UBC Theses and Dissertations
Identification and characterization of active enhancers in the developing cerebellum Ramirez, Castel Miguel
Brain development is a complex process requiring precise coordination of molecular processes across a multitude of cell-types. Underpinning these events are gene expression programs which require intricate regulation by regulatory DNA sequences known as enhancers. Transcription factors bind to enhancers in a temporal and cell-type specific manner resulting in the activation of target gene expression. These sequences are enriched for variants associated with neurodevelopmental and psychiatric disorders, highlighting their importance in development. However, a detailed understanding of the enhancers that govern changes in gene expression during embryonic and early postnatal cerebellum development remains elusive. In this dissertation, we take another step toward interpreting the genetic variation found within these sequences by defining molecular pathways under enhancer regulation in the developing cerebellum. As a comprehensive overview of the genetics of mouse and human cerebellar development and to establish a catalogue of cerebellar genes that can be targeted by enhancer elements, we constructed the Cerebellar Gene Database. Active enhancers in the mouse cerebellum at embryonic and postnatal ages were then identified by conducting chromatin immunoprecipitation followed by sequencing for enhancer associated histone marks H3K4me1 and H3K27ac and Cap Analysis of Gene Expression followed by sequencing. We found that the majority of cerebellar enhancers have dynamic activity between embryonic and postnatal development. Cerebellar enhancers with temporally specific activity were enriched for neural transcription factor motifs, many of which are understudied in the context of cerebellar development. Putative gene targets displayed spatially restricted expression patterns and regulate a multitude of processes spanning several developmental epochs such as specification, differentiation and maturation. To validate these findings, activity was profiled for enhancers predicted to regulate Nfib, a gene critical for granule cell development, using in situ hybridization of transcribed enhancer RNAs. We used these analyses to discover two novel regulators of cerebellar development: Pax3, a marker of GABAergic progenitors and interneuron precursors and Bhlhe22, a regulator of postnatal granule cell migration. This dissertation provides insight into the dynamics of gene expression regulation by enhancers in the developing brain and delivers a rich resource from which novel genes can be discovered.
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