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.
Although all different types of human cells share the same genome, their gene expression is spatially and temporally distinct. Establishing a tissue-specific transcriptional program during development is required for the unique function of each tissue. Enhancers are key regulatory elements that control this process. They consist of short DNA regions, usually located far from gene promoters, can be occupied by co-activators and other transcription factors, and function as
Although the role of individual enhancers in cell-type specific gene expression has been studied for a long time, it is only recently that genome-wide analysis has allowed the identification of all enhancers in the mammalian genome. This was achieved by using various histone markers that recognize enhancer regions, as well as the development of whole-genome sequencing techniques (Heintzman et al., 2009; Hon et al., 2009; Shlyueva et al., 2014). Enhancers are mainly enriched with H3K4me1 and H3K27ac, and do not contain H3K4me3 as a promoter marker, but H3K27ac-enriched enhancers in the active state can be distinguished from poised enhancers occupied with H3K4me1 only (Fig. 1). Poised enhancers are not yet functionally active; however, enrichment in the H3K27ac marker could be observed since they are active to regulate target gene expression. Global analyses by ChIP-seq to detect lineage-specific transcription factors and histone modification markers identified enhancer clusters known as super-enhancers, which are located close to each other in the genome (Fig. 1) (Chapuy et al., 2013; Hnisz et al., 2013; Kitagawa et al., 2017; Le Noir et al., 2017; Liang et al., 2016; Loven et al., 2013; Niederriter et al., 2015; Shin et al., 2016; Teppo et al., 2016; Whyte et al., 2013). Super-enhancers contain multiple hotspots co-occupied by lineage-specific transcription factors and mediators within a certain region, which typically spans tens of kilobases covered with extended histone modification markers of active enhancers. Therefore, H3K27ac and MED1, lineage-specific factors, are commonly used to identify tissue-specific super-enhancers; combinations of transcription factors and H3K27ac are also used for advanced analysis.
Super-enhancers control tissue-specific gene regulation in mouse stem cells, hair follicle stem cells, macrophages, adipocytes, and mammary epithelium (Adam et al., 2015; Gosselin et al., 2014; Shin et al., 2016; Siersbaek et al., 2014; Whyte et al., 2013). Several key features enable super-enhancers to regulate tissue-specific gene expression. First, genes associated with super-enhancers are expressed at higher levels than genes associated with typical enhancers, which are single enhancer regions controlling target gene expression. For example, in mouse embryonic stem cells, super-enhancers were found to regulate the key regulatory transcription factors Oct4, Sox2, and Nanog, which were ranked based on MED1 signals (Whyte et al., 2013). The expression of genes associated with super-enhancers such as
Determination of the molecular mechanism of long noncoding RNA (Mattick and Makunin, 2006), identification of enhancer RNA (eRNA), and bi-directionally transcribed RNA in enhancer regions (Andersson et al., 2014; Kim et al., 2010; Lam et al., 2014; Murakawa et al., 2016), raised questions regarding enhancer establishment, enhancer target-gene regulation, and chromosome looping structure (Cheng et al., 2015; Kaikkonen et al., 2013; Kim et al., 2010; Li et al., 2013; 2015; Mousavi et al., 2013; Pnueli et al., 2015; Schaukowitch et al., 2014; Shibayama et al., 2014; Wu et al., 2014). Global run-on sequencing (GRO-seq) and total RNA-seq are typically applied to identify global eRNAs in the whole genome. Although eRNA is not essential for all enhancers, it affects the regulation of active-enhancer transcription for gene expression (Cheng et al., 2015; Kim et al., 2010). Moreover, it was previously shown that eRNA affects cell-type-specific transcriptional regulation (Mousavi et al., 2013; Wu et al., 2014). eRNA is thought to function in enhancer–promoter interactions directly or indirectly to regulate gene expression by transcription pre-initiation complexes RNA pol II, DNA- and RNA-binding transcription factor, or co-factor of DNA- and RNA-binding transcription factor (Fig. 1) (Andersson, 2015; Beagrie and Pombo, 2016; Li et al., 2016). The formation of genomic looping structures, where enhancers interact with promoters, require an eRNA, mediator, and cohesion (Fig. 1) (Shibayama et al., 2014). However, it has recently been reported that eRNA could be a byproduct of the co-occupation of RNA PolII driven by enhancer-promoter looping (Kron et al., 2014). The function of eRNA during chromatin looping is still unknown, and further studies on the topic are required.
