Sp1 phosphorylation and its regulation of gene transcription.
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ABSTRACT:
SUBMITTER: Tan NY
PROVIDER: S-EPMC2682032 | biostudies-literature | 2009 May
REPOSITORIES: biostudies-literature
Publications
Sp1 phosphorylation and its regulation of gene transcription.
Tan Nicole Y NY Khachigian Levon M LM
Molecular and cellular biology 20090309 10
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Project description:Neph3 (filtrin) is expressed in the glomerular podocytes where it localizes at the specialized cell adhesion structures of the foot processes called slit diaphragms which form the outermost layer of the glomerular filtration barrier. Neph3 protein shows homology and structural similarity to Neph1, Neph2 and nephrin, which all are crucial for maintaining the normal glomerular ultrafiltration function. The exact function of Neph3 in the kidney is not known but we have previously shown that the level of Neph3 mRNA is decreased in proteinuric diseases. This suggests that Neph3 may play a role in the pathogenesis of kidney damage, and emphasizes the need to analyze the regulatory mechanisms of Neph3 gene. In this study we investigated the transcriptional regulation of Neph3 gene by identifying transcription factors that control Neph3 expression.We cloned and characterized approximately 5 kb fragment upstream of the Neph3 gene. Neph3 proximal promoter near the transcription start site was found to be devoid of TATA and CAAT boxes, but to contain a highly GC-rich area. Using promoter reporter gene constructs, we localized the main activating regulatory region of Neph3 gene in its proximal promoter region from -105 to -57. Within this region, putative transcription factor binding sites for NF-kappaB and Sp1 were found by computational analysis. Mutational screening indicated that NF-kappaB and Sp1 response elements are essential for the basal transcriptional activity of the Neph3 promoter. Co-transfection studies further showed that NF-kappaB and Sp1 regulate Neph3 promoter activity. In addition, overexpression of NF-kappaB increased endogenous Neph3 gene expression. Chromatin immunoprecipitation assay using cultured human podocytes demonstrated that both NF-kappaB and Sp1 interact with the Neph3 promoter.Our results show that NF-kappaB and Sp1 are key regulators of Neph3 expression at the basal level in podocytes, therefore providing new insight into the molecular mechanisms that contribute to the expression of Neph3 gene.
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Project description:BackgroundAdenocarcinoma of the pancreas is one of the most aggressive tumor diseases affecting the human body. The oncogenic potential of pancreatic cancer is mainly characterized by extremely rapid growth triggered by the activation of oncogenic signaling cascades, which suggests a change in the regulation of important transcription factors. Amongst others, NFAT transcription factors are assumed to play a central role in the carcinogenesis of pancreatic cancer. Recent research has shown the importance of the transcription factor Sp1 in the transcriptional activity of NFATc2 in pancreatic cancer. However, the role of the interaction between these two binding partners remains unclear. The current study investigated the role of Sp1 proteins in the expression of NFATc2 target genes and identified new target genes and their function in cells. A further objective was the domain of the Sp1 protein that mediates interaction with NFATc2. The involvement of Sp1 proteins in NFATc2 target genes was shown by means of a gene expression profile analysis, and the results were confirmed by quantitative RT-PCR. The functional impact of this interaction was shown in a thymidine incorporation assay. A second objective was the physical interaction between NFATc2 and different Sp1 deletion mutants that was investigated by means of immunoprecipitation.ResultsIn pancreatic cancer, the proto-oncogene c-Fos, the tumor necrosis factor TNF-alpha, and the adhesion molecule integrin beta-3 are target genes of the interaction between Sp1 and NFATc2. Loss of just one transcription factor inhibits oncogenic complex formation and expression of cell cycle-regulating genes, thus verifiably decreasing the carcinogenic effect. The current study also showed the interaction between the transcription factor NFATc2 and the N-terminal domain of Sp1 in pancreatic cancer cells. Sp1 increases the activity of NFATc2 in the NFAT-responsive promoter.ConclusionsThe regulation of gene promotors during transcription is a rather complex process because of the involvement of many proteins that - as transcription factors or co-factors - regulate promotor activity as required and control cell function. NFATc2 and Sp1 seem to play a key role in the progression of pancreatic cancer.
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Project description:BackgroundPodocalyxin (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.ResultsThe 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.ConclusionOur 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.
