Exploring a Functional Role of Chromosome Loop Extrusion Direction on Regulating Genome Biology

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ABSTRACT/PROJECT SUMMARY Elucidating mechanisms that control chromosome topology is of much interest as ~10% of human diseases arise from changes in genomic architecture. The Cohesin and CTCF proteins establish and revise chromosome loops to direct promoter/enhancer contacts that mediate tissue- and developmental stage-specific gene transcription. In a cell population, loops can form through uni- or bi-directional cohesin-mediated loop extrusion between CTCF binding elements (CBEs) bound by CTCF. Yet, any potential role for the directionality of loop formation has not been considered. Antigen receptor (AgR) loci are great models for studying functions of chromosome topology because changes in their architectures during lymphocyte development help establish AgR repertoires vital for immunity. The recombination of AgR locus variable (V), diversity (D), and joining (J) gene segments produces AgR diversity. Thus, studying AgR loci also yields mechanistic insights into how chromosome topology controls the initiation of V(D)J recombination and the cellular response to DNA damage that suppresses transformation of all cells. AgR loci contain many V segments and CBEs spanning vast genomic distances located far upstream of (D)J clusters flanked by CBEs of convergent orientation with V CBEs. When an AgR locus activates, the RAG endonuclease binds (D)J segments to establish a recombination center (RC) and CTCF and Cohesin form loops that reposition all V segments within similar spatial proximity to the RC. Early studies formulated a model wherein Cohesin/CTCF-mediated loops between V and RC CBEs generate compacted locus structures that drive long- distance V-to-RC recombination by increasing the chance for diffusion-based collisions between V segments and the RC (structural synapsis). This mechanism would support recombination by deletion or inversion. More recent next generation sequencing (NGS) studies of Igh and Tcra/d show that cohesin-directed loop extrusion from the RC directs V-to-RC recombination by allowing RAG to unidirectionally scan the locus and capture a V RSS (scanning synapsis). This mechanism dictates that recombination occurs only by deletion and uses V CBEs to impede loop extrusion. While scanning synapsis might mediate V-to-RC rearrangements by deletion at all loci, it cannot direct long-range V-to-RC rearrangements that occur through inversion in Tcrb and Igk loci. Based on differences between published Igh studies and unpublished Tcrb data of the applicant, he hypothesizes that the direction of cohesin-mediated loop extrusion across a locus determines whether scanning or structural synapsis mediates long-range rearrangement. To test this hypothesis, the applicant proposes to: Aim 1) determine the extents that scanning and structural synapsis operate within Tcrb and Aim 2) elucidate the impacts of Vb CBE modifications on long-range synapsis by each mechanism. The expected outcomes would yield a major advance by providing strong evidence that the direction of chromosome loop extrusion dictates the underlying mechanism of long-range V-to-RC recombination. This would serve as a foundation for experiments to determine the function of loop extrusion direction in regulating gene expression and repair, replication, and segregation of genomes.