CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning

Abstract

RAG endonuclease initiates V(D)J recombination in progenitor (pro)-B cells 1 . Upon binding a recombination center (RC)-based J H , RAG scans upstream chromatin via loop extrusion, potentially mediated by cohesin, to locate Ds and assemble a DJ H -based RC 2 . CTCF looping factor-bound elements (CBEs) within IGCR1 upstream of Ds impede RAG-scanning 3 – 5 ; but their inactivation allows scanning to proximal V H s where additional CBEs activate rearrangement and impede scanning any further upstream 5 . Distal V H utilization is thought to involve diffusional RC access following large-scale Igh locus contraction 6 – 8 . Here, we test the potential of linear RAG-scanning to mediate distal V H usage in G1-arrested v-Abl- pro-B cell lines 9 , which undergo robust D-to-J H but little V H -to-DJ H rearrangements, presumably due to lack of locus contraction 2 , 5 . Through an auxin-inducible approach 10 , we degrade the cohesin-component Rad21 10 – 12 or CTCF 12 , 13 in these G1-arrested lines. Rad21 degradation eliminated all V(D)J recombination and RAG-scanning-associated interactions, except RC-located DQ52-to-J H joining in which synapsis occurs by diffusion 2 . Remarkably, while CTCF degradation suppressed most CBE-based chromatin interactions, it promoted robust RC interactions with, and robust V H -to-DJ H joining of, distal V H s, 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.

Direct observation of RAG recombinase recruitment to chromatin and theIgHlocus in live pro-B cells

“…Chromatin loop extrusion has recently been shown to be the main process directing RAG-mediated V(D)J recombination (25,26). The first step of IgH gene assembly occurs in a 60kbp domain that includes D H gene segments, and the recombination center (25).…”

Gradual opening of Smc arms in prokaryotic condensin

“…SMC protein complexes are ancient enzymes with a unique architecture that organize chromosomal DNA molecules, presumably by DNA loop extrusion (Yatskevich et al, 2019). In 4 eukaryotes, cohesin folds DNA into loop domains to regulate gene expression and to direct 5 DNA recombination (Ba et al, 2020;Merkenschlager and Nora, 2016). By a distinct mechanism, cohesin also holds sister chromatids together (Yatskevich et al, 2019).…”

Dynamic 3D Locus Organization and Its Drivers Underpin Immunoglobulin Recombination

“… (A) The loop extrusion model for D H -J H , proximal V H -DJ H and distal V H -DJ H recombination. Cohesin-mediated RAG-scanning leads to the extrusion of progressively larger loops, until loops are stalled by convergent CBEs ( 53 , 54 ). (B) In the phase separation model for Igh recombination, recombination occurs near the boundary of the gel phase (gray ovals) ( 55 ).…”

“…Clarification of the roles of CTCF and cohesin in RAG scanning by loop extrusion has come from degradation of Rad21 and CTCF in v-Abl-transformed pro-B cell lines, using an auxin-inducible degron system ( 54 ). These cell lines have impaired locus contraction and favor proximal over distal V H -DJ H recombination compared to primary pro-B cells, despite the distal V H genes being highly transcribed; the underlying defect is not known ( 54 ). Rad21 degradation led to: disruption of looping in the 3’ region; severe reduction in D H -J H recombination (DQ52 could still recombine through diffusion based mechanisms); and total abrogation of V H -DJ H recombination.…”