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Oral squamous cell carcinoma (OSCC) is the leading cause of death in patients with head and neck cancer. Reliable biomarkers to guide treatment decisions for OSCC remain scarce. The purpose of this study was to identify novel prognostic markers regulated by super enhancers in OSCC. Eight modules were obtained by weighted gene co-expression network analysis (WGCNA), among which MEblue module had the highest correlation with tumor stage, alcohol consumption and smoking. There were 41 genes regulated by super enhancers in MEblue module. Functional analysis showed that 41 super enhancer-regulated genes were involved in cancer progression. A total of twenty transcription factors of the 41 genes were predicted. Prognostic analysis of the 41 genes and the top 5 transcription factors showed that patients with high expression of AHCY, KCMF1, MANBAL and TFDP1 had a poor prognosis. Immunohistochemical analysis showed that AHCY, KCMF1 and MANBAL were highly expressed in OSCC tissue. Cellular experiment demonstrated that TFDP1 promoted AHCY, KCMF1 and MANBAL expression by binding to the super enhancers of these genes. Knockdown of TFDP1, AHCY, KCMF1 and MANBAL inhibited the proliferation of OSCC cells. In conclusion, AHCY, KCMF1 and MANBAL were recognized as super enhancer-regulated prognostic biomarkers regulated by TFDP1 in OSCC.

Aberrant expression of long non-coding RNAs (lncRNAs) has been reported in multiple cancers. However, the underlying mechanisms mediated by super-enhancers remain elusive. Here we sought to define the role of a novel lncRNA termed lncRNA-DAW in tumorigenesis. Our results revealed that lncRNA-DAW was driven by a liver-specific super-enhancer and transcriptionally activated by HNF4G, leading to frequent elevation in hepatocellular carcinoma (HCC) specimens. Ectopic expression of lncRNA-DAW promoted both in vivo and in vitro tumor growth. By using RNA sequencing, Wnt2 was screened out as a downstream effector of lncRNA-DAW. We next found that lncRNA-DAW physically interacted with EZH2, a negative regulator of Wnt2. This interplay subsequently potentiated CDK1-EZH2 interaction, leading to the phosphorylation and ubiquitination of EZH2. The lncRNA-DAW-mediated EZH2 degradation facilitated the de-repression of Wnt2 transcription, which eventually activated the Wnt/β-catenin pathway. Furthermore, we verified that Wnt2 potentiated in vitro and in vivo cancer cell growth by activating the Wnt/β-catenin pathway. Finally, Wnt2 amplification was confirmed as a common event in liver cancer, and the expression of lncRNA-DAW was positively correlated with Wnt2 in HCC specimens. Collectively, we are the first to identify lncRNA-DAW as a novel candidate oncogene in liver cancer, and this lncRNA may serve as a novel clinical diagnosis biomarker for liver cancer., Graphical abstract, lncRNA-DAW was driven by a liver-specific super-enhancer and frequently upregulated in liver cancer. lncRNA-DAW diminished PRC2-mediated repression of Wnt2 by antagonizing the function of EZH2, eventually leading to the activation of the Wnt/β-catenin pathway and cancer cell proliferation.

Objective: PD-L1 and PD-L2 expression levels determine immune evasion and the therapeutic efficacy of immune checkpoint blockade. The factors that drive inducible PD-L1 expression have been extensively studied, but mechanisms that result in constitutive PD-L1 expression in cancer cells are largely unknown. Methods: DNA elements were deleted in cells by CRISPR/Cas9-mediated knockout. Protein function was inhibited by chemical inhibitors. Protein levels were examined by Western blot, mRNA levels were examined by real-time RT-PCR, and surface protein expression was determined by cellular immunofluorescence and flow cytometry. Immune evasion was examined by in vitro T cell-mediated killing. Results: We determined the core regions (chr9: 5, 496, 378–5, 499, 663) of a previously identified PD-L1L2-super-enhancer (SE). Through systematic analysis, we found that the E26 transformation-specific (ETS) variant transcription factor (ETV4) bound to this core DNA region but not to DNA surrounding PD-L1L2SE. Genetic knockout of ETV4 dramatically reduced the expressions of both PD-L1 and PD-L2. ETV4 transcription was dependent on ERK activation, and BRAF/TAK1-induced ERK activation was dependent on extracellular signaling from αvβ3 integrin, which profoundly affected ETV4 transcription and PD-L1/L2 expression. Genetic silencing or pharmacological inhibition of components of the PD-L1L2-SE-associated pathway rendered cancer cells susceptible to T cell-mediated killing. Conclusions: We identified a pathway originating from the extracellular matrix that signaled via integrin/BRAF/TAK1/ERK/ETV4 to PD-L1L2-SE to induce PD-L1-mediated immune evasion. These results provided new insights into PD-L1L2-SE activation and pathways associated with immune checkpoint regulation in cancer.

Many enhancers exist as clusters in the genome, which has led to the coining of the term ‘super-enhancer’. In this Viewpoint, five experts discuss our biological understanding of enhancer clusters, how we can responsibly study their functions, and their opinions on whether names for enhancer clusters are an informative reflection of their functional properties. They also provide their thoughts on key unanswered questions and future directions in the field. In this Viewpoint, five experts discuss our biological understanding of super-enhancers, how we can responsibly study their functions, and their opinions on whether names for enhancer clusters are an informative reflection of their functional properties.

Acute and chronic inflammation is a basic pathological event that contributes to atherosclerosis, cancer, infectious diseases, and immune disorders. Inflammation is an adaptive process to both external and internal stimuli experienced by the human body. Although the mechanism of gene transcription is highly complicated and orchestrated in a timely and spatial manner, recent developments in next-generation sequencing, genome-editing, cryo-electron microscopy, and single cell-based technologies could provide us with insights into the roles of super enhancers (SEs). Initially, SEs were implicated in determining cell fate; subsequent studies have clarified that SEs are associated with various pathological conditions, including cancer and inflammatory diseases. Recent technological advances have unveiled the molecular mechanisms of SEs, which involve epigenetic histone modifications, chromatin three-dimensional structures, and phase-separated condensates. In this review, we discuss the relationship between inflammation and SEs and the therapeutic potential of SEs for inflammatory diseases.

Background Transcriptional programs control cell fate, and identifying their components is critical for understanding diseases caused by cell lesion, such as podocytopathy. Although many transcription factors (TFs) are necessary for cell-state maintenance in glomeruli, their roles in transcriptional regulation are not well understood. Methods The distribution of H3K27ac histones in human glomerulus cells was analyzed to identify superenhancer-associated TFs, and ChIP-seq and transcriptomics were performed to elucidate the regulatory roles of the TFs. Transgenic animal models of disease were further investigated to confirm the roles of specific TFs in podocyte maintenance. Results Superenhancer distribution revealed a group of potential TFs in core regulatory circuits in human glomerulus cells, including FOXC1/2, WT1, and LMX1B. Integration of transcriptome and cistrome data of FOXC1/2 in mice resolved transcriptional regulation in podocyte maintenance. FOXC1/2 regulated differentiation-associated transcription in mature podocytes. In both humans and animal models, mature podocyte injury was accompanied by deregulation of FOXC1/2 expression, and FOXC1/2 overexpression could protect podocytes in zebrafish. Conclusions FOXC1/2 maintain podocyte differentiation through transcriptional stabilization. The genome-wide chromatin resources support further investigation of TFs' regulatory roles in glomeruli transcription programs.

