| Summary: | V(D)J recombination generates a diverse repertoire of immunoglobulin and T cell receptor genes by mixing and matching individual V, D and J gene segments. A critical step in this process is the correct recruitment of two gene segments into a synaptic complex, but how these gene segments, which can be can be up to 3 Mb apart in the genome, are brought together for recombination is poorly understood. An ideal model to monitor gene segment interaction is the murine immunoglobulin lambda locus (Igλ) as 70% of recombination occurs between just two gene segments, Vλ1 and Jλ1. Here, I describe the optimisation of two transgenic mouse models in which Igλ recombination can be studied to address these questions. To monitor the dynamics of these gene segments interacting for recombination, transcription activator-like effector (TALE) probes linked to fluorescent proteins and Gaussia luciferase were developed. As a prerequisite for this, I show that TALEs can bind to their target site, even when the binding site is within transcriptionally inactive chromatin. However, monitoring gene segment interactions using TALEs was unsuccessful due to high background fluorescence. Therefore, I investigated how Igλ is activated and folds to facilitate gene segment interaction by performing chromatin immunoprecipitation (ChIP) and chromosome conformation capture (3C) experiments. CTCF, cohesin and YY1 have been shown to be involved in the formation of long-range chromosome interactions and I present evidence that the lambda locus folds into a series of loops prior to the activation of recombination. Furthermore, I present evidence that co-ordinate activation of parallel enhancers could lead to the co-ordinate activation of non-coding transcription, a key regulator of V(D)J recombination, through the Vλ1 and Jλ1 gene segments. This work provides the first evidence for a model of how Igλ is activated and folds to facilitate gene segment interaction to enable V(D)J recombination.
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