Investigating Cis and Trans Regulation of Complex Phenotypes

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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.