Collaborative Research: RoL: Revealing a new mechanism of action for eukaryotic transcriptional activation domains

这个生命规则项目旨在发现在真核细胞中打开基因的新分子机制。 几十年来的研究已经导致了传统的信念，即负责开启或激活基因的蛋白质被锁定在刚性形状中。 这些形状允许蛋白质与DNA或其他蛋白质相互作用，就像钥匙插入锁一样。 相比之下，该项目将探索一种新的想法，即激活蛋白质的形状不是刚性结构，这种刚性的缺乏使蛋白质能够在极其拥挤的细胞空间中运作，并以灵活的方式与基因DNA相互作用。 这一想法将使用实验室实验和计算方法相结合进行测试。 这些结果将提高对分子如何在细胞中相互作用的基本理解，从而为先进的基因编辑，个性化医疗或药物开发等应用提供概念基础。 该项目还将通过在巴特勒大学和普渡大学提供多学科的研究生和本科生STEM培训，并通过参与和组织系列研讨会和讲习班，在印第安纳州和其他邻近州建立生物信息学研究社区网络，从而产生教育影响。人们普遍认为，转录因子中的转录激活结构域通过直接招募共激活因子和转录起始来发挥作用复杂的组件。 已知转录激活结构域在其氨基酸序列中是非常异质的，它们具有固有的无序结构，并且它们具有低亲和力和特异性。 然而，其作用机制的细节仍然难以捉摸。 该项目将测试新的想法，即当在同一个转录因子中与高亲和力DNA结合结构域偶联时，转录激活结构域通过与核小体的低亲和力相互作用来触发染色质重塑，然后使核小体重塑和转录共激活因子的随后招募成为可能。 实验的目的是区分所谓的“核小体洗涤剂”模型和传统的直接招聘模型。 基因激活机制将通过创建和筛选除细胞存活所必需的关键基因激活剂的短（10-20个氨基酸）转录激活结构域之外的所有参数相同的大型复杂细胞文库来研究。 将在体内筛选单个文库，使用下一代测序技术在DNA水平上测序，并利用生物信息学和机器学习进行分析。 与关键文库携带基因激活子的分子内环境的改变或报告基因环境的改变相关的修饰将有助于提取转录激活结构域的关键特征和真核基因激活的生物学规则。该奖项由分子和细胞生物科学部的遗传机制项目、生物基础设施部的生物研究基础设施创新项目、该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准。

This Rules of Life project seeks to discover new molecular mechanisms for turning genes on in eukaryotic cells. Decades of previous research have led to the conventional belief that the proteins responsible for turning on, or activating, genes are locked into rigid shapes. These shapes allow the proteins to interact with DNA, or with other proteins, like a key fits into a lock. By contrast, this project will explore a new idea that the shape of activating proteins is not a rigid structure and that this lack of rigidity gives the proteins the ability to operate in extremely crowded cellular space and to interact in flexible ways with gene DNA. This idea will be tested using a combination of laboratory experiments and computational approaches. The results will improve fundamental understanding of how molecules interact with each other in the cell, thereby providing a conceptual foundation for applications such as advanced gene editing, personalized medicine or drug development. The project will also have educational impact by providing multidisciplinary graduate and undergraduate STEM training at Butler University and Purdue University and by building a network of bioinformatics research communities in Indiana and other neighboring states through participation and organization of seminar series and workshops.Transcriptional activation domains in transcription factors are widely believed to work via direct recruitment of coactivators and transcription initiation complex components. Transcriptional activation domains are known to be very heterogeneous in their amino acid sequences, they have intrinsically disordered structure, and they have low affinity and specificity. However, details of their mechanism of action remain elusive. This project will test the novel idea that when coupled in the same transcription factor with a high-affinity DNA binding domain, transcriptional activation domains function to trigger chromatin remodeling through low-affinity interactions with nucleosomes, which then enable subsequent recruitment of nucleosome remodelers and transcriptional co-activators. Experiments are designed to discriminate between the so-called "nucleosome detergent" model and the traditional direct recruitment model. Gene activation mechanisms will be studied by the creation and screening of large complexity libraries of cells identical in all parameters other than the short (10-20 amino acids) transcription activation domain of the key gene activator, that is necessary for cell survival. Individual libraries will be screened in vivo, sequenced at the DNA level using next generation sequencing techniques, and analyzed utilizing bioinformatics and machine learning. Modifications related to the change of the intramolecular context of the key library-carrying gene activator, or the change of the reporter gene context, will help to extract key features of the transcriptional activation domains and the biological rules of eukaryotic gene activation. This award was jointly funded by the Genetic Mechanisms Program in the Division of Molecular and Cellular Biosciences, by the Infrastructure Innovations for Biological Research Program in the Division of Biological Infrastructure, and by the Rules of Life initiative of the Division of Emerging Frontiers in the Biological Sciences Directorate.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.