Extended Data Fig. 9: Working model for loop-extrusion-mediated RAG downstream scanning.
From: The fundamental role of chromatin loop extrusion in physiological V(D)J recombination
a–i, Model for cohesin-mediated loop extrusion of chromatin past nascent Igh RC in JHΔ v-Abl lines based on RAG2-deficient background analyses. For all examples, increased interactions of impediment sites with RC targets scanning activity in RAG-sufficient cells. a, Cohesin (red rings) are loaded at multiple sites in the RC–3′ CBEs Igh subdomain. Illustrations show cohesin loading at the region downstream of RC. b, Cohesin-mediated extrusion promotes a linear interaction between the nascent RC and downstream regions. c, Robust transcription (green arrow) across the Iγ2b–Sγ2b impedes loop extrusion. d, In a subset of cells, loop extrusion proceeds past the Iγ2b–Sγ2b impediment to the 3′ CBEs loop anchor. e–i, Loop extrusion in JHΔ-dCas9-Sγ1-sgRNA lines is impeded, directly or indirectly, by the dCas9-bound Sγ1. As dCas9 impediment is not a complete block, loop extrusion in a subset of cells proceeds downstream, allowing dynamic formation of sub-loops of RC with Iγ2b-Sγ2b or 3′ CBEs. j–l, In RAG-sufficient cells, RC-bound RAG might enhance the dCas9-bound Sγ1 extrusion impediment. m–p, Elimination of Iγ2b-promoter-driven transcription permits unimpeded RAG-bound RC extrusion to the 3′ CBEs anchor, increasing RAG scanning activity there. q, r, 3C-HTGTS analysis of RC interactions with DH and flanking regions in the JHΔ-dCas9 line (q) and DH–JH+/− line (r). DpnII (n = 4, biological replicates) and NlaIII (n = 3, biological replicates) digestions are shown for the JHΔ-dCas9 line. The NlaIII digestion more clearly reveals an interaction peak near DH3-2 owing to a paucity of DpnII sites in that region. NlaIII digestion of the DH–JH+/− line shows a similar RC interaction pattern to that of the JHΔ-dCas9 line (r, n = 2 technical repeats). Bar graphs show relative RC interaction of the 25-kb intervening DH region (from DH2-3 to DH2-8) versus that of the same-sized neighbouring regions (n as indicated above). Data represent mean ± s.d. (q) or mean (r). P values calculated via two-tailed paired t-test.
