Comprehensive identification of E3 ubiquitin ligases that degrade heart, lung, and blood-relevant transcription factors

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Project Summary Faithful development and maintenance of individual cell types in the heart, lungs, and blood (HLB) are dependent upon cell type-specific decoding of the genome. These cell type-specific transcriptional programs are the result of the coordinated actions of many transcription factors (TFs), which regulate transcriptional output in a temporal and locus specific fashion. Due to their central role in HLB biology, a comprehensive molecular understanding of TF function would reveal the contribution of TFs to maintaining homeostasis, as well as inform how disruption of this balance can lead to the progression of HLB disorders. While methods to understand how TFs regulate gene expression exist, we lack methods and analytical tools to assay how TFs themselves are regulated post-translationally. By narrowly focusing on well-known phenotypes (DNA-binding) with well-developed methods (CHIP-seq, CUT&Tag), we fail to capture biological information at other regulatory levels, such as the post-translational control of TF degradation by E3 ubiquitin ligases. Importantly, this failure is not one of interest, but rather stems from the dearth of facile, scalable methods to study post-translation regulation of TFs. To address this critical shortcoming, I will develop a novel assay capable of measuring the effect of genetic perturbations on TF protein abundance at a scale that is not possible with current methods (Aim 1). I will initially apply this method to a library of 127 TFs previously demonstrated to have tissue specific expression in heart, lung, blood, bone marrow, or lymph node 1, and then subsequently scale the method to study the effect of genetic perturbation on 2000 TFs to comprehensively discover E3 ubiquitin ligases that regulate TF abundance. (Aim 2). Finally, I will map the minimal peptides necessary for observed changes in protein levels (Aim 3). This study will provide a target-linked map of E3-TF pairs, and lead to a broader understanding of the factors controlling transcription factor protein levels. Further, we will gain insights into the role of post-translational TF regulation in homeostasis as well as the mechanisms by which this homeostasis becomes dysregulated. Importantly, the E3-TF pairs discovered in this project will be of interest as therapeutic targets, with each pair representing a novel protein-protein interaction to target for stabilization or ablation. As a part of the fellowship, I will also undergo a training plan aimed at expanding my abilities as an experimental biologist as well as improving my knowledge of computational methods and statistics. The research and training plan will be performed in collaboration with my sponsor and co-sponsor, Dr. Jay Shendure and Dr. Ning Zheng, both at the University of Washington. My advisors were specifically chosen because they each have outstanding records of performing cutting edge research and training the next generation of scientific thought leaders.