Investigating Cis and Trans Regulation of Complex Phenotypes

项目概要/摘要 尽管它的重要性，我们不了解的影响，在调控基因组的复杂性， 性状目前，该领域缺乏系统地解决这种关系所必需的遗传模型， 已知基因调控中的数量变异对数量表型输出的影响。番茄 开发提供了一个高通量的遗传系统，以解决这一差距;在番茄增强子的变化， 产生基因表达谱和定量表型输出。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-顺式调节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博士一起进行定量培训。 在K99阶段，使用CRISPR碱基编辑在顺式调控基因中产生深度序列变异， 基序，并用RNA测序表征变体的分子和表型结果， 定量表型分析和基因型-表型建模。在目标2中，Zebell博士将使用上位性分析 研究工程和天然顺式和反式调节之间的数量遗传关系， 变异，用分子生物学补充遗传实验，以评估转录因子-DNA结合 对基序变体的亲和力，包括大规模多平行滴定曲线和微量热泳。在 在R 00阶段（目标3），Zebell博士将利用目标2中描述的遗传相互作用， 使用CRISPR介导的抑制子的不同家族的转录因子DNA结合结构域的变化 筛选方法以进化有效结合顺式调节基序变体的因子。目标3将采用SNP- SELEX评估天然和进化的转录因子与全基因组顺式调节SNP的结合。 候选人索菲亚·泽贝尔博士接受过转录因子分子生物学方面的培训， 自2013年以来出版。职业发展计划将使Zebell博士在高等教育中获得新的技术技能。 通过指导研究，生产遗传学和基因组学、组织培养和转化， 课程，并通过讲习班和实践经验的职业技能。导师扎卡里·李普曼博士是一位 遗传学专家，培养了10名富有成效的博士后，顾问委员会成员乔伊斯货车埃克博士 Yiping Qi博士（基因组编辑），Yuval Esed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。高度协作的世界级 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 地方来接受这种训练。总之，本提案提出了一个研究培训和职业生涯计划 发展建立博士. Zebell作为一个独立的调查员，同时提供深入了解一个被低估的 基因组中的一个区域，对多种人类疾病具有重要意义。

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.