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Enhancers not only activate target promoters to stimulate messenger RNA (mRNA) synthesis, but they themselves also undergo transcription to produce enhancer RNAs (eRNAs), the significance of which is not well understood. Transcription at the participating enhancer-promoter pair appears coordinated, but it is unclear why and how. Here, we employ cell-free transcription assays using constructs derived from the human GREB1 locus to demonstrate that transcription at an enhancer and its target promoter is interdependent. This interdependence is observable under conditions where direct enhancer-promoter contact (EPC) takes place. We demonstrate that transcription activation at a participating enhancer-promoter pair is dependent on i) the mutual availability of the enhancer and promoter, ii) the state of transcription at both the enhancer and promoter, iii) local abundance of both eRNA and mRNA, and iv) direct EPC. Our results suggest transcriptional interdependence between the enhancer and the promoter as the basis of their transcriptional concurrence and coordination throughout the genome. We propose a model where transcriptional concurrence, coordination and interdependence are possible if the participating enhancer and promoter are entangled in the form of EPC, reside in a proteinaceous bubble, and utilize shared transcriptional resources and regulatory inputs.

Enhancer RNAs (eRNAs), a class of non-coding RNAs (ncRNAs) transcribed from enhancer regions, serve as a type of critical regulatory element in gene expression. There is increasing evidence demonstrating that the aberrant expression of eRNAs can be broadly detected in various human diseases. Some studies also revealed the potential clinical utility of eRNAs in these diseases. In this review, we summarized the recent studies regarding the pathological mechanisms of eRNAs as well as their potential utility across human diseases, including cancers, neurodegenerative disorders, cardiovascular diseases and metabolic diseases. It could help us to understand how eRNAs are engaged in the processes of diseases and to obtain better insight of eRNAs in diagnosis, prognosis or therapy. The studies we reviewed here indicate the enormous therapeutic potency of eRNAs across human diseases.

Enhancers confer precise spatiotemporal patterns of gene expression in response to developmental and environmental stimuli. Over the last decade, the transcription of enhancer RNAs (eRNAs) - nascent RNAs transcribed from active enhancers - has emerged as a key factor regulating enhancer activity. eRNAs are relatively short-lived RNA species that are transcribed at very high rates but also quickly degraded. Nevertheless, eRNAs are deeply intertwined within enhancer regulatory networks and are implicated in a number of transcriptional control mechanisms. Enhancers show changes in function and sequence over evolutionary time, raising questions about the relationship between enhancer sequences and eRNA function. Moreover, the vast majority of single nucleotide polymorphisms associated with human complex diseases map to the non-coding genome, with causal disease variants enriched within enhancers. In this Primer, we survey the diverse roles played by eRNAs in enhancer-dependent gene expression, evaluating different models for eRNA function. We also explore questions surrounding the genetic conservation of enhancers and how this relates to eRNA function and dysfunction., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Infectious diseases are spreading in to previously unreported geographical regions, and are reappeared in regions 75 or 100 years after their last reported case, as a result of environmental changes caused by anthropogenic activities. A pathogen, vector/host monitoring methodology is therefore indispensable in identifying potential transmission sites, providing early warnings and evaluating the human health risks of these infectious diseases in a given area. Recently, environmental DNA (eDNA) and environmental RNA approach (eRNA) have become widespread in monitoring organisms in the environment due to advantages like lower cost, time, and labour requirements. However, eDNA/eRNA based monitoring of pathogens and vectors/hosts using aquatic samples is limited to very few studies. In this review, we summarized the currently available eDNA/eRNA based human and non-human pathogens and vectors/hosts detection studies in aquatic samples. Species-specific shedding, transport, and decay of eDNA/eRNA in aquatic environments which is essential in estimating the abundance of pathogen, vectors/host in focus is also summarized. We also suggest the usage of eDNA/eRNA approach in urban aquatic samples like runoff in identifying the disease vectors/hosts inhabiting in locations which are not accessible easily., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Current advancements in environmental RNA (eRNA) exploit its relatively fast turnover rate relative to environmental DNA (eDNA) to assess 'metabolically active' or temporally/spatially recent community diversity. However, this focus significantly underutilizes the trove of potential ecological information encrypted in eRNA. Here, we argue for pushing beyond current species-level eDNA detection capabilities by using eRNA to detect any organisms with unique eRNA profiles, potentially including different life-history stages, sexes, or even specific phenotypes within a species. We also discuss the future of eRNA as a means of assessing the physiological status of organisms and the ecological health of populations and communities, reflecting ecosystem-level conditions. We posit that eRNA has the potential to significantly improve the resolution of organism detection, biological monitoring, and biomonitoring applications in ecology., Competing Interests: Declaration of Interests None declared by authors., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Recent studies have reported widespread stimulus-dependent transcription of mammalian enhancers into noncoding enhancer RNAs (eRNAs), some of which have central roles in the enhancer-mediated induction of target genes and modulation of phenotypic outcomes during development and disease. In cerebral ischemia, the expression and functions of eRNAs are virtually unknown. Here, we applied genome-wide H3K27ac ChIP-seq and genome-wide RNA-seq to identify enhancer elements and stroke-induced eRNAs, respectively, in the mouse cerebral cortex during transient focal ischemia. Following a 1-h middle cerebral artery occlusion (MCAO) and 6 h of reperfusion, we identified 77 eRNAs that were significantly upregulated in stroke as compared to sham, of which 55 were exclusively expressed in stroke. The knockdown of two stroke-induced eRNAs in the mouse brain resulted in significantly larger infarct volumes as compared to controls, suggesting that these eRNAs are involved in the post-stroke neuroprotective response. A preliminary comparison of eRNA expression in the male versus female cortices revealed sex-dependent patterns that may underlie the physiological differences in response to stroke between the two sexes. Together, this study is the first to illuminate the eRNA landscape in the post-stroke cortex and demonstrate the significance of an eRNA in modulating post-stroke cortical brain damage.

