Role Of Sp1 Research Articles

Structural and functional characterization of the human and mouse fibulin-1 gene promoters: role of Sp1 and Sp3.

Fibulin-1 is a multifunctional extracellular protein involved in diverse biological processes including cardiovascular development, haemostasis and cancer. To investigate the transcriptional regulation of the gene encoding fibulin-1 we cloned and analysed about 4.0 kb of the 5'-flanking regions of both the human and mouse fibulin-1 genes. The human and mouse fibulin-1 promoters share little sequence similarity except for a short region of approx. 150-170 bp immediately upstream of the translation start site. The conserved region contains a TATA-like sequence (ATAATT) and multiple consensus binding sites for Sp1 and activator protein 2 (AP-2). That the short conserved region in each gene confers basal promoter activity is demonstrated by transient transfections of promoter deletion constructs for both the human and mouse genes into cells that express fibulin-1 constitutively. Co-transfections of promoter constructs with expression plasmids for Sp1, Sp3 and Sp4 into Drosophila SL2 cells indicate that Sp1 and Sp3 are essential for transcriptional activation and that these two factors act synergistically. Electrophoretic mobility-shift assays show that Sp1 and Sp3, but not AP-2, bind to the basal promoter of the human fibulin-1 gene. The results demonstrate the functional importance of Sp1 and Sp3 in regulating the expression of the fibulin-1 gene.

Basal and parathyroid hormone induced expression of the human 25-hydroxyvitamin D 1α-hydroxylase gene promoter in kidney AOK-B50 cells: role of Sp1, Ets and CCAAT box protein binding sites

The regulation of the gene for renal 25-hydroxyvitamin D 1α- hydroxylase (1α(OH)ase; CYP27B1) by parathyroid hormone (PTH) under hypocalcemic conditions is fundamentally important for the maintenance of calcium and phosphate homeostasis. The molecular mechanism that underlies this hormonal response is of current interest and has been investigated in the present study by transfection analysis of the human 1α(OH)ase promoter in kidney AOK-B50 cells. We have shown that the first 305 bp of promoter can be induced by hormone in transient transfection assays and also within a chromatin environment when stably integrated. Mutagenesis of possible transcription factor binding sites within this promoter length has shown that three sites clustered within the region from −66 to −135 contribute to basal expression. A likely Sp1 and a CCAAT box site are particularly important for basal expression although these sites are not likely to functionally cooperate in a major way. Mutagenesis of the CCAAT box site consistently reduced PTH induction although mutagenesis of the Sp1, Ets and other possible binding sites in the 305 bp of promoter has no significant effect on the level of PTH induction. Other experiments showed that PTH induction but not basal expression was sensitive to the protein kinase inhibitor H89. We have therefore identified for the first time the sites in the 1α(OH)ase promoter responsible for basal expression and provide evidence for the role of a CCAAT box binding protein in a PTH mechanism of induction that involves an H89 sensitive step.

Blocking Sp1 Transcription Factor Broadly Inhibits Extracellular Matrix Gene Expression In Vitro and In Vivo: Implications for the Treatment of Tissue Fibrosis

Fibrosis is a consequence of injury characterized by accumulation of excess collagen and other extracellular matrix components, resulting in the destruction of normal tissue architecture and loss of function. Sp1 was originally described as a ubiquitous transcription factor. It is involved in the basal expression of extracellular matrix genes and may, therefore, be important in fibrotic processes. To evaluate the effect of Sp1 blockade on the expression of extracellular matrix genes, clones of NIH 3T3 fibroblasts stably transfected with an anti-sense Sp1 expression vector. Simultaneously reduced expression of several extracellular matrix genes as compared with mock-transfected clones was noted using differential hybridization of cDNA microarrays, without significant alteration in cell growth. Transfection of human dermal fibroblasts with several extracellular matrix gene (COL1A1, COL1A2, COL3A1, COL5A2, COL7A1, TIMP-1, and decorin) promoter/reporter constructs demonstrated that anti-sense Sp1-induced reduction of extracellular matrix gene mRNA steady-state levels results from transcriptional repression, consistent with the role of Sp1 as a transcription factor. Decoy Sp1 binding oligonucleotides inhibited COL1A2 promoter activity both in cultured fibroblasts and in vivo, in the skin of transgenic mice, which have integrated a mouse COL1A2 promoter/luciferase reporter gene construct. These results indicate that targeting Sp1 efficiently blocks extracellular matrix gene expression, and suggest that such an approach may represent an interesting therapeutic alternative toward the treatment of fibrotic disorders.

