Allosteric Regulation of V(D)J Recombination by the Active Chromatin Mark H3K4me3
Bettridge, John Thomas
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V(D)J recombination is initiated by the RAG recombinase; a heterotetrameric complex consisting of two RAG-1 and two RAG-2 subunits. The susceptibility of gene segments to cleavage by RAG is associated with histone modifications characteristic of active chromatin, including pan histone acetylation, and trimethylation of histone H3 at lysine 4 (H3K4me3). Binding of H3K4me3 by a plant homeodomain (PHD) in RAG-2 stimulates both DNA substrate binding and catalysis of the RAG-1 subunit. This has suggested an allosteric mechanism in which information regarding occupancy of the RAG-2 PHD is transmitted to RAG-1, thereby altering RAG-1’s activity. To determine whether the conformational distribution of RAG is altered by H3K4me3, we mapped changes in solvent accessibility of cysteine thiols by differential isotopic chemical footprinting using tandem liquid chromatography mass spectrometry. Binding of H3K4me3 to the RAG-2 PHD induces conformational changes in RAG-1 within the DNA-binding domains and in the ZnH2 domain, which acts as a scaffold for the catalytic center. These conformational changes are dependent on an intact PHD finger: mutation of tryptophan W453 in RAG-2 to alanine disrupts the RAG-2 PHD-H3K4me3 interacton, and renders RAG-2 unable to stimulate conformational changes in RAG-1. Thus, engagement of H3K4me3 by an intact RAG-2 PHD is associated with dynamic conformational changes in RAG-1, consistent with allosteric control by active chromatin. To reveal the interface through which RAG-2 transmits this allosteric signal to RAG-1, we generate chimeric murine RAG-2 proteins bearing plant homeodomains derived from distantly related shark RAG-2 PHD fingers. One of these chimeric RAG-2 proteins, bearing a PHD finger derived from C. punctatum, exhibits a loss of function similar to W453A, but retained the ability to bind H3K4me3. Selective reversion of amino acid residues 417 through 433 in the C. punctatum PHD finger back to murine sequence was sufficient to rescue the W453A-like phenotype, indicating the allosteric transmission interface lies within this region of RAG-2. These data support a model where RAG-2 engagement of H3K4me3 produces conformational changes within the surface of the RAG-2 PHD finger, which in turn directly stimulate the activity of RAG-1, “priming” the RAG-1 subunit for interactions with DNA.