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ere we describe the rat counterpart as well as a newly identified variant with the exon 3-3 alignment in cultured rat cells. A qualitative evaluation of such alignments in poly(A)(+) RNA-rich preparation showed that both alignments arose from trans-splicing rather than circularization of a single strand. The identification of the trans-spliced products in both rat and human raises the possibility that trans-splicing on Sp1 pre-mRNA is rather common to mammals. It was observed that the level of the trans-spliced variants varies in different rat organs.

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oter is the most conserved noncoding region within the Cx40-gene known thus far and contains five potential binding sites for Sp-family transcription factors. The binding of both Sp1 and Sp3 to each of these DNA elements was demonstrated by EMSA. Luciferase assays of the natural rCx40 proximal promoter or mutated derivatives in Cx40-expressing (NCM, primary rat neonatal cardiomyocytes and A7r5, rat smooth muscle embryonic thoracic aorta cells) and -nonexpressing cells (N2A, mouse neuroblastoma cells) revealed that all sites are contributing to basal promoter activity. Trans-activation assays in Drosophila Schneider line 2 cells demonstrated that Sp1 and Sp3 activate the rCx40 proximal promoter in a dose-dependent and additive manner.

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1 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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ight:700;'>Sp1 into PML NBs, leading to the reduction of Sp1 transactivation function. Specifically, Chromatin immunoprecipitation (ChIP) assay demonstrated that induced expression of PML significantly diminishes the amount of Sp1 binding to its target gene promoter, immunofluorescence staining showed dramatic increase in the co-localization between PML and Sp1 upon induction of PML expression, moreover, PML and Sp1 co-fractionated in the core nuclear matrix. Our study further showed that PML promotes SUMOylation of Sp1 in a RING-motif-dependent manner, SUMOylation of Sp1 facilitates physical interaction between Sp1 and PML and recruitment of Sp1 into the PML NBs, the SUMO binding motif of PML was also important for its interaction with Sp1. The results of this study demonstrate a novel mechanism by which PML regulates gene expression through sequestration of the transcription factor into PML NBs.

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ents that were critical for the transcriptional activation of MALAT1 gene. Reporter gene assays were employed to analyze the effect of Sp1 on the promoter activity of MALAT1 gene. The binding activity of Sp1 with the promoter of MALAT1 gene was examined by EMSA and ChIP assay. Effects of Sp1 on regulation of MALAT1 were analyzed by RNA interference in vitro and in vivo mouse model. RESULTS: By means of luciferase assay, Sp1 was found to activate the promoter of the human MALAT1 gene. The binding of Sp1 to this region was also detected by electrophoretic mobility shift and chromatin immunoprecipitation assays. Sp1 knockdown also decreased the MALAT1 and inhibited A549 lung cancer cells' growth and invasion in vitro. Furthermore, knockdown of Sp1 also mimicked the inhibition of MALAT1 in A549 lung cancer cells' growth and metastasis in vivo. CONCLUSIONS: Taken together, our data suggest that upregulation of MALAT1 was mediated by the transcription factor Sp1 in A549 lung cancer cells, and Sp1 could be therapeutic target for cancer.

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olytic properties of the enzyme. By in situ hybridization we found the prominent expression of the mRNA in the epithelial layer of the small intestinal upper villi and of the colon, where cells are loosely attached to the basement membrane. When MT-SP1 was expressed in Caco-2, a colonic carcinoma cell line, the protein was localized exclusively on the basolateral side. A secreted form of the enzyme produced in COS-1 cells digested fibronectin and laminin. These findings suggest that MT-SP1 participates in the control of intestinal epithelial turnover by regulating the cell-substratum adhesion.

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weight:700;'>sp1 expression is mediated by YY1 is not well understood. RESULTS: We excised YY1 in the murine embryos using Sox2-cre and found that Mesp1 was down-regulated in the embryonic day (E) 7.5 mutant embryos. Also, YY1 activated the 6 kb Mesp1 regulatory element fused to a luciferase reporter. We identified two putative YY1 binding sites in the proximal promoter region of Mesp1 gene, and found that mutation of these sites significantly reduced YY1-induced activation of the Mesp1 promoter. We also uncovered one cognitive site for SP1, one of the earliest binding partners of YY1 identified. Mutation of this SP1 site repressed SP1-induced activation of the Mesp1 promoter. Moreover, YY1 and SP1 synergistically activated the Mesp1 promoter. Consistently, while Lacz expression driven by the wild-type 6 kb regulatory element of Mesp1 gene was robust in E7.5 mouse embryos, the mutation of these binding sites in the context of this 6 kb sequence substantially reduced the LacZ expression during embryogenesis. CONCLUSIONS: YY1 and SP1 independently and cooperatively govern the Mesp1 expression during embryogenesis. Developmental Dynamics 245:379-387, 2016. (c) 2015 Wiley Periodicals, Inc.

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ensus sequence as probe, two major and four minor bands were seen. Three bands were present at all stages of differentiation, two are lost during terminal differentiation and one is present only in late differentiating cells. The octamer probe yielded three bands, which co-migrate with Oct2, Oct3 and Oct7. The exact identity of these factors has not been established. The Oct3-like band was detected only in epithelial cell extracts, the other two bands were also found in fibre cell extracts, whereby the intensity of the Oct2-like band decreased relative to that of the Oct7-like band during differentiation. In transfection studies, a rat gamma D-crystallin promoter in which the proximal activator has been replaced by a dimer of the Sp1-consensus sequence showed a gradual increase in activity with differentiation, in contrast, a similar construct with an octamer dimer was active only during late differentiation and mimicked the pattern of activation of the parental gamma D-crystallin promoter.

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cs factors have been implicated in other populations of patients with osteoporosis. Polymorphism at the Sp1 binding site of the collagen type I A1 (COLIA1) gene is thought to be an important factor in the development of osteoporosis. METHODS: Alleles S and s, detected by presence of a G or T nucleotide, respectively in a regulatory site of the COLIA1 gene were investigated in 37 beta-thalassemia major patients with osteoporosis and 92 controls without osteoporosis or osteopenia using polymerase chain reaction-restriction fragment length polymorphism. RESULTS: Fifteen and nine beta-thalassemia major patients displayed SS and Ss genotypes, respectively, whereas 13 were found to have an ss genotype. The mean BMD of the beta-thalassemia major patients with ss genotype was similar to those with the Ss and SS genotypes. In the control group, 77 and 15 subjects had SS and Ss genotypes, respectively, with no ss genotype. Allelic and genotypic distribution in patients were significantly different from controls. CONCLUSION: Determining base substitutions at the Sp1 binding site on the COLIA1 gene in early years may be important in preventing osteoporosis in children with beta-thalassemia major.

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p1 and Egr-1 can specifically bind to these overlapping elements, that this binding appears to be mutually exclusive, and that binding site occupancy is dependent upon the concentration of each factor and its binding affinity for each site. Egr-1 binds to the -165 bp site with relatively high affinity (IC50 = 14 nM) and to the -45 bp site with relatively low affinity (IC50 = 1360 nM), whereas Sp1 binds to both sites with intermediate affinities (IC50 = 210 and 140 nM, respectively). Consistent with the DNA-binding data, Egr-1 stimulates PNMT promoter activity primarily through interaction with the -165 bp site, while Sp1 stimulates PNMT promoter activity by interacting with both the -45 bp and the -165 bp sites. These results show that Sp1 and Egr-1 are capable of differentially activating PNMT gene expression, thereby suggesting that different stimuli may control the activity of the PNMT gene by selectively regulating Sp1 and/or Egr-1.

