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Enhancers are cis-regulatory elements that control the establishment of cell identities during development. In mammals, enhancer activation is tightly coupled with DNA demethylation. However, whether this epigenetic remodeling is necessary for enhancer activation is unknown. Here, we adapted single-molecule footprinting to measure chromatin accessibility and transcription factor binding as a function of the presence of methylation on the same DNA molecules. We leveraged natural epigenetic heterogeneity at active enhancers to test the impact of DNA methylation on their chromatin accessibility in multiple cell lineages. Although reduction of DNA methylation appears dispensable for the activity of most enhancers, we identify a class of cell-type-specific enhancers where DNA methylation antagonizes the binding of transcription factors. Genetic perturbations reveal that chromatin accessibility and transcription factor binding require active demethylation at these loci. Thus, in addition to safeguarding the genome from spurious activation, DNA methylation directly controls transcription factor occupancy at active enhancers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Gene activation requires the cooperative activity of multiple transcription factors at cis-regulatory elements (CREs). Yet, most transcription factors have short residence time, questioning the requirement of their physical co-occupancy on DNA to achieve cooperativity. Here, we present a DNA footprinting method that detects individual molecular interactions of transcription factors and nucleosomes with DNA in vivo. We apply this strategy to quantify the simultaneous binding of multiple transcription factors on single DNA molecules at mouse CREs. Analysis of the binary occupancy patterns at thousands of motif combinations reveals that high DNA co-occupancy occurs for most types of transcription factors, in the absence of direct physical interaction, at sites of competition with nucleosomes. Perturbation of pairwise interactions demonstrates the function of molecular co-occupancy in binding cooperativity. Our results reveal the interactions regulating CREs at molecular resolution and identify DNA co-occupancy as a widespread cooperativity mechanism used by transcription factors to remodel chromatin., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)