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S. Rowan, T. Siggers, S. A. Lachke, Y. Yue, M. L. Bulyk, R. L. Maas; Use of Lower-Affinity Transcription Factor Binding Sites to Control Temporal Expression of Pax6 During Mammalian Lens Development. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6457.
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The mammalian developmental program is based on precisely modulated spatial and temporal levels of key transcriptional regulators. What is not well understood is how regulators biophysically interpret the affinities and composition of their DNA binding sites to mediate quantitative responses. Pax6 is an exquisitely dosage-sensitive transcriptional regulator that is essential for eye, brain, and pancreas development, and its expression is under precise transcriptional control during mammalian embryogenesis, particularly during lens induction.
We utilized a genetic approach to identify factors that control Pax6 expression in the developing lens, protein-binding microarrays to identify putative binding sites, and transgenic reporter assays to functionally assay identified binding sites.
We identified the Prep1 (pKnox1) homeodomain transcription factor as a critical and dose-dependent upstream regulator of Pax6 during lens formation. By utilizing protein-binding microarrays, we identified a pair of phylogenetically-conserved lower-affinity Prep1 binding sites, each uniquely required for Pax6 lens enhancer activity during development. We showed using both in vivo experimental data and mathematical modeling that Prep1 bound to the two sites works synergistically and not additively to regulate Pax6 transcription in response to the changing physiological concentration of Prep1. Furthermore, increasing the affinity of both sites led to premature high-level reporter activity, demonstrating that the affinity of the Prep1 binding sites dictates the timing of Pax6 lens enhancer activation.
We demonstrate in the novel context of mammalian development, an affinity-dependent mechanism, defined within the constraints of a simple biophysical model that controls the timing of gene expression. We propose that binding site affinity may be utilized broadly as a tunable mechanism to interpret changing levels of key regulators during mammalian development.
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