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on levels of Sp1 in 227 GC patients using immune-histochemical staining and fluorescence in situ hybridization. Data on clinicopathological features and relevant prognostic factors in these patients were analyzed. Of the 227 gastric cancer samples, 11.89% were positive for HER-2 overexpression/amplification under the new scoring system, and the frequency of negative, weak positive and strong positive expression of Sp1 was 14.98%, 48.01% and 37.0% respectively. No statistically positive correlation was observed between the expression levels of HER-2 and Sp1 in GC tissues. HER-2 overexpression was closely correlated to the Lauren type, degree of differentiation, tumor size and lymph node metastasis, and Sp1 as well (P < 0.05). Overexpression of HER-2 and Sp1 predicted poor survival in univariate analysis as well as in a Cox proportional hazards model.

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immortalized or malignant, regardless of their lineage or tissue origin. This promoter normally drives the expression of the Pem homeobox gene in specific cell types in ovary and placenta but is aberrantly expressed in lymphomas, neuroblastomas, retinoblastomas, carcinomas, and sarcomas. By deletion analysis we identified a region between nucleotides -80 and -104 that was absolutely critical for the expression from this distal Pem promoter (Pem Pd). Site-specific mutagenesis and transfection studies revealed that this region contains two consensus Ets sites and a single Sp1 site that were necessary for Pem Pd expression. Gel shift analysis showed that Ets and Sp1 family members bound to these sites. Transfection studies demonstrated that the Ets family members Elf1 and Gabp and the Sp1 family members Sp1 and Sp3 transactivated the Pem Pd. Surprisingly, we found that Sp3 was a more potent activator of the Pem Pd than was Sp1; this is unusual, because Sp3 is either a weak activator or a repressor of most other promoters. Activation by either Elf1 or Gabp required an intact Sp1 family member binding site, suggesting that Ets and Sp1 family members cooperate to activate Pem Pd transcription. Expression from the Pem Pd (either transiently transfected or endogenous) depended on the Ras pathway, which could explain both its Ets- and Sp1-dependent expression in normal cells and its aberrant expression in tumor cells, in which ras protooncogenes are frequently mutated. We suggest that the Pem Pd may be a useful model system to understand the molecular mechanism by which a tissue-specific promoter can be corrupted in tumor cells.

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h the Sp1 site) in subjects with LDD. DESIGN: Blood was collected from 24 Greek army recruits, aged 29+/-7.6 years, with LDD, and 66 healthy men, aged 26+/-4.38 years, matched for body mass index (BMI) and age, with normal BMD and with no history of trauma or fractures, who served as controls. DNA was extracted and the COL1A1 gene was sequenced. Of the control subjects, 12 were army recruits and 54 were selected from the general population. RESULTS: The four base-pair insertion polymorphism in the COL1A1 gene analyzed by polymerase chain reaction amplification of DNA produces two different fragments (alleles A1 and A2): 14 patients (58.3%) were homozygous for A2A2, versus 35 controls (53%), while 3 patients (12.5%) were A1A1, and 8 of the control subjects (12%) had this genotype. There were no statistically significant differences in the presence of the two alleles of this polymorphism between patients with LDD and control subjects. CONCLUSIONS: A four base-pair insertion polymorphism of the COL1A1 gene is not associated with the presence of LDD in young males, unlike the Sp1 site polymorphism of the same gene. These data reinforce the association between LDD and the functional polymorphisms of the Sp1 site by showing that other polymorphic sites of the of the COL1A1 gene in the same population of patients are not linked to the disease.

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ption from the MLP via a DE sequence-dependent mechanism. Here we use a transient expression approach to show that L4-22K binds to an additional site downstream of the MLP start site, the so-called R1 region, which includes the major late first leader 5' splice site. Binding of L4-22K to R1 has a suppressive effect on MLP transcription. L4-22K binds to the distal part of R1 and stimulates the recruitment of Sp1 and other cellular factors to a site overlapping the first leader 5' splice site. Binding of Sp1 to the 5' splice site region had an inhibitory effect on L4-22K-activated MLP transcription.

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ransporter and aminopeptidase N messenger RNA in the intestinal mucosa were upregulated during starvation. Therefore, we selected and studied three transcriptional factors: activator protein (AP)-1, Sp1, and hepatocyte nuclear factor (HNF)-1, which not only play important roles for enterocytes proliferation, but also exist in promoter lesions of the brush border enzymes and peptide transporter. METHODS: In the present study, we performed electrophoretic mobility shift assays employing AP-1, Sp1, and HNF-1, and evaluated the changes in the DNA binding activities in rat jejunum during starvation and refeeding. RESULTS: Two days after starvation, the Sp1 binding activity was significantly decreased to 61.8% as compared with the control level, whereas AP-1 was 121.4% and HNF-1 was 77.5%. Two hours after refeeding, the AP-1 activity was significantly increased to 175.0% as compared with the control level, and the HNF-1 activity was significantly increased to 180.2%. In contrast, the decreased SP1 level did not recover until 24 h after refeeding. CONCLUSIONS: The DNA binding activities of these three transcriptional factors were significantly changed in the rat jejunum during starvation and refeeding. Our results provide insight into the molecular mechanisms of the transcriptional regulations associated with mucosal atrophy, regrowth, and functional changes of the jejunal epithelium in response to starvation and refeeding.

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le='font-weight:700;'>Sp1 is itself regulated at different levels, for example, by glycosylation, acetylation, and phosphorylation by kinases such as the atypical protein kinase C-zeta. Although Sp1 controls the basal and inducible regulation of many genes, the posttranslational processes regulating its function and their relevance to pathology are not well understood. Here we have used a variety of approaches to identify 3 amino acids (Thr668, Ser670, and Thr681) in the zinc finger domain of Sp1 that are modified by PKC-zeta and have generated novel anti-peptide antibodies recognizing the PKC-zeta-phosphorylated form of Sp1. Angiotensin II, which activates PKC-zeta phosphorylation (at Thr410) via the angiotensin II type 1 receptor, stimulates Sp1 phosphorylation and increases Sp1 binding to the platelet-derived growth factor-D promoter. All 3 residues in Sp1 (Thr668, Ser670, and Thr681) are required for Sp1-dependent platelet-derived growth factor-D activation in response to angiotensin II. Immunohistochemical analysis revealed that phosphorylated Sp1 is expressed in smooth muscle cells of human atherosclerotic plaques and is dynamically expressed together with platelet-derived growth factor-D in smooth muscle cells of the injured rat carotid artery wall. This study provides new insights into the regulatory mechanisms controlling the PKC-zeta-phospho-Sp1 axis and angiotensin II-inducible gene expression.

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ticipating with its promoter elements in rat brain astrocytes (RBA-1). CORM-2-induced HO-1 expression was mediated through superoxide, p38 mitogen-activated protein kinase(MAPK), extracellular signal-regulated protein kinases 1 and 2 (Erk1/2), protein tyrosine kinase 2 (Pyk2), platelet-derived growth factor receptor (PDGFR), and phosphatidylinositol 3'-kinase (PI3K/Akt), revealed by the pharmacological inhibitors or knockdown of these signaling molecules. CORM-2-enhanced HO-1 promoter activity was inhibited by co-transfection with small interfering RNA (siRNA) of c-Jun, specificity protein 1 (Sp1), or nuclear factor-erythroid 2-related factor 2 (Nrf2). Immunoprecipitation assay showed that CORM-2 increased the association of nuclear Nrf2 with Sp1 and c-Jun. Furthermore, chromatin immunoprecipitation (ChIP) assay confirmed that Nrf2, Sp1, and c-Jun are associated with the proximal ARE binding site on HO-1 promoter, suggesting that Nrf2/Sp1/c-Jun cooperations are key transcription factors modulating HO-1 expression. Mechanistically, CORM-2-induced ARE promoter activity was reduced by the inhibitors of reactive oxygen species (ROS), p38 MAPK, Pyk2, MAPK/ERK kinases 1 and 2 (MEK1/2), PDGFR, and PI3K/Akt or the siRNAs of c-Jun, SP1, and Nrf2. These findings suggested that CORM-2 increases formation of c-Jun, Sp1, and Nrf2 complex and binding with ARE1 binding site, which is mediated through both ROS/p38 MAPK and Pyk2-dependent PDGFR/PI3K/Akt/Erk1/2 pathways, resulting in HO-1 expression in RBA-1 cells.

