Mechanisms of pathogenic gene activation by aberrant transcriptional hubs formed by mutant ENL

突变ENL形成的异常转录中心激活致病基因的机制

• 批准号: 10750194
• 负责人: Kaeli Marie Mathias
• 金额: $ 4.77万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10750194
• 关键词: Acetylation; Affect; Behavior; Binding; Biological Assay; Biological Models; Cell Line; Cell Nucleus; Cells; Chromatin; Complex; DNA; DNA Polymerase II; Defect; Development; Disease; Elements; Embryo; Engineering; Ensure; GAS41 gene; Gene Activation; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomics; Goals; Health; Histone Acetylation; Human; Image; Imaging Techniques; Kinetics; Link; MLLT3 gene; Malignant Neoplasms; Measurable; Mediating; Messenger RNA; Methods; Modeling; Molecular; Molecular Conformation; Monitor; Mutagenesis; Mutation; Nature; Nephroblastoma; Oncogenic; Organism; Outcome; Output; Pathogenicity; Pathologic; Pattern; Phosphorylation; Physical condensation; Positioning Attribute; Process; Production; Property; Protein Overexpression; Proteins; Proteomics; RNA; Reader; Regulation; Regulator Genes; Resolution; Role; Series; Site; Specificity; System; Techniques; Technology; Testing; Time; Transcription Elongation; Transcription Regulatory Protein; Transcriptional Activation; Transcriptional Regulation; Variant; Visualization; Work; cellular imaging; gain of function; gene correction; genetic regulatory protein; human disease; in vivo; in vivo imaging; insight; interest; kidney cell; leukemia; molecular dynamics; molecular imaging; molecular scale; mutant; novel; novel therapeutic intervention; recruit; residence; single molecule; virulence gene

PROJECT SUMMARY Transcription is an essential and tightly regulated process that requires the coordination of many factors to ensure proper gene expression. Current models of transcription are predicated on stable, hierarchical interactions. These models have been challenged through recent developments in in vivo imaging, which have revealed that many transcriptional regulatory proteins interact transiently with chromatin. Instead of relying on stability, occupancy at target loci is achieved through more frequent interactions resulting from the formation of high local-concentration assemblies within nuclei, called hubs. Little is known about the functional impacts of hub formation on transcription, how hubs alter the kinetics of regulatory proteins and how hubs function in cancer, human expansion repeat disease, and other diseases. Previous studies largely rely on the ectopic overexpression of proteins of interest and qualitative assays to study hub function and there is a of lack of both specific strategies to perturb hub formation/properties with a measurable functional output and application of suitable technologies to look at protein kinetics in vivo. The goal of this project is to use oncogenic mutations found in the chromatin reader protein, ENL, to elucidate the mechanisms by which hubs impact transcription. ENL mutations are among the first examples of pathogenic mutations that result in aberrant hub formation. Importantly, such hub formation is functionally required for hyper-activation of target genes. The high specificity and gain-of-function nature of ENL mutations make them a powerful system to study both the mechanisms of hub formation as well as how aberrant hubs contribute to human disease. I hypothesize that ENL mutant proteins promote the clustering of multiple elements, both genomic and proteomic, to alter transcription at target loci. In Aim 1, I will combine advanced imaging techniques, including single molecule tracking and live imaging of transcription, to determine the effect of hub formation on the molecular kinetics of incorporated proteins and transcription dynamics. In Aim 2, I will investigate the effect of hub formation on the spatial proximity of target genes using DNA-FISH and live imaging to determine if hubs drive genome reorganization for coordinated expression of target loci. Completion of this project will offer novel insights as to how pathogenic mutations result in aberrant hub formation and affect transcriptional dynamics to drive disease. More broadly, this work will advance our understanding of hub-mediated gene regulation, revealing the potential for novel therapeutic strategies to target gene dysregulation in disease.

项目摘要 转录是一个必不可少且受严格调节的过程，需要许多因素协调 确保适当的基因表达。当前的转录模型基于稳定的层次结构 互动。这些模型通过在体内成像的最新发展中受到了挑战，这些模型具有 表明许多转录调节蛋白与染色质瞬时相互作用。而不是依靠 稳定性，目标基因座的占用是通过形成的更频繁的相互作用来实现的 核内的高局部浓度组件，称为集线器。关于功能影响的知之甚少 轮毂在转录时形成，集线器如何改变调节蛋白的动力学以及集线器在癌症中的作用， 人类扩张重复疾病和其他疾病。先前的研究很大程度上依赖于异位 对研究中心功能的感兴趣蛋白质和定性测定的过表达，并且缺乏两者 具有可测量的功能输出和应用的特定策略 合适的技术，可以在体内查看蛋白质动力学。该项目的目的是使用致癌突变 在ENL的染色质读取器蛋白中发现，以阐明轮毂影响转录的机制。 ELL突变是致病突变的第一个例子之一，导致异常中心形成。 重要的是，这种枢纽形成在功能上是靶基因过度激活所必需的。高特异性 ELL突变的功能性质使它们成为研究两种机制的强大系统 集线器形成以及异常中心如何促进人类疾病。我假设ELL突变蛋白 促进基因组和蛋白质组学多种元素的聚类，以改变目标基因座的转录。在 AIM 1，我将结合先进的成像技术，包括单分子跟踪和现场成像 转录，确定枢纽形成对掺入蛋白质和的分子动力学的影响 转录动力学。在AIM 2中，我将研究中心形成对目标空间接近的影响 使用DNA-FISH和LIVE成像的基因来确定HUBS是否驱动基因组重组以协调 目标基因座的表达。该项目的完成将提供有关致病性突变的新见解 在异常中心的形成中，影响转录动态以驱动疾病。更广泛地，这项工作将 促进我们对集线器介导的基因调节的理解，揭示了新型治疗的潜力 针对疾病中基因失调的策略。