Molecular Basis for Differential V(D)J Recombination Mechanisms in Immunoglobulin Loci

Abstract

V(D)J recombination assembles variable region exons in antigen receptor loci and contributes to the exceptional diversity of immunoglobulins and T cell receptors. V(D)J recombination is initiated by the dedicated Recombination Activating Gene (RAG) endonuclease, which recognizes and cleaves at recombination signal sequences (RSSs) flanking each variable (V), diversity (D) and joining (J) gene segment. In the often multi-mega-base antigen receptor loci, how V(D)J recombination machinery coordinates with chromatin structural elements to ensure accurate and ordered rearrangements is an overarching question of the field. Over the past decade, studies focusing on the immunoglobulin heavy chain locus (Igh) demonstrated that cohesin loop extrusion-mediated RAG chromatin scanning is the underlying mechanism of strictly deletional rearrangements in Igh. However, how the immunoglobulin κ light chain locus (Igκ) enables both deletional and inversional Vκ-to-Jκ rearrangements remained, until recently, a mystery. This dissertation presents findings in mouse models and cell lines to support the model that orientation-independent Igκ primary recombination occurs through a short-range diffusion process dependent on the CTCF-based Cer/Sis element which separates the Vκ and Jκ domains. Importantly, strong Igκ RSSs are crucial for programming such diffusional Vκ-to-Jκ rearrangements, in contrast to the generally weaker Igh RSSs that rely on adjacent loop extrusion impediments for robust utilization during RAG scanning. After primary Vκ-to-Jκ joining-mediated removal of Cer/Sis, RAG linearly scans the newly proximal Vκ domain for predominantly deletional Igκ secondary recombination. However, unlike Igh, Vκ segments with strong RSSs can also be robustly utilized for inversional Igκ secondary recombination. In summary, the body of work presented in this dissertation elucidated the long-sought molecular mechanisms governing physiologically-important Igκ primary and secondary recombination. These findings also suggest that differential RSS strength can mediate distinct V(D)J recombination mechanisms in Igκ versus Igh and can potentially serve as an additional layer of regulation in other antigen receptor loci.