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ranscription assays suggest that this gene is subject to transcriptional control since no activity of its promoter is detected in nonproducing cells. The gene specifies two mRNAs that encode the same protein and originate from transcription initiation at alternative sites. Expression analysis of hybrid genes bearing deletions of the gene's 5'-flanking region fused to a reporter gene defines a promoter region within the gene-proximal 260 base pairs of DNA. The cis-acting elements that control promoter activity include binding sites for transcription factors Sp1 and alphaCbf, a 60-kDa CCAAT box-binding protein. The gene promoter, however, functions not only in exocrine pancreatic cells but also in cells of other origin. No cell-specific transcriptional control element was detected in as much as 10 kilobases of 5'-flanking region. We discuss models of how the cell-specific expression of the endogenous ptf1-p48 gene might be established during development of the animal.

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f CYP1B1 in cancer progression, we investigated the action of CYP1B1 in cells with increased CYP1B1 via the inducer 7,12-dimethylbenz[alpha]anthracene (DMBA) or an overexpression vector, in addition to decreased CYP1B1 via the inhibitor tetramethoxystilbene (TMS) or siRNA knockdown. We observed that CYP1B1 promoted cell proliferation, migration, and invasion in MCF-7 and MCF-10A cells. To understand its molecular mechanism, we measured key oncogenic proteins including beta-catenin, c-Myc, ZEB2, and matrix metalloproteinases following CYP1B1 modulation. CYP1B1 induced epithelial-mesenchymal transition (EMT) and activated Wnt/beta-catenin signaling via upregulation of CTNNB1, ZEB2, SNAI1, and TWIST1. Sp1, a transcription factor involved in cell growth and metastasis, was positively regulated by CYP1B1, and suppression of Sp1 expression by siRNA or DNA binding activity using mithramycin A blocked oncogenic transformation by CYP1B1. Therefore, we suggest that Sp1 acts as a key mediator for CYP1B1 action. Treatment with 4-hydroxyestradiol (4-OHE2), a major metabolite generated by CYP1B1, showed similar effects as CYP1B1 overexpression, indicating that CYP1B1 activity mediated various oncogenic events in cells. In conclusion, our data suggests that CYP1B1 promotes cell proliferation and metastasis by inducing EMT and Wnt/beta-catenin signaling via Sp1 induction.

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the most important feature of SCLC. In this study, we investigated the involvement of E2F1 in the process of invasion and metastasis in SCLC by regulating the expression of matrix metalloproteinases (MMPs).
METHODS: Immunohistochemistry was performed to evaluate the expression of E2F1 and MMPs in SCLC samples in a Chinese Han population. The impact of E2F1 on invasion and metastasis was observed by transwell and wound healing experiments with depletion of E2F1 by specific siRNA. The target genes regulated by E2F1 were identified by chromatin immunoprecipitation (ChIP)-to-sequence, and the expressions of target genes were detected by real time PCR and western blotting. The dual luciferase reporter system was performed to analyze the regulatory relationship between E2F1 and MMPs.
RESULTS: E2F1 is an independent and adverse prognosis factor that is highly expressed in SCLC in a Chinese Han population. Knockdown of E2F1 by specific siRNA resulted in the downregulation of migration and invasion in SCLC. The expressions of MMP-9 and -16 in SCLC were higher than other MMPs, and their expressions were most significantly reduced after silencing E2F1. ChIP-to-sequence and promoter-based luciferase analysis demonstrated that E2F1 directly controlled MMP-16 expression via an E2F1 binding motif in the promoter. Although one E2F1 binding site was predicted in the MMP-9 promoter, luciferase analysis indicated that this binding site was not functionally required. Further study demonstrated that E2F1 transcriptionally controlled the expression of Sp1 and p65, which in turn enhanced the MMP-9 promoter activity in SCLC cells. The associations between E2F1, Sp1, p65, and MMP-9 were validated by immunohistochemistry staining in SCLC tumors.
CONCLUSIONS: E2F1 acts as a transcriptional activator for MMPs and directly enhances MMP transcription by binding to E2F1 binding sequences in the promoter, or indirectly activates MMPs through enhanced Sp1 and NF-kappa B as a consequence of E2F1 activation in SCLC.

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egulation of TLR4 expression through promoter methylation and histone modification between transcriptional regulation and silencing of the TLR4 gene in gastric cancer cells. Chromatin immunoprecipitation was carried out to screen for factors related to TLR4 methylation such as MeCP2, HDAC1, and Sp1 on the TLR4 promoter. Moreover, DNA methyltransferase inhibitor 5-aza-deoxycytidine (5-aza-dC) induced demethylation of the TLR4 promoter and increased H3K4 trimethylation and Sp1 binding to reactivate silenced TLR4. In contrast, although the silence of TLR4 activated H3K9 trimethylation and MeCP2 complex, combined treatment with TLR4 agonist and 5-aza-dC upregulated H3K4 trimethylation and activated with transcription factors as Sp1 and NF-kappaB. This study demonstrates that recruitment of the MeCP2/HDAC1 repressor complex increases the low levels of TLR4 expression through epigenetic modification of DNA and histones on the TLR4 promoter, but Sp1 activates TLR4 high expression by hypomethylation and NF-kappaB signaling in gastric cancer cells.

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of its functional role and the underlying mechanisms in modulation of myofibroblast (a key effector of hypertrophic scar formation) differentiation remains unclear. Here, we identified that eIF6 is a novel regulator of transforming growth factor-beta1 (TGF-beta1) expression at transcription level, which plays a key role in myofibroblast differentiation. Mechanistically, this effect is associated with eIF6 altering the occupancy of the TGF-beta1 promoter by H2A.Z (Swiss-Prot P0C0S6) and Sp1. Accordingly, modulation of eIF6 expression in myofibroblasts signi fi cantly affects their differentiation via the TGF-beta/Smad signaling pathway, which was verified in vivo by the observation that heterozygote eIF6(+/-) mice exhibited enhanced TGF-beta1 production coupled with increased alpha-smooth muscle actin (alpha-SMA)(+) myofibroblasts after skin injury. Overall, our data reveal a novel transcriptional regulatory mechanism of eIF6 that acts on facilitating Sp1 recruitment to TGF-beta1 promoter via H2A.Z depletion and thus results in increased TGF-beta1 transcription, which contributes to myofibroblast differentiation.

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(PDGFR-alpha) are poorly understood. Here we demonstrate that PDGFR-alpha protein and transcriptional regulation in SMCs is under the positive regulatory influence of the zinc finger nuclear protein, Sp1. Electrophoretic mobility shift, competition, and supershift analysis revealed the existence of an atypical G-rich Sp1-binding element located in the PDGFR-alpha promoter -61 to -52 bp upstream of the transcriptional start site. Mutation of this sequence ablated endogenous Sp1 binding and activation of the PDGFR-alpha promoter. PDGFR-alpha transcription, mRNA, and protein expression were repressed in SMCs exposed to fibroblast growth factor-2 (FGF-2). This inhibition was rescued by the blockade of extracellular signal-regulated kinase-1/2 (ERK1/2). FGF-2 repression of PDGFR-alpha transcription was abrogated upon mutation of this Sp1-response element. FGF-2 stimulated Sp1 phosphorylation in an ERK1/2- but not p38-dependent manner, the growth factor enhancing Sp1 interaction with the PDGFR-alpha promoter. Mutation of residues Thr(453) and Thr(739) in Sp1 (amino acids phosphorylated by ERK) blocked FGF-2 repression of PDGFR-alpha transcription. These findings, taken together, demonstrate that FGF-2 stimulates ERK1/2-dependent Sp1 phosphorylation, thereby repressing PDGFR-alpha transcription via the -61/-52 element in the PDGFR-alpha promoter. Phosphorylation triggered by FGF-2 switches Sp1 from an activator to a repressor of PDGFR-alpha transcription, a finding previously unreported in any Sp1-dependent gene.