Role of Sp1 and Sp3 in the Transcriptional Regulation of the Rat Fatty Acid Synthase Gene

Inspection of the 5′ region of the sequence of the rat fatty acid synthase (FAS) gene revealed a high GC content between −900 and +500, implying several binding sites for members of the Sp1 family of transcription factors. Using SL2 and H4IIE cells in conjunction with FAS promoter/luciferase constructs either successively deleted or containing defined deletions we characterized six GC boxes—GC-I to GC-VI—locatedbetween −557 and −83 and discovered a seventh, GC-VII, in the first intron. In vitro DNAse I- footprinting, electrophoretic mobility shift assays, and the yeast one-hybrid system indicated that Sp1 as well as Sp3 interacts with GC-I to GC-VII. Each of the GC boxes conferred Sp1-dependent transcription on the FAS-Mini promoter and in the case of GC-I, Sp1, and Sp3 exert an additive effect on FAS promoter activity.

IL-4-induced Oxidative Stress Upregulates VCAM-1 Gene Expression in Human Endothelial Cells

Vascular cell adhesion molecule-1 (VCAM-1) is expressed in early stages of atherosclerosis; however, the mechanisms of its upregulation are not fully understood. In the present study, we examined the effects of interleukin-4 (IL-4) on VCAM-1 gene expression and its transcriptional regulatory mechanism in human umbilical vein endothelial cells (HUVEC). Reverse transcription-polymerase chain reaction showed that VCAM-1 mRNA was induced in IL-4-treated HUVEC in a time- and dose-dependent manner. Among known transcription factors that have binding sites in the promoter region of the VCAM-1 gene, IL-4 activated only SP-1. In contrast, nuclear factor- κ B (NF- κ B), activator protein-1 (AP-1) and interferon regulatory factor-1 (IRF-1), which also have consensus binding sequences in the 5-flanking region of the human VCAM-1 gene, were not activated. The role of SP-1 in IL-4-induced VCAM-1 expression was confirmed in HUVEC transfected with a reporter construct of the VCAM-1 promoter with mutated SP-1 binding site. As IL-4 treatment of HUVEC enhanced the intracellular oxidizing potential, as indicated by an increase in 2,7-dichlorofluorescein (DCF) fluorescence, we studied the effect of antioxidants on IL-4-induced VCAM-1 expression. Pretreatment of HUVEC with pyrrolidine dithiocarbamate (PDTC) or N-acetylcysteine (NAC) completely prevented IL-4-induced VCAM-1 expression. In addition, PDTC inhibited IL-4-related activation of SP-1. These results suggest that IL-4-induced oxidative stress upregulates the expression of VCAM-1 gene in HUVEC at transcriptional levels via activation of SP-1 transcription factor. In contrast, NF- κ B, AP-1 or IRF-1 do not appear to be involved in the signal transduction cascade.

A Role for Sp1 in the Transcriptional Regulation of Hepatic Triacylglycerol Hydrolase in the Mouse

Microsomal triacylglycerol hydrolase (TGH) hydrolyzes stored triacylglycerol in cultured hepatoma cells (Lehner, R., and Vance, D. E. (1999) Biochem. J. 343, 1-10). We studied expression of TGH in murine liver and found both protein and mRNA increased dramatically at 27 days after birth. Nuclear run-on assays demonstrated that this was due to increased transcription. We cloned 542 base pairs upstream of the transcriptional start site of the murine TGH gene. Electrophoretic mobility shift assays demonstrated enhanced binding of hepatic nuclear proteins from 27-day-old mice to the murine TGH promoter, yielding three differentially migrating complexes. DNase I footprint analysis localized these complexes to two distinct regions: site A contains a putative Sp binding site, and site B contains a degenerate E box. We transfected primary murine hepatocytes with a series of 5'-deletion constructs upstream of the reporter luciferase cDNA. Positive control elements were identified in a segment containing site A. Competitive electrophoretic mobility shift assays and supershift assays demonstrated that site A binds Sp1 and Sp3. Transcriptional activation assays in Schneider SL-2 insect cells demonstrated that Sp1 is a potent activator of the TGH promoter. These experiments directly link increased TGH expression at the time of weaning to transcriptional regulation by Sp1.

Sp1 is a key regulator of the PDGF beta-receptor transcription.