In both humans and mice, CTCF-binding elements form a series of interacting loops across the MHC class II (MHC-II) locus, and CTCF is required for maximal MHC-II gene expression. In humans, a CTCF-bound chromatin insulator termed XL9 and a super enhancer (SE) DR/DQ-SE situated in the intergenic region between HLA-DRB1 and HLA-DQA1 play critical roles in regulating MHC-II expression. In this study, we identify a similar SE, termed IA/IE-SE, located between H2-Eb1 and H2-Aa of the mouse that contains a CTCF site (C15) and a novel region of high histone H3K27 acetylation. A genetic knockout of C15 was created and its role on MHC-II expression tested on immune cells. We found that C15 deletion did not alter MHC-II expression in B cells, macrophages, and macrophages treated with IFN-γ because of functional redundancy of the remaining MHC-II CTCF sites. Surprisingly, embryonic fibroblasts derived from C15-deleted mice failed to induce MHC-II gene expression in response to IFN-γ, suggesting that at least in this developmental lineage, C15 was required. Examination of the three-dimensional interactions with C15 and the H2-Eb1 and H2-Aa promoters identified interactions within the novel region of high histone acetylation within the IA/IE-SE (termed N1) that contains a PU.1 binding site. CRISPR/Cas9 deletion of N1 altered chromatin interactions across the locus and resulted in reduced MHC-II expression. Together, these data demonstrate the functional redundancy of the MHC-II CTCF elements and identify a functionally conserved SE that is critical for maximal expression of MHC-II genes.

Transcriptional addiction plays a pivotal role in maintaining the hallmarks of cancer cells. Thus, targeting super-enhancers (SEs), which modulate the transcriptional activity of oncogenes, has become an attractive strategy for cancer therapy. As yet, however, the molecular mechanisms of this process in bladder cancer (BC) remain to be elucidated. Here, we aimed to provide detailed information regarding the SE landscape in BC and to investigate new potential pharmaceutical targets for BC therapy. We employed THZ1 as a potent and specific CDK7 inhibitor. In vitro and in vivo studies were carried out to investigate the anticancer and apoptosis-inducing effects of THZ1 on BC cells. Whole-transcriptome sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) were performed to investigate the mechanism and function of SE-linked oncogenic transcription in BC cells. We found that THZ1 serves as an effective and potent inhibitor with suppressive activity against BC cells. An integrative analysis of THZ1-sensitive and SE-associated oncogenes yielded potential new pharmaceutical targets, including DDIT4, B4GALT5, PSRC1 and MED22. Combination treatment with THZ1 and the DDIT4 inhibitor rapamycin effectively suppressed BC cell growth. In addition, we found that THZ1 and rapamycin sensitized BC cells to conventional chemotherapy. Our data indicate that exploring BC gene regulatory mechanisms associated with SEs through integrating RNA-seq and ChIP-seq data improves our understanding of BC biology and provides a basis for innovative therapies.

MLL is an aggressive subtype of leukemia with a poor prognosis that mostly affects pediatric patients. MLL-rearranged fusion proteins (MLLr) induce aberrant target gene expression resulting in leukemogenesis. MLL and its fusions are tethered to chromatin by LEDGF/p75, a transcriptional co-activator that specifically recognizes H3K36me2/3. LEDGF/p75 is ubiquitously expressed and associated with regulation of gene expression, autoimmune responses, and HIV replication. LEDGF/p75 was proven to be essential for leukemogenesis in MLL. Apart from MLL, LEDGF/p75 has been linked to lung, breast, and prostate cancer. Intriguingly, LEDGF/p75 interacts with Med-1, which co-localizes with BRD4. Both are known as co-activators of super-enhancers. Here, we describe LEDGF/p75-dependent chemoresistance of MLLr cell lines. Investigation of the underlying mechanism revealed a role of LEDGF/p75 in the cell cycle and in survival pathways and showed that LEDGF/p75 protects against apoptosis during chemotherapy. Remarkably, LEDGF/p75 levels also affected expression of BRD4 and Med1. Altogether, our data suggest a role of LEDGF/p75 in cancer survival, stem cell renewal, and activation of nuclear super enhancers. ispartof: Cancer Gene Therapy vol:29 issue:2 pages:133-140 ispartof: location:England status: published

Esophageal squamous cell carcinoma (ESCC) is the predominant subtype of esophageal cancer with a particularly high prevalence in certain geographical regions and a poor prognosis with a 5-year survival rate of 15–25%. Despite numerous studies characterizing the genetic and transcriptomic landscape of ESCC, there are currently no effective targeted therapies. In this study, we used an unbiased screening approach to uncover novel molecular precision oncology targets for ESCC and identified the bromodomain and extraterminal (BET) family member bromodomain testis-specific protein (BRDT) to be uniquely expressed in a subgroup of ESCC. Experimental studies revealed that BRDT expression promotes migration but is dispensable for cell proliferation. Further mechanistic insight was gained through transcriptome analyses, which revealed that BRDT controls the expression of a subset of ΔNp63 target genes. Epigenome and genome-wide occupancy studies, combined with genome-wide chromatin interaction studies, revealed that BRDT colocalizes and interacts with ΔNp63 to drive a unique transcriptional program and modulate cell phenotype. Our data demonstrate that these genomic regions are enriched for super-enhancers that loop to critical ΔNp63 target genes related to the squamous phenotype such as KRT14, FAT2, and PTHLH. Interestingly, BET proteolysis-targeting chimera, MZ1, reversed the activation of these genes. Importantly, we observed a preferential degradation of BRDT by MZ1 compared with BRD2, BRD3, and BRD4. Taken together, these findings reveal a previously unknown function of BRDT in ESCC and provide a proof-of-concept that BRDT may represent a novel therapeutic target in cancer.

Background Super-enhancer-associated long noncoding RNAs (SE-lncRNAs) have been reported to play essential roles in tumorigenesis, but the fundamental mechanism of SE-lncRNAs in colorectal cancer (CRC) remains largely unknown. Methods A microarray was performed to identify the differentially expressed SE-lncRNAs between CRC tissues and peritumoral tissues. A novel SE-lncRNA, AC005592.2, was selected from these differentially expressed SE-lncRNAs to explore its effects on CRC development. Fluorescence quantitative real-time PCR (qRT-PCR) was used to assay the expression of AC005592.2 in CRC tissues and cell lines. Functional assays were applied to identify the biological effects of AC005592.2 in CRC cells. Furthermore, RNA-seq was employed to predict potential targets of AC005592.2. Results AC005592.2 was significantly increased in CRC tissues and cells. High expression of AC005592.2 was significantly associated with TNM stage and tumor differentiation in CRC patients. Knockdown of AC005592.2 suppressed CRC cell proliferation, invasion and migration but promoted apoptosis, while AC005592.2 overexpression exerted the opposite effects on CRC cells. In addition, AC005592.2 positively regulated the expression of olfactomedin 4 (OLFM4), which was also upregulated in CRC tissues. Conclusion The findings suggested that AC005592.2 is a crucial promoter of CRC progression and may serve as an attractive therapeutic target for CRC.

Super-enhancers (SEs) mediate high transcription levels of target genes. Previous studies have shown that SEs recruit transcription complexes and generate enhancer RNAs (eRNAs). We characterized transcription at the human and murine β-globin locus control region (LCR) SE. We found that the human LCR is capable of recruiting transcription complexes independently from linked globin genes in transgenic mice. Furthermore, LCR hypersensitive site 2 (HS2) initiates the formation of bidirectional transcripts in transgenic mice and in the endogenous β-globin gene locus in murine erythroleukemia (MEL) cells. HS2 3′eRNA is relatively unstable and remains in close proximity to the globin gene locus. Reducing the abundance of HS2 3′eRNA leads to a reduction in β-globin gene transcription and compromises RNA polymerase II (Pol II) recruitment at the promoter. The Integrator complex has been shown to terminate eRNA transcription. We demonstrate that Integrator interacts downstream of LCR HS2. Inducible ablation of Integrator function in MEL or differentiating primary human CD34+ cells causes a decrease in expression of the adult β-globin gene and accumulation of Pol II and eRNA at the LCR. The data suggest that transcription complexes are assembled at the LCR and transferred to the globin genes by mechanisms that involve Integrator mediated release of Pol II and eRNA from the LCR.

