Investigating Cis and Trans Regulation of Complex Phenotypes

研究复杂表型的顺式和反式调节

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要/摘要尽管它的重要性，但我们不了解调节基因组变异的影响特质。目前，该领域缺乏系统地解决这一关系所需的遗传模型，因此很少已经知道基因调控中定量变异对定量表型输出的影响。番茄开发提供了一个高通量遗传系统来解决这一差距。番茄增强剂的变化产生基因表达和定量表型输出的光谱。研究的理由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-CIS调节基序结合的界面上的变化。在AIM 1中，申请人Sophia Zebell博士将与导师Zachary Lippman博士进行培训。 K99阶段的遗传学，使用CRISPR基础编辑来产生顺式调节的深层序列变化主题，并表征带有RNA测序的变体的分子和表型后果，定量表型和基因型对表型建模。在AIM 2中，Zebell博士将使用上学分析研究工程和自然顺式和反式调节之间的定量遗传关系变异，补充分子生物学的遗传实验，以评估转录因子-DNA结合对基序变体的亲和力，包括大量的多行滴定曲线和微观嗜热。在 R00阶段（AIM 3）Zebell博士将利用AIM 2中特征的遗传相互作用来解决遗传使用CRISPR介导的抑制剂的转录因子DNA结合域的转录因子DNA结合域的变化筛选方法进化有效结合顺式调节基序变体的因素。 AIM 3将采用SNP- SELEX评估天然和进化的转录因子与泛基因组范围顺式调节SNP的结合。候选人Sophia Zebell博士接受了转录因子的分子生物学训练，有六个自2013年以来的出版物。职业发展计划将允许Zebell博士获得高级的新技术技能通过指导的研究和课程工作以及通过研讨会和实践经验的职业技能。导师Zachary Lippman博士是遗传学专家已经培训了10个生产性博士后，顾问委员会成员乔伊斯·范·埃克（Joyce Van Eck）博士（转换），Yiping Qi博士（基因组编辑），Yuval Eshed博士（基因调节）和Justin Kinney博士（计算生物学/生物物理学）提供互补的专业知识。高度协作的世界一流冷春港实验室和利普曼实验室的定量遗传学研究环境是理想的选择获得此培训的地方。总而言之，该提案介绍了研究培训和职业的计划开发以建立Zebell博士为独立研究人员，同时提供对研究不足的见解基因组的区域对各种人类疾病具有重要意义。