Role Of Sp1 Research Articles

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.

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 2100 bp 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 fi2473 to fi2088. 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 fi2473}fi2216 and fi2140}fi2088, 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

Cell-growth regulation of the hamster dihydrofolate reductase gene promoter by transcription factor Sp1.

The dihydrofolate reductase (DHFR) gene (dhfr) promoter contains cis-acting elements for the transcription factors Sp1 and E2F. Given the ability of Sp1 to activate the dhfr promoter, we have evaluated the contribution of Sp1 to the cell-growth regulation of the dhfr gene. Using gel-mobility assays performed with DNA probes from the minimal promoter of the hamster dhfr gene and nuclear extracts from cultured hamster cells (CHO K1) we show that the binding of Sp1 to the dhfr promoter is cell-growth-phase regulated. Accordingly, dhfr transcription and mRNA levels in K1 cells increase upon serum stimulation. Cytological detection of Sp1 by immunofluorescence reveals a decrease of this protein in the process leading to the G0 state, and an increase upon serum stimulation of quiescent cells. These results were confirmed by western blot analysis. It is concluded that Sp1 progressively binds to the hamster dhfr promoter after stimulation of cell proliferation, which can account for the transcriptional regulation of the dhfr gene during the cell cycle. The role of Sp1 in the specific control of dhfr during the cell cycle was confirmed in vivo using cell lines derived from dhfr-negative cells transfected with dhfr plasmids carrying either the wild-type or mutated Sp1-binding or E2F-binding sites in the dhfr minimal promoter.

18 Role of Sp1 and Sp3 in basal and hormone-induced transcriptional activity of the human t-PA gene promoter

18 Role of Sp1 and Sp3 in basal and hormone-induced transcriptional activity of the human t-PA gene promoter

Induction of Vascular Endothelial Growth Factor by Tumor Necrosis Factor α in Human Glioma Cells: POSSIBLE ROLES OF SP-1

The expression of vascular endothelial growth factor (VEGF) has been implicated in brain tumor angiogenesis, and the promoter region for the VEGF gene contains several SP-1 and AP-1 (c-Fos and c-Jun) binding motifs. Among eight human glioma cell lines, cellular mRNA levels of transcription factors SP-1 and AP-1 (c-Fos and c-Jun) were found to be closely correlated with those of VEGF. VEGF expression appears to be highly susceptible to hypoxia or exogenous cytokines and growth factors. Of various cytokines and growth factors, basic fibroblast growth factor (bFGF), tumor necrosis factor alpha (TNF-alpha), and interleukin 1 most potently enhanced VEGF mRNA levels of a glioma cell line, U251. Incubation of the glioma cells with bFGF or TNF-alpha increased both VEGF and SP-1 mRNA at 30 min and c-Fos mRNA at 1-3 h, over 5-fold. Nuclear run-on assays showed an apparent increase of the transcription of the VEGF gene as well as the SP-1 gene by bFGF or TNF-alpha. Gel mobility shift assays demonstrated that only SP-1 binding activity was increased 1 h after exposure to bFGF or TNF-alpha, and also that AP-1, but not SP-1, activity was significantly activated by hypoxia. Mithramycin, an inhibitor of SP-1, at 1-10 nM inhibited activation of the VEGF gene by bFGF or TNF-alpha but not that by hypoxia. Western blot analysis also demonstrated an increase in cellular amounts of VEGF by TNF-alpha and a decrease by co-administration with mithramycin. The promoter activity of the VEGF gene, which contains five SP-1 binding sites and one AP-1 binding site but not hypoxia regulatory elements, was enhanced by bFGF or TNF-alpha but not by hypoxia. The chloramphenicol acetyltransferase assay with VEGF promoter deletion constructs demonstrated that four clusterized SP-1 binding sites in the proximal promoter were essential for the basal transcription and the TNF-alpha-dependent activation. These data indicated that the expression of the VEGF gene enhanced by bFGF or TNF-alpha appeared to be mediated in part through the transcription factor SP-1, suggesting a different mechanism from that for hypoxia-induced activation of the VEGF gene.

Role of Sp1 in Transcriptional Activation of Human Nitric Oxide Synthase Type III Gene

Endothelial nitric oxide synthase (eNOS or NOS-III) is constitutively expressed. To elucidate the mechanism by which the basal expression of NOS-III gene is activated, we constructed in a luciferase vector, pXP1, serial 5′-deletion mutants of a 1.3-kb 5′-flanking fragment and transiently expressed them in cultured human endothelial cells. The promotor activity was detected in the −198/+22 region which contains several putative Sp1 binding sites. DNase I footprinting assays coupled with gel shift assays revealed the GC box (−104/−90) to be the Sp1 binding site. Site-directed mutation of 4 crucial bases in this site reduced the promotor activity by > 90%. These findings provide strong evidence that binding of Sp1 or closely related protein to this site is required for the activation of basal NOS-III transcription.