Super-enhancers (SEs) are congregated enhancer clusters with high level of loading of transcription factors (TFs), cofactors and epigenetic modifications. Through direct co-occupancy at their own SEs as well as each other's, a small set of so called "master" TFs form interconnected core regulatory circuitry (CRCs) to orchestrate transcriptional programs in both normal and malignant cells. These master TFs can be predicted mathematically using epigenomic methods. In this Review, we summarize the identification of SEs and CRCs in cancer cells, the mechanisms by which master TFs and SEs cooperatively regulate cancer-type-specific expression programs, and the cancer-type- and subtype-specificity of CRC and the significance in cancer biology.

Meningiomas are the most common primary intracranial tumor with current classification offering limited therapeutic guidance. Here, we interrogated meningioma enhancer landscapes from 33 tumors to stratify patients based upon prognosis and identify novel meningioma-specific dependencies. Enhancers robustly stratified meningiomas into three biologically distinct groups (adipogenesis/cholesterol, mesodermal, and neural crest) distinguished by distinct hormonal lineage transcriptional regulators. Meningioma landscapes clustered with intrinsic brain tumors and hormonally responsive systemic cancers with meningioma subgroups, reflecting progesterone or androgen hormonal signaling. Enhancer classification identified a subset of tumors with poor prognosis, irrespective of histologic grading. Superenhancer signatures predicted drug dependencies with superior in vitro efficacy to treatment based upon the NF2 genomic profile. Inhibition of DUSP1, a novel and druggable meningioma target, impaired tumor growth in vivo. Collectively, epigenetic landscapes empower meningioma classification and identification of novel therapies. Significance: Enhancer landscapes inform prognostic classification of aggressive meningiomas, identifying tumors at high risk of recurrence, and reveal previously unknown therapeutic targets. Druggable dependencies discovered through epigenetic profiling potentially guide treatment of intractable meningiomas. This article is highlighted in the In This Issue feature, p. 1611

Estrogen receptor α (ER) acts as an oncogenic signal in endometrial endometrioid carcinoma. ER binding activity largely depends on chromatin remodeling and recruitment of transcription factors to estrogen response elements. A deeper understanding of these regulatory mechanisms may uncover therapeutic targets for ER-dependent endometrial cancers. We show that estrogen induces accessible chromatin and ER binding at a subset of enhancers, which form higher-order super enhancers that are vital for ER signaling. ER positively correlates with active enhancers in primary tumors, and tumors were effectively classified into molecular subtypes with chromatin accessibility dynamics and ER-dependent gene signature. ARID1A binds within ER-bound enhancers and regulates ER-dependent transcription. Knockdown of ARID1A or fulvestrant treatment profoundly affects the gene-expression program, and inhibits cell growth phenotype by affecting the chromatin environment. Importantly, we found dysregulated expression of circadian rhythms genes by estrogen in cancer cells and in primary tumors. Knockdown of ARID1A reduces the chromatin accessibility and ER binding at enhancers of the circadian gene ARNTL and BHLHE41, leading to a decreased expression of these genes. Altogether, we uncover a critical role for ARID1A in ER signaling and therapeutic target in ER-positive endometrial cancer.

Next generation antiandrogens such as enzalutamide (Enz) are effective initially for the treatment of castration-resistant prostate cancer (CRPC). However, the disease often relapses and the underlying mechanisms remain elusive. By performing H3-lysine-27 acetylation (H3K27ac) ChIP-seq in Enz-resistant CRPC cells, we identified a group of super enhancers (SEs) that are abnormally activated in Enz-resistant CRPC cells and associated with enhanced transcription of a subset of tumor promoting genes such as CHPT1, which catalyzes phosphatidylcholine (PtdCho) synthesis and regulates choline metabolism. Increased CHPT1 conferred CRPC resistance to Enz in vitro and in mice. While androgen receptor (AR) primarily binds to a putative CHPT1 enhancer and mediates androgen-dependent expression of CHPT1 gene in Enz-sensitive prostate cancer cells, AR binds to a different enhancer within the CHPT1 SE locus and facilities androgen-independent expression of CHPT1 in Enz-resistant cells. We further identified a long-non coding RNA transcribed at CHPT1 enhancer (also known as enhancer RNA) that binds to the H3K27ac reader BRD4 and participates in regulating CHPT1 SE activity and CHPT1 gene expression. Our findings demonstrate that aberrantly activated SE upregulates CHPT1 expression and confers Enz resistance in CRPC, suggesting that SE-mediated expression of downstream effectors such as CHPT1 can be viable targets to overcome Enz resistance in PCa.

Background Lung tumors and normal lung tissues show large differences in epigenetic modification which can affect the chromosome structure and expression of genes. However, the epigenetic reprogramming in lung adenocarcinoma remains unclear. Methods and results With the bioinformatics analysis, we found that some activated super-enhancers (SEs) only appear in lung adenocarcinoma cells, and 781 abnormal activated super-enhancers (AASEs) were found. Not only are the traditional oncogenes found to be activated by AASEs, such as MET and SLC2A1, but also some new genes were activated by AASEs, which probably contributes to the carcinogenic process in lung cancer. The enrichment analysis of the genes activated by AASEs shows that the glycolysis process and cell proliferation were enhanced and the apoptotic process was negatively regulated. Two AASEs were separately knockout by CRISPR/Cas9 in A549, PC-9, and H1299 cell lines and the expression of target genes decreased. The motif of CTCF, SMARCA1, SOX4, FOXM1, IRF3, IRF7, and STAT2 was enriched in AASEs, supporting that the chromosome structure changed and these transcription factors would be the master regulators on the formation of AASEs. Conclusion This study provided comprehensive insight into the mechanisms of SEs, as well as a potential therapeutic target for lung cancer.

Owing to bursts in the expression of thousands of germline-specific genes, the testis has the most diverse and complex transcriptome of all organs. By analyzing the male germline of mice, we demonstrate that the genome-wide reorganization of super-enhancers (SEs) drives bursts in germline gene expression after the mitosis-to-meiosis transition. SE reorganization is regulated by two molecular events: the establishment of meiosis-specific SEs via A-MYB (MYBL1), a key transcription factor for germline genes, and the resolution of SEs in mitotically proliferating cells via SCML2, a germline-specific Polycomb protein required for spermatogenesis-specific gene expression. Before entry into meiosis, meiotic SEs are preprogrammed in mitotic spermatogonia to ensure the unidirectional differentiation of spermatogenesis. We identify key regulatory factors for both mitotic and meiotic enhancers, revealing a molecular logic for the concurrent activation of mitotic enhancers and suppression of meiotic enhancers in the somatic and/or mitotic proliferation phases. The mapping of super-enhancers (SEs) in four stages of spermatogenesis reveals how meiotic enhancers remain ‘poised’ for activation as spermatogonia enter meiosis.

