The development of next-generation sequencing (NGS) methods revolutionized the study of genomic transcriptionally active regions. NGS now makes it possible to catalog transcripts from coding sequences and also allows the discovery of RNA species that are not templates for protein synthesis.
Several classes of non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and long non-coding RNAs (ncRNAs), are already known to play diverse roles in post-transcriptional regulation of mRNA stability and epigenetic control of chromatin activities.
Recently a new class of RNAs called enhancer RNAs or eRNAs has been identified {Kim et al. 2010}. However, it remains an open question whether eRNAs are just transcriptional noise or have a relevant biologically function. As usually, to clarify this, more research is needed.
Figure 1: Overview of a typical gene transcribed by RNA polymerase II.
A typical gene transcribed by RNA polymerase II has a promotor that extends upstream from the site where transcription is initiated (Figure 1). (See Lewin, Benjamin; Genes VII chapter 20, 2000). The promotor contains several short sequence elements that bind transcription factors, up to 10 base pairs (bp) in length, and promotors are dispersed over a sequence region that can be greater than 200 bp. Enhancers contain a more closely packed array of elements that also bind transcription factors. Enhancer regions may be located at a distance of several kilobases (kb). The DNA duplex may be coiled or rearranged such that transcription factors at the promotor and the enhancer interact to form a large protein-DNA complex.
What are eRNAs?
Enhancer RNA or eRNA are RNAs transcribed by RNA polymerase II (RNAPII) from the domain of transcription enhancers. eRNAs are a class of short non-coding RNAs, 50 to 2,000 nucleotides in length, transcribed from DNA enhancer regions. eRNAs stimulate gene expression, but the precise mechanisms how they function remains unclear. Enhancers are intergenic DNA elements that regulate transcription of target genes in response to signaling pathways by interacting with promotors over large genomic distances. Enhancers contain binding sites for transcription factors that promote RNA polymerase II (RNAPII) recruitment and transcription activation. Also, enhancers carry unique epigenetic marks that distinguish them from promoters. Additionally, these regulatory elements have an open chromatin conformation that increases accessibility to transcription factors and RNAPII. Enhancers are bi-directionally transcribed into eRNAs {Kim et al. 2010}.
Rahman et al. recently reported that eRNAs are localized exclusively in the nucleus and the induction of eRNAs occurs with similar kinetics as that of target mRNAs. eRNAs are mostly nascent at enhancers however their steady-state levels remain lower than those of their cognate mRNAs at the single-allele level. eRNAs are rarely co-expressed with their target loci. It appears that active gene transcription does not require the continuous transcription of eRNAs or their accumulation at enhancers.
Genome-wide sequencing methods allowed studying stimulus-dependent enhancer functions in tissues cells. Kim et al. in 2010 found that the level of eRNA expression at neuronal enhancers positively correlates with the level of mRNA synthesis in nearby genes. Their findings suggest that eRNA synthesis occurs specifically at enhancers that actively promote mRNA synthesis and the mechanism of enhancer activation involves RNAPII binding and eRNA synthesis.
Levels of eRNA expression correlate with mRNA levels of the corresponding enhancers target gene. eRNAs can be identified by a specific chromatin signature: H3K4me1 and H3K27ac. Furthermore, enhancers are often transcribed and bound by RNA polymerase II. eRNAs appear to be rarely spliced, are not polyadenylated, and are often transcribed in both directions. So far, the mechanisms by which eRNAs facilitate enhancer function remain unclear. More research to clarify this is needed.
Kim et al. suggested that eRNA synthesis is required to establish and maintain a chromatin landscape at enhancers needed for enhancer function. It is also possible that the eRNA transcripts are functionally important by themselves.
Enhancers are classified as cis-regulatory genetic elements that controll temporal and cell-type specific patterns of gene expression. Active enhancers have been found to generate bi-directional non-coding RNA transcripts called enhancer eRNAs. Enhancers contain bidirectional elements that allow assisting initiation and the activity of a promotor is increased by the presence of an enhancer. The enhancer is located distinct from the promotor and the position of the enhancer relative to the promotor can vary substantially. An enhancer can stimulate any promotor placed in its vicinity. Enhancers often show redundancy in function. DNA must be able to form a loop structure if proteins bound at an enhancer several kb distant from a promotor need to interact directly with proteins bound in the vicinity of the starting point such that the enhancer and promotor are closely located to each other. Enhancers may function by bringing proteins into the vicinity of the promotor. A model for eRNAs and transcriptional activation of a typical gene is illustrated in figure 2.
Figure 2: Model for eRNAs, enhancer derived RNAs, and transcriptional activation of a gene (Feng Liu, 2017). The structure of a typical gene is illustrated. The gene is associated with two cis-regulatory elements, (i) a promotor, located in proximity, and (ii) an enhancer, located distal to the transcription start site of the gene. In general, when its enhancer is inactivae the gene is turned “off” (A). When an enhancer is activated by transcription factors, it can loop towards the promotor and turn “on” the transcription of the gene (B). Both, enhancers and promotors are classified as non-coding elements.
A more detailed model can be reviewed in the paper published by Kim et al. in 2015 in which the loop structure is shown together with the transcription machinery.
To find and varify the presence of eRNAs, Espinosa suggested in 2015 to design a couple of CRISPRs, one to delete the lncRNA "promotor" and a second one to induce a pol(A) cassette early in the path of RNAPII. If the first genome edit affects expression but the second one doesn't it is an "e" or eRNA.
Reference
Espinosa JM; Revisiting lncRNAs: How Do You Know Yours Is Not an eRNA? Mol Cell. 2016 Apr 7;62(1):1-2. doi: 10.1016/j.molcel.2016.03.022.
Giles et al. 2015. ncRNA function in chromatin organization., in Epigenetic Gene Expression and Regulation}.
Kim, T.-K., Hemberg, M., Gray, J. M., Costa, A. M., Bear, D. M., Wu, J., … Greenberg, M. E. (2010). Widespread transcription at neuronal activity-regulated enhancers. Nature, 465(7295), 182–187. http://doi.org/10.1038/nature09033. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE21161.
Kim, T-K., Hemberg, M., and Gray J.M.; 2015. Enhancer RNAs: A class of long noncoding RNAs synthesized at enhancers. Cold Spring Harb. Perspect 2015,7:a018622, 1-3.
Lewin, Benjamin; Genes VII chapter 20, 2000
Feng Liu; Enhancer-derived RNA: A Primer. Genomics Proteomics Bioinformatics 15 (2017) 196-200.
Rahman, S., Zorca, C. E., Traboulsi, T., Noutahi, E., Krause, M. R., Mader, S., & Zenklusen, D. (2017). Single-cell profiling reveals that eRNA accumulation at enhancer–promoter loops is not required to sustain transcription. Nucleic Acids Research, 45(6), 3017–3030. http://doi.org/10.1093/nar/gkw1220.
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