Genomic context sensitizes regulatory elements to genetic disruption

Abstract

SUMMARYEnhancer 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 murineIgf2/H19locus, a paradigmatic model of enhancer selectivity. We assembled payloads containing a 157-kb functionalIgf2/H19locus and engineered mutations to genetically direct CTCF occupancy at the imprinting control region (ICR) that switches the target gene of theH19enhancer cluster. Contrasting the activity of payloads delivered to the endogenous locus or to a safe harbor locus (Hprt) revealed that theIgf2/H19locus includes additional, previously unknown long-range regulatory elements. Exchanging components of theIgf2/H19locus with the well-studiedSox2locus showed that theH19enhancer cluster functioned poorly out of context, and required its native surroundings to activateSox2expression. Conversely, theSox2locus control region (LCR) could activate bothIgf2andH19outside 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 theH19enhancers are tightly coordinated within their native locus, theSox2LCR 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.