Synthetic regulatory genomics uncovers enhancer context dependence at the Sox2 locus

• DOI: 10.1016/j.molcel.2023.02.027
• Publication Date: 2023-03-16T14:53:13Z
• Authors: Ran Brosh; Camila Coelho; André M. Ribeiro-...; Gwen Ellis; Megan S. Hogan; Hannah J. Ashe; Nicolette Somogyi; Raquel Ordoñez; Raven D. Luther; Emily Huang; Jef D. Boeke; Matthew T. Maurano
• Keywords: 0301 basic medicine; Mice; 03 medical and health sciences; Enhancer Elements, Genetic; Gene Expression Regulation; SOXB1 Transcription Factors; Animals; Genomics; Regulatory Sequences, Nucleic Acid; Transcription Factors

AbstractExpression of Sox2 in mouse embryonic stem cells (mESCs) depends on a distal regulatory cluster of DNase I hypersensitive sites (DHSs), but their individual contributions and degree of independence remain a mystery. Here, we comprehensively analyze the regulatory architecture of Sox2 at its endogenous locus using Big-IN to scarlessly integrate DNA payloads ranging up to 143 kb. We analyzed 83 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 to establish the necessity and sufficiency of genomic features for Sox2 expression. We found that two LCR DHSs comprising a handful of key TF recognition sequences were each sufficient to autonomously sustain significant expression in mESCs. However, three additional LCR DHSs were entirely context-dependent, in that they showed no activity alone but could dramatically augment activity of the core DHSs. Our synthetic regulatory genomics approach demonstrates that composite regulatory elements can be reduced to a tractable set of essential sequence features, and is readily scalable to investigate regulatory architecture at other key loci genome-wide.