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的和严格调控的过程，需要许多因素的协调， 确保基因正确表达。当前的转录模型是基于稳定的、分层的 交互.这些模型已经通过体内成像的最新发展受到挑战， 揭示了许多转录调控蛋白与染色质瞬时相互作用。而不是依靠 稳定性，在靶基因座的占据是通过更频繁的相互作用来实现的，所述相互作用是由 原子核内的高局部浓度聚集体，称为枢纽。关于这些功能的影响知之甚少， 枢纽在转录中的形成，枢纽如何改变调节蛋白的动力学以及枢纽如何在癌症中发挥作用， 人类扩张重复疾病和其他疾病。以前的研究主要依赖于异位 目的蛋白的过表达和研究枢纽功能的定性分析，但两者都缺乏 用可测量的功能输出干扰轮毂形成/性能的具体策略，以及 合适的技术来观察体内蛋白质动力学。这个项目的目标是利用致癌突变 在染色质阅读器蛋白ENL中发现，以阐明枢纽影响转录的机制。 ENL突变是导致异常枢纽形成的致病性突变的第一个例子。 重要的是，这样的枢纽形成是靶基因的超活化在功能上所需的。的高特异性 ENL突变的功能获得性质使其成为研究ENL突变机制的强大系统。 枢纽形成以及异常枢纽如何导致人类疾病。我假设ENL突变蛋白 促进基因组和蛋白质组多个元件的聚集，以改变目标基因座的转录。在 目标1，我将联合收割机结合先进的成像技术，包括单分子跟踪和实时成像， 转录，以确定枢纽形成对掺入的蛋白质的分子动力学的影响， 转录动力学在目标2中，我将研究枢纽形成对目标空间邻近度的影响 基因使用DNA-FISH和实时成像，以确定枢纽是否驱动基因组重组， 靶基因座的表达。该项目的完成将为致病突变如何导致 并影响转录动力学以驱动疾病。更广泛地说，这项工作将 推进我们对枢纽介导的基因调控的理解，揭示新的治疗潜力， 针对疾病中基因失调的策略。