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Enhancers as non-coding RNA transcription units: recent insights and future perspectives

Key Points

  • Enhancers are distal regulatory genomic elements that determine spatiotemporal and quantitative gene transcription programmes in response to developmental or environmental cues. Global transcriptomic and (epi)genomic advances have found that enhancers are pervasively transcribed into non-coding RNAs (ncRNAs), which are named enhancer RNAs (eRNAs).

  • The expression levels of eRNAs are highly correlated with the activity of the functional enhancers, both in developmental and rapid signal-regulated transcriptional programmes. eRNAs bear several common and unique features compared to other long ncRNAs or coding mRNAs.

  • Heterogeneity of eRNAs exists in terms of both transcriptional processes and RNA properties, which might be linked to their functional diversity. There is evidence supporting a functional role of both the eRNA transcripts per se, and the act of transcription, in the activation of target coding genes. However, for the large majority of eRNAs in the mammalian genome, evidence of their functional roles is still limited.

  • Mechanisms underlying the roles of specific eRNAs include modulating the chromatin accessibility of cognate promoters, stabilizing enhancer–promoter interactions, trapping transcription factors on genomic sites, and regulating the RNA polymerase II pause release at cognate promoters. The act of enhancer transcription may facilitate histone modifications, remodel large areas of chromatin and induce transcriptional interference.

  • Enhancer transcription may take part in generating several important biological phenomena, including transcription-dependent R-loops, genomic mutations and instability at enhancers, and potentially facilitate new gene birth during evolution.

  • Further evidence is needed to fully understand the functions of different categories of enhancers as transcription units and the roles of eRNAs per se, in gene regulation, development and disease.

Abstract

Networks of regulatory enhancers dictate distinct cell identities and cellular responses to diverse signals by instructing precise spatiotemporal patterns of gene expression. However, 35 years after their discovery, enhancer functions and mechanisms remain incompletely understood. Intriguingly, recent evidence suggests that many, if not all, functional enhancers are themselves transcription units, generating non-coding enhancer RNAs. This observation provides a fundamental insight into the inter-regulation between enhancers and promoters, which can both act as transcription units; it also raises crucial questions regarding the potential biological roles of the enhancer transcription process and non-coding enhancer RNAs. Here, we review research progress in this field and discuss several important, unresolved questions regarding the roles and mechanisms of enhancers in gene regulation.

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Figure 1: Activation of an enhancer.
Figure 2: Enhancer transcription process and enhancer RNA processing.
Figure 3: Functional roles of enhancer transcription in gene regulation.
Figure 4: Biological significance of enhancer transcription and enhancer RNAs.

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Acknowledgements

The authors thank M. Chuang for her helpful comments on the manuscript. This work was supported by the US Department of Defense breast cancer research programme postdoctoral fellowships (BC110381 to W.L. and BC103858 to D.N.) and grants from the US National Institutes of Health to M.G.R. M.G.R. is an investigator with the Howard Hughes Medical Institute. The authors apologize for not being able to cite many important other publications owing to the imposed limit.

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Glossary

Non-coding

DNA regions or RNA transcripts that do not code for proteins.

Long non-coding RNAs

(lncRNAs). Non-coding RNAs that are longer than 200 nucleotides.

Hypersensitivity

Chromatin regions such as enhancers and other regulatory elements often display extra sensitivity to DNase treatment, reflecting the openness of the regions.

Locus control regions

(LCRs). Genomic elements that elevate expression of linked genes through long-range regulation, in a copy number-dependent and tissue-specific manner. A well-studied example is the LCR upstream of the β-globin gene in erythroid cells.

Super enhancers

A group of active enhancers densely clustered in a ~10–30 kb region, highly associated with cell identity genes and disease-associated genomic variations. Also known as stretch enhancers.

Shadow enhancers

A term coined by Michael Levine and colleagues to describe the phenomenon of having another enhancer in the vicinity (or sometimes scattered across larger chromosomal domains) in addition to a primary enhancer to control the expression pattern of an important developmental gene; their biological roles are still under investigation, but, at least in some cases, shadow enhancers act to confer phenotypic robustness under environmental and genetic variability.

Regulatory archipelagos

A term denoting the presence of multiple enhancers in the Hox gene loci during limb development, with each of them playing quantitative or qualitative roles for Hox gene transcription.

