Enhancer RNAs Research Articles

Assessment of the Cardiac Noncoding Transcriptome by Single-Cell RNA Sequencing Identifies FIXER, a Conserved Profibrogenic Long Noncoding RNA.

Cardiac fibroblasts have crucial roles in the heart. In particular, fibroblasts differentiate into myofibroblasts in the damaged myocardium, contributing to scar formation and interstitial fibrosis. Fibrosis is associated with heart dysfunction and failure. Myofibroblasts therefore represent attractive therapeutic targets. However, the lack of myofibroblast-specific markers has precluded the development of targeted therapies. In this context, most of the noncoding genome is transcribed into long noncoding RNAs (lncRNAs). A number of lncRNAs have pivotal functions in the cardiovascular system. lncRNAs are globally more cell-specific than protein-coding genes, supporting their importance as key determinants of cell identity. In this study, we evaluated the value of the lncRNA transcriptome in very deep single-cell RNA sequencing. We profiled the lncRNA transcriptome in cardiac nonmyocyte cells after infarction and probed heterogeneity in the fibroblast and myofibroblast populations. In addition, we searched for subpopulation-specific markers that can constitute novel targets in therapy for heart disease. We demonstrated that cardiac cell identity can be defined by the sole expression of lncRNAs in single-cell experiments. In this analysis, we identified lncRNAs enriched in relevant myofibroblast subpopulations. Selecting 1 candidate we named FIXER (fibrogenic LOX-locus enhancer RNA), we showed that its silencing limits fibrosis and improves heart function after infarction. Mechanitically, FIXER interacts with CBX4, an E3 SUMO protein ligase and transcription factor, guiding CBX4 to the promoter of the transcription factor RUNX1 to control its expression and, consequently, the expression of a fibrogenic gene program.. FIXER is conserved in humans, supporting its translational value. Our results demonstrated that lncRNA expression is sufficient to identify the various cell types composing the mammalian heart. Focusing on cardiac fibroblasts and their derivatives, we identified lncRNAs uniquely expressed in myofibroblasts. In particular, the lncRNA FIXER represents a novel therapeutic target for cardiac fibrosis.

Identification and characterization of transcribed enhancers during cerebellar development through enhancer RNA analysis

BackgroundThe development of the brain requires precise coordination of molecular processes across many cell-types. Underpinning these events are gene expression programs which require intricate regulation by non-coding regulatory sequences known as enhancers. In the context of the developing brain, transcribed enhancers (TEs) regulate temporally-specific expression of genes critical for cell identity and differentiation. Transcription of non-coding RNAs at active enhancer sequences, known as enhancer RNAs (eRNAs), is tightly associated with enhancer activity and has been correlated with target gene expression. TEs have been characterized in a multitude of developing tissues, however their regulatory role has yet to be described in the context of embryonic and early postnatal brain development. In this study, eRNA transcription was analyzed to identify TEs active during cerebellar development, as a proxy for the developing brain. Cap Analysis of Gene Expression followed by sequencing (CAGE-seq) was conducted at 12 stages throughout embryonic and early postnatal cerebellar development.ResultsTemporal analysis of eRNA transcription identified clusters of TEs that peak in activity during either embryonic or postnatal times, highlighting their importance for temporally specific developmental events. Functional analysis of putative target genes identified molecular mechanisms under TE regulation revealing that TEs regulate genes involved in biological processes specific to neurons. We validate enhancer activity using in situ hybridization of eRNA expression from TEs predicted to regulate Nfib, a gene critical for cerebellar granule cell differentiation.ConclusionsThe results of this analysis provide a valuable dataset for the identification of cerebellar enhancers and provide insight into the molecular mechanisms critical for brain development under TE regulation. This dataset is shared with the community through an online resource (https://goldowitzlab.shinyapps.io/trans-enh-app/).

Identification of a ΔNp63-Dependent Basal-Like A Subtype-Specific Transcribed Enhancer Program (B-STEP) in Aggressive Pancreatic Ductal Adenocarcinoma.

Subtype-specific enhancer activity analysis via detection of eRNAs on a single-cell level in patient material can be used as a potential tool for treatment stratification.

DeepITEH: a deep learning framework for identifying tissue-specific eRNAs from the human genome.

