Quantitative modulation of a spatial enhancer through the biophysical properties of a transcription factor binding site

Fine-tuning of gene expression by transcription factors (TFs) is essential for normal development, but how TF binding is converted into precise expression levels is poorly understood. We used a <i>Drosophila</i> enhancer responding to the TF Distal-less (Dll) to investigate this relationship. By combining computational structure analysis and quantifying relative TFBS affinity and enhancer activity, we describe how TF concentration is converted into precise expression across the developing <i>Drosophila</i> wing. We show that, although the regulatory function of the enhancer generally follows the classical Hill equation, several additional terms are needed to describe the effects of affinity, orientation, sequence, and wing region on the maximum achievable expression and position of transcriptional activity in a tissue. We also found that Dll relies on two distinct sets of amino acid-DNA contacts for binding to higher affinity sites, but contacts are more evenly distributed at lower affinities. Our work integrates information at molecular and tissue scales to describe how the modulation of a single TFBS determines the regulatory function of an enhancer in a complex in vivo environment.