The hyperactivation and overexpression of critical oncogenes is a common occurrence in multiple types of malignant tumors. Recently, the abnormal activation mechanism of an oncogene by a super-enhancer (SE) has attracted significant attention. A series of changes (insertion, deletion, translocation and rearrangement) in the genome occurring in cancer cells may generate new SEs, leading to the overexpression of SE-driven oncogenes. SEs are composed of typical enhancers densely loaded with mediator complexes, transcription factors, and chromatin regulators, and drive the overexpression of oncogenes associated with cellular identity and disease. Cyclin-dependent kinase 7 (CDK7) and bromodomain protein 4 (BRD4) are critical mediator complexes associated with SE-mediated transcription. Clinical trials have shown that emerging small-molecule inhibitors (CDK7 and BRD4 inhibitor), targeting the SE exert a notable effect on cancer treatment. Increasing evidences has illustrated that the SE and its associated complexes play a critical role in the development of various types of cancer. The present review discusses the composition, function and regulation of SEs and their contribution to oncogenic transcription. In addition, creative therapeutic approaches that target SE, their advantages and disadvantages, as well as the problems with their clinical application are discussed. It was found that targeting SE may be used in conventional treatment and establish more access for patients with cancer.

Fixed-tissue ChIP-seq for H3K27 acetylation (H3K27ac) profiling (FiTAc-seq) is an epigenetic method for profiling active enhancers and promoters in formalin-fixed, paraffin-embedded (FFPE) tissues. We previously developed a modified ChIP-seq protocol (FiT-seq) for chromatin profiling in FFPE. FiT-seq produces high-quality chromatin profiles particularly for methylated histone marks but is not optimized for H3K27ac profiling. FiTAc-seq is a modified protocol that replaces the proteinase K digestion applied in FiT-seq with extended heating at 65 °C in a higher concentration of detergent and a minimized sonication step, to produce robust genome-wide H3K27ac maps from clinical samples. FiTAc-seq generates high-quality enhancer landscapes and super-enhancer (SE) annotation in numerous archived FFPE samples from distinct tumor types. This approach will be of great interest for both basic and clinical researchers. The entire protocol from FFPE blocks to sequence-ready library can be accomplished within 4 d. This protocol describes a ChIP-seq procedure optimized for profiling H3K27 acetylation in archived formalin-fixed, paraffin-embedded (FFPE) tissues sampled through whole or macrodissected sectioning or from punched cores.

Estrogen receptor α (ERα) modulates gene expression by interacting with chromatin regions that are frequently distal from the promoters of estrogen-regulated genes. Active chromatin-enriched “super-enhancer” (SE) regions, mainly observed in in vitro culture systems, often control production of key cell type–determining transcription factors. Here, we defined super-enhancers that bind to ERα in vivo within hormone-responsive uterine tissue in mice. We found that SEs are already formed prior to estrogen exposure at the onset of puberty. The genes at SEs encoded critical developmental factors, including retinoic acid receptor α (RARA) and homeobox D (HOXD). Using high-throughput chromosome conformation capture (Hi-C) along with DNA sequence analysis, we demonstrate that most SEs are located at a chromatin loop end and that most uterine genes in loop ends associated with these SEs are regulated by estrogen. Although the SEs were formed before puberty, SE-associated genes acquired optimal ERα-dependent expression after reproductive maturity, indicating that pubertal processes that occur after SE assembly and ERα binding are needed for gene responses. Genes associated with these SEs affected key estrogen-mediated uterine functions, including transforming growth factor β (TGFβ) and LIF interleukin-6 family cytokine (LIF) signaling pathways. To the best of our knowledge, this is the first identification of SE interactions that underlie hormonal regulation of genes in uterine tissue and optimal development of estrogen responses in this tissue.

Long noncoding RNAs (lncRNAs) have important regulatory roles in cancer biology. Although some lncRNAs have well‐characterized functions, the vast majority of this class of molecules remains functionally uncharacterized. To systematically pinpoint functional lncRNAs, a computational approach was proposed for identification of lncRNA‐mediated competing endogenous RNAs (ceRNAs) through combining global and local regulatory direction consistency of expression. Using esophageal squamous cell carcinoma (ESCC) as model, we further identified many known and novel functional lncRNAs acting as ceRNAs (ce‐lncRNAs). We found that most of them significantly regulated the expression of cancer‐related hallmark genes. These ce‐lncRNAs were significantly regulated by enhancers, especially super‐enhancers (SEs). Landscape analyses for lncRNAs further identified SE‐associated functional ce‐lncRNAs in ESCC, such as HOTAIR, XIST, SNHG5, and LINC00094. THZ1, a specific CDK7 inhibitor, can result in global transcriptional downregulation of SE‐associated ce‐lncRNAs. We further demonstrate that a SE‐associated ce‐lncRNA, LINC00094 can be activated by transcription factors TCF3 and KLF5 through binding to SE regions and promoted ESCC cancer cell growth. THZ1 downregulated expression of LINC00094 through inhibiting TCF3 and KLF5. Our data demonstrated the important roles of SE‐associated ce‐lncRNAs in ESCC oncogenesis and might serve as targets for ESCC diagnosis and therapy., We developed the GloceRNA method for the identification of functional ce‐lncRNAs based on merging global and local regulatory direction consistency of expression associated with ceRNAs. GloceRNA identified many known and novel functional ce‐lncRNAs, which regulated the expression of a large number of cancer hallmark genes. We further identified novel SE‐associated ce‐lncRNAs and demonstrated their important roles in ESCC.

Super-enhancers (SEs) consist of a cluster of many enhancers bound to a great number of transcription factors. They are critical cis-regulatory elements that determine the identity of various human cell types. During tumorigenesis, DNA mutations and indels, chromosomal rearrangements, three-dimensional chromatin structural changes, and viral infections mediate oncogenic SE activation, and activated SEs have been found to regulate the expression of oncogenic genes. Inhibition specifically targeted to oncogenic SE assembly and activation provides a novel powerful therapeutic strategy for various cancers. In this paper, we first introduce the current understanding of oncogenic SE assembly and activation and then summarize the pathogenic factors and mechanism of oncogenic SE activation. Next, we elaborate on the oncogenic functions of SEs in cancers and the application of SEs as therapeutic targets. Finally, we turn our focus to the use of SEs in basic research and clinical trials., Cancer: Enhancing the expression of cancer-causing genes Drugs that block the assembly and activation of large DNA segments involved in enhancing gene expression could help in the treatment of cancer. Faqing Tang of Hunan Cancer Hospital in Changsha, China, and colleagues review the ways in which cancer cells hijack clusters of gene-regulating sequences known as super-enhancers, regulatory gene regions that normally help determine a cell’s unique identity, to drive the aberrant gene activity that fuels tumor growth. The researchers describe how numerous factors, ranging from internal DNA alterations, both large and small, to viral infections and other external assaults, can spur the formation of cancer-causing super-enhancers, leading to out-of-control gene expression. Therapies that selectively target these super-enhancers are now in early clinical testing. However, more studies of super-enhancers and their role in cancer development are needed to inform future drug development.

Molecular endocrinology of vitamin D is based on the activation of the transcription factor vitamin D receptor (VDR) by the vitamin D metabolite 1α,25-dihydroxyvitamin D3. This nuclear vitamin D-sensing process causes epigenome-wide effects, such as changes in chromatin accessibility as well as in the contact of VDR and its supporting pioneer factors with thousands of genomic binding sites, referred to as vitamin D response elements. VDR binding enhancer regions loop to transcription start sites of hundreds of vitamin D target genes resulting in changes of their expression. Thus, vitamin D signaling is based on epigenome- and transcriptome-wide shifts in VDR-expressing tissues. Monocytes are the most responsive cell type of the immune system and serve as a paradigm for uncovering the chromatin model of vitamin D signaling. In this review, an alternative approach for selecting vitamin D target genes is presented, which are most relevant for understanding the impact of vitamin D endocrinology on innate immunity. Different scenarios of the regulation of primary upregulated vitamin D target genes are presented, in which vitamin D-driven super-enhancers comprise a cluster of persistent (constant) and/or inducible (transient) VDR-binding sites. In conclusion, the spatio-temporal VDR binding in the context of chromatin is most critical for the regulation of vitamin D target genes.

