Abstract A033: Mechanisms that mediate intralocus and interlocus regulation of V(D)J recombination at immunoglobulin light chain loci

The N-terminal variable regions of immunoglobulin (Ig) heavy (IgH) and light (IgL) chains are involved in specific antigen binding and assembled from germline variable (V), diversity (D), and joining (J) gene segments through a somatic recombination reaction known as V(D)J recombination. V(D)J recombination is initiated by RAG endonuclease and completed by non-homologous DNA end-joining. V(D)J recombination is tightly regulated in the contexts of order, lineage, and allelic exclusion. For IgL this regulation additionally involves isotypic exclusion, in which most B cells express Ig molecules containing only one of the two IgL isotypes, κ and λ. Controlled V(D)J recombination is important beyond its role in generating diverse antibody repertoires against diverse antigens, since dysregulation of V(D)J recombination can not only underlie various immune disorders, but lead to oncogenic translocations that are frequently found in human B and T cell leukemias and lymphomas. Many of such translocations involve IgL chain loci. To provide new insights into mechanisms of chromosomal translocations involving IgL chain loci, we sought to investigate mechanisms of normal V(D)J recombination at IgL loci by developing and applying multiple new approaches. In this study, we employed high-throughput genome-wide translocation sequencing (HTGTS) to identify RAG activities at Igκ and Igλ loci in v-Abl transformed progenitor B (pro-B) line. Remarkably, we found such RAG activities were largely confined to unanticipated distinct chromatin topologically associated loop domains within Igκ and Igλ loci, adding a new layer of regulation of gene rearrangements at IgL loci. Furthermore, we developed a highly sensitive and unbiased assay, referred to as HTGTS repertoire sequencing (HTGTS-Rep-seq), to quantify Igκ and Igλ repertoires in mouse bone marrow precursor B (pre-B) and splenic B cell populations. HTGTS-Rep-seq detected diverse Igκ and Igλ VJ rearrangements involving individual functional Jκ and Jλ segments, respectively. Currently we are integrating such enormous Igκ and Igλ repertoire data with long-range chromatin interaction map data revealed by Hi-C and our recently developed high-resolution HTGTS chromosome conformation capture sequencing (HTGTS-3C-seq) methods to further address the mechanisms of gene rearrangement regulation by chromatin loop domains at these loci. In addition, to address the mechanisms of isotypic exclusion that has been debated, we generated germline mouse models harboring endogenously-generated Igκ deletions and a productively assembled IgH variable region exon by generating induced-pluripotent stems cells (iPSCs) from peripheral B cells. We then applied global nuclear run-on sequencing (GRO-seq) to bone marrow pre-B cells purified from iPSC-generated mice in a RAG-deficient background (which abrogates V(D)J recombination) to elucidate transcriptional features at Igκ and Igλ loci and specifically to reveal potential transcriptional alterations at Igλ in the context of Igκ deletion. Compared with Igκ-intact RAG-deficient pre-B cells, Igκ-deleted RAG-deficient pre-B cells showed remarkably increased transcription across the whole Igλ locus, indicating specific Igκ-deletion-triggered transcriptional activation at Igλ. Citation Format: Zhaoqing Ba, Jiazhi Hu, Zhou Du, Sherry G. Lin, Duane R. Wesemann, Frederick W. Alt. Mechanisms that mediate intralocus and interlocus regulation of V(D)J recombination at immunoglobulin light chain loci [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A033.