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Results for “Sp1 Transcription Factor”

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At least 19 recordsLinked to original sources

PubMed1993

Alteration of DNA binding specificity by nickel (II) substitution in three zinc (II) fingers of transcription factor Sp1.

Transcription factor Sp1, which has a DNA binding domain composed of three zinc fingers, binds to GC box (consensus sequence, G/T-GGGCGG-G/A-G/A-C/T) and activates the transcription by RNA polymerase II. Metal substitution of nickel(II) for zinc(II) in Sp1 causes no differences in the mode of protein-DNA interaction. However, sequence preference of Ni(II)Sp1 changes from 5'-GGGGCGGGGC to 5'-GGGGCGTGGC, and is distinct from that of Zn(II)Sp1. The result indicates an important effect of metal-induced folding on sequence-specific recognition of DNA by zinc-finger proteins.

Base Sequence↗

PubMed1999

Nuclear localization of transcription factor Sp1.

Transcription factor Sp1 is importantly related to expression of many cellular genes. In order to identify the nuclear localization signal of Sp1, various truncated regions of Sp1 were fused to green fluorescent protein (GFP) and were expressed in HeLa cell. The C-terminal region are required to localize Sp1 in the nucleus of HeLa cell.

Cell Nucleus↗

PubMed1998

Unique DNA binding mode of the N-terminal zinc finger of transcription factor Sp1.

Transcription factor Sp1 has three tandem repeats of a Cys2His2-type zinc finger motif and specifically binds to GC box DNA. Although the DNA binding mode of the three zinc fingers of Sp1 is predicted to be similar to that of Zif268, this model does not explain the DNA binding property of the N-terminal zinc finger (finger 1). To understand the DNA recognition mode of Sp1, we have performed detailed analyses for the contribution of finger 1 to the high-affinity binding to the GC box DNA and for the interaction mechanism between finger 1 and DNA. Results of electrophoretic analyses using finger-deleted mutants of Sp1 and GC box mutants in the finger-contacting subsite demonstrate that the contribution of finger 1 to the total DNA binding affinity is lower than that of the C-terminal finger 3 but is dispensable for the high-affinity binding. The DNA sequence selectivity of finger 1 at the 3'-portion of the GC box is lower than that of fingers 2 and 3 at the 5'-portion. Alanine scanning mutagenesis in the alpha-helix of finger 1 reveals that Lys-1 immediately preceding the helix is important for the recognition of the two guanine bases, but other putative key amino acids do not affect the DNA binding. These results demonstrate that (1) the contribution of finger 1 to the DNA binding affinity and the sequence selectivity of Sp1 is smaller than that of fingers 2 and 3 and (2) the interaction mechanism between finger 1 and DNA is different from the Zif268 model. DNA interaction of Sp1 finger 1 has also been discussed in connection with that of TFIIIA or WT1.

Amino Acid Sequence↗

PubMed2000

Zn finger and nuclear localization of transcription factor Sp1.

Transcription factor Sp1 is located in the nucleus of a mammalian cell and importantly related to expression of many cellular genes. In order to elucidate the nuclear localization mechanism of Sp1, various truncated fragments of Sp1 were fused to green fluorescent protein (GFP) and expressed in HeLa cell. The results show significance of the DNA binding region, especially, zinc finger (Zn finger) domain for nuclear localization of Sp1 in HeLa cell.

Binding Sites↗

PubMed1989

Evidence that the upstream stimulatory factor and the Sp1 transcription factor bind in vitro to the promoter of the human-growth-hormone gene.

Expression of the human-growth-hormone gene is restricted to pituitary somatotrophs. Two protein-DNA complexes that are specific to the pituitary, and two that are not, had been demonstrated in vitro on the promoter of this gene. The two pituitary-specific footprints had been ascribed to a single protein called growth hormone factor 1. We have now characterized the factors responsible for the two other footprints by means of deoxyribonuclease-I protection and gel-retardation experiments. The first footprint, located between -257 and -290 relative to the transcription initiation site, involves at least two factors present in pituitary cells. One of these factors binds between nucleotides -257 and -267, and is indistinguishable from the upstream stimulatory factor, also called major late transcription factor or upstream element factor, initially described in HeLa cells. Earlier work by others had shown that the activator protein 2 purified from HeLa cells can bind to nucleotides -263 and -290. Our experiments suggest that a factor different from activator protein 2 is involved in the protection of this region against deoxyribonuclease I. The second footprint, located between nucleotides -116 and -140, involves only one factor. This factor, present in pituitary cells, recognizes a GC box and is indistinguishable from transcription factor Sp1, previously described in HeLa cells. The human-growth-hormone gene is therefore a candidate for regulation by these factors in vivo.

