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Principal Investigator: David G. Schatz
Organization: YALE UNIVERSITY
Fiscal Year: 2024
Award: $563,811
Funding agency: National Institute of Allergy and Infectious Diseases
Despite four decades of study, major gaps remain in our understanding of key molecular and cellular processes that orchestrate the action of the RAG1/RAG2 endonuclease during antigen receptor (AgR) gene assembly by V(D)J recombination. V(D)J recombination occurs in focal regions of RAG binding in AgR loci known as recombination centers (RCs) but virtually nothing is known about the composition or organizing principles of RCs. Our recent findings demonstrate that recombinase function is modulated by nuclear compartmentalization of RAG1 and that RAG1's large N-terminal region (NTR) controls RAG activity, trafficking, chromatin binding, gene segment repertoire, and interaction with a newly discovered recombination factor, Spin1. However, RAG nuclear cell biology is poorly understood and the RAG1 NTR is largely a black box, with almost no understanding of its structure, the residues that mediate its many functions, or how it works together with the remainder of the RAG enzyme. Indeed, no structures are available for the holo-RAG complex, severely limiting insight into cooperation between the catalytic and regulatory domains. The central objective of our proposed experiments is to fill these gaps in understanding through a systematic dissection of RCs and RAG nuclear cell biology, the function of the RAG1 NTR, and the structure of the holo-RAG-nucleosome complex. We will use complementary biochemical, imaging, molecular, structural, and genetic approaches to achieve the following aims:
Aim 1. Delineate RAG nuclear cell biology and the contribution of the RAG1 NTR. Our findings reveal a repressive association of RAG1 with nucleoli and repositioning of RAG1 adjacent to nuclear speckles when recombination is activated. We will systematically characterize RAG nuclear compartmentalization, function, and entry into RCs, determine how each parameter is controlled by binding partners and the RAG1 NTR, and establish the physiological role of RAG1 nucleolar association in a novel RAG1 mutant mouse. Aim 2. Determine components of and regulatory mechanisms in RCs. We will identify the components of RCs using in vivo proximity labeling and quantitative proteomics, establish the mechanism by which Spin1 interacts with RAG1 and stimulates recombination, and determine how the RAG1 NTR dictates gene segment repertoire through control of the balance between short- and long-range modes of recombination. Aim 3. Determine the structural basis of holo-RAG-nucleosome interactions. Building on extensive preliminary data, we will use: i) X-ray crystallography to determine the structure of a RAG1 NTR fragment free and bound to histone tails; ii) cryo-electron microscopy to determine the structure of RAG1/2 bound to DNA and the nucleosome core particle; and iii) cryo-electron tomography to determine the structure of holo RAG bound to chromatin in situ in the nucleus-thereby providing unprecedented insight into the quaternary structure of RAG, its mechanism of recruitment to chromatin, and the function of its regulatory domains.
Terms: <Adaptive Immune System><Address><Alleles><Allelomorphs><Antigen Receptors><B-Cell Development><Binding><Biochemical><Black Box><Blood leukocyte><Cell Communication and Signaling><Cell Function><Cell Nucleolus><Cell Nucleus><Cell Physiology><Cell Process><Cell Signaling><Cellular Function><Cellular Physiology><Cellular Process><Cellular biology><Chromatin><Chromosomal Deletion><Chromosomal dislocation><Chromosomal translocation><Chromosome Deletion><Complex><Core Particle><Cryo-electron Microscopy><Cryo-electron tomography><Cryoelectron Microscopy><DNA><DNA Recombination><Data><Deoxyribonucleic Acid><Development><Dissection><Electron Cryomicroscopy><Enzyme Gene><Enzymes><Equilibrium><Exclusion><Funding><Genes><Genetic Processes><Genetic Recombination><Genetic Translocation><Genetics-Mutagenesis><Genome><Genome Stability><Genomic Stability><Goals><Hematopoietic Cell Tumor><Hematopoietic Malignancies><Hematopoietic Neoplasms><Hematopoietic Neoplasms including Lymphomas><Hematopoietic Tumor><Hematopoietic and Lymphoid Cell Neoplasm><Hematopoietic and Lymphoid Neoplasms><Histone Code><Histones><Human Chromosomes><Image><Immune Globulins><Immunoglobulin V(D)J Rearrangement><Immunoglobulins><In Situ><In Vitro><Intracellular Communication and Signaling><Investigators><J segment gene><Knowledge><Label><Leukocytes><Leukocytes Reticuloendothelial System><Lymphatic cell><Lymphocyte><Lymphocytic><MHC Receptor><Major Histocompatibility Complex Receptor><Malignant Hematopoietic Neoplasm><Maps><Marrow leukocyte><Mediating><Methods><Mice><Mice Mammals><Modification><Molecular><Molecular Interaction><Murine><Mus><Mutagenesis><Mutagenesis Molecular Biology><Mutant Strains Mice><Mutate><Mutation Analysis><N-terminal><NH2-terminal><Nuclear><Nucleosome Core><Nucleosome Core Particle><Nucleosomes><Nucleus><Partial Monosomy><Peptide Signal Sequences><Peptides><Physiologic><Physiological><Plasmosome><Play><Position><Positioning Attribute><Proteomics><RNA Processing><Rag1><Rag1 Mouse><Reaction><Reader><Receptor Gene><Recombination><Regulation><Research Personnel><Researchers><Rest><Role><Scanning><Series><Signal Peptide><Signal Sequences><Signal Transduction><Signal Transduction Systems><Signaling><Single Crystal Diffraction><Structure><Subcellular Process><T-Cell Antigen Receptors><T-Cell Lymphoma><T-Cell Non-Hodgkin's Lymphoma><T-Cell NonHodgkins Lymphoma><T-Cell Receptor><T-Cell and NK-Cell Non-Hodgkin's Lymphoma><Tail><Testing><V(D)J Rearrangement><V(D)J Recombination><VDJ rearrangement><VDJ recombination><White Blood Cells><White Cell><Work><X Ray Crystallographies><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray/Neutron Crystallography><Xray Crystallography><acquired immune system><antagonism><antagonist><balance><balance function><biological signal transduction><blood cancer><cancer of blood><cancer of the blood><cell biology><chromosome dislocation><chromosome translocation><cohesin><cryo-EM><cryo-EM tomography><cryoEM><cryoEM tomography><cryoelectron tomography><cryogenic electron microscopy><developmental><electron cryo-tomography><endonuclease><experiment><experimental research><experimental study><experiments><gene locus><genetic approach><genetic locus><genetic strategy><genomic location><genomic locus><imaging><in vivo><insight><leukemia/lymphoma><lymph cell><lymphoma/leukemia><mouse mutant><novel><nucleolus><particle><prevent><preventing><protein signal sequence><recombinase><recruit><social role><superresolution microscopy><trafficking><virtual><white blood cell><white blood corpuscle>