TY - JOUR TI - Sp3 represses gene expression via the titration of promoter-specific transcription factors AU - Kennett, SB AU - Moorefield, KS AU - Horowitz, JM T2 - JOURNAL OF BIOLOGICAL CHEMISTRY AB - We have determined previously that Sp3 encodes three distinct gene products as follows: a full-length protein (Sp3) that is an activator of transcription and two isoforms (M1 and M2) derived via internal translational initiation that function as transcriptional repressors. To identify amino acids and functions required for transcriptional repression, we employed PCR-directed mutagenesis to create a panel of mutated M2 proteins. Biochemical and functional analyses of these mutated proteins indicate that functions encoded by the M2 carboxyl terminus, such as DNA binding activity and the capacity to form multimeric complexes, are not required or sufficient for transcriptional repression. Instead, a 93-amino acid portion of the trans-activation domain was shown to be the minimal portion of M2 required to block Sp-dependent gene expression. Transcriptional analysis of three Sp-dependent promoters showed that mutations sustained by many M2 proteins result in promoter-specific effects. Regions of M2 required for physical interactions with five TATA box-associated factors (TAFIIs) were mapped, and mutations that disrupt the interaction of M2 with two of these proteins, TAFII70 and TAFII40, were identified. We conclude that Sp3- mediated transcriptional repression is due, at least in part, to competition for promoter-specific transcription factors. We have determined previously that Sp3 encodes three distinct gene products as follows: a full-length protein (Sp3) that is an activator of transcription and two isoforms (M1 and M2) derived via internal translational initiation that function as transcriptional repressors. To identify amino acids and functions required for transcriptional repression, we employed PCR-directed mutagenesis to create a panel of mutated M2 proteins. Biochemical and functional analyses of these mutated proteins indicate that functions encoded by the M2 carboxyl terminus, such as DNA binding activity and the capacity to form multimeric complexes, are not required or sufficient for transcriptional repression. Instead, a 93-amino acid portion of the trans-activation domain was shown to be the minimal portion of M2 required to block Sp-dependent gene expression. Transcriptional analysis of three Sp-dependent promoters showed that mutations sustained by many M2 proteins result in promoter-specific effects. Regions of M2 required for physical interactions with five TATA box-associated factors (TAFIIs) were mapped, and mutations that disrupt the interaction of M2 with two of these proteins, TAFII70 and TAFII40, were identified. We conclude that Sp3- mediated transcriptional repression is due, at least in part, to competition for promoter-specific transcription factors. TATA box-associated factors glutathione S-transferase dihydrofolate reductase hemagglutinin 6-(2-deoxy-β-d-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-c][1,2]oxazin-7-one triphosphate Sp1 is the founding member of a family of five transcription factors, Sp1–5, that govern the expression of a wide variety of mammalian genes (for review, see Ref. 1Phillipsen S. Suske G. Nucleic Acids Res. 1991; 27: 2991-3000Crossref Scopus (537) Google Scholar). Sp1 encodes a ubiquitously expressed nuclear phosphoprotein that has been divided into five sub-domains based upon their respective functions (2Pascal E. Tjian R. Genes Dev. 1991; 5: 1646-1656Crossref PubMed Scopus (356) Google Scholar, 3Courey A.J. Tjian