PMC:1702488 / 14729-18258 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/1702488","sourcedb":"PMC","sourceid":"1702488","source_url":"http://www.ncbi.nlm.nih.gov/pmc/1702488","text":"The Sp-binding site and NRSE synergistically repress the expression of MOR gene\nIndependent studies have shown that Sp1 has functional implication in expression of some of NRSF target genes including N-methyl-d-aspartate receptors, dynamin I, synaptophysin and glutamate receptor ionotropic kainate-5 genes (22–24). For MOR gene regulation, Sp1 factor acts as a major positive regulator by binding two cis-elements (distal and proximal promoters) located at −340 through −300 residues from the translation start site while Sp3 has a minor effect by binding to one of these elements (25). As mentioned above, a computer search identified a G/C box sequence at the downstream of NRSE region. To verify the functionality of this motif in MOR transcription, we generated various MOR/luciferase reporter constructs containing pGL4.7NRSP (wild type), pGL4.7NRmSP (mutated NRSE and wild-type Sp-binding site), pGL4.7NRSPm (wild-type NRSE and mutated Sp-binding site), pGL4.7NRmSPm (mutated both in NRSE and Sp-binding site) (Figure 2A). All the constructs had been generated in more than 4.7 kb MOR promoter region containing distal and proximal elements and possibly other unidentified elements that might have roles in the promoter activity. Thus, the promoter assay in the current study may accurately reveal the role of newly identified GC box in the MOR gene regulation.\nThe constructs were transiently transfected in NS20Y and PC12 cells separately and the promoter activity was examined by measuring the signal of the reporter gene. As expected, the mutation in either NRSE or GC box sequence increased the expression of reporter gene in NS20Y cells (Figure 2B). Interestingly, when both elements were mutated at the same time, the promoter activity (14-fold in NS20Y cell) was repeatedly greater than the sum of activities from the constructs where each element was mutated singly (4-fold in Sp site mutation and 6-fold in NRSE mutation). Therefore, these data indicate that NRSE and Sp-binding site may cooperate to repress the MOR promoter activity. Meanwhile, the activities of the same reporter constructs in the NRSF negative PC12 cells (judged from the western blot in Figure 2C) had little effect (Figure 2B) on the promoter activity. These data suggest that a putative Sp-binding site acts as a synergic repressor with NRSE in NS20Y cells and NRSF and NRSE plays an important role to decide the function of the newly identified Sp-binding site as a repressive cis-element.\nIn addition, we also investigated whether the transcriptional repression activity of the putative Sp-binding site in the MOR promoter can be relieved in the NS20Y cells after mithramycin A treatment. NS20Y cells were transiently transfected either with pGL4.7NRSP or pNRSPm constructs (Figure 3A). Twenty-four hours after transfection, the cells were treated with either 100 or 400 nM mithramycin A. When the wild-type promoter sequence (pGL4.7NRSP) was transfected, a dose-dependent increase in the promoter activity was observed (Figure 3B). In contrast, when the putative Sp-binding site was mutated (pGL4.7NRSPm), the high promoter activity was detected even without treatment of mithramycin A strongly supporting the notion that this putative Sp-binding site downregulates expression of the MOR gene. Interestingly, at the highest concentration, mithramycin A started to decrease the promoter activity. At this condition, mithramycin A could interfere the binding of positive transcriptional regulators such as Sp1 within the promoter region.","divisions":[{"label":"Title","span":{"begin":0,"end":79}}],"tracks":[{"project":"WikiPainGoldStandard","denotations":[{"id":"T77","span":{"begin":45,"end":52},"obj":"NEGREGULATION"},{"id":"T78","span":{"begin":57,"end":67},"obj":"GENEXPRESSION"},{"id":"T79","span":{"begin":135,"end":146},"obj":"REGULATION"},{"id":"T80","span":{"begin":150,"end":160},"obj":"GENEXPRESSION"},{"id":"T81","span":{"begin":324,"end":339},"obj":"GENEXPRESSION"},{"id":"T82","span":{"begin":368,"end":386},"obj":"POSREGULATION"},{"id":"T83","span":{"begin":539,"end":545},"obj":"REGULATION"},{"id":"T84","span":{"begin":739,"end":752},"obj":"TRANSCRIPTION"},{"id":"T85","span":{"begin":3246,"end":3259},"obj":"NEGREGULATION"},{"id":"T86","span":{"begin":3260,"end":3270},"obj":"GENEXPRESSION"},{"id":"T87","span":{"begin":71,"end":79},"obj":"Protein