Locus contraction at the Ig kappa locus

Ig kappa 基因座的基因座收缩

• 批准号: 9231524
• 负责人: ANN J FEENEY
• 金额: $ 48.13万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9231524
• 关键词: 3-Dimensional; Abelson murine leukemia virus; Affect; Antibodies; Antibody Repertoire; Architecture; B-Lymphocytes; Binding; Binding Sites; Biological Assay; CRISPR/Cas technology; Cell Line; Cells; ChIP-seq; Characteristics; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Contracts; DNA Double Strand Break; Data; Development; Elements; Employee Strikes; Enhancers; Epigenetic Process; Family; Frequencies; Gene Targeting; Genes; Genetic Enhancer Element; Health; Histones; Immune system; Immunoglobulin Genes; In Vitro; Indium; Individual; Light; Location; Mediator of activation protein; Molecular Conformation; Movement; Mus; Mutant Strains Mice; Mutate; Mutation; Nucleic Acid Regulatory Sequences; Oncogenes; Pattern; Play; Post-Translational Protein Processing; Process; Publishing; Regulation; Regulatory Element; Research; Role; STI571; Site; Sorting - Cell Movement; Specificity; Staging; Structure; System; TCF3 gene; Technology; Testing; Time; V(D)J Recombination; base; combat; complement C4c; deep sequencing; genome editing; histone modification; in vitro Assay; insight; interest; kinase inhibitor; leukemia/lymphoma; novel; pathogen; three dimensional structure; transcription factor; zygote

 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.

 描述（由申请人提供）：Igκ 基因座产生多种轻链重排，但如何调节这一复杂过程尚不清楚。 3-D 结构的重大变化促进了多样化 Igκ 库的创建。 Igκ 基因座在重排时发生，称为“基因座收缩”，这种压缩使整个基因座中的 Vκ 基因有机会接近其中一个 Vκ 所在的 Jκ 基因。然而，对于协调前 B 细胞中 Igκ 基因座的 3D 结构和远程循环相互作用的重要因素，3D 拓扑如何影响 Vκ 基因尚不清楚。我们在 RAG-/-μ pre-B 细胞中进行了 ChIP-seq，检测各种组蛋白 + 修饰，包括增强子的特征性表观遗传标记 H3K4me1。 Vκ 位点内有很多 H3K4me1 峰，其中大部分不存在于原 B 细胞中。H3K4me1 区域的这种发育阶段特异性表明它们可能在调节 Vκ 重排中发挥作用。我们发现，Vκ 基因座内的一些区域是与 iEκ（位于 4 个 Jκ 基因下游的增强子）以及 3'Eκ 进行长程相互作用的优先位点。最重要的远程相互作用中心的特征是高 H3K4me1 和 H3K27ac 的广泛区域，ChIP-seq 揭示这些中心表现出多个关键转录因子 (TF) 的结合，因此我们追求其中一些新型增强子元件将。在协调收缩 Igκ 基因座的 3D 配置中发挥重要作用，并进一步追求这一点 为了验证我们的假设，我们使用 CRISPR/Cas9 基因组编辑技术删除 Abl pre-B 细胞系中的这些潜在调控区域。我们已经删除了 Abl 细胞系中最重要的这些元件，并且重排模式与亲本系显着不同。在这些体外研究中，我们在这里建议通过使用 CRISPR 技术对小鼠进行基因靶向删除最有趣的部分，对分选的前 B 细胞和前 B 细胞进行种系删除，并对其进行深入分析。我们将通过 3C 和 4C 分析这些增强子样元件的缺失对长程相互作用的动态影响，通过 3D-FISH 分析对基因座收缩的影响，并通过深度测序分析 Igκ 库的变化。准确地说，增强子样中枢的删除不仅会影响 Igκ 位点的 3D 构象，而且还会深刻影响由此产生的 Igκ 库的组成。我们还确定了 Igκ 位点内的 CTCF 位点。 混合方向，我们探索的两个可能对 Igκ 拓扑至关重要，我们将研究它们，这些研究可能会深入了解 Vκ 基因利用不平等的原因，并且很可能更普遍地适用于理解远程活性。的增强剂。