R. Cell. 1988; 55: 887-898Abstract Full Text PDF PubMed Scopus (1079) Google Scholar). The Sp1trans-activation domain is composed of three sub-domains termed A–C, each of which is capable of stimulating transcription if tethered to DNA via a DNA-binding domain. Sub-domains A and B are composed by serine- and threonine-rich regions as well as glutamine-rich regions. The glutamine-rich portions of A and B are believed to be required for trans-activation, whereas the function(s) of the serine/threonine-rich sub-regions is(are) less well understood. Domain C carries a number of charged amino acids and weakly stimulates transcription in the absence of domains A or B. Carboxyl-terminal to the domain C is a region featuring three Cys2-His2 zinc “fingers” required for sequence-specific DNA binding to GC-rich promoter elements. A carboxyl-terminal domain, termed D, facilitates protein multimerization and is essential for synergistic trans-activation of promoters with multiple Sp-binding sites. Sp1 associates with a large number of transcription-associated proteins, including components of the basal transcription complex (e.g.hTAFII130/dTAFII110 and hTAFII55; Refs. 4Chiang C.-M. Roeder R.G. Science. 1995; 267: 531-536Crossref PubMed Scopus (352) Google Scholar, 5Dynlacht B.D. Hoey T. Tjian R. Cell. 1991; 66: 563-576Abstract Full Text PDF PubMed Scopus (485) Google Scholar, 6Rojo-Niersbach E. Furukawa T. Tanese N. J. Biol. Chem. 2000; 274: 33778-33784Abstract Full Text Full Text PDF Scopus (31) Google Scholar, 7Gill G. Pascal E. Tseng Z.H. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 192-196Crossref PubMed Scopus (473) Google Scholar), sequence-specific DNA-binding proteins (e.g.E2F, YY1, p53, and AP-2; Refs. 8Pena P. Reutens A.T. Albanese C. D'Amico M. Watanabe G. Donner A. Shu I.W. Williams T. Pestell R.G. Mol. Endocrinol. 2001; 13: 1402-1416Crossref Scopus (49) Google Scholar, 9Lin S.-Y. Rhys Black A. Kostic D. Pajovic S. Hoover C.N. Azizkhan J.C. Mol. Cell. Biol. 1996; 16: 1668-1675Crossref PubMed Scopus (252) Google Scholar, 10Karlseder J. Rotheneder H. Wintersberger E. Mol. Cell. Biol. 1996; 16: 1659-1667Crossref PubMed Scopus (315) Google Scholar, 11Lee J.-S. Galvin K.M. Shi Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6145-6149Crossref PubMed Scopus (276) Google Scholar, 12Gualberto A. Baldwin Jr., A.S. J. Biol. Chem. 1995; 270: 19680-19683Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 13Seto E. Lewis B. Shenk T. Nature. 1993; 365: 462-464Crossref PubMed Scopus (249) Google Scholar), and transcriptional regulators (e.g. p107, HDAC-1, and VHL-1; Refs. 14Chang Y.-C. Illenye S. Heintz N.H. Mol. Cell. Biol. 2001; 21: 1121-1131Crossref PubMed Scopus (60) Google Scholar, 15Datta P.K. Raychaudhuri P. Bagchi S. Mol. Cell. Biol. 1995; 15: 5444-5452Crossref PubMed Scopus (91) Google Scholar, 16Mukhopadhyay D. Knebelmann B. Cohen H.T. Ananth S. Sukhatme V.P. Mol. Cell. Biol. 1997; 17: 5629-5639Crossref PubMed Scopus (309) Google Scholar, 17Venepally P. Waterman M.R. J. Biol. Chem. 1995; 270: 25402-25410Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 18Daniel S. Kim K.