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Project description:BackgroundThe family of MAGE genes is well known due to the majority of MAGE genes expressing specifically in tumor tissues while restrictedly in normal tissues. MAGE-D4 is one of the MAGE family and considered as a promising target for glioma immunotherapy because of its overexpression in glioma and restricted expression in normal tissues. Whereas the mechanism of MAGE-D4 heterogeneous expression in glioma has not yet been elucidated. In this study, the transcriptional regulation mechanism of MAGE-D4 in glioma is focused from the perspectives of promoter methylation and SP1.MethodsDual-luciferase reporter assay was performed to identify the core promoter of MAGE-D4 gene. Mass spectrometry was applied to quantify the methylation status of MAGE-D4 promoter in 50 glioma and 9 normal brain tissues. The influence of methylation and SP1 on MAGE-D4 transcriptional activity was evaluated by dual-luciferase reporter assay, qRT-PCR, western blot and ChIP-qPCR. Decitabine, an epigenetic drug, was used to treat the glioma cells. Then the treated cells were evaluated the influence of demethylation on SP1 binding to MAGE-D4 promoter.ResultsThe -358 to +172 bp region was identified as the core promoter of MAGE-D4 gene which demonstrated hypomethylated and negative correlation between methylation level and MAGE-D4 mRNA expression in glioma tissues. For single CpG unit analysis, 8 CpG units (CpG unit 1, 2, 3, 4, 5, 6, 9 and 12) in MAGE-D4 core promoter showed hypomethylated in glioma and the methylation level of CpG unit 6 was positively associated with the prognosis of glioma patients. Furthermore, the methylation level of CpG unit 1 and 6 was negative negatively correlated with MAGE-D4 mRNA expression. Then, the results demonstrated that the promoter activity of MAGE-D4 was decreased by methylation in glioma cell lines. In addition, SP1 can binds directly to the MAGE-D4 promoter leading to up-regulation of MAGE-D4 mRNA through activation of its promoter. Finally, demethylation of MAGE-D4 promoter could benefit the SP1 binding and resulting co-activation of MAGE-D4 promoter by demethylation and SP1 in glioma cell lines.ConclusionThese findings indicate that the synergies of promoter hypomethylation and SP1 up-regulated MAGE-D4 transcription in glioma, which implies a potential approach to resolve the heterogeneous expression of MAGE-D4 in order to establish foundation for the MAGE-D4 based glioma therapy.
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Project description:Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) gene plays a crucial role in maintaining genomic stability, tumorigenesis and myogenesis. However, little is known about the regulatory elements governing the transcription of porcine ROCK1 gene. In the current study, the transcription start site (TSS) was identified by 5'-RACE, and was found to differ from the predicted one. The region in ROCK1 promoter which is critical for promoter activity was investigated via progressive deletions. Site-directed mutagenesis indicated that the region from -604 to -554 bp contains responsive elements for Sp1. Subsequent experiments showed that ROCK1 promoter activity is enhanced by Sp1 in a dose-dependent manner, whereas treatment with specific siRNA repressed ROCK1 promoter activity. Electrophoretic mobility shift assay (EMSA), DNA pull down and chromatin immunoprecipitation (ChIP) assays revealed Sp1 can bind to this region. qRT-PCR and Western blotting research followed by overexpression or inhibition of Sp1 indicate that Sp1 can affect endogenous ROCK1 expression at both mRNA and protein levels. Overexpression of Sp1 can promote the expression of myogenic differentiation 1(MyoD), myogenin (MyoG), myosin heavy chain (MyHC). Taken together, we conclude that Sp1 positively regulates ROCK1 transcription by directly binding to the ROCK1 promoter region (from -604 to -532 bp) and may affect the process of myogenesis.
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Project description:BackgroundLRP5, a member of the low density lipoprotein receptor superfamily, regulates diverse developmental processes in embryogenesis and maintains physiological homeostasis in adult organisms. However, how the expression of human LRP5 gene is regulated remains unclear.ResultsIn order to characterize the transcriptional regulation of human LRP5 gene, we cloned the 5' flanking region and evaluated its transcriptional activity in a luciferase reporter system. We demonstrated that both KLF15 and Sp1 binding sites between -72 bp and -53 bp contribute to the transcriptional activation of human LRP5 promoter. Chromatin immunoprecipitation assay demonstrated that the ubiquitous transcription factors KLF15 and Sp1 bind to this region. Using Drosophila SL2 cells, we showed that KLF15 and Sp1 trans-activated the LRP5 promoter in a manner dependent on the presence of Sp1-binding and KLF15-binding motifs.ConclusionsBoth KLF15 and Sp1 binding sites contribute to the basal activity of human LRP5 promoter. This study provides the first insight into the mechanisms by which transcription of human LRP5 gene is regulated.
