Abstract
Oct-3/4 is a key transcriptional factor whose expression level governs the fate of primitive inner cell mass and embryonic stem (ES) cells. Previously, an upstream 3.3-kb distal enhancer (DE) fragment was identified to be responsible for the specific expression of mouse Oct-3/4 in the inner cell mass and ES cells. However, little is known about the cis-elements and trans-factors required for DE activity. In this study, we identified a novel cis-element, called Site 2B here, located approximately 30 bp downstream from Site 2A, which was previously revealed in DE by an in vivo chemical modification experiment. Using the luciferase reporter assay, we demonstrated that both Site 2A and Site 2B are necessary and sufficient for activating DE in the contexts of both the native Oct-3/4 promoter and the heterologous thymidine kinase minimal promoter. In an electrophoretic mobility shift assay we showed that Site 2B specifically binds to Oct-3/4 and Sox2 when ES-derived cell extracts were used, whereas Site 2A binds to a factor(s) present in both ES and NIH 3T3 cells. Furthermore, we showed that the physiological level of Oct-3/4 in ES cells is required for Site 2B-mediated DE activity using the inducible knock-out system of Oct-3/4 in ES cells. These results indicate that Oct-3/4 is a member of the gene family regulated by Oct-3/4 and Sox2, as reported before for the FGF-4, UTF1, Sox2, and Fbx15 genes. Thus, Oct-3/4 and Sox2 comprise a regulatory complex that controls the expression of genes important for the maintenance of the primitive state, including themselves. This autoregulatory circuit of the Sox2.Oct-3/4 complex may contribute to maintaining robustly the precise expression level of Oct-3/4 in primitive cells.
Highlights
Totipotent cells are defined by their ability to produce both somatic and germ cells as well as extraembryonic tissues in mammals
In an electrophoretic mobility shift assay we showed that Site 2B binds to Oct-3/4 and Sox2 when embryonic stem (ES)-derived cell extracts were used, whereas Site 2A binds to a factor(s) present in both ES and NIH 3T3 cells
Together with the results obtained with mutants lacking Site 2A or Site 2B (Figs. 1 and 2), these results clearly demonstrated that both Sites 2A and 2B are necessary and sufficient for the specific activation of the native Oct-3/4 promoter and the heterologous minimal thymidine kinase (TK) promoter in ES cells
Summary
Totipotent cells are defined by their ability to produce both somatic and germ cells as well as extraembryonic tissues in mammals. Mouse Oct-3/4 is expressed during early development in cells that have totipotent or pluripotent differentiation ability (1, 6 –9). Oct-3/4 is expressed in undifferentiated embryonic stem (ES) and embryonal carcinoma (EC) cells and is down-regulated when these cells are induced to differentiate by retinoic acid (RA) treatment or the removal of leukemia inhibitory factor (2, 7). These remarkable expression patterns of Oct-3/4 during early mouse development and the undifferentiated cell lines suggest that Oct-3/4 plays an important role in maintaining the toti- or pluripotency of cells. POUS and POUH bind to the two halves of the consensus sequence, ATGC and AAAT, respectively (13)
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