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ation of energy homeostasis in preovulatory granulosa cells (GCs). In this study, we hypothesized that FSH may regulate IRS-2 expression and thereby maintain the energy balance in GCs. We demonstrate here that FSH specifically increases IRS-2 expression in human and rat GCs. FSH-stimulated IRS-2 expression was inhibited by actinomycin D or cycloheximide. Furthermore, FSH decreases IRS-2 mRNA degradation indicating post-transcriptional stabilization. Herein, we demonstrate a role of cAMP pathway in the activation of IRS-2 expression by FSH. Scan and activity analysis of IRS-2 promoter demonstrated that FSH regulates IRS-2 expression through SP1 binding sites. FSH stimulates SP1 translocation into nucleus and its binding to IRS-2 promoter. These results are corroborated by the fact that siRNA mediated knockdown of IRS-2 decreased the FSH-stimulated PI3K activity, p-Akt levels, GLUT4 translocation and glucose uptake. However, FSH was not able to increase IRS-2 expression in GCs from PCOS women undergoing IVF. Interestingly, IRS-2 mRNA expression was downregulated in GCs from the PCOS rat model. Taken together, our findings establish that FSH induces IRS-2 expression and thereby activates PI3K, Akt and glucose uptake. Crucially, our data confirms a molecular defect in FSH action in PCOS GCs which may cause deceleration of metabolism and follicular growth leading to infertility. These results lend support for a therapeutic potential of IRS-2 in the management of PCOS.

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ood. In this study, we show that Gata3 activates transcription of parathyroid hormone (PTH), which is secreted from parathyroid glands and is critical for regulating serum calcium and phosphate homeostasis. Gata3 interacted with Gcm2 and MafB, two known transcriptional regulators of parathyroid development, and synergistically stimulated the PTH promoter. An SP1-binding element (GC box) located within the PTH-promoter proximal region was critical for activating transcription by Gata3. In addition, the ubiquitous transcription factor SP1 also interacted with Gata3 as well as MafB and Gcm2, and HDR syndrome-associated Gata3 mutants were defective in activating the PTH promoter. These results suggest that Gata3 is a critical regulator of PTH gene expression.

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reporters containing 160 kb of human genomic sequence containing the hTERT gene. Upon chromosomal integration, the bacterial artificial chromosomes recapitulated endogenous hTERT expression, contrary to transient reporters. Sp1/Sp3 expression did not correlate with hTERT promoter activity, and these TFs bound to the hTERT promoters in both telomerase-positive and telomerase-negative cells. Mutation of the proximal GC-box resulted in a dramatic decrease of hTERT promoter activity, and mutations of all five GC-boxes eliminated its transcriptional activity. Neither mutations of GC-boxes nor knockdown of endogenous Sp1 impacted promoter binding by other TFs, including E-box-binding proteins, and histone acetylation and trimethylation of histone H3K9 at the hTERT promoter in telomerase-positive and -negative cells. The result indicated that promoter binding by Sp1/Sp3 was essential, but not a limiting step, for hTERT transcription. hTERT transcription required a permissive chromatin environment. Importantly, our data also revealed different functions of GC-boxes and E-boxes in hTERT regulation; although GC-boxes were essential for promoter activity, factors bound to the E-boxes functioned to de-repress hTERT promoter.

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nal role of EP4 in the occurrence and progression of non small cell lung cancer (NSCLC) remained to be elucidated. METHODS: Cell viability was measured by MTT assays. Western blot was performed to measure the phosphorylation and protein expression of PI3-K downstream effector Akt, transcription factors SP1, p65, and EP4. Quantitative real-time PCR (qRT-PCR) was used to examine the mRNA levels of EP4 gene. Exogenous expression of SP1, p65, and EP4 genes was carried out by transient transfection assays. EP4 promoter activity was measured by Dual Luciferase Reporter Kit. RESULTS: We showed that solamargine inhibited the growth of lung cancer cells. Mechanistically, we found that solamargine decreased the phosphorylation of Akt, the protein, mRNA expression, and promoter activity of EP4. Moreover, solamargine inhibited protein expression of SP1 and NF-kappaB subunit p65, all of which were abrogated in cells transfected with exogenous expressed Akt. Intriguingly, exogenous expressed SP1 overcame the effect of solamargine on inhibition of p65 protein expression, and EP4 protein expression and promoter activity. Finally, exogenous expressed EP4 feedback reversed the effect of solamargine on phosphorylation of Akt and cell growth inhibition. CONCLUSION: Our results show that solamargine inhibits the growth of human lung cancer cells through inactivation of Akt signaling, followed by reduction of SP1 and p65 protein expression. This results in the inhibition of EP4 gene expression. The cross-talk between SP1 and p65, and the positive feedback regulatory loop of PI3-K/Akt signaling by EP4 contribute to the overall responses of solamargine in this process. This study unveils a novel mechanism by which solamargine inhibits growth of human lung cancer cells.

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cp2 exon 1 from a rat liver cDNA library and identified cis-acting regulatory elements. The transcriptional start site was identified as "C," -359 bp from the ATG codon. A reporter gene assay showed that deletion of the nucleotide sequence between -264 and -60 bp resulted in a significant decrease in promoter activity in HepG2 and H4IIE cells. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) revealed that the increase in promoter activity is related to an enhanced ability of Sp1 to bind to its motifs at -84 to -61 bp within the ucp2 proximal promoter. Overexpression of exogenous Sp1 in H4IIE cells also increased the promoter activity. We demonstrated that the expression of UCP2 mRNA and protein is markedly increased in rats with nonalcoholic steatohepatitis (NASH). Coincidently, levels of Sp1 binding to -84/-61 bp were also increased. Overall, our data indicate that the Sp1-binding site located at the proximal promoter is involved in the regulation of rat UCP2 expression.

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n of the calmodulin gene (Majumdar, G., Harmon, A., Candelaria, R., Martinez-Hernandez, A., Raghow, R., and Solomon, S. S. (2003) Am. J. Physiol. 285, E584-E591). Here we report the temporal dynamics of accumulation of total, O-GlcNAc-modified, and phosphorylated Sp1 in H-411E hepatoma cells by immunohistochemistry with monospecific antibodies, confocal microscopy, and matrix-assisted laser desorption and ionization-time of flight mass spectrometry. Insulin elicited sequential and reciprocal post-translational modifications of Sp1. The O-glycosylation of Sp1 and its nuclear accumulation induced by insulin peaked early (approximately 30 min), followed by a steady decline of O-GlcNAc-modified Sp1 to negligible levels by 240 min. The accumulation of phosphorylated Sp1 in the nuclei of insulin-treated cells showed an opposite pattern, increasing steadily until reaching a maximum around 240 min after treatment. Analyses of the total, O-GlcNAc-modified, or phosphorylated Sp1 by Western blot and mass spectrometry corroborated the sequential and reciprocal control of post-translational modifications of Sp1 in response to insulin. Treatment of cells with streptozotocin (a potent inhibitor of O-GlcNAcase) led to hyperglycosylation of Sp1 that failed to be significantly phosphorylated. The mass spectrometry data indicated that a number of common serine residues of Sp1 undergo time-dependent, reciprocal O-glycosylation and phosphorylation, paralleling its rapid translocation from cytoplasm to the nucleus. Later, changes in the steady state levels of phosphorylated Sp1 mimicked the enhanced steady state levels of calmodulin mRNA seen after insulin treatment. Thus, O-glycosylation of Sp1 appears to be critical for its localization into the nucleus, where it undergoes obligatory phosphorylation that is needed for Sp1 to activate calmodulin gene expression.