Enhancers are noncoding DNA elements that are present upstream or downstream of a gene to control its spatial and temporal expression. Specific histone modifications, such as monomethylation on histone H3 lysine 4 (H3K4me1) and H3K27ac, have been widely used to assign enhancer regions in mammalian genomes. In recent years, emerging evidence suggests that active enhancers are bidirectionally transcribed to produce enhancer RNAs (eRNAs). This finding not only adds a new reliable feature to define enhancers but also raises a fundamental question of how eRNAs function to activate transcription. Although some believe that eRNAs are merely transcriptional byproducts, many studies have demonstrated that eRNAs execute crucial tasks in regulating chromatin conformation and transcription activation. In this review, we summarize the current understanding of eRNAs from their biogenesis, functions, and regulation to their pathological significance. Additionally, we discuss the challenges and possible mechanisms of eRNAs in regulated transcription., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

It is now evident that transcriptional gene regulation usually requires the re-organization of chromatin architecture. Increasing evidence suggested various kinds of RNAs are involved in this process. Especially the nascent RNAs retained at their site of transcription can serve as a scaffold for organizing transcriptionally either favorable or unfavorable chromatin structures. An emerging concept of phase separation explains how these chromatin structures can be maintained as physically discrete subcompartments within membrane-less nucleoplasm. Evidences that support the crucial role of nascent RNAs in the formation of phase-separated condensates are now rapidly growing.

Since the discovery that enhancers can support transcription, the roles of enhancer RNAs have remained largely elusive. We identified that enhancer RNAs interact with and augment bromodomain engagement with acetylated chromatin. Here, we discuss our recent findings and the potential mechanisms underlying the regulation and functions of enhancer RNA-bromodomain associations.

Biological roles for extracellular RNA (eRNA) have become apparent. For example, eRNA can induce contact activation in blood via activation of the plasma proteases factor XII (FXII) and factor XI (FXI). We sought to reveal the biological role of the secretory enzyme ribonuclease 1 (RNase 1) in an organismal context by generating and analyzing RNase 1 knockout ( Rnase1 -/- ) mice. We found that these mice are viable, healthy, and fertile, though larger than Rnase1 plasma contains more RNA than does the plasma of +/+ mice. Rnase1 -/- plasma contains more RNA than does the plasma of Rnase1 mice, and is consistent with the known effects of eRNA on FXII activation. The apparent activity of FXI in the plasma of +/+ mice was 1000-fold higher when measured in an assay triggered by a low concentration of tissue factor than in assays based on recalcification, consistent with eRNA enhancing FXI activation by thrombin. These findings suggest that one of the physiological functions of RNase 1 is to degrade eRNA in blood plasma. Loss of this function facilitates FXII and FXI activation, which could have effects on inflammation and blood coagulation. We anticipate that Rnase1 mice will be a useful tool for evaluating other hypotheses about the functions of RNase 1 and of eRNA in vivo.-/- mice clots more rapidly than does wild-type plasma. This phenotype appeared to be due to increased levels of the active form of FXII (FXIIa) in the plasma of Rnase1 -/- mice compared to Rnase1 +/+ mice, and is consistent with the known effects of eRNA on FXII activation. The apparent activity of FXI in the plasma of Rnase1 -/- mice was 1000-fold higher when measured in an assay triggered by a low concentration of tissue factor than in assays based on recalcification, consistent with eRNA enhancing FXI activation by thrombin. These findings suggest that one of the physiological functions of RNase 1 is to degrade eRNA in blood plasma. Loss of this function facilitates FXII and FXI activation, which could have effects on inflammation and blood coagulation. We anticipate that Rnase1 -/- mice will be a useful tool for evaluating other hypotheses about the functions of RNase 1 and of eRNA in vivo., (© 2019 Garnett et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

