Abstract
The bulk of gene expression regulation in most organisms is accomplished through the action of transcription factors (TFs) on cis-regulatory elements (CREs). In eukaryotes, these CREs are generally characterized by nucleosomal depletion and thus higher physical accessibility of DNA. Many methods exploit this property to map regions of high average accessibility, and thus putative active CREs, in bulk. However, these techniques do not provide information about coordinated patterns of accessibility along the same DNA molecule, nor do they map the absolute levels of occupancy/accessibility. SMF (Single-Molecule Footprinting) fills these gaps by leveraging recombinant DNA cytosine methyltransferases (MTase) to mark accessible locations on individual DNA molecules. In this chapter, we discuss current methods and important considerations for performing SMF experiments.
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Acknowledgements
The authors thank members of the Greenleaf, Bintu and Kundaje labs for many helpful discussions. This work was supported by NIH grants (P50HG007735, RO1 HG008140, U19AI057266, and UM1HG009442 to W.J.G., 1UM1HG009436 to W.J.G. and A.K., 1DP2OD022870-01 and 1U01HG009431 to A.K., and HG006827 to C.H.), the Rita Allen Foundation (to W.J.G.), the Baxter Foundation Faculty Scholar Grant, and the Human Frontiers Science Program grant RGY006S (to W.J.G). W.J.G is a Chan Zuckerberg Biohub investigator and acknowledges grants 2017-174468 and 2018-182817 from the Chan Zuckerberg Initiative. Fellowship support provided by the Stanford School of Medicine Dean’s Fellowship (G.K.M.).
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Hinks, M., Marinov, G.K., Kundaje, A., Bintu, L., Greenleaf, W.J. (2023). Single-Molecule Mapping of Chromatin Accessibility Using NOMe-seq/dSMF. In: Marinov, G.K., Greenleaf, W.J. (eds) Chromatin Accessibility. Methods in Molecular Biology, vol 2611. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2899-7_8
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DOI: https://doi.org/10.1007/978-1-0716-2899-7_8
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