Genes under control of super-enhancers are expressed at extremely high levels and are frequently associated with nuclear speckles. Recent data suggest that the high concentration of unphosphorylated RNA polymerase II (Pol II) and Mediator recruited to super-enhancers create phase-separated condensates. Transcription initiates within or at the surface of these phase-separated droplets and the phosphorylation of Pol II, associated with transcription initiation and elongation, dissociates Pol II from these domains leading to engagement with nuclear speckles, which are enriched with RNA processing factors. The transitioning of Pol II from transcription initiation domains to RNA processing domains effectively co-ordinates transcription and processing of highly expressed RNAs which are then rapidly exported into the cytoplasm.

Background: Genome-wide association studies (GWAS) have identified dozens of loci associated with colon and rectal adenocarcinoma risk. As tissue-specific super-enhancers (SE) play important roles in tumorigenesis, we systematically investigate SEs and inner variants in established GWAS loci to decipher the underlying biological mechanisms. Methods: Through a comprehensive bioinformatics analysis on multi-omics data, we screen potential single-nucleotide polymorphisms (SNP) in cancer-specific SEs, and then subject them to a two-stage case–control study containing 4,929 cases and 7,083 controls from the Chinese population. A series of functional assays, including reporter gene assays, electrophoretic mobility shift assays (EMSA), CRISPR–Cas9 genome editing, chromosome conformation capture (3C) assays, and cell proliferation experiments, are performed to characterize the variant's molecular consequence and target genes. Results: The SNP rs11064124 in 12p13.31 is found significantly associated with the risk of colon and rectal adenocarcinoma with an odds ratio (OR) of 0.87 [95% confidence interval (CI), 0.82–0.92, P = 8.67E-06]. The protective rs11064124-G weakens the binding affinity with vitamin D receptor (VDR) and increases the enhancer's activity and interactions with two target genes' promoters, thus coactivating the transcription of CD9 and PLEKHG6, which are both putative tumor suppressor genes for colon and rectal adenocarcinoma. Conclusions: Our integrative study highlights an SE polymorphism rs11064124 and two susceptibility genes CD9 and PLEKHG6 in 12p13.31 for colon and rectal adenocarcinoma. Impact: These findings suggest a novel insight for genetic pathogenesis of colon and rectal adenocarcinoma, involving transcriptional coactivation of diverse susceptibility genes via the SE element as a gene regulation hub.

seRNA is a noncoding RNA (ncRNA) transcribed from active super-enhancer (SE), through which SE exerts biological functions and participates in various physiological and pathological processes. seRNA recruits cofactor, RNA polymerase II and mediator to constitute and stabilize chromatin loop SE and promoter region, which regulates target genes transcription. In tumorigenesis, DNA insertion, deletion, translocation, focal amplification and carcinogen factor mediate oncogenic SE generation, meanwhile, oncogenic SE transcribes into tumor-related seRNA, termed as oncogenic seRNA. Oncogenic seRNA participates in tumorigenesis through activating various signal-pathways. The recent reports showed that oncogenic seRNA implicates in a widespread range of cytopathological processes in cancer progression including cell proliferation, apoptosis, autophagy, epithelial-mesenchymal transition, extracellular matrix stiffness and angiogenesis. In this article, we comprehensively summarized seRNA’s characteristics and functions, and emphatically introduced inducible formation of oncogenic seRNA and its functional mechanisms. Lastly, some research strategies on oncogenic seRNA were introduced, and the perspectives on cancer therapy that targets oncogenic seRNA were also discussed.

Histone modifications affect transcription by changing the chromatin structure. In particular, histone H3 lysine 4 trimethylation (H3K4me3) is one of the most recognized epigenetic marks of active transcription. While many studies have provided evidence of the correlation between H3K4me3 and active transcription, details regarding the mechanism involved remain unclear. The first study on the broad H3K4me3 domain was reported in 2014; subsequently, the function of this domain has been studied in various cell types. In this review, we summarized the recent studies on the role of the broad H3K4me3 domain in transcription, development, memory formation, and several diseases, including cancer and autoimmune diseases. The broadest H3K4me3 domains are associated with increased transcriptional precision of cell-type-specific genes related to cell identity and other essential functions. The broad H3K4me3 domain regulates maternal zygotic activation in early mammalian development. In systemic autoimmune diseases, high expression of immune-responsive genes requires the presence of the broad H3K4me3 domain in the promoter-proximal regions. Transcriptional repression of tumor-suppressor genes is associated with the shortening of the broad H3K4me3 domains in cancer cells. Additionally, the broad H3K4me3 domain interacts with the super-enhancer to regulate cancer-associated genes. During memory formation, H3K4me3 breadth is regulated in the hippocampus CA1 neurons. Taken together, these findings indicate that H3K4me3 breadth is essential for the regulation of the transcriptional output across multiple cell types.

Rationale: The forkhead box A1 (FOXA1) is a crucial transcription factor in initiation and development of breast, lung and prostate cancer. Previous studies about the FOXA1 transcriptional network were mainly focused on protein-coding genes. Its regulatory network of long non-coding RNAs (lncRNAs) and their role in FOXA1 oncogenic activity remains unknown. Methods: The Cancer Genome Atlas (TCGA) data, RNA-seq and ChIP-seq data were used to analyze FOXA1 regulated lncRNAs. RT-qPCR was used to detect the expression of DSCAM-AS1, RT-qPCR and Western blotting were used to determine the expression of FOXA1, estrogen receptor α (ERα) and Y box binding protein 1 (YBX1). RNA pull-down and RIP-qPCR were employed to investigate the interaction between DSCAM-AS1 and YBX1. The effect of DSCAM-AS1 on malignant phenotypes was examined through in vitro and in vivo assays. Results: In this study, we conducted a global analysis of FOXA1 regulated lncRNAs. For detailed analysis, we chose lncRNA DSCAM-AS1, which is specifically expressed in lung adenocarcinoma, breast and prostate cancer. The expression level of DSCAM-AS1 is regulated by two super-enhancers (SEs) driven by FOXA1. High expression levels of DSCAM-AS1 was associated with poor prognosis. Knockout experiments showed DSCAM-AS1 was essential for the growth of xenograft tumors. Moreover, we demonstrated DSCAM-AS1 can regulate the expression of the master transcriptional factor FOXA1. In breast cancer, DSCAM-AS1 was also found to regulate ERα. Mechanistically, DSCAM-AS1 interacts with YBX1 and influences the recruitment of YBX1 in the promoter regions of FOXA1 and ERα. Conclusion: Our study demonstrated that lncRNA DSCAM-AS1 was transcriptionally activated by super-enhancers driven by FOXA1 and exhibited lineage-specific expression pattern. DSCAM-AS1 can promote cancer progression by interacting with YBX1 and regulating expression of FOXA1 and ERα.

