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Basal Promoter Activity (basal + promoter_activity)
Selected AbstractsCharacterization of core promoter elements for ecdysone receptor isoforms of the silkworm, Bombyx moriINSECT MOLECULAR BIOLOGY, Issue 2 2007H. Shirai Abstract Two ecdysone receptor (EcR) isoforms, EcR-A and EcR-B1, are expressed in a tissue- and stage-specific manner, although the details of their transcription mechanisms are unknown. We determined the transcription start sites of EcR-A and EcR-B1 isoforms of Bombyx mori and found that both core promoter regions consist of initiator (Inr) and downstream promoter elements (DPE) but not TATA boxes. Promoter truncation analysis performed using the luciferase reporter assays and BmN cells showed that, in both isoforms, the regions ,296 to ,74 for BmEcR-B1, ,104 to ,61 for BmEcR-A and downstream regions of +1 are essential for basal transcriptional activity. Mutation experiments revealed that both DPE and its 5,-flanking CGCGCG sequence are crucial but DPE of BmEcR-B1 is not important for BmEcR-A transcription. These results indicate that the basal promoter activities differ between the two BmEcR isoforms. [source] Identification and characterization of a novel progesterone receptor-binding element in the mouse prostaglandin E receptor subtype EP2 geneGENES TO CELLS, Issue 9 2003Sohken Tsuchiya Background:, Gene expression of prostaglandin E receptor EP2 is induced in the luminal epithelium of the mouse uterus during peri-implantation period (day-5 of pseudopregnancy), suggesting the involvement of progesterone and its receptor (PR) in this expression. However it remains unclear whether PR affects EP2 gene expression through its binding. Results:, We investigated transcriptional regulation of EP2 gene expression with reporter gene analysis using HeLa cells with or without expression of the PR. The 5,-flanking region (,3260 to ,27, upstream of the translation initiation site) exhibited progesterone-induced promoter activation and basal promoter activity in the presence of PR. Using successive deletion analysis, we determined the six regulatory regions in the EP2 gene. Three regions were found to be involved in progesterone-induced promoter activation, whereas the other three regions were involved in basal promoter activity in the presence of PR. We identified a novel PR-binding sequence, 5,-G(G/A)CCGGA-3,, in the two basal promoter regions and Sp1- and Sp3-binding in the other basal promoter region. Conclusions:, We identified a novel PR-binding sequence, which may be involved in the regulation of basal promoter activity in the EP2 gene. [source] Expression of Acid-Sensing Ion Channel 3 (ASIC3) in Nucleus Pulposus Cells of the Intervertebral Disc Is Regulated by p75NTR and ERK Signaling,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2007Yoshiyasu Uchiyama Abstract Although a recent study has shown that skeletal tissues express ASICs, their function is unknown. We show that intervertebral disc cells express ASIC3; moreover, expression is uniquely regulated and needed for survival in a low pH and hypoeromsotic medium. These findings suggest that ASIC3 may adapt disc cells to their hydrodynamically stressed microenvironment. Introduction: The nucleus pulposus is an avascular, hydrated tissue that permits the intervertebral disc to resist compressive loads to the spine. Because the tissue is hyperosmotic and avascular, the pH of the nucleus pulposus is low. To determine the mechanisms by which the disc cells accommodate to the low pH and hypertonicity, the expression and regulation of the acid sensing ion channel (ASIC)3 was examined. Materials and Methods: Expression of ASICs in cells of the intervertebral disc was analyzed. To study its regulation, we cloned the 2.8-kb rat ASIC3 promoter and performed luciferase reporter assays. The effect of pharmacological inhibition of ASICs on disc cell survival was studied by measuring MTT and caspase-3 activities. Results: ASIC3 was expressed in discal tissues and cultured disc cells in vitro. Because studies of neuronal cells have shown that ASIC3 expression and promoter activity is induced by nerve growth factor (NGF), we examined the