The role of non-coding RNA transcription in immunoglobulin rearrangement and antibody diversity

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Recombination of multiple genes in the immunoglobulin and T cell receptor DNA loci generates the vast repertoire of antibodies and T cell receptors required for a functional immune system. The focus of our research is to understand the chromatin remodelling mechanisms that open up these antigen receptor loci, to facilitate V(D)J recombination in lymphocytes. Since these DNA loci are the largest in the genome, containing hundreds of genes, they have evolved several dynamic processes to ensure appropriate chromatin opening, including nuclear relocalisation (movement within the nucleus), extensive histone modification, non-coding RNA transcription, 3D DNA looping (to bring distal genes together). Thus they also provide an excellent paradigm for chromatin regulation of all multigene loci. In particular, eukaryotic genomes have recently been shown to produce an enormous number of non-coding RNAs, which are thought to play a vital role in many nuclear processes, including developmental regulation of gene expression. However, the mechanisms are poorly understood. We have shown that large non-coding RNA transcripts are generated in the immunoglobulin heavy chain V (variable) region prior to V to DJ recombination (Bolland et al 2004, Nature Immunology 5; 630-637), and in the D (diversity) region prior to D to J recombination (Bolland et al 2007, Mol Cell Biol 27:5523-33). We are testing the hypothesis that this transcription opens up closed immunoglobulin chromatin to make it accessible to the recombinase enzymes for V(D)J recombination. We have generated mouse models in which this transcription is interrupted. The aim of this project will be to determine the effect of transcription loss on D to J and V to DJ recombination, and to discover which of the processes above are inhibited. State-of-the-art techniques including high-throughput FISH (fluorescence in situ hybridization), chromatin immunoprecipitation and next generation sequencing will be used.

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