P19 Cell Neural Differentiation Model NSPc1 Of Target Genes Identified

NSPc1is a Polycomb Group protein highly expressed in neural system, and highly homologous to the well-known stem cell maintenance factor Bmi1. NSPc1can endow embryonic stem cell (ESC) with partial capacity to self-renew independent of LIF (Leukemic inhibitory factor); our previous study showed that its expression was much higher in neural stem cells (NSC) than in terminally differentiated neurons; knockout of NSPc1decreased the proliferation of the induced neural differentiation processes of the P19and NT2cell lines, and meanwhile enhanced their differentiation tendency. NSPc1was seen as a new stem cell maintenance factor, especially in nervous system. However, we do not know much about its target gene profiles so far.In our study, we firstly established the P19cell neural differentiation model under induction of1μM retinoid acid, which is a mature model for in vitro study of neural stem cell differentiation. Western Blot was used to test the expression change of NSPcl protein in this model. Result showed that in the P19neural differentiation model, the NSPc1protein expression in Day6(neuron) was significantly lower than that in Day0(undifferentiated stem cell).Next, ChIP-on-chip was introduced to screen for NSPc1target gene spectrum in P19cells, and the functional features of them was analyzed with bio-informatics. We found out that the1280NSPcl target genes screened out were clustered in "cellular, metabolic, developmental" biological processes, gathered in the molecular functional groups of "binding and transcriptional regulator", and located mainly in "cell parts or organelles". Moreover, Gene Ontology terms associated with "differentiation, development, transcription and its regulation, neurogenesis" were of significantly higher frequency with NSPc1target genes than their random frequency across the entire genome.Lastly, after comprehensive consideration of the ChIP-chip result, previous expression array analysis result, and literature reports on the possible functions of the genes, we chose7candidate target genes from the whole target gene pool. Real-time PCR was used to detect the mRNA level of NSPc1and these target genes during the differentiation process. The7target genes are:Cdk9, Hmgb2, Hnrnpl, Rgmb, Rnf10, Tnk2and Zfp148. According to our Real-time PCR result, the NSPc1mRNA level was lowered in Day6, while re-elevated above original level in Day10, i.e. it was downregulated during the "neural stem cell-neuronal precursor cell-inmature neuron" process, while upregulated again in the process of neuronal maturation. The candidate target genes tested got different results:Cdk9and Tnk2levels decrease from Day0to6, while Hnrnpl and Rnf10increase.Therefore, we concluded that NSPcl’s own expression in the P19neural differentiation model is dynamically changing, and so are its regulations of the large amount of neural proliferation, differentiation and maturation related target genes. From the relative expression changing of NSPc1and the target genes tested, we can see that the regulative mechanism can be very complicated, and may involve lots of multilaminal and networking processes.