Synthetic regulatory genomics uncovers enhancer context dependence at the Sox2 locus

• Published in: Molecular cell Vol. 83; no. 7; pp. 1140 - 1152.e7
• Main Authors: Brosh, Ran, Coelho, Camila, Ribeiro-dos-Santos, André M., Ellis, Gwen, Hogan, Megan S., Ashe, Hannah J., Somogyi, Nicolette, Ordoñez, Raquel, Luther, Raven D., Huang, Emily, Boeke, Jef D., Maurano, Matthew T.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 06.04.2023
• Subjects: Animals; CTCF; Enhancer Elements, Genetic; enhancers; Gene Expression Regulation; gene regulation; genetic engineering; genome writing; Genomics; Mice; Regulatory Sequences, Nucleic Acid - genetics; SOXB1 Transcription Factors - metabolism; stem cells; synthetic regulatory genomics; Transcription Factors - genetics; Transcription Factors - metabolism; genome writing; stem cells; enhancers; gene regulation; synthetic regulatory genomics; CTCF; genetic engineering
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2023.02.027

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. [Display omitted] •Synthetic regulatory genomics enables dissection of enhancer necessity and sufficiency•Replacing the Sox2 locus control region with a single DHS retains 30% of its activity•Neighboring DHSs synergistically modulate each other’s activity•Context-dependent DHSs cannot work alone but double the activity of neighboring DHSs Enhancer clusters are hallmarks of tissue-specific regulation, but the contributions of individual enhancers and their degree of interdependence remain unclear. Brosh et al. use synthetic regulatory genomics to repeatedly rewrite the Sox2 locus, dissecting its overall architecture and precisely delineating distinct roles for individual regulatory elements.