Regulation of B cell Class Switching by Histone H2A Deubiquitinating Enzymes

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Introduction: Antibody production is one of the central mechanisms of immune response. The generation of antibody diversity takes place through the processes of V(D)J recombination, class switch recombination (CSR), and somatic hypermutation that involve the introduction of programmed DNA damage into the B cell genome at the antibody-encoding genes. Resolution of this DNA damage is critical for B cell survival, genomic stability, and effective immune defenses.*** Regulation of DNA packaging plays a central role in DNA repair, and is controlled through chemical modifications of histone proteins. This is exemplified by histone H2AX phosphorylation (H2AX-pSer139) that is a prominent early mark of DNA damage. More recently the key role of histone H2A ubiquitination by the enzymes RNF168 and RNF8 in DNA repair has been uncovered. Importantly, loss of either RNF168 or RNF8 impairs B cell class-switching, indicating that regulation of histone H2A ubiquitination is essential in this specialized DNA repair pathway in B cells.*** The role of histone H2A deubiquitinating enzymes (H2A-DUBs) in DNA repair is less well understood. Multiple H2A-DUBs have been identified in different studies, including BRCC36, USP3, USP44, USP16, and BAP1. However their roles in the regulation of CSR and other specialized DNA repair processes in the immune system remain unknown.******Hypothesis: DUBs targeting ubiquitinated forms of histone H2A (H2A-DUBs) regulate B cell class-switching. At the molecular level they control DNA packaging during the DNA repair reaction, ensuring effective DNA rejoining of the antibody-encoding genes. On the physiological level they impact immune system functions, genomic-stability, and ultimately the fitness and survival of the organism.****Immediate Goals: The primary goal of this research program is to identify the H2A-DUB-regulators of B cell class switching, through knockdown studies in the CH12F3 B cell line and studies with primary B cells from novel conditional-knockout mouse models.***Long-term Goals: In the long-term the research program will provide detailed characterization of the functions of these DUBs in the DNA damage response and repair network of activated B cells, and their cross-talk with other chromatin modifiers and DNA repair factors. It will also allow further analyses of these H2A-DUBs in various specialized physiological DNA repair processes, such as V(D)J recombination, somatic hypermutation, and the systemic regulation of genomic stability in diverse tissues.***Significance: The program will provide important insights into the physiological functions of the poorly characterized H2A-DUB proteins. It will advance our knowledge of the fundamental mechanisms of B cell mediated immunity, and will contribute to the growing understanding of specializations and variations in DNA damage response and genome-maintenance in different cell types.**