ABSTRACT
Transcription factor FOXM1 plays a critical role in maintenance of stem cell pluripotency through stimulating the transcription of pluripotency-related genes in mouse pluripotent stem cells. In this study, we have found that the repression of FOXM1 expression is mediated by FOXM1 3'UTR during retinoic acid-induced differentiation of human pluripotent NT2/D1 embryonal carcinoma cells. FOXM1 3'UTR contains a microRNA response element (MRE) for miR-134, which has been shown to attenuate the expression of pluripotency-related genes post-transcriptionally during mouse embryonic stem cell differentiation. We have determined that miR-134 is induced during RA-induced differentiation of NT2/D1 cells and the overexpression of miR-134 represses the expression of FOXM1 protein but not FOXM1 mRNA. Furthermore, the expression of OCT4 is diminished by FOXM1 knockdown and the OCT4 promoter is regulated directly by FOXM1, suggesting that FOXM1 is required for maintaining the expression of OCT4 in NT2/D1 cells. Together, our results suggest that FOXM1 is essential for human pluripotent stem cells and miR-134 attenuates its expression during differentiation.
Subject(s)
Carcinoma, Embryonal/pathology , Embryonal Carcinoma Stem Cells/cytology , Forkhead Transcription Factors/genetics , MicroRNAs/genetics , Octamer Transcription Factor-3/genetics , Pluripotent Stem Cells/cytology , Animals , Antineoplastic Agents/pharmacology , Carcinoma, Embryonal/genetics , Cell Differentiation , Cell Line, Tumor , Forkhead Box Protein M1 , Forkhead Transcription Factors/biosynthesis , HEK293 Cells , Humans , Mice , Octamer Transcription Factor-3/biosynthesis , Promoter Regions, Genetic/genetics , RNA Interference , RNA, Messenger/biosynthesis , RNA, Small Interfering , Response Elements/genetics , Tretinoin/pharmacologyABSTRACT
FoxA1 belongs to the fork head/winged-helix transcription factor family and participates in stimulating neuronal differentiation of pluripotent stem cells at early stages. To explore the biological roles of FoxA1 during this process, the stable expression of a GFP-FoxA1 fusion protein was established in P19 pluripotent embryonal carcinoma cells. Although they still express pluripotency-related transcription factors such as Oct4, Nanog, and Sox2, the generated P19 GFPFoxA1 cells exhibited a decreased activity of alkaline phosphatase and an increased expression of SSEA-3 compared with P19 cells. Elevated levels of nestin expression and prominin-1+ populations were observed in P19 GFPFoxA1 cells, implicating that the stable expression of FoxA1 promoted P19 cells to gain partial characteristics of neural stem cells. Furthermore, the promoter of nestin was confirmed to be bound and activated by FoxA1 directly. The expression of neuron-specific marker tubulin betaIII also existed in P19 GFPFoxA1 cells. P19 GFPFoxA1 cells showed an earlier onset of differentiation during RA-induced neuronal differentiation, evidenced by a more rapid change on the Nanog decrease and the tubulin betaIII increase. Thus, overexpression of FoxA1 alone may promote pluripotent P19 cells to become neural stem-like cells.
Subject(s)
Embryonic Stem Cells/metabolism , Gene Expression Regulation, Developmental , Hepatocyte Nuclear Factor 3-alpha , Neoplastic Stem Cells/metabolism , Neural Stem Cells/metabolism , Pluripotent Stem Cells/metabolism , AC133 Antigen , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Antigens, Tumor-Associated, Carbohydrate/genetics , Antigens, Tumor-Associated, Carbohydrate/metabolism , Biomarkers/metabolism , Cell Differentiation/drug effects , Cell Differentiation/genetics , Embryonic Stem Cells/cytology , Embryonic Stem Cells/drug effects , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Glycoproteins/genetics , Glycoproteins/metabolism , Hepatocyte Nuclear Factor 3-alpha/genetics , Hepatocyte Nuclear Factor 3-alpha/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Intermediate Filament Proteins/genetics , Intermediate Filament Proteins/metabolism , Mice , Nanog Homeobox Protein , Neoplastic Stem Cells/cytology , Neoplastic Stem Cells/drug effects , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nestin , Neural Stem Cells/cytology , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Peptides/genetics , Peptides/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/drug effects , Promoter Regions, Genetic , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism , Stage-Specific Embryonic Antigens/genetics , Stage-Specific Embryonic Antigens/metabolism , Tretinoin/pharmacology , Tubulin/genetics , Tubulin/metabolismABSTRACT
Transcription factor Foxm1 plays a critical role during embryonic development and its expression is repressed during retinoic acid (RA)-induced differentiation of pluripotent P19 embryonal carcinoma cells at the early stage, correlated with downregulation of expression of pluripotency markers. To study whether Foxm1 participates in the maintenance of pluripotency of stem cells, we knock down Foxm1 expression in P19 cells and identify that Oct4 are regulated directly by Foxm1. Knockdown of Foxm1 also results in spontaneous differentiation of P19 cells to mesodermal derivatives, such as muscle and adipose tissues. Maintaining Foxm1 expression prevents the downregulation of pluripotency-related transcription factors such as Oct4 and Nanog during P19 cell differentiation. Furthermore, overexpression of FOXM1 alone in RA-differentiated P19 cells (4 days) or human newborn fibroblasts restarts the expression of pluripotent genes Oct4, Nanog and Sox2. Together, our results suggest a critical involvement of Foxm1 in maintenance of stem cell pluripotency.
