1. K. Manoj, D. Jatkar, M. Ali Al-Radhawi, E.D. Sontag, and D. Del Vecchio. Paradoxical gene regulation explained by competition for genomic sites. 2026. Note: Submitted. Also bioRxiv 2025.11.27.691022. Keyword(s): resource competition, systems biology, synthetic biology, gene regulation. Abstract:

   Understanding how opposing regulatory factors shape gene expression is essential for understanding complex biological systems. A motivating observation, drawn from cancer epigenetics, is that removing an activating factor can sometimes lead to higher, not lower, expression of a gene that is also subject to a repressing factor. Prior theoretical work explained this counterintuitive behavior by competition of repressors and activators for genomic binding sites. However, it has been difficult to test this directly in natural systems, where layers of regulation obscure causal relationships. This paper introduces a fully synthetic, tunable genetic platform in a prokaryotic model system that reconstitutes this competition mechanism in a controlled and isolated setting. The genetic platform contains a target gene with binding sites for both an activator and a repressor, together with separate overlapping decoy binding sites for the same regulators. Activator and repressor functions are implemented using CRISPRa and CRISPRi, which permit independent control of regulator expression levels, design of the binding sites, and modulation of the binding affinities. Using this minimal system, we demonstrate that increasing activator expression level can reduce expression of the target gene when both regulators are present, consistent with the hypothesis that additional activator molecules displace the repressor from decoy sites, which becomes available to repress the target. By demonstrating how competition for genomic binding sites can invert expected regulatory responses, this synthetic framework provides a system for understanding similar paradoxical behaviors in natural regulatory networks and establishes a foundation for future studies in more complex mammalian contexts.

1. M. Ali Al-Radhawi, K. Manoj, D. Jatkar, A. Duvall, D. Del Vecchio, and E.D. Sontag. Competition for binding targets results in paradoxical effects for simultaneous activator and repressor action. In Proc. 63rd IEEE Conference on Decision and Control (CDC), pages 5579-5585, 2024. [PDF] Keyword(s): resource competition, epigenetics, systems biology, synthetic biology, gene regulatory systems. Abstract:

   In the context of epigenetic transformations in cancer metastasis, a puzzling effect was recently discovered, in which the elimination (knock-out) of an activating regulatory element leads to increased (rather than decreased) activity of the element being regulated. It has been postulated that this paradoxical behavior can be explained by activating and repressing transcription factors competing for binding to other possible targets. It is very difficult to prove this hypothesis in mammalian cells, due to the large number of potential players and the complexity of endogenous intracellular regulatory networks. Instead, this paper analyzes this issue through an analogous synthetic biology construct which aims to reproduce the paradoxical behavior using standard bacterial gene expression networks. The paper first reviews the motivating cancer biology work, and then describes a proposed synthetic construct. A mathematical model is formulated, and basic properties of uniqueness of steady states and convergence to equilibria are established, as well as an identification of parameter regimes which should lead to observing such paradoxical phenomena (more activator leads to less activity at steady state). A proof is also given to show that this is a steady-state property, and for initial transients the phenomenon will not be observed. This work adds to the general line of work of resource competition in synthetic circuits.

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