Locus folding mechanisms determine modes of antigen receptor gene assembly (ChIP-seq)

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 bi-directional 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'Vß segment (Trbv1) impaired loop extrusion originating locally and extending to DßJß 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 DßJß-bound RAG as the sole mechanism of Vß recombination, distinguishing it from Igh. Our data indicate that CBE-dependent generation of loops cooperates with promoter-mediated activation of chromatin to juxtapose Vß and DßJß 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. Overall design: ChIP Seq was performed in DN thymocytes from ~5 Rag1-/- mice per experiment with either a WT Tcrb locus or with the indicated mutations with the indicated antibodies.