TY - JOUR T1 - Dissection of a complex enhancer cluster at the <em>Sox2</em> locus JF - bioRxiv DO - 10.1101/2022.06.20.495832 SP - 2022.06.20.495832 AU - Ran Brosh AU - Camila Coelho AU - André M. Ribeiro-dos-Santos AU - Megan S. Hogan AU - Hannah J. Ashe AU - Gwen Ellis AU - Nicolette Somogyi AU - Raquel Ordoñez AU - Raven Luther AU - Emily Huang AU - Jef D. Boeke AU - Matthew T. Maurano Y1 - 2022/01/01 UR - http://biorxiv.org/content/early/2022/06/21/2022.06.20.495832.abstract N2 - A complex landscape of genomic regulatory elements underpins patterns of metazoan gene expression, yet it has been technically difficult to disentangle composite regulatory elements within their endogenous genomic context. Expression of the Sox2 transcription factor (TF) in mouse embryonic stem cells (mESCs) depends on a distal regulatory cluster of DNase I hypersensitive sites (DHSs), but the contributions of individual DHSs and their degree of independence remain a mystery. Here, we comprehensively analyze the regulatory architecture of the Sox2 locus in mESCs using Big-IN to scarlessly deliver payloads ranging up to 143 kb, permitting deletions, rearrangements and inversions of single or multiple DHSs, and surgical alterations to individual TF recognition sequences. Multiple independent mESC clones were derived for each payload, extensively sequence-verified, and profiled for expression of Sox2 specifically from the engineered allele. We found that a single core DHS comprising a handful of key TF recognition sequences is sufficient to sustain significant expression in mESCs, though its contribution is modulated by additional DHSs. Moreover, their overall activity was influenced by specific DHS order and/or orientation effects. We built a highly predictive model for locus regulation which includes nonlinear components indicating both synergy and redundancy among key DHSs. Our results suggest that composite regulatory elements and their influence on gene expression can be resolved to a tractable set of sequence features using synthetic regulatory genomics.Competing Interest StatementJ.D.B. is a Founder and Director of CDI Labs, Inc., a founder of and consultant to Neochromosome, Inc, a founder, SAB member of and consultant to ReOpen Diagnostics, LLC and serves or served on the Scientific Advisory Board of the following: Sangamo, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Tessera Therapeutics, Inc. and the Wyss Institute. ER -