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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要转录是一个重要且严格调控的过程，需要许多因素的协调确保基因正确表达。当前的转录模型基于稳定的、分层的互动。这些模型受到体内成像最新发展的挑战，这些发展已研究表明许多转录调节蛋白与染色质短暂相互作用。而不是依赖稳定性，通过形成更频繁的相互作用来实现目标位点的占据原子核内的高局部浓度组件，称为中心。对于功能影响知之甚少转录中的中枢形成、中枢如何改变调节蛋白的动力学以及中枢如何在癌症中发挥作用，人类扩张重复疾病和其他疾病。以往的研究很大程度上依赖于异位目的蛋白的过度表达和研究中枢功能的定性测定，两者都缺乏通过可测量的功能输出和应用来扰乱枢纽形成/属性的具体策略研究体内蛋白质动力学的合适技术。该项目的目标是利用致癌突变在染色质读取蛋白 ENL 中发现，用于阐明中枢影响转录的机制。 ENL 突变是导致异常枢纽形成的致病性突变的首批例子之一。重要的是，这种枢纽的形成是靶基因过度激活的功能所必需的。特异性高 ENL 突变的功能获得性和功能获得性使它们成为研究这两种机制的强大系统中枢的形成以及异常中枢如何导致人类疾病。我假设 ENL 突变蛋白促进基因组和蛋白质组等多个元件的聚集，以改变目标位点的转录。在目标1，我将结合先进的成像技术，包括单分子跟踪和实时成像转录，以确定中心形成对掺入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使用 DNA-FISH 和实时成像来确定中枢是否驱动基因组重组以协调基因组目标位点的表达。该项目的完成将为致病突变如何产生提供新的见解异常枢纽形成并影响转录动力学以驱动疾病。更广泛地说，这项工作将增进我们对中枢介导的基因调控的理解，揭示新型治疗的潜力针对疾病中基因失调的策略。