Pp2a

Transcriptional control of activation induced deaminase (AID) function

SUMMARY Somatic hypermutation (SHM) and class switch recombination (CSR) are vital for the generation of high affinity antibodies with appropriate effector function, protection against infection, and vaccine efficiency. They are initiated when the activation induced deaminase (AID) deaminates cytidines in single-stranded DNA in the context of transcription by RNA polymerase 2 (Pol2). Aberrant DNA deamination by AID is an important driver of genetic instability and the development of B cell malignancies. Understanding the factors and mechanisms that coordinate AID-mediated deamination with Pol2 transcription is an important objective in the study of humoral immunity and the central goal of research under this grant. Our preliminary data demonstrate that Pol2 pause factor NELF, Super Elongation Complex (SEC) components MLLT1/3, and the phosphatase module of the Integrator-protein phosphatase complex (INT-PP2A) are required for SHM, with MLLT1/3 but not NELF being required for AID binding to its chromatin targets. Our findings yield a new conceptual framework and model for AID-Pol2 collaboration in which NELF and a balance between kinase and phosphatase activities of SEC and INT-PP2A regulate Pol2 pausing/elongation to generate the critical stalled Pol2 complex on which AID acts. Further, our work has yielded major methodological advances that allow us to overcome obstacles that have stymied progress in the field. In this proposal, we take advantage of these conceptual and technical advances to pursue our central goal through the following two aims: Aim 1: Determine the molecular mechanisms by which NELF and other Pol2 regulatory factors enable AID-Pol2 collaboration and SHM/CSR. It has previously been very difficult to assess the role of cell-essential factors in SHM. By combining our new Rapid Assay for SHM (RASH) cells with degron technology, we will determine the mechanism of action of our newly discovered regulators of SHM using genomic, transcriptomic, and interaction assays that assess Pol2 distribution, phosphorylation, and activity, and the chromatin binding profiles of and interactions between AID and components of NELF, SEC, and INT-PP2A. AID and MLLT1 appear to co-associate in a complex and we will test for a direct interaction between AID and MLLT1/3. Factors will be tested for roles in CSR and validated in human cell line and germinal center B cell models and in mice. Aim 2: Hypothesis testing and deep mechanistic analysis through perturbation of the balance between Pol2 pause/arrest and elongation. We will rigorously test our new model for AID-Pol2 collaboration using degron, reconstitution, mutagenesis, and small molecular inhibitor approaches to perturb the balance between Pol2 pausing and elongation, revealing how altering NELF-Pol2 interactions and the balance between SEC kinase and INT-PP2A phosphatase activities influences SHM efficiencies and AID binding. Together, our proposed studies are significant for the development of new technologies and for understanding mechanisms of antibody gene diversification and causes of genome instability and cancer.