Targeting of somatic hypermutation in the genome

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SUMMARY Somatic hypermutation (SHM) generates point mutations in immunoglobulin (lg) genes and is vital for the generation of high affinity antibodies, vaccine efficacy, and protection against infection. The reaction is initiated when the activation induced deaminase (AID) deaminates cytidines in single-stranded DNA in the context of transcription by RNA polymerase 2 (Pol2). Mis-targeting of SHM contributes to genetic instability and the development of a range of B cell malignancies. Understanding the factors and mechanisms that regulate and target AID/SHM is one of the most important objectives in the study of humoral immunity and is the central goal of research under this grant Our recent studies have yielded a new conceptual framework for SHM targeting in which genome looping processes bring SHM targeting ("DIVAC") elements with their bound transcription factors (TFs) to the target locus where they stall Pol2, thereby enabling AID action. Further, our preliminary 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: Gene Discovery-Identify new factors involved in SHM and that bind DIVAC elements in vivo. Few factors involved in SHM targeting are known and no SHM CRISPR/Cas9 screens have been reported. Furthermore, the TFs that bind DIVAC in vivo to mediate SHM are unknown. We will use novel Rapid Assay for SHM (RASH) cell lines to perform CRISPR knockout and activation screens to identify SHM factors, and in vivo proximity labeling and quantitative proteomics to identify factors that associate with active DIVAC elements. Factors identified will be validated in multiple human cell line models and in mice. Aim 2: Hypothesis Testing and Mechanism-Determine the contribution and mechanism of action of chromatin architecture factors, TFs, and TF activation domains in SHM. It has not previously been possible to assess the role of cell essential factors in SHM. By combining our new Precision Assessment of AID Targeting (PAAT) assay with degron technology, we will rigorously test the role of chromatin architectural proteins, major B cell TFs, and factors identified in Aim 1 for their role in SHM across the entire genome. Mechanism of action will be assessed through genome wide assays that assess parameters of transcription, epigenetic marks, factor binding, and chromatin looping. Genome "AID activity maps" will reveal the contribution of each factor to AID/SHM targeting at lg and non-lg loci. Finally, we take advantage of a novel SHM reconstitution system to test the hypothesis that DIVAC-bound TFs target SHM using intrinsically disordered activation domains to create a phase-separated condensate. 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.