Enhancer RNAs, or eRNAs, are small molecules of non-coding RNA that are transcribed from enhancer loci. They are involved in the regulation of gene transcription and can be used to treat disease.
2010 saw the discovery of enhancer RNAs using high-throughput sequencing to locate stimuli-dependent enhancers.
This demonstrated the RNA polymerase II dependence of enhancer RNA transcription. These are typically between 0.5 and 2 kb in size.
In addition, the expression level of enhancer RNAs is often within the same range as that of their parent enhancers, indicating a correlation between enhancer function and enhancer RNA expression.
Enhancer RNAs Detection
To find enhancer RNAs, Next Generation Sequencing (NGS) with a high coverage is necessary. This is due to the fact that the expression level of enhancer RNA is 19–34 fold lower than that of gene transcripts.
The stability of enhancer RNAs is also significantly lower than that of mRNA. Therefore, technologies such as global nuclear run-on sequencing (GRO-seq) and precision nuclear run-on sequencing (PRO-seq) or sensitive methods such as cap analysis of gene expression sequencing are necessary.
Chromatin immunoprecipitation, in situ hybridization, and the BruUV-seq approach, which uses UV light for deep sequencing of nascent RNAs, are further techniques for enhancing the presence and detection of specific RNAs.
Due to the fact that enhancers are only functionally active at particular times and locations, these strategies may be most effective only when enhancers are functionally active.
Functions of enhancer RNAs
Indicators of active enhancers: Enhancer RNAs can be utilised to assess whether an enhancer is inactive or active, according to several studies. A study revealed that the presence of enhancer RNAs rather than epigenetic markers determines the tissue-specific expression of enhancers.
Transcriptional regulation of target genes: Initially, it was believed that enhancer RNAs are by-products of transcription when regulating the expression of target genes through transcriptional control. Nevertheless, further research has demonstrated that enhancer RNAs can influence the expression of several genes by binding to them. Large-scale studies demonstrate a positive correlation between the expression of enhancer RNAs and target gene expression. One of the ways in which this can occur is by its effect on RNA Polymerase II. Furthermore, enhancers physically interact with promoters to generate E-P loops. Additionally, research has demonstrated that enhancer RNA can reinforce the E-P looping and upregulate the expression of particular genes.
Biogenesis of Enhancer RNAs
DNA sequences upstream and downstream of extragenic enhancer regions are translated into eRNAs.
Several model enhancers have showed the ability to directly bind RNA Pol II and general transcription factors to create the pre-initiation complex (PIC) in advance of the transcription start point in gene promoters.
Activated enhancers in particular cell types have been shown to both recruit RNA Pol II and serve as a template for active transcription of their local sequences.
Enhancer areas produce two distinct forms of non-coding transcripts, 1D-eRNAs and 2D-eRNAs, depending on the directionality of transcription.
The type of eRNAs produced may be determined by the composition of the pre-initiation complex and specific transcription factors recruited to the enhancer. The vast majority of eRNAs stay in the nucleus following transcription.
eRNAs are typically extremely unstable and are actively destroyed by the nuclear exosome. Not all enhancers are transcribed, with non-transcribed enhancers in every cell type outnumbering transcribed enhancers by an order of magnitude of tens of thousands.
Biogenesis of Enhancer RNAs | Pclermont, CC BY-SA 3.0 https://creativecommons.org/licenses/by-sa/3.0, via Wikimedia Commons
1D eRNAs
In most instances, unidirectional transcription of enhancer regions creates polyadenylated eRNAs that are >4 kb in length.
Enhancers that produce polyA+ eRNAs have a lower H3K4me1/me3 ratio than 2D-eRNAs in their chromatin signature.
PolyA+ eRNAs differ from lengthy multiexonic poly transcripts (meRNAs) produced by transcription initiation at intragenic enhancers.
These lengthy noncoding RNAs, which perfectly replicate the structure of the host gene except for the alternate first exon, have a low potential for coding.
Consequently, polyA+ 1D-eRNAs may be a mixture of genuine enhancer-templated RNAs and multiexonic RNAs.
2D eRNAs
Bidirectional transcription at enhancer sites creates eRNAs that are relatively shorter (0.5-2kb) and non-polyadenylated.