We observed increased expression of ADAMDEC1 RNA in monocytes from patients with systemic lupus erythematosus. The precise role of ADAMDEC1 is uncertain and uniquely among metalloproteinases it utilizes a zinc-coordinating aspartic acid residue which allows it to escape inhibition by tissue inhibitor of metalloprotease-3 (TIMP-3). A closely related gene encodes the protein ADAM28, which is not up-regulated in lupus. We leveraged the ability to look at both gene's promoters and enhancers simultaneously. ADAMDEC1 was up-regulated by LPS while ADAM28 was not upregulated in the short term. We identified MAP kinases and NFκB as critical cell pathways regulating the expression of ADAMDEC1. These same pathways were implicated in driving the expression of the ADAMDEC1 upstream enhancer RNAs. We demonstrated that binding of the enhancer RNAs produced from the upstream enhancer were critically important and that p300 bound to both the RNA from the enhancer and the DNA at the enhancer. P300 binding to the enhancer was dependent on NFκB. These data define the critical pathways regulating the expression of ADAMDEC1 and extend our knowledge of the roles of enhancer RNAs and mechanistically links p300 and enhancer RNAs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Enhancers can act as cis-regulatory elements (CREs) to control development and cellular function by regulating gene expression in a tissue-specific and ubiquitous manner. However, the regulatory network and characteristic of different types of enhancers (e.g., transcribed/non-transcribed enhancers, tissue-specific/ubiquitous enhancers) across multiple tissues are still unclear. Results: Here, a total of 53,924 active enhancers and 10,307 enhancer-associated RNAs (eRNAs) in 10 tissues (adrenal, brain, breast, heart, liver, lung, ovary, placenta, skeletal muscle and kidney) were identified through the integration of histone modifications (H3K4me1, H3K27ac and H3K4me3) and DNase I hypersensitive sites (DHSs) data. Moreover, 40,101 tissue-specific enhancers (TS-Enh), 1,241 ubiquitously expressed enhancers (UE-Enh) as well as transcribed enhancers (T-Enh), including 7,727 unidirectionally transcribed enhancers (1D-Enh) and 1,215 bidirectionally transcribed enhancers (2D-Enh) were defined in 10 tissues. The results show that enhancers exhibited high GC content, genomic variants and transcription factor binding sites (TFBS) enrichment in all tissues. These characteristics were significantly different between TS-Enh and UE-Enh, T-Enh and NT-Enh, 2D-Enh and 1D-Enh. Furt hermore, the results showed that enhancers obviously upregulate the expression of adjacent target genes which were remarkably correlated with the functions of corresponding tissues. Finally, a free user-friendly tissue-specific enhancer database, TiED (http://lcbb.swjtu.edu.cn/TiED), has been built to store, visualize, and confer these results. Conclusion: Genome-wide analysis of the regulatory network and characteristic of various types of enhancers showed that enhancers associated with TFs, eRNAs and target genes appeared in tissue specificity and function across different tissues., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Enhancer-derived RNAs (eRNAs) are a group of RNAs transcribed by RNA polymerase II from the domain of transcription enhancers, a major type of cis-regulatory elements in the genome. The correlation between eRNA production and enhancer activity has stimulated studies on the potential role of eRNAs in transcriptional regulation. Additionally, eRNA has also served as a marker for global identification of enhancers. Here I review the brief history and fascinating properties of eRNAs., (Copyright © 2017 The Author. Production and hosting by Elsevier B.V. All rights reserved.)

Monomethylation of histone H3 at lysine 4 (H3K4me1) and acetylation of histone H3 at lysine 27 (H3K27ac) are correlated with transcriptionally engaged enhancer elements, but the functional impact of these modifications on enhancer activity is not well understood. Here we used CRISPR/Cas9 genome editing to separate catalytic activity-dependent and independent functions of Mll3 (Kmt2c) and Mll4 (Kmt2d, Mll2), the major enhancer H3K4 monomethyltransferases. Loss of H3K4me1 from enhancers in Mll3/4 catalytically deficient cells causes partial reduction of H3K27ac, but has surprisingly minor effects on transcription from either enhancers or promoters. In contrast, loss of Mll3/4 proteins leads to strong depletion of enhancer Pol II occupancy and eRNA synthesis, concomitant with downregulation of target genes. Interestingly, downregulated genes exhibit reduced polymerase levels in gene bodies, but not at promoters, suggestive of pause-release defects. Altogether, our results suggest that enhancer H3K4me1 provides only a minor contribution to the long-range coactivator function of Mll3/4., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Tissue-specific transcription is critical for normal development, and abnormalities causing undesirable gene expression may lead to diseases such as cancer. Such highly organized transcription is controlled by enhancers with specific DNA sequences recognized by transcription factors. Enhancers are associated with chromatin modifications that are distinct epigenetic features in a tissue-specific manner. Recently, super-enhancers comprising enhancer clusters co-occupied by lineage-specific factors have been identified in diverse cell types such as adipocytes, hair follicle stem cells, and mammary epithelial cells. In addition, noncoding RNAs, named eRNAs, are synthesized at super-enhancer regions before their target genes are transcribed. Many functional studies revealed that super-enhancers and eRNAs are essential for the regulation of tissue-specific gene expression. In this review, we summarize recent findings concerning enhancer function in tissue-specific gene regulation and cancer development.

Enzalutamide is a second-generation anti-androgen for treatment of castration-resistant prostate cancer (CPRC). It prolongs survival of CRPC patients, but its overall survival benefit is relatively modest (4.8 months) and by 24 months most patients progress on enzalutamide. To date, however, the molecular mechanisms underlying enzalutamide resistance remain elusive. Herein, we report enzalutamide treatment-induced alterations of androgen receptor (AR)-regulated enhancer RNAs (AR-eRNAs) and their roles in enzalutamide-resistant growth and survival of CRPC cells. AR chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) and RNA-seq analyses revealed that 188 and 227 AR-eRNAs were differentially expressed in enzalutamide-resistant LNCaP and C4-2 cells, respectively. The AR-eRNAs upregulated in C4-2 cells and downregulated in LNCaP cells were selected through meta-analysis. Expression of AR-eRNAs and related mRNAs in the loci of FTO, LUZP2, MARC1 and NCAM2 were further verified by real-time RT-PCR. Silencing of LUZP2 inhibited, but silencing of MARC1 increased the growth of enzalutamide-resistant C4-2 cells. Intriguingly, meta-analysis showed that expression of LUZP2 mRNA increased in primary tumors compared to normal prostate tissues, but decreased again in metastatic CRPC. Our findings suggest that eRNA alteration profiling is a viable new approach to identify functional gene loci that may not only contribute to enzalutamide-resistant growth of CRPC, but also serve as new targets for CRPC therapy., Competing Interests: The authors declare no conflicts of interest.