Enhancers are vital cis-regulatory elements that regulate gene expression. Enhancer RNAs (eRNAs), a type of long noncoding RNAs, are transcribed from enhancer regions in the genome. The tissue-specific expression of eRNAs is crucial in the regulation of gene expression and cancer development. The methods that identify eRNAs based solely on genomic sequence data have high error rates because they do not account for tissue specificity. Specific histone modifications associated with eRNAs offer valuable information for their identification. However, identification of eRNAs using histone modification data requires the use of both RNA-seq and histone modification data. Unfortunately, many public datasets contain only one of these components, which impedes the accurate identification of eRNAs. We introduce DeepITEH, a deep learning framework that leverages RNA-seq data and histone modification data from multiple samples of the same tissue to enhance the accuracy of identifying eRNAs. Specifically, deepITEH initially categorizes eRNAs into two classes, namely, regularly expressed eRNAs and accidental eRNAs, using histone modification data from multiple samples of the same tissue. Thereafter, it integrates both sequence and histone modification features to identify eRNAs in specific tissues. To evaluate the performance of DeepITEH, we compared it with four existing state-of-the-art enhancer prediction methods, SeqPose, iEnhancer-RD, LSTMAtt, and FRL, on four normal tissues and four cancer tissues. Remarkably, seven of these tissues demonstrated a substantially improved specific eRNA prediction performance with DeepITEH, when compared with other methods. Our findings suggest that DeepITEH can effectively predict potential eRNAs on the human genome, providing insights for studying the eRNA function in cancer. The source code and dataset of DeepITEH have been uploaded to https://github.com/lyli1013/DeepITEH.

Enhancer RNAs in transcriptional regulation: recent insights.

Enhancers are a class of cis-regulatory elements in the genome that instruct the spatiotemporal transcriptional program. Last decade has witnessed an exploration of non-coding transcripts pervasively transcribed from active enhancers in diverse contexts, referred to as enhancer RNAs (eRNAs). Emerging evidence unequivocally suggests eRNAs are an important layer in transcriptional regulation. In this mini-review, we summarize the well-established regulatory models for eRNA actions and highlight the recent insights into the structure and chemical modifications of eRNAs underlying their functions. We also explore the potential roles of eRNAs in transcriptional condensates.

Control of enhancer and promoter activation in the type I interferon response by the histone demethylase Kdm4d/JMJD2d.

Transcriptional activation depends on the interplay of chromatin modifiers to establish a permissive epigenetic landscape. While histone 3 lysine 9 (H3K9) methylation has long been associated with gene repression, there is limited evidence to support a role for H3K9 demethylases in gene activation. We leveraged knockdown and overexpression of JMJD2d / Kdm4d in mouse embryonic fibroblasts, coupled with extensive epigenomic analysesm to decipher the role of histone 3 lysine 9 demethylases in the innate immune response. Here we describe the H3K9 demethylase Kdm4d/JMJD2d as a positive regulator of type I interferon responses. In mouse embryonic fibroblasts (MEFs), depletion of JMJD2d attenuates the transcriptional response, conferring increased viral susceptibility, while overexpression of the demethylase results in more robust IFN activation. We find that the underlying mechanism of JMJD2d in type I interferon responses consists of an effect both on the transcription of enhancer RNAs (eRNAs) and on dynamic H3K9me2 at associated promoters. In support of these findings, we establish that JMJD2d is associated with enhancer regions throughout the genome prior to stimulation but is redistributed to inducible promoters in conjunction with transcriptional activation. Taken together, our data reveal JMJD2d as a chromatin modifier that connects enhancer transcription with promoter demethylation to modulate transcriptional responses.

Epigenetic Alterations in DCIS Progression: What Can lncRNAs Teach Us?

Some transcripts that are not translated into proteins can be encoded by the mammalian genome. Long noncoding RNAs (lncRNAs) are noncoding RNAs that can function as decoys, scaffolds, and enhancer RNAs and can regulate other molecules, including microRNAs. Therefore, it is essential that we obtain a better understanding of the regulatory mechanisms of lncRNAs. In cancer, lncRNAs function through several mechanisms, including important biological pathways, and the abnormal expression of lncRNAs contributes to breast cancer (BC) initiation and progression. BC is the most common type of cancer among women worldwide and has a high mortality rate. Genetic and epigenetic alterations that can be regulated by lncRNAs may be related to early events of BC progression. Ductal carcinoma in situ (DCIS) is a noninvasive BC that is considered an important preinvasive BC early event because it can progress to invasive BC. Therefore, the identification of predictive biomarkers of DCIS-invasive BC progression has become increasingly important in an attempt to optimize the treatment and quality of life of patients. In this context, this review will address the current knowledge about the role of lncRNAs in DCIS and their potential contribution to the progression of DCIS to invasive BC.

