Epigenetic regulation and transcription factor programming enhances neurogenesis in neural stem cells.

摘要

In this thesis, we questioned how neuronal and glial phenotypes become specialized. Epigenetic chromatin modifiers and transcription factors were investigated for their roles in programming and maintaining neural lineage restriction. A relatively homogeneous population of cells was generated by deriving immortalizing neural clones from embryonic rat forebrains. Three phenotypes; neuronal, glial and multipotential (GE6, GE2, CTX8), provided contrasting lineages to probe the factors responsible for shaping cell fate. One particular clone, GE6, differentiated into a functional inhibitory like interneuron. Gene expression analysis showed several genes such as Ascl1, Dlx1, Dlx5, may be responsible for the interneuronal specificity. Epigenetic regulation through histone modifications is believed to be an essential component within the developing nervous system, ultimately affecting cell fate. Testing chromatin signatures on specific neural genes with permissive and repressive histone "marks" shows that chromatin state in undifferentiated precursors correlates with current and predicts downstream gene expression. These results suggest that cell fate may already be predetermined. Furthermore, ChIP sequencing reveals global differences between the representative clones. Extrinsic growth factors, such as BMP2 promotes the neuronal and glial phenotypes in the multipotential cell CTX8. BMP2 asserts its phenotypic response in part by regulating global acetylation enrichment in specific neural gene networks, providing a mechanism to promote and maintain cell fate. Directly altering chromatin marks using a histone deacetylase inhibitor, valproic acid (VPA), globally acetylates the chromatin of CTX8 cells and enhances neurogenesis. VPA treatment was also confirmed to maintain and increase acetylation in specific neuronal genes, such as Ascl1. In addition, several microRNAs thought to play a role in neurogenesis were epigenetically regulated after VPA treatment. Finally, through the combination of gene expression and epigenetic analyses, direct programming through exogenous expression of Ascl1, Dlx1 and Dlx5 enhanced neurogenesis in CTX8. Gene expression and epigenetic signature mapping provides us with a deeper understanding of how lineage restriction occurs. Learning the programming rules will assist in directing homogeneous populations of neuronal cells to further probe the mechanisms of neurodegenerative diseases.