Novel regulatory regions within the immunoglobulin kappa locus

描述（由申请人提供）：Ig？基因座产生一系列的Ig kappa轻链重排，但这个复杂的过程是如何调控的尚不清楚。创造一个多样化的Ig？由于Ig？位点被称为“位点收缩”发生在重排的时候。这种压缩允许V？整个基因座的基因有机会接近J？哪一个V基因？基因会在每个前b细胞中重新排列。然而，在协调Ig的3D结构和远程循环相互作用方面，重要的因素是什么？在前b细胞中的位点尚不清楚。此外，三维结构如何影响V？基因利用尚不清楚。我们在RAG-/-¿+前b细胞中对多种组蛋白翻译后修饰进行了ChIP-seq，包括H3K4me1，这是增强子的特征表观遗传标记。令人惊讶的是，在V？基因座，其中大部分不存在于前b细胞中。H3K4me1区域的发育阶段特异性表明，这些区域可能在调节V？重排。已发表的研究表明，在V？这些位点是与iE长期相互作用的优先位点。，增强子位于4j ？基因。我们发现，最主要的远程相互作用枢纽具有广泛的高H3K4me1区域，并且ChIP-seq显示这些枢纽可以结合多种关键转录因子（TF），如Pax5， EBF, E2A, Ikaros， PU.1或YY1。因此，我们假设这些假定的新调控元件中的一些在调节Ig？曲目，可能是通过编排的3D配置

DESCRIPTION (provided by applicant): The Ig? locus produces a wide array of Ig kappa light chain rearrangements, but how this complex process is regulated is not known. The creation of a diverse Ig? repertoire is facilitated by major changes in the 3-dimensional structure of the Ig? locus called "locus contraction" that occur at the time of rearrangement. This compaction allows V? genes throughout the locus a chance to come into proximity with the J? genes to which one V? 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 Ig? locus in pre-B cells are not known. Furthermore, how the 3D structure affects V? gene utilization is not known. We have performed ChIP-seq in RAG-/-¿+ pre-B cells for a variety of histone posttranslational modifications, including H3K4me1, the characteristic epigenetic mark of enhancers. It was surprising to observe that there were so many peaks of H3K4me1 within the V? locus, most of which are not present in pro-B cells. This developmental stage-specificity of the H3K4me1 regions suggests that these regions may play a role in regulating V? rearrangement. Published studies indicate that there are some regions within the V? locus that are preferential sites of long-range interactions with iE?, the enhancer located just downstream of the 4 J? genes. We found that the most predominant long-range interaction hubs are characterized by broad regions of high H3K4me1, and ChIP-seq reveals that these hubs demonstrate binding of multiple key transcription factors (TF) such as Pax5, EBF, E2A, Ikaros, PU.1 or YY1. We therefore hypothesize that some of these putative novel regulatory elements will be important in regulating the composition of the Ig? repertoire, possibly by orchestrating the 3D configuration of the contracted Ig? locus. We will assay these regions for traditional enhancer activity in a pGL3 luciferase vector. Specific TF binding sites within the enhancer regions will be mutagenized to determine which TF are essential for enhancer activity. To test our hypothesis that these regulatory regions will influence V gene repertoire composition, possibly by altering the 3D structure of the locus, we will delete putative regulatory regions, and also mutate individual TF binding sites within these regions, in an Abelson pre-B cell line, using CRISPR/Cas9 PrecisionX homologous recombination genome editing technology. Culture of the Abl pre-B cell line with the Abl-kinase inhibitor STI571 for 48 hours robustly induces a diverse repertoire of Ig? rearrangements. The effects of the deletions or mutations on long-range interactions will be assayed by 3C, and changes in the Ig? repertoire will be assayed by TaqMan real time PCR and by deep sequencing. We hypothesize that deletion of key TF binding sites and/or larger regulatory regions with binding sites for several key TF will profoundly affect the resulting Ig? repertoire. Whether this effect will be predominantly of genes in the vicinity of each enhancer/TF binding site cluster, or more globally, will be determined. This would demonstrate a highly novel function for these elements bearing the epigenetic characteristics of traditional enhancers.