Dynamic modulation of genomic enhancer elements in the suprachiasmatic nucleus, the site of the mammalian circadian clock.

The mammalian suprachiasmatic nucleus (SCN), located in the ventral hypothalamus, synchronizes and maintains daily cellular and physiological rhythms across the body, in accordance with environmental and visceral cues. Consequently, the systematic regulation of spatiotemporal gene transcription in the SCN is vital for daily timekeeping. So far, the regulatory elements assisting circadian gene transcription have only been studied in peripheral tissues, lacking the critical neuronal dimension intrinsic to the role of the SCN as central brain pacemaker. By using histone-ChIP-seq, we identified SCN-enriched gene regulatory elements that associated with temporal gene expression. Based on tissue-specific H3K27ac and H3K4me3 marks, we successfully produced the first-ever SCN gene-regulatory map. We found that a large majority of SCN enhancers not only show robust 24-h rhythmic modulation in H3K27ac occupancy, peaking at distinct times of day, but also possess canonical E-box (CACGTG) motifs potentially influencing downstream cycling gene expression. To establish enhancer-gene relationships in the SCN, we conducted directional RNA-seq at six distinct times across the day and night, and studied the association between dynamically changing histone acetylation and gene transcript levels. About 35% of the cycling H3K27ac sites were found adjacent to rhythmic gene transcripts, often preceding the rise in mRNA levels. We also noted that enhancers encompass noncoding, actively transcribing enhancer RNAs (eRNAs) in the SCN, which in turn oscillate, along with cyclic histone acetylation, and correlate with rhythmic gene transcription. Taken together, these findings shed light on genome-wide pretranscriptional regulation operative in the central clock that confers its precise and robust oscillation necessary to orchestrate daily timekeeping in mammals.

882 Enhancer RNA (eRNA) profiling to understand transcription regulation in keratinocytes

882 Enhancer RNA (eRNA) profiling to understand transcription regulation in keratinocytes

Bi-allelic variants in INTS11 are associated with a complex neurological disorder

The Integrator complex is a multi-subunit protein complex that regulates the processing of nascent RNAs transcribed by RNA polymerase II (RNAPII), including small nuclear RNAs, enhancer RNAs, telomeric RNAs, viral RNAs, and protein-coding mRNAs. Integrator subunit 11 (INTS11) is the catalytic subunit that cleaves nascent RNAs, but, to date, mutations in this subunit have not been linked to human disease. Here, we describe 15 individuals from 10 unrelated families with bi-allelic variants in INTS11 who present with global developmental and language delay, intellectual disability, impaired motor development, and brain atrophy. Consistent with human observations, we find that the fly ortholog of INTS11, dIntS11, is essential and expressed in the central nervous systems in a subset of neurons and most glia in larval and adult stages. Using Drosophila as a model, we investigated the effect of seven variants. We found that two (p.Arg17Leu and p.His414Tyr) fail to rescue the lethality of null mutants, indicating that they are strong loss-of-function variants. Furthermore, we found that five variants (p.Gly55Ser, p.Leu138Phe, p.Lys396Glu, p.Val517Met, and p.Ile553Glu) rescue lethality but cause a shortened lifespan and bang sensitivity and affect locomotor activity, indicating that they are partial loss-of-function variants. Altogether, our results provide compelling evidence that integrity of the Integrator RNA endonuclease is critical for brain development.

Abstract 4913: Enhancer RNA profiles predict sensitivity to a novel GPX4 inhibitor

