The P19 Cellular Neural Induction Of Differentiation In Ngn1 Gene Expression Mechanisms Of Epigenetic Regulation

Neurogenesis is a complicated multiple steps event.In each step,certain specific genes are induced and others are repressed.It has been found that epigenetic regualtion is involved in regulating the expression of many genes during neurogenesis.Basic helix-loop-helix(bHLH) proteins are expressed in most stages in neural lineages during development and play crucial role in the determination of cell fates and differentiation. Neurogenin1(Ngn1) is an important member of bHLH proteins,which induces neurogenesis and prohibits differentiation of neural stem cells into astrocytes.Ngn1 is induced in neuronal precursor cells,but the mechanism of its regulation is still unclear.To investigate the regulation of ngn1 gene during neurogenesis,RA(all-trans retinoic acid)-induced P19 embryonal carcinoma cells were used as a neural differentiation model in vitro.In this study,epigenetic changes and mechanisms of histone modifications, SWI/SNF chromatin remodeling complex and other transcription related-factors in the regulation of Ngn1 gene were carried out.1.The establishment of RA-incduce neural differentiation in P19 cells and analysis of ngn1 expressionP19 cells were exposed to RA for 4 days and cultured in medium without RA for another 3 or 4 days to differentiate into neuron-like cells with the formation of neurite networks.Most differentiated P19 cells showed positive immunostaining for neuron specific classâ…¢-tubulin(TuJ1),and a dramatically increased protein level of TuJ1 in Western blot was observed.Quantitative RT-PCR assay showed that ngn1 mRNA greatly increased after 2 days of RA treatment.Simultaneously,RNA polymeraseâ…¡(RNA Polâ…¡) was enriched on the promoter of ngn1 analyzed by chromatin immunoprecipitation (ChIP) assay.These results suggested the status of ngn1 transcription was switched from repression to activation by RA induction.During the differentiation of P19 cells,changes of the expression of several epigenetic regulation factors were observed by RT-PCR and Western blot assays,suggesting epigenetic regulation might play key role in this process.2.RA treatment induced changes of histone modifications in ngn1 gene region in P19 cellsCovalent modifications of histones are key regulations of chromatin dynamics,and can effectively regulate genes expression.The N-terminal tail of core histones,as well as positions in the globular domain,can carry post-translational modifications such as acetylation,phosphorylation,ubiquitination,methylation,SUMOylation and ADP-ribosylation.Such modifications can alter DNA-histone interactions within and between nucleosomes and affect higher-order chromatin structures.ChIP assays were performed to investigate changes of histone modifications in ngn1 gene region during RA treatment.It was found that acetylation of histone H3K9 significantly increased,especially in the proximal promoter region and exon.region of ngn1 gene.Another key target of acetylation,H3K14 was also analyzed and showed limited increase of acetylation..In addition,treatment with a histone deacetylase(HDAC) inhibitor Trichostatin A(TSA) caused increased expression of ngn1,suggesting histone acetylation could promote the expression of ngn1.Histone acetyltransferases p300 and PCAF are coactivators and able to acetylate histones and other factors.It was observed that p300 bound on ngn1 promoter,with little change during RA treatment in P19 cells.But PCAF appeared on the promoter of ngn1only after 2 days of RA treatment,suggesting that PCAF might participate in the histone acetylation of ngn1 promoter.Methylation is another important modification of histones,which is considered as pivotal in epigenetic regulation.We analysed the methylation of histone H3K9,H3K27 and H3K4 in P19 cells during RA treatment by ChIP assays.It was found that dimethylation of H3K9 first declined and then increased,whereas,the trimethylation of H3K9 had no significant change.Trimethylation of H3K27 increased greatly after 1d of RA treatment,and then decreased gradually.Trimethylation of H3K4,as an activation mark,increased steadily,especially in the proximal promoter region and exon.region of the gene.These results suggested a significant change of histone methylation in ngn1 expression.3.The participation of SWI/SNF chromatin remodeling complex in the activation of ngn1ATP-dependent chromatin remodeling complex can hydrolyze ATP and restructure and mobilize nucleosomes,allowing regulated exposure of DNA in chromatin.The ATPase subunits of mammal SWI/SNF complex are Brg1 and Brm.The role of Brm in neural differentiation has been unclear.Brm was undetectable before RA treatment by Western blot,and its expression was induced by RA.Binding of Brm on ngn1 promoter was observed after 3 days of RA treatment by ChIP assay,suggesting chromatin remodeling complex SWI/SNF with Brm as the ATPase subunit was involved in activation of ngn1.4.The interaction of Sox6 with the promoter of ngn1 in RA induced p19 cellsSox6 is a member of SRY-related high-mobility-group box(Sox) proteins,and plays an important role in the development of the central nervous system(CNS).However,the mechanism of the regulation of Sox6 in the development of the CNS is still unclear.The expression of Sox6 increased when treated with RA,and then decreased during the neuronal differentiation.By bioinformatic method,several predicted binding sites of Sox proteins were found on the promoter of ngn1.We showed that Sox6 could bind to 2 HMG consensus sites on ngn1 promoter with EMSA and ChIP assay,and the data from coIP asssay suggested an interaction between Sox6 and PCAF.Our results suggested that Sox6 directly binding to the promoter and activate the expression of ngn1 during the neural differentiation of P19 cells.In summary,results on the changes of acetylation and methylation of core histones surrounding the promoter region of ngn1 gene enriched our knowledge on the regulatory mechanism of the gene during neural differentiation,and provided new information on 'histone code'.The induced expression of Brm and its binding on ngn1 promoter are novel clues for an important role of Brm in neural differentiation.We demonstrated for the first time that Sox6 directly binds the promoter of ngn1,which provided a novel insight into the function of Sox6 in neuronal differentiation.These findings are helpful for us to understand details of epigenetic regulations in neural development,and may provide novel clue for future therapy in neurological diseases.