Domains of transcription factor Sp1 required for synergistic activation with sterol regulatory element binding protein 1 of low density lipoprotein receptor promoter.

Feedback regulation of transcription from the low density lipoprotein (LDL) receptor gene is fundamentally important in the maintenance of intracellular sterol balance. The region of the LDL receptor promoter responsible for normal sterol regulation contains adjacent binding sites for the ubiquitous transcription factor Sp1 and the cholesterol-sensitive sterol regulatory element-binding proteins (SREBPs). Interestingly, both are essential for normal sterolmediated regulation of the promoter. The cooperation by Sp1 and SREBP-1 occurs at two steps in the activation process. SREBP-1 stimulates the binding of Sp1 to its adjacent recognition site in the promoter followed by enhanced stimulation of transcription after both proteins are bound to DNA. In the present report, we have defined the protein domains of Sp1 that are required for both synergistic DNA binding and transcriptional activation. The major activation domains of Sp1 that have previously been shown to be essential to activation of promoters containing multiple Sp1 sites are required for activation of the LDL receptor promoter. Additionally, the C domain is also crucial. This slightly acidic approximately 120-amino acid region is not required for efficient synergistic activation by multiple Sp1 sites or in combination with other recently characterized transcriptional regulators. We also show that Sp1 domain C is essential for full, enhanced DNA binding by SREBP-1. Taken together with other recent studies on the role of Sp1 in promoter activation, the current experiments suggest a unique combinatorial mechanism for promoter activation by two distinct transcription factors that are both essential to intracellular cholesterol homeostasis.

A Novel cis-Acting Element Controlling the Rat CYP2D5 Gene and Requiring Cooperativity Between C/EBPβ and an Sp1 Factor

The rat CYP2D5 gene encodes a cytochrome P450 and is expressed in liver cells. Its expression commences a few days after birth, and maximal mRNA levels are achieved when animals reach puberty. Transfection and DNA binding studies were performed to investigate the mechanism controlling developmentally programmed, liver-specific expression of CYP2D5. Transfection studies using a series of CYP2D5 upstream DNA chloramphenicol acetyltransferase gene fusion constructs identified a segment of DNA between nucleotides -55 and -156 that conferred transcriptional activity in HepG2 cells. Activity was markedly increased by cotransfection with a vector expressing C/EBP beta but was unaffected by vectors producing other liver-enriched transcription factors (C/EBP alpha, HNF-1 alpha, and DBP). DNase I footprinting revealed a region protected by both HepG2 and liver cell nuclear extracts between nucleotides -83 and -112. This region displayed some sequence similarity to the Sp1 consensus sequence and was able to bind the Sp1 protein, as assessed by a gel mobility shift assay. The role of Sp1 in CYP2D5 transcription was confirmed by trans activation of the 2D5-CAT construct in Drosophila melanogaster cells by using an Sp1 expression vector. C/EBP beta alone was unable to directly bind the -83 to -112 region of the promoter but was able to produce a ternary complex when combined with HepG2 nuclear extracts or recombinant human Sp1. C/EBP alpha was unable to substitute for C/EBP beta in forming this ternary complex. A poor C/EBP binding site is present adjacent to the Sp1 site, and mutagenesis of this site abolished formation of the ternary complex with the CYP2D5 regulatory region. These result establish that two transcription factors can work in conjunction, possibly by protein-protein interaction, to activate the CYP2D5 gene.

A novel cis-acting element controlling the rat CYP2D5 gene and requiring cooperativity between C/EBP beta and an Sp1 factor.

The rat CYP2D5 gene encodes a cytochrome P450 and is expressed in liver cells. Its expression commences a few days after birth, and maximal mRNA levels are achieved when animals reach puberty. Transfection and DNA binding studies were performed to investigate the mechanism controlling developmentally programmed, liver-specific expression of CYP2D5. Transfection studies using a series of CYP2D5 upstream DNA chloramphenicol acetyltransferase gene fusion constructs identified a segment of DNA between nucleotides -55 and -156 that conferred transcriptional activity in HepG2 cells. Activity was markedly increased by cotransfection with a vector expressing C/EBP beta but was unaffected by vectors producing other liver-enriched transcription factors (C/EBP alpha, HNF-1 alpha, and DBP). DNase I footprinting revealed a region protected by both HepG2 and liver cell nuclear extracts between nucleotides -83 and -112. This region displayed some sequence similarity to the Sp1 consensus sequence and was able to bind the Sp1 protein, as assessed by a gel mobility shift assay. The role of Sp1 in CYP2D5 transcription was confirmed by trans activation of the 2D5-CAT construct in Drosophila melanogaster cells by using an Sp1 expression vector. C/EBP beta alone was unable to directly bind the -83 to -112 region of the promoter but was able to produce a ternary complex when combined with HepG2 nuclear extracts or recombinant human Sp1. C/EBP alpha was unable to substitute for C/EBP beta in forming this ternary complex. A poor C/EBP binding site is present adjacent to the Sp1 site, and mutagenesis of this site abolished formation of the ternary complex with the CYP2D5 regulatory region. These result establish that two transcription factors can work in conjunction, possibly by protein-protein interaction, to activate the CYP2D5 gene.

