Locus folding mechanisms determine modes of antigen receptor gene assembly

This study demonstrates that the precise mechanisms used to fold antigen receptor loci to position their V and (D)J gene segments in close spatial proximity determine how RAG-mediated synapsis and recombination of these segments occur across vast linear genomic distances. The dynamic folding of genomes regulates numerous biological processes, including antigen receptor (AgR) gene assembly. We show that, unlike other AgR loci, homotypic chromatin interactions and bidirectional chromosome looping both contribute to structuring Tcrb for efficient long-range V(D)J recombination. Inactivation of the CTCF binding element (CBE) or promoter at the most 5 ' V beta segment (Trbv1) impaired loop extrusion originating locally and extending to D beta J beta CBEs at the opposite end of Tcrb. Promoter or CBE mutation nearly eliminated Trbv1 contacts and decreased RAG endonuclease-mediated Trbv1 recombination. Importantly, Trbv1 rearrangement can proceed independent of substrate orientation, ruling out scanning by D beta J beta-bound RAG as the sole mechanism of V beta recombination, distinguishing it from Igh. Our data indicate that CBE-dependent generation of loops cooperates with promoter-mediated activation of chromatin to juxtapose V beta and D beta J beta segments for recombination through diffusion-based synapsis. Thus, the mechanisms that fold a genomic region can influence molecular processes occurring in that space, which may include recombination, repair, and transcriptional programming.