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

项目总结 转录是一个基本的和严格调控的过程，需要许多因素的协调来 确保适当的基因表达。目前的转录模型是基于稳定的、层次分明的 互动。这些模型受到了体内成像技术的最新发展的挑战，这些技术已经 揭示了许多转录调节蛋白与染色质短暂地相互作用。与其依赖于 稳定性，在目标位置的占有率是通过形成更频繁的相互作用而实现的 原子核内的高局部浓度聚集，称为中枢。人们对它的功能影响知之甚少 Hub在转录上的形成，Hub如何改变调节蛋白的动力学，Hub在癌症中如何发挥作用， 人类不断膨胀，反复患病，以及其他疾病。以前的研究在很大程度上依赖于异位 过表达感兴趣的蛋白质和定性分析来研究中枢功能，但两者都缺乏 利用可测量的功能输出和应用来扰动轮毂形成/特性的特定策略 研究体内蛋白质动力学的合适技术。这个项目的目标是利用致癌基因突变 在染色质阅读器蛋白ENL中发现，以阐明中枢影响转录的机制。 ENL突变是导致异常中枢形成的第一批致病突变之一。 重要的是，这种中枢的形成在功能上是超激活靶基因所必需的。高特异度 而ENL突变的功能获得性使其成为研究两者机制的有力系统。 中枢的形成以及异常中枢如何导致人类疾病。我推测ENL突变蛋白 促进多种元素的聚集，包括基因组和蛋白质组，以改变目标基因座的转录。在……里面 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HUB形成对被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接近度的影响 使用DNA-FISH和实时成像来确定枢纽是否驱动协调的基因组重组 靶基因座的表达。该项目的完成将为致病基因突变的结果提供新的见解 在异常的中枢形成中，并影响转录动力学以驱动疾病。更广泛地说，这项工作将 促进我们对HUB介导的基因调控的理解，揭示新的治疗方法的潜力 针对疾病中基因失调的策略。

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.