Thursday, April 3, 2025
4/3/2025
Mechanisms of shadow enhancer robustness during development - Development is a remarkably robust process. Developmental gene regulatory networks can reproducibly pattern the embryo in spite of genetic and environmental variation and the inherent molecular noise in the system. Shadow enhancers – sets of enhancers that control a single gene and drive an overlapping spatio-temporal expression pattern – are a pervasive feature of developmental networks. Despite their apparent redundancy, case studies from Drosophila and mice show that shadow enhancers are required to drive normal development in the face of temperature stress or developmental gene knockouts. Systematic surveys find shadow enhancers control most developmental genes in fruit flies, mice, and humans. Despite their importance, we lack a complete picture of the features of shadow enhancers that allow them to drive robust developmental patterning. Many developmental diseases, e.g., autism or diabetes, seem to be caused by a combination of mutations and environmental influences. Since shadow enhancers seem vital for buffering the effects of genetic and environmental variation, uncovering their mechanisms may provide insight certain non- coding mutations combined with environmental stress lead to aberrant expression and disease. Our long-term goals are to find features of developmental networks that drive robust patterning and to learn why some mutations in enhancers cause disease while others are harmless. The central aim of this proposal is to test hypotheses about how the types, numbers, and organization of transcription factor (TF) binding sites among shadow enhancers impact their ability to drive consistent gene expression and, by extension, normal developmental patterning. We propose three Aims. Aim 1: To find functional requirements of shadow enhancers, we will build new sets of shadow enhancers and measure their mRNA dynamics. Aim 2: To generate predictive models of enhancer function, we will systematically perturb enhancers in vitro. Aim 3: To reveal the evolutionary trajectories of shadow enhancers, we will categorize the ages and sequence origins of shadow enhancers. These aims will connect the organization of TF binding sites in shadow enhancers to their function, enabling a deeper understanding of how a gene’s regulatory DNA affects its sensitivity to perturbation. We will use a variety of innovative approaches to achieve our goals, including novel synthetic transcription factors and high throughput enhancer assays. Given the prevalence of shadow enhancers in controlling diverse developmental processes, understanding their architecture and origins may rationalize why some developmental systems, e.g., cranio-facial patterning, are more fragile than others.
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