Elucidating The Regulatory Role Of 3d Genome Folding During Neural Differentiation And Synaptic Activation

The causal link between the three-dimensional conformation of the genome and spatiotemporal control of gene regulation has long been studied in the form of enhancer-promoter interactions. Only recently have advances in molecular biology and next generation sequencing allowed higher-order chromatin folding to be queried genome-wide at ultra-high-resolution. In this thesis we leverage Chromosome Conformation Capture Carbon Copy (5C) along with RNA-seq and ChIP-seq to elucidate how the genome is reconfigured during neural development, cellular reprogramming, and synaptic activation. We observe that the first step in neural differentiation is accompanied by a bulk decommissioning of nearly half of the architectural protein CTCF’s binding sites in the pluripotent genome, a trend which continues throughout terminal neuronal differentiation and results in the dissolution of many chromatin loops present in embryonic stem cells (ESCs). We identify another zinc finger protein, Yin Yang 1 (YY1), at the base of looping interactions between neural progenitor cell (NPC) specific genes and enhancers; siRNA knockdown of YY1 specifically disrupts interactions between key NPC enhancers and their target genes. Additionally, we find that many of the CTCF sites that are decommissioned during neural lineage commitment are not efficiently restored during cellular reprogramming of NPCs to induced pluripotent stem cells (iPSCs). CTCF sites that do not successfully regain binding in iPSCs underlie incompletely reprogrammed chromatin architecture, resulting in an iPSC genome folding and transcriptional signature that resembles an intermediate state between ESCs and NPCs. Culture in 2i media conditions restores the CTCF binding, genome folding, and gene expression of iPSCs to patterns resembling those of ESCs. Finally, we find that a large subset of chromatin loops surrounding select neuronal activity response genes (ARGs) are induced de novo during cortical neuron activation. We observe a striking correlation between the number, length, and kinetics of loops an ARG forms and how much time that ARG takes to be upregulated in response to neuronal activity. Additionally, we find that common single nucleotide variants (SNVs) associated with Autism Spectrum Disorder connect activity-inducible enhancers to upregulated genes, whereas Schizophrenia SNVs anchor pre-existing loops connecting activity-decommissioned enhancers to activity-downregulated genes. Altogether this work begins to elucidate how the 3-D genome orchestrates cellular state and function decisions during mammalian brain development from the earliest neural lineage commitment through the refinement of connections between terminally differentiated neurons.