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ested the hypothesis that Sp1 interactions are facilitated by an IRBP. Binding of nuclear extracts to region V probes was reduced by mutational or chemical interference with the AT-rich element. Blocking the AT site also reduced interactions of Sp1 with region V in vitro and blunted transactivation of region V reporter constructs by Sp1 in vivo. Sp1 binding was enhanced by small quantities of hepatic nuclear extracts, but enhancement was reduced by the AT mutation and abolished by a 5-base pair insertion between the AT-rich and GC-rich sites, and transactivation by Sp1 in vivo was diminished by inserting bases between the AT-rich and GC-rich elements. However, treating cells with insulin increased the ability of nuclear extracts to enhance Sp1 binding. These findings indicate that the presence of the AT-rich element is essential for the actions of Sp1 in vitro and in vivo, and the combination of both spacing requirements and insulin responsiveness suggests that IRBP may interact directly with Sp1.

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regulatory mechanism of rat GLUT1 (rGLUT1) gene expression, we transfected rGLUT1 promoter-chloramphenicol acetyltransferase constructs into Rcho-1 cells. Deletion analysis of the rGLUT1 promoter suggested that the region -76/-53 bp was essential for basal transcriptional activity. Electrophoretic mobility shift assays showed that transcription factors Sp1 and Sp3 bound two GC boxes in the region -99/-33 bp of the rGLUT1 promoter. Mutation analysis of the Sp1 binding sites revealed that the promoter-proximal site located between -76 and -53 bp was essential for basal rGLUT1 promoter activity. Furthermore, the decreased level of GLUT1 may result from a decreased level of Sp1 during differentiation. These findings suggest that Sp1 is involved in the regulation of rGLUT1 gene expression during rat trophoblast differentiation.

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-PCR in human BC cell lines and clinical samples and the prognostic potential of miR-200b expression was further evaluated. In vitro, effects of miR-200b on BC cell proliferation, apoptosis and cell cycle distribution were tested by CCK-8 kit, flow cytometric analysis respectively. Luciferase assay and Western blot analysis were performed to validate the potential targets of miR-200b after the preliminary screening by employing open access software. We found that miR-200b was significantly down-regulated in both BC tissues and cell lines. The low expression of miR-200b was correlated with late TNM stage, negative oestrogen receptor and positive HER-2 status. Multivariate analysis showed that miR-200b expression was an independent prognostic predictor for BC patients. Integrated analysis identified Sp1 as a direct and functional target of miR-200b. Knockdown of Sp1 inhibited cell proliferation, induce apoptosis and act on cell cycle resembling that of miR-200b high expression. Our data demonstrates that miR-200b has potential to serve as prognostic biomarker and tumour suppressor for BC patients. As a direct and functional target of miR-200b, Sp1 and miR-200b both could be an exciting target for BC treatment strategy.

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-flanking region were cloned into pGL3-BASIC upstream of the luciferase reporter gene and transfected into 3T3-F442A adipocytes. Analysis of a 4.3-kb DNA fragment at the 5'-flanking region of this gene revealed that the Gpc4 promoter is a TATA-less promoter with a large cluster of GC boxes. Competitive electrophoretic mobility shift and supershift assays identified a cluster of nine functional GC boxes binding Sp1 and Sp3 in this region. Transactivation experiments in insect cells showed that both Sp1 and Sp3 are major activators of the Gpc4 promoter. Gpc4 is expressed in adipocytes where its expression is highest in confluent 3T3-F442A adipoblasts and decreases dramatically as cells differentiate. Sp protein analyses demonstrated a major decrease in Sp3 protein in differentiated adipocytes as compared to undifferentiated adipoblasts. These experiments show that Gpc4 is developmentally regulated in 3T3-F442A adipocytes and suggest that Sp transcription factors play a significant role in the regulated expression of Gpc4.

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t the G2 phase through a mechanism that was dependent on the JNK pathway. Microarrays further showed that PP2A inhibitors induced expression changes in multiple genes that participate in cell cycle transition. To verify whether these expression changes were executed in a PP2A-dependent manner, we targeted the PP2A catalytic subunit (PP2Ac) using siRNA and evaluated gene expression with a microarray. After the cross comparison of these microarray data, we identified that CDK1 was potentially the same target when treated with either PP2A inhibitors or PP2Ac siRNA. In addition, we found that the down-regulation of CDK1 occurred in a JNK-dependent manner. Luciferase reporter gene assays demonstrated that repression of the transcription of CDK1 was executed through the JNK-dependent activation of the Sp1 transcription factor. By constructing deletion mutants of the CDK1 promoter and by using ChIP assays, we identified an element in the CDK1 promoter that responded to the JNK/Sp1 pathway after stimulation with PP2A inhibitors. Cantharidin and OA also up-regulated the expression of p21, an inhibitor of CDK1, via autophagy rather than PP2A/JNK pathway. Thus, this present study found that the PP2A/JNK/Sp1/CDK1 pathway and the autophagy/p21 pathway participated in G2/M cell cycle arrest triggered by PP2A inhibitors.

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und 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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a-N-acetylglucosamine (O-GlcNAc) moieties has been implicated in the nuclear transport of some proteins, including transcription factor Sp1. The nucleocytoplasmic enzymes O-beta-N-acetylglucosaminyltransferase (OGT) and O-beta-N-acetylglucosaminidase (O-GlcNAcase) adds or remove O-GlcNAc moieties, respectively. If O-GlcNac moieties contribute to the nuclear targeting of alpha4, a decrease in O-GlcNAcylation (e.g., by inhibition of OGT) may redistribute alpha4 to the cytosol. The present study showed that alpha4 and Sp1 were both O-GlcNAcylated in quiescent and PRL-treated Nb2 cells. PRL alone or PRL + streptozotocin (STZ; an O-GlcNAcase inhibitor) significantly (P Sp1 protein levels, caused a significant decrease in the GlcNAc/Sp1 ratio (P Sp1 to the cytosol. Finally, a 50% downregulation of OGT gene expression by small interfering RNA (i.e., siOGT) partially redistributed both alpha4 and Sp1 to the cytosol. The alpha4 protein partner PP2Ac had no detectable O-GlcNAc moieties and its nuclear distribution was not affected by siOGT. In summary, alpha4 and Sp1 contained O-GlcNAc moieties, which contributed to their nuclear targeting in Nb2 cells.

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mited examples of the SENP regulation of SUMO2/3-targeted proteolysis mediated by RNF4. The present study investigated the role of SENP3 in the global protein turnover related to SUMO2/3-targeted ubiquitination and focused in particular on the SENP3 regulation of the stability of Sp1. Our data demonstrated that SENP3 impaired the global ubiquitination profile and promoted the accumulation of many proteins. Sp1, a cancer-associated transcription factor, was among these proteins. SENP3 increased the level of Sp1 protein via antagonizing the SUMO2/3-targeted ubiquitination and the consequent proteasome-dependent degradation that was mediated by RNF4. De-conjugation of SUMO2/3 by SENP3 attenuated the interaction of Sp1 with RNF4. In gastric cancer cell lines and specimens derived from patients and nude mice, the level of Sp1 was generally increased in parallel to the level of SENP3. These results provided a new explanation for the enrichment of the Sp1 protein in various cancers, and revealed a regulation of SUMO2/3 conjugated proteins whose levels may be tightly controlled by SENP3 and RNF4.

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sociating with malignant phenotypes. However, little was known about its expression and function in PDAC. We showed that PLD1 was elevated in both the cell lines and clinical samples of PDAC, and it positively correlated with vascular invasion (p = 0.041) and responsible for a poor prognosis (p = 0.009). Meanwhile, we also found Sp1 to be elevated in the disease, correlating with vascular invasion (p = 0.007). Moreover, the correlation assay suggested that PLD1 positively correlated with Sp1 in the clinical sample (r = 0.390; p < 0.001) and the cell lines. Finally, we showed that co-high expression of both the factors confers the poorest prognosis for the patients, and that their simultaneous high expression might be an independent prognostic factor (p = 0.001; HR = 3.427; 95% CI 1.629-7.211).

