| Mouse embryonic stem (ES) cells are pluripotent cell lines isolated from the inner cell mass of blastulas which can proliferate unlimitedly and have ability of self-renewal. It is a hot spot for studying the molecular mechanisms of its developmental pluripotency. Tle4 gene is a member of Groucho/Tle gene family which includes five domains: the highly conserved glutamine (Q) rich, the WD-repeat domain, GP domain, CcN motif and SP domain. Mouse Tle4 gene is located in chromosome 19 that its full length is 234.45kb with 20 exons and 19 introns. It encodes 773 amino acid. The result from RNA interference library indicates Tle4 gene is a target gene that can maintain ES cells pluripotency. In the present study, we have systematically investigated the transcriptional regulation of Tle4 gene and its role in ES cells. The contents and results were summarized as followings:(1)At first, It has found that Tle4 protein contain seven conserved WD40 domains by bioinformatic analysis. At the same time, it was high expressed in tumor cells, immunity cells and ES cells, which indicates that it was related to cell differentiation and proliferation.(2)Characterization and identification of 5'-flanking region of Tle4 gene was analyzed by bioinformatic information. Seven overlapping genomic fragments from the 5'-flanking region of Tle4 gene were coloned into pGL3-basic vector to construct Tle4 promoter reporters. The transcriptional activities of these promoter fragments were tested in ES cells, F9 cells by dual luciferase assay. The results of software suggested that the -2137bp～-1794bp region (the first A nueleotide of the translation initiation codon ATG was numbered as+l) was the critical region, which contains many putative transcription factor binding sites and important element for transcriptional activation. In addition, the -2521bp～-2137bp region may contain negative regulatory elements.(3)Three different fragments of -2137bp～-1794bp region have been coloned into pGL3-basic vector for further analysis. The results suggested that transcriptional activity of -2027bp～-1927bp region was strongest and it was the core regulation region. Many transcription factor binding sites, such as Tcf1, Lef1, Sp1, et al, were found, which may be involved in the transcriptional control of Tle4 gene.(4) Analysis of site-directed mutations in Tle4 gene core regulation region. The site-directed mutations of Tcf1 fragment was coloned into pGL3-basic vector to detect. The transcriptional activity of mutation Tcf1-binding sites was repressed to some extent, but not significantly. This indicated that Tcf1 may play certain roles, but was not necessary.(5)Tle4 gene was expressed in blastulas, fetus, EB of 2d and 6d. It was not expressed in MEF.(6) According to the sequence of Tle4 gene, three pairs of siRNA fragments and one unspecific siRNA fragment were synthesized. These siRNA fragments were transfected with Lipofectamine 2000 into mouse ES cells. Total RNA was isolated from ES cells after 24 hours transfection by total RNA kit. The high efficient siRNA that significantly down-regulated Tle4 gene expression was selected by RT-PCR analysis. ES cells were transfected by Lipofectamine 2000 with the selected siRNA fragments. But the morphous, alkaline phosphatase activity, differentiation activity of ES cells were not changed and ES cells still maintained pluripotency after transfection.(7) The full-length mRNA sequence of Tle4 gene was cloned into vector for over-expression. ES cells were disintegrated after transfection with over-expression vector. The vector for subcelluar localization pEGFP-N1-Tle4 was also construed and Tle4 protein was localized to cell nucleus in ES cells.In summary, the study on transcriptional control and the role in ES cells of Tle4 gene can contribute to the understanding of the molecular function and mechanism of this gene. It has active theory and actual significance.
|
PDF Full Text Request