Content area
Regulatory elements in the genome, such as enhancers and promoters, encode sequences that orchestrate when, in which cell types, and to what extent all human genes are expressed. The activities of these sequences are fine-tuned by their endogenous chromatin context, which is crucial for quantitative, spatiotemporal regulation of gene expression. However, mapping and decoding the sequences in regulatory elements responsible for cell type-specific gene expression remains challenging due to the lack of high-throughput tools for precisely measuring the quantitative effects of sequence edits directly on gene expression in endogenous context.
In Chapter 1, I provide a concise introduction of the intricate relationship between regulatory DNA sequence, chromatin context, and cell type-specific gene regulation, and the capabilities and limitations of the technologies currently used to study them. In Chapter 2, I describe Variant-EFFECTS, a transformative technology we developed to introduce hundreds of designed edits to endogenous regulatory DNA and quantify their effects on gene expression. Through a series of proof-of-concept screens, we systematically dissected and reprogrammed 3 regulatory elements for 2 genes in 2 cell types. These data revealed endogenous binding sites with effects specific to genomic context, transcription factor motifs with cell type-specific activities, and limitations of computational models for predicting the effect sizes of variants. We identified small edits that can tune gene expression over a large dynamic range, suggesting new possibilities for prime editing-based therapeutics targeting regulatory DNA. Variant-EFFECTS provides a generalizable tool to dissect regulatory DNA and to identify genome editing reagents that tune gene expression in an endogenous context.