Person:

Dring, Edward

RAG endonuclease initiates V(D)J recombination in progenitor (pro)-B cells1. Upon binding a recombination center (RC)-based JH, RAG scans upstream chromatin via loop extrusion, potentially mediated by cohesin, to locate Ds and assemble a DJH-based RC2. CTCF looping factor-bound elements (CBEs) within IGCR1 upstream of Ds impede RAG-scanning3-5; but their inactivation allows scanning to proximal VHs where additional CBEs activate rearrangement and impede scanning any further upstream5. Distal VH utilization is thought to involve diffusional RC access following large-scale Igh locus contraction6-8. Here, we test the potential of linear RAG-scanning to mediate distal VH usage in G1-arrested v-Abl-pro-B cell lines9, which undergo robust D-to-JH but little VH-to-DJH rearrangements, presumably due to lack of locus contraction2,5. Through an auxin-inducible approach10, we degrade the cohesin-component Rad2110-12 or CTCF12,13 in these G1-arrested lines. Rad21 degradation eliminated all V(D)J recombination and RAG-scanning-associated interactions, except RC-located DQ52-to-JH joining in which synapsis occurs by diffusion2. Remarkably, while CTCF degradation suppressed most CBE-based chromatin interactions, it promoted robust RC interactions with, and robust VH-to-DJH joining of, distal VHs, with patterns similar to those of "locus-contracted" primary pro-B cells. Thus, down-modulation of CTCF-bound scanning-impediment activity promotes cohesin-driven RAG-scanning across the 2.7Mb Igh locus.

RAG endonuclease initiates IgH locus (Igh) V(D)J assembly in progenitor (pro)-B cells by joining Ds to JHs, before joining upstream VHs to DJH intermediates1. In mouse pro-B cells, the CTCF-binding element (CBE)-anchored chromatin loop domain2 at the 3’end of Igh contains an internal sub-domain spanning the 5’CBE anchor (IGCR1)3, the DHs, and a RAG-bound recombination center (RC)4. The RC comprises JH-proximal D (DQ52), 4 JHs, and the intronic enhancer (“iE”)5. Robust RAG cleavage is restricted to paired V(D)J segments flanked by complementary recombination signal sequences (12RSSs and 23RSSs)6. Ds are flanked downstream and upstream by 12RSSs that, respectively, mediate deletional joining with convergently-oriented JH-23RSSs and VH-23RSSs6. Despite 12/23 compatibility, inversional D to JH joining via upstream D-12RSSs is rare7,8. Plasmid-based assays attributed lack of inversional D to JH joining to sequence-based preference for downstream D-12RSSs9, as opposed to putative linear scanning mechanisms10,11. Given recent findings that RAG linearly scans convergent CBE-anchored chromatin loops4,12-14, potentially formed by cohesin-mediated loop extrusion15-18, we revisited a scanning role. Here, we report that JH-23RSS chromosomal orientation programs RC-bound RAG to linearly scan upstream chromatin in the 3’Igh sub-domain for convergently-oriented D-12RSSs and, thereby, to mediate deletional joining of all Ds, except RC-based DQ52 that joins by a diffusion-related mechanism. In a DQ52-based RC, formed in the absence of JHs, RAG bound by the downstream DQ52-RSS scans the downstream constant region exon-containing 3’Igh sub-domain in which scanning can be impeded by targeted nuclease-dead Cas9 (dCas9) binding, by transcription through repetitive Igh switch sequences, and by the 3’Igh CBE-based loop anchor. Notably, each scanning impediment focally increases RAG activity on potential substrate sequences within the impeded region. High resolution mapping of RC chromatin interactions reveals that such focal RAG targeting is associated with corresponding impediments to the loop extrusion process that drives chromatin past RC-bound RAG.