@article{ca179534c8f848758158bccf861457a8,
title = "Synthetic regulatory genomics uncovers enhancer context dependence at the Sox2 locus",
abstract = "Sox2 expression in mouse embryonic stem cells (mESCs) depends on a distal cluster of DNase I hypersensitive sites (DHSs), but their individual contributions and degree of interdependence remain a mystery. We analyzed the endogenous Sox2 locus using Big-IN to scarlessly integrate large DNA payloads incorporating deletions, rearrangements, and inversions affecting single or multiple DHSs, as well as surgical alterations to transcription factor (TF) recognition sequences. Multiple mESC clones were derived for each payload, sequence-verified, and analyzed for Sox2 expression. We found that two DHSs comprising a handful of key TF recognition sequences were each sufficient for long-range activation of Sox2 expression. By contrast, three nearby DHSs were entirely context dependent, showing no activity alone but dramatically augmenting the activity of the autonomous DHSs. Our results highlight the role of context in modulating genomic regulatory element function, and our synthetic regulatory genomics approach provides a roadmap for the dissection of other genomic loci.",
keywords = "CTCF, enhancers, gene regulation, genetic engineering, genome writing, stem cells, synthetic regulatory genomics",
author = "Ran Brosh and Camila Coelho and Ribeiro-dos-Santos, {Andr{\'e} M.} and Gwen Ellis and Hogan, {Megan S.} and Ashe, {Hannah J.} and Nicolette Somogyi and Raquel Ordo{\~n}ez and Luther, {Raven D.} and Emily Huang and Boeke, {Jef D.} and Maurano, {Matthew T.}",
note = "Funding Information: We thank Brendan Camellato, Leslie Mitchell, Sudarshan Pinglay, Weimin Zhang, Yu Zhao, and Yinan Zhu for their help with the yeast assembly. This work was partially funded by National Institutes of Health (NIH) grants RM1HG009491 (to J.D.B.) and R35GM119703 (to M.T.M.). R.B. A.M.R.-d.-S. M.S.H. J.D.B. and M.T.M. designed experiments. R.B. M.S.H. G.E. R.O. and R.D.L. performed experiments. C.C. M.S.H. N.S. R.B. and G.E. assembled DNA payloads. M.S.H. H.J.A. E.H. G.E. and R.D.L. performed sequencing. A.M.R.-d.-S. and M.T.M. performed computational analyses. R.B. A.M.R.-d.-S. and M.T.M. wrote the manuscript. R.B. J.D.B. and M.T.M. are listed as inventors on a patent application describing Big-IN. J.D.B. is a founder and Director of CDI Labs, Inc. a founder of and consultant to Neochromosome, Inc. a founder of, SAB member of, and consultant to ReOpen Diagnostics, LLC, and serves or served on the Scientific Advisory Board of the following: Logomix, Inc. Sangamo, Inc. Modern Meadow, Inc. Rome Therapeutics, Inc. Sample6, Inc. Tessera Therapeutics, Inc. and the Wyss Institute. Funding Information: We thank Brendan Camellato, Leslie Mitchell, Sudarshan Pinglay, Weimin Zhang, Yu Zhao, and Yinan Zhu for their help with the yeast assembly. This work was partially funded by National Institutes of Health (NIH) grants RM1HG009491 (to J.D.B.) and R35GM119703 (to M.T.M.). Publisher Copyright: {\textcopyright} 2023 Elsevier Inc.",
year = "2023",
month = apr,
day = "6",
doi = "10.1016/j.molcel.2023.02.027",
language = "English (US)",
volume = "83",
pages = "1140--1152.e7",
journal = "Molecular Cell",
issn = "1097-2765",
publisher = "Cell Press",
number = "7",
}