Two different, overlapping pathways of transcription initiation are active on the TATA-less human androgen receptor promoter. The role of Sp1

In this study, the minimal promoter requirements of the TATA-less human androgen receptor (hAR) gene promoter are described. The hAR promoter is characterized by a short GC-box (-59/-32) and a long homopurine stretch (-117/-60). Two major transcription initiation sites, AR transcription initiation site I (AR-TIS I, (+1/2/3)) and AR transcription initiation site II (AR-TIS II, (+12/13)) are located in a 13-base pair region (Faber, P. W., van Rooij, H. C. J., van der Korput, J. A. G. M., Baarends, W. M., Brinkmann, A. O., Grootegoed, J. A., and Trapman, J. (1991) J. Biol. Chem. 266, 10743-10749). Transient transfection of COS cells with hAR promoter deletion and mutant constructs, followed by RNA isolation and S1 nuclease protection analysis showed that the process of transcription initiation through AR-TIS I and AR-TIS II is regulated by different promoter sequences. The GC-box directed initiation from AR-TIS II but did not affect AR-TIS I utilization, which is dependent upon sequences between positions -5 and +57. Band shift analysis identified the transcription factor Sp1 as the protein interacting with the GC-box. A single Sp1 binding sequence was found to be present in the GC-box. Footprint analysis confirmed the interaction of Sp1 with this sequence. The differential initiation through AR-TIS I and AR-TIS II was substantiated by the introduction of point mutations in the Sp1 binding sequence: only mutations that specifically abolished Sp1 binding interfered with AR-TIS II utilization, but all mutations left AR-TIS I initiation intact.

Pregnancy-specific beta 1-glycoprotein concentrations throughout pregnancy in the rhesus monkey.

Proteins that are immunologically related to human pregnancy-specific beta 1-glycoprotein (hSP1) have been detected in serum samples from pregnant subhuman primates. However, little information is available concerning the concentrations or secretory dynamics of SP1 in nonhuman primate pregnancy. To examine serum SP1 concentrations throughout rhesus monkey gestation, a heterologous RIA for rhesus SP1 (rSP1) was developed which used an antiserum against rSP1 and purified human SP1 as the standard and for iodination. No rSP1 was detected in serum samples from 8 normal male monkeys, 8 castrated females, or 10 regularly cycling females. Levels of rSP1 were measured in 260 serum samples from 43 pregnant monkeys. rSP1 was first detected on day 14 after mating, and all monkeys sampled 25 or more days after mating had detectable serum rSP1 levels. Serum rSP1 rose exponentially from day 14 to about day 50, followed by a more gradual rise until term. This pattern of secretion was in marked contrast to that found for macaque CG, which was already detectable 10 days after mating, peaked at 20 days, and returned to nonpregnant levels by day 40. The disappearance of rSP1 after delivery was best fit by a 2-component 4-parameter model with a mean residence time of 48 h. These data indicate that the pattern of rSP1 secretion throughout pregnancy is qualitatively similar to that found for hSP1, as is the long half-life of the protein in the circulation after delivery. rSP1 measurements may provide a useful index of placental function during rhesus pregnancy and serve as a model to study the biological role of SP1 in primates.

Serum levels of a placental protein during gestation in the baboon

A placental specific protein (Sp-1) isolated from human placentas was used to establish a radioimmunoassay for measurement of a baboon protein in the sera of pregnant baboons. No reaction was observed with any known pituitary or placental hormone in this assay system. Levels of Sp-1 were detected at 17 to 18 days of gestation and reached high levels by the end of the first trimester of pregnancy. A slow increase in serum levels continued thereafter until parturition. Disappearance of Sp-1 post partum was very slow and required 26 to 34 days to become undetectable in the serum. No function or role of Sp-1 in pregnancy has been suggested, but possible use of its measurement for detecting abnormal placental function deseves investigation.