-H. J. Biol. Chem. 1996; 271: 1385-1392Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). As might be expected given the variety of proteins with which it interacts, protein-binding sites have been identified throughout Sp1. For example, hTAFII130/dTAFII110 interact with Sp1 via itstrans-activation domain; hTAFII55 binds the Sp1 zinc fingers, and E2F requires the zinc finger and D sub-domains of Sp1 for protein-protein interactions. A wide variety of extracellular stimuli have been shown to induce gene expression via discrete promoter elements bound by Sp1 and Sp3 (17–29). Moreover, subtle mutations that negate the association of Sp1/Sp3 with their cognate binding sites completely block the induction of such genes by their respective inducing agents. Although GC-rich elements within the promoters of many Sp1/Sp3-regulated genes have been identified and their necessity for induced transcription has been noted, it remains largely unclear how extracellular stimuli activate Sp1/Sp3-dependent genes. For example, in most instances treatment of cells with inducing agents does not lead to consistent alterations in (i) the abundance or subcellular localization of Sp1/Sp3, (ii) the affinity of Sp1/Sp3 for DNA, (iii) the formation of Sp1/Sp3 multimers, nor (iv) the post-translational modification Sp1/Sp3. Instead, extracellular stimuli may activate Sp-dependent genes via alterations in protein-protein interactions. For example, transforming growth factor-β induces p15Ink4B transcription by catalyzing the formation of protein complexes between Sp1 and members of the Smad family of transcription factors (30Feng X.-H. Lin X. Derynck R. EMBO J. 2000; 19: 5178-5193Crossref PubMed Scopus (350) Google Scholar). Whether regulated interactions between Sp family members and other factors account for the induced transcription of additional Sp-dependent genes remains to be determined. Several years ago we identified two novel Sp3-derived proteins, termed M1 and M2, that arise by internal translational initiation within the region of Sp3 mRNA that encodes the Sp3 B domain (31Kennett S.B. Udvadia A.J. Horowitz J.M. Nucleic Acids Res. 1997; 25: 3110-3117Crossref PubMed Scopus (233) Google Scholar). Sp3, M1, and M2 appear to be expressed in all mammalian cells and tissues at approximately equivalent levels independent of growth status or induction by extracellular stimuli. In contrast to full-length Sp3, M1 and M2 function as potent repressors of Sp-mediated transcription, and Sp3 is at least 10-fold more sensitive to M1/M2-mediated repression than is Sp1. Given that Sp3 encodes proteins with opposing activities, we reasoned that understanding their differential regulation may shed light on mechanisms governing the activity of Sp-dependent promoters. To understand further the mechanism(s) by which M1/M2 repress transcription, we prepared a panel of M2 proteins carrying a limited number of random amino acid substitutions and examined their capacity to function as transcriptional regulators of three Sp-regulated promoters: DHFR, p21, andMDR-1. Additionally, we examined each of these mutated proteins for their capacity to bind DNA, to form multimeric complexes with Sp family members, and to bind components of the basal transcription complex. These studies have resulted in the following observations. 1) Random mutagenesis generated a panel of mutated M2 proteins that carry “loss-of-function” and “gain-of-function” mutations. 2) Many amino acid substitutions affect M2-mediated repression in a promoter-specific fashion. 3) DNA binding activity and the capacity to multimerize are not required or sufficient for M2-mediated repression. 4) The minimal region required for transcriptional repression by M2 consists of 93 amino acids of the B domain. 5) Several components of the TAFII complex bind Sp1, Sp3, and M2 in vitro. 