Enhancer RNA transcription near segmentation gene enhancers can be analyzed in situ using FISH.

Enhancer RNAs (eRNAs) are non-coding transcripts produced at enhancer regions, which appear to be involved in transcriptional regulation. Up to date, these have been primarily investigated using labor- and cost-intensive genomic techniques. However, the precise mechanisms by which eRNA transcription or eRNA transcripts themselves mediate transcriptional regulation remain unclear. Here, we present a novel experimental approach that allows us to analyze characteristics of eRNA transcription in fixed and live whole Drosophila melanogaster embryos. We employ the anterior-posterior patterning genes as a model system to investigate the dynamics of eRNA expression, utilizing an imaging-based approach. We combined high-sensitivity fluorescence in situ hybridization chain reaction with high-resolution confocal microscopy to detect eRNA and mRNA molecules. Through this experimental assay, we identified foci of elevated transcriptional activity that generate eRNA transcripts correlated with mRNA production at the same gene locus. We could show that this eRNA transcription is independent of promoter activity. Additionally, we demonstrate that insulators can influence eRNA transcription, resulting in loss of eRNA transcription. Moreover, we observe that eRNAs can originate both within classical enhancer regions and outside of them, including from foreign bacterial sequences when these are placed near enhancer sequences, underscoring the strong influence of local regulatory context on eRNA initiation. In live embryos using MS2-MCP live imaging, our analysis of insulators showed a modest reduction in mRNA burst intensity accompanied by a slight increase in burst frequency. Overall, our imaging-based approach offers a novel platform for dissecting enhancer-eRNA interactions and could be adapted for wider applications.