Highly occupied target regions

(HOT regions; also known as hotspots). Genomic regions that associate with multiple transcription factors, either simultaneously or sequentially, and that are usually uncovered by chromatin immunoprecipitation followed by sequencing. They are identified in multiple organisms and cell types.

TF collective or MegaTrans enhancer

Highly active enhancers that are bound by multiple transcription factors simultaneously (similar to the definition of HOT regions and super enhancers). However, these two terms have been used to describe a situation in which one (MegaTrans) or several (TF collective) major transcription factors bind target enhancers in cis (that is, direct association through a specific DNA motif), which then act to tether other transcription factors in trans (that is, bind the major transcription factor through protein–protein interaction).

Promoter upstream transcripts

(PROMPTs). Primary transcripts that are generated pervasively from gene promoters but are transcribed in the opposite direction from the sense strand (that is, mRNAs). PROMPTs generally display low stability, lack of splicing and polyadenylation; very similar to enhancer RNAs in many aspects. Also known as upstream antisense RNAs.

General transcription factors

Transcription factors that work together with RNA polymerase II to form the pre-initiation complex at transcription start sites to initiate transcription. They consist of TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH.

Bidirectional transcripts

The two transcripts initially observed to be generated by some coding gene promoters that go to either a sense or an antisense direction, which produces the mRNAs and the promoter upstream transcripts, respectively. A similar phenomenon is now observed to exist for some enhancers.

DRB

(5,6-dichloro-1-β-D-ribofuranosylbenzimidazole). An adenosine analogue that acts as an inhibitor of cyclin-dependent kinases needed for efficient RNA polymerase II elongation.

RNA exosome

A multi-protein complex capable of degrading various types of RNA molecules in the cytoplasm, nucleus and the nucleolus; it bears both exo- and endo-ribonucleolytic functions in eukaryotes.

RNAPII carboxy-terminal domain

(RNAPII CTD). An evolutionary conserved tandem repeat of heptapeptides Y1S2P3T4S5P6S7 that is present in the C terminus of RPB1, the largest subunit of RNA polymerase II (RNAPII).

Small nuclear RNAs

A class of small RNAs in the nucleus of eukaryotic cells that have been found to largely take part in regulating splicing (for example, U1 and U2 RNAs) and occasionally in transcriptional control of RNA polymerase II (for example, 7SK RNA).

Enhancer–promoter inter-regulation

A hypothesis in which active enhancers affect promoter expression, and some promoters also control enhancer transcription.

Transcriptional noise

A term used to denote non-productive and perhaps random transcriptional activity of an RNA polymerase on a DNA template, which is proposed to take place due to open chromatin regions.

De novo enhancers

A group of enhancers that were not marked by any epigenomic marks during cell lineage determination, but rather are generated acutely in a mature cell type after treatment with acute stimulation; they are also known as latent enhancers.

NELF complex

(Negative elongation factor complex). A four-subunit complex consisting of NELF-A, NELF-B, NELF-E and either NELF-C or NELF-D. As denoted by the name, it negatively affects transcription by RNA polymerase II.

Myogenic differentiation 1

(MyoD1). A gene encoding a key transcription factor that promotes muscle-specific gene transcription programmes that are required for myogenic determination.

Genomic variations

The varied DNA sequences in alleles of certain genes carried by individuals within and among populations, which may or may not result in phenotypic variations.

R-loops

RNA/DNA hybrid structures in the genome, in which nascent RNA binds the transcribing DNA strand through sequence complementarity, leaving the non-bound single-strand DNA displaced and prone to damage. Once thought to be transcriptional by-products, R-loops have now been found to be involved in the regulation of gene expression, epigenetic modifications, DNA replication and genome stability.

Somatic hypermutation

A biological process mainly conducted by activation-induced cytidine deaminase in activated B cells, in which the immunoglobulin genes are highly mutated to generate a library of diversified antibodies.

Class switch recombination

A biological mechanism enabling B cells to switch their production of immunoglobulin from one type to another (for example, IgM to IgG), which involves an activation-induced cytidine deaminase-mediated specific DNA double-strand break and recombination.

Single nucleotide polymorphisms

(SNPs). Genomic variations that involve a single nucleotide.

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Li, W., Notani, D. & Rosenfeld, M. Enhancers as non-coding RNA transcription units: recent insights and future perspectives. Nat Rev Genet 17, 207–223 (2016). https://doi.org/10.1038/nrg.2016.4

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