Genomic context sensitizes regulatory elements to genetic disruption

Enhancer function is frequently investigated piecemeal using truncated reporter assays or single deletion analysis. Thus it remains unclear to what extent enhancer function at native loci relies on surrounding genomic context. Using the Big-IN technology for targeted integration of large DNAs, we analyzed the regulatory architecture of the murine Igf2 / H19 locus, a paradigmatic model of enhancer selectivity. We assembled payloads containing a 157-kb functional Igf2 / H19 locus and engineered mutations to genetically direct CTCF occupancy at the imprinting control region (ICR) that switches the target gene of the H19 enhancer cluster. Contrasting activity of payloads delivered at the endogenous Igf2 / H19 locus or ectopically at Hprt revealed that the Igf2 / H19 locus includes additional, previously unknown long-range regulatory elements. Exchanging components of the Igf2 / H19 locus with the well-studied Sox2 locus showed that the H19 enhancer cluster functioned poorly out of context, and required its native surroundings to activate Sox2 expression. Conversely, the Sox2 locus control region (LCR) could activate both Igf2 and H19 outside its native context, but its activity was only partially modulated by CTCF occupancy at the ICR. Analysis of regulatory DNA actuation across different cell types revealed that, while the H19 enhancers are tightly coordinated within their native locus, the Sox2 LCR acts more independently. We show that these enhancer clusters typify broader classes of loci genome-wide. Our results show that unexpected dependencies may influence even the most studied functional elements, and our synthetic regulatory genomics approach permits large-scale manipulation of complete loci to investigate the relationship between locus architecture and function. HIGHLIGHTS ### Competing Interest Statement R.B., J.D.B. and M.T.M are listed as inventors on a patent application describing Big-IN. Jef Boeke is a founder and Director of CDI Labs, Inc., a founder of and consultant to Opentrons LabWorks/Neochromosome, Inc, and serves or served on the Scientific Advisory Board of the following: CZ Biohub New York, LLC, Logomix, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sangamo, Inc., Tessera Therapeutics, Inc. and the Wyss Institute.