubation in zymogram developing buffer, the gels were stained with 0.5% Coomassie, and then destained. Gelatinolytic activities were detected as transparent bands (31Huhtala P. Tuuttila A. Chow L.T. Lohi J. Keski-Oja J. Tryggvason K. J. Biol. Chem. 1991; 266: 16485-16490Abstract Full Text PDF PubMed Google Scholar). MMP-9 Enzyme-linked Immunosorbent Assay—Total human MMP-9 levels were measured from cultured BOEC supernatants by enzyme-linked immunosorbent assay (R&D Biosystems, Minneapolis, MN) based on the manufacturer's instructions. Cell Motility—Chemotaxis was measured by using a modified Boyden chamber assay (8-μm pore size, Neuro Probes, Inc., Gaithersburg, MD) as we described previously (32Lee J.S. Kang Decker N. Chatterjee S. Yao J. Friedman S. Shah V. Am. J. Pathol. 2005; 166: 1861-1870Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Briefly, the bottom wells of the chamber were filled with 26 μl of serum-free media containing 10 ng/ml VEGF, and covered with a polycarbonated filter. 2 × 104 serum-starved cells were added into the upper chamber of each well. Cells were incubated for 6 h at 37 °C with CDCA or vehicle to stimulate FXR-dependent gene transcription. Migrated cells at the lower surface of filters were fixed and stained using Hema3 stain (Biochemical Sciences, Inc., Swedesboro, NJ). Cellular migration was determined by counting the number of stained cells on membranes in five randomly selected, non-overlapping high power fields. Data are presented as percent change in VEGF-induced chemotaxis. Luciferase Reporter Assay—HepG2 cells were transfected with wild-type or mutated Sp1 human MMP-9-promoter-luciferase reporter constructs (33Sato H. Seiki M. Oncogene. 1993; 8: 395-405PubMed Google Scholar) (Dr. H. Sato, Japan) and 0.01 μg of Renilla luciferase reporter vector to control for transfection efficiency (pRL-TK) using Lipofectamine 2000 (Invitrogen). 10 h later, culture medium was changed, and cells were cultured for an additional day then stimulated with vehicle or 50 μm CDCA for 6 h. Luciferase assays were conducted using a dual luciferase kit (Promega, Madison, WI) as described previously (16Cao S. Fernandez-Zapico M.E. Jin D. Puri V. Cook T.A. Lerman L.O. Zhu X.Y. Urrutia R. Shah V. J. Biol. Chem. 2005; 280: 1901-1910Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Electrophoretic Mobility Shift Assay—5.0 μg of nuclear extract from vehicle or CDCA (50 μm)-stimulated cells was incubated in a binding buffer (50 mm Hepes, pH 7.9, 1.0 mm EDTA, 100 mm KCl, 20% glycerol, 2.5 mm MgCl2, 0.5 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 1.5 μg of poly(dI-dC)) for 10 min at room temperature. The 32P-labeled probe encoding the Sp1 binding site of MMP-9 promoter 5′-ATTCCTTCCGCCCCCAGATG-3′ was added for 20 min. In some experiments, an excess of cold probe, at the indicated dilutions, was added concomitant with the addition of radiolabeled probe. The mixture was electrophoresed at 12.5 V/cm on 4% nondenaturing polyacrylamide gel in 0.5 × TBE (Tris borate-EDTA). Gels were vacuum-dried and autoradiographed (16Cao S. Fernandez-Zapico M.E. Jin D. Puri V. Cook T.A. Lerman L.O. Zhu X.Y. Urrutia R. Shah V. J. Biol. Chem. 2005; 280: 1901-1910Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Chromatin Immunoprecipitation Assay—Cells were transfected with either control pcDNA or pSP2 or pKLF6 and then incubated with vehicle or CDCA (50 μm). Cells were cross-linked with formaldehyde for 10 min at 37 °C, then harvested in SDS lysis buffer (Upstate Biotechnology) and sheared to fragment DNA. Samples were then immunoprecipitated using agarose-conjugated antibodies to SP2 or KLF6 (Santa Cruz Biotechnology, Santa, Cruz, CA), control (IgG) antibody, or agarose beads alone. After removal of the cross-links, immunoprecipitated DNA was purified using phenol/chloroform extraction (500 μl) and ethanol precipitation. DNA was used for PCR with sense primer 5′-ATT CAG CCT GCG GAA GAC AGG G-3′ and antisense primer 5′-TGA TGG AAG ACT CCC TGA GAC TTC-3′ encoding the Sp1 binding site of MMP-9 promoter and detected by visualizing PCR products on an agarose gel as we previously described (16Cao S. Fernandez-Zapico M.E. Jin D. Puri V. Cook T.A. Lerman L.O. Zhu X.Y. Urrutia R. Shah V. J. Biol. Chem. 2005; 280: 1901-1910Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Co-immunoprecipitation and Western Blot Analysis—Cells overexpressing rSHP-His were lysed in a buffer (50 mm Tris, pH 7.5/150 mm NaCl/3 mm MgCl2/1 mm EDTA/0.5% Nonidet P-40/10% glycerol). Lysates were precleared with control IgG and Protein A-Sepharose beads, and then incubated overnight with Anti-His antibody (Santa Cruz Biotechnology). After incubation with Protein A-Sepharose beads and washing, samples were separated by SDS-PAGE, transferred to nylon membranes, and immunoblotted with SP2 or KLF6 antibody. To