Investigating Cis and Trans Regulation of Complex Phenotypes

研究复杂表型的顺式和反式调节

• 批准号: 10644267
• 负责人: Sophia Zebell
• 金额: $ 11.89万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10644267
• 关键词: Address; Affect; Affinity; Alleles; Architecture; Area; Award; Binding; Binding Proteins; Biological Assay; Biophysics; Breeding; Cell Culture Techniques; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Complex; Computational Biology; DNA; DNA Binding; DNA Binding Domain; DNA-Protein Interaction; Dependence; Development; Development Plans; Directed Molecular Evolution; Disease; Educational workshop; Engineering; Enhancers; Environment; Experimental Genetics; Family; Gene Expression; Gene Expression Regulation; Genetic; Genetic Epistasis; Genetic Models; Genetic Polymorphism; Genetic Research; Genetic Techniques; Genetic Variation; Genome; Genomics; Genotype; Health; Human; In Vitro; Individual; Laboratories; Learning; Linear Models; Link; Maps; Measurement; Mediating; Mentors; Modeling; Molecular; Molecular Biology; Molecular Genetics; Morphology; Outcome; Output; Pathway interactions; Phase; Phenotype; Physical assessment; Plants; Postdoctoral Fellow; Productivity; Professional Competence; Publications; Qi; Quantitative Genetics; Quantitative Trait Loci; Regulation; Regulator Genes; Research; Research Personnel; Research Training; Role; Site; Specificity; System; Technical Expertise; Testing; Titrations; Tomatoes; Training; Untranslated RNA; Variant; Whole Organism; Work; base editing; career; career development; cell transformation; dosage; experience; experimental study; flexibility; genetic resource; genetic variant; genome editing; genome wide association study; genome-wide; genomic variation; human disease; in vivo; inflorescence; insertion/deletion mutation; insight; member; mutant; pan-genome; prime editing; programs; therapeutic development; tissue culture; trait; transcription factor; transcription factor USF; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT Despite its importance, we do not understand the impact of variation in the regulatory genome on complex traits. Currently, the field lacks the genetic models necessary to address this relationship systematically, so little is known about the effect of quantitative variation in gene regulation on quantitative phenotypic output. Tomato development offers a high-throughput genetic system to address this gap; variation in a tomato enhancer produces a spectrum of gene expression and quantitative phenotypic output. The rationale of the Research Training Plan is to use this system to characterize the phenotypic and molecular consequences of genomic variation at the interface of transcription factor-cis regulatory motif binding. In Aim 1, the applicant Dr. Sophia Zebell will train with mentor Dr. Zachary Lippman in quantitative genetics during the K99 phase, using CRISPR base editing to generate deep sequence variation in cis-regulatory motifs, and characterizing the molecular and phenotypic consequences of variants with RNA-sequencing, quantitative phenotyping, and genotype-to-phenotype modeling. In Aim 2, Dr. Zebell will use epistasis analysis to investigate the quantitative genetic relationship between engineered and natural cis and trans regulatory variation, complementing genetic experiments with molecular biology to assess transcription factor-DNA binding affinities to motif variants, including massively multiparallel titration curves and microscale thermophoresis. In the R00 phase (Aim 3) Dr. Zebell will leverage the genetic interactions characterized in Aim 2 to address genetic variation in transcription factor DNA-binding domains of different families using a CRISPR-mediated suppressor screen approach to evolve factors that effectively bind cis-regulatory motif variants. Aim 3 will employ SNP- SELEX to assess binding of native and evolved transcription factors to pan-genome-wide cis-regulatory SNPs. Candidate Dr. Sophia Zebell is trained in the molecular biology of transcription factors, with six publications since 2013. The Career Development Plan will allow Dr. Zebell to gain new technical skills in high throughput genetics and genomics, tissue culture and transformation through mentored research and coursework, and career skills through workshops and practical experience. Mentor Dr. Zachary Lippman is an expert in genetics who has trained 10 productive postdocs, and Advisory Board members Dr. Joyce Van Eck (transformation), Dr. Yiping Qi (genome editing), Dr. Yuval Eshed (gene regulation), and Dr. Justin Kinney (computational biology/biophysics) offer complementary expertise. The highly collaborative, world-class quantitative genetics research environment of Cold Spring Harbor Laboratory and the Lippman lab are the ideal place to acquire this training. In summary, this proposal presents a plan for Research Training and Career Development to establish Dr. Zebell as an independent investigator while offering insights into an understudied area of the genome with important implications for a wide variety of human diseases.

项目概要/摘要 尽管它很重要，但我们不了解调控基因组的变异对复杂的影响。 特征。目前，该领域缺乏系统地解决这种关系所需的遗传模型，因此很少 已知基因调控的数量变异对数量表型输出的影响。番茄 开发提供了高通量遗传系统来弥补这一差距；番茄增强剂的变异 产生一系列基因表达和定量表型输出。研究的基本原理 培训计划是使用该系统来表征基因组的表型和分子后果 转录因子-顺式调控基序结合界面的变异。 在目标 1 中，申请人 Sophia Zebell 博士将与导师 Zachary Lippman 博士一起进行定量方面的培训 K99阶段的遗传学，使用CRISPR碱基编辑在顺式调控中产生深度序列变异 基序，并通过 RNA 测序表征变异的分子和表型后果， 定量表型分析和基因型到表型建模。在目标 2 中，Zebell 博士将使用上位分析 研究工程和天然顺式和反式调节之间的定量遗传关系 变异，用分子生物学补充遗传实验来评估转录因子-DNA 结合 对基序变体的亲和力，包括大规模多重平行滴定曲线和微尺度热泳。在 R00 阶段（目标 3）Zebell 博士将利用目标 2 中描述的遗传相互作用来解决遗传问题 使用 CRISPR 介导的抑制器改变不同家族的转录因子 DNA 结合域的变化 筛选方法来进化有效结合顺式调控基序变体的因子。目标 3 将采用 SNP- SELEX 用于评估天然和进化转录因子与全基因组顺式调节 SNP 的结合。 候选人 Sophia Zebell 博士接受过转录因子分子生物学方面的培训，拥有 6 个转录因子 自 2013 年以来发表的出版物。职业发展计划将使 Zebell 博士能够获得高新的技术技能 通过指导研究和指导进行通量遗传学和基因组学、组织培养和转化 通过研讨会和实践经验学习课程作业和职业技能。导师 Zachary Lippman 博士是 遗传学专家 Joyce Van Eck 博士培养了 10 名富有成效的博士后，顾问委员会成员 Dr. Joyce Van Eck （转化）、齐一平博士（基因组编辑）、Yuval Eshed 博士（基因调控）和 Justin Kinney 博士 （计算生物学/生物物理学）提供互补的专业知识。高度协作、世界一流 冷泉港实验室和李普曼实验室的数量遗传学研究环境是理想的 获得此培训的地方。总之，该提案提出了研究培训和职业计划 将 Zebell 博士确立为独立研究者，同时提供对未受研究对象的见解 对多种人类疾病具有重要影响的基因组区域。