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ty is stimulated by carcinogens and oncogenes, suggesting the involvement of EK in carcinogenesis. Currently, little is known about EK transcriptional regulation by endogenous or exogenous signals, and the ek gene promoter has never been studied. METHODOLOGY/PRINCIPAL FINDINGS: In this report, we mapped the important regulatory regions in the human ek1 promoter. 5' deletion analysis and site-directed mutagenesis identified a Sp site at position (-40/-31) that was essential for the basal transcription of this gene. Treatment of HCT116 cells with trichostatin A (TSA), a histone deacetylase inhibitor, significantly upregulated the ek1 promoter activity through the Sp(-40/-31) site and increased the endogenous expression of ek1. Chromatin immunoprecipitation assay revealed that TSA increased the binding of Sp1, Sp3 and RNA polymerase II to the ek1 promoter in HCT116 cells. The effect of TSA on ek1 promoter activity was cell-line specific as TSA treatment did not affect ek1 promoter activity in HepG2 cells. CONCLUSION/SIGNIFICANCE: In conclusion, we showed that Sp1 and Sp3 are not only essential for the basal transcription of the ek1 gene, their accessibility to the target site on the ek1 promoter is regulated by histone protein modification in a cell line dependent manner.

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ich this occurs remain to be elucidated. We have shown previously that GH3 cells transfected with the rat GnRH receptor cDNA (GGH3-1' cells) support the expression of a cotransfected fusion gene composed of 797 base pairs of rat LHbeta gene 5'-flanking sequence and the first 5 base pairs of the 5'-untranslated region fused to a luciferase reporter (-797/+5LHbetaLUC) and respond to a GnRH agonist with a 10-fold stimulation of activity. Furthermore, we have shown that DNA sequences at -490/-352 confer GnRH responsiveness to the rat LHbeta gene. We have now identified two putative binding sites for Sp1, a three-zinc-finger transcription factor, within this region. Using electrophoretic mobility shift assay, DNase I footprinting, and methylation interference assays, we demonstrate that Sp1 can bind to these sites and that Sp1 is responsible for DNA-protein complexes formed using GGH3-1' and alphaT3-1 nuclear extracts. Mutations of the Sp1 binding sites, which block binding of Sp1, blunt the stimulation of the LHbeta gene promoter by GnRH. These data define GnRH-responsive elements in the LHbeta 5'-flanking sequence and suggest that Sp1 plays an important role in conferring GnRH responsiveness to the LHbeta subunit gene.

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weight:700;'>Sp1) protein expression with clinicopathological significance and disease prognosis in NPC patients receiving radiotherapy. A total of 82 NPC patients (55 males and 27 females, median age: 48 years old) were enrolled and received radiotherapy between September 2011 and March 2014. Tumor tissue and grossly adjacent normal mucosa were obtained in each patient. Sp1 expression was detected by western blot and immunohistochemical analysis, and the associations with clinicopathological status and radiotherapy response were analyzed. Our Results showed Sp1 protein expression was higher in CNE-1 and CNE-2 nasopharyngeal cancer cells than in normal nasopharyngeal mucosal NP69 cells. All 82 patients' tissue sections were stained positive for the Sp1 protein, and 39 (47.6%) patients showed higher level than adjacent normal mucosa. Sp1-overexpression in the tumor tissue was correlated with a higher tumor stage, nodal status, clinical stage and distant metastasis (P < 0.01). Patients with higher Sp1 expression in pretreatment biopsies had a lower radiotherapy response compared to those with lower expression. In conclusion, Sp1 may play roles in radioresistance of nasopharyngeal cancer which attributes to tumor invasiveness, and serve as a novel prognostic marker of NPC radiotherapy. However, further studies are required to validate our findings in larger samples and explore more detailed mechanisms underlying radioresistance of Sp1.

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tro Cell. Dev. Biol. Anim. 2014). However, the mechanism of the Col11a1 gene regulation in chondrocytes has not been fully elucidated. In this study, we further characterized the proximal promoter activity of the mouse Col11a1 gene in chondrocytes. Cell transfection experiments with deletion and mutation constructs indicated that the downstream region of the NF-Y binding site (-116 to +1) is also necessary to regulate the proximal promoter activity of the mouse Col11a1 gene. This minimal promoter region has no TATA box and GC-rich sequence; we therefore examined whether the GC-rich sequence (-96 to -67) is necessary for the transcription regulation of the Col11a1 gene. Luciferase assays using a series of mutation constructs exhibited that the GC-rich sequence is a critical element of Col11a1 promoter activity in chondrocytes. Moreover, in silico analysis of this region suggested that one of the most effective candidates was transcription factor Sp1. Consistent with the prediction, overexpression of Sp1 significantly increased the promoter activity. Furthermore, knockdown of Sp1 expression by siRNA transfection suppressed the proximal promoter activity and the expression of endogenous transcript of the mouse Col11a1 gene. Taken together, these results indicate that the transcription factor Sp1 upregulates the proximal promoter activity of the mouse Col11a1 gene in chondrocytes.

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tumorigenesis of liver cancer. However, the regulation of TIAM2S ectopic expression in HCC cells remains largely unknown. This study aimed to identify the mechanism underlying the ectopic expression of TIAM2S in liver cancer cells. In this report, we provide evidence illustrating that Sp1 binds directly to the GC box located in the TIAM2S core promoter. We further demonstrated that overexpression of Sp1 in HepaRG cells promotes endogenous TIAM2S mRNA and protein expressions, and knockdown of Sp1 in 2 HCC cell lines, HepG2 and PLC/PRF/5, led to a substantial reduction in TIAM2S mRNA and protein in these cells. Of 60 paired HCC samples, 70% showed a significant increase (from 1.1- to 3.6-fold) in Sp1 protein expression in the tumor cells. The elevated Sp1 expression was highly correlated with both TIAM2S mRNA and protein expressions in these samples. Together, these results illustrate that Sp1 positively controls TIAM2S transcription and that Sp1-mediated transcriptional activation is essential for TIAM2S ectopic expression in liver cancer cells.

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gamma1 chain gene (LAMC1) promoter contains several GC/GT-rich motifs including the bcn-1 element. Using the bcn-1 element as a bait in the yeast one-hybrid screen, we cloned the gut-enriched Kruppel-like factor (GKLF or KLF4) from a rat mesangial cell library. We show that GKLF binds bcn-1, but this binding is not required for the GKLF-mediated activation of the LAMC1 promoter. The activity of GKLF is dependent on a synergism with another Kruppel-like factor, Sp1. The LAMC1 promoter appears to have multiple GKLF- and Sp1-responsive elements which may account for the synergistic activation. We provide evidence that the synergistic action of GKLF and Sp1 is dependent on the promoter context and the integrity of GKLF activation and DNA-binding domain. GKLF is thought to participate in the switch from cell proliferation to differentiation. Thus, the Sp1-GKLF synergistic activation of the LAMC1 promoter may be one of the avenues for expression of laminin gamma1 chain when laminin is needed to regulate cell differentiation.

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or-1 (PAI-1) gene promoter through the transcription factor, Sp1; and that the glutamine:fructose-6-phosphate amidotransferase inhibitor, 6-diazo-5-oxonorleucine, inhibited the effect of high glucose, but not that of glucosamine. Here, we examined the role of protein kinase C (PKC) isoforms in the regulation of the PAI-1 promoter and Sp1 transcriptional activity by the HBP. In transient transfections, exposure to 2 mm glucosamine or 20 mm glucose for 4 days increased the activities of a PAI-1 promoter-luciferase reporter gene as well as the Sp1 transcriptional activation domain fused to the GAL4 DNA-binding domain cotransfected with a GAL4 promoter-luciferase reporter. Cotransfected dominant negative PKC-betaI and -delta completely blocked the induction of PAI-1 promoter transcription by both sugars, whereas only dominant negative PKC-betaI interfered with Sp1-GAL4 activation. Both glucosamine and high glucose stimulated the in vitro kinase activity of immunoprecipitated PKC-betaI and -delta. Furthermore, 6-diazo-5-oxonorleucine suppressed high glucose-induced PKC kinase activity and Sp1-GAL4 transcriptional activation. These findings demonstrate a requirement for the PKC-betaI and -delta signal transduction pathways in HBP-induced transcription.