effect of NGF on nucleus pulposus cells. Surprisingly, ASIC3 promoter activity did not increase after NGF treatment. The absence of induction was linked to nonexpression of tropomyosin-related kinase A (TrkA), a high-affinity NGF receptor, although a modest expression of p75NTR was seen. When treated with p75NTR antibody or transfected with dominant negative-p75NTR plasmid, there was significant suppression of ASIC3 basal promoter activity. To further explore the downstream mechanism of control of ASIC3 basal promoter activity, we blocked p75NTR and measured phospho extracellular matrix regulated kinase (pERK) levels. We found that DN-p75NTR suppressed NGF mediated transient ERK activation. Moreover, inhibition of ERK activity by dominant negative-mitogen activated protein kinase kinase (DN-MEK) resulted in a dose-dependent suppression of ASIC3 basal promoter activity, whereas overexpression of constitutively active MEK1 caused an increase in ASIC3 promoter activity. Finally, to gain insight in the functional importance of ASIC3, we suppressed ASIC activity in nucleus pulposus cells. Noteworthy, under both hyperosmotic and acidic conditions, ASIC3 served to promote cell survival and lower the activity of the pro-apoptosis protein, caspase-3. Conclusions: Results of this study indicate that NGF serves to maintain the basal expression of ASIC3 through p75NTR and ERK signaling in discal cells. We suggest that ASIC3 is needed for adaptation of the nucleus pulposus and annulus fibrosus cells to the acidic and hyperosmotic microenvironment of the intervertebral disc. [source] Craniosynostosis-Associated Gene Nell-1 Is Regulated by Runx2,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2007Thien Truong Abstract We studied the transcriptional regulation of NELL-1, a craniosynostosis-related gene. We identitifed three OSE2 elements in the NELL-1 promoter that are directly bound and transactivated by Runx2. Forced expression of Runx2 induces NELL-1 expression in rat calvarial cells. Introduction: We previously reported the upregulation of NELL-1 in human craniosynostosis and the overexpression of Nell-1 in transgenic animals that induced premature suture closure associated with increased osteoblast differentiation. To study the transcriptional regulation of NELL-1, we analyzed the 5, flanking region of the human NELL-1 gene. We identified three osteoblast specific binding elements 2 (OSE2) sites (A, B, and C) within 2.2 kb upstream of the transcription start site and further studied the functionality of these sites. Materials and Methods: An area of 2.2 kb and a truncated 325 bp, which lacked the three OSE sites, were cloned into a luciferase reporter gene, and co-transfected with Runx2 expression plasmid. The three OSE2 sites were individually mutated and co-transfected with Runx2 expression plasmid into Saos2 cells. Gel shifts and supershifts with Runx2 antibodies were used to determine specific binding to OSE2 sites. CHIP assays were used to study in vivo binding of Runx2 to the Nell-1 promoter. Runx2 expression plasmid was transfected into wildtype and Runx2,/, calvarial cells. Nell-1, osteocalcin, and Runx2 expression levels were measured using RT-PCR. Results: Addition of Runx2 dose-dependently increased the luciferase activity in the human NELL-1 promoter-luciferase p2213. The p325 truncated NELL-1 construct showed significantly lower basal level of activity. Nuclear extract from Saos2 cells formed complexes with site A, B, and C probes and were supershifted with Runx2 antibody. Mutation of sites A, B, and C significantly decreased basal promoter activity. Furthermore, mutation of sites B and C had a blunted response to Runx2, whereas mutation of site A had a lesser effect. Runx2 bound to NELL-1 promoter in vivo. Transfection of Runx2 in rat osteoblasts upregulated Nell-1 and Ocn expression, and in Runx2 null calvarial cells, both Nell-1 and Ocn expression were rescued. Conclusions: Runx2 directly binds to the OSE2 elements and transactivates the human NELL-1 promoter. These results suggest that Nell-1 is likely a downstream target of Runx2. These findings may also extend our understanding of the molecular mechanisms governing the pathogenesis of