The mouse PDGF beta-receptor promoter is tightly controlled by NF-Y that binds to a CCAAT box located upstream of the initiation site [1, 2]. In this report, we show that Sp1 plays an essential role in the PDGF beta-receptor transcription. Within the upstream GC rich area there are two Sp1 binding sites located in close proximity to the CCAAT box. Deletion of the GC rich region resulted in a 50% decrease of the transcriptional activity of the promoter, and a complete loss of its responsiveness to over-expression of Sp1. There was an additive effect between NF-Y and Sp I in reporter activity when they were co-transfected together with the promoter-reporter construct. Furthermore, transfection of NF-Y failed to enhance transcriptional activity when the Sp1 binding sites were deleted from the promoter, suggesting an important role for Sp1 in this NF-Y controlled transcription. We have recently reported that c-Myc represses PDGF beta-receptor transcription through its interference with the transactivation activity of NF-Y [3]. In the case of p21(wafl/cip1) transcription, c-Myc was shown to repress its transcription by sequestering Sp1 [4]. However, we could not find any effect of Sp1 in the c-Myc-mediated repression on the PFDGF beta-receptor promoter, since the deletion of SpI binding sites could not attenuate the repression by c-Myc on the promoter activity.

TUMOR NECROSIS FACTOR-α REPRESSES ANDROGEN SENSITIVITY IN THE LNCaP PROSTATE CANCER CELL LINE

TUMOR NECROSIS FACTOR-α REPRESSES ANDROGEN SENSITIVITY IN THE LNCaP PROSTATE CANCER CELL LINE

Regulation of Na,K-ATPase β1 Subunit Gene Transcription by Low External Potassium in Cardiac Myocytes: ROLE OF Sp1 AND Sp3

Expression of Na,K-ATPase activity is up-regulated in cells incubated for extended intervals in the presence of low external K(+). Our previous data showed that exposure of cardiac myocytes to low K(+) increased the steady-state abundance of Na,K-ATPase beta1 subunit mRNA. In the present study we determined that incubation of primary cultures of neonatal rat cardiac myocytes with low K(+) augmented Na,K-ATPase beta1 gene expression at a transcriptional level and that this effect required extracellular Ca(2+). The stimulatory effect of low K(+) on Na,K-ATPase beta1 gene transcription was not dependent on increased contractile activity of cardiac myocytes. Na,K-ATPase beta1 5'-flanking region deletion plasmids used in transient transfection analysis demonstrated that the region between nucleotides -62 to -42 of the beta1 promoter contained a low K(+) response element. Site-directed mutagenesis of a potential GC box core motif GCG in the -58/-56 region of the beta1 promoter decreased basal and low K(+)-mediated transcription. Mutation of the core sequence of a putative GC box element located between nucleotides -101 and -99 further decreased the low K(+) effect on beta1 gene transcription. Electrophoretic mobility shift assays using oligonucleotides spanning the proximal and distal GC box elements of the beta1 promoter showed enhanced binding of two complexes in response to low K(+). The inclusion of a consensus GC box sequence as a competitor in gel shift analysis reduced factor binding to the low K(+) response elements. Antibodies to transcription factors Sp1 and Sp3 interacted with components of both DNA-binding complexes and binding of nuclear factors was abolished in gel shift studies using GC box mutants. Together these data indicate that enhanced binding of Sp1 and Sp3 to two GC box elements in the rat Na,K-ATPase beta1 subunit gene promoter mediates beta1 gene transcription up-regulation in neonatal rat cardiac myocytes exposed to low external K(+).

The human immunodeficiency virus type 1 Tat protein up-regulates the promoter activity of the beta-chemokine monocyte chemoattractant protein 1 in the human astrocytoma cell line U-87 MG: role of SP-1, AP-1, and NF-kappaB consensus sites.

It has been shown that the human immunodeficiency virus type 1 (HIV-1) Tat protein can specifically enhance expression and release of monocyte chemoattractant protein 1 (MCP-1) from human astrocytes. In this study, we show evidence that Tat-induced MCP-1 expression is mediated at the transcriptional level. Transient transfection of an expression construct encoding the full-length Tat into the human glioblastoma-astrocytoma cell line U-87 MG enhances reporter gene activity from cotransfected deletion constructs of the MCP-1 promoter. HIV-1 Tat exerts its effect through a minimal construct containing 213 nucleotides upstream of the translational start site. Site-directed mutagenesis studies indicate that an SP1 site (located between nucleotides -123 and -115) is critical for both constitutive and Tat-enhanced expression of the human MCP-1 promoter, as mutation of this SP1 site significantly diminished reporter gene expression in both instances. Gel retardation experiments further demonstrate that Tat strongly enhances the binding of SP1 protein to its DNA element on the MCP-1 promoter. Moreover, we also observe an increase in the binding activities of transcriptional factors AP1 and NF-kappaB to the MCP-1 promoter following Tat treatment. Mutagenesis studies show that an upstream AP1 site and an adjacent NF-kappaB site (located at -128 to -122 and -150 to -137, respectively) play a role in Tat-mediated transactivation. In contrast, a further upstream AP1 site (-156 to -150) does not appear to be crucial for promoter activity. We postulate that a Tat-mediated increase in SP1 binding activities augments the binding of AP1 and NF-kappaB, leading to synergistic activation of the MCP-1 promoter.

