The Studies On The Roles Of Foxm1Transcription Factor In Maintenance Of Stem Cells Pluripotency

Pluripotent stem cells are undifferentiated cells with indefinite replication potential(self-renewal)and the ability to differentiate into all types of cells and can form chimeric animals(differentiaton). Several transcription factors are required for maintenance of pluripotency, such as Oct4, Nanog, and Sox2, and the inactivation of these genes leads to loss of pluripotency and aberrant differentiation. Transcription factor Foxml belongs to the fork head/winged-helix family of transcription factors and plays important roles in cell proliferation, DNA damage repair, and in tumorigenesis. This study focuses on the function and molecular mechanisms of Foxml in maintenance of stem cell pluripotency:We have used the model of RA-induced P19cell neural differentiation to study on the relationship between expression levels of Foxml and pluripotency, to test how Foxml regulates the transcription of pluripotency related genes, and to confirm that Foxml is required for maintenance of stem cell pluripotency. The results of this study are summarized as following:1. The teratomas formed by P19cells contained tissues of all three germ layers including neural rosette (ectoderm), cuboidal epithelium (endoderm), and muscle (mesoderm), revealing that P19cells possess the pluripotency. We confirmed that RA-induced P19cell neural differentiation displayed at least three phases:the first phase in which P19cells lost their pluripotency(0-24h); the second phase in which certain percentage of P19cells committed to the neural differentiation and neural stem-like cells appeared(1-3d); and the terminal differentiation phase(6-7days following RA treatment) in w;hich differentiated neuronal cell populations appeared. Transcription factor Foxm1highly expressed in P19pluripotent cells and its expression decreased dramatically between day1to day2following RA treatment. The reduction of Foxml expression post RA treatment was correlated with the decreased expression of many of the known pluripotency-related genes, such as Oct4, Nanog, Sox2, and Utfl. Interestingly, the down-regulation of Foxml occurred before the decrease of Oct4expression during differentiation, implicating that Foxml might be one of the upstream controlling factors of Oct4expression in the maintainance of stem cell pluripotency.2. The inhibition of Foxm1expression by AdFoxm1siRNA resulted in the decreased activity of alkaline phosphatase in P19cells,and also abolished the expression of Oct4,Nanog, Sox2and Utfl in P19cells pluripotency of P19cells, implying its role in maintaining the pluripotency of stem cells. knockdown of Foxml results in lost of pluripotency of P19cells. The inhibition of Foxm1expression by AdFoxm1siRNA also abolished the expression of Oct4and Nanog in P19cells,implicating that the levels of Oct4and Nanog in pluripotent stem cells rely on the Foxm1expression. Taken together, our data reveal that Foxml highly expressed in the undifferentiated P19pluripotent cells and was downregulated by the differentiation process. Importantly, inhibition of its expression caused the lost of pluripotency of P19cells, implying its role in maintaining the pluripotency of stem cells.3.Knockdown of Foxml resulted in spontaneous differentiation to cardiomyocytes in vivo resulted in a rapid increase of Gata4expression. hematoxylin and eosin-stained sections from teratomas of AdFoxm1siRNA-infected P19cells, in which only mesodermal derivatives such as muscle and adipose connective tissues were observed, implying its role in maintaining the pluripotency of stem cells.4. Foxml blinds and stimulates Oct4promoter. the Oct4gene was one of the direct transcriptional targets of Foxml in pluripotent stem cells. found multiple tandem Foxml putative binding sites at the regions of-3434to-3415bp,-2895to-2880bp, and-1328to-1313bp in the Oct4promoter. Chromatin Immunoprecipitation (ChIP) assays were found obvious binding activity of Foxml on the Oct4promoter region around-3kb but not the-1.3kb region. These results confirmed that Foxml bound directly to endogenous Oct4promoter-3kb upstream region in pluripotent stem cells. Cotransfection of Foxml expression vector caused a significant increase in-3kb Oct4promoter activity.5. Maintained expression of FOXM1prevented the decrease of pluripotency genes such as Oct4, Nanog, Sox2, and endogenous Foxml during P19cell differentiation.The expression of exogenous FOXM1in the differentiated P19cells (RA/EB4day) re-started expression of Oct4, Nanog, Sox2, and endogenous Foxm1. ndicated a potential of Foxml to induce the expression of pluripotency-related genes in differentiated cells.6. The overexpression of FOXM1activated the expression of pluripotency-related genes in human embryonic fibroblasts (HEFs). HEFs were infected3times by AdFOXM1(100pfu/cell) at4days interval between each infection. We noticed that the multiple AdFOXM1infections caused dramatic morphology changes of HEFs, which formed cell aggregates and colonies afterwards, Moreover, we found that the colonies formed by AdFOXM1-infected HEFs possessed high levels of alkaline phosphatase activity, suggesting that these colonies started to have some characteristics similar to pluripotent stem cells. Next, the HEFs infected with AdFOXM1were analyzed for pluripotency markers by RT-PCR. We found that the overexpression of FOXM1re-activated expression of OCT4, NANOG, and SOX2in HEFs. suggested that FOXM1might be one of the candidates for reprogramming differentiated somatic cells to iPS cells.