During FY12 we accomplished the following: 1. Completed analyses of chromatin structure and epigenetic state of DJH recombined IgH alleles. We found that DJH junctions were selectively marked by multiple forms of activation-associated chromatin modifications including histone modifications, DNA demethylation and nuclease sensitivity. These changes, which were induced by recombination, did not extend to un-rearranged DH gene segments located within 4kb 5 of the DJH junction. Importantly, recruitment of recombinase proteins, RAG1 and RAG2, closely mirrored the active chromatin state. We validated these results in primary pro-B cells purified from mouse bone marrow. These observations were incorporated into a unified model that explains three universal features of VH recombination. 2. We completed an analysis of bivalent chromatin generation and resolution during hematopoiesis to T lymphocyte lineage. We answered three outstanding questions in this study: 1) What precursor states do bivalent genes come from, and does this vary with developmental stage, 2) What state(s) do bivalent genes resolve to and does this vary with developmental state, and 3) Is bivalency a transitional intermediate between functionally opposing forms of histone modifications? Our observations highlight the complex dynamics of bivalent chromatin during hematopoeisis. We hypothesize that bivalency provides developmental flexibility at key decision-making junctures to divert hematopoiesis towards one or the other lineages in response to environmental or pathogen challenge. 3. We extended micro-ChIP studies to hematopoietic differentiation to the B lineage. Common lymphoid progenitors (CLP), pro-B and pre-B cell developmental stages were purified from bone marrow and assayed for genome-wide distribution of H3K4me3, H3K27me3 and H3K36me3. Computational analyses are underway. 4. We carried out micro-ChIP, analyses of T cell precursors obtained from the thymus of TCF1-deficient mice (collaboration with Jyoti Sen, NIA/IRP). Computational analyses are underway. 5. We re-calibrated use of DNase I for genome-wide studies of DNase I hypersensitive sites (DHS) in pro-B cell nuclei. Deep sequencing experiments carried out in FY11 indicated that DNase concentrations used were too high for assaying DHSs. Unfortunately, the optimal DNase I concentration could only be determined after going through the full experimental plan. Thus, we spent considerable time re-titrating the dose of DNase I required to obtain optimal information from these cells. 6. We used three different assays to determine the frequency of DH gene segment utilization in the first step of IgH gene assembly. All assays demonstrated highest frequency of DFL16.1 rearrangements, which is the DH gene segment that lies furthest (50 kb) from the JH gene segments. Utilization of intervening DSP gene segments fell with increasing distance from DFL16.1, despite gradually reducing the distance to JH. Thus, linear disposition along the chromosome does not determine frequency of DH recombination. Rather, we propose that spatial configuration of the pre-rearrangement IgH locus regulates DH gene usage.
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