Shared retinoic acid responsive enhancers coordinately regulate nascent transcription of Hoxb coding and non-coding RNAs in the developing mouse neural tube

Signaling pathways regulate the patterns of Hox gene expression that underlie their functions in the specification of axial identity. Little is known about the properties of cis-regulatory elements and underlying transcriptional mechanisms that integrate graded signaling inputs to coordinately control Hox expression. Here, we optimized a single molecule fluorescent in situ hybridization (smFISH) technique with probes spanning introns to evaluate how three shared retinoic acid response element (RARE)-dependent enhancers in the Hoxb cluster regulate patterns of nascent transcription in vivo at the level of single cells in wild-type and mutant embryos. We predominately detect nascent transcription of only a single Hoxb gene in each cell, with no evidence for simultaneous co-transcriptional coupling of all or specific subsets of genes. Single and/or compound RARE mutations indicate that each enhancer differentially impacts global and local patterns of nascent transcription, suggesting that selectivity and competitive interactions between these enhancers is important to robustly maintain the proper levels and patterns of nascent Hoxb transcription. This implies that rapid and dynamic regulatory interactions potentiate transcription of genes through combined inputs from these enhancers in coordinating the retinoic acid response.

Automated summary

Signaling pathways regulate the patterns of Hox gene expression in axial identity specification. Little is known about the properties of cis-regulatory elements and transcriptional mechanisms that control Hox expression through graded signaling inputs. This study optimized a single molecule fluorescent in situ hybridization (smFISH) technique to evaluate the role of three retinoic acid response element (RARE)-dependent enhancers in regulating nascent transcription patterns of Hoxb genes. The results suggest that these enhancers have differential impacts on global and local patterns of transcription, and their competitive interactions are important for maintaining proper levels and patterns of Hoxb transcription.