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

基因组的三维结构是发育动态的，并已被证明对基因调控具有关键影响。在动物中，这种空间组织反映在错综复杂的相互作用中，使线性基因组中广泛分离的基因和调控元件在物理上接近，并将基因组的部分隔离到兆基规模的拓扑相关区域中。最近，转录抑制因子CCCTC结合因子(CTCF)由于其在体内以定向依赖的方式二聚化的能力而成为介导3D基因组组织的关键角色。CTCF基因敲除会导致基因组拓扑结构的丧失和致死性。尽管CTCF在双边支系中高度保守，但越来越多的证据表明，CTCF在脊索动物进化过程中的某个时刻获得了连接遥远基因组区域的特性，伴随着非编码调控基因组的扩张。目前尚不清楚CTCF是如何促进脊椎动物基因组拓扑结构的，而它在昆虫中的主要功能仍然是在短距离尺度上的相互作用和基因调节。我们提出了一种创新的多物种方法，使用在3D染色质组织中具有不同角色的生物来从功能上剖析其不同的模式，以及在苍蝇、小鼠和海鞘中介导CTCF功能的机制。利用CRISPR/Cas基因组编辑，我们将在体内物种之间交换CTCF蛋白，以评估其在不同调控环境中的功能及其对空间基因组组织和转录程序的影响。利用创新的蛋白质组学策略，我们将剖析物种和上下文特定的CTCFs蛋白-蛋白质相互作用，然后直接测试调控模式要求。该项目将促进我们对3D染色质组织的进化起源、其对发育基因调控的功能重要性以及不同CTCFs作用的分子机制的理解。

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.