Overexpression of c-fos enhances the transcription of human arachidonate 12-lipoxygenase in A431 cells

Abstract The effect of transient transfection with expression vector of c-Fos on the expression of 12-lipoxygenase in human epidermoid carcinoma A431 cells was studied. Overexpression of c-Fos increased the expression of 12-lipoxygenase mRNA and enzyme activity, and also activated the promoter activity of 12-lipoxygenase gene in a dose-dependent manner. Co-transfection with c-Fos and c-Jun expression vectors in cells synergistically increased the promoter activity of 12-lipoxygenase. With the aid of additional 5′-deletion and site-directed mutagenesis, the downstream and middle Sp1 sites residing at −123 to −114 bp and −158 to −150 bp were found to be critical for the c-Fos response of activating the transcription of human 12-lipoxygenase gene. Furthermore, the specific role of Sp1 in c-Fos response was confirmed by using the reporter plasmid driven by SV40 early promoter. These results indicate that the requirement of Sp1-binding sites in the promoter region of 12-lipoxygenase gene for c-Fos response is similar to that previously observed in EGF response.

Composite action of three GC/GT boxes in the proximal promoter region is important for gastrin gene transcription

The proximal region of the human gastrin gene promoter contains three GC/GT boxes at positions −140 to −134 bp, −108 to −102 bp and −67 to −61 bp. In this study we have examined the significance of the three elements, and their role in Sp1 and Sp3 mediated gastrin transcription. In AGS cells, mutation of each of the boxes caused a moderate decrease in promoter activity from 33 to 63%, whereas double or triple mutations reduced activity to 3–12%. In Drosophila cells Sp1 activated the promoter, mainly through the distal GC box. Similarly, co-transfection of heterologous promoter constructs revealed that only the distal GC box increased activation by Sp1. The effect of Sp3 was cell-line dependent, since Sp3 inhibited the gastrin promoter activity in AGS cells and caused a synergistic activation of the Sp1 stimulated gastrin promoter in Drosophila cells. Both effects were dependent on the C-terminal DNA binding domain of Sp3. The results indicates that the combined effect of the GC/GT boxes and the ratio between Sp1 and Sp3 are important for gastrin gene expression.

Involvement of Sp1 in basal and retinoic acid induced transcription of the human tissue-type plasminogen activator gene

Transcription of the human tissue-type plasminogen activator (t-PA) gene is regulated by a multi-hormonal responsive enhancer at −7 kb. Transient co-transfections of Drosophila SL2 and human HT1080 fibrosarcoma cells with t-PA reporter constructs showed that Sp1 and Sp3 activate the t-PA promoter. Moreover Sp1 (but not Sp3) binding to the promoter is involved in induction by retinoic acid (RA), a response mediated through the enhancer. The role of Sp1 is specific, since mutation of the CRE element in the promoter did not affect response to RA. In contrast, the glucocorticoid induction mediated by the enhancer is independent of these Sp1 and CRE elements.

Functional studies by site-directed mutagenesis on the role of Sp1 in the expression of the pyruvate kinase M and aldolase A genes

During the cell cycle of mitogen stimulated rat thymocytes, an 8–10-fold induction of glycolytic enzymes and a corresponding increase in the mRNA levels has been observed. This prompted us to study the transcriptional regulation of the rat aldolase A and pyruvate kinase M genes. cis-Regulatory elements of both promoters were evaluated by site-directed mutagenesis of promoter/luciferase constructs and transient transfections of rat hepatoma FTO2B cells. Furthermore, the binding proteins were identified by mobility shift assays in the presence of specific antibodies. In the aldolase AH1 promoter, five binding sites for Sp1 and Sp3 and a TPA responsive element were identified as essential for transcriptional regulation. Most of the promoter activity can be attributed to these regulatory elements. In the pyruvate kinase M promoter three out of five binding sites of Sp1 and Sp3 (B box and GC boxes 1 and 3) turned out to be functional in the transfection assays whereas the disruption of GC box 2 had no effect, and the disruption of the GC box 4 had only a minor effect on the promoter activity. Both promoters are stimulated by Sp1 as well as Sp3, as judged by cotransfection experiments of Drosophila SL2 cells. Therefore, the Sp1- and Sp3-directed transcription provides a means for common regulatory mechanism of the aldolase A and the pyruvate kinase M genes.