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cancer remains poorly understood. In the present study, we report that HBXIP is able to up-regulate FGF4 to enhance the migration of breast cancer cells. Immunohistochemistry staining showed that HBXIP and FGF4 were highly expressed in clinical metastatic lymph nodes of breast tumor. The expression levels of HBXIP were positively related to those of FGF4 in clinical breast cancer tissues. Then, we validated that HBXIP up-regulated the expression of FGF4 at the levels of promoter, mRNA and protein by luciferase reporter gene assays, reverse transcription-polymerase chain reaction and Western blot analysis. Moreover, we found that HBXIP was able to activate FGF4 promoter through transcriptional factor Sp1 by luciferase reporter gene assays. Chromatin immunoprecipitation assays confirmed that HBXIP coactivated Sp1 to stimulate FGF4 promoter. In function, we showed that HBXIP promoted breast cancer cell migration through FGF4 by wound healing and transwell cell migration assays. Thus, we conclude that the oncoprotein HBXIP up-regulates FGF4 through activating transcriptional factor Sp1 to promote the migration of breast cancer cells. Therapeutically, HBXIP may serve as a novel target in breast cancer.

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velopment; however, its target genes and molecular mode of action remain to be elucidated. Here we show that SCL and the c-Kit receptor are coexpressed in hematopoietic progenitors at the single-cell level and that SCL induces c-kit in chromatin, as ectopic SCL expression in transgenic mice sustains c-kit transcription in developing B lymphocytes, in which both genes are normally down-regulated. Through transient transfection assays and coimmunoprecipitation of endogenous proteins, we define the role of SCL as a nucleation factor for a multifactorial complex (SCL complex) that specifically enhances c-kit promoter activity without affecting the activity of myelomonocytic promoters. This complex, containing hematopoietic-specific (SCL, Lim-only 2 (LMO2), GATA-1/GATA-2) and ubiquitous (E2A, LIM- domain binding protein 1 [Ldb-1]) factors, is tethered to DNA via a specificity protein 1 (Sp1) motif, through direct interactions between elements of the SCL complex and the Sp1 zinc finger protein. Furthermore, we demonstrate by chromatin immunoprecipitation that SCL, E2A, and Sp1 specifically co-occupy the c-kit promoter in vivo. We therefore conclude that c-kit is a direct target of the SCL complex. Proper activation of the c-kit promoter depends on the combinatorial interaction of all members of the complex. Since SCL is down-regulated in maturing cells while its partners remain expressed, our observations suggest that loss of SCL inactivates the SCL complex, which may be an important event in the differentiation of pluripotent hematopoietic cells.

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s nucleosomal rearrangement suggesting that they participate in turning-off the viral promoters. In aiming to elucidate the role of the capsid proteins in gene regulation, we studied the interaction between VP3, an internal capsid protein, and the cellular transcription factor Sp1, a major regulator of both the early and late viral promoters. Our results showed that VP3 repressed transcription from the viral early promoter in vitro. We found significant cooperativity between Sp1 and VP3 in specific DNA-binding to the Sp1 binding site. In addition, protein-protein interactions between VP3 and Sp1 in the absence of DNA were observed. These findings have led us to conclude that the novel host-viral Sp1-VP3 complex down regulates viral transcription and further suggest that Sp1 participates in recruiting VP3 to the SV40 minichromosome in SV40 assembly.

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f telomere uncapping using the TRF2(¿B¿M)-induced senescence model in human diploid fibroblasts. We observed that the expression of Sp1 is down-regulated in the TRF2(¿B¿M)-induced senescence, which was mediated by ATM and p38 MAPK. In addition, overexpression of Sp1 prevented the TRF2(¿B¿M)-induced senescence. Among transcriptional targets of Sp1, expression levels of nuclear transport genes such as karyopherin a, Nup107, and Nup50 were down-regulated in the TRF2(¿B¿M)-induced senescence, which was prevented by Sp1 overexpression. Moreover, inhibition of the nuclear transport by wheat germ agglutinin (an import inhibitor) and leptomycin B (an export inhibitor) induced premature senescence. These results suggest that Sp1 is an anti-senescence transcription factor in the telomere uncapping-induced senescence and that down-regulation of Sp1 leads to the senescence via down-regulation of the nuclear transport.

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ith iron transport-related genes by a hypoxia-inducible transcription factor, Hif2alpha. In the current study, we sought to test the role of Sp1 in transcriptional regulation of Atp7a expression during iron deprivation/hypoxia. Initial studies in IEC-6 cells showed that mithramycin, an Sp1 inhibitor, reduced expression of Atp7a and iron transport-related genes (Dmt1, Dcytb, and Fpn1) and blocked their induction by CoCl2, a hypoxia mimetic. Consistent with this, overexpression of Sp1 increased endogenous Atp7a mRNA and protein expression and stimulated Atp7a, Dmt1, and Dcytb promoter activity. Site-directed mutagenesis and functional analysis of a basal Atp7a promoter construct revealed four functional Sp1 binding sites that were necessary for Hif2alpha-mediated induction of promoter activity. Furthermore, chromatin immunoprecipitation (ChIP) assays confirmed that Sp1 specifically interacts with the Atp7a promoter in IEC-6 cells and in rat duodenal enterocytes. This investigation has thus revealed a novel aspect of Atp7a gene regulation in which Sp1 may be necessary for the HIF-mediated induction of gene transcription during iron deficiency/hypoxia. Understanding regulation of Atp7a expression may help further clarify the physiological role of copper in the maintenance of iron homeostasis. Furthermore, this Sp1/Hif2alpha regulatory mechanism may have broader implications for understanding the genetic response of the intestinal epithelium to maintain whole-body iron homeostasis during states of deficiency.

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HCN2 and HCN4 genes in the heart, plays an important role in rhythmogenesis. Their expressions reportedly increase in hypertrophic and failing hearts, contributing to arrhythmogenesis under these conditions. However, how their expressions are controlled remained unclear. We performed a study to characterize the regulatory elements and transcriptional control of HCN2 and HCN4 genes. We identified the transcription start sites by 5'RACE and core promoter regions of these genes using luciferase reporter assay, and revealed the ubiquitous Sp1 protein as a common transactivator of HCN2 and HCN4 genes. We further unraveled robust increases in HCN2/HCN4 transcripts and protein levels, using real-time RT-PCR and Western blot analyses, in a rat model of left ventricular hypertrophy and in angiotensin II-induced neonatal ventricular hypertrophy. The upregulation of HCN2 and HCN4 transcription was accompanied by pronounced elevations of Sp1 and silencing of Sp1 by siRNA prevented overexpression of HCN2/HCN4 in hypertrophic cardiomyocytes. Our data indicate that Sp1 drives HCN2/HCN4 transcription and determines the functional level of HCN2/HCN4 mRNAs, and upregulation of Sp1 underlie the abnormal re-expression of HCN2/HCN4 genes in hypertrophied myocytes. This study also provides the first evidence for the role of Sp1 in the reactivation of 'fetal' cardiac genes, HCN2 and HCN4, in ventricular myocytes, and thereby in the pathological electrical remodeling in hypertrophied myocytes.

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ogesterone and histone deacetylase (HDAC) inhibitors have also been reported to promote the glial cell differentiation with the enhancement of serotonin-stimulated brain-derived neurotrophic factor gene transcription through the production of 5alpha-reduced neurosteroids, thus suggesting that glial cell differentiation is probably implicated in the protection and survival of neuronal cells in the brain. Therefore, the expression of 5alpha-R gene in glial cells seems physiologically important in maintaining the neural function in the brain, but little is known about the mechanism underlying the regulation of 5alpha-R gene transcription. In the present study, the effect of a HDAC inhibitor trichostatin A (TSA) on 5alpha-R gene transcription in the glioma cells was examined, and TSA was shown to induce the elevation of 5alpha-R mRNA levels through the activation of the 5alpha-R promoter via a mechanism involving Sp1 and Sp3 transcription factors in a time- and concentration-dependent manner. Thus, both Sp1 and Sp3 are considered to play a physiological role in the regulation of 5alpha-R gene expression, and hence the production of 5alpha-reduced neurosteroids in glial cells.