study the endogenous interaction of SHP with SP2/KLF6, nuclear extract of cells treated with either vehicle or CDCA (50 μm) were immunoprecipitated with SP2 or KLF6 antibody (Santa Cruz Biotechnology) and immunoblotted with SHP antibody (Santa Cruz Biotechnology) as described above. Proteins were also probed separately with human FXR, SP2, KLF6, and Histone H1 (Santa Cruz Biotechnology), MMP-9 (BD Biosciences), and β-actin (Sigma). Statistical Analysis—The data in the bar graphs represent the mean ± S.D. of at least three independent experiments, each performed with duplicate samples. Blots, autoradiographs, and micrographs represent typical experiments reproduced at least three times with similar results. Statistical analyses were performed using a Student's t test, with a two-tailed value of p < 0.05 considered significant. FXR Regulates Cell Motility via the Up-regulation of MMP-9—Our work initiated from the observation that FXR is expressed in BOECs and regulates cell motility via the up-regulation of MMP-9, a key cellular protease important in cell motility (Fig. 1, A–E, and supplemental Fig. S1A). Initially, the FXR-mediated up-regulation of MMP-9 was identified using a pathway-specific array approach (Fig. 1A). These results were confirmed by qRT-PCR, which showed that the FXR ligand, CDCA, induced a concentration-dependent up-regulation of MMP-9 mRNA levels, with a 6-fold increase observed in response to 10 μm CDCA (Fig. 1B). In addition to these observations at the mRNA level, we also found that FXR activation resulted in a 2-fold increase in MMP-9 enzymatic activity and 3-fold increase in MMP-9 protein levels as measured by gelatin zymography (Fig. 1C) and enzyme-linked immunosorbent assay, respectively (Fig. 1D). Moreover, similar results were obtained with the synthetic FXR agonist, 6-ECDCA (Fig. 1, B–D). Lastly, cells were incubated with the natural FXR ligand, CDCA, under the same conditions used above to stimulate FXR-dependent gene transcription, and then chemotaxis was examined in response to the chemotactic agent, VEGF. Interestingly, cells incubated with CDCA evidenced enhanced migration compared with those incubated with vehicle (Fig. 1E). To more firmly establish the MMP-9 protease in cell migration, we silenced MMP-9 using a specific siRNA. MMP-9 protein levels were markedly reduced in the presence of MMP-9 siRNA as observed by Western blot (supplemental Fig. S1B). Also, MMP-9 mRNA levels were significantly reduced by 80% in the presence of MMP-9 siRNA, whereas GAPDH expression remained unchanged as observed by qRT-PCR (supplemental Fig. S1, C and D). Interestingly, under these conditions migration was not observed in cells transfected with MMP-9 siRNA indicating that this molecule is required to trigger FXR-dependent cell motility. In addition to the pharmacological experiments shown above, a series of molecular gain- and loss-of-function approaches were also used to confirm that CDCA activation of MMP-9 gene transcription was indeed mediated directly by FXR. First, cells were transfected with FXR siRNA or scrambled siRNA. A concentration-dependent decrease in FXR mRNA levels was observed in response to FXR siRNA in cells with 30 nm FXR siRNA effectively knocking down 85% of FXR mRNA levels (supplemental Fig. S2, A–C). CDCA increased MMP-9 mRNA levels in cells transfected with scrambled siRNA but not in cells transfected with FXR siRNA as assessed by qRT-PCR (Fig. 2A). Furthermore, retroviral FXR (rFXR) overexpression rescued the MMP-9-silencing effect of FXR siRNA both basally as well as in response to CDCA (Fig. 2A). In control experiments, rFXR robustly increased both FXR mRNA and protein levels by severalfold (supplemental Fig. S2D). Lastly, siRNA transfection of BOECs also suppressed BOEC migration indicating that the effects of CDCA on cell motility were also occurring through an FXR-dependent mechanism (Fig. 2B). Therefore, these studies demonstrate that FXR regulates cell motility through an MMP-9-dependent mechanism. FXR Activation of MMP-9 Gene Transcription Requires SHP—To search for biochemical mechanisms used by FXR to regulate gene transcription, we used HepG2 cells, a commonly used cell for transcription mechanism studies relating to FXR (4Denson L.A. Sturm E. Echevarria W. Zimmerman T.L. Makishima M. Mangelsdorf D.J. Karpen S.J. Gastroenterology. 