Enhancers that produce polyA-eRNAs have a chromatin signature with a larger H3K4me1/me3 ratio than those that generate 1D-eRNAs.
In general, enhancer transcription and the synthesis of bidirectional eRNAs have a substantial association with gene transcription activity.
Enhancer RNAs in disease
Enhancer RNA and inflammation
IL1, an enhancer RNA, is implicated in the NF-KB signalling pathway and modulates the proinflammatory response, according to studies.
This enhancer RNA knockdown could lessen the inflammatory response.
Enhancer RNA and neurodegenerative disorders
Enhancers may harbour mutations associated with neurodevelopmental and neuropsychiatric diseases.
Studies have identified more than a hundred enhancers in the brain, the majority of which are translated into enhancer RNAs in the striatum of mouse brains.
Enhancer RNAs include genetic variations related with autism spectrum diseases.
Enhancer RNA and cancer
In prostate cancer, hundreds of enhancer RNAs were revealed to be differently expressed.
The estrogen-induced transcription of eRNA was discovered to be increased in MCF-7 breast cancer cells. Another study revealed that enhancer RNAs are drastically diminished in throat cancer.
Enhancer RNA in diabetes
Rosiglitazone, an anti-diabetic medication, has been demonstrated to increase insulin sensitivity via regulating blood glucose.
This medicine also increased the quantities of enhancer RNAs at certain places, indicating that enhancer RNA has a function in regulating glucose levels and diabetes.
Mechanisms of Enhancer RNAs
The assumption that unique eRNAs perform separate and significant biological roles is supported by the facts that not all enhancers are transcribed at the same time and that eRNA transcription correlates with enhancer-specific activity.
The role of eRNAs in biology is still being debated. The fact that eRNAs are so susceptible to degradation via exosomes and nonsense-mediated decay further reduces their utility as transcriptional regulators.
There are now four primary hypotheses for how eRNAs work, and they are all backed by various pieces of experimental evidence.
Mechanisms of Enhancer RNAs | Pclermont, CC BY-SA 3.0 https://creativecommons.org/licenses/by-sa/3.0, via Wikimedia Commons
Transcriptional Noise
Multiple studies have demonstrated that RNA Pol II is present in a wide variety of extragenic locations, suggesting that eRNAs may merely reflect the byproduct of random “leaky” transcription and have no functional importance.
Since RNA Pol II is a highly promiscuous RNA polymerase, it is possible that it could generate extragenic transcriptional noise at places with open, transcriptionally competent chromatin.
Since open sites also vary depending on the type of cell they are located in, this could account for the fact that eRNA expression varies by tissue.
Transcription-dependent effects
Gene transcription driven by RNA Pol II leads to euchromatin development by recruiting histone acetyltransferases and other histone modifiers.
Enhancer regions, which are often found in far-flung places but can be recruited to target genes by DNA looping, may also benefit from the presence of these enzymes, as an opening of chromatin has been hypothesised for these areas.
So, in this scenario, eRNAs are expressed in response to RNA Pol II transcription but serve no biological purpose.
Functional activity in cis
This process proposes that eRNAs are functional molecules that display cis activity, whereas the two prior hypotheses suggested that eRNAs were not functionally relevant.
According to this theory, eRNAs can bring in regulatory proteins close to where they are made. Histone acetyltransferases are thought to be recruited via transcripts arising from enhancers upstream of the Cyclin D1 gene.
Depletion of these eRNAs was observed to result in transcriptional silencing of Cyclin D1.
Functional activity in trans
In the most recent theory, eRNAs located in different parts of the genome regulate transcription.
The transcriptional competence of individual loci can be altered by eRNAs due to the differential recruitment of protein complexes. As an example of a trans regulatory eRNA, Evf-2 works by increasing the expression of Dlx2, which in turn increases the activity of the Dlx5 and Dlx6 enhancers.
It is possible that eRNAs that act in trans can also function in cis, and vice versa.
References
Jeong, M., & Goodell, M. A. (2016). Noncoding Regulatory RNAs in Hematopoiesis. Hematopoiesis, 245–270. doi:10.1016/bs.ctdb.2016.01.006
Ye R, Cao C, Xue Y. Enhancer RNA: biogenesis, function, and regulation. Essays Biochem. 2020 Dec 7;64(6):883-894. doi: 10.1042/EBC20200014. PMID: 33034351.