Abstract Inhibition of the GPX4 enzyme leads to the rapid accumulation of toxic lipid peroxides and cell death through a process known as ferroptosis. This mechanism has recently garnered significant attention in the field of oncology because many difficult-to-treat tumor types are particularly susceptible to this non-canonical form of cell death. However, robust biomarkers that predict sensitivity to ferroptosis are needed to help select well-defined tumor subtypes and patient segments that are likely to respond to GPX4-targeting. Our nascent RNA platform measures the active transcription of not only protein-coding transcripts, but also transcripts initiating from enhancer regions (enhancer RNAs) critical for defining cell type. We reasoned that these data could afford the unique opportunity to identify non-coding transcripts that could serve as novel biomarkers of sensitivity to ferroptosis. We recently discovered a potent and selective GPX4 inhibitor and profiled the compound for its ability to induce ferroptosis across a diverse group of cancer cell types. We chose several of the most sensitive and resistant cell lines for further examination using our nascent RNA platform and identified novel relationships between sensitivity to ferroptosis and cell-specific enhancer RNA profiles. Importantly, enhancer RNA profiles were more effective at stratifying sensitive and resistant cell lines than protein-coding gene expression. Using enhancer RNA expression profiles as biomarkers of drug sensitivity represents a novel strategy that could ultimately improve our ability to select patient cohorts more likely to benefit from emerging drug candidates and established therapeutics. Citation Format: Timothy Read, David Simpson, Jenna Rimel, Christy Rhine, Casey Pham, Cole Liechty, Eric Martin, Joey Azofeifa. Enhancer RNA profiles predict sensitivity to a novel GPX4 inhibitor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4913.

CRNDE, an enhancer RNA of prognostic value in glioma, correlates with immune infiltration: A pan-cancer analysis

Background: Recent evidence has emerged regarding the modification of the relationship between the enhancer RNA (eRNA) colorectal neoplasia differentially expressed (CRNDE) and the prognosis of patients with cancer. Nevertheless, the role of CRNDE in pan-cancer and tumour immune microenvironment (TIME) remains largely unexplored. Methods: Multiple bioinformatic methods were employed to unravel the role of elncRNA CRNDE in a wide range of cancers from the perspective of the expression patterns, prognosis, tumour immunity, and the association between CRNDE and its target gene IRX5 based on the public database. A nomogram was created by integrating independent factors. Gene set enrichment analysis (GSEA) was used to determine the potential signalling pathway mechanisms. Molecular biology experiments including RT-qPCR, MTT, Transwell, and wound-healing analyses were performed to evaluate the proliferation, migration, and invasion of glioma cells. Finally, RT-qPCR and immunohistochemistry were performed to analyse the expression and function of CRNDE in gliomas of different grades. Nissl staining was used to evaluate the potential effects of CRNDE knockdown in glioblastoma tissues. Results: CRNDE was dysregulated and associated with clinical features in 15 of the 33 cancer types. The survival analysis showed that high CRNDE expression might lead to a worsened prognosis among the six cancer types. CRNDE might be involved in immune regulation in multiple cancer types. CRNDE correlated with multiple tumours and immune-related pathways. Additionally, CRNDE knockdown inhibited glioma cell proliferation, migration, and invasion. Nissl staining confirmed that CRNDE siRNA notably decreased tumour growth. A stronger correlation between immune cells and CRNDE/IRX5 in lower-grade glioma than in glioblastoma multiforme tissues, and an association with IRX5 and immune checkpoint gene NRP1 were observed. Conclusions: An Enhancer RNA CRNDE may serve as a biomarker for cancer immunologic infiltration and poor prognosis, providing a new approach for cancer treatment.

The epigenetic function of androgen receptor in prostate cancer progression.

Androgen and androgen deprivation (castration) therapies, including androgen receptor antagonists, are clinically used to treat patients with prostate cancer. However, most hormone-dependent prostate cancer patients progress into a malignant state with loss of hormone-dependency, known as castration (drug)-resistant prostate cancer (CRPC), after prolong androgen-based treatments. Even in the CRPC state with irreversible malignancy, androgen receptor (AR) expression is detectable. An epigenetic transition to CRPC induced by the action of AR-mediated androgen could be speculated in the patients with prostate cancer. Androgen receptors belongs to the nuclear receptor superfamily with 48 members in humans, and acts as a ligand-dependent transcriptional factor, leading to local chromatin reorganization for ligand-dependent gene regulation. In this review, we discussed the transcriptional/epigenetic regulatory functions of AR, with emphasis on the clinical applications of AR ligands, AR protein co-regulators, and AR RNA coregulator (enhancer RNA), especially in chromatin reorganization, in patients with prostate cancer.

Interactive enhancer hubs (iHUBs) mediate transcriptional reprogramming and adaptive resistance in pancreatic cancer

ObjectivePancreatic ductal adenocarcinoma (PDAC) displays a remarkable propensity towards therapy resistance. However, molecular epigenetic and transcriptional mechanisms enabling this are poorly understood. In this study, we aimed to identify novel...