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Project description:APOBEC3G (A3G), a member of the recently discovered family of human cytidine deaminases, is expressed in peripheral blood lymphocytes and has been shown to be active against HIV-1 and other retroviruses. To gain new insights into the transcriptional regulation of this restriction factor, we cloned and characterized the promoter region of A3G. Transcriptional start sites were identified by 5'-rapid amplification of cDNA ends analysis. Luciferase reporter assays demonstrated that a 1025 bp A3G promoter sequence (from -959 to +66 relative to the major transcriptional start site) displayed constitutive promoter activity. In T cells, the A3G promoter was not inducible by mitogenic stimulation, interferon treatment or expression of HIV-1 proteins. Using a series of 5' deletion promoter constructs in luciferase reporter assays, we identified a 180 bp region that was sufficient for full promoter activity. Transcriptional activity of this A3G core promoter was dependent on a GC-box (located at position -87/-78 relative to the major transcriptional start site) and was abolished after mutation of this DNA element. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that the identified GC-box represented a binding site for the ubiquitous transcription factors specificity protein (Sp) 1 and Sp3.
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Project description:Defects in human DNA mismatch repair have been reported to underlie a variety of hereditary and sporadic cancer cases. We characterized the structure of the MSH6 promoter region to examine the mechanisms of transcriptional regulation of the MSH6 gene. The 5'-flanking region of the MSH6 gene was found to contain seven functional Sp1 transcription factor binding sites that each bind Sp1 and Sp3 and contribute to promoter activity. Transcription did not appear to require a TATA box and resulted in multiple start sites, including two major start sites and at least nine minor start sites. Three common polymorphisms were identified in the promoter region (-557 T-->G, -448 G-->A, and -159 C-->T): the latter two were always associated, and each of these functionally inactivated a different Sp1 site. The polymorphic allele -448 A -159 T was demonstrated to be a common Caucasian polymorphism found in 16% of Caucasians and resulted in a five-Sp1-site promoter that had 50% less promoter activity and was more sensitive to inactivation by DNA methylation than the more common seven Sp1 site promoter allele, which was only partially inactivated by DNA methylation. In cell lines, this five-Sp1-site polymorphism resulted in reduced MSH6 expression at both the mRNA and protein level. An additional 2% of Caucasians contained another polymorphism, -210 C-->T, which inactivated a single Sp1 site that also contributes to promoter activity.
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Project description:The ubiquitous transcription factor specificity protein 1 (SP1) is heavily modified posttranslationally. These modifications are critical for switching its functions and modulation of its transcriptional activity and DNA binding and stability. However, the mechanism governing the stability of SP1 by cellular signaling pathways is not well understood. Here, we provide biochemical and functional evidence that SP1 is an integral part of the Wnt signaling pathway. We identified a phosphodegron motif in SP1 that is specific to mammals. In the absence of Wnt signaling, glycogen synthase kinase 3β (GSK3β)-mediated phosphorylation and β-TrCP E3 ubiquitin ligase-mediated ubiquitination are required to induce SP1 degradation. When Wnt signaling is on, SP1 is stabilized in a β-catenin-dependent manner. SP1 directly interacts with β-catenin, and Wnt signaling induces the stabilization of SP1 by impeding its interaction with β-TrCP and axin1, components of the destruction complex. Wnt signaling suppresses ubiquitination and subsequent proteosomal degradation of SP1. Furthermore, SP1 regulates Wnt-dependent stability of β-catenin and their mutual stabilization is critical for target gene expression, suggesting a feedback mechanism. Upon stabilization, SP1 and β-catenin cooccupy the promoters of TCFL2/β-catenin target genes. Collectively, this study uncovers a direct link between SP1 and β-catenin in the Wnt signaling pathway.
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Project description:Dmrt1 is a recently described gene that is specifically expressed in the gonads and is required for postnatal testis differentiation. Here, we describe the transcriptional mechanisms regulating the Dmrt1 proximal promoter in testicular Sertoli cells. A genomic clone containing exon 1 of the rat Dmrt1 gene and more than 9 kilobases of 5' flanking sequence was isolated and characterized. Several prominent transcriptional start sites were identified, with the major site located 102 bases from the translational start. The Dmrt1 5' flanking region from -5000 to +74 was transcriptionally active in primary Sertoli cells, and deletion analysis of this fragment identified 2 major regions needed for full Dmrt1 promoter function. These regions were located between -3200 and -2000 base pairs (bp) and downstream of -150 bp relative to the major transcriptional start site. DNase I footprint analysis of the region downstream of -150 bp revealed 3 regions that are bound by proteins from Sertoli cell nuclear extracts. Site-directed mutagenesis of these regions identified 2 elements that activate the Dmrt1 promoter and 2 that repress it. The positive elements bind the transcription factors Sp1, Sp3, and Egr1, suggesting that these transcription factors play a critical role in Dmrt1 regulation in the testis.
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