Novel regulatory regions within the immunoglobulin kappa locus

免疫球蛋白 kappa 基因座内的新调控区域

• 批准号: 8755573
• 负责人: ANN J FEENEY
• 金额: $ 28.43万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8755573
• 关键词: Abelson murine leukemia virus; Affect; Alleles; Antibodies; Antibody Repertoire; B-Lymphocytes; Binding; Binding Sites; Biological Assay; Cell Line; Cells; ChIP-seq; Characteristics; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Contracts; DNA Double Strand Break; DNA Sequence Rearrangement; Data; Development; Elements; Enhancers; Epigenetic Process; Future; Genes; Genetic Enhancer Element; Genome; Histones; Hour; IGK@ gene cluster; Immune system; Immunoglobulin Genes; Individual; Light; Luciferases; Mature B-Lymphocyte; Mediator of activation protein; Movement; Multiple Myeloma; Mutate; Mutation; Nucleic Acid Regulatory Sequences; Oncogenes; Play; Post-Translational Protein Processing; Process; Publishing; Regulation; Regulatory Element; Role; STI571; Site; Specificity; Staging; System; T-Cell Lymphoma; TCF3 gene; Technology; Testing; Time; V(D)J Recombination; cell type; combat; complement C4c; deep sequencing; homologous recombination; interest; kinase inhibitor; leukemia/lymphoma; novel; pathogen; promoter; public health relevance; research study; screening; three dimensional structure; transcription factor; vector

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.

描述（由申请人提供）：IG？基因座产生广泛的IG κ轻链重排，但这一复杂过程是如何调节的尚不清楚。创造一个多样化的IG？剧目是促进了重大变化的三维结构的IG？在重排时发生的称为“位点收缩”的位点。这种压缩允许V？整个基因座的基因有机会接近J？哪个基因是V？基因会在每个前B细胞中重排。然而，这是重要的因素，在编排的三维结构和长程循环的相互作用的IG？前B细胞中的基因座未知。此外，3D结构如何影响V？基因利用尚不清楚。我们已经在RAG-/-<$+前B细胞中进行了ChIP-seq，用于各种组蛋白翻译后修饰，包括H3 K4 me 1，增强子的特征性表观遗传标记。令人惊讶的是，观察到有这么多的峰H3 K4 me 1内的V？基因座，其中大部分不存在于pro-B细胞。这种发展阶段特异性的H3 K4 me 1地区表明，这些地区可能发挥作用，在调节V？重排发表的研究表明，有一些地区内的V？是与iE？发生长程相互作用的优先位点，增强子位于下游的4 J？基因.我们发现，最主要的长程相互作用中心的特征是广泛的高H3 K4 me 1区域，ChIP-seq显示这些中心显示出多种关键转录因子（TF）的结合，如Pax 5，EBF，E2 A，Ikaros，PU.1或YY 1。因此，我们推测，这些推定的新的调控元件中的一些将是重要的调节组成的IG？剧目，可能通过编排的3D配置， 收缩的IG？基因座我们将在pGL 3荧光素酶载体中测定这些区域的传统增强子活性。将对增强子区域内的特异性TF结合位点进行诱变，以确定哪些TF对于增强子区域内的TF结合是必需的。 活动为了验证我们的假设，即这些调控区可能通过改变基因座的3D结构来影响V基因库的组成，我们将删除推定的调控区。 在Abelson pre-B细胞系中，使用CRISPR/Cas9 PrecisionX同源重组基因组编辑技术，在这些区域内突变单个TF结合位点。Abl前B细胞系与β-激酶抑制剂STI 571培养48小时，强烈诱导了IG？重新安排将通过3C分析缺失或突变对远程相互作用的影响，并分析IG的变化？将通过TaqMan真实的时间PCR和深度测序来测定库。我们假设，删除关键TF结合位点和/或较大的调控区域与结合位点的几个关键TF将深刻影响所产生的IG？保留曲目。将确定这种效应是否主要是每个增强子/TF结合位点簇附近的基因，或更全面地影响。这将证明这些元件具有传统增强子的表观遗传特征的高度新颖的功能。