biostudies-literatureFallon CUSC SBIR/STTR Planning AwardEuropean Molecular Biology OrganizationGraduate School of Quantitative Biosciences MunichDeutsche ForschungsgemeinschaftEuropean Fund for Regional DevelopmentNational Institutes of Healtheadz5902https://www.ebi.ac.uk/biostudies/studies/S-EPMC12778045biostudies-literatureUnknown12(2)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 transcrScience advancesQuantitative modulation of a spatial enhancer through the biophysical properties of a transcription factor binding site.PMC12778045#10377R35GM118336Funding to Light Microscopy Facility (LMF) of the Center for Molecular and Cellular Bioengineering of the Technical University DresdenQBMR35GM13037610377GO2495/11-1Rohs RGompel NKribelbauer-Swietek JFCeolin SFylaktakis APVeselinovic DLing LKothari MMann RSCooper BHGlassford WJDurmaz ADPapadopoulos DKMuhling BSrinivasan VFallon CChiu TPMitra RfalseQuantitative 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 transcr2026-01-01T00:00:00Z2026 Jan2026-06-09T05:27:46.848Z2026-06-09T03:07:47.285ZS-EPMC127780454149951310.1126/sciadv.adz5902