entrezID:18390"},{"id":"T88","span":{"begin":116,"end":119},"obj":"Protein entrezID:20683"},{"id":"T89","span":{"begin":232,"end":241},"obj":"Protein entrezID:13429"},{"id":"T90","span":{"begin":243,"end":256},"obj":"Protein entrezID:20977"},{"id":"T91","span":{"begin":261,"end":300},"obj":"Protein entrezID:14809"},{"id":"T92","span":{"begin":341,"end":344},"obj":"Protein entrezID:20683"},{"id":"T93","span":{"begin":320,"end":323},"obj":"Protein entrezID:18390"},{"id":"T94","span":{"begin":523,"end":526},"obj":"Protein entrezID:20687"},{"id":"T95","span":{"begin":735,"end":738},"obj":"Protein entrezID:18390"},{"id":"T96","span":{"begin":3278,"end":3286},"obj":"Protein entrezID:18390"}],"relations":[{"id":"R83","pred":"REPORTED THEME","subj":"T77","obj":"T78"},{"id":"R84","pred":"REPORTED THEME","subj":"T78","obj":"T87"},{"id":"R85","pred":"REFERENCED THEME","subj":"T79","obj":"T80"},{"id":"R86","pred":"REFERENCED THEME","subj":"T79","obj":"T80"},{"id":"R87","pred":"REFERENCED THEME","subj":"T79","obj":"T80"},{"id":"R88","pred":"REFERENCED THEME","subj":"T80","obj":"T89"},{"id":"R89","pred":"REFERENCED THEME","subj":"T80","obj":"T90"},{"id":"R90","pred":"REFERENCED THEME","subj":"T80","obj":"T91"},{"id":"R91","pred":"REFERENCED THEME","subj":"T81","obj":"T93"},{"id":"R92","pred":"REFERENCED THEME","subj":"T81","obj":"T93"},{"id":"R93","pred":"REFERENCED THEME","subj":"T82","obj":"T81"},{"id":"R94","pred":"REFERENCED THEME","subj":"T83","obj":"T81"},{"id":"R95","pred":"REFERENCED THEME","subj":"T84","obj":"T95"},{"id":"R96","pred":"REPORTED THEME","subj":"T85","obj":"T86"},{"id":"R97","pred":"REPORTED THEME","subj":"T86","obj":"T96"},{"id":"R98","pred":"REFERENCED CAUSE","subj":"T88","obj":"T79"},{"id":"R99","pred":"REFERENCED CAUSE","subj":"T88","obj":"T79"},{"id":"R100","pred":"REFERENCED CAUSE","subj":"T88","obj":"T79"},{"id":"R101","pred":"REFERENCED CAUSE","subj":"T92","obj":"T82"},{"id":"R102","pred":"REFERENCED CAUSE","subj":"T94","obj":"T83"}],"attributes":[{"subj":"T77","pred":"source","obj":"WikiPainGoldStandard"},{"subj":"T78","pred":"source","obj":"WikiPainGoldStandard"},{"subj":"T79","pred":"source","obj":"WikiPainGoldStandard"},{"subj":"T80","pred":"source","obj":"WikiPainGoldStandard"},{"subj":"T81","pred":"source","obj":"WikiPainGoldStandard"},{"subj":"T82","pred":"source","obj":"WikiPainGoldStandard"},{"subj":"T83","pred":"source","obj":"WikiPainGoldStandard"},{"subj":"T84","pred":"source","obj":"WikiPainGoldStandard"},{"subj":"T85","pred":"source","obj":"WikiPainGoldStandard"},{"subj":"T86","pred":"source","obj":"WikiPainGoldStandard"},{"subj":"T87","pred":"source","obj":"WikiPainGoldStandard"},{"subj":"T88","pred":"source","obj":"WikiPainGoldStandard"},{"subj":"T89","pred":"source","obj":"WikiPainGoldStandard"},{"subj":"T90","pred":"source","obj":"WikiPainGoldStandard"},{"subj":"T91","pred":"source","obj":"WikiPainGoldStandard"},{"subj":"T92","pred":"source","obj":"WikiPainGoldStandard"},{"subj":"T93","pred":"source","obj":"WikiPainGoldStandard"},{"subj":"T94","pred":"source","obj":"WikiPainGoldStandard"},{"subj":"T95","pred":"source","obj":"WikiPainGoldStandard"},{"subj":"T96","pred":"source","obj":"WikiPainGoldStandard"}]},{"project":"2_test","denotations":[{"id":"17130167-11533047-76660569","span":{"begin":308,"end":310},"obj":"11533047"},{"id":"17130167-12492469-76660569","span":{"begin":308,"end":310},"obj":"12492469"},{"id":"17130167-12425938-76660569","span":{"begin":308,"end":310},"obj":"12425938"},{"id":"17130167-9765304-76660570","span":{"begin":583,"end":585},"obj":"9765304"}],"attributes":[{"subj":"17130167-11533047-76660569","pred":"source","obj":"2_test"},{"subj":"17130167-12492469-76660569","pred":"source","obj":"2_test"},{"subj":"17130167-12425938-76660569","pred":"source","obj":"2_test"},{"subj":"17130167-9765304-76660570","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"WikiPainGoldStandard","color":"#b193ec","default":true},{"id":"2_test","color":"#93ec97"}]}]}}