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not been identified. We studied the regulation of UbC expression in L6 muscle cells because dexamethasone stimulates the transcription of this gene and others encoding components of the ubiquitin-proteasome pathway. Results of in vivo genomic DNA footprinting experiments indicate that a protein(s) binds to Sp1 sites approximately 50 bp upstream from the UbC transcription start site; dexamethasone changes the methylation pattern at these sites. Sp1 binds to DNA probes corresponding to the rat or human UbC promoter, and treating cells with dexamethasone increases this binding. Deletion and mutation analyses of the rat and human UbC promoters are consistent with an important role of Sp1 in UbC induction by glucocorticoids. Dexamethasone-induced ubiquitin expression is blocked by mithramycin, an inhibitor of Sp1 binding. UO126, a pharmacologic inhibitor of MEK1, also blocks UbC transcriptional activation by dexamethasone; L6 cells transfected to express constitutively active MEK1 exhibit increased UbC promoter activity. Thus, glucocorticoids increase UbC expression in muscle cells by a novel transcriptional mechanism involving Sp1 and MEK1.

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rowth factor B-chain (PDGF-B). The regulatory mechanisms underlying pVHL suppression of PDGF-B expression, however, are completely unknown. This understanding would shed vital light on the control of growth factor gene expression by this tumor suppressor. Here we report that pVHL can repress both endogenous steady-state PDGF-B mRNA expression and PDGF-B promoter-dependent transcription in WKY12-22 cells. Transient transfection analysis utilizing PDGF-B promoter-chloramphenicol acetyl transferase (CAT) reporter constructs revealed that pVHL inhibition of PDGF-B expression is mediated via the Sp1-binding element in the proximal region of the PDGF-B promoter. Recent studies have demonstrated a physical interaction between pVHL and Sp1, which activates the PDGF-B promoter. We show that Sp1 can rescue PDGF-B promoter activity and endogenous PDGF-B mRNA expression from pVHL repression. These findings thus demonstrate a pivotal role for Sp1 in pVHL inhibition of PDGF-B transcription.

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with a feature of C-U deamination. In this study, we identified a new member of cytidine deaminase family, NYD-SP15. Previous work showed that the deduced structure of the protein contained two dCMP_cyt_deam domains, which were involved in zinc ion binding. NYD-SP15 was expressed variably in a wide range of tissues, indicating its worthy biological function and creative significances. Sequence analysis, RT-PCR, western blot, flow cytometry, direct-site mutation and GST pull-down assay were performed to analyze the construction and function of NYD-SP15. The results in our studies showed that NYD-SP15 was closely related to deoxycytidylate deaminase and cytidine deaminase, with authentic cytidine deaminase activity in vivo and vitro as well as homo dimerization effects. NYD-SP15 contained nuclear localization sequence (NLS) and nuclear export-signal (NES) and could dynamically shuttle between the nucleus and cytoplasm. Furthermore, NYD-SP15 gene over-expression reduced the cells growth and blocked G1 to S phase, which implied a potential inhibition effect on cell growth.

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es have indicated that established hr-HPV-positive cell lines display a reduced expression of ISGs, which correlates with decreased levels of interferon kappa (IFN-kappa), a type I IFN constitutively expressed in keratinocytes. Prior studies have also suggested that IFN-beta has anti-hr-HPV activity but the underlying mechanisms are not well understood. The downregulation of IFN-kappa by hr-HPV raises the possibility that IFN-kappa has anti-HPV activity. Using doxycycline-inducible IFN-kappa expression in CIN612-9E cells, which maintain extrachromosomally replicating HPV31 genomes, we demonstrated that IFN-kappa inhibits the growth of these cells and reduces viral transcription and replication. Interestingly, the initiation of viral early transcription was already inhibited at 4 to 6 h after IFN-kappa expression. This was also observed with recombinant IFN-beta, suggesting a common mechanism of IFNs. Transcriptome sequencing (RNA-seq) analysis identified 1,367 IFN-kappa-regulated genes, of which 221 were modulated >2-fold. The majority of those (71%) matched known ISGs, confirming that IFN-kappa acts as a bona fide type I IFN in hr-HPV-positive keratinocytes. RNA interference (RNAi) and cotransfection experiments indicated that the inhibition of viral transcription is mainly due to the induction of Sp100 proteins by IFN-kappa. Consistent with published data showing that Sp100 acts as a restriction factor for HPV18 infection, our results suggest that hr-HPV target IFN-kappa to prevent Sp100 expression and identify Sp100 as an ISG with anti-HPV activity. IMPORTANCE: High-risk HPV can establish persistent infections which may progress to anogenital cancers. hr-HPV interfere with the expression of interferon (IFN)-stimulated genes (ISGs), which is due to reduced levels of IFN-kappa, an IFN that is constitutively expressed in human keratinocytes. This study reveals that IFN-kappa rapidly inhibits HPV transcription and that this is due to the induction of Sp100 proteins. Thus, Sp100 represents an ISG for hr-HPV.

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genotyped 139 unrelated non-diabetic Czech females (49.5 +/- 13.3 years, average BMI at baseline 32.2 +/- 4.6 kg/m(2). Biochemical and anthropometrical measurements were performed before and after ten weeks of lifestyle intervention. CONCLUSION: Overweight/obese female carriers of the NYD-SP18 rs6971091 GG genotype exhibited a more beneficial response to the intensive lifestyle intervention than others.

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roversial. In this study, we utilized the monocytic cell line THP-1 as an in vitro latency model for human cytomegalovirus infection (HCMV). Characterization of THP-1 cells by immunofluorescence andWestern blot analysis confirmed the expression of all major ND10 components. THP-1 cells with a stable, individual knockdown of PML, hDaxx or Sp100 were generated. Importantly, depletion of the major ND10 proteins did not prevent the terminal cellular differentiation of THP-1 monocytes. After construction of a recombinant, endotheliotropic human cytomegalovirus expressing IE2-EYFP, we investigated whether the depletion of ND10 proteins affects the onset of viral IE gene expression. While after infection of differentiated, THP-1-derived macrophages as well as during differentiation-induced reactivation from latency an increase in the number of IE-expressing cells was readily detectable in the absence of the major ND10 proteins, no effect was observed in non-differentiated monocytes. We conclude that PML, hDaxx and Sp100 primarily act as cellular restriction factors during lytic HCMV replication and during the dynamic process of reactivation but do not serve as key determinants for the establishment of HCMV latency.

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, absent tissue plasma cells and hepatic veno-occlusive disease. This is the first report of the involvement of a nuclear body protein in a human primary immunodeficiency and of high-penetrance genetic mutations in hepatic veno-occlusive disease.

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ion and induce antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) against NSCLC in vitro. We generated recombinant adenovirus of Ad-SP17 and control Ad-null. Infection with Ad-SP17, but not control, induced higher levels of SP17 expression in UCB-derived DC-Ad-SP17. Infection with Ad-SP17 significantly increased the frequency of CD80(+), CD83(+), CD86(+), and HLA-DR(+) DC that produced higher levels of IL-12, but lower IL-10. Co-culture of DC-Ad-SP17 with autologous UCB lymphocytes induced high frequency of IFNgamma(+) CD8(+) CTLs, which had selective cytotoxicity against SP17(+) lung cancer CRL-5922 cells in a HLA-I restrictive manner. Thus, UCB-derived DC modulated for SP17 expression induced antigen-specific anti-tumor immunity against SP17(+) NSCLC, and SP17 may be a valuable target for development of immunotherapy against SP17(+) NSCLC.