Genes encoding B and T cell antigen receptors are assembled by DNA recombination in somatic cells. Functional immunoglobulin heavy chain (IgH) gene assembly requires two regulated recombination events. The first juxtaposes a DH to a JH gene segment; VH gene recombination follows, with VH gene segments rearranging to the pre-formed DJH junction. We have proposed that stepwise rearrangements of DH and VH gene segments is regulated by sequential changes in chromatin structure that occur accross the locus. ? ? The following studies were conducted during this fiscal year:? 1) To confirm, or refute, the hypothesis that intervening DH gene segments undergo repeat-induced gene silencing. We used the following approaches in parallel: a) down-regulation of RNAi components in pro-B cells using siRNAs; b) expression of 5'hExo in pro-B cells using transient and stable transfections; c) utilization of single-, and double strand-, specific RNases to detect DH-spcific ds RNAs in pro-B cells; d) analyses of the DH region in TSA-treated pro-B cells; e) analyses of DH transcripts in bone marrow pro-B cells from Dicer-deficient mice (in collaboration with Drs. K. Rajewsky and S. Muljo).? 2) To determine whether polycomb-mdeiated gene silencing affects interveninf DH gene segments. We developed chromatin immunoprecipitation assays using anti-H3K27me-, me2- and me3- specific antibodies. Mono- and dimethylated H3K27, but not tri-methylated H3K27, was detected at the intervening DH gene segments. ? 3) To quantitate the utilization of DH gene segments in primary recobination events. Two parallel approaches were utilized: first, we transiently expressed the RAG2 gene in a RAG2-deficient pro-B cell line, and analyzed recombination events as a function of time; second, we purified bone marrow precursor populations by flow cytometry (CLPs, Hardy fractions A+B, C and D) and quantitated DJH recombination in these cells.? 4) To examine the state of DNA methylation before, during and after DH to JH recombination. We used bisulfite modification to analyse CpG methylation across 50kb of the DH-Cm locus in primary bone marrow cells that represent different stages of B cell differentiation. Spleen B cells were taken as the final developmental stage. We also examined CpG methylation in DJH recombinants in CD4+CD8+ thymocytes.? 5) To conduct mechanistic studies on enhancer function and chromatin structure in-vitro. Chromatin substrates were generated using a fully recombinanat system with proteins expressed in bacteria and baculovirus. Recombinanat enhancer binding proteins were purified from bacteria and chromatin remodelling complexes from mammalian cells. We also studied VDJ recombination in this system using RAG1 and RAG2 purified from bacteria and 293T cells, respectively. ? ? The combination of in-vivo and in-vitro analyses is expected to provide a comprehensive view of genome activation, a crucial feature of normal human development and one that is often disrupted in diseased states.
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