Sartorelli_2020: Enhancer RNAs are an important regulatory layer of the epigenome

keywords

Enhancer RNA (eRNA)

Enhancer RNAs

Introduction

eRNAs

A recent addition to the expanding list of regulatory ncRNAs is the emerging class of enhancer RNAs (eRNAs), which are transcribed from enhancers in a tissue-specific manner.

Regulatory Non-coding RNAs that are transcribed from enhancers in a tissue specific manner

RNA-mediate gene regulation

Enhancer definition

Enhancers are classically defined as DNA sequences that regulate the gene expression networks underlying distinct cellular identities and cellular responses to environmental cues

• ENCODE estimates >400,00 putative enhancers in the human genome

• DNA Sequencing have made it easier to predict enhancer location and activity, however there are still lots of challenges

  • Enhancers functions are still challenging because they can act over long and variable distances on their target genes

  • Individual enhancers also have a tendency to regulate multiple genes is another challenge

  • To add to the list of challenges, enhancer activity is dynamic and restricted to particular cell types/tissues and environmental signals

  • Enhancers have modest sequence conservation across species

Many different models for how enhancers function in gene control have been proposed since their initial discovery nearly four decades ago

• There is considerable evidence demonstrating that looping of distal enhancers to their target promoters is a required for enhancer function.

  • Does this cause an issue with affecting multiple genes at once?

Key study revealed that experimental induction of Chromatin Looping between the mouse β-globin (Hbb) Promoter and its associated enhancer region results in transcriptional activation of the Hbb gene

Additional analyses of the forced looping of the Hbb enhancer and promoter regions revealed that enhancer–promoter contacts affect transcription by supporting an increase in the transcriptional burst fraction (number of transcribing alleles), although the burst size (number of transcripts produced) was unaltered

• One role of looping increasing transcription may be due to binding transcription factors, cofactors, and the general transcription machinery gets bound to enhancers which just raised the local abundance of the transcription machinery within the vicinity of specific target genes.

• Enhancer looping has also been RNA polymerase II (RNAPII)-mediated transcriptional elongation. Specifically, the LIM domain-binding protein 1 (LDB1)

Enhancers may also regulate target genes via transcripts produced from the enhancer regions themselves

• eRNA production has been shown to play a part in the regulation of gene expresion in multiple cell types, in response to various stimuli (So maybe jsut stimuli not just at rest?)

• eRNAs may dynamically remodel cellular transcriptomes and add a new layer of complexity to gene regulation

However, an issue that remains to be resolved is whether eRNAs have direct roles in gene control, as current efforts to determine this face the challenge of uncoupling eRNA function from the act of enhancer transcription. Thus, it remains necessary to develop tools to experimentally manipulate and model the direct functions of eRNAs

Enhancers as functional noncoding RNA transcription units

• Discovered nearly a decade ago(Written in 2020) two studies discovered eRNAs

• eRNA is synthesized in a cell-type and signal-dependent manner

Because eRNAs are not readily detectable in steady-state RNA-sequencing data, their annotation depends on sequencing nascent RNA using approaches that include GRO-seq, precision run-on nuclear sequencing (PRO-seq) and Cap Analysis Gene Expression (CAGE) These nascent transcription assays have been instrumental in uncovering a wide array of ncRNAs, including long noncoding RNAs, enhancer RNAs, promoter upstream transcripts and upstream antisense RNAs. Indeed, global annotation analyses have revealed that eRNA transcripts account for a large proportion of initiation events in the transcriptome, with approximately 40,000–65,000 eRNAs expressed in human cells. The annotation of such transcripts in Drosophila melanogaster and Caenorhabditis elegans reinforces the finding that eRNAs are a common feature of active enhancers in metazoans

eRNAs are produced from active enhancers that share several features:

1. An open chromatin state, reflected by the presence of DNase hypersensitive sites (DHSs)

2. Binding of transcription factors and cofactors, including the Histone Acetyltransferase p300 and cAMP response element–binding protein (CBP)

3. the co-occurrence of histone H3 lysine 4 monomethylation (H3K4me1) and histone H3 lysine 27 acetylation (H3K27ac)

• These may have guided enhancer identification, not all active enhancers share these features, they may not have eRNA production

• Not all active enhancer have comparable levels of transcriptional activity

  • Higher levels of eRNA synthesis have been shown to correlate with a higher ratio of H3K4me3 to H3K4me1 and increased levels of RNAPII occupancy

    • This suggests that measuring H3K4me1 may underestimate the number of functional enhancers, and H3K4me3 may be a better predictor of enhancer activity

• So identifying addition features to predict enhancers will improve the identification of functionally active enhancers and real eRNAs. eRNAs may actually be a reliable predictor of enhancer activity.

