Pre-B Cell Receptor Signaling Induces Immunoglobulin Kappa Locus Accessibility by Functional Redistribution of Enhancer-mediated Chromatin Interactions

During B cell development the precursor B cell receptor (pre-BCR) checkpoint is thought to increase immunoglobulin kappa light chain (Igk) locus accessibility to the V(D)J recombinase. Accordingly, pre-B cells lacking the pre-BCR signaling molecules Btk or Slp65 showed reduced germline Vk transcription. To investigate whether pre-BCR signaling modulates Vk accessibility through enhancer-mediated Igk locus topology, we performed chromosome conformation capture and sequencing analyses. These revealed that already in pro-B cells the intronic and 3'k enhancers robustly interact with the 3.2 Mb Vk region and its flanking sequences. Analyses in wild-type, Btk and Slp65 single and double-deficient mice demonstrated that pre-BCR signaling reduces interactions of both enhancers with sequences flanking the Igk locus and increases interactions of the 3'k enhancer with Vk genes. Remarkably, pre-BCR signaling does not significantly affect interactions between the intronic enhancer and Vk genes, which are already robust in pro-B cells. Both enhancers interact most frequently with highly used Vk genes, which are often marked by transcription factor E2A. From these findings, we conclude that Igk locus contraction occurs already in pro-B cells and that pre-BCR signaling induces accessibility through functional redistribution of long-range chromatin interactions within the Vk region, whereby the two enhancers play distinct roles.