Animals↗

PubMed1991

The Sp1 transcription factor gene (SP1) and the 1,25-dihydroxyvitamin D3 receptor gene (VDR) are colocalized on human chromosome arm 12q and rat chromosome 7.

By means of somatic cell hybrids segregating either human or rat chromosomes, the genes encoding the transcription factor Sp1 (SP1) and the 1,25-dihydroxyvitamin D3 receptor (VDR) were both assigned to human chromosome arm 12q and to rat chromosome 7. This result implies that the locus for the clinical disorder vitamin D dependency rickets type II maps on 12q. The phenylalanine hydroxylase (PAH) and the retinoic acid receptor-gamma (RARG) genes also map on human chromosome arm 12q and rat chromosome 7, indicating that a synteny group is conserved on these chromosomes.

Animals↗

PubMed1986

Activation of the AIDS retrovirus promoter by the cellular transcription factor, Sp1.

The nature and position of transcriptional control elements responsible for the expression of genes encoded by the retrovirus associated with acquired immune deficiency syndrome (AIDS) have not been precisely defined. In this study it is shown that the mammalian Sp1 transcription factor binds to promoter sequences within the AIDS retrovirus long terminal repeat (LTR) and activates RNA synthesis five- to eightfold in reconstituted reactions in vitro. Experiments in which regions of DNA were protected from added reagents by specifically bound proteins (footprinting) indicated that the upstream promoter region of the AIDS virus LTR lies between -45 and -77 (relative to the RNA start site, +1) and contains three tandem, closely spaced SP1 binding sites of variable affinity. Base-substitution mutations targeted to one or all three Sp1 binding sites were found both to eliminate the binding of Sp1 and to cause up to a tenfold reduction in transcriptional efficiency in vitro. These findings suggest that one important component of the AIDS virus transcriptional control region interacts with a cellular transcription factor, Sp1, and that this factor must function in conjunction with transcriptional elements located downstream of the RNA cap site to mediate the response of the LTR to viral trans-activation.

Acquired Immunodeficiency Syndrome↗

PubMed1994

Nucleosome binding by the constitutive transcription factor Sp1.

The constitutive transcription factor Sp1 plays a role in the transcription of numerous viral and cellular genes including constitutive "housekeeping" genes and inducible genes. Sp1 has also been implicated in the formation of a nucleosome-free region in Simian virus 40 (SV40) minichromosomes. To investigate the potential functions of Sp1 in remodeling chromatin structures, Sp1 was analyzed for the ability to bind its recognition sites (GC boxes) in DNA fragments reconstituted into nucleosome cores. Sp1 was found to bind to the GC boxes of the SV40 early promoter when this element was reconstituted into nucleosome cores. The affinity of Sp1 for the nucleosomal SV40 early promoter DNA was reduced approximately 10-20-fold relative to naked DNA. A peptide containing only the zinc fingers of Sp1 was also capable of binding nucleosomal DNA, indicating that the glutamine-rich and serine/threonine-rich domains of Sp1 are not required for nucleosome binding. The binding of Sp1 to nucleosome cores resulted in the formation of a ternary Sp1-nucleosome complex.

Animals↗

PubMed2000

Transglutaminase-dependent modulation of transcription factor Sp1 activity.

Modification of transcription factors would result in significant changes in the expression of related genes. Recently, the presence of transglutaminase (TGase) has been reported in nuclei, the biological significances of which have attracted a great concern. In this study, we tested the possibility that nuclear TGase would crosslink and regulate the activity of a glutamine-rich transcription factor Sp1. The addition of purified guinea pig liver TGase increased the binding activity of Sp1 to the target DNA sequence by gel electrophoretic mobility shift assay. The activity of the human p21WAF1 promoter containing six Sp1 binding sites was increased by the cotransfection of the TGase 2 gene, and two Sp1 sites at -82 and -69, relative to the transcription start site, were essential for the increased activity in human renal embryonic 293T cells. The activity of a minimal promoter containing three consensus Sp1 binding sites was increased by co-transfection of human TGase 2 gene. The amount of Sp1 protein was increased dramatically in TGase 2-transfected 293T cells and the Sp1 protein itself from HeLa cell nuclear extracts was crosslinked readily by purified TGase at 37 degrees C in the presence of Ca2+. These results suggest that the nuclear TGase might modulate the activity of the Sp1 transcription factor probably via the posttranslational or transcriptional modification of the factor by TGase.