Regulation of enhancer transcription

• Enhancers and promoters share similar properties and rules for transcription initiation

  • TATA box

  • Nucleosome spacing

  • Assembly of general transcription factors (TFIID/RNAPII)

  • Cofactors (Mediator, p300)

  • Transcription occurs in a bidirectional manner

• Enhancers and promoters have been shown to be functionally interchangeable in supporting RNAPII initiation

Studies in Drosophila have also revealed that bidirectionally transcribed enhancers behave as weak promoters and, conversely, that bidirectionally transcribed promoters can function as strong enhancers. These findings blur the classical definitions of promoters and enhancers and raise the possibility that noncoding transcripts generated at both regulatory regions may be functional

There are discernible differences between the transcriptional elongation phases of eRNA and mRNA transcript production

• They primarily reflect the well-defined cycles of RNAPII, which correlate with phosphorylation and dephosphorylation of carboxyl-terminal domain (CTD)

  • The CTD domain consists of a heptameric sequence (YSPTSPS) repeated 52 times that is phosphorylated at S2, S5, S7, T4 and Y1

  • The general model of CTD phosporylation is that CTD becomes enriched with S5P at the 5' ends of

  coding regions, and as RNAPII elongates that transcript, S2P levels increase and S5p levels decrease.

• Elogation of enhancer transcripts is distinguished by low levels of the S2P form of RNAPII and minimal levels of the elgation-specific histone mark H3K36me3, both of which are enriched in gene bodies of lncRNAs and mRNAs.

Tyr1P

• Prevalence of Tyr1P coincides with the production of eRNAs and promoter-directed upstream antisense transcripts (PROMPTs), both are relatively unstable due to exosome-mediated degradation

• Tyr1P is required for transcription termination control in yeast

• Has been shown to prevent transcription read through at mammalian gene ends.

• No apparent contribution of Tyr1P to the regulation of transcription readthrough is observed at enhancers

• Tyr1P may contribute to different functional consequences at promoters and enhancers

Classification of noncoding enhancer RNAs

• eRNAs don't have a comprehensive definition yet. They've been described as both:

  • short, bidirectional, non-polyadenylated, non-spliced and unstable

  • Unidirectionally transcribed, spliced, polyadenylated and stable

• Majority of eRNAs are not polyadenylated or spliced, they tend to be capped, shown by 5'GRO-seq and CAGE

• eRNAs are predominately localized in the nucleus and chromatin-bound fractions

• early transcription termination of eRNAs is regulated by the Integrator complex in a manner that is probably dependent on termination-triggering polyadenylation (pA)-like signals

  • High turnover rate of eRNAs is mediated by the nuclear RNA exosome complex

• Length of eRNA transcripts is predicted to be less than 150 nucleotides based on transcription termination sites

  • Current predictions are coming from PRO-seq and Start-seq, and do not exclude the possibility of longer transcripts

• Current predicitons of eRNA length as a distinct family of lncRNAs, the difference being that lncRNA are >200 nucleotides and are processed making them more stable. However, the definitions are not mutually exclusive and there's probably some overlap.

• The inherent complexity in classifying eRNAs is because of the difficulty in uncoupling specific lncRNAs from their associated enhancer elements

• a recent study found that genomic regions defined by bidirectional transcription and enhancer features (termed RNA-producing centers, or EPCs) are located in proximity to the transcription start sites of lncRNAs

  • The ability of the EPCs to drive lncRNA production was shown to be correlated with higher enhancer activity and is in part linked to lncRNA maturation and the presence of evolutionarily conserved U1 splicing motifs