Activation of Sp1 and Its Functional Co-operation with Serum Amyloid A-activating Sequence Binding Factor in Synoviocyte Cells Trigger Synergistic Action of Interleukin-1 and Interleukin-6 in Serum Amyloid A Gene Expression

The serum amyloid A (SAA) protein has been implicated in the progression and pathogenesis of rheumatoid arthritis through induction of collagenase activity in synovial fibroblast cells that line the joint tissues. We demonstrate that SAA is synergistically induced in synovial cells by interleukin (IL)-1 and IL-6 that are present at significantly high level in the synovial fluid of arthritis patients. These cytokines induced phenotypic changes in synovial cells, promoting protrusion and increased cellular contact. Induction of SAA under this condition is mediated by promoter elements located between -254 and -226, which contains binding sites for transcription factors Sp1 and SAA activating sequence binding factor (SAF). Mutation of these sequences abolishes SAA promoter response to IL-1 and IL-6. The role of Sp1 in SAA induction was demonstrated by increased DNA binding activity, phosphorylation, and increased protein content of Sp1 during cytokine treatment. Sp1 interacts with the SAA promoter in association with SAF as an SAF. Sp1 heteromeric complex. Furthermore, using a phosphatase inhibitor, we demonstrated increased transactivation potential of both Sp1 and SAF as a consequence of a phosphorylation event. These results provide first evidence for cytokine-mediated activation of Sp1 in synovial fibroblast cells and its participation in regulating SAA expression by acting in conjunction with SAF.

On the role of the general transcription factor Sp1 in the activation and repression of diverse mammalian oxidative phosphorylation genes.

To gain insight into the role of the general transcription factor, Sp1, in the expression of nuclear genes involved in mitochondrial biogenesis, we investigated Sp1 activation of the adenine nucleotide translocator 2, cytochrome c1, F1-ATPase beta subunit, and the mitochondria transcription factor (mtTFA) promoters transfected into Drosophila cell lines. The numbers and organization of GC elements vary in the four promoters, but the magnitude of activation by coexpressed human Sp1 was similar. A feature common to the four promoters is the presence of multiple, proximal Sp1-activating elements that account for 50% or more of the transcription activation by Sp1. The distribution and function of individual distal Sp1 elements is less defined and appear to be more promoter-specific. Finally, data from transfected Drosophila cells provide the first direct proof for the involvement of Sp1 in the negative regulation of the ANT2 promoter and as a possible participant in repression of the beta-subunit promoter. The role of Sp1 in both the positive and negative regulation of OXPHOS promoters is unique.

Transcriptional regulation of the human MnSOD gene: The role of SP1 and AP-2: [formula omitted] and Daret K. St. Clair. Graduate Center for Toxicology, University of Kentucky, Lexington KY 40536

Transcriptional regulation of the human MnSOD gene: The role of SP1 and AP-2: [formula omitted] and Daret K. St. Clair. Graduate Center for Toxicology, University of Kentucky, Lexington KY 40536

