Promoter- and Enhancer-Dependent Cohesin Loading Initiates Chromosome Looping to Fold Tcrb Loci for Long-Range Recombination [Gro-Seq]

Cohesin-mediated chromosome looping regulates diverse processes, including antigen receptor (AgR) gene assembly by V(D)J recombination. To understand mechanisms that coordinate genome topologies, we focused on a genetically tractable AgR locus, Tcrb. Cohesin loading and initiating loop extrusion (LE) from a nearby CTCF-binding element (CBE) required the promoter of the most 5’Vb segment, creating long-range contacts with target downstream DJb segments within the recombination center (RC). CBEs flanking the RC have multiple functions: terminators of LE originating in the Vb cluster, initiators of LE in the RC, and insulation of enhancer activity. Deletion of the Tcrb super-enhancer abolished loop extrusion from the neighboring RC but spared longrange contacts, indicating that unidirectional loop extrusion from upstream Vb segments was sufficient. Thus, Vb promoter- or enhancer-dependent cohesin loading initiates LE in opposite directions across the locus to assemble a broad Tcrb repertoire, a finding that has broad implications for genomic architecture and function. Gro-Seq was performed on sorted (B220-, CD19-, CD11b-, CD11c-, NK1.1-, TER119-, TCRβ-, TCRγ/δ-) DN thymocytes from Rag1-/- mice with either a WT Tcrb locus or with the indicated mutations.