2001; 121: 140-147Abstract Full Text PDF PubMed Scopus (370) Google Scholar). First, we co-transfected HepG2 cells with a human wild-type MMP-9 promoter and either FXR siRNA or scrambled siRNA and then incubated cells with vehicle or 50 μm CDCA. CDCA significantly increased relative luciferase activity in HepG2 cells co-transfected with scrambled siRNA, as compared with FXR siRNA (Fig. 2C). Next, we overexpressed FXR or retrovirus encoding lacZ in HepG2 cells transfected with wild-type MMP-9 promoter and then incubated cells with vehicle or CDCA. Interestingly, the relative luciferase activity was significantly increased by >2.5-fold in HepG2 cells overexpressing FXR as compared with lacZ control, both in the presence and absence of CDCA (Fig. 2D). One prototypical pathway by which FXR regulates target genes is through heterodimerization with nuclear receptors such as retinoic acid receptor and binding to consensus IR-1 repeats located within target promoters (11Zavacki A.M. Lehmann J.M. Seol W. Willson T.M. Kliewer S.A. Moore D.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7909-7914Crossref PubMed Scopus (88) Google Scholar, 12Zhao A. Lew J.L. Huang L. Yu J. Zhang T. Hrywna Y. Thompson J.R. de Pedro N. Blevins R.A. Pelaez F. Wright S.D. Cui J. J. Biol. Chem. 2003; 278: 28765-28770Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Another indirect pathway occurs through transcriptional activation of the atypical non-DNA binding nuclear receptor SHP (13Boulias K. Katrakili N. Bamberg K. Underhill P. Greenfield A. Talianidis I. EMBO J. 2005; 24: 2624-2633Crossref PubMed Scopus (120) Google Scholar). To differentiate between the roles of these two pathways, we performed computer-assisted analysis of the published nucleotide sequence upstream of the transcriptional start site of the human MMP-9 gene, which failed to identify an IR-1 repeat (data not shown). Consequently, we focused our attention on SHP as a mediator of FXR activation of MMP-9 gene transcription. For this purpose, first, we measured mRNA and protein levels of SHP in response to increasing concentrations of CDCA. A concentration-dependent increase in SHP mRNA levels was observed in cells incubated with CDCA as analyzed by qRT-PCR (Fig. 3A). A concurrent concentration-dependent increase in SHP protein levels were also observed as analyzed by Western blot (Fig. 3B). In addition, CDCA induced up-regulation of SHP mRNA levels was not observed in cells transfected with FXR siRNA, confirming SHP as an FXR target gene (Fig. 3C). Interestingly, an increase in SHP mRNA levels (2.26-fold) was also observed in response to MMP-9 siRNA (supplemental Fig. S3A). However, a time kinetic analysis using qRT-PCR revealed that the CDCA-induced increase in SHP mRNA levels preceded the increase in MMP-9 mRNA levels (Fig. 3D). To determine if SHP is required for CDCA activation of MMP-9 gene transcription, we next examined if gene silencing of SHP could block CDCA-induced MMP-9 up-regulation. Indeed, MMP-9 mRNA levels did not increase in response to CDCA in BOECs transfected with SHP siRNA (Fig. 4A). Furthermore, retroviral SHP (rSHP) overexpression rescued the MMP-9-silencing effect of FXR siRNA both basally as well as in the presence of CDCA (Fig. 4A). In control experiments, a concentration-dependent decrease in SHP mRNA level was observed in cells transfected with SHP siRNA with 30 nm SHP siRNA reducing SHP mRNA levels by 70% and blocking CDCA induced up-regulation of SHP mRNA (supplemental Fig. S3, B and C). Next, overexpression of SHP mRNA levels was pursued using a retroviral system (rSHP), which increased SHP mRNA levels in transduced cells by >4-fold (supplemental Fig. S3D). Overexpression potentiated both basal and CDCA-induced up-regulation of MMP-9 mRNA levels by 2-fold (Fig. 4B). Subsequently, these results were corroborated using the MMP-9 promoter in HepG2 cells. siRNA-based silencing of SHP abolished the CDCA-induced increase in MMP-9 promoter activity (Fig. 4C). Additionally, transduction experiments using rSHP significantly increased the relative MMP-9 luciferase activity by >2-fold both basally and in response to CDCA (Fig. 4D). These studies establish that FXR activates MMP-9 gene transcription through SHP and led us to further identify the components of this pathway. SHP-mediated Antagonism of SP2 and KLF6 Repression Is Required for the Up-regulation of MMP-9 by FXR—The MMP-9 promoter contains an Sp1 binding site and indeed prior studies have demonstrated that Sp1 is important for regulating MMP-9 gene transcription (33Sato H. Seiki M. Oncogene. 1993; 8: 395-405PubMed Google Scholar). Interestingly, the expanding family of Sp/KLF proteins regulates target genes through DNA binding to Sp1 motifs. Furthermore, intersection of FXR signaling with Sp/KLF protein gene regulation is unexplored. To examine this further, we transfected HepG2 cells with wild-type reporter luciferase construct or one with a point mutation in the Sp1 site. The Sp1 mutant construct evidenced a severalfold increase in MMP-9 promoter activity, suggesting that repressor protein binding to the Sp1 motif of the MMP-9 promoter may regulate MMP-9 gene transcription (Fig. 5A). Because Sp1-like proteins may function as repressors, we comprehensively examined the
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