Locus contraction at the Ig kappa locus

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 DESCRIPTION (provided by applicant): The Igκ locus produces a wide array of light chain rearrangements, but how this complex process is regulated is not known. The creation of a diverse Igκ repertoire is facilitated by major changes in the 3-D structure of the Igκ locus calld "locus contraction" that occurs at the time of rearrangement. This compaction allows Vκ genes throughout the locus a chance to come into proximity with the Jκ genes to which one Vκ gene will rearrange in each pre-B cell. However, the factors which are important in orchestrating the 3D structure and long-range looping interactions of the Igκ locus in pre-B cells are not known. Furthermore, how the 3D topology affects Vκ gene utilization is not known. We have performed ChIP-seq in RAG-/-µ pre-B cells for a variety of histone + modifications, including H3K4me1, the characteristic epigenetic mark of enhancers. It was surprising to observe that there were so many peaks of H3K4me1 within the Vκ locus, most of which are not present in pro- B cells. This developmental stage-specificity of the H3K4me1 regions suggests that they may play a role in regulating Vκ rearrangement. Published studies indicate that there are some regions within the Vκ locus that are preferential sites of long-range interactions with iEκ, the enhancer located jut downstream of the 4 Jκ genes, and with 3'Eκ. We found that the most predominant long-range interaction hubs are characterized by broad regions of high H3K4me1 and H3K27ac, and ChIP-seq reveals that these hubs demonstrate binding of multiple key transcription factors (TF). We therefore hypothesized that some of these novel enhancer elements will be important in orchestrating the 3D configuration of the contracted Igκ locus, and further hypothesized that this will affect the composition of the Igκ repertoire. To test our hypotheses, we are deleting these potential regulatory regions in an Abl pre-B cell line using CRISPR/Cas9 genome editing. Culture of the Abl pre-B cells with STI571 robustly induces a diverse repertoire of Igκ rearrangements. We have already deleted the most prominent of these elements in the Abl cell line, and the pattern of rearrangement is strikingly different from that of the parental line. Base on these in vitro studies, we propose here to delete the most interesting ones by gene targeting in mice using CRISPR technology to make germline deletions of individual novel enhancer elements. Sorted pro-B cells and pre-B cells will be analyzed in depth, and the dynamic effects of the deletions of these enhancer-like elements on long-range interactions will be assayed by 3C and 4C, the effects on locus contraction by 3D-FISH, and the changes in the Igκ repertoire by deep sequencing. We hypothesize that deletion of enhancer-like hub regions will not only affect the 3D conformation of the Igκ locus, but will also profoundly affect the composition of th resulting Igκ repertoire. We have also determined that the CTCF sites within the Igκ locus are in mixed orientations, and there are two that we hypothesize may be critical for Igκ topology, and we will investigate them. These studies may provide insight into the reasons for unequal Vκ gene utilization, and may well be more generally applicable to understanding long-range activity of enhancers.