CBE Doctoral Dissertation Defense: “Molecular Mechanisms of Spatiotemporal Gene Control in Early Embryonic Development”

Development of a newly fertilized embryo is a dynamic, complex, and highly coordinated process that requires precise genetic regulation and control. As the embryo develops, DNA regulatory elements known as enhancers drive gene expression patterns in specific regions of an organism at a particular time. Mis-regulation of gene expression due to mutations in enhancers can result in severe disease phenotypes and developmental defects. However, understanding the fundamental mechanisms that activate and regulate precise transcription remains challenging. This thesis aims to elucidate the molecular mechanisms of enhancer-mediated transcriptional regulation in high spatiotemporal resolution in different genetic contexts.

Early development is largely controlled by maternally deposited proteins, while the zygotic genome remains transcriptionally silent. The transition from maternal to zygotic control has been extensively studied, yet we still lack a comprehensive understanding of the processes that result in zygotic genome activation. In Chapter 2, we identify distinct yet overlapping mechanisms of nuclear to cytoplasmic (N/C) ratio control on transcription driven by various enhancers under different genetic perturbations.

Enhancers contain unique binding sites for various proteins, known as transcription factors (TFs), that regulate spatiotemporal expression of a target gene. Many enhancers contain multiple binding sites for the same TF and the specific contribution of the various TF binding sites to the overall expression of a target gene is unclear. In Chapter 3, we characterize enhancer-mediated gene expression upon systematic modulation of TF binding sites. We find that mutating a single TF binding site results in a dramatic reduction in mRNA production. Through thermodynamic modeling, we uncover the synergistic capabilities of each binding site to the total transcriptional dynamics.

In this thesis we use a combination of live imaging, CRISPR/Cas9 genome editing technology, quantitative analysis, and mathematical modeling to explore a new world of gene regulation and transcriptional dynamics. By understanding the fundamental mechanisms that spatiotemporally control and modulate the expression of essential developmental genes, we can gain insights into gene mis-regulation and serious diseases.