A new paradigm has emerged in recent years characterizing transcription initiation as a bidirectional process encompassing a larger proportion of the genome than previously thought. Past concepts of coding genes thinly scattered among a vast background of transcriptionally inert noncoding DNA have been abandoned. A richer picture has taken shape, integrating transcription of coding genes, enhancer RNAs (eRNAs), and various other noncoding transcriptional events. In this review we give an overview of recent studies detailing the mechanisms of RNA polymerase II (RNA Pol II)-based transcriptional initiation and discuss the ways in which transcriptional direction is established as well as its functional implications., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Active transcriptional enhancers are often transcribed to eRNAs, whose changing levels mirror those of the target gene mRNA. We discuss some of the reported functions of these eRNAs and their likely diversity to allow utilization of distinct cis regulatory regions to enhance transcription in diverse developmental and cellular contexts.

The circadian oscillator is based on transcription-translation feedback loops that generate 24 h oscillations in gene expression. Although circadian regulation of mRNA expression at the transcriptional level is one of the most important steps for the generation of circadian rhythms within the cell, multiple lines of evidence point to a disconnect between transcript oscillation and protein oscillation. This can be explained by regulatory RNA-binding proteins acting on the nascent transcripts to modulate their processing, export, translation and degradation rates. In this chapter we will review what is known about the different steps involved in circadian gene expression from transcription initiation to mRNA stability and translation efficiency. The role of ribonucleoprotein particles in the generation of rhythmic gene expression is only starting to be elucidated, but it is likely that they cooperate with the basal transcriptional machinery to help to maintain the precision of the clock under diverse cellular and environmental conditions.

Following reports by ENCyclopedia Of DNA Elements (ENCODE; GENCODE) Consortium and others, it is now fairly evident that the majority (70-80%) of the mammalian genome has the potential to be transcribed into non-protein-coding RNAs (ncRNAs). Critical to our understanding of genetic processes is the mechanism by which ncRNAs exert their roles. Accordingly, ncRNAs are shown to regulate the expression of protein-coding loci (i.e., genes) at the transcriptional as well as post-transcriptional stages. We recently reported on a widespread transcription at the DNA enhancer elements in myogenic cells. In our study, we found certain enhancer RNAs (eRNAs) regulate chromatin accessibility of the transcriptional machinery at loci encoding master regulators of myogenesis (i.e., MyoD/MyoG), thus suggesting their significance and site-specific impact in cellular programming. Here, we examine recent discoveries pertinent to the proposed role(s) of eRNAs in regulating gene expression. We will highlight consistencies, discuss confounding observations, and consider a lack of critical information in a way to prioritize future objectives.

Here, we used RNA capture-seq to identify a large population of lncRNAs that are expressed in the infralimbic prefrontal cortex of adult male mice in response to fear-related learning. Combining these data with cell-type-specific ATAC-seq on neurons that had been selectively activated by fear extinction learning, we find inducible 434 lncRNAs that are derived from enhancer regions in the vicinity of protein-coding genes. In particular, we discover an experience-induced lncRNA we call ADRAM (activity-dependent lncRNA associated with memory) that acts as both a scaffold and a combinatorial guide to recruit the brain-enriched chaperone protein 14-3-3 to the promoter of the memory-associated immediate-early gene Nr4a2 and is required fear extinction memory. This study expands the lexicon of experience-dependent lncRNA activity in the brain and highlights enhancer-derived RNAs (eRNAs) as key players in the epigenomic regulation of gene expression associated with the formation of fear extinction memory.

Smoking and HPV infection are known causes for the vast majority of head and neck squamous cell carcinomas (HNSCC) due to their likelihood of causing gene dysregulation and genomic alterations. Enhancer RNAs (eRNAs) are non-coding RNAs that are known to increase nearby and target gene expression, and activity that has been suggested to be affected by genetic and epigenetic alterations. Here we sought to identify the effects of smoking and HPV status on eRNA expression in HNSCC tumors. We focused on four patient cohorts including smoking/HPV+, smoking/HPV-, non-smoking/HPV+, and non-smoking/HPV- patients. We used TCGA RNA-seq data from cancer tumors and adjacent normal tissue, extracted eRNA read counts, and correlated these to survival, clinical variables, immune infiltration, cancer pathways, and genomic alterations. We found a large number of differentially expressed eRNA in each patient cohort. We also found several dysregulated eRNA correlated to patient survival, clinical variables, immune pathways, and genomic alterations. Additionally, we were able to find dysregulated eRNA nearby seven key HNSCC-related oncogenes. For example, we found eRNA chr14:103272042-103272430 (eRNA-24036), which is located close to the TRAF3 gene to be differentially expressed and correlated with the pathologic N stage and immune cell populations. Using a separate validation dataset, we performed differential expression and immune infiltration analysis to validate our results from the TCGA data. Our findings may explain the association between eRNA expression, enhancer activity, and nearby gene dysregulation.

Approaches involving environmental DNA/RNA (eDNA/eRNA) – collectively eNA – can be used for pathogen discovery, ecology studies, surveillance, disease risk assessment, and early warning systems, after appropriate validation. eNA can provide evidence of the presence of pathogens in a system, and increases the spatiotemporal scope and efficiency of surveillance efforts – but it cannot, on its own, provide confirmatory evidence of infection of local hosts. The 'pathogen eNA continuum' provides a framework for designing pathogen eDNA/eRNA investigations, to elucidate pathogen life cycles, host reservoirs and vectors, and to link environmental detection to actual infections. Pathogen eRNA may provide additional insight into pathogen viability and activity, and can be used alternatively or in addition to eDNA. eNA approaches can be developed for predictive spatiotemporal modelling of pathogen distribution and impacts. Detection of pathogens, parasites, and other symbionts in environmental samples via eDNA/eRNA (collectively eNA) is an increasingly important source of information about their occurrence and activity. There is great potential for using such detections as a proxy for infection of host organisms in connected habitats, for pathogen monitoring and surveillance, and for early warning systems for disease. However, many factors require consideration, and appropriate methods developed and verified, in order that eNA detections can be reliably interpreted and adopted for surveillance and assessment of disease risk, and potentially inclusion in international standards, such as the World Organisation for Animal Health guidelines. Disease manifestation results from host–symbiont–environment interactions between hosts, demanding a multifactorial approach to interpretation of eNA signals. [ABSTRACT FROM AUTHOR]

