Cornwall Scoones, Jake Sorel; (2025) Reconstructing gene regulation in neural development: a constructionist approach. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Stem cells possess a remarkable ability to differentiate into various cell types directed by external signals. Decoding these signals relies on cis-regulatory elements (CREs) controlling the activity of target genes in response to appropriate combinations and thresholds of signals. While differential transcription factor binding is known to control signal-dependent gene regulation, how CREs integrate the amount and combination of inputs to secure stable and accurate fate choices remains unclear. This gap in knowledge hinders our ability to engineer precise cellular responses to external signals. Here, I developed a high-throughput combinatorial screening strategy, termed NeMECIS, to investigate signal-dependent synthetic CREs (synCREs) by concatenating fragments of functional CREs. I used this constructionist approach to test whether synCRE activity could be decomposed into individual modular contributions and whether pairwise synergies were important. My exploration of synCRE design space revealed surprising flexibility and compatibility among regulatory sequence fragment combinations. Through statistical modelling, I established quantitative rules governing signal-dependent CRE activity: regulatory sequence fragments exhibit characteristic activities that multiply to achieve strong, signal-dependent CREs; these activities are tuned by synergies among fragment pairs; and these synergies often depend on spacing. These findings provide a predictive framework for CRE redesign. As a proof of concept, I predictably re-engineered a CRE involved in neural development, and used these synthetic regulatory elements to reprogramme motor neuron patterning and differentiation. The rules established in this study lay the foundation for creating synthetic DNA sequences to rewire GRNs and reprogramme stem cell differentiation, advancing our ability to manipulate cellular fate decisions in regenerative medicine and tissue engineering applications.

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