6) The binding of two TAFII1 proteins, TAFII70 and TAFII40, is compromised in several M2 mutants. We conclude from these observations that M1/M2-mediated repression occurs at least in part via the titration of one or more transcription factors that may be required in a promoter-specific fashion. C-33A cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA), and DrosophilaSchneider line-2 (SL2) cells were a gift of Dr. Cheaptip Benyajati (University of Rochester, Rochester, NY). C-33A and SL2 cells were cultured as described previously (31Kennett S.B. Udvadia A.J. Horowitz J.M. Nucleic Acids Res. 1997; 25: 3110-3117Crossref PubMed Scopus (233) Google Scholar, 32Udvadia A.J. Rogers K.T. Horowitz J.M. Cell Growth Differ. 1992; 3: 597-608PubMed Google Scholar, 33Udvadia A.J. Templeton D.J. Horowitz J.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3953-3957Crossref PubMed Scopus (199) Google Scholar). Rabbit anti-Sp3 was prepared against a GST fusion protein containing the amino-terminal 300 amino acids of Sp3, and its preparation and characterization have been described (31Kennett S.B. Udvadia A.J. Horowitz J.M. Nucleic Acids Res. 1997; 25: 3110-3117Crossref PubMed Scopus (233) Google Scholar). Affinity-purified mouse anti-HA.11 antibody was obtained from a commercial supplier (Covance Research Products, Richmond, CA). pPacSp1 was obtained from Dr. Robert Tjian (University of California, Berkeley; see Ref. 3Courey A.J. Tjian R. Cell. 1988; 55: 887-898Abstract Full Text PDF PubMed Scopus (1079) Google Scholar). pPacSp3, pPacM1, pPacM2, pBSK-Sp3/flu, pCR-M1/flu, and pCR-M2/flu were prepared as described (31Kennett S.B. Udvadia A.J. Horowitz J.M. Nucleic Acids Res. 1997; 25: 3110-3117Crossref PubMed Scopus (233) Google Scholar, 33Udvadia A.J. Templeton D.J. Horowitz J.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3953-3957Crossref PubMed Scopus (199) Google Scholar). Constructions employed for in vitro translation of human and DrosophilaTAFII proteins were obtained from Dr. Robert Tjian.DHFR-CAT has been described (34Swick A.G. Blake M.C. Kahn J.W. Azizkhan J.C. Nucleic Acids Res. 1989; 17: 9291-9304Crossref PubMed Scopus (91) Google Scholar). To generate aDHFR-luciferase construct, DHFR-CAT, 5′ (5′-GGAGATCTAGCGCGCGGCTGTACTAC-3′) and 3′ primers (5′-GGAAGCTTGCAGCCTGTACGCTGTGC-3′), and the PCR were employed to amplify DHFR promoter sequences. A resulting 175-bp promoter fragment was subcloned in plasmid pRL (Promega, Inc., Madison, WI). pgpLuc-B carries a 1320-bp portion of the human MDR-1promoter and was obtained from Dr. Kathleen Scotto (Memorial Sloan-Kettering Cancer Center, NY; see Ref. 35Ince T.A. Scotto K.W. J. Biol. Chem. 2001; 270: 30249-30252Abstract Full Text Full Text PDF Scopus (138) Google Scholar). p21P93-S carries a 44-bp portion of the human p21 promoter and was obtained from Dr. Xiao-Fan Wang (Duke University Medical Center; see Ref.19Datto M.B. Yu Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 20Li J.-M. Nichols M.A. Changrasekharan S. Xiong Y. Wang X.-F. J. Biol. Chem. 1995; 270: 26750-26753Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 21Biggs J.R. Kudlow J.E. Kraft A.S. J. Biol. Chem. 1996; 271: 901-906Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 22Greenwel P. Inagaki Y. Hu W. Walsh M. Ramirez F. J. Biol. Chem. 1997; 272: 19738-19745Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Mutagenesis of M2 was performed using methods described by Zaccolo et al. (44Zaccolo M. Williams D.M. Brown D.M. Gherardi E. J. Mol. Biol. 1996; 255: 589-603Crossref PubMed Scopus (275) Google Scholar). Reactions employed pGEX-M2 (see below) as template, 5′ (5′-CCTGACTTCATGTTGTATGAC-3′) and 3′ primers (5′-CAGTCACGATGAATTCTCGAGAATCCCTAGCTAGCGTAATCTG-3′), andTaq polymerase (Invitrogen). Three PCR cycles (92 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min) were performed in a 20-μl reaction containing 4 ng of linearized