biostudies-literatureDoughty BRNHLBI NIH HHSNHGRI NIH HHSNINDS NIH HHSNIGMS NIH HHS745-754https://www.ebi.ac.uk/biostudies/studies/S-EPMC12326879biostudies-literatureUnknown636(8043)The binding of multiple transcription factors (TFs) to genomic enhancers drives gene expression in mammalian cells¹. However, the molecular details that link enhancer sequence to TF binding, promoter state and transcription levels remain unclear. Here we applied single-molecule footprinting^2,3 to measure the simultaneous occupancy of TFs, nucleosomes and other regulatory proteins on engineered enhancer-promoter constructs with variable numbers of TF binding sites for both a synthetic TF and an endogenous TF involved in the type I interferon response. Although TF binding events on nucleosome-free DNA are independent, activation domains recruit cofactors that destabilize nucleosomes, driving observed TF binding cooperativity. Average TF occupancy linearly determines proNatureSingle-molecule states link transcription factor binding to gene expression.PMC12326879T32 GM145402R01 HL171611R35 GM128947P50 HG007735R01 HG013317DP1 HG013599UM1 HG009436R01 NS128028Parks BEMarklund EGreenleaf WJTan YDubocanin DBintu LMarinov GKDoughty BRHinks MMSchaepe JMRios-Martinez CThurm ARfalseSingle-molecule states link transcription factor binding to gene expression.The binding of multiple transcription factors (TFs) to genomic enhancers drives gene expression in mammalian cells¹. However, the molecular details that link enhancer sequence to TF binding, promoter state and transcription levels remain unclear. Here we applied single-molecule footprinting^2,3 to measure the simultaneous occupancy of TFs, nucleosomes and other regulatory proteins on engineered enhancer-promoter constructs with variable numbers of TF binding sites for both a synthetic TF and an endogenous TF involved in the type I interferon response. Although TF binding events on nucleosome-free DNA are independent, activation domains recruit cofactors that destabilize nucleosomes, driving observed TF binding cooperativity. Average TF occupancy linearly determines pro2024-01-01T00:00:00Z2024 Dec2026-06-06T07:19:13.727Z2026-06-06T03:06:53.885ZS-EPMC123268793956768310.1038/s41586-024-08219-w