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Background: Unveiling pro-proliferation genes involved in crosstalk between pulmonary artery endothelial cells and pulmonary artery smooth muscle cells (PASMCs) are important to improving the therapeutic outcome of pulmonary hypertension (PH). Although growing studies have shown that super-enhancers (SEs) play a pivotal role in pathological and physiological processes, the SE-associated genes in PH and their impact on PASMC proliferation remain largely unexplored., Methods and Results: We used serotype 5 adenovirus-associated virus to interfere with syndecan-4 and constructed an SU5416 combined with hypoxia-PH model. Chromatin immunoprecipitation sequencing analysis, chromatin immunoprecipitation quantitative polymerase chain reaction, and bioinformatics were used to confirm early growth response 1 was involved in regulating syndecan-4-associated SE in PASMCs. The effects of syndecan-4 and its underlying mechanisms were subsequently elucidated using Western blot, coimmunoprecipitation, and cell coculture assays. Herein, we identified a novel SE-associated gene, syndecan-4, in hypoxia-exposed PASMCs. Syndecan-4 was transcriptionally driven by early growth response 1 via an SE and was significantly overexpressed in hypoxic PASMCs and plasma from patients with PH. Mechanism studies revealed that syndecan-4 induces PASMC proliferation by interacting and regulating protein kinase C α ubiquitination. In addition, syndecan-4 was enriched in exosomes secreted from hypoxic PASMCs, which subsequently transported and led to pulmonary artery endothelial cell dysfunction. Syndecan-4 inhibition in hypoxia by serotype 5 adenovirus-associated virus treatment attenuated the pulmonary artery remodeling and development of PH in vivo., Conclusions: Taken together, our results demonstrate that an SE-driven syndecan-4 modulates crosstalk of PASMCs and pulmonary artery endothelial cells and promotes vascular remodeling via the protein kinase C α and exosome pathway, thus providing potential targets for the early diagnosis and treatment of hypoxic PH.

Gallbladder cancer (GBC) is a highly aggressive malignancy lacking clinically available targeted therapeutic agents. Super-enhancers (SEs) are crucial epigenetic cis-regulatory elements whose extensive reprogramming drives aberrant transcription in cancers. To study SE in GBC, the genomic distribution of H3K27ac is profiled in multiple GBC tissue and cell line samples to establish the SE landscape and its associated core regulatory circuitry (CRC). The biliary lineage factor SOX9 and Wnt pathway effector TCF7L2, two master transcription factor (TF) candidates identified by CRC analysis, are verified to co-occupy each other's SE region, forming a mutually autoregulatory loop to drive oncogenic SE reprogramming in a subset of GBC. The SOX9/TCF7L2 double-high GBC cells are highly dependent on the two TFs and enriched of SE-associated gene signatures related to stemness, ErbB and Wnt pathways. Patients with more such GBC cells exhibited significantly worse prognosis. Furthermore, SOX9/TCF7L2 double-high GBC preclinical models are found to be susceptible to SE-targeted CDK7 inhibition therapy in vitro and in vivo. Together, this study provides novel insights into the epigenetic mechanisms underlying the oncogenesis of a subset of GBCs with poorer prognosis and illustrates promising prognostic stratification and therapeutic strategies for treating those GBC patients in future clinical trials., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Super enhancers (SEs) are large clusters of transcriptional enhancers driving the expression of genes crucial for defining cell identity. In cancer, tumor-specific SEs activate key oncogenes, leading to tumorigenesis. Identifying SE-driven oncogenes in tumors and understanding their functional mechanisms is of significant importance. In this study, a previously unreported SE region is identified in T-cell acute lymphoblastic leukemia (T-ALL) patient samples and cell lines. This SE activates the expression of interferon regulatory factor 2 binding protein 2 (IRF2BP2) and is regulated by T-ALL master transcription factors (TFs) such as ETS transcription factor ERG (ERG), E74 like ETS transcription factor 1 (ELF1), and ETS proto-oncogene 1, transcription factor (ETS1). Hematopoietic system-specific IRF2BP2 conditional knockout mice is generated and showed that IRF2BP2 has minimal impact on normal T cell development. However, in vitro and in vivo experiments demonstrated that IRF2BP2 is crucial for T-ALL cell growth and survival. Loss of IRF2BP2 affects the MYC and E2F pathways in T-ALL cells. Cleavage under targets and tagmentation (CUT&Tag) assays and immunoprecipitation revealed that IRF2BP2 cooperates with the master TFs of T-ALL cells, targeting the enhancer of the T-ALL susceptibility gene recombination activating 1 (RAG1) and modulating its expression. These findings provide new insights into the regulatory network within T-ALL cells, identifying potential new targets for therapeutic intervention., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Gastric cancer (GC) is a major cause of cancer-related mortality worldwide. Despite the widespread recognition of tumor immunotherapy in treating unresectable GC, challenges, including ineffective immunotherapy and drug resistance, persist. Therefore, understanding the regulatory mechanisms of PD-L1, particularly in the context of super-enhancers (SEs) and