template, 0.5 μm primers, 500 μm each dNTP, and 500 μm dPTP (Amersham Biosciences). An aliquot of this reaction was subsequently employed as template for an additional 22 rounds of amplification in the absence of dPTP. PCR products were digested with DpnI, purified, and cloned in pCR-BluntII-TOPO. Expression plasmids carrying mutated M2 cDNAs were prepared by subcloning inserts from pCR-BluntII-TOPO into pPac (3Courey A.J. Tjian R. Cell. 1988; 55: 887-898Abstract Full Text PDF PubMed Scopus (1079) Google Scholar). Sequencing of M2 mutants was performed by the North Carolina State University sequencing facility using a PerkinElmer Life Sciences AB1377 sequenator or Sequenase version 2.0 DNA polymerase following a protocol supplied by the manufacturer (Amersham Biosciences). pGEX-Sp1, a bacterial expression construct that carries the Sp1 coding region fused in-frame with glutathione S-transferase (GST), has been described (36Murata Y. Kim H.G. Rogers K.T. Udvadia A.J. Horowitz J.M. J. Biol. Chem. 1994; 269: 20674-20681Abstract Full Text PDF PubMed Google Scholar). pGEX-M2 was prepared by subcloning the M2 cDNA carried by pCR-M2/flu in pGEX-2TK (Amersham Biosciences). GST-Sp3 was prepared using pBSK-Sp3/flu, 5′ (5′-GGGGGATCCGCCACCATGAATTCCGGGCCATCGCCG-3′), and 3′ primers (5′-GGAATTCCTCCATTGTCTCATTTCCAG-3′), and the PCR. A 2142-bp amplified fragment carrying the entire Sp3 cDNA was subcloned in pGEX-2TK. pGEX-FSH15 has been described previously (36Murata Y. Kim H.G. Rogers K.T. Udvadia A.J. Horowitz J.M. J. Biol. Chem. 1994; 269: 20674-20681Abstract Full Text PDF PubMed Google Scholar). To create GST expression plasmids carrying M2 cDNAs terminating at amino acids 103, 197, or 353, M2 cDNAs were amplified from pPacM2 using a 5′ primer (5′-GGGGGATCCATGGATAGTTCAGACAATTCA-3′), one of three 3′ primers (5′-CTACTAGACTCCTTGAAGTTG-3′, 5′-CTAACCAAGTGTGAGGGTTTC-3′, or 5′-TCAGTTAACAAACAAAAGGGCG-3′), and Taq polymerase. Amplified M2 cDNAs carrying premature termination codons were subcloned in pGEX-2TK. Mutated M2 GST fusion proteins were prepared by amplification from pPacM2 plasmids using 5′ (5′-GGGGGATCCATGGATAGTTCAGACAATTCA-3′) and 3′ primers (5′-GGAATTCCTCCATTGTCTCATTTCCAG-3′) andTaq polymerase. Amplified cDNAs were subcloned in pGEX-2TK. Baculovirus stocks encoding Sp1, Sp3, M1, and M2 were prepared using the PCR, appropriate primers, and pCMV4-Sp1/flu (37Udvadia A.J. Rogers K.T. Higgins P.D.R. Murata Y. Martin K.H. Humphrey P.A. Horowitz J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3265-3269Crossref PubMed Scopus (187) Google Scholar), pBSK-Sp3/flu, pCR-M1/flu, and pCR-M2/flu as substrates. Amplified cDNAs were subcloned in pFASTBacHTA and used to prepare virus stocks according to methods supplied by the manufacturer (Invitrogen). Transient transfections for transcription assays were performed by calcium phosphate precipitation as described (31Kennett S.B. Udvadia A.J. Horowitz J.M. Nucleic Acids Res. 1997; 25: 3110-3117Crossref PubMed Scopus (233) Google Scholar, 33Udvadia A.J. Templeton D.J. Horowitz J.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3953-3957Crossref PubMed Scopus (199) Google Scholar). Cell extracts were prepared for analysis 48 h after transfection. The Dual-Luciferase Reporter Assay System (Promega, Inc.) was employed to quantify luciferase activity precisely as recommended by the manufacturer. Luminescence was detected in a Lumat LB 9507 luminometer (EG & G Berthold, Bad Wildbad, Germany), and results were normalized against total cell protein concentration. To prepare Drosophila SL2 extracts for Western blotting or protein/DNA binding