A previous study reported a correlation between super-enhancers and eRNA, which also coordinated collective transcription factor binding (Hah et al., 2015). Even though not all eRNAs were expressed at super-enhancer regions, both super-enhancers and eRNA were affected by genomic damage (Qian et al., 2014). Genomic instability can influence the super-enhancer establishment and eRNA expression and as a result, it could affect gene expression. In B cells, many super-enhancers contained activation-induced cytidine deaminase (AID) sequences, even for non-AID target genes. Consequently, AID-induced damage of enhancer regions could affect B cell function. Additionally, it could control chromatin looping by damaging regions involved in promoter-enhancer interactions (Qian et al., 2014). A correlation between loss or gain of super-enhancer formation and eRNA expression was clearly observed (Hah et al., 2015; Schmidt et al., 2015). Hah et al. (Hah et al., 2015) observed that during toll-like receptor 4 (TLR4) signaling in macrophages, ~93% of intergenic super-enhancers were associated with eRNAs, whereas only ~30% of intergenic typical enhancers were associated with eRNAs, suggesting a correlation between super-enhancer formation and eRNA transcription. Accordingly, in those intergenic super-enhancer regions, tissue-specific transcription factor binding and gene expression were much higher. It is possible that eRNA expression in super-enhancers controls transcription factor binding and consequently, regulates inflammatory gene expression. Interestingly, it also has been found that some lncRNAs called x-eRNA were targeted by the RNA exosome at super-enhancers (Pefanis et al., 2015). Since eRNAs generate complexes with single-stranded DNA for protection from genomic instability by the rapid degradation activity of the RNA exosome complex, it could be suggested that super-enhancers might be connected with their target genes through RNA exosome-mediated transcription regulation. However, it is unclear whether the mechanism is dependent on direct RNA-protein complex at enhancer-promoter interaction yet.
It is important to identify cancer-related epigenetic regulators and their functions to understand cancer pathogenesis. Oncogenes were shown to be regulated by super-enhancers (Chapuy et al., 2013; Loven et al., 2013; Pott and Lieb, 2015), and aberrant regulation of eRNA is closely related to tumorigenesis (Li et al., 2013; Teppo et al., 2016). Herein, recent discoveries of aberrant super-enhancers and eRNAs in various cancer types will be discussed (Table 1).
Cancer-related super-enhancers were found in various cancers such as colorectal cancer, small cell lung cancer, T cell acute lymphoblastic leukemia, and acute myeloid leukemia (Christensen et al., 2014; Hnisz et al., 2015; Mansour et al., 2014; Pelish et al., 2015). Among the many cancer-related super-enhancers, those related to the
The transcriptional regulatory mechanism of the
Pelish et al. identified mediator-associated cyclin-dependent kinase 8 (CDK8) as a negative regulator of super-enhancer associated transcription in acute myeloid leukemia (AML) cells (Pelish et al., 2015). ChIP-seq binding profiles at putative enhancer elements of CCAAT/enhancer binding protein, alpha (CEBPA) showed that CDK8 most closely related to MED1, followed by BRD4 and H3K27ac (Pelish et al., 2015). Furthermore, treatment with cortistatin A (CA), which inhibits mediator kinases including CDK8, increased the expression of tumor suppressor genes regulated by super-enhancers and inhibited AML progression in a xenograft model
eRNAs are transcribed from enhancer regions, but their role in regulating enhancer activity remains unknown. Li et al. (2013) performed global run-on sequencing (GRO-seq) in 17β-estradiol (E2)-treated human breast cancer cell line. They identified that E2-associated enhancers induced eRNA transcription from enhancers adjacent to E2-activated genes, and that knockdown of
eRNA transcription in human breast cancer is induced not only by estrogen but also by nutlin-3a, an activator of p53 (Leveille et al., 2015). Leveille et al. (2015) performed GRO-seq in MCF-7 cells treated with nutlin-3a to identify p53-regulated eRNAs and their role in p53-regulated enhancer activation. They discovered that lncRNA activator of enhancer domains (LED) is a p53-induced target, which binds to p53-regulated enhancer regions, inducing transcription of eRNA, such as