Nuclear receptors induce both transcriptional activation and repression programs responsible for development, homeostasis, and disease. Here, we report a previously overlooked enhancer decommissioning strategy underlying a large estrogen receptor alpha (ERα)-dependent transcriptional repression program. The unexpected signature for this E2-induced program resides in indirect recruitment of ERα to a large cohort of pioneer factor basally active FOXA1-bound enhancers that lack cognate ERα DNA-binding elements. Surprisingly, these basally active estrogen-repressed (BAER) enhancers are decommissioned by ERα-dependent recruitment of the histone demethylase KDM2A, functioning independently of its demethylase activity. Rather, KDM2A tethers the E3 ubiquitin-protein ligase NEDD4 to ubiquitylate/dismiss Pol II to abrogate eRNA transcription, with consequent target gene downregulation. Thus, our data reveal that Pol II ubiquitylation/dismissal may serve as a potentially broad strategy utilized by indirectly bound nuclear receptors to abrogate large programs of pioneer factor-mediated, eRNA-producing enhancers.

Although the use and development of molecular biomonitoring tools based on environmental nucleic acids (eDNA and eRNA; collectively known as eNAs) have gained broad interest for the quantification of biodiversity in natural ecosystems, studies investigating the impact of site‐specific physicochemical parameters on eNA‐based detection methods (particularly eRNA) remain scarce. Here, we used a controlled laboratory microcosm experiment to comparatively assess the environmental degradation of eDNA and eRNA across an acid–base gradient following complete removal of the progenitor organism (Daphnia pulex). Using water samples collected over a 30‐day period, eDNA and eRNA copy numbers were quantified using a droplet digital PCR (ddPCR) assay targeting the mitochondrial cytochrome c oxidase subunit I (COI) gene of D. pulex. We found that eRNA decayed more rapidly than eDNA at all pH conditions tested, with detectability—predicted by an exponential decay model—for up to 57 h (eRNA; neutral pH) and 143 days (eDNA; acidic pH) post organismal removal. Decay rates for eDNA were significantly higher in neutral and alkaline conditions than in acidic conditions, while decay rates for eRNA did not differ significantly among pH levels. Collectively, our findings provide the basis for a predictive framework assessing the persistence and degradation dynamics of eRNA and eDNA across a range of ecologically relevant pH conditions, establish the potential for eRNA to be used in spatially and temporally sensitive biomonitoring studies (as it is detectable across a range of pH levels), and may be used to inform future sampling strategies in aquatic habitats. [ABSTRACT FROM AUTHOR]

Growing interest and affordability of environmental DNA and RNA (eDNA and eRNA) approaches for biodiversity assessments and monitoring of complex ecosystems have led to the emergence of manifold protocols for nucleic acids (NAs) isolation and processing. Although there is no consensus on a standardized workflow, the common practice for water samples is to concentrate NAs via filtration using varying pore size membranes. Using the smallest pore is assumed to be most efficient for NAs capture from a wide range of material (including sub‐cellular particles); however, a trade‐off must occur between detection of a meaningful molecular signal and cost/time effort when processing samples using fine pore membranes.Comparative studies involving formal efficiency assessments are lacking, which restricts informed decision‐making around an optimized sampling approach for applications such as biosurveillance (i.e. detection and monitoring of target taxa—nuisance organisms, endangered and indicator taxa or other species of economic or cultural importance). Here, we present an experimental study using an easily cultured microalgal species (Alexandrium pacificum) to test different filter membranes for capturing NAs in the context of cost/time effort and cell fractions encountered in nature (whole cells, partially lysed and naked NAs).The results showed no statistically significant difference between membrane types for capturing target eDNA signal from intact and partially lysed cell treatments. In terms of time effort and volume processed, higher efficiency ratings were obtained with the larger pore size (5 μm) cellulose membranes. Positively charged nylon demonstrated enhanced capture of naked NAs, and especially eRNA signal, across treatments.Our findings support using coarse pore size filters for adequate capture of target NA signal (from both eDNA and eRNA) with less processing time. The framework presented here can provide a quick and robust feasibility check and comparative assessment of new and existing NA processing technologies, and allows sufficient control over multiple parameters, including physical–chemical water properties, temporal scales, and concentration and type of input material. [ABSTRACT FROM AUTHOR]

Simple Summary: This review focuses on eRNAs and the several mechanisms by which they can regulate gene expression. In particular we describe here the most recent examples of eRNAs dysregulated in cancer or involved in the immune escape of tumor cells. Enhancer RNAs (eRNAs) are non-coding RNAs (ncRNAs) transcribed in enhancer regions. They play an important role in transcriptional regulation, mainly during cellular differentiation. eRNAs are tightly tissue- and cell-type specific and are induced by specific stimuli, activating promoters of target genes in turn. eRNAs usually have a very short half-life but in some cases, once activated, they can be stably expressed and acquire additional functions. Due to their critical role, eRNAs are often dysregulated in cancer and growing number of interactions with chromatin modifiers, transcription factors, and splicing machinery have been described. Enhancer activation and eRNA transcription have particular relevance also in inflammatory response, placing the eRNAs at the interplay between cancer and immune cells. Here, we summarize all the possible molecular mechanisms recently reported in association with eRNAs activity. [ABSTRACT FROM AUTHOR]