assays, transient transfections were performed using SuperFect Transfection Reagent (Qiagen Inc., Hilden, Germany). Cell extracts were prepared 48 h after transfection. Nuclear extracts were prepared using methods described by Lee et al. (38Lee K.A. Bindereif A. Green M.R. Gene Anal. Tech. 1988; 5: 22-31Crossref PubMed Scopus (394) Google Scholar). Oligonucleotides were synthesized on an automated DNA synthesizer, deprotected, and partially purified through Sephadex G-25 spin columns. Radiolabeled probes for standard protein/DNA binding assays were prepared from the following oligonucleotides and their complements: GT box (39Kingsley C. Winoto A. Mol. Cell. Biol. 1992; 12: 4251-4261Crossref PubMed Scopus (490) Google Scholar), 5′-AGCTTCCGTTGGGGTGTGGCTTCACGTCGA-3′; p21 (19Datto M.B. Yu Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar), 5′-GAGCCGGGGTCCCGCCTCCTTGAGGCGGGCCC-3′; and MDR-1 (35Ince T.A. Scotto K.W. J. Biol. Chem. 2001; 270: 30249-30252Abstract Full Text Full Text PDF Scopus (138) Google Scholar), 5′-CAGGAACAGCGCCGGGGCGTGGGCTAGC-3′. For quantitative protein/DNA binding assays, six double-stranded 60-mers were synthesized each carrying a single promoter-derived Sp-binding site flanked by common nucleotide sequences. The promoter-derived sequences utilized for these experiments are as follows: p21, 5′-CCCGCCTCCT-3′; MDR-1, 5′-CGCCGGGGCGTGGGC-3′; DHFR-1, 5′-AGGGCGTGGC-3′; DHFR-2, 5′-GAGGCGGGGC-3′; DHFR-3, 5′-GAGGCGGAGT-3′; and DHFR-4, 5′-TGGGCGGGGC-3′. Annealed and complementary oligonucleotides were radiolabeled and purified as described previously (31Kennett S.B. Udvadia A.J. Horowitz J.M. Nucleic Acids Res. 1997; 25: 3110-3117Crossref PubMed Scopus (233) Google Scholar, 32Udvadia A.J. Rogers K.T. Horowitz J.M. Cell Growth Differ. 1992; 3: 597-608PubMed Google Scholar). Protein/DNA binding assays were performed as described previously (31Kennett S.B. Udvadia A.J. Horowitz J.M. Nucleic Acids Res. 1997; 25: 3110-3117Crossref PubMed Scopus (233) Google Scholar, 32Udvadia A.J. Rogers K.T. Horowitz J.M. Cell Growth Differ. 1992; 3: 597-608PubMed Google Scholar), and complexes were visualized by autoradiography. For quantitative protein/DNA binding assays, whole cell protein extracts prepared from baculovirus-infected Sf9 cells were incubated with a radiolabeled probe derived from the c-fos gene (5′-CCCTTGCGCCACCCCTCT-3′; see Ref. 32Udvadia A.J. Rogers K.T. Horowitz J.M. Cell Growth Differ. 1992; 3: 597-608PubMed Google Scholar), and the resulting protein-DNA complexes were quantified in situ using an InstantImager (Packard Instrument Co.). Volumes of these extracts that led to half-maximal binding of this probe were then employed in assays performed in triplicate with Sp-binding sites derived from theDHFR, p21, and MDR-1 genes and quantified in situ. Whole cell or nuclear extracts were resolved on denaturing polyacrylamide gels and transferred to nitrocellulose using a semi-dry transfer apparatus. Nitrocellulose filters were incubated with 5% milk in TBS-T (2.42 g/liter Tris, 8 g/liter NaCl, pH 7.6, 1 ml/liter Tween 20) from 1 h to overnight. Primary antibodies were diluted in TBS-T (anti-Sp3 at 1:2000 and anti-HA.11 at 1:1000), incubated with filters for 1 h at room temperature, and washed with TBS-T. Filters were incubated with horseradish peroxidase-conjugated secondary antibodies diluted in TBS-T (anti-mouse, 1:10,000, Amersham Biosciences, or anti-rabbit, 1:40,000, Invitrogen) for 1 h at room temperature with gentle agitation and washed in TBS-T, and antigen-antibody complexes were detected using ECL Western blotting Detection Reagents (Amersham Biosciences). In vitro