craniosynostosis. [source] RANKL Treatment Releases the Negative Regulation of the Poly(ADP-Ribose) Polymerase-1 on Tcirg1 Gene Expression During Osteoclastogenesis,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2006Guillaume E Beranger Abstract The Tcirg1 gene encodes the osteoclast-specific a3 isoform of the V-ATPase a subunit. Using the mouse osteoclastic model RAW264.7 cells, we studied Tcirg1 gene expression, and we identified PARP-1 as a transcriptional repressor negatively regulated by RANKL during osteoclastogenesis. Introduction: The TCIRG1 gene encodes the a3 isoform of the V-ATPase a subunit, and mutations at this locus account for ,60% of infantile malignant osteopetrosis cases. Using RAW264.7 cells as an osteoclastic differentiation model, we undertook a transcriptional study of the mouse Tcirg1 gene focused on the 4-kb region upstream of the transcription starting point. Materials and Methods: The promoter activity of serial-deletion fragments of the Tcirg1 gene promoter was monitored throughout the RAW264.7 cell differentiation process. We next performed EMSA, UV cross-linking, affinity purification, mass spectrometry analysis, gel supershift, and siRNA transfection experiments to identify the factor(s) interacting with the promoter. Results: The ,3946/+113 region of the mouse Tcirg1 gene displayed a high basal promoter activity, which was enhanced by RANKL treatment of RAW264.7 cells. Constructs deleted up to ,1589 retained this response to RANKL. A deletion up to ,1402 induced a 3-fold enhancement of the basal activity, whereas RANKL response was not affected. EMSA experiments led us to identify within the ,1589/,1402 region, a 10-nucleotide sequence, which bound a nuclear protein present in nondifferentiated RAW264.7 cells. This interaction was lost using nuclear extracts derived from RANKL-treated cells. Affinity purification followed by mass spectrometry analysis and gel supershift assay allowed the identification of poly(ADP-ribose) polymerase-1 (PARP-1) as this transcriptional repressor, whereas Western blot experiments revealed the cleavage of the DNA-binding domain of PARP-1 on RANKL treatment. Finally, both PARP-1 depletion after siRNA transfection and RAW264.7 cell treatment by an inhibitor of PARP-1 activity induced an increase of a3 mRNA expression. Conclusions: We provide evidence that the basal transcription activity of the Tcirg1 gene is negatively regulated by the binding of PARP-1 protein to its promoter region in mouse pre-osteoclast. On RANKL treatment, PARP-1 protein is cleaved and loses its repression effect, allowing an increase of Tcirg1 gene expression that is critical for osteoclast function. [source] Transcriptional regulation of human excitatory amino acid transporter 1 (EAAT1): cloning of the EAAT1 promoter and characterization of its basal and inducible activity in human astrocytesJOURNAL OF NEUROCHEMISTRY, Issue 6 2003Seon-Young Kim Abstract Excitatory amino acid transporter 1 (EAAT1) is one of the two glial glutamate transporters that clear the extracellular glutamate generated during neuronal signal transmission. Here, we cloned and characterized a 2.1-kb promoter region of human EAAT1 and investigated its function in the transcriptional regulation of the EAAT1 gene in human primary astrocytes. The full-length promoter region lacked TATA and CCAAT boxes and an initiator element, it contained several potential transcription factor-binding sites and it exhibited promoter activity in primary astrocytes and in several types of transformed cells. Consecutive 5,-deletion analysis of the EAAT1 promoter indicated the presence of negative and positive regulatory regions and a putative core promoter between ,57 bp and +20 bp relative to the transcription start site (TSS). The core promoter contained a single GC-box in position ,52/,39 and one E-box near the TSS and the GC-box site that was responsible for 90% of the basal promoter activity as determined by mutational analysis. Electrophoretic mobility shift, supershift and competition assays demonstrated binding of stimulating proteins (Sp) 1 and 3 to the GC-box and upstream stimulating factor (USF) 1 to the E-box. Treatment of primary human astrocytes with