Cloning And Identification Of Core Area Of Mouse TLE4 Gene Promoter

TLE4 gene is Fracture4(E(spl) Homolog,Drosophila)transduction protein-like Enhancer,which is a member of Grouchio Total inhibitor famaly.The full-length of TLE4 gene is 149.912kb,which is composed of 20 extrons and 19 introns The coding protein is a Transcriptional corepressor complex,which contains a Domain of SP and WD40.RT-PCR results showed that TLE4 gene was expressed in all tissues,with the highest expression in brain,expecially in caudate nucleus.Some researches showed that TLE4 gene is involved in differentiation of embryonic stem cell,playing a role in inhibiting transcriptional activation through coactivating with some other transcription factors.This inhibitory effect is dependent on PAX5 gene,CTNNB1 gene and TCF family members,which are associated with Wnt signaling pathway.The expression reglution of TLE4 gene plays an important role in cell fate decision of embryonic development.However,there is no report about transcriptional regulation of TLE4 gene.This research aims at mouse TLE4 gene promoter sequence.In this research,we cloned the 2849bp TLE4 upstream regulatory sequences, including the 5' UTR(untranslation region) and partial coding sequence.Feature of 5'-flanking region of TLE4 gene was analyzed by bioinformatics method.The potential transcriptional start site and transcriptional factor binding sites in the TLE4' regulatory region were predicted.The relative luciferase activities were determined by transfecting the mouse F9 teratoma cells and the mouse embryonic stem cells with several promoter fragments controlling luciferase reporter plasmids.The main results were listed as follow:1 The 2849 bp TLE4 gene regulatory sequences,including the 5'flanking sequence and partial coding sequence,were cloned and secquenced.Blast results indicated that the sequences were consistent with mouse TLE4 sequences in GenBank(No.AC000041).2 Mouse F9 teratoma cells and embryonic stem cells were cultured in vitro,which could grow normally in vitro,and passaged stablely.3 The expression of TLE4 gene was detected in mouse embryonic stem cells(ES) and mouse teratoma cells(F9) using RT-PCR(reversed transcription-PCR).The results showed that TLE4 Gene was expressed in both of the two types of cells.4 The features of TLE4 gene regulatory region were analyzed by bioinformatics.The transcription factor binding sites in TLE4' upsteam regulatory region(-2027bp～1927bp) were predicted.5 Seven promotor fragments with different length were obtained by walking deletion and then cloned into luciferase report gene expression vectors.The relative activities were determined by transfecting the mouse F9 teratoma cells and the mouse embryonic stem cells with the constructed dual-luciferase vectors.The promoter activity rusults in the two cell cultures indicated that negative regulation elements may exist in the promotor region from -2521bp to-2137bp,and further deletion analysis found that the region from -2137 bp to -1794bp in TLE4 promotor showed higher activity than other regions.In addition,a functional HSF regulation element was predicted in this region.6 Three different DNA fragments deletion luciferase vector were constructed further from promoter region(-2137bp～-1794bp) By transiently transfecting mouse embryonic stem cells(ES), a 100-nt-long(-2027bp～-1927bp) core promoter region was obtained,which was consistent with bioinformatics prediction results.