Simple Summary: In cancer, regulatory regions of the genome are hijacked by the tumor cells for the activation of oncogenes that lead to cancer initiation and progression. One of the most salient regulatory elements of the genome are called enhancers, which are characterized by their ability to increase the expression of their target genes. In this study, we identified a novel enhancer that drives the expression of the oncogene CSF1 in triple-negative breast cancer patients, the most aggressive subtype of breast cancer. We demonstrate that this enhancer is specifically active in triple-negative breast cancer patients compared to other breast cancer subtypes and show that its target genes portend a worse clinical outcome in patients. We then use innovative CRISPR-based genome engineering techniques to systematically perturb various features of this enhancer to elucidate its mechanisms of action and determine the consequences on tumor cell growth. Furthermore, we test our model for CSF1 enhancer function in ovarian cancer cells and demonstrate that our findings can apply to other cancer types. These results demonstrate the significant impact of enhancers in cancer biology and highlight their potential as tractable targets for therapeutic intervention. Enhancers are critical regulatory elements in the genome that help orchestrate spatiotemporal patterns of gene expression during development and normal physiology. In cancer, enhancers are often rewired by various genetic and epigenetic mechanisms for the activation of oncogenes that lead to initiation and progression. A key feature of active enhancers is the production of non-coding RNA molecules called enhancer RNAs, whose functions remain unknown but can be used to specify active enhancers de novo. Using a combination of eRNA transcription and chromatin modifications, we have identified a novel enhancer located 30 kb upstream of Colony Stimulating Factor 1 (CSF1). Notably, CSF1 is implicated in the progression of breast cancer, is overexpressed in triple-negative breast cancer (TNBC) cell lines, and its enhancer is primarily active in TNBC patient tumors. Genomic deletion of the enhancer (via CRISPR/Cas9) enabled us to validate this regulatory element as a bona fide enhancer of CSF1 and subsequent cell-based assays revealed profound effects on cancer cell proliferation, colony formation, and migration. Epigenetic silencing of the enhancer via CRISPR-interference assays (dCas9-KRAB) coupled to RNA-sequencing, enabled unbiased identification of additional target genes, such as RSAD2, that are predictive of clinical outcome. Additionally, we repurposed the RNA-guided RNA-targeting CRISPR-Cas13 machinery to specifically degrade the eRNAs transcripts produced at this enhancer to determine the consequences on CSF1 mRNA expression, suggesting a post-transcriptional role for these non-coding transcripts. Finally, we test our eRNA-dependent model of CSF1 enhancer function and demonstrate that our results are extensible to other forms of cancer. Collectively, this work describes a novel enhancer that is active in the TNBC subtype, which is associated with cellular growth, and requires eRNA transcripts for proper enhancer function. These results demonstrate the significant impact of enhancers in cancer biology and highlight their potential as tractable targets for therapeutic intervention. [ABSTRACT FROM AUTHOR]

The FIMO software in the MEME suite (http://meme-suite.org/doc/fimo.html?man%5ftype=web) was used to obtain human TF motif data from the Jaspar database (http://jaspar.genereg.net/), and TF-binding sites/motifs were predicted based on the enhancer loci data corresponding to the eRNAs obtained from the analysis.[28],[29] SEs are cis-regulatory elements with determinative regulatory effects on cell fate. Analysis of SLE-Related Master TFs of eRNAs SEs consist of multiple enhancer-like elements that are densely occupied by master TFs.[14] TFs play an important role in mediating eRNA biogenesis;[9] however, SLE-related TFs of eRNAs have not been fully unraveled. Keywords: enhancer RNA; eRNA; systemic lupus erythematosus; SLE; PBMCs; transcription factors; TFs; super-enhancer EN enhancer RNA eRNA systemic lupus erythematosus SLE PBMCs transcription factors TFs super-enhancer 775 791 17 05/17/22 20220201 NES 220201 Introduction Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by loss of immune tolerance, immune cell activation, and pathogenic autoantibody production.[1] The pathology of SLE, which is a highly clinically heterogeneous disease, remains largely unclear, but involves the dysregulation of multiple immune cell lineages and genes. Various studies have demonstrated the critical roles of eRNAs in target gene transcription.[10],[15],[16] A recent study showed that eRNAs exert key roles in cancer pathogenesis and are potential therapeutic targets worthy of further investigation.[11] However, the relationships between eRNAs, associated SEs, and TFs and SLE pathogenesis in PBMCs have not been explored. [Extracted from the article]

A large portion of the human genome is transcribed into RNAs without known protein-coding functions, far outnumbering coding transcription units. Extensive studies of long noncoding RNAs (lncRNAs) have clearly demonstrated that they can play critical roles in regulating gene expression, development, and diseases, acting both as transcriptional activators and repressors. More recently, enhancers have been found to be broadly transcribed, resulting in the production of enhancer-derived RNAs, or eRNAs. Here, we review emerging evidence suggesting that at least some eRNAs contribute to enhancer function. We discuss these findings with respect to potential mechanisms of action of eRNAs and other ncRNAs in regulated gene expression.