Animals↗

PubMed2004

Celecoxib inhibits vascular endothelial growth factor expression in and reduces angiogenesis and metastasis of human pancreatic cancer via suppression of Sp1 transcription factor activity.

The aggressive biology of human pancreatic adenocarcinoma has been linked with overexpression of vascular endothelial growth factor (VEGF). Constitutive activation of the transcription factor Sp1 plays a critical role in VEGF overexpression. Recent studies indicated that celecoxib, a selective cyclooxygenase-2 inhibitor, exhibits potent antitumor activity. However, the underlying molecular mechanisms of this activity remain unclear. In the present study, we used a pancreatic cancer model to determine the role of Sp1 in the antitumor activity of celecoxib. Treatment of various pancreatic cancer cells with celecoxib suppressed VEGF expression at both the mRNA and protein level in a dose-dependent manner. VEGF promoter deletion and point mutation analyses indicated that a region between nucleotide -109 and -61 and its intact Sp1-binding sites were required for the inhibition of VEGF promoter activity by celecoxib. Also, celecoxib treatment reduced both Sp1 DNA binding activity and transactivating activity. This decreased activity correlated with reduced Sp1 protein and its phosphorylation as determined using Western blot analysis. Furthermore, in an orthotopic pancreatic cancer animal model, celecoxib treatment inhibited tumor growth and metastasis. The antitumor activity was consistent with inhibition of angiogenesis as determined by evaluating tumor microvessel formation, which correlated with decreased Sp1 activity and VEGF expression. Collectively, our data provide a novel molecular mechanism for the antitumor activity of celecoxib and may help further improve its effectiveness in controlling pancreatic cancer growth and metastasis.

Adenocarcinoma↗

PubMed2006

Downregulation of transcription factor, Sp1, during cellular senescence.

We found that the protein level of Sp1 transcription factor decreases as normal human fibroblasts undergo replicative aging. Sp1 also undergoes a rapid decrease in the protein level and activity in MCF-7 cells that are induced to a state of cellular senescence. In the cells treated with other DNA damaging chemicals such as actinomycin D and H(2)O(2), the Sp1 level decreased progressively as well. Inhibition of ATM/ATR kinases prevented this downregulation, suggesting that DNA damage signaling is involved in the regulation of the Sp1. This decrease in Sp1 protein level is due to the accelerated proteasomal degradation since a proteasome inhibitor, ALLN, blocked this downregulation. Therefore, the global decrease in gene transcription frequently reported in aging cells and tissues could be attributed at least in part to the decrease in Sp1 level.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2006

Role of transcription factor Sp1 and CpG methylation on the regulation of the human podocalyxin gene promoter.

BACKGROUND: Podocalyxin (podxl) is a heavily glycosylated transmembrane protein mainly found on the apical membrane of rat podocytes and also in endothelial, hematopoietic, and tumor cells. Despite of its interest no much is known about the transcriptional regulation of podxl in different cells. Thus, we aimed at studying the functional features of the 5'-regulatory region of the human Podxl gene. RESULTS: The promoter region of the human Podxl gene has been cloned and its structure and function were analyzed. The primary DNA sequence is rich in G+C and is devoid of TATA or CAAT boxes. The sequence contains recognition sites for several putative transcription factors; however, the basic promoter activity seems to rely entirely on Sp1 transcription factor since supershift analysis was positive only for this factor. The region encompassed by 66 to -111 nts conferred the minimal transcriptional activity that increases as the number of Sp1 sites augmented with the length of the promoter fragment. In Sp1-lacking insect cells the Podxl promoter constructs showed activity only if cotransfected with an Sp1 expression plasmid. Finally, mutation of the Sp1 sites reduced the promoter activity. We analyzed whether methylation of the CpG dinucleotides present in the first approximately 600 nts of the promoter region of Podxl could explain the variable rates of expression in different types of cells. Inactivation of methyltransferases by 5'-aza-2'deoxicitidine showed a dose-dependent increase in the podxl content. Moreover, in vitro methylation of the promoter constructs -111,-181 and -210 led to an almost complete reduction of the promoter activity. A correlation was found between the degree of methylation of the CpG promoter dinucleotides and the rate of podxl expression in different cell lines. CONCLUSION: Our results indicate that transcriptional regulation of Podxl is supported primarily by Sp1 site(s) and that DNA-methylation of the CpG promoter islands contributes to control the tissue specific expression of podxl.

