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. Studies during this fiscal year analysed the detailed chromatin structure of the DH-Cmu domain within which the first steps of recombination occur. We found three interesting features. First, the domain boundaries are unlike those previously described; second, there is strikling chromatin structural hetrogeneity amongst the twelve DH gene segments; third, we identified a novel state of gene activation characterized by tissue-specific transcripts from an otherwise inactive locus. These observations have implications for the regulation of V(D)J recombination. We used in-vitro chromatin assembly, together with IgH enhancer-binding proteins, to understand how regulatory proteins affect chromatin structure of the IgH locus. We obtained the first evidence for the combinatorial control of chromatin structure by enhancer binding proteins. Specifically, we found that generation of a DNase1 hypersensitive site depends on the constellation, but not the number, of proeins that bind to plasmid DNA assembled into nucleosomes. The ability of proteins to generate, or disrupt, hypersensitivity was shown to depend on specific chromatin re-modelling activities that they recruited to the template DNA. 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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