transcribed/translated proteins were produced using a coupled reticulocyte lysate system (TNT; Promega, Inc.) with T3 or T7 RNA polymerase and l-[35S]methionine (Tran35S-label; ICN). pBSK-Sp1/flu, pBSK-Sp3/flu, pCR-M2/flu, PCR-amplified mutated M2 cDNAs, and TAFIIconstructs were employed as templates for these reactions. Mutated M2 cDNAs in pPac were amplified and prepared for in vitrotranscription/translation using a 5′ T7 promoter-containing primer, a 3′ primer, and the PCR. Protein binding assays were performed as described (40Sterner J.M. Dew-Knight S. Musahl C. Kornbluth S. Horowitz J.M. Mol. Cell. Biol. 1998; 18: 2748-2757Crossref PubMed Scopus (165) Google Scholar). GST bead-bound proteins were resolved on denaturing polyacrylamide gels and visualized by autoradiography. We have shown previously that Sp1 and Sp3 stimulate transcription of theDHFR promoter and that Sp1/Sp3-mediated transcription is repressed by two isoforms of Sp3, termed M1 and M2, that arise via internal translational initiation (31Kennett S.B. Udvadia A.J. Horowitz J.M. Nucleic Acids Res. 1997; 25: 3110-3117Crossref PubMed Scopus (233) Google Scholar, 33Udvadia A.J. Templeton D.J. Horowitz J.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3953-3957Crossref PubMed Scopus (199) Google Scholar, 37Udvadia A.J. Rogers K.T. Higgins P.D.R. Murata Y. Martin K.H. Humphrey P.A. Horowitz J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3265-3269Crossref PubMed Scopus (187) Google Scholar). To determine whether these results were likely to reflect a general mechanism of transcriptional regulation, we extended our analyses to thep21 and MDR-1 promoters, two well characterized Sp-dependent promoters of physiologic and therapeutic interest. The p21-luciferase construct employed in these studies includes an Sp protein-binding site that is required for transcriptional stimulation by transforming growth factor-β, calcium, or sodium butyrate, as well as a second Sp protein-binding site that is also required for induction by sodium butyrate (19Datto M.B. Yu Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 23Nakano K. Mizuno T. Sowa Y. Orita T. Yoshino T. Okuyama Y. Fujita T. Ohtani-Fujita N. Matsukawa Y. Tokino T. Yamagishi H. Oka T. Nomura H. Sakai T. J. Biol. Chem. 1997; 272: 22199-22206Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 24Discher D.J. Bishopric N.H. Wu X. Peterson C.A. 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TheMDR-1-luciferase construct employed includes an Sp protein-binding site that is required for induction by serum or the c-Raf kinase (42Cornwell M.M. Smith D.E. J. Biol. Chem. 1993; 268: 15347-15350Abstract Full Text PDF PubMed Google Scholar, 43Miltenberger R.J. Cortner J. Farnham P.J. J. Biol. Chem. 1993; 268: 15674-15680Abstract Full Text PDF PubMed Google Scholar). As illustrated in Fig. 1 A, the ectopic expression of Sp3 in Drosophila SL2 cells stimulated transcription of the DHFR, p21, andMDR-1 promoters to varying degrees. p21transcription was stimulated 40-fold more effectively thanMDR-1 and 12-fold more than the DHFR promoter. Analogous results were obtained for activation of each of these promoters by Sp1 (data not shown). Consistent with results reported previously for the DHFR promoter, co-expression of M1 or M2 with Sp1 or Sp3 resulted in repression of p21 andMDR-1 transcription (Fig. 1 B and data not shown). Interestingly, the sensitivity of each promoter to M1/M2-mediated repression was noted to be promoter-specific. The promoter most acutely sensitive to activation by Sp1/Sp3, p21, exhibited the least sensitivity to M1/M2-mediated repression. In