Enhancer RNA IRS2e is essential for IRS2 expression and the oncogenic properties in oral squamous cell carcinoma.

The crucial roles of enhancer RNAs (eRNAs) in the regulation of gene expression in human diseases have drawn wider and wider attention in recent years. However, the specific expression profile and function of eRNAs are still rarely discussed in oral squamous cell carcinoma (OSCC), the most common subtype of head and neck squamous cell carcinoma (HNSC). In this study, we aimed to investigate the expression and function of an uncharacterized eRNA, insulin receptor substrate 2 enhancer RNA (IRS2e), in OSCC. We found that IRS2e was overexpressed in HNSC and its overexpression was positively correlated with a poor prognosis. The downregulation of IRS2e by short hairpin RNA significantly inhibited cell growth and induced cellular apoptosis and cell-cycle arrest in OSCC cells. Furthermore, the ablation of IRS2e inhibited tumor growth in vivo. Mechanically, IRS2e is essential for the expression of insulin receptor substrate 2 (IRS2), an oncogene nearby IRS2e in chromosome 13. Altogether, our study demonstrated that IRS2e is a novel oncogenic eRNA required for oncogene IRS2 expression in OSCC.

EaQTLdb: An atlas of enhancer activity quantitative trait loci across cancer types.

Enhancers are key regulatory elements that exert crucial roles in diverse biological processes, including tumorigenesis and cancer development. Active enhancers could produce transcripts termed enhancer RNAs (eRNAs), which could be used as an index of enhancer activity. Here, we present a versatile data portal, enhancer activity quantitative trait loci database (eaQTLdb; http://www.bioailab.com:3838/eaQTLdb), for exploring the effects of genetic variants on enhancer activity and prioritizing candidate variants across different cancer types. By leveraging the accumulated multiomics data, we systematically identified genetic variants which influence enhancer activity in different cancer types, termed as eaQTLs. We have linked the eaQTLs to hallmarks of cancer and patients' overall survival to illustrate their potential biological roles in cancer development and progression. Notably, eaQTLs associated with the infiltration abundance of 24 different immune cell types were identified and incorporated into eaQTLdb. In addition, we applied colocalization analyses to examine 59 complex diseases and traits to identify eaQTLs colocalized with diseases/traits GWAS signals. Overall, eaQTLdb, incorporating a rich resource for exploration of eaQTLs in different cancer types, will not only benefit users in prioritizing candidate genetic variants and enhancers, but also help researchers decipher the roles of eaQTLs in the dysregulated pathways of cancer and tumor immune microenvironment, opening new diagnostic and therapeutic avenues in precise medicine.

A genome-wide CRISPR activation screen identifies SCREEM a novel SNAI1 super-enhancer demarcated by eRNAs.

The genome is pervasively transcribed to produce a vast array of non-coding RNAs (ncRNAs). Long non-coding RNAs (lncRNAs) are transcripts of >200 nucleotides and are best known for their ability to regulate gene expression. Enhancer RNAs (eRNAs) are subclass of lncRNAs that are synthesized from enhancer regions and have also been shown to coordinate gene expression. The biological function and significance of most lncRNAs and eRNAs remain to be determined. Epithelial to mesenchymal transition (EMT) is a ubiquitous cellular process that occurs during cellular migration, homeostasis, fibrosis, and cancer-cell metastasis. EMT-transcription factors, such as SNAI1 induce a complex transcriptional program that coordinates the morphological and molecular changes associated with EMT. Such complex transcriptional programs are often subject to coordination by networks of ncRNAs and thus can be leveraged to identify novel functional ncRNA loci. Here, using a genome-wide CRISPR activation (CRISPRa) screen targeting ∼10,000 lncRNA loci we identified ncRNA loci that could either promote or attenuate EMT. We discovered a novel locus that we named SCREEM (SNAI1 cis-regulatory eRNAs expressed in monocytes). The SCREEM locus contained a cluster of eRNAs that when activated using CRISPRa induced expression of the neighboring gene SNAI1, driving concomitant EMT. However, the SCREEM eRNA transcripts themselves appeared dispensable for the induction of SNAI1 expression. Interestingly, the SCREEM eRNAs and SNAI1 were co-expressed in activated monocytes, where the SCREEM locus demarcated a monocyte-specific super-enhancer. These findings suggest a potential role for SNAI1 in monocytes. Exploration of the SCREEM-SNAI axis could reveal novel aspects of monocyte biology.