Osteosarcoma is an aggressive malignancy with poor prognosis. Super-enhancers (SE) have been highlighted as critical oncogenic elements required for maintaining the cancer cell characteristics. However, the regulatory role of SEs in osteosarcoma properties has not yet been elucidated. In the current study, we found that osteosarcoma cells and clinical specimens shared a significant fraction of SEs. Moreover, leukemia-inhibitory factor (LIF) was identified as an essential factor under the control of osteosarcoma-specific SE. The expression of LIF was positively correlated with the stem cell core factor genes in osteosarcoma. Furthermore, LIF recombinant protein–treated osteosarcoma cells displayed enhanced stem cell–like characteristics, such as increased sphere-forming potential, stimulated self-renewal, upregulated metastasis ability, and increased stemness-related gene expression. Notably, the histone 3 lysine 27 tri-methylation (H3K27me3) demethylase UTX was found as a key activator of LIF transcription in osteosarcoma. The UTX inhibitor, GSK-J4, induced H3K27me3 accumulation and impaired histone 3 lysine 27 acetylation (H3K27ac) at LIF gene locus, leading to LIF signaling pathway inhibition. GSK-J4 treatment resulted in profound defects in stem cell-like characteristics and stemness-related gene activation in osteosarcoma by modulating the H3K27ac of NOTCH1 signaling pathway gene loci. The NOTCH1 inhibitor Crenigacestat (TargetMol, T3633) repressed LIF-mediated activation of the stemness-related genes in osteosarcoma patient-derived primary tissues. Implications: This study reveals osteosarcoma SE profiles and uncovers a distinct tumor–stemness epigenetic regulatory mechanism in which an osteosarcoma-specific SE-mediated factor, LIF, promotes osteosarcoma stemness gene activation via NOTCH1 signaling pathway.

Background and Aims: Aberrant transcriptional programs are highly regulated processes that play important roles in the development and progression of hepatocellular carcinoma (HCC). Emerging evidence suggests that super-enhancers (SEs) often drive critical oncogene expression. However, SE-associated genes in HCC pathogenesis are still poorly understood. Methods: We performed integrative ChIP-seq and Hi-C analyses of HCC cells and identified ajuba LIM protein (AJUBA) as a SE-associated gene. We evaluated AJUBA expression in HCC using immunohistochemistry, immunoblotting, and qRT-PCR. ChIP and luciferase reporter assays were performed to demonstrate that transcription factor 4 (TCF4) bound to AJUBA-associated SEs. We then assessed the role of AJUBA in HCC using both in vitro and in vivo assays. Epithelial-mesenchymal transition (EMT) was examined using immunofluorescence and immunoblotting assays. Furthermore, we used immunoprecipitation and BiFC assays to explore the underlying mechanisms. Results: We identified AJUBA as a SE-associated oncogene in HCC regulated by TCF4. High AJUBA expression was related to an aggressive phenotype and unfavorable outcome in HCC patients. AJUBA knockdown significantly reduced cell migration and invasion capacities both in vitro and in vivo. Furthermore, AJUBA overexpression in HCC recruited tumor necrosis factor associated factor 6 (TRAF6), enhancing the phosphorylation of Akt and increasing Akt activity toward GSK-3β, thus promoting EMT. Conclusions: Our results provide functional and mechanistic links between the SE-associated gene AJUBA and tumor EMT in aggressive HCC.

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.

Purpose Super-enhancer (SE)-associated oncogenes extensively potentiate the uncontrolled proliferation capacity of cancer cells. In this study, we aimed to identify the SE-associated hub genes associated with the clinical characteristics of chronic myeloid leukemia (CML). Methods Eigengenes from CML clinical modules were determined using weighted gene co-expression network analysis (WGCNA). Overlapping genes between eigengenes and SE-associated genes were used to construct protein-protein interaction (PPI) networks and annotate for pathway enrichment analysis. Expression patterns of the top-ranked SE-associated hub genes were further determined in CML patients and healthy controls via real-time PCR. After treatment of K562 cells with the BRD4 inhibitor, JQ1, for 24 hrs, mRNA and protein levels of SE-associated hub genes were evaluated using real-time PCR and Western blotting, respectively. H3K27ac, H3K4me1 and BRD4 ChIP-seq signal peaks were used to predict and identify SEs visualized by the Integrative Genomics Viewer. Results The yellow module was significantly related to the status and pathological phase of CML. SE-associated hub candidate genes were mainly enriched in the cell cycle pathway. Based on the PPI networks of hub genes and the top rank of degree, five SE-associated genes were identified: specifically, BUB1, CENPO, KIF2C, ORC1, and RRM2. Elevated expression of these five genes was not only related to CML status and phase but also positively regulated by SE and suppressed by the BRD4 inhibitor, JQ1, in K562 cells. Strong signal peaks of H3K27ac, H3K4me1 and BRD4 ChIP-seq of the five genes were additionally observed close to the predicted SE regions. Conclusion This is the first study to characterize SE-associated genes linked to clinical characteristics of CML via weighted gene co-expression network analysis. Our results support a novel mechanism involving aberrant expression of hub SE-associated genes in CML patients and K562 cells, and these genes will be potential new therapeutic targets for human leukemia.

WNT signaling activates MYC expression in cancer cells. Here we report that this involves an oncogenic super-enhancer-mediated tethering of active MYC alleles to nuclear pores to increase transcript export rates. As the decay of MYC transcripts is more rapid in the nucleus than in the cytoplasm, the oncogenic super-enhancer-facilitated export of nuclear MYC transcripts expedites their escape from the nuclear degradation system in colon cancer cells. The net sum of this process, as supported by computer modeling, is greater cytoplasmic MYC messenger RNA levels in colon cancer cells than in wild type cells. The cancer-cell-specific gating of MYC is regulated by AHCTF1 (also known as ELYS), which connects nucleoporins to the oncogenic super-enhancer via β-catenin. We conclude that WNT signaling collaborates with chromatin architecture to post-transcriptionally dysregulate the expression of a canonical cancer driver. Oncogenic MYC expression involves super-enhancer-mediated tethering of MYC alleles to nuclear pores, thus increasing messenger RNA export. This is regulated by AHCTF1 and β-catenin.

Summary DNA double-strand breaks (DSBs) are deleterious and tumorigenic but could also be essential for DNA-based processes. Yet the landscape of physiological DSBs and their role and repair are still elusive. Here, we mapped DSBs at high resolution in cancer and non-tumorigenic cells and found a transcription-coupled repair mechanism at oncogenic super-enhancers. At these super-enhancers the transcription factor TEAD4, together with various transcription factors and co-factors, co-localizes with the repair factor RAD51 of the homologous recombination pathway. Depletion of TEAD4 or RAD51 increases DSBs at RAD51/TEAD4 common binding sites within super-enhancers and decreases expression of related genes, which are mostly oncogenes. Co-localization of RAD51 with transcription factors at super-enhancers occurs in various cell types, suggesting a broad phenomenon. Together, our findings uncover a coupling between transcription and repair mechanisms at oncogenic super-enhancers, to control the hyper-transcription of multiple cancer drivers., Graphical Abstract, Highlights • Physiological DSBs are enriched at highly active oncogenic super-enhancers (SEs) • RAD51 co-localizes with transcription factors at SE in various cells • TOP1 mediates DSBs at SEs that are repaired by a RAD51-dependent mechanism • Depletion of RAD51 increases DSBs at SEs and decreases expression of related oncogenes., By mapping physiological double-strand breaks (DSBs) of tumorigenic and non-tumorigenic cells Hazan et al. uncover a coupled transcription-repair mechanism at oncogenic super-enhancers in which RAD51 of the homologous recombination pathway plays a key role supporting the hyper-transcription of related oncogenes.

