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

Fine-tuning of gene expression by transcription factors (TF) is essential for normal development, but how TF binding is converted into precise expression levels is poorly understood. We used a Drosophila enhancer responding to the TF Distal-less (Dll) to investigate this relationship. By combining computational structural analysis and quantifying relative TFBS affinity and enhancer activity we describe how TF concentration is converted into precise expression across developing Drosophila wings. We show that the regulatory function of the enhancer closely follow the classical Hill equation, and we describe the effects of affinity, orientation, sequence and wing-region on the maximum achievable expression and the position of transcriptional activity in a tissue. We also found that Dll relies on two distinct sets of amino acid-DNA contacts for 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 TF binding site determines the regulatory function of an enhancer in a complex in vivo environment.