Contributions of BRWD1-mediated cohesin conversion to B cell biology - PROJECT SUMMARY In developing B cells, and in the germinal center (GC), genomic mechanisms of diversity must be strictly coordinated with those of proliferative selection. Failure to do so risks genomic instability and leukemic transformation. In the bone marrow (BM), this is done by ordering B lymphopoiesis into alternating and mutually exclusive states of either stochastic immunoglobulin gene (Ig) recombination or cell proliferation with selection. Likewise, GC B cells are divided into three subpopulations that occupy different niches and compartmentalize the incompatible functions of proliferation, somatic hypermutation and selection. In both the BM and GC, transit between proliferation and DNA mutation states requires large reordering of both genomic accessibility and transcription. In the BM, the epigenetic reader BRWD1 orchestrates the radical change in enhancer landscapes when cells exit proliferation and initiate Igk recombination. We have recently demonstrated that it does this by converting static to dynamic cohesin at topologically associating domain (TAD) boundaries. Dynamic cohesin then extrudes DNA across TADs to appose promoters and enhancers for gene activation and to contract Igk for recombination. We now provide preliminary evidence suggesting that BRWD1 is recruited to these TAD boundaries by specialized CTCF sites flanked by DNA GAGA sequences and that GAGA motifs are important for both BRWD1 recruitment and function. Finally, we demonstrate that deleting Brwd1 in GCs leads to disordered subsets and diminished function. Overall, our findings support a model in which BRWD1 orchestrates essential B cell functions, in both the BM and GC, by regulating dynamic cohesin-mediated chromatin topology to both determine enhancer landscapes and poise immunoglobulin genes for recombination. Specific Aims: Aim 1. To define BRWD1 GC functions and determine if these are associated with cohesin conversion. Hypothesis: We hypothesize that BRWD1 and cohesin conversion are used, and reused, to rewire B cell gene topology across cell state transitions. Aim 2: Determine the mechanisms and functional importance of BRWD1-mediated GAGA motif recognition. Hypothesis: BRWD1 recognizes GAGA motifs and this is important for both BRWD1 chromatin binding and chromatin remodeling. Aim 3. Determine how the recruitment of BRWD1 and cohesin are coordinated for cohesin conversion. Hypothesis: CTCF assembles BRWD1 with cohesin at TAD boundaries to orchestrate cohesin conversion.