FT-6876, a Potent and Selective Inhibitor of CBP/p300, is Active in Preclinical Models of Androgen Receptor-Positive Breast Cancer.

Patients with triple-negative breast cancer (TNBC) expressing the androgen receptor (AR) respond poorly to neoadjuvant chemotherapy, although AR antagonists have shown promising clinical activity, suggesting these tumors are AR-dependent. cAMP responsive element binding protein (CREB)-binding protein (CBP) and p300 are transcriptional co-activators for the AR, a key driver of AR+ breast and prostate cancer, and may provide a novel therapeutic target in AR+ TNBC. The aim of this study was to determine the therapeutic potential of FT-6876, a new CBP/p300 bromodomain inhibitor, in breast cancer models with a range of AR levels in vitro and in vivo. Effects of FT-6876 on the CBP/p300 pathway were determined by combining chromatin immunoprecipitation (ChIP) with precision run-on sequencing (PRO-seq) complemented with H3K27 acetylation (Ac) and transcriptional profiling. The antiproliferative effect of FT-6876 was also measured in vitro and in vivo. We describe the discovery of FT-6876, a potent and selective CBP/p300 bromodomain inhibitor. The combination of ChIP and PRO-seq confirmed the reduction in H3K27Ac at specific promoter sites concurrent with a decrease in CBP/p300 on the chromatin and a reduction in nascent RNA and enhancer RNA. This was associated with a time- and concentration-dependent reduction in H3K37Ac associated with a decrease in AR and estrogen receptor (ER) target gene expression. This led to a time-dependent growth inhibition in AR+ models, correlated with AR expression. Tumor growth inhibition was also observed in AR+ tumor models of TNBC and ER+ breast cancer subtypes with consistent pharmacokinetics and pharmacodynamics. Our findings demonstrate FT-6876 as a promising new CBP/p300 bromodomain inhibitor, with efficacy in preclinical models of AR+ breast cancer.

BAP18 facilitates CTCF-mediated chromatin accessible to regulate enhancer activity in breast cancer.

The estrogen receptor alpha (ERα) signaling pathway is a crucial target for ERα-positive breast cancer therapeutic strategies. Co-regulators and other transcription factors cooperate for effective ERα-related enhancer activation. Recent studies demonstrate that the transcription factor CTCF is essential to participate in ERα/E2-induced enhancer transactivation. However, the mechanism of how CTCF is achieved remains unknown. Here, we provided evidence that BAP18 is required for CTCF recruitment on ERα-enriched enhancers, facilitating CTCF-mediated chromatin accessibility to promote enhancer RNAs transcription. Consistently, GRO-seq demonstrates that the enhancer activity is positively correlated with BAP18 enrichment. Furthermore, BAP18 interacts with SMARCA1/BPTF to accelerate the recruitment of CTCF to ERα-related enhancers. Interestingly, BAP18 is involved in chromatin accessibility within enhancer regions, thereby increasing enhancer transactivation and enhancer-promoter looping. BAP18 depletion increases the sensitivity of anti-estrogen and anti-enhancer treatment in MCF7 cells. Collectively, our study indicates that BAP18 coordinates with CTCF to enlarge the transactivation of ERα-related enhancers, providing a better understanding of BAP18/CTCF coupling chromatin remodeling and E-P looping in the regulation of enhancer transcription.

ERNA profiling uncovers the enhancer landscape of oesophageal adenocarcinoma and reveals new deregulated pathways.

Cancer is driven by both genetic and epigenetic changes that impact on gene expression profiles and the resulting tumourigenic phenotype. Enhancers are transcriptional regulatory elements that are key to our understanding of how this rewiring of gene expression is achieved in cancer cells. Here, we have harnessed the power of RNA-seq data from hundreds of patients with oesophageal adenocarcinoma (OAC) or its precursor state Barrett's oesophagus coupled with open chromatin maps to identify potential enhancer RNAs and their associated enhancer regions in this cancer. We identify ~1000 OAC-specific enhancers and use these data to uncover new cellular pathways that are operational in OAC. Among these are enhancers for JUP, MYBL2, and CCNE1, and we show that their activity is required for cancer cell viability. We also demonstrate the clinical utility of our dataset for identifying disease stage and patient prognosis. Our data therefore identify an important set of regulatory elements that enhance our molecular understanding of OAC and point to potential new therapeutic directions.