5' Flanking Region↗

PubMed1988

Sp1 transcription factor binds DNA and activates transcription even when the binding site is CpG methylated.

In vertebrates, a negative correlation between gene activity and CpG methylation of DNA, notably in the promoter region, is well established. Therefore, it is conceivable that differential binding of transcription factors to methylated versus unmethylated binding sites is crucial for gene activity. Since the consensus binding site of transcription factor Sp1 contains a central CpG, we have investigated the binding of Sp1 factor to unmethylated and synthetically CpG-methylated DNA. A strong Sp1 binding site was methylated on both strands at two CpG positions, located in the center and at the periphery of the recognition sequence. Our studies show that neither binding in vitro, nor transcription in vivo and in vitro are affected by methylation of the Sp1 binding site. We discuss the possibility that binding of Sp1 factor, which is often associated with promoters of housekeeping genes, prevents CpG methylation.

Animals↗

PubMed2003

Characterization of the neuropilin-1 promoter; gene expression is mediated by the transcription factor Sp1.

Neuropilin-1 (NRP1) is a receptor for the vascular endothelial growth factor (VEGF) family of angiogenesis factors and for the semaphorin family of secreted neuronal guidance polypeptides. Very little is known, however, about how NRP1 gene expression is regulated. In this study, it was demonstrated that the tumor promoter, TPA (12-O-tetradecanoylphorbol-13-acetate) significantly up-regulated NRP1 mRNA levels by increasing its gene transcription rate in a manner dependent on de novo protein synthesis. To determine which elements regulate functional NRP1 expression, the promoter regions of human and mouse NRP1 genes were cloned and characterized. Promoter-reporter gene transfection experiments using deletion and point mutations demonstrated that two Sp1 elements are major contributors to both the constitutive and TPA-induced activity of the NRP1 promoter. Gel shift analysis showed a specific binding of the Sp1 transcription factor to those elements. Further mutational analysis revealed that an AP-1, and a CCAAT box also contributed to NRP1 constitutive and TPA-induced promoter activity. It was concluded that NRP1 expression is regulated by the cooperation of several regulatory elements including AP-1, Sp1, and a CCAAT box.

Animals↗

PubMed1995

Coexpression of NF-kappa B/Rel and Sp1 transcription factors in human immunodeficiency virus 1-induced, dendritic cell-T-cell syncytia.

Productive infection of T cells with human immunodeficiency virus 1 (HIV-1) typically requires that the T cells be stimulated with antigens or mitogens. This requirement has been attributed to the activation of the transcription factor NF-kappa B, which synergizes with the constitutive transcription factor Sp1 to drive the HIV-1 promoter. Recently, we have found that vigorous replication of HIV-1 takes place in nonactivated memory T cells after syncytium formation with dendritic cells (DCs). These syncytia lack activated cells as determined by an absence of staining for Ki-67 cell cycle antigen. The expression and activity of NF-kappa B and Sp1 were, therefore, analyzed in isolated T cells and DCs from humans and mice. We have used immunolabeling, Western blot analysis, and electrophoretic mobility shift and supershift assays. T cells lack active NF-kappa B but express Sp1 as expected. DCs express high levels of all known NF-kappa B and Rel proteins, with activity residing primarily within RelB, p50, and p65. However, DCs lack Sp1, which may explain the failure of HIV-1 to replicate in purified DCs. Coexpression of NF-kappa B and Sp1 occurs in the heterologous DC-T-cell syncytia that are induced by HIV-1. Therefore, HIV-1-induced cell fusion brings together factors that upregulate virus transcription. Since DCs and memory T cells frequently traffic together in situ, these unusual heterologous syncytia could develop in infected individuals and lead to chronic HIV-1 replication without ostensible immune stimulation.

Animals↗

PubMed2000

A set of proteins interacting with transcription factor Sp1 identified in a two-hybrid screening.