Trophectoderm (TE) lineage development is pivotal for proper implantation, placentation, and healthy pregnancy. However, only a few TE-specific transcription factors (TFs) have been systematically characterized, hindering our understanding of the process. To elucidate regulatory mechanisms underlying TE development, here we map super-enhancers (SEs) in trophoblast stem cells (TSCs) as a model. We find both prominent TE-specific master TFs (Cdx2, Gata3, and Tead4), and >150 TFs that had not been previously implicated in TE lineage, that are SE-associated. Mapping targets of 27 SE-predicted TFs reveals a highly intertwined transcriptional regulatory circuitry. Intriguingly, SE-predicted TFs show 4 distinct expression patterns with dynamic alterations of their targets during TSC differentiation. Furthermore, depletion of a subset of TFs results in dysregulation of the markers for specialized cell types in placenta, suggesting a role during TE differentiation. Collectively, we characterize an expanded TE-specific regulatory network, providing a framework for understanding TE lineage development and placentation., Trophectoderm lineage development is essential for implantation, placentation, and healthy pregnancy. Here the authors map super-enhancers (SEs) in trophoblast stem cells and find both TE-specific master regulators and 150 previous uncharacterised transcription factors that are SE-associated, providing insight into trophectoderm-specific regulatory networks.

Widespread aberrant gene expression is pathological hallmark of polycystic kidney disease (PKD). Numerous pathogenic signaling cascades, including c-Myc, Fos, and Jun are transactivated. However, the underlying epigenetic regulators are poorly defined. Here we show that H3K27ac, a histone modification that marks active enhancers, is elevated in mouse and human ADPKD samples. Using comparative H3K27ac ChIP-Seq analysis, we mapped >16000 active intronic and intergenic enhancer elements in Pkd1-mutant mouse kidneys. We find that the cystic kidney epigenetic landscape resembles that of a developing kidney, and >90% of upregulated genes in Pkd1-mutant kidneys are co-housed with activated enhancers in the same topologically associated domains. Furthermore, we identify an evolutionarily-conserved enhancer cluster downstream of the c-Myc gene and super-enhancers flanking both Jun and Fos loci in mouse and human ADPKD models. Deleting these regulatory elements reduces c-Myc, Jun, or Fos abundance and suppresses proliferation and 3D cyst growth of Pkd1-mutant cells. Finally, inhibiting glycolysis and glutaminolysis or activating Ppara in Pkd1-mutant cells lowers global H3K27ac levels and on c-Myc enhancers. Thus, our work suggests that epigenetic rewiring mediates the transcriptomic dysregulation in PKD, and the regulatory elements can be targeted to slow cyst growth.

SummaryThe contractile properties of adult myofibers are shaped by their Myosin heavy chain (MYH) isoform content. We identify by snATAC-seq a 42kb super-enhancer (SE) at the locus regrouping the fast Myh (fMyh) genes. By 4C-seq we show that active fMyh promoters interact with the SE by DNA looping, leading to the activation of a single promoter per nucleus. A rainbow mouse transgenic model of the locus including the SE recapitulates the endogenous spatio-temporal expression of adult fMyh genes. In situ deletion of the SE by CRISPR/Cas9 editing demonstrates its major role in the control of associated fMyh genes, and deletion of two fMyh genes at the locus reveals an active competition of the promoters for the shared SE. Last, by disrupting the organization of fMyh, we uncover positional heterogeneity within limb skeletal muscles that may underlie selective muscle susceptibility to damage in certain myopathies.

Background Increased breast cancer screening over the past four decades has led to a substantial rise in the diagnosis of ductal carcinoma in situ (DCIS). Although DCIS lesions precede invasive ductal carcinoma (IDC), they do not always transform into cancer. The current standard-of-care for DCIS is an aggressive course of therapy to prevent invasive and metastatic disease resulting in over-diagnosis and over-treatment. Thus, there is a critical need to identify functional determinants of progression of DCIS to IDC to allow discrimination between indolent and aggressive disease. Recent studies show that super-enhancers, in addition to promoting other gene transcription, are themselves transcribed producing super-enhancer associated long noncoding RNAs (SE-lncRNAs). These SE-lncRNAs can interact with their associated enhancer regions in cis and influence activities and expression of neighboring genes. Furthermore, they represent a novel, untapped group of therapeutic targets. Methods With an integrative analysis of enhancer loci with global expression of SE-lncRNAs in the MCF10A progression series, we have identified differentially expressed SE-lncRNAs which can identify mechanisms for DCIS to IDC progression. Furthermore, cross-referencing these SE-lncRNAs with patient samples in the The Cancer Genome Atlas (TCGA) database, we have unveiled 27 clinically relevant SE-lncRNAs that potentially interact with their enhancer to regulate nearby gene expression. To complement SE-lncRNA expression studies, we conducted an unbiased global analysis of super-enhancers that are acquired or lost in progression. Results Here we designate SE-lncRNAs RP11-379F4.4 and RP11-465B22.8 as potential markers of progression of DCIS to IDC through regulation of the expression of their neighboring genes (RARRES1 and miR-200b, respectively). Moreover, we classified 403 super-enhancer regions in MCF10A normal cells, 627 in AT1, 1053 in DCIS, and 320 in CA1 cells. Comparison analysis of acquired/lost super-enhancer regions with super-enhancer regions classified in 47 ER positive patients, 10 triple negative breast cancer (TNBC) patients, and 11 TNBC cell lines reveal critically acquired pathways including STAT signaling and NF-kB signaling. In contrast, protein folding, and local estrogen production are identified as major pathways lost in progression. Conclusion Collectively, these analyses identify differentially expressed SE-lncRNAs and acquired/lost super-enhancers in progression of breast cancer important for promoting DCIS lesions to IDC.

Background A new class of regulatory elements called super-enhancers, comprised of multiple neighboring enhancers, have recently been reported to be the key transcriptional drivers of cellular, developmental, and disease states. Results Here, we defined super-enhancer RNAs as highly expressed enhancer RNAs that are transcribed from a cluster of localized genomic regions. Using the cap analysis of gene expression sequencing data from FANTOM5, we systematically explored the enhancer and messenger RNA landscapes in hundreds of different cell types in response to various environments. Applying non-negative matrix factorization (NMF) to super-enhancer RNA profiles, we found that different cell types were well classified. In addition, through the NMF of individual time-course profiles from a single cell-type, super-enhancer RNAs were clustered into several states with progressive patterns. We further investigated the enriched biological functions of the proximal genes involved in each pattern, and found that they were associated with the corresponding developmental process. Conclusions The proposed super-enhancer RNAs can act as a good alternative, without the complicated measurement of histone modifications, for identifying important regulatory elements of cell type specification and identifying dynamic cell states.

Trophoblast stem cells (TSCs) are derived from blastocysts and the extra-embryonic ectoderm (ExE) of post-implantation embryos and play a significant role in fetal development, but the roles that TSCs play in the earlier status of fetal diseases need further exploration. Super enhancers (SEs) are dense clusters of stitched enhancers that control cell identity determination and disease development and may participate in TSC differentiation. We identified key cell identity genes regulated by TSC-SEs via integrated analysis of H3K27ac and H3K4me1 chromatin immunoprecipitation sequencing (ChIP-seq), RNA-sequencing (RNA-seq) and ATAC-sequencing (ATAC-seq) data. The identified key TSC identity genes regulated by SEs, such as epidermal growth factor receptor (EGFR), integrin β5 (ITGB5) and Paxillin (Pxn), were significantly upregulated during TSC differentiation, and the transcription network mediated by TSC-SEs enriched in terms like focal adhesion and actin cytoskeleton regulation related to differentiation of TSCs. Additionally, the increased chromatin accessibility of the key cell identity genes verified by ATAC-seq further demonstrated the regulatory effect of TSC-SEs on TSC lineage commitment. Our results illustrated the significant roles of the TSC-SE-regulated network in TSC differentiation, and identified key TSC identity genes EGFR, ITGB5 and Pxn, providing novel insight into TSC differentiation and lays the foundation for future studies on embryo implantation and related diseases.

