Decoding Influences of Genetic Variation on Gene Regulation in Blood and Immune Cell Development

Abstract

Common and rare genetic variants often impact DNA-binding regulatory proteins such as transcription factors or cis-regulatory elements to predispose to human disease. Tremendous progress has been made in associating thousands of genetic variants with diverse phenotypes and diseases. However, dissecting the underlying causal mechanism of these variants remains one of the greatest challenges of the post-genomic era, significantly hindering opportunities for personalized medicine and the identification of novel therapeutic targets. Two main factors contribute to this challenge: (1) there is a lack of high-throughput assays to recreate large numbers of variants in the important setting of primary cells; (2) our understanding of the connectivity between cis-regulatory elements, transcription factors, and their target genes is limited. In this dissertation, I sought to tackle these obstacles directly in primary human hematopoietic cells, which comprise >93% of our bodies’ cells and are the target of numerous curative therapies. In Chapter 2, we develop massively parallel variants screens in primary human hematopoiesis. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. In Chapter 3, we present a framework to systematically infer gene regulation across single cells at steady-state, grounded in the stochasticity of transcriptional bursting and the time-shifted nature of regulatory kinetics. In Chapter 4, we develop a strategy to couple pooled perturbations of key hematopoietic transcription factors in primary hematopoiesis with single-cell gene expression and chromatin accessibility readouts. We discover that variants within transcription factor-sensitive networks contribute disproportionately to the heritability of blood cell phenotypes, remarkably higher than any other cell-state-dependent chromatin elements active throughout hematopoietic differentiation. Collectively, our work contributes to addressing one of the fundamental questions of population genetics and the post-genome-wide association study era: the leap from association to function.