The two-hybrid system was used to isolate cDNA clones encoding polypeptides that interact with the N-terminal region (activation domains A, B and C) of the Sp1 transcription factor. Among the 65 collected clones, 43 contained cDNA fragments with open reading frames. They corresponded to 13 genes encoding proteins of known function and to 15 genes, the proteins of which have no known function. Six overlapping cDNA clones corresponded to the Hsc70 protein. Host cell factor (HCF-1) and the KIAA0461 gene (encoding a putative Zn-finger protein of unknown function) were both identified through the isolation of three overlapping cDNA clones. Two cDNA fragments encoding the same region of the SREBP-2 transcription factor were independently selected and two overlapping cDNA clones corresponded to the splicing factor SF3A120. Two different cDNA clones encoded the N- and C-terminal region of the Oct-1 transcription factor. Transcription factors Elf-1 and TIEG, as well as HSph2, the putative human homologue of a murine polyhomeotic gene, were each represented by a single clone. Noticeably, for the four identified transcription factors, the DNA-binding domain was excluded from the selected polypeptides. In vitro binding of the selected polypeptides to the Sp1 protein was demonstrated for the four transcription factors and for the SF3A120, Hsc70, HCF-1, HSph2 and pKIAA0461(245) proteins. Four other cDNA clones encoding polypeptides of unknown function were tested in the in vitro binding assay. All four polypeptides were found to interact with Sp1 in this assay.

Animals↗

PubMed2006

Estrogen receptor regulates insulin-like growth factor-I receptor gene expression in breast tumor cells: involvement of transcription factor Sp1.

The insulin-like growth factors, IGF-I and IGF-II are a family of mitogenic polypeptides with important roles in growth and differentiation. The biological actions of the IGFs are mediated by the IGF-I receptor (IGF-IR), a cell-surface tyrosine kinase, whose activation by serum IGF-I seems to be a key step in breast cancer initiation. Evidence accumulated indicates that estrogens stimulate the expression and activity of IGF axis components. The aim of our study was to examine the transcriptional mechanisms involved in regulation of IGF-IR gene expression by the estrogen receptor (ER). For this purpose, transient transfections using an IGF-IR promoter-luciferase reporter plasmid were performed in breast cancer-c derived ER-positive MCF-7 cells and isogenic ER-negative C4 cells. To examine the potential involvement of zinc-finger nuclear proteins in the transactivating effect of estrogens, chromatin immunoprecipitation (ChIP) experiments were performed using an Sp1 antibody, along with the Sp1-family-binding inhibitor Mithramycin A. The results obtained indicate that basal IGF-IR promoter activity was 5.8-fold higher in MCF-7 than in C4 cells. Estradiol treatment significantly activated the IGF-IR promoter in MCF-7, but not in C4 cells. Furthermore, the estrogen responsive region in the IGF-IR promoter was mapped to a GC-rich sequence located between nucleotides -40 and -188 in the 5' flanking region. ChIP experiments revealed that at least part of the estrogen effect on IGF-IR expression was mediated through activation of the Sp1 transcription factor. In summary, our studies demonstrate that IGF-IR gene transcription in breast cancer cells is controlled by interactions between ERalpha and Sp1. Dysregulated expression of the IGF-IR gene may have pathologic consequences with relevance in breast cancer etiology.

5' Flanking Region↗

PubMed2005

Stage-specific expression of myelin basic protein in oligodendrocytes involves Nkx2.2-mediated repression that is relieved by the Sp1 transcription factor.

The homeodomain-containing protein Nkx2.2 is critical for the development of oligodendrocyte lineage cells, but the target genes of Nkx2.2 regulation have not been identified. In the present study, we found that the myelin basic protein gene is one of the genes that is regulated by Nkx2.2. Expression of Nkx2.2 represses the expression of myelin basic protein in oligodendrocyte progenitors. Two regulatory elements in the myelin basic protein promoter were identified and found to interact with Nkx2.2 in vitro. Despite their sequence divergence, both sites were involved in the Nkx2.2-mediated repression of the myelin basic protein promoter. Binding of Nkx2.2 also blocked and disrupted the binding of the transcriptional activator Puralpha to the myelin basic protein promoter. Additionally Nkx2.2 recruited a histone deacetylase 1-mSin3A complex to the myelin basic protein promoter. We also found that the transcription factor Sp1 was able to compete off the binding of Nkx2.2 to its consensus binding site in vitro and reversed the repressive effect of Nkx2.2 in vivo. Our data revealed a novel role for Nkx2.2 in preventing the precocious expression of myelin basic protein in immature oligodendrocytes. Based on this study and our previous reports, a model for myelin basic protein gene control is proposed.

Animals↗