RAG1/RAG2 (RAG) endonuclease-mediated assembly of antigen receptor genes in developing B and T cells is essential for adaptive immunity. Yet, this process has fatal risks as evidenced by generation of self-reactive receptors and formation of oncogenic antigen receptor locus translocations. The RAG proteins are comprised of core domains essential for recombination and non-core regions that control the induction and repair of RAG DNA double strand breaks (DSBs). In 2000, mutations of the RAG1 N-terminus non-core region were found to cause Omenn Syndrome, a B and T lymphocyte immunodeficiency linked with autoimmunity. Since then, the RAG1 N-terminus has been discovered to contain an ubiquitin ligase and interact with another ubiquitin ligase and a kinase. However, it remains unknown how the RAG1 N-terminus promotes lymphocyte differentiation and tolerance by mechanisms beyond modulating RAG activity. Ig? and Ig? B cells develop from pre-B cells. The applicant has established mouse models and a primary pre-B cell culture system to elucidate functions of the RAG1 N-terminus in B cell development. RAG DSBs induced during Ig? recombination in pre-B cells signal transcriptional activation of the cytoplasmic Pim2 kinase, which promotes survival of pre-B cells with RAG Ig? DSBs to promote Ig? B cell development. The applicant now demonstrates that Rag1C/C mice lacking the Rag1 N-terminus have impaired development of Ig? B cells and decreased stability and expression of Pim2 protein induced by RAG Ig? DSBs. He shows that Rag1PG/PG mice lacking Rag1 ubiquitin ligase activity have a similar defect in Ig? B cell development, implying that RAG1-mediated protein ubiquitination is required for RAG DSBs to inhibit proteasome-mediated Pim2 degradation. The PI hypothesizes that RAG1 ubiquitin ligase activity at RAG Ig? DSBs triggers intracellular signals that post-transcriptionally shape the pre-B cell DSB response and thereby orchestrate B cell development. The applicant proposes to test fundamental aspects of this hypothesis by determining how the RAG1 N-terminus stabilizes Pim2 protein (Aim 1) and post-transcriptionally shapes the pre-B cell proteome to modulate expression of proteins that control B cell development (Aim 2). This study will cement the innovative concept that RAG1 has critical functions that transcend its role in cleaving and helping repair DNA as a RAG subunit. The results should yield novel insights into mechanisms that coordinate Ig? and Ig? recombination, pre-B cell survival, and B cell receptor signaling to create broad protective antigen receptor repertoires and prevent autoimmunity. Considering that autoimmunity in Omenn Syndrome mainly manifests in the T cell compartment, knowledge and approaches from this project will lay the strong foundation for a future RO1 grant and other avenues of basic and translational immunology research.
It is not known how the RAG1 N-terminus promotes lymphocyte development and prevents autoimmunity. The applicant shows evidence that the RAG1 N-terminus enhances Ig? B cell development by signaling to inhibit degradation of the pro-survival Pim2 protein. The applicant proposes to elucidate mechanisms and determine the extent to which the RAG1 N-terminus shapes the pre-B cell proteome to regulate B cell development.
