The 3D-Structures of the Immunoglobulin Heavy Chain Locus

免疫球蛋白重链基因座的 3D 结构

• 批准号: 10364676
• 负责人: CORNELIS MURRE
• 金额: $ 38.16万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10364676
• 关键词: Address; Adopted; Affect; Amino Acids; Antibodies; Antigen Receptors; Architecture; B-Cell Development; B-Lymphocytes; Back; Binding; Binding Proteins; Binding Sites; Cell Line; Cells; Chromatin; Chromatin Structure; Code; Color; Crowding; DNA; Data; Development; Diffuse; Diffusion; Disease; Distal; Elements; Enhancers; Environment; Epigenetic Process; Frequencies; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic Recombination; Genetic Transcription; Genome; Genomic Segment; Genomics; Grant; Health; Heavy-Chain Immunoglobulins; Histone Acetylation; Histones; IGH@ gene cluster; Image; Label; Location; Maps; Measures; Monitor; Motion; Nuclear; Nuclear Lamina; Nucleic Acid Regulatory Sequences; Pattern; Phase; Positioning Attribute; Proteins; Receptor Gene; Regulatory Element; Role; S-nitro-N-acetylpenicillamine; Single Nucleotide Polymorphism; Site; Specificity; Speed; TCF3 gene; Time; Transferase; V(D)J Recombination; Virus; bcr-abl Fusion Proteins; chromatin remodeling; crosslink; experimental study; insight; mutant; novel strategies; promoter; recruit; stem cells; three dimensional structure; transcription factor

Antibodies are generated by somatic recombination involving variable (V), diversity (D) and joining (J) gene segments. During the previous grant cycle, we initiated studies aimed to visualize VH-DHJH interactions in live cells. Using single color labeling we were able to track the trajectories adopted by VH and DHJH elements. We found that VH and DHJH elements were subjected to fractional Langevin motion, in which VH and DHJH elements bounce back and forth in a spring-like fashion. However, since only a single genomic region was marked, this approach did not permit us to directly monitor VH-DHJH interactions. To directly visualize VH- DHJH encounters we developed a novel approach. We generated BCR-ABL transformed pro- B cell lines that carried tandem arrays of wild-type TET-operator and mutant TET-operator binding sites in the VH and DHJH regions, respectively. To mark the VH and DHJH regions these cells were transduced with virus expressing WT-TET GFP and MUT-TET SNAP-TAG. The results were surprising. VH-DHJH motion was severely sub-diffusive. We found that VH regions were trapped in distinct chromatin configurations that were remarkably stable (<60 minutes). Only VH regions located nearby DHJH regions had a chance for a VH-DHJH encounter. Comparison of simulated and experimental data suggested that such severely sub-diffusive motion was imposed by geometric confinement (loop domains) and phase separation/gelation (reversible cross-links within VH-DHJH and DH-JH loop domains). A caveat of these studies is that they were performed using BCR-ABL transformed pro-B cells. Here we propose to perform and extend these studies using primary pre-pro-B and pro-B cells. Specifically, we would track VH-DHJH motion but now in primary B cell progenitors. We would describe VH-DHJH motion in physical terms, including diffusion coefficients, scaling exponents, velocities and spatial confinement. We would image across long-time scales to examine how long VH and DHJH regions are trapped in distinct configurations and determine when (timing) and how (speed) such configurations change. We would measure first-encounter times as well as pairing times. We would examine whether transcriptional regulators and chromatin remodelers modulate VH-DHJH motion and first-encounter times. We would identify critical residues in RAG1 that would dictate pairing times. We would visualize VDJ recombination in live cells. Data obtained from these experiments would reveal whether and how space and time intersect to modulate the motion of paired coding and regulatory elements.

抗体通过涉及可变（V）、多样性（D）和可变性（V）的体细胞重组产生。 连接（J）基因片段。在上一个赠款周期，我们启动了旨在 可视化活细胞中的VH-DHJH相互作用。使用单色标签，我们能够跟踪 VH和DHJH元素采用的轨迹。我们发现VH和DHJH元件是 进行分数朗之万运动，其中VH和DHJH元件反弹， 以春天般的姿态出现。然而，由于仅标记了单个基因组区域， 方法不允许我们直接监测VH-DHJH相互作用。为了直接可视化VH- DHJH遇到我们开发了一种新的方法。我们产生了BCR-ABL转化的亲- 携带野生型TET操纵基因和突变型TET操纵基因串联阵列的B细胞系 分别在VH和DHJH区中的结合位点。为了标记VH和DHJH区域， 用表达WT-TETGFP和MUT-TETSNAP-TAG的病毒转导这些细胞。 结果令人惊讶。VH-DHJH运动严重亚扩散。我们发现， 区域被困在显着稳定（<60 分钟）。只有位于DHJH区域附近的VH区域才有机会与VH-DHJH结合。 遭遇模拟和实验数据的比较表明，这种严重 亚扩散运动是由几何约束（环域）和相位 分离/凝胶化（VH-DHJH和DH-JH环结构域内的可逆交联）。的还有告诫 这些研究的一个重要特点是使用BCR-ABL转化的pro-B细胞进行。这里我们 建议使用原代前原B和原B细胞进行和扩展这些研究。 具体来说，我们将跟踪VH-DHJH运动，但现在是在原代B细胞祖细胞中。我们 用物理术语描述VH-DHJH运动，包括扩散系数，标度指数， 速度和空间限制。我们将在长时间尺度上进行成像， 长VH和DHJH区域被捕获在不同的构型中，并决定何时（定时） 以及这些配置如何（速度）变化。我们也会测量第一次相遇的时间 作为配对时间。我们将研究转录调节因子和染色质 重塑者调节VH-DHJH运动和首次相遇时间。我们将确定关键的 RAG 1中决定配对时间的残基。我们可以想象VDJ重组， 活细胞从这些实验中获得的数据将揭示空间和时间是否以及如何 交叉以调节成对编码和调节元件的运动。