zinc finger protein 36 ring finger protein-like 1 (ZFP36L1) RNA-binding protein, is crucial. In this study, we performed H3K27ac Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing, investigated the heterogeneity of SEs between two GC subtypes with differential growth patterns, and revealed the immune escape signatures driven by ZFP36L1-SE in infiltrative GC through SEs inhibitors treatment. The regulation of ZFP36L1 to PD-L1 was evaluated by quantitative PCR, western blot, flow cytometry, and immunohistochemistry. Furthermore, we explored its regulatory mechanisms using a combination of molecular biology techniques, including luciferase reporter assay, GST/RNA pull-down, chromatin immunoprecipitation (ChIP)/RIP experiments, and in vivo functional assays. We demonstrated that ZFP36L1, driven by an SE, enhances IFN-γ-induced PD-L1 expression, with SPI1 identified as the specific transcription factor binding to ZFP36L1-SE. Mechanistically, ZFP36L1 binds to the adenylate uridylate-rich element in the 3' untranslated region (3'UTR) of HDAC3 mRNA, exacerbating its mRNA decay, and thereby facilitating PD-L1 abnormal transcriptional activation. Collectively, our findings provide mechanistic insights into the role of the SPI1-ZFP36L1-HDAC3-PD-L1 signaling axis in orchestrating immune escape mechanisms in GC, thereby offering valuable insights into the potential targets for immune checkpoint therapy in GC management., Competing Interests: XW, JL, JC, WC, WX, KW, LL, JH, JC, HZ No competing interests declared, (© 2024, Wei, Liu et al.)

Background: Super-enhancers (SEs) typically govern the expression of critical oncogenes and play a fundamental role in the initiation and progression of cancer. Focusing on genes that are abnormally regulated by SE in cancer may be a new strategy for understanding pathogenesis. In the context of this investigation, we have identified a previously unreported SE-driven gene IRF2BP2 in neuroblastoma (NB)., Methods: The expression and prognostic value of IRF2BP2 were detected in public databases and clinical samples. The effect of IRF2BP2 on NB cell growth and apoptosis was evaluated through in vivo and in vitro functional loss experiments. The molecular mechanism of IRF2BP2 was investigated by the study of chromatin regulatory regions and transcriptome sequencing., Results: The sustained high expression of IRF2BP2 results from the activation of a novel SE established by NB master transcription factors MYCN, MEIS2, and HAND2, and they form a new complex that regulates the gene network associated with the proliferation of NB cell populations. We also observed a significant enrichment of the AP-1 family at the binding sites of IRF2BP2. Remarkably, within NB cells, AP-1 plays a pivotal role in shaping the chromatin accessibility landscape, thereby exposing the binding site for IRF2BP2. This orchestrated action enables AP-1 and IRF2BP2 to collaboratively stimulate the expression of the NB susceptibility gene ALK, thereby upholding the highly proliferative phenotype characteristic of NB., Conclusions: Our findings indicate that SE-driven IRF2BP2 can bind to AP-1 to maintain the survival of tumor cells via regulating chromatin accessibility of the NB susceptibility gene ALK., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Chemoresistance remains a formidable challenge in pancreatic ductal adenocarcinoma (PDAC) treatment, necessitating a comprehensive exploration of underlying molecular mechanisms. This work aims to investigate the dynamic epigenetic landscape during the development of gemcitabine resistance in PDAC, with a specific focus on super-enhancers and their regulatory effects. We employed well-established gemcitabine-resistant (Gem-R) PDAC cell lines to perform high-throughput analyses of the epigenome, enhancer connectome, and transcriptome. Our findings revealed notable alterations in the epigenetic landscape and genome architecture during the transition from gemcitabine-sensitive to -resistant PDAC cells. Remarkably, we observed substantial plasticity in the activation status of super-enhancers, with a considerable proportion of these cis-elements becoming deactivated in chemo-resistant cells. Furthermore, we pinpointed the NDRG1 super-enhancer (NDRG1-SE) as a crucial regulator in gemcitabine resistance among the loss-of-function super-enhancers. NDRG1-SE deactivation induced activation of WNT/β-catenin signaling, thereby conferring gemcitabine resistance. This work underscores a NDRG1 super-enhancer deactivation-driven β-catenin pathway activation as a crucial regulator in the acquisition of gemcitabine-resistance. These findings advance our understanding of PDAC biology and provide valuable insights for the development of effective therapeutic approaches against chemoresistance in this malignant disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Super-enhancers (SEs) have been recognized as key epigenetic regulators underlying cancer stemness and malignant traits by aberrant transcriptional control and promising therapeutic targets against human cancers. However, the SE landscape and their roles during head and neck squamous cell carcinoma (HNSCC) development especially in cancer stem cells (CSCs) maintenance remain underexplored yet. Here, we identify leukemia inhibitory factor (LIF)-SE as a representative oncogenic SE to activate LIF transcription in HNSCC. LIF secreted from cancer cells and cancer-associated fibroblasts promotes cancer stemness by driving SOX2 transcription