Locus contraction at the Ig kappa locus

 描述(申请人提供)：Igκ基因座产生一系列广泛的轻链重排，但这一复杂过程是如何调控的尚不清楚。在重排时，Igκ基因座的三维结构发生了重大变化，从而促进了多样化的Igκ谱系的创建。这种紧凑使整个座位的Vκ基因有机会接近Jκ基因，在每个前B细胞中，一个Vκ基因将重排到J DNA基因附近。然而，在协调前B细胞中Igκ基因座的3D结构和长程环状相互作用中起重要作用的因素尚不清楚。此外，3D拓扑对Vκ基因利用的影响尚不清楚。我们已经在RAG-/-µPre-B细胞中进行了各种组蛋白+修饰的芯片序列，包括H3K4me1，增强剂的特征表观遗传标记。令人惊讶的是，在V-κ基因座上有如此多的H3K4me1峰，其中大多数在Pro-B细胞中不存在。H3K4me1区的这种发育阶段特异性提示它们可能在调节Vκ重排中发挥作用。已发表的研究表明，Vκ基因座中有一些区域是与增强子Ieκ和3‘Eκ发生长程相互作用的优先区域。Ie RNAs是位于4个Jκ基因下游的增强子。我们发现，最主要的远程相互作用中心的特点是广泛的高H3K4me1和H3K27ac区域，芯片序列显示这些中心展示了多个关键转录因子(Tf)的结合。因此，我们假设其中一些新的增强子元件在协调收缩的Igκ位点的3D构型方面将是重要的，并进一步假设这 将影响Igκ曲目的组成。为了验证我们的假设，我们使用CRISPR/Cas9基因组编辑删除了Abl前B细胞系中这些潜在的调节区。用STI571培养ABLPre-B细胞可强烈诱导一系列不同的Igκ重排。我们已经删除了Abl细胞系中最突出的这些元件，重排模式与亲本系的重排模式明显不同。在这些体外研究的基础上，我们建议通过在小鼠中使用CRISPR技术进行基因打靶来删除最感兴趣的基因，从而对单个新的增强子元件进行种系删除。将深入分析分选的Pro-B细胞和前B细胞，并通过3C和4C分析这些增强子样元件缺失对远程相互作用的动态影响，通过3D-FISH分析对位点收缩的影响，并通过深度测序分析Igκ谱系的变化。我们推测，增强子样枢纽区域的缺失不仅会影响Igκ基因座的3D构象，而且还会深刻影响由此产生的Igκ谱系的组成。我们还确定了免疫球蛋白κ基因座内的ctcf位点在 混合取向，我们假设有两个可能对Igκ拓扑至关重要，我们将对它们进行研究。这些研究可能提供对Vκ基因利用不平等的原因的洞察，并且很可能更普遍地适用于理解增强子的长程活性。

 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.