Genomic context sensitizes regulatory elements to genetic disruption.

Genomic context critically modulates regulatory function but is difficult to manipulate systematically. The murine insulin-like growth factor 2 (Igf2)/H19 locus is a paradigmatic model of enhancer selectivity, whereby CTCF occupancy at an imprinting control region directs downstream enhancers to activate either H19 or Igf2. We used synthetic regulatory genomics to repeatedly replace the native locus with 157-kb payloads, and we systematically dissected its architecture. Enhancer deletion and ectopic delivery revealed previously uncharacterized long-range regulatory dependencies at the native locus. Exchanging the H19 enhancer cluster with the Sox2 locus control region (LCR) showed that the H19 enhancers relied on their native surroundings while the Sox2 LCR functioned autonomously. Analysis of regulatory DNA actuation across cell types revealed that these enhancer clusters typify broader classes of context sensitivity genome wide. These results show that unexpected dependencies influence even well-studied loci, and our approach permits large-scale manipulation of complete loci to investigate the relationship between regulatory architecture and function.

Enhancer selectivity across cell types delineates three functionally distinct enhancer-promoter regulation patterns.

BACKGROUND: Multiple enhancers co-regulating the same gene is prevalent and plays a crucial role during development and disease. However, how multiple enhancers coordinate the same gene expression across various cell types remains largely unexplored at genome scale. RESULTS: We develop a computational approach that enables the quantitative assessment of enhancer specificity and selectivity across diverse cell types, leveraging enhancer-promoter (E-P) interactions data. We observe two well-known gene regulation patterns controlled by enhancer clusters, which regulate the same gene either in a limited number of cell types (Specific pattern, Spe) or in the majority of cell types (Conserved pattern, Con), both of which are enriched for super-enhancers (SEs). We identify a previously overlooked pattern (Variable pattern, Var) that multiple enhancers link to the same gene, but rarely coexist in the same cell type. These three patterns control the genes associating with distinct biological function and exhibit unique epigenetic features. Specifically, we discover a subset of Var patterns contains Shared enhancers with stable enhancer-promoter interactions in the majority of cell types, which might contribute to maintaining gene expression by recruiting abundant CTCF. CONCLUSIONS: Together, our findings reveal three distinct E-P regulation patterns across different cell types, providing insights into deciphering the complexity of gene transcriptional regulation.

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.