Study On The Role Of Negative Regularion Of Mouse Nanog Gene

Embryonic stem cells(ESCs), derived from the inner cell mass(ICM) of mammalian embryos, have the unique ability to grow indefinitely in culture while retaining their pluripotency. In terms of this differentiation potential into derivatives of all three embryonic germ layers, it is supposed to be utilized in regenerative medicine. However, the exact regulation mechanisms of pluripotency maintenance and differentiation of ESCs are still blur. For this reason, it is hard for clinical application of ESCs. Thus, it is of great significance to investigate the mechanism of ESC pluripotency maintenance and differentiation. Nanog is a homeodomain transcription factor, and important for sustaining ESC pluripotency and selfrenewal. To release the suppression factors on Nanog is helpful to enhance the efficiency of iPS cell production. Therefore, to uncover the negative regulation role of Nanog is helpful for iPS cell production. In this study, we detected the expression of genes by real-time PCR, Western blot, immunofluorescence stain, gene microarray, small RNA sequencing(RNA-seq). We detected the level of gene regulation by dual luciferase reporter assay and chromatin immunoprecipitation(ChIP) assay. We also used in vivo biotinylation system to investigate the interactome of Foxc1, and Co-IP assay was carried out to detect the relationship of two proteins on whether they could interact with each other. In this study, we investigated the negative regulation role of Nanog at transcriptional and post-transcriptional levels. The results of our study are the following:1. Nanog expression was repressed after the differentiation of F9 cells. We found that pluripotent factors such as Nanog were downregulated, and differentiation-associated transcription factors were upregulated after the differentiation of F9 cells treated with retinoic acid(RA). We also found the reduction activity of alkaline phosphatase in F9 cells after RA treatment. The expression levels of Foxc1 and Nanog were detected by real-time PCR in NIH/3T3, F9 and J1 ESCs. We found the higher level of Foxc1 in NIH/3T3 cells in contrast with the expression levels in F9 cells and J1 ESCs. However, the expression of Nanog was opposite to Foxc1 expression in NIH/3T3 cells.2. Foxc1 repressed Nanog gene expression. We first confirmed the proper expression of p3×Flag-Foxc1 vector by real-time PCR and Western blot, and we found that Nanog mRNA and protein expression level were repressed after overexpression of Foxc1 in F9 EC cells. The consistent results were also obtained after knockdown Foxc1 by siRNA against Foxc1. However, the expression of pluripotent factor Oct4 was not influenced by Foxc1. These results indicated that the repression of Naong by Foxcl was not due to the differentiation of F9 EC cells. We also found that Nanog promoter activity was repressed by Foxc1, which was detected by dual luciferase reporter assay. And the depletion of-3128/-2224 in Naong promoter could rescue the Nanog promoter activity by 1.5. These results implied there exist negative regulation element in-3128/-2224 region in Naong promoter. Chip assay confirmed that Foxc1 can bind to Nanog promoter, and influence Nanog expression.3. Identification interactome of Foxc1. We constructed HEK293 T cells stably expressing biotinylated Foxc1 and named as 293T-Biotin-Foxc1, and control cells 293T-Biotin. We isolated Foxc1 protein complexes by streptavidin-mediated affinity purification and identified them by SDS-PAGE-mass-spectrometer. We found that p53, Carm1, Ehmt2, Wdr77, Gata6, Hdac2, Nedd8 were the potential interacting proteins of Foxc1. p53, Gata6, Carm1 have been reported to be able to influence the expression of Nanog. Therefore, these results provided useful basis for studying Nanog expression regulated by Foxc1. Moreover, histone modification related proteins such as Ehmt2 and Hdac2 are also the potential interacting proteins. These results implying that epigenetic regulation might participate the regulation relationship between Foxc1 and Nanog.4. Foxc1 interacting protein Tet3 inhibited the expression of Nanog. Overexpression of Foxc1 promoted the expression of Tet3 in F9 cells. Knockdown Foxc1 by siRNA against Foxc1 could result in the reduction expression of Tet3. Moreover, overexpression of Tet3 could induce the expression of Foxc1, but repressed the expression of Nanog. Tet3 and Foxc1 could interact with each other as demonstrated by Co-IP assay. Therefore, Foxc1 could cooperate with its interacting protein Tet3 to regulate the expression of Nanog.5. Suppression of FGF-ERK signal pathway promoted the expression of Nanog. One of the key factors to establish the ground state of m ESCs is to repress FGF-ERK signal. In this study, we used gene microarray to detect the whole expression of mRNAs after suppression of FGF-ERK by PD0325901 in mouse J1 ESCs. We found that the addition of PD0325901 could influence the pluripotent factors and differentiation related genes. It also promoted the formation of typical ESC morphology as cultured in gelatin coated dishes. In addition, the expression of Nanog was changed from low to high expression level. Small RNA sequencing was used to detect the whole microRNA(miRNA) expression levels after the suppression of FGF-ERK signal pathway. We found that some upregulated miRNAs were helpful for the production of iPS cells. Meanwhile most of differential miRNAs were downregulated.6. FGF-ERK signal pathway related miRNAs regulated Naong expression, and promoted homogeneous ESCs. miR-296 repressed the expression of Nanog in J1 ESCs, while miR-296 was repressed by PD0325901.