The Epigenome is not a static entity, but is dramatically altered in response to environmental and developmental cues throughout the lifetime of an organism. In the case of the developing central nervous system, multipotent neural progenitor cells (NPCs) undergo marked reconfiguration of epigenetic marks as they exit the cell cycle and differentiate into the diverse neural and glial phenotypes that make up the mammalian brain. The dynamic nature of the Epigenome is poorly understood, particularly at short time scales, due to severe limitations in heterogeneity and asynchrony of the large cell populations typically required for genomics assays. Here we propose to overcome these technical challenges by building powerful new optical tools for the precise control of transcription dynamics in the context of the three-dimensional nucleus. Specifically, we aim to build modular, light-inducible architectural proteins for precise spatiotemporal control over the dynamics of 3-D interactions between distal enhancers and promoters of genes essential for neural lineage commitment. A recent study described the striking observation that three basic helix-loop-helix transcription factors are dynamically expressed in an oscillatory manner in proliferating, self-renewing NPCs, whereas only one of these factors transitions to sustained expression upon terminal differentiation. Our own preliminary studies reveal that genes encoding these candidate oscillatory factors are (1) often localized at boundaries between topological sub-domains (sub-TADs) and (2) connected to distal enhancers throughout both adjacent sub-TADs through long-range 3-D interactions. We hypothesize that dynamic 3-D interactions between distal enhancers and promoters of neural-specific bHLH transcription factors might govern the oscillatory expression of these genes. We will test our hypothesis by generating high-resolution maps of higher-order genome folding in NPCs and NPC-derived terminally differentiated neurons and astrocytes around candidate oscillatory factors. We will employ a pipeline of customized computational algorithms to integrate genome folding maps with an annotated catalogue of cell type-specific enhancers to create predictive models for the propensity of an individual enhancer to form 3-D interactions with developmentally regulated neural genes. In parallel, we will build modular, light-activated looping systems with CRISPR/Cas9 gene targeting in combination with proteins that are able to undergo light-inducible dimerization. We will then undertake a series of studies exploring looping dynamics to understand the organizing principles governing neural cell fate commitment. This work is innovative because it studies Epigenome dynamics with a cross- disciplinary approach that combines CRISPR/Cas9 genome editing, principles from optogenetics and genomics tools for mapping 3-D genome topology (i.e. Chromosome-Conformation-Capture and deep sequencing). Discoveries made by this work should yield an unprecedented view into the currently unknown dynamic behavior of genome structure and how it is linked to cell fate transitions in the developing brain.
Emerging evidence suggests that epigenetic modifications on top of the DNA sequence are dynamically altered to control gene expression during mammalian brain development. Here we propose to build a new set of light-inducible tools for the precise spatiotemporal control over 3-D looping interactions between regulatory sequences and their developmentally regulated target genes. We plan to use our `3-D optoepigenetic' tools to understand transcription dynamics during healthy brain development, with an eye toward our long-term goal to understand how epigenetic mechanisms go awry during the onset and/or progression of neurodegenerative and neurodevelopmental disease states. Answers to these questions are fundamental to numerous nuclear functions across a vast range of biological systems, and should shed transformative new light into the organizing principles governing the mechanistic link between 3-D genome folding, transcription and cell fate in the developing brain.
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