Abstract
Oct4 plays a critical role both in maintaining pluripotency and the cell fate decision of embryonic stem (ES) cells. Nonetheless, in the determination of the neuroectoderm (NE) from ES cells, the detailed regulation mechanism of the Oct4 gene expression is poorly understood. Here, we report that crosstalk between Oct4 and Meis1a, a Pbx-related homeobox protein, is required for neural differentiation of mouse P19 embryonic carcinoma (EC) cells induced by retinoic acid (RA). During neural differentiation, Oct4 expression was transiently enhanced during 6–12 h of RA addition and subsequently disappeared within 48 h. Coinciding with up-regulation of Oct4 expression, the induction of Meis1a expression was initiated and reached a plateau at 48 h, suggesting that transiently induced Oct4 activates Meis1a expression and the up-regulated Meis1a then suppresses Oct4 expression. Chromatin immunoprecipitation (ChIP) and luciferase reporter analysis showed that Oct4 enhanced Meis1a expression via direct binding to the Meis1 promoter accompanying histone H3 acetylation and appearance of 5-hydoxymethylcytosine (5hmC), while Meis1a suppressed Oct4 expression via direct association with the Oct4 promoter together with histone deacetylase 1 (HDAC1). Furthermore, ectopic Meis1a expression promoted neural differentiation via formation of large neurospheres that expressed Nestin, GLAST, BLBP and Sox1 as neural stem cell (NSC)/neural progenitor markers, whereas its down-regulation generated small neurospheres and repressed neural differentiation. Thus, these results imply that crosstalk between Oct4 and Meis1a on mutual gene expressions is essential for the determination of NE from EC cells.
Highlights
Cell fate decisions are fundamental for development, but we do not know how core pluripotency circuit genes, including Oct4, Sox2, Nanog, Klf4/5, and Tbx3, reorganize transition from a pluripotent to a differentiated cell state [1]
We previously reported that the neural cell fate decision in P19 cells is carried out during 2 days of retinoic acid (RA) addition, suggesting that Meis1a, but not Meis1b, is deeply involved in neural differentiation
In this study, based on the analysis of Oct4 and Meis1 promoter activities, we found the possibility that up-regulated Oct4 during 12 h of the immediate-early stages of RA-primed P19 embryonic carcinoma (EC) cell neural differentiation dose-dependently stimulates Meis1a gene expression accompanying acetylated histone H3 (AcH3) and appearance of 5hmC (Fig. 3), while the Oct4-induced Meis1a suppresses Oct4 expression via direct binding to the distal Meis1a-BEs3/4 of the Oct4 promoter together with histone deacetylase 1 (HDAC1) (Fig. 4)
Summary
Cell fate decisions are fundamental for development, but we do not know how core pluripotency circuit genes, including Oct, Sox, Nanog, Klf4/5, and Tbx, reorganize transition from a pluripotent to a differentiated cell state [1]. It has been demonstrated that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts are achieved by introducing four factors, Oct, Sox, Klf, and c-Myc [5,6]. Kim et al reported that Oct is sufficient to generate iPS cells from adult mouse neural stem cells [7]. Critical expression levels of Oct mRNA in ES and embryonic carcinoma (EC) cell lines such as P19 and F9 cells are rapidly down-regulated by differentiation induced with retinoic acid (RA) [8,9]. The expression level of Oct is crucial for the maintenance of pluripotency and for early cell differentiation decisions [10]
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