The Function Research On Cell Division Cycle Associated 8 (CDCA8) Promoter

Chromosome passenger complex (CPC) is an important dynamic structure during cell division. It plays an important role in ranking of the metaphase chromosomes, separation of the sister chromatid and cytokinesis. Human cell division cycle associated 8 (CDCA8) encodes a protein, termed Borealin/Dasra B, which is one of the four components of CPC. Functional studies revealed that Borealin was required for targeting of the CPC to centromeres, correction of kinetochore attachment errors and stability of the bipolar spindle in human cells.Expression analysis demonstrated that CDCA8 expressed highly in human cancer cells and undifferentiated embryonic stem cells (hESCs), compared with normal cells. In order to resolve the regulatory mechanism of differential expression and in-depth understand the gene function, CDCA8 promoter and its associated transcription factors were studied.In preliminary studies, Dr. Miao Congxiu Cloned and functionally analyzed the CDCA8 promoter in vitro:by 5'-RACE, three transcription start sites of CDCA8 were found at 200 bp,194 bp and 175 bp upstreame of translation start site, one of which is alternative splicing; the location of the promoter was defined by transient transfecting the Hela cells with reporter gene driven by the 5'-Truncated promoter fragments. Transient expression assays in different types of cells revealed that CDCA8 promoter is significantly activated in undifferentiated hESCs and cancer cell lines but repressed in differentiated hESCs and normal primary cells. This suggested the regulation of differential expression of CDCA8 gene was mainly on transcriptional level.In this study, to confirm the promoter activity in vivo, "promoter-EGFP" fragment was microinjected into mouse zygotes and expression of EGFP directed by the promoter was observed in developing embryos; "promoter-Luciferase" transgenic mice were established by microinjection and were identified by PCR and Southern Blot. In vivo imaging, multi-organization Luciferase assay, immunofluorescence and immunohistochemistry were used to test the ability of the promoter to drive gene expression in each issue.In order to explore the regulatory mechanism and possible reasons for the differential promoter activity, here, bisulfite sequencing PCR (BSP) was used firstly to compare the methylation status of basic promoter between undifferentiated and differentiated hESCs. Subsequent analysis was conducted on the level of transcription factors (TFs):First, the potential binding sites for known TFs on CDCA8 promoter were predicted by computer analysis; then the TFs binding to the promoter in hESCs, NTERA-2 and hEF cells were compared using Pull-down and Protein/DNA arrays, and confirmed with EMSA and Western Blot; site-specific mutagenesis and over-expression assay were used to test the effect of TFs on promoter activity.The results are as follows:Expression of EGFP directed by CDCA8 promoter was observed in 2-cell,4-cell and morula of early developing mouse embryos. Both in vivo imaging and multi-organization luciferase assay showed that strong luciferase expression was detected in the testes of adult transgenic mice. Immunohistochemistry and immunofluorescence staining revealed that luciferase expression was restricted to the subpopulation of spermatogonia, adjacent to the basement membrane of seminiferous tubules. The expression was disappeared along with the cell differentiation/spermiogenesis toward the lumen, which agreed with the promoter activity in vitro. BSP showed there was no change in methylation of CpG islands in the basic promoter during differentiation of hESCs. The TFs, whose binding amount on promoter had more than two times changes in hESCs and NTERA-2, compared with hEF, were found by Pull-down and Protein/DNA arrays. Among them, the changes of NFY were further confirmed by EMSA and Western Blot. Moreover, the site-specific mutagenesis and over-expression assay showed the binding of NFY was very important to the promoter activity. Therefore, the changes of binding amount of NFY could considerably responsible for the differential promoter activity in different cell types.Overall, the main characterization of CDCA8 promoter was defined in this study, including promoter location, promoter activity in vitro and in vivo, and TFs involved. These findings are of great significance and establish the basis for further research on transcription regulatory and function of CDCA8 gene.