in an autocrine/paracrine manner, respectively. Mechanistically, enhancer elements E1, 2, 4 within LIF-SE recruit SOX2/SMAD3/BRD4/EP300 to facilitate LIF transcription; LIF activates downstream LIFR-STAT3 signaling to drive SOX2 transcription, thus forming a previously unknown regulatory feedback loop (LIF-SE-LIF/LIFR-STAT3-SOX2) to maintain LIF overexpression and CSCs stemness. Clinically, increased LIF abundance in clinical samples correlate with malignant clinicopathological features and patient prognosis; higher LIF concentrations in presurgical plasma dramatically diminish following cancer eradication. Therapeutically, pharmacological targeting LIF-SE-LIF/LIFR-STAT3 significantly impairs tumor growth and reduces CSC subpopulations in xenograft and PDX models. Our findings reveal a hitherto uncharacterized LIF-SE-mediated auto-regulatory loop in regulating HNSCC stemness and highlight LIF as a novel noninvasive biomarker and potential therapeutic target for HNSCC., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Tumor immune microenvironment is crucial for diffuse large B-cell lymphoma (DLBCL) development. However, the mechanisms by which super-enhancers (SEs) regulate the interactions between DLBCL cells and tumor-infiltrating immune cells remains largely unknown. This study aimed to investigate the role of SE-controlled genes in regulating the interactions between DLBCL cells and tumor-infiltrating immune cells. Single-cell RNA-seq, bulk RNA-seq and H3K27ac ChIP-seq data were downloaded from the Heidelberg Open Research Data database and Gene Expression Omnibus database. HOMER algorithm and Seurat package in R were used for bioinformatics analysis. Cell proliferation and lactate dehydrogenase (LDH) release was detected by MTS and LDH release assays, respectively. Interaction between B cell cluster and CD8 + T cell and NK cell cluster was most obviously enhanced in DLBCL, with CD70-CD27, MIF-CD74/CXCR2 complex, MIF-CD74/CD44 complex and CCL3-CCR5 interactions were significantly increased. NK cell sub-cluster showed the strongest interaction with B cell cluster. ZZZ3 upregulated the transcription of CD70 by binding to its SE. Silencing CD70 in DOHH2 cells significantly promoted the proliferation of co-cultured NK92 cells and LDH release from DOHH2 cells, which was counteracted by ZZZ3 overexpression in DOHH2 cells. CD70 silencing combined with PD-L1 blockade promoted LDH release from DOHH2 cells co-cultured with NK92 cells. In conclusion, DLBCL cells inhibited the proliferation and killing of infiltrating NK cells by regulating ZZZ3/CD70 axis. Targeting ZZZ3/CD70 axis combined with PD-L1 blockade is expected to be a promising strategy for DLBCL treatment., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2024 Li, Cui, Wang, Cao and Liu.)

Dysregulated super-enhancer (SE) results in aberrant transcription that drives cancer initiation and progression. SEs have been demonstrated as novel promising diagnostic/prognostic biomarkers and therapeutic targets across multiple human cancers. Here, we sought to develop a novel prognostic signature derived from SE-associated genes for head and neck squamous cell carcinoma (HNSCC). SE was identified from H3K27ac ChIP-seq datasets in HNSCC cell lines by ROSE algorithm and SE-associated genes were further mapped and functionally annotated. A total number of 133 SE-associated genes with mRNA upregulation and prognostic significance was screened via differentially-expressed genes (DEGs) and Cox regression analyses. These candidates were subjected for prognostic model constructions by machine learning approaches using three independent HNSCC cohorts (TCGA-HNSC dataset as training cohort, GSE41613 and GSE42743 as validation cohorts). Among dozens of prognostic models, the random survival forest algorithm (RSF) stood out with the best performance as evidenced by the highest average concordance index (C-index). A prognostic nomogram integrating this SE-associated gene signature (SEAGS) plus tumor size demonstrated satisfactory predictive power and excellent calibration and discrimination. Moreover, WNT7A from SEARG was validated as a putative oncogene with transcriptional activation by SE to promote malignant phenotypes. Pharmacological disruption of SE functions by BRD4 or EP300 inhibitor significantly impaired tumor growth and diminished WNT7A expression in a HNSCC patient-derived xenograft model. Taken together, our results establish a novel, robust SE-derived prognostic model for HNSCC and suggest the translational potentials of SEs as promising therapeutic targets for HNSCC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Our research endeavored to develop a robust predictive signature grounded in super-enhancer-related genes (SERGs), with the dual objectives of forecasting survival outcomes and evaluating the tumor immune microenvironment (TiME) in hepatocellular carcinoma (HCC)., Methods: HCC RNA-sequencing data were retrieved from The Cancer Genome Atlas (TCGA), and 365 patients were randomly assigned to training or testing sets in 1:1 ratio. SERGs of HCC were downloaded from Super-Enhancer Database (SEdb). On the basis of training set, a SERGs signature was identified, and its prognostic value was confirmed by internal and external validation (GSE14520) sets. We subsequently examined the model for potential functional enrichment and the degree of tumor immune infiltration. Additionally, we carried out in vitro experiments to delve into the biological functions