Enhancer RNAs

Enhancer RNAs (eRNAs) represent a class of relatively short non-coding RNA molecules (50-2000 nucleotides) transcribed from the DNA sequence of enhancer regions. They were first detected in 2010 through the use of genome-wide techniques such as RNA-seq and ChIP-seq. eRNAs can be subdivided into two main classes: 1D eRNAs and 2D eRNAs, which differ primarily in terms of their size, polyadenylation state, and transcriptional directionality. The expression of a given eRNA seems to correlate with the activity of its corresponding enhancer in a context-dependent fashion. Increasing evidence suggests that eRNAs actively play a role in transcriptional regulation in cis and in trans, and while their mechanisms of action remain unclear, a few models have been proposed. Enhancer RNAs (eRNAs) represent a class of relatively short non-coding RNA molecules (50-2000 nucleotides) transcribed from the DNA sequence of enhancer regions. They were first detected in 2010 through the use of genome-wide techniques such as RNA-seq and ChIP-seq. eRNAs can be subdivided into two main classes: 1D eRNAs and 2D eRNAs, which differ primarily in terms of their size, polyadenylation state, and transcriptional directionality. The expression of a given eRNA seems to correlate with the activity of its corresponding enhancer in a context-dependent fashion. Increasing evidence suggests that eRNAs actively play a role in transcriptional regulation in cis and in trans, and while their mechanisms of action remain unclear, a few models have been proposed. Enhancers as sites of extragenic transcription were initially discovered in genome-wide studies that identified enhancers as common regions of RNA polymerase II (RNA pol II) binding and non-coding RNA transcription. The level of RNA pol II–enhancer interaction and RNA transcript formation were found to be highly variable among these initial studies. Using explicit chromatin signature peaks, a significant proportion (~70%) of extragenic RNA Pol II transcription start sites were found to overlap enhancer sites in murine macrophages. Out of 12,000 neuronal enhancers in the mouse genome, almost 25% of the sites were found to bind RNA Pol II and generate transcripts. These eRNAs, unlike messenger RNAs (mRNAs), lacked modification by polyadenylation, were generally short and non-coding, and were bidirectionally transcribed. Later studies revealed the transcription of another type of eRNAs, generated through unidirectional transcription, that were longer and contained a poly A tail. Furthermore, eRNA levels were correlated with mRNA levels of nearby genes, suggesting the potential regulatory and functional role of these non-coding enhancer RNA molecules. eRNAs are transcribed from DNA sequences upstream and downstream of extragenic enhancer regions. Previously, several model enhancers have demonstrated the capability to directly recruit RNA Pol II and general transcription factors and form the pre-initiation complex (PIC) prior to the transcription start site at the promoter of genes. In certain cell types, activated enhancers have demonstrated the ability to both recruit RNA Pol II and also provide a template for active transcription of their local sequences. Depending on the directionality of transcription, enhancer regions generate two different types of non-coding transcripts, 1D-eRNAs and 2D-eRNAs. The nature of the pre-initiation complex and specific transcription factors recruited to the enhancer may control the type of eRNAs generated. After transcription, the majority of eRNAs remain in the nucleus. In general, eRNAs are very unstable and actively degraded by the nuclear exosome. Not all enhancers are transcribed, with non-transcribed enhancers greatly outnumbering the transcribed ones in the order of magnitude of dozens of thousands in every given cell type. In most cases, unidirectional transcription of enhancer regions generates long (>4kb) and polyadenylated eRNAs. Enhancers that generate polyA+ eRNAs have a lower H3K4me1/me3 ratio in their chromatin signature than 2D-eRNAs. PolyA+ eRNAs are distinct from long multiexonic poly transcripts (meRNAs) that are generated by transcription initiation at intragenic enhancers. These long non-coding RNAs, which accurately reflect the host gene’s structure except for the alternative first exon, display poor coding potential. As a result, polyA+ 1D-eRNAs may represent a mixed group of true enhancer-templated RNAs and multiexonic RNAs. Bidirectional transcription at enhancer sites generates comparatively shorter (0.5-2kb) and non-polyadenylated eRNAs. Enhancers that generate polyA- eRNAs have a chromatin signature with a higher H3K4me1/me3 ratio than 1D-eRNAs. In general, enhancer transcription and production of bidirectional eRNAs demonstrate a strong correlation of enhancer activity on gene transcription. The notions that not all enhancers are transcribed at the same time and that eRNA transcription correlates with enhancer-specific activity support the idea that individual eRNAs carry distinct and relevant biological functions. However, there is still no consensus on the functional significance of eRNAs. Furthermore, eRNAs can easily be degraded through exosomes and nonsense-mediated decay, which limits their potential as important transcriptional regulators. To date, four main models of eRNA function have been proposed, each supported by different lines of experimental evidence. Since multiple studies have shown that RNA Pol II can be found at a very large number of extragenic regions, it is possible that eRNAs simply represent the product of random “leaky” transcription and carry no functional significance. The non-specific activity of RNA Pol II would therefore allow extragenic transcriptional noise at sites where chromatin is already in an open and transcriptionally competent state. This would explain even tissue-specific eRNA expression as open sites are tissue-specific as well. RNA Pol II-mediated gene transcription induces a local opening of chromatin state through the recruitment of histone acetyltransferases and other histone modifiers that promote euchromatin formation. It was proposed that the presence of these enzymes could also induce an opening of chromatin at enhancer regions, which are usually present at distant locations but can be recruited to target genes through looping of DNA. In this model, eRNAs are therefore expressed in response to RNA Pol II transcription and therefore carry no biological function.