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

这个生命法则项目试图发现在真核细胞中开启基因的新分子机制。此前数十年的研究使人们普遍认为，负责启动或激活基因的蛋白质被锁定在僵硬的形状中。这些形状允许蛋白质与DNA或其他蛋白质相互作用，就像钥匙可以插入锁中一样。相比之下，这个项目将探索一种新的想法，即激活蛋白质的形状不是一个刚性结构，这种刚性的缺乏使蛋白质能够在极其拥挤的细胞空间中操作，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的方式进行测试。这些结果将提高对分子在细胞内如何相互作用的基本理解，从而为先进的基因编辑、个性化药物或药物开发等应用提供概念基础。该项目还将产生教育影响，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，并通过参与和组织研讨会系列和工作坊，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。转录因子中的转录激活域被广泛认为是通过直接招募辅助激活因子和转录起始复合体组件发挥作用的。已知转录激活结构域的氨基酸序列具有很强的异质性，具有内在的无序结构，亲和力和特异性低。然而，他们的行动机制的细节仍然难以捉摸。该项目将测试这一新想法，即当与相同的转录因子与高亲和力DNA结合域结合时，转录激活域通过与核小体的低亲和力相互作用来触发染色质重塑，从而使随后能够招募核小体重构体和转录共激活子。实验旨在区分所谓的核小体洗涤剂模式和传统的直接招募模式。基因激活机制将通过创建和筛选除关键基因激活剂的短(10-20个氨基酸)转录激活域以外的所有参数相同的大型复杂细胞文库来研究，这是细胞生存所必需的。单个文库将在体内进行筛选，使用下一代测序技术在DNA水平上进行测序，并利用生物信息学和机器学习进行分析。与携带关键文库的基因激活子的分子内上下文的变化或报告基因上下文的变化相关的修饰将有助于提取转录激活域的关键特征和真核基因激活的生物学规律。该奖项由分子和细胞生物科学部的遗传机制计划、生物基础设施部的生物研究基础设施创新计划和生物科学总监新兴前沿部门的生命规则倡议共同资助。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.