Alternative Splicing Directed Epigenetic Regulation in Brain Development

PROJECT SUMMARY/ABSTRACT Brain development requires intricate regulation of transcription, splicing, translation, and chromatin remodeling, but how these processes interconnect is not understood. We propose to elucidate how posttranscriptional alternative splicing regulates the epigenetic programs of brain development. During brain development, alternative splicing of a subunit of the chromatin remodeling BAF (BRG1/BRM-associated factor) complex, DPF2, switches from the canonical DPF2-Short (S) to a longer brain-specific DPF2-Long (L) isoform due to the inclusion of a highly conserved exon 7. This alternative splicing switch impacts transcriptional regulatory programs in embryonic stem cells (ESCs) and neural progenitor cells (NPCs), where DPF2-S and -L isoforms exhibit discrete binding preferences to chromatin regions marked by different histone modifications, selectively recruiting the BAF complex to specific genomic enhancers or promoters. Failure to switch from DPF2-S to -L during ESC differentiation into glutamatergic neurons promotes the proliferation of non-neuronal cells and reduces differentiation into neurons. However, how the brain-specific DPF2-L isoform functions in the epigenetic regulation of the brain remains unknown. We propose to determine how the splicing switch of DPF2 controls transcriptional and chromatin regulatory programs and neuronal maturation during brain development. By utilizing CRISPR/Cas9-mediated genome-edited ESC lines and mouse models expressing either DPF2-S or DPF2-L, we aim to determine the heterogeneity of different neuronal and non-neuronal lineages and the transcriptional regulatory programs driven by alternative DPF2 isoforms in in vitro glutamatergic differentiation cultures, as well as in mouse brains. Because DPF2 functions as part of the BAF complex, we propose to map genome-wide BAF complex recruitment and accessible open chromatin regions at different developmental time points from embryonic to adult brains. We recently discovered that DPF2-associated BAF complexes reorganize into smaller subcomplexes in older adult brains, and now propose to determine the subunit composition and modular organization of these subcomplexes and map their chromatin recruitment to understand their specific roles in regulating epigenetic programs associated with brain aging.