turn, a promoter that is only modestly activated by Sp1/Sp3, MDR-1, exhibited the greatest sensitivity to M1/M2-mediated repression. Sensitivity ofDHFR to Sp-mediated activation and repression was noted to fall between these two extremes. Given the results presented in Fig. 1, we conclude that intrinsic differences between Sp-dependent promoters influence the degrees to which they are activated by Sp1/Sp3 and repressed by M1/M2. Because Sp proteins activated and repressed the DHFR, p21, and MDR-1to varying degrees, we wished to determine whether these effects might be accounted for by inherent differences in their capacity to bind DNA. For example, the relative sensitivity of p21 to Sp3-mediated transcription and insensitivity to M2-mediated repression might be explained if Sp3 and M2 bind the p21 promoter with substantially different affinities. To address this issue, we performed a series of quantitative protein/DNA binding assays using radiolabeled DNA probes carrying Sp-binding sites from the DHFR,p21, and MDR-1 promoters. Recombinant baculovirus stocks encoding Sp1, Sp3, M1, or M2 proteins were prepared; Sf9 cells were infected with these viruses, and protein extracts from infected cells were normalized for DNA binding activity using a well characterized Sp protein-binding site derived from the mouse c-fos promoter. The volume of each protein extract that bound 50% of the c-fos probe was then employed in protein/DNA binding assays with six Sp protein-binding sites derived from the DHFR, p21, and MDR-1promoters. Each assay was performed in triplicate and quantitatedin situ. As shown in Table I, Sp proteins bound to each probe, and the capacity of a given Sp protein to bind these DNAs varied over a 3-fold range. Interestingly, little correlation was noted between the relative capacity of Sp proteins to bind these DNAs in vitro and the efficiencies with which they activate or repress transcription in vivo. For example, Sp1 and Sp3 bound the Sp-binding site derived from the MDR-1promoter more efficiently than a site derived from the p21promoter, yet MDR-1 is activated significantly less efficiently by both proteins in vivo. Similarly, Sp protein-binding sites derived from all three promoters were bound equivalently by M2, yet their sensitivity to M2-mediated repression varies over a 13-fold range in vivo. We conclude from these studies that the relative capacity of Sp proteins to stimulate or repress transcription is not directly correlated with the efficiency with which they interact with their cognate promoter binding sitesin vitro.Table IRelative binding of recombinant Sp proteins to oligonucleotides carrying Sp-binding sites derived from three Sp-dependent promotersSp proteinPromoterp21MDR-1DHFRDHFR-1DHFR-2DHFR-3DHFR-4Sp112.52.21.813.0Sp311.22.51.91.23.0M111.51.71.20.62.1M211110.51.4Protein extracts were prepared from Sf9 cells infected with baculovirus stocks encoding Sp family members, and the volume of each extract required to bind 50% of a radiolabeled oligonucleotide probe derived from the c-fos promoter was determined. This volume of cell extract was later employed in protein/DNA binding assays with oligonucleotides derived from the DHFR, p21, andMDR-1 promoters. Binding assays were performed in triplicate and quantified in situ. DNA binding activities for each protein were normalized to the amount of binding activity detected for each protein on the p21 oligonucleotide. Open table in a new tab Protein extracts were prepared from Sf9 cells infected with b DA - 2002/3/22/ PY - 2002/3/22/ DO - 10.1074/jbc.M108661200 VL - 277 IS - 12 SP - 9780-9789 SN - 0021-9258 ER -