Increased androgen receptor (AR) expression is critical for castration-resistant prostate cancer (CRPC) progression (van der Steen et al., 2013). Therefore, it is important to elucidate the AR regulatory mechanism. Zhao et al. (2016) identified a group of AR-regulated enhancer RNAs (AR-eRNAs) in CRPC cells, including the
As described above, dysregulated super-enhancers and eR-NAs are closely related to cancer development, hence, the therapeutic effects of targeting aberrant super-enhancers or eRNAs have been studied (Table 2). Yokoyama et al. provided evidence suggesting that epigenetic regulators may be used as therapeutic targets in epithelial ovarian cancer (EOC) stem-like cells (CSCs) characterized by increased aldehyde dehydrogenase (ALDH) activity (Landen et al., 2010; Steg et al., 2012; Yokoyama et al., 2016). Because increased ALDH activity enhances stem-related gene expression and chemotherapy resistance in EOC (Landen et al., 2010), therapeutic strategies targeting ALDH are important. Yokoyama et al. showed that the bromodomain and extraterminal (BET) inhibitor JQ1 suppressed
Jiang et al. (2016) found that the known small-molecule specific CDK7 inhibitor THZ1 is a potent drug against oesophageal squamous cell carcinoma (OSCC). THZ1 injection reduced OSCC tumor weight and distal OCSS cell metastasis to the lungs (Jiang et al., 2016). Interestingly, treatment with low THZ1 doses decreased the levels of oncogenic transcripts, such as runt related transcription factor 1 (
Despite massive efforts to study the function of enhancers, our overall understanding of how enhancers regulate tissue-specific gene expression was limited. The development of high-throughput sequencing technologies allowed the identification of complete transcription factor landscapes and histone modification marks in the whole genome. Comparative analysis between various tissues can lead to a comprehensive understanding of enhancers as master regulators during development. Numerous ChIP-seq data, describing enhancer epigenetic features in a tissue-specific manner, suggest that super-enhancers are involved in tissue-specific gene regulation. Functional studies through CRISPR/Cas9 revealed that individual enhancer elements in super-enhancers have unique functions. The next question to be considered is which elements are required to maintain the unique functions of enhancers, and further research is required to show whether enhancer functions are determined by the DNA sequence itself or the structure of the super-enhancer unit. Enhancers recruit transcription factors and mediators, but this might not be sufficient to enhance target gene expression. Many enhancers are co-occupied by RNA Pol II, leading to eRNA transcription. eRNA plays a role in enhancing target gene expression through the formation of enhancer-promoter looping. The mechanisms underlying eRNA function are not yet clear, but aberrant eRNAs have been identified during tumorigenesis, suggesting that epi-genetic regulators could be potential therapeutic targets.
Super-enhancers and enhancer RNAs in cancer
|Cancer type||Sample||Super-enhancer associated genes||References|
|Colorectal cancer||HCT-116||Hnisz et al., 2015|
|Heyn et al., 2016|
|Small cell lung cancer||NCI-H69||Christensen et al., 2014|
|T cell acute lylmphoblastic leukemia||Jurkat||Mansour et al., 2014|
|Acute myeloid leukemia||MOLM-14||Pelish et al., 2015|
|Cancer type||Sample||Enhancer RNAs||References|
|Breast cancer||MCF-7||(E2-induced eRNAs) ||Li et al., 2013|
|(p53-regulated eRNAs) ||Leveille et al., 2015|
|Castration-resistant prostate cancer||LNCaP, C4-2||(AR-regulated eRNAs)||Zhao et al., 2016b|
|(enzalutamide resistance) ||Zhao et al., 2016a|
Therapeutic effect targeted by super-enhancers or eRNAs
|Ovarian cancer||ALDH||BET inhibitor (JQ1)||Reduction of tumor size under combination treatment of JQ1 and cisplatin||Yokoyama et al., 2016|
|Oesophageal squamous cell carcinoma||RUNX1, YAP1, DNAJB1, STEBF2, PAK4||CDK7 inhibitor (THZ1)||Reduction of OSCC tumor weight and distal OSCC cell metastasis||Jiang et al., 2016|