Responses to developmental and environmental cues depend on precise spatiotemporal control of gene transcription. Enhancers, which comprise DNA elements bound by regulatory proteins, can activate target genes in response to these external signals. Recent studies have shown that enhancers are transcribed to produce enhancer RNAs (eRNAs). Do eRNAs play a functional role in activating gene expression or are they non-functional byproducts of nearby transcription machinery? The unstable nature of eRNAs and over-reliance on knockdown approaches have made elucidating the possible functions of eRNAs challenging. We focus here on studies using cloned eRNAs to study their function as transcripts, revealing roles for eRNAs in enhancer–promoter looping, recruiting transcriptional machinery, and facilitating RNA polymerase pause–release to regulate gene expression. Enhancers are regulatory regions in the genome that bind sequence-specific DNA-binding transcription factors. They are transcribed to produce enhancer RNAs (eRNAs). Current studies have relied on depletion of eRNAs ('subtraction' experiments) rather than on expressing them as physical entities from cloned cDNAs ('addition' experiments). This has left open questions about whether these non-coding transcripts have functions in regulating gene expression or whether they are merely transcriptional noise. A limited number of studies that have expressed eRNAs from cloned cDNAs suggest that they function by facilitating enhancer–promoter looping, stimulating the recruitment and activity of regulatory proteins, establishing a permissive chromatin environment for gene activation, and aiding in the release of paused RNA polymerase II. Future studies using genome-targeting approaches to study eRNAs in endogenous chromatin will further define the functions of eRNAs in the regulation of target gene expression. [ABSTRACT FROM AUTHOR]

The regulation of gene expression is a fundamental cellular process and its misregulation is a key component of disease. Enhancers are one of the most salient regulatory elements in the genome and help orchestrate proper spatiotemporal gene expression during development, in homeostasis, and in response to signaling. Notably, molecular aberrations at enhancers, such as translocations and single nucleotide polymorphisms, are emerging as an important source of human variation and susceptibility to disease. Herein we discuss emerging paradigms addressing how genes are regulated by enhancers, common features of active enhancers, and how non-coding enhancer RNAs (eRNAs) can direct gene expression programs that underlie cellular phenotypes. We survey the current evidence, which suggests that eRNAs can bind to transcription factors, mediate enhancer-promoter interactions, influence RNA Pol II elongation, and act as decoys for repressive cofactors. Furthermore, we discuss current methodologies for the identification of eRNAs and novel approaches to elucidate their functions. [ABSTRACT FROM AUTHOR]

Background: The estrogen receptor (ER) is a ligand-dependant transcription factor expressed in many breast cancers and is the target of many endocrine-based cancer therapies. Genome-wide studies have shown that the ER binds to gene-specifc enhancer regions in response to β-estradiol (E2) which undergo transcription producing noncoding enhancer RNA (eRNA). While eRNAs are important for transcriptional activation of neighboring genes, the mechanism remains poorly understood. Results: Using ChIP-Seq we generate a global profle of thymine DNA glycosylase (TDG), an ER coactivator that plays an essential role in DNA demethylation, in response to E2 in the MCF7 breast cancer cell line. Remarkably, we found that in response to E2 TDG localized to enhancers which also recruit ERα, RNA Pol II and other coregulators and which are marked by histone modifcations indicative of active enhancers. Importantly, depletion of TDG inhibits E2-mediated transcription of eRNAs and transcription of ER-target genes. Functionally, we fnd that TDG both sensitizes MCF7 cells to tamoxifen-mediated cytostasis and increases migration and invasion of MCF7 cells. Conclusions: Taken together we fnd that TDG plays a central role in mediating transcription at a subset of enhancers and governs how MCF7 cells respond to both estrogenic and anti-estrogenic compounds and may be an efective therapeutic target. [ABSTRACT FROM AUTHOR]

Gene transcription is strictly controlled by the interplay of regulatory events at gene promoters and gene-distal regulatory elements called enhancers. Despite extensive studies of enhancers, we still have a very limited understanding of their mechanisms of action and their restricted spatio-temporal activities. A better understanding would ultimately lead to fundamental insights into the control of gene transcription and the action of regulatory genetic variants involved in disease. Here, I review and discuss pros and cons of state-of-the-art genomics methods to localize and infer the activity of enhancers. Among the different approaches, profiling of enhancer RNAs yields the highest specificity and may be superior in detecting in vivo activity. I discuss their apparent similarities to promoters, which challenge the established view of enhancers and promoters as distinct entities, and present a unifying model of regulatory elements in transcriptional regulation, in which activity, transcriptional output and regulatory function is context specific. [ABSTRACT FROM AUTHOR]

Environmental (e)DNA metabarcoding holds great promise for biomonitoring and ecotoxicological applications. However, few studies have compared the performance of eDNA versus eRNA metabarcoding in assessing organismal response to marine pollution, in experimental conditions. Here, we performed a chromium (Cr)-spiked mesocosm experimental test on benthic foraminiferal community to investigate the effects on species diversity by analysing both eDNA and eRNA metabarcoding data across different Cr concentrations in the sediment. Foraminiferal diversity in the eRNA data showed a significant negative correlation with the Cr concentration in the sediment, while a positive response was observed in the eDNA data. The foraminiferal OTUs exhibited a higher turnover rate in eRNA than in the eDNA-derived community. Furthermore, in the eRNA samples, OTUs abundance was significantly affected by the Cr gradient in the sediment (Pseudo-R2 = 0.28, p = 0.05), while no significant trend was observed in the eDNA samples. The correlation between Cr concentration and foraminiferal diversity in eRNA datasets was stronger when the less abundant OTUs (<100 reads) were removed and the analyses were conducted exclusively on OTUs shared between eRNA and eDNA datasets. This indicates the importance of metabarcoding data filtering to capture ecological impacts, in addition to using the putatively active organisms in the eRNA dataset. The comparative analyses on foraminiferal diversity revealed that eRNA-based metabarcoding can better assess the response to heavy metal exposure in presence of subtle concentrations of the pollutant. Furthermore, our results suggest that to unlock the full potential for ecosystem assessment, eDNA and eRNA should be studied in parallel to control for potential sequence artifacts in routine ecosystem surveys. [Display omitted] • eDNA and eRNA biomonitoring potential was assessed in mesocosm experiment. • Chromium concentration negatively influenced organismal diversity in eRNA samples. • eDNA-derived diversity was not affected by chromium levels in the sediment. • eDNA-informed filtering improved assessment of response signals in eRNA samples. [ABSTRACT FROM AUTHOR]

