Mechanisms that underpin immunoglobulin gene rearrangement

/Abstract Antibodies are encoded by three loci: (i) the immunoglobulin heavy chain (Igh) locus, (ii) the immunoglobulin kappa (Igk locus and (iii) the immunoglobulin lambda (Igl locus. The Igh locus is segregated into variable (V), diversity (D), joining (J) and constant (C) regions. The Igk and Igl are comprised of V, J and C regions but lack D elements. The Igh and Igk loci utilize different mechanisms to generate VHDHJH or VkJk joints. Igh locus rearrangement is instructed by RAG scanning involving loop extrusion that pulls DHJH elements along chromatin until they align with VH gene segments to undergo VH-DHJH rearrangement. Vk-Jk locus rearrangement is regulated by changes in chromatin folding that brings distally-located Vk genes within close spatial proximity of Jk gene segments. The joining of Vk and Jk elements is regulated by enhancers, including the intronic Igk locus enhancer (iEκ), located in a genomic region separating Jk from Ck, and an enhancer, E34, that is positioned in the Igk variable region. iEk facilitates Vk-Jk joining involving Vk gene segments that span the entire Igk locus. E34 instructs Vk- Jk rearrangements involving Vk genes that are located nearby. We recently demonstrated that the E34 enhancer repositions a loop domain that harbors a subset of Vk genes into a recombination hub that is shared with Jk gene segments and the iEk enhancer. We showed the E34 deposits H3K27Ac across the enhancer region. We postulated that E34-mediated deposition of H3K27Ac constructs a confined spatial environment to prevent displacement of Vk from Jk gene segments (sub-diffusive motion). Here, we seek to determine how enhancers and epigenetics regulate Vk-Jk chromatin dynamics. In aim 1 we would develop a strategy to determine in quantitative terms, whether and how enhancers orchestrate Vk-Jk chromatin dynamics and instruct a sub-diffusive environment to enrich for Vk-Jk encounters. In aim 2 we would seek to determine whether epigenetic marks deposited across the E34 enhancer dictate Vk-Jk sub-diffusive motion and spatial confinement to increase Vk-Jk interaction- and Vk-Jk rearrangement- frequencies. The proposed method would enable us to determine how in 4D space enhancers and epigenetic modifications enrich for Vk-Jk encounters to generate diverse antibody repertoires. Project Number: 1R21AI193549-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: CORNELIS MURRE | Institution: UNIVERSITY OF CALIFORNIA, SAN DIEGO, LA JOLLA, CA | Award Amount: $438,500 | Activity Code: R21 | Study Section: Molecular and Structural Immunology Study Section[MSI] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21AI19354901