Evolution of 3D chromatin architecture: The role of CTCF across taxa

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The three-dimensional organization of the genome is developmentally dynamic and has been shown to critically affect gene regulation. In animals, this spatial organization is reflected by intricate interactions that bring genes and regulatory elements separated widely in the linear genome into close physical proximity and by sequestering sections of the genome into megabase-scale topologically associated domains. Recently, the transcriptional repressor CCCTC-binding factor (CTCF) has emerged as a key player in mediating 3D genome organization due to its capacity to dimerize in vivo in an orientation-dependent manner. CTCF knock out causes loss of genome topology and lethality. Though CTCF is deeply conserved among the bilaterian clade, mounting evidence demonstrates that the property of CTCF to interconnect distant genomic regions has been acquired at some point during the evolution of the chordates, concomitant with the expansion of the non-coding regulatory genome. It remains unresolved how CTCF promotes genome topology in vertebrates, while its primary function in insects, for example, remains refined to interactions and gene regulation on the short-distance scale. We propose an innovative multi-species approach using organisms with distinct roles of CTCF in 3D chromatin organization to functionally dissect its divergent modes, as well as the mechanisms that mediate CTCF function in flies, mice, and ascidians. Using CRISPR/Cas genomic editing, we will exchange CTCF proteins between species in vivo to assess its function in different regulatory contexts and its impact on spatial genome organization and transcriptional programs. Using innovative proteomic stratagems, we will then dissect species- and context-specific CTCF protein-protein interactions to then directly test regulatory mode requirements.The proposed project will advance our understanding of the evolutionary origins of 3D chromatin organization, its functional importance for developmental gene regulation, and the molecular mechanisms by which divergent CTCFs act.