Assessing the Function and Dynamics of Spatial Genome Architecture during Embryogenesis
Gene expression is initiated through the action of enhancer elements, which are dispersed throughout the genome, often at great distances from the target genes they regulate. How enhancers in one part of a chromosome can impart regulatory information to another distal region is a long-stranding question in genome regulation. Despite having direct implications for development, evolution and disease, the underlying mechanism and realtime dynamics of enhancer-promoter topologies remain unknown. To address this, we will take an interdisciplinary approach, combining genetic dissection in both cis and trans, quantitative single cell imaging, genome engineering, and state-of-the-art live imaging during embryogenesis. Many of the general principles underlying genome regulation are highly conserved from Drosophila to humans. We will leverage the ease of genome engineering in Drosophila, and the wealth of information about developmental enhancers to functionally dissect the inherent properties of genome architecture.
This proposal has three complementary aims: (1) To dissect the requirement of genome architecture for enhancer function, we will genetically delete all interacting regions within three chromatin domains and assess their functional impact on genome architecture, gene expression and embryonic development in homozygous embryos. (2) To understand how genome architecture is first established during development, we will deplete different factors from early embryos in trans. The combination of aims 1 and 2 will provide one of the most comprehensive functional dissections of the role of 3D genome architecture during embryogenesis to date. (3) To measure the dynamics and order of events in genome regulation, we will engineer embryos with tagged enhancers, promoters and nascent transcripts at two loci to quantify the dynamics of distal enhancer-promoter interactions during embryogenesis and determine how that relates to functional output (i.e. nascent transcription). The combined results from all three aims will provide unique functional, quantitative and dynamic insights, providing the missing data to move from correlation to causation in genome regulation.