Ezh2 In Ra Induced Neuronal Differentiation In P19 Cells

In the mammalian development, the development of nervous system is a very complex process. Neural development includes a cascade of genetic programs that precisely control stage-specific gene activities required for neural patterning, cell migration, and neural connectivity. Proper regulation of stage-secific gene expression in the nervous system is not only controlled by the classical genetic pathway, but is also subject to modulation by epigenetic mechanisms such as DNA and protein methylation, histone modification, nucleosome, and chromatin remodeling. Epigenetic regulation plays important roles in the maintenance of the identity and the determination of the differentiation.Neurogeninl is a kind of BHLH protein and it is induced to express in the neuronal precursors. In the neural development, Ngnl induces the neurogenesis and prohibits differentiation of neural stem cells into astrocytes.Covalent modifications of histone are key regulations of chromatin dynamics, and can effectively regulate genes expression. The posttranslational modifications include methylation, acetylation, and so on. Such modifications can alter DNA-histone interactions within and between nucleosomes and affect higher order chromatin structures. Several sites that can be methylated exits in the N-terminal of histone H3 H3(K4, K9, K27, K36) and H4 (K20). Trimethylation of H3K27 is catalysed by Ezh2In Drosophila and mammalian cells, H3K27me3 correlates with transcription repression. Ezh2 is important in the renewal and maintenance of the stem cells.Based on the laboratory's foundation, we continued to investigate the regulation of Ngnl gene during neurogenesis, especially the mechanism of the epigenetic regulation, such as histone modification.1,The establishment of all-trans RA-induced neural differentiation in P19 cells and analysis of Ngnl expressionWe cultured P19 cell in medium with RA for 4 days and then in medium without RA for another 4 days. In the first 4 days, the cells began to form aggregates and after RA is removed, P19 began to differentiate and formed neurite networks. Western blot against TuJ1 showed that TuJ1 increased gradually, illustrated the successful induction of neural differentiation. Simultaneously, western blot against Ngnl showed that Ngnl appeared after 1 days of RA induction,and its expression increased as the induction continued.2,Expression of Ngnl, and the analysis of its promoterExpression of Ngn1 increased after RA induction, and binding sites for RA receptor existes in the promoter.We speculate the expression of Ngnl can be regulated by the RA receptor directly.3,RA treatment induced recruitment of the histone modification of H3K27 and Ezh2 on the Ngnl geneChIP assay was performed to investigate the changes of H3K27me3 in Ngnl gene region. It was found that trimethylation of H3K27 increased greatly after RA treatment and then decreased. Then ChIP assay was performed to investigate the combination of Ezh2 to Ngnlgene region. It was found the combination of Ezh2 increased after RA induction, and then decreased.Our results suggest that Ezh2 is involved in the induction of P19 differentiation.4,The mutation of Ser21 disturbs the recruitment of Ezh2 and NgnlAkt can interact with Ezh2, and phosphate Ser21. Ser21's phosphorylation decreased its affinity with histone H3. We transfected the expression plasmid of Ezh2 into P19 cell. It was found that the recruitment of Ezh2 to Ngnl increased first and the decreased after induction.The variation was the same as the recruitment of Ezh2 with Ngn1 in vivo. Then we mutated Ser21 to asp to imitate its phosphorylation.We transfected the mutated expression vector S21D-Ezh2 to P19, and it was found the recruitment of Ezh2 with Ngnl was destroyed seriously.5,Ezh2 plays an important role in the maintenance of P19 cell pluripotencyWestern blot analysis showed that expression of Ezh2 was high in the uninduced P19 cell, but decreased greatly in the fourth day of induction.These results showed in the neural development Ezh2 and H3K27me3 fine regulate Ngnl to express at the right time and special cell. And we also propose PI3K/Akt is involved in the regulation of Ngnl.