As a critical part of adaptive immunity, generation of a highly diverse antibody repertoire begins with efficient assembly of the immunoglobulin heavy chain locus (IgH) through V(D)J recombination during early B cell development. At IgH locus, hundreds of VHs are widely-spread within a 2.4 Mb upstream region and multiple DHs and JHs are located within a downstream 0.3 Mb domain. The lymphoid-specific RAG1/RAG2 (RAG) endonuclease initiates V(D)J recombination by cutting paired recombination signal sequences (RSSs) flanking V, D, and J segments. A major knowledge gap is what mechanisms bring the RSS pairs, which are widely separated in chromosomal distance, into spatial proximity for RAG cleavage. Although 3D genome topology and long-range chromatin interaction at IgH locus has been implicated to play important roles in directing RAG targeting, understanding the underlying mechanisms is greatly hindered due to lack of high-resolution chromatin interaction maps and efficient systems to comprehensively characterize the putative regulatory elements. The overall objective of this proposal is to determine the molecular mechanisms driving functional chromatin interactions mediating IgH RAG targeting. We have recently revealed a novel mechanism driving D to JH recombination, whereby cohesin-mediated chromatin loop extrusion propels RAG scanning within D-JH domain to promote physiologically deletional D-to-JH joining. With new compelling evidence, we propose that this cohesin-mediated dynamic chromatin looping also operates during V to DJH recombination to ensure generation of a diverse VH repertoire. With ultra-sensitive chromatin interaction and V(D)J recombination assays combined with comprehensive genetic studies, in Aim1, we will address the functional importance of chromatin loop extrusion in V to DJH recombination.
In Aim2, we will determine the molecular mechanisms underlying cohesin-mediated IgH long-range looping.
In Aim3, we will identify and characterize novel IgH cis- regulatory elements in directing RAG long-range targeting. These studies will be greatly facilitated by a novel v-Abl pro-B cell line we generated that shows efficient IgH long-range chromatin looping and diverse VH utilization across IgH locus. This will provide a flexible cell-based system to systematically characterize complex interplay between IgH cis-regulatory elements and trans-acting factors, which is difficult to achieve in animal models. Completion of this project will provide new mechanistic insights on how the dynamic 3D genome topology harnesses a major immune process for generation of diverse antibody repertoires.
V(D)J recombination is a fundamental immune process that protects us from pathogens by generating diverse antibody repertoires, but can also make errors by targeting undesired genomic loci. This study aims to investigate the key mechanisms that ensure properly targeted V(D)J recombination activity for generation of antibodies, and thereby suppress unwanted genomic lesions underlying lymphoid malignancies.
