Genomic context sensitizes regulatory elements to genetic disruption

Abstract

Enhancer function is frequently investigated piecemeal using truncated reporter assays or single deletion analysis, thus it remains unclear to what extent their function is influenced by surrounding genomic context. Using our Big-IN technology for targeted integration of large DNAs, we analyzed the regulatory architecture of the Igf2/H19 locus, a paradigmatic model of enhancer selectivity. We assembled payloads containing a 157-kb functional Igf2/H19 locus, engineering mutations to genetically direct CTCF occupancy at the imprinting control region (ICR) that switches the target gene of the H19 enhancer cluster. Contrasting their activity when delivered to the endogenous locus or to a safe harbor locus (Hprt) revealed that the functional elements comprising the Igf2/H19 locus are highly sensitive to their native context. Exchanging components of the Igf2/H19 locus with the well-studied Sox2 locus showed that the H19 enhancer cluster in particular functions poorly out of context, and required its native surroundings to activate Sox2. Conversely, the Sox2 locus control region (LCR) could activate Igf2 and H19, but its activity was only partially modulated by CTCF occupancy at the ICR. Analysis of regulatory DNA actuation across cell types showed that, while H19 is tightly coordinated within its native locus, the Sox2 LCR is more independent. We show that these enhancer clusters typify broader classes of loci genome-wide. Our synthetic regulatory genomics approach shows that unexpected dependencies may influence even the most studied functional elements and permits large-scale manipulation of complete loci to investigate how locus architecture relates to function.