Investigating the role of enhancer–promoter proximity in regulating gene expression

The development of an organism requires the expression of each gene to be precisely orchestrated in space and time. This spatiotemporal control is partly achieved through enhancers: cis-regulatory sequences that can modulate transcription at their cognate promoters, even from millions of base pairs away. Despite the important regulatory role of enhancers, the mechanisms by which enhancers and promoters interact to ex- change regulatory information is still debated. Specifically, it is unclear whether close spatial proximity between regulatory elements is required for transcription. Adding another layer of complexity, recent studies have revealed multiway enhancer-promoter (E-P) interactions that appear to drive cell type-specific gene expression. However, the frequency of these multiway interactions in single cells remains under-explored. To address these questions, we combined DNA and RNA fluorescence in situ hybridization (FISH), super-resolution microscopy, and Tri-C to investigate changes in E-P distances during the transition from naive to primed pluripotency in mouse. To this end, we developed NOVA-FISH - a method capable of visualizing small regulatory elements in close genomic proximity. We then used NOVA-FISH, together with Oligopaint, to examine pairwise and multiway E-P interactions for five genes that are differentially expressed during the naive-to-primed transition: Nanog, Dppa3, Sox2, Dnmt3a and Prdm14. For Nanog and Dppa3, we additionally correlated E-P distance with nascent transcription. Despite transcriptional changes of several orders of magnitude, we found that changes in pairwise E-P distances during the naive-to-primed transition are highly locus-dependent. Tri-C data at the Nanog locus revealed a weak enrichment of multiway contacts when Nanog was highly expressed in the naive state, but not in the primed state, when Nanog was downregulated. As transcription often occurs in transient bursts within a subset of cells, we combined RNA and DNA FISH to identify active alleles. We observed a positive correlation between shorter E-P distances and transcription at the Nanog and Dppa3 loci. Together, our data support models of dynamic contact, in which shorter E–P distances are transiently stabilized during transcriptional initiation, and multiway hubs may contribute to regulating cell type–specific gene expression.