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

通过必需转录因子的定向诱变揭示糖皮质激素反应表型背后的新基因调控机制

• 批准号: 10449610
• 负责人: Graham Johnson
• 金额: $ 14.36万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-09 至 2022-09-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10449610
• 关键词: ATAC-seq; Address; Advisory Committees; Applications Grants; Award; Benign; Binding Proteins; Biochemical; Biological Assay; CRISPR/Cas technology; Cell Line; Cell physiology; Cells; ChIP-seq; Clustered Regularly Interspaced Short Palindromic Repeats; Code; DNA biosynthesis; Data; Development; Disease; Dissection; Endocrine; Endocrine System Diseases; Engineering; Enhancers; Feedback; Foundations; Funding; Future; Gene Expression; Genes; Genetic; Genetic Screening; Genetic Variation; Genomic approach; Genomics; Genotype; Glucocorticoid Receptor; Glucocorticoids; Goals; Health; Hormonal; Human; Individual; Institutes; Job Application; Journals; Laboratories; Leadership; Manuscripts; Measures; Mediator of activation protein; Mentors; Metabolic; Metabolic Diseases; Mutagenesis; Mutation; Nuclear Receptors; Nucleotides; Pathogenicity; Pathologic; Pathway interactions; Pharmacologic Substance; Phenotype; Physiological; Population; Positioning Attribute; Post-Translational Protein Processing; Postdoctoral Fellow; Prevalence; Proteins; Publications; Publishing; Publishing Peer Reviews; RNA; Regulator Genes; Reporter; Research; Research Personnel; Research Training; Resolution; Risk; SET Domain; Scientist; Secure; Signal Pathway; Signal Transduction; Statistical Data Interpretation; Stimulus; Technology; Testing; Tissues; Training; Transcript; United States National Institutes of Health; Untranslated RNA; Variant; Work; career; career development; clinical effect; clinically relevant; exhaustion; experience; functional genomics; genome editing; genome-wide; glucocorticoid receptor alpha; graduate student; high throughput screening; high throughput technology; hormonal signals; hormone therapy; immune function; individual response; loss of function; mutant; new technology; novel; programs; response; reverse genetics; single cell analysis; single cell technology; single-cell RNA sequencing; skills; steroid hormone; tenure track; transcription factor; transcriptome; transcriptome sequencing; whole genome

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.

摘要 激素信号和内分泌治疗对人类健康至关重要。遗传变异改变内分泌 这些反应会增加患上内分泌失调和代谢疾病的风险。主导 内分泌应答改变的潜在机制涉及转录因子（TF）的编码变体 调节这些信号通路。表征TF编码变体的调节作用， 仍然具有挑战性，从而限制了我们对关键内分泌途径如何控制的理解。改变 这个范例，这个建议的目的是确定致病性编码变异如何在 糖皮质激素受体（GR）是类固醇激素信号传导的主要和代表性调节因子，它改变了糖皮质激素受体的表达。 对糖皮质激素（GC）的基因组应答。GR中有数千种已知的突变， 还有更多的有待观察。对于几乎所有这些变体，对GR活性的影响是 未知此外，特定GR变体的患病率在不同人群中不同，并且可能导致GR基因突变。 对药物和生理刺激的各种个体反应通过GR发出信号。 该提案通过测试我的研究，直接解决了变异（GR突变体）如何影响表型（基因表达）的问题。 一个中心假设，即GR中的遗传变异通过修饰 与其他TF和辅助因子的相互作用。在初步数据的指导下，我将通过以下方式来检验我的中心假设： 实现两个具体目标。在目标1中，我将在一次试验中同时测量194 通过高通量反向遗传学筛选突变型GR基因型对GC反应基因表达的影响 平台我将测试GR编码变异，这些变异要么是致病的，排除翻译后修饰，要么是在 积极或消极选择的极端。GC响应转录组的变化将识别缺失- 功能缺失、亚病理性和良性编码突变，从而产生以下疾病的经验致病性评分： 每个变种在目标2中，我将测量特定的临床相关GR突变如何改变基因组应答 GCs，并最终扰乱三个独立的纯合标记的突变GR细胞系的细胞功能。 这些目标利用了我现有的技能和领域专业知识， 技术和统计分析，我需要实现科学独立。帮助我过渡到 作为一名独立的调查员，我组建了一个跨学科的咨询委员会， 我研究所的顶尖科学家这个委员会将对我的研究和专业进展提出建议， 包括提供关于终身教职工作申请和赠款的反馈。我会用我的成就 这个奖项是为了在高影响力的期刊上发表同行评审的手稿， R1研究所，并获得独立的NIH资助。完成本建议书中概述的研究培训 是我朝着领导一个独立实验室的长期目标迈出的重要一步， 确定遗传变异如何影响基因表达对内分泌信号的反应。