Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
5907635 | Gene | 2008 | 9 Pages |
Abstract
Early ocular development is controlled by a complex network of transcription factors, cell cycle regulators, and diffusible signalling molecules. Together, these molecules regulate cell proliferation and apoptosis, and specify retinal fate. NKX5-3 is a homeobox transcription factor implicated in eye development. The analysis of the 5'-flanking region of the mouse Nkx5-3 gene revealed a predicted TATA-less promoter sequence between â 416 and â 166 of the translation start site. To functionally characterise Nkx5-3 promoter activity, serial deletions of the promoter sequence were introduced in pGL-3 basic vector and promoter activity of these 5'- and 3'-deleted constructions was tested in HeLa and CHO cells. Transactivation assays identified a region between â 350 and â 296 exhibiting promoter-like activity. Combined analysis by deletions and point mutations showed that this sequence, containing multiple Sp1 binding sites was necessary to promote transcriptional activity. Binding of Sp1 to this region was confirmed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation, using an antibody specific for Sp1. Altogether, these results demonstrated that the immediate upstream region of Nkx5-3 gene possessed a strong intrinsic promoter activity in vitro, suggesting a potential role in Nkx5-3 transcription in vivo.
Keywords
PBSMUTAP-2ChrSV40Activator protein 2TBSTFBSDTToligodeoxyribonucleotideTSSTransactivation assayDMEMcDNADNA complementary to RNADulbecco's modified Eagle's mediumSp1Electrophoretic mobility shift assayEDTAESTethylenediamine tetraacetic acidoligoTris-buffered salineExpressed Sequence Tagbase pairmutateddithiothreitoltranscription start sitetranscription factor binding siteEMSA یا electrophoretic mobility shift assay Sp1 transcription factorPhosphate-buffered salineSpecificity protein 1UTR یا untranslated regions untranslated regionwild typeNucleotidepolymerase chain reactionPCRSimian virus 40TATA-less promoterCHiPChromosome
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Authors
Olivia Nichini, Daniel F. Schorderet,