Abstract
Distal enhancer elements in DNA enable higher-order chromatin interactions that facilitate gene expression programs and thus contribute to cellular phenotype and function. In the brain, enhancer-promoter interactions help to ensure cell- and tissue-specific gene expression profiles, defining which genes are active during neuronal specification and which genes remain accessible in adult neurons. In addition to their close links to gene activation, enhancer elements themselves are subject to widespread, bidirectional transcription that yield non-coding enhancer RNA (eRNA). However, although eRNAs are correlated with overall enhancer activity, the precise function of eRNAs remains controversial. Here, we examined the function of eRNAs arising from multiple enhancers near the well-characterized immediate early gene Fos (also known as c-Fos). We show that eRNA transcription from Fos enhancers is dynamically modulated by various forms of neuronal activity, requires RNA polymerase II, and precedes activity-dependent induction of Fos mRNA. Visualization of Fos eRNA transcripts on a single cell level using single molecule fluorescent in situ hybridization revealed localization within the cell nucleus. Anti-sense based Fos eRNA knockdown decreased Fos mRNA expression, whereas mRNA knockdown did not affect eRNA levels. Targeted stimulation of eRNA synthesis from Fos enhancers using CRISPR-dCas9 fusion proteins produced corresponding increases in Fos mRNA expression, with limited cross-talk between enhancers. Similarly, CRISPR-targeted delivery of eRNA to a Fos enhancer elevated mRNA induction following neuronal depolarization. Finally, we show that anti-sense based knockdown of a single Fos eRNA is sufficient to alter neuronal physiology. Together, these results suggest that RNAs transcribed from neuronal enhancers are important regulators of enhancer-driven gene regulatory programs and neuronal function.
