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

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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.