| Summary: | 博士 === 國防醫學院 === 生命科學研究所 === 99 === Neurons and astrocytes are generated from common neural progenitors, yet neurogenesis precedes astrocyte formation during embryogenesis. The mechanisms of neural development underlying suppression and de-suppression of differentiation- related genes for cell fate specifications are not well understood.
By using an in vitro system in which NTera-2 cells were induced to differentiate into an astrocyte lineage, we demonstrated that astrocyte-specific markers, glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS), were expressed. In addition, olig2 was exported from nucleus to cytoplasm which induced phosphorylation of STAT3 and the accumulation of STAT3-p300 complex in the nucleus.
In contrast to previous studies, upon induction of cell fate specification toward an astrocytic lineage, the CpG islands at GFAP promoter and coding region of GFAP were not demethylated, based on bisulfite sequencing and DNA methylation immunoprecipitation assay. Moreover, profiling of phosphoproteins using a newly developed label-free LC-MS/MS quantitation technology was performed to facilitate the identification of specific regulators involved in astrocyte differentiation. Of the proteins that showed temporal changes in phosphorylation state upon induction of differentiation, it was found that many were found to have functions in cell development, differentiation and transcriptional regulation. Furthermore, we revealed a novel role of Sin3A in maintaining the suppression of GFAP in NTera-2 cells. Sin3A coupled with MeCP2 binds to the GFAP promoter, and their level of occupancy correlates with repression of GFAP transcription. The repression by Sin3A and MeCP2 may be an essential mechanism underlying the inhibition of cell differentiation. Upon commitment toward an astrocyte lineage, Sin3A-MeCP2 departs from the promoter, and activated STAT3 binds to the promoter and exon 1 of GFAP simultaneously; meanwhile, olig2 is exported from nuclei to the cytoplasm. This suggests that a three-dimensional or higher-order structure is induced by STAT3 binding between the promoter and proximal coding regions. STAT3 then recruits CBP/p300 to exon 1 and targets the promoter for histone H3K9 and H3K14 acetylation. CBP/p300-mediated histone modification further facilitates chromatin remodeling, thereby enhancing H3K4 trimethylation and subsequent recruitment of RNA polymerase II to activate GFAP gene transcription. These results provide evidence that exchange of repressor for activator complexes and epigenetic modifications are both critical for cellular differentiation and lineage-specific gene expression.
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