cellular modulators 8-bromo cyclic AMP and epidermal growth factor increased EAAT1 promoter activity in transient transfection assays and increased cellular EAAT1 mRNA expression and glutamate uptake by astrocytes. Conversely, tumor necrosis factor-, reduced both EAAT promoter activity and cellular EAAT1 mRNA expression. These results enable studies of transcriptional regulation of EAAT1 gene at the promoter level. [source] Transcription factors NF-,B and Sp1 are major determinants of the basal promoter activity of the rat GD3-synthase geneJOURNAL OF NEUROCHEMISTRY, Issue 2002G. Zeng GD3-synthase is one of the key sialyltransferases responsible for synthesis of ganglioside GD3, the substrate for initiation of the ,b' and ,c' series ganglioside synthesis. We have previously cloned the rat GD3-synthase gene promoter, and preliminary characterization has identified a minimal 0.5-kb region that has a strong basal promoter activity, and is GC-rich and has no CAAT or TATA boxes. In this study, we showed that the Sp1 and NF-,B sites in this region significantly contributed to basal GD3-synthase promoter activity. When either the Sp1 or NF-,B sites were deleted, a 50% decrease in promoter activity was observed. The same results were obtained by a decoy strategy using oligonucleotides containing the Sp1 or NF-,B sites. The binding to the Sp1 and NF-,B sites was confirmed by electrophoretic mobility shift assay (EMSA), competition and supershift EMSA. In addition, cell-type specific activation of the promoter was also determined. The promoter was highly activated in the GD3-expressing F-11 cells while repressed in NG-108 cells in which GD3 is almost undetectable. An additional band of NF-,B family was identified only in the F-11 nuclear extract using the NF-,B consensus probe by EMSA. DNA pull-down assays were further carried out to screen proteins that bound to the promoter including the basal region and the potential negative-regulatory region between ,526 and ,769. More than 10 major binding proteins were pulled down, some of which were present only in the F-11 or NG-108 nuclear extracts. Our data demonstrate that NF-,B and Sp1 are the major determinants for the basal promoter activity and some factors such as NF-,B may be involved in cell type-specific expression of the gene. [source] Hypoxia and glucocorticoid signaling converge to regulate macrophage migration inhibitory factor gene expressionARTHRITIS & RHEUMATISM, Issue 8 2009Laura M. Elsby Objective Macrophage migration inhibitory factor (MIF) is a proinflammatory mediator involved in the pathogenesis of rheumatoid arthritis. This study was undertaken to identify the MIF promoter elements responsible for regulating gene expression. Methods Luciferase reporter gene assays were used to identify the MIF promoter sequence responsible for basal activity. Bioinformatic analysis was used to predict transcription factor binding sites, and electrophoretic mobility shift assay (EMSA) was used to demonstrate transcription factor binding. Chromatin immunoprecipitation (ChIP) was used to demonstrate transcription factor loading on the MIF promoter. Results We identified the minimal promoter sequence required for basal MIF promoter activity that was also capable of conferring glucocorticoid-dependent inhibition in a T lymphocyte model cell line. Deletion studies and EMSA revealed 2 elements in the MIF promoter that were responsible for basal promoter activity. The 5, element binds CREB/activating transcription factor 1, and the 3, element is a functional hypoxia-responsive element binding hypoxia-inducible factor 1,. Further studies demonstrated that the cis elements are both required for glucocorticoid-dependent inhibition. ChIP demonstrated glucocorticoid-dependent recruitment of glucocorticoid receptor , to the MIF promoter in lymphocytes within 1 hour of treatment and a concomitant decrease in acetylated histone H3. Conclusion Our findings indicate that hypoxia and glucocorticoid signaling converge on a single element regulating MIF; this regulatory unit is a potential interacting node for microenvironment sensing of oxygen tension and glucocorticoid action in foci of inflammation. [source] | |||||||||||||