Sp1 and Egr-1 Have Opposing Effects on the Regulation of the RatPgp2/mdr1b Gene

The promoter of the rat pgp2/mdr1b gene has a GC-rich region (pgp2GC) that is highly conserved in mdr genes and contains an consensus Sp1 site. Sp1's role in transactivation of the pgp2/mdr1b promoter was tested in Drosophila Schneider cells. The pgp2/mdr1b promoter was strongly activated by co-transfected wild type Sp1 but not mutant Sp1 and mutation of the Sp1 site abrogated Sp1-dependent transactivation. In gel shift assays, the same mutations abolished Sp1-DNA complex formation. Moreover, basal activity of the pgp2/mdr1b Sp1 mutant promoter was dramatically lower. Enforced ectopic overexpression of Sp1 in H35 rat hepatoma cells revealed that cell lines overexpressing Sp1 had increased endogenous pgp2/mdr1b mRNA, demonstrating that Sp1 activates the endogenous pgp2/mdr1b gene. Pgp2GC oligonucleotide also bound Egr-1 in gel shift assays and Egr-1 competitively displaced bound Sp1. In transient transfections of H35 cells (and human LS180 and HepG2 cells) Egr-1 potently and specifically suppressed pgp2/mdr1b promoter activity and mutations in the Egr-1 site decreased Egr-1 binding and correlated with pgp2/mdr1b up-regulation. Ectopic overexpression of Egr-1 in H35 cells decreased Pgp expression and selectively increased vinblastine sensitivity. In conclusion, Sp1 positively regulates while Egr-1 negatively regulates the rat pgp2/mdr1b gene. Moreover, competitive interactions between Sp1 and Egr-1 in all likelihood determine the constitutive expression of the pgp2/mdr1b gene in H35 cells.

Regulation of the human protein C inhibitor gene expression in HepG2 cells: role of Sp1 and AP2.

Protein C inhibitor (PCI) is the plasma inhibitor of activated protein C, which is the main protease of the anticoagulant protein C pathway. In this study the transcriptional regulation of human PCI gene in the human hepatoma cell line, HepG2, was characterized by evaluating the transient expression of a luciferase reporter gene. The 5' flanking region (residues -1587 to +2) of the PCI gene showed an adequate transcriptional activity, the maximum transcriptional activity being in a region between residues -452 and -94, which contains an Sp1-binding site, two AP2-binding sites and an inverted AP2-binding site. Transient expression assays with various deletion mutants and site-directed mutants showed that the Sp1-binding site (residues -302 to -294) has a potent promoter activity and that the upstream AP2-binding site (residues -350 to -343) has a potent enhancer activity; no activity was detected in the inverted (residues -413 to -404) and downstream (residues -136 to -127) AP2-binding sites. In addition, a region of the PCI gene (residues -452 to -414) containing the STATx-binding site, the A-activator (AA)-binding site, and the interferon alpha (IFN-alpha) response element, and another region of the PCI gene (residues -176 to -147) containing the GATA-1 and the IFN-gamma response element showed potent silencer activities. Gel mobility-shift assays with various DNA fragments indicated that the Sp1-binding site, the upstream AP2-binding site, the AA-binding site and the IFN-gamma response element interact with nuclear protein(s) of HepG2 cells. These findings suggest that the Sp1-binding site is the promoter, the AP2-binding site (residues -350 to -343) the enhancer, and both the AA-binding site and the IFN-gamma response element are the silencers of human PCI gene expression in HepG2 cells.

Characterization of human involucrin promoter distal regulatory region transcriptional activator elements-a role for Sp1 and AP1 binding sites.

Human involucrin (hINV) is an important precursor of the keratinocyte cornified envelope that is specifically expressed in the suprabasal layers of stratifying epithelia. Previous truncation and mutagenesis experiments have shown that an activator protein 1 (Ap1) site, AP1-5, located 2100bp upstream of the transcription start site, is required for optimal promoter activity. These previous studies suggest that AP1-5 is part of a distal regulatory region spanning nucleotides -2473 to -2088. In the present report, we study the distal regulatory region (DRR), which surrounds AP1-5. Our studies show that this region contains weak and strong activator elements spanning nucleotides -2473/-2216 and -2140/-2088, respectively. The strong activator element contains AP1-5 and an adjacent specificity protein 1 (Sp1) site. The AP1-5 site is absolutely required for DRR activity, as its mutation reduces transcription to basal levels. Mutagenesis studies of the AP1-5 and Sp1 sites in the presence or absence of the weak activator element indicate that the Sp1 site and the weak activator element synergistically activate the AP1-5 site-dependent transcription. The cooperation between the Sp1 and AP1-5 sites is also observed in the context of the full-length promoter. Gel mobility shift and supershift studies show that Sp1, but not Sp2, Sp3 or Sp4 binds to the Sp1 site. When the Sp1 site is mutated or the distance between the AP1-5 and Sp1 site is increased, the binding of AP1 factors to AP1-5 is markedly reduced. Surprisingly, gel shift studies suggest that activation does not require the formation of a stable AP1/Sp1/DNA ternary complex. These studies suggest that the AP1-5 site is absolutely required for transcriptional activation, that the weak activator element and Sp1 sites serve to enhance this activation, and that the Sp1 site is required for optimal AP1 factor binding at the AP1-5 site.