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

Ralph Stadhouders, Marjolein De Jong - de Bruijn, Magdalena Rother, Saravanan Pillai, Claudia Ribeiro de Almeida, Petros Kolovos, Menno van Zelm, Wilfred van Ijcken, Frank Grosveld, Eric Soler, Rudi Hendriks

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Abstract

During B cell development, the precursor B cell receptor (pre-BCR) checkpoint is thought to increase immunoglobulin light chain (Ig) locus accessibility to the V(D)J recombinase. Accordingly, pre-B cells lacking the pre-BCR signaling molecules Btk or Slp65 showed reduced germline V transcription. To investigate whether pre-BCR signaling modulates V accessibility through enhancer-mediated Ig locus topology, we performed chromosome conformation capture and sequencing analyses. These revealed that already in pro-B cells the enhancers robustly interact with the approximate to 3.2 Mb V region and its flanking sequences. Analyses in wild-type, Btk, and Slp65 single- and double-deficient pre-B cells demonstrated that pre-BCR signaling reduces interactions of both enhancers with Ig locus flanking sequences and increases interactions of the 3 enhancer with V genes. Remarkably, pre-BCR signaling does not significantly affect interactions between the intronic enhancer and V genes, which are already robust in pro-B cells. Both enhancers interact most frequently with highly used V genes, which are often marked by transcription factor E2a. We conclude that the enhancers interact with the V region already in pro-B cells and that pre-BCR signaling induces accessibility through a functional redistribution of long-range chromatin interactions within the V region, whereby the two enhancers play distinct roles. Author Summary B lymphocyte development involves the generation of a functional antigen receptor, comprising two heavy chains and two light chains arranged in a characteristic Y shape. To do this, the receptor genes must first be assembled by ordered genomic recombination events, starting with the immunoglobulin heavy chain (IgH) gene segments. On successful rearrangement, the resulting IgH protein is presented on the cell surface as part of a preliminary version of the B cell receptorthe pre-BCR. Pre-BCR signaling then redirects recombination activity to the immunoglobulin light chain gene. The activity of two regulatory enhancer elements is known to be crucial for opening up the gene, but it remains largely unknown how the hundred or so Variable (V) segments in the locus gain access to the recombination system. Here, we studied a panel of pre-B cells from mice lacking specific signaling molecules, reflecting absent, partial, or complete pre-BCR signaling. We identify gene regulatory changes that are dependent on pre-BCR signaling and occur via long-range chromatin interactions between the enhancers and the V segments. Surprisingly the light chain gene initially contracts, but the interactions then become more functionally redistributed when pre-BCR signaling occurs. Interestingly, we find that the two enhancers play distinct roles in the process of coordinating chromatin interactions towards the V segments. Our study combines chromatin conformation techniques with data on transcription factor binding to gain unique insights into the functional role of chromatin dynamics.
Original languageUndefined/Unknown
JournalPLoS Biology (print)
Volume12
Issue number2
DOIs
Publication statusPublished - 2014

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