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（由适用提供）：IGκ基因座会产生各种光链重排，但是如何调节这种复杂过程。通过在重排时发生的Igκ基因座调用“基因座收缩”的3-D结构的重大变化制备了发散的Igκ曲目。这种压实使整个基因座的Vκ基因有机会与Jκ基因接近一个Vκ基因将在每个前B细胞中重新排列。然而，尚不清楚在策划3D结构和Igκ基因座的远程循环相互作用方面重要的因素。此外，3D拓扑如何影响Vκ基因的利用率。我们在抹布 - / - μPre-b细胞中进行了chip-seq，以进行多种组蛋白 +修饰，包括H3K4ME1，这是增强子的特征表观遗传标记。令人惊讶的是，观察到Vκ基因座中H3K4me1的峰值很多，其中大多数不存在于proB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重排方面发挥作用。已发表的研究表明，Vκ基因座的某些区域是与IEκ相互作用的优先位点，即4Jκ基因下游的增强子（增强子）以及3'Eκ。我们发现，最主要的远程相互作用中心的特征是高H3K4ME1和H3K27AC的广泛区域，而CHIP-SEQ则表明，这些枢纽表现出了多个关键转录因子（TF）的结合。因此，我们假设这些新型增强子元素中的某些对于策划了合同的Igκ基因座的3D配置，并进一步假设这一点 将影响Igκ曲目的组成。为了检验我们的假设，我们使用CRISPR/CAS9基因组编辑在ABL前B细胞系中删除了这些潜在的调节区域。具有STI571的ABL PER-B细胞的培养可牢固地诱导IGκ重排的不同曲目。我们已经删除了ABL单元线中这些元素中最突出的元素，并且重排的模式与父母系列的模式有很大的不同。基于这些体外研究，我们在这里建议通过使用CRISPR技术在小鼠中靶向基因来删除最有趣的研究，从而对单个新型增强子元素进行种系缺失。将深入分析排序的Pro-B细胞和PER-B细胞，并将通过3C和4C分配这些增强子样品对远程相互作用的缺失的动态效应，3D-FISH对基因座收缩的影响，以及通过深度测序对IGκ库的影响。我们假设删除增强子样中心区域不仅会影响Igκ基因座的3D构象，而且还会深刻影响所得IGκ库的组成。我们还确定Igκ基因座中的CTCF位点在 混合方向，我们假设有两个对于IGκ拓扑至关重要，我们将研究它们。这些研究可能会洞悉不平等基因利用的原因，并且很可能通常适用于理解增强子的远程活性。