Uncovering novel gene regulatory mechanisms underlying glucocorticoid response phenotypes through targeted mutagenesis of an essential transcription factor

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ABSTRACT Hormone signaling and endocrine therapies are critical to human health. Genetic variation altering endocrine responses contributes to the risk of developing endocrine disorders and metabolic diseases. A predominant mechanism underlying altered endocrine responses involves coding variants in the transcription factors (TF) regulating these signaling pathways. Characterizing the regulatory effects of TF coding variants en masse remains challenging, thus limiting our understanding of how key endocrine pathways are controlled. To change this paradigm, the objective of this proposal is to determine how pathogenic coding variants in the glucocorticoid receptor (GR), a major and representative regulator of steroid hormone signaling, alter the genomic response to glucocorticoids (GCs). There are thousands of known mutations in the GR, and likely many more that have yet to be observed. For nearly all of those variants, the effects on GR activity are unknown. Further, the prevalence of specific GR variants differs across populations and may contribute to the diverse range of individual responses to pharmaceutical and physiological stimuli signaled though the GR. This proposal directly addresses how variation (GR mutants) impacts phenotype (gene expression) by testing my central hypothesis that genetic variation in the GR alters subsets of the genomic GC response by modifying interactions with other TFs and co-factors. Guided by preliminary data, I will test my central hypothesis by completing two Specific Aims. In Aim 1, I will simultaneously measure, in a single assay, the effects of 194 mutant GR genotypes on GC-responsive gene expression via a high-throughput reverse genetic screening platform. I will test GR coding variants that are either pathogenic, preclude post-translational modification, or at the extremes of positive or negative selection. Changes in the GC-responsive transcriptome will identify loss- of-function, sub-pathological, and benign coding mutations thus yielding an empirical pathogenicity score for each variant. In Aim 2, I will measure how specific clinically relevant GR mutations alter the genomic response to GCs and ultimately perturb cellular function in three independent homozygous tagged mutant GR cell lines. These Aims leverage my existing skill sets and domain expertise against training in genome editing, single-cell technologies, and statistical analyses I require to achieve scientific independence. To aid my transition to becoming an independent investigator, I have formed an interdisciplinary Advisory Committee composed of leading scientists at my institute. This committee will advise on my research and professional progress, including providing feedback on tenure-track job applications and grants. I will use my accomplishments under this award to publish peer-reviewed manuscripts in high impact journals, secure a tenure track position at an R1 institute, and obtain independent NIH funding. Completion of the research training outlined in this proposal is a significant step towards my long-term goal of leading an independent laboratory focused on determining how genetic variation influences gene expression responses to endocrine signals.