Super-enhancers (SEs) play essential roles in colorectal cancer (CRC) progression. However, how the SE landscape is orchestrated by transcriptional regulators and evolves is not clear. Using de novo motif analysis, we show that the hepatocyte nuclear factor 1 (HNF1)-binding motif is enriched in SEs in cell lines derived from liver metastases, but not in those from primary tumors. This finding was further validated by extending the method to pancreatic cancer and a pair of isogenic CRC lines. Next, we revealed HNF1-alpha (HNF1A) was majorly expressed and upregulated in CRC liver metastatic cell lines. Clinically, HNF1A was remarkably upregulated in synchronous liver metastases as compared to localized tumors. Collectively, our study implicates HNF1A as a key regulator in shaping the SE landscape in CRC liver metastasis.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy with high relapse/refractory rate. Genetic and epigenetic abnormalities are driving factors for leukemogenesis. RUNX1 and RUNX2 from the Runt-related transcription factor (RUNX) family played important roles in AML pathogenesis. However, the relationship between RUNX3 and AML remains unclear. Here, we found that RUNX3 was a super-enhancer-associated gene and highly expressed in AML cells. The Cancer Genome Atlas (TCGA) database showed high expression of RUNX3 correlated with poor prognosis of AML patients. We observed that Runx3 knockdown significantly inhibited leukemia progression by inducing DNA damage to enhance apoptosis in murine AML cells. By chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we discovered that RUNX3 in AML cells mainly bound more genes involved in DNA-damage repair and antiapoptosis pathways compared to that in normal bone marrow cells. Runx3 knockdown obviously inhibited the expression of these genes in AML cells. Overall, we identified RUNX3 as an oncogene overexpressed in AML cells, and Runx3 knockdown suppressed AML progression by inducing DNA damage and apoptosis.

Liquid–liquid phase‐separated (LLPS) transcriptional factor assemblies at super‐enhancers (SEs) provide a conceptual framework for underlying transcriptional control in mammal cells. However, the mechanistic understanding of LLPS in aberrant transcription driven by dysregulation of SEs in human malignancies is still elusive. By integrating SE profiling and core regulatory circuitry (CRC) calling algorithm, the CRC of metastatic and chemo‐resistant osteosarcoma is delineated. CRC components, HOXB8 and FOSL1, produce dense and dynamic phase‐separated droplets in vitro and liquid‐like puncta in cell nuclei. Disruption of CRC phase separation decreases the chromatin accessibility in SE regions and inhibits the release of RNA polymerase II from the promoter of SE‐driven genes. Importantly, absence of CRC key component causes a reduction in osteosarcoma tumor growth and metastasis. Moreover, it is shown that CRC condensates can be specifically attenuated by the H3K27 demethylase inhibitor, GSK‐J4. Pharmacological inhibition of the CRC phase separation results in metastasis suppression and re‐sensitivity to chemotherapy drugs in patient‐derived xenograft model. Taken together, this study reveals a previously unknown mechanism that CRC factors formed LLPS condensates, and provides a phase separation‐based pharmacological strategy to target undruggable CRC components for the treatment of metastatic and chemo‐resistant osteosarcoma., Core regulatory circuitry (CRC) factors forms liquid–liquid phase‐separated condensates at super‐enhancers to regulate chromatin accessibility and oncogenic transcription. Pharmacological inhibition of the CRC phase separation by GSK‐J4 suppresses metastasis and chemoresistance in osteosarcoma patient‐derived xenograft model.

Aims: The genetic association between Behcet's disease susceptibility and IL-10 has been confirmed in multiple cohorts, but the underlying mechanism of this association remains unclear. Materials & methods: We combined public resources and laboratory experiments (electrophoretic mobility shift assays, chromatin immunoprecipitation, luciferase reporter gene and CRISPR/Cas9 genome editing) to analyze transcription factor binding and enhancer activity controlling IL-10 expression. Results & conclusion: The T allele of noncoding rs3024490 within super-enhancer elements is able to specifically bind TBX1 and, in turn, promotes the enhancer activity and increased expression of IL-10. However, a relative deficiency in TBX1 in Behcet's disease patients leads to the low expression of IL-10 and increased risk of developing Behcet's disease.Lay abstract Behcet's disease is an important cause of blindness. Previous reports show that genetic factors are linked with this disease, although the exact genetic mechanism is unclear. Many of these genetic factors are involved in the control of the immune response, including a large family of proteins known as cytokines. Some of the cytokines are proinflammatory while others can dampen the inflammatory response. An example of the latter is the IL-10 cytokine. We found that individuals carrying a specific site in the noncoding region of the IL-10 gene had a higher risk of Behcet's disease than other noncarriers. This study was designed to further investigate the biological mechanisms explaining the role of the specific site in the development of Behcet's disease. The results show that this specific site affects the binding of an important transcription factor, TBX1, which reduces IL-10 production. The dysregulated control of IL-10 explains why individuals with this genetic trait are more susceptible to developing Behcet's disease.

Chromosomal rearrangements are a frequent cause of oncogene deregulation in human malignancies. Overexpression of EVI1 is found in a subgroup of acute myeloid leukemia (AML) with 3q26 chromosomal rearrangements, which is often therapy resistant. In AMLs harboring a t(3;8)(q26;q24), we observed the translocation of a MYC super-enhancer (MYC SE) to the EVI1 locus. We generated an in vitro model mimicking a patient-based t(3;8)(q26;q24) using CRISPR-Cas9 technology and demonstrated hyperactivation of EVI1 by the hijacked MYC SE. This MYC SE contains multiple enhancer modules, of which only one recruits transcription factors active in early hematopoiesis. This enhancer module is critical for EVI1 overexpression as well as enhancer-promoter interaction. Multiple CTCF binding regions in the MYC SE facilitate this enhancer-promoter interaction, which also involves a CTCF binding site upstream of the EVI1 promoter. We hypothesize that this CTCF site acts as an enhancer-docking site in t(3;8) AML. Genomic analyses of other 3q26-rearranged AML patient cells point to a common mechanism by which EVI1 uses this docking site to hijack enhancers active in early hematopoiesis., Chromosome rearrangements can be a cause of altered oncogene expression in cancer, such as a 3q26 translocation in some acute myeloid leukemias (AML) that leads to overexpression of EVI1. Here the authors engineer this rearrangement in a cell line and show that EVI1 overexpression is a result of ‘enhancer hijacking’ of the MYC superenhancer, which is facilitated by CTCF-mediated loops.

Relapse of patients with chronic myelogenous leukemia (CML) may occur at least partially because leukemia stem cells (LSCs) lack sensitivity to tyrosine kinase inhibitors (TKIs) such as imatinib. The precise regulation of LSC stemness is incompletely understood. Given that traits of LSCs are subject to epigenetic regulation, we hypothesized that LSCs might be dependent on continuous active transcription of genes associated with super-enhancers (SEs), which might, in turn, suggest an opportunity for intervention. In this study, we tested this hypothesis and delineated the SE landscape in LSCs from patients with CML. Disruption of the SE-associated gene transcription by THZ1, a covalent cyclin-dependent kinase 7 (CDK7) inhibitor, efficiently eradicated LSCs in retroviral BCR-ABL–driven CML mice while sparing normal hematopoietic stem cells. Furthermore, we found that X-box binding protein 1 (