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.

抽象的 激素信号传导和内分泌疗法对人类健康至关重要。遗传变异改变内分泌 反应有助于患上内分泌疾病和代谢疾病的风险。主要的 内分泌反应改变的机制涉及转录因子（TF）中的编码变体 调节这些信号通路。表征TF编码变体的调节效应。 仍然具有挑战性，从而限制了我们对如何控制关键内分泌途径的理解。改变 该范式是该提案的目的是确定致病性编码方式如何 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剂，改变了 对糖皮质激素（GCS）的基因组反应。 GR中有成千上万的已知突变，可能 还有更多尚待观察的东西。对于几乎所有这些变体，对GR活动的影响是 未知。此外，特定GR变体的流行率在人群之间有所不同，可能有助于 通过GR表示，对药物和生理刺激的个人反应范围各不相同。这 提案直接解决了通过测试我的变异（GR突变体）如何影响表型（基因表达） 中心假设是，GR中的遗传变异通过修饰来改变基因组GC反应的子集 与其他TF和共同因素的相互作用。在初步数据的指导下，我将通过 完成两个具体目标。在AIM 1中，我将在单个测定中同时测量194的影响 通过高通量反向遗传筛选的GC响应基因表达上的突变GR基因型 平台。我将测试致病性，排除翻译后修改或在AT的GR编码变体 积极或负选择的极端。 GC响应转录组的变化将确定损失 - 功能，下病理和良性编码突变，从而产生了经验的致病性评分 每个变体。在AIM 2中，我将衡量特定临床相关的GR突变如何改变基因组反应 在三个独立的纯合标签突变型GR细胞系中，对GCS并最终扰动细胞功能。 这些目标利用我现有的技能和领域专业知识，而不是基因组编辑中的培训 我需要实现科学独立性的技术和统计分析。帮助我过渡到 成为独立调查员，我成立了一个跨学科咨询委员会 我研究所的主要科学家。该委员会将为我的研究和专业进步提供建议， 包括提供有关终身任务申请和赠款的反馈。我会在下面利用我的成就 该奖项旨在在高影响期刊上发布同行评审的手稿，并在 R1研究所，并获得独立的NIH资金。该提案中概述的研究培训的完成 是朝着领导专注于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。