Simple Summary: It is well established that tobacco smoke is the key player in lung squamous cell carcinoma (LUSC) pathogenesis, and there is growing evidence that electronic cigarette (e-cigarette) vapor may also cause LUSC. Recently, several studies have associated tobacco smoke with differential enhancer RNA (eRNA) expression. However, the effects of tobacco smoke and e-cigarette vapor on eRNA expression in correlation to LUSC outcomes have not been fully elucidated. This study demonstrates that tobacco smoke and e-cigarette vapor may decrease DNA methylation and increase chromosomal alterations at key sites, which ultimately upregulate the expression of oncogenic eRNAs and downregulate the expression of tumor-suppressing eRNAs. Subsequently, we demonstrate that these eRNAs may have altered interactions with immune cells to promote LUSC pathogenesis and reduced patient survival. We hope our results can be validated in future studies, and the key eRNAs we identified may be used as effective targets for more specialized treatments for smoking-mediated LUSC. Tobacco is the primary etiologic agent in worsened lung squamous cell carcinoma (LUSC) outcomes. Meanwhile, it has been shown that etiologic agents alter enhancer RNAs (eRNAs) expression. Therefore, we aimed to identify the effects of tobacco and electronic cigarette (e-cigarette) use on eRNA expression in relation to LUSC outcomes. We extracted eRNA counts from RNA-sequencing data of tumor/adjacent normal tissue and before/after e-cigarette tissue from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO), respectively. Tobacco-mediated LUSC eRNAs were correlated to patient survival, clinical variables, and immune-associated elements. eRNA expression was also correlated to mutation rates through the Repeated Evaluation of Variables Conditional Entropy and Redundance (REVEALER) algorithm and methylated sites through methylationArrayAnalysis. Differential expression analysis was then completed for the e-cigarette data to compare with key tobacco-mediated eRNAs. We identified 684 downregulated eRNAs and 819 upregulated eRNAs associated with tobacco-mediated LUSC, specifically, with the cancer pathological stage. We also observed a decrease in immune cell abundance in tobacco-mediated LUSC. Yet, we found an increased association of eRNA expression with immune cell abundance in tobacco-mediated LUSC. We identified 16 key eRNAs with significant correlations to 8 clinical variables, implicating these eRNAs in LUSC malignancy. Furthermore, we observed that these 16 eRNAs were highly associated with chromosomal alterations and reduced CpG site methylation. Finally, we observed large eRNA expression upregulation with e-cigarette use, which corresponded to the upregulation of the 16 key eRNAs. Our findings provide a novel mechanism by which tobacco and e-cigarette smoke influences eRNA interactions to promote LUSC pathogenesis and provide insight regarding disease progression at a molecular level. [ABSTRACT FROM AUTHOR]

Enhancers are critical genomic elements that can cooperate with promoters to regulate gene transcription in both normal and cancer cells. Recent studies reveal that enhancer regions are transcribed to produce a class of noncoding RNAs referred to as enhancer RNAs (eRNAs). Emerging evidence shows that eRNAs play important roles in enhancer activation and enhancer-driven gene regulation, and the expression of eRNAs may be a critical factor in tumorigenesis. The important roles of eRNAs in cancer signaling pathways are also gradually unveiled, providing a new insight into cancer therapy. Here, we review the roles of eRNAs in regulating cancer signaling pathways and discuss the potential of eRNA-targeted therapy for human cancers. [ABSTRACT FROM AUTHOR]

In deep-sea environments, which have low densities of large benthic animals, meiofauna are a useful indicator of biodiversity. However, in low-latitudes and in low-productivity areas, meiofaunal density is low, but is higher than that of megafaunal or macrofaunal density. Therefore, it is difficult to collect a sufficient number of specimens for statistical analysis. In addition, because faunal classification has been based on conventional microscopic observations, the use of meiofauna to estimate biodiversity in deep-sea environments is time-consuming. However, metabarcoding analyses focused on environmental DNA (eDNA) or RNA (eRNA) have recently been used to examine deep-sea eukaryotic diversity and communities. Here, we examined meiofaunal assemblages using microscopic, eDNA-, and eRNA-based methods at Xufu Guyot (JA06 Seamount), off the southeastern coast of Minami-Torishima Island in the North Pacific Ocean. Microscopic analysis failed to detect a significant difference in diversity or community structure between the seamount terrace and base. This was likely because of the low abundance of meiofauna, which was caused by the low surface productivity at the study area. However, eDNA/eRNA-based metabarcoding analyses revealed spatial variations in diversity and community structures within a single seamount. Therefore, metabarcoding analysis might be useful to elucidate meiofaunal assemblages in areas with low productivity and low faunal density. • Meiofaunal assemblages at JA06 Seamount were examined using the conventional and metabarcoding methods. • The conventional methods failed to detect the heterogeneity of meiofaunal assemblages. • The metabarcoding methods provided invaluable information on meiofaunal assemblages. • The metabarcoding analysis is useful to reveal meiofaunal assemblages in areas with low productivity and low faunal density. [ABSTRACT FROM AUTHOR]