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

摘要 激素信号和内分泌治疗对人类健康至关重要。基因变异改变内分泌 应对措施会增加内分泌紊乱和代谢性疾病的风险。占主导地位的 内分泌反应改变的机制涉及转录因子(Tf)的编码变体 调节这些信号通路。表征Tf编码变异体的整体调控效应 仍然具有挑战性，从而限制了我们对关键内分泌途径是如何控制的理解。去改变 这一范例，这项建议的目标是确定致病基因编码变体如何在 糖皮质激素受体(GR)是类固醇激素信号的主要和代表性调节因子，它改变 基因组对糖皮质激素(GC)的反应。在GR中有数千个已知的突变，很可能 还有更多的问题有待观察。对于几乎所有这些变体，对GR活性的影响是 未知。此外，特定的GR变异体在不同人群中的流行率不同，可能导致 不同个体对药物和生理刺激的反应通过GR发出信号。这 提案通过测试My来直接解决变异(GR突变体)如何影响表型(基因表达) 中心假说，GR中的遗传变异通过改变基因组GC反应的亚群来改变 与其他转录因子和辅助因子的相互作用。在初步数据的指导下，我将通过以下方式检验我的中心假设 完成两个具体目标。在目标1中，我将在一次测试中同时测量194个 高通量反向遗传筛选突变型GR基因对GC反应基因表达的影响 站台。我将测试GR编码变体，这些变体要么是致病的，要么是排除翻译后修饰的，或者是 积极选择或消极选择的极端。GC反应转录组的变化将识别丢失- 功能、亚病理和良性编码突变，从而产生经验致病性分数 每一个变种。在目标2中，我将测量特定的临床相关GR突变如何改变基因组反应 并最终扰乱三个独立的纯合子标记突变GR细胞系的细胞功能。 这些目的是利用我现有的技能和领域专业知识来对抗基因组编辑，单细胞 技术，以及我实现科学独立所需的统计分析。来帮助我过渡到 作为一名独立调查员，我成立了一个跨学科咨询委员会，由 我所在研究所的顶尖科学家。这个委员会将就我的研究和专业进展提供建议， 包括就终身教职申请和补助金提供反馈。我将利用我的成就在 这一奖项是为了在影响较大的期刊上发表同行评议的手稿，确保在 R1研究所，并获得独立的NIH资金。完成本提案中概述的研究培训 是朝着我领导一个独立实验室的长期目标迈出的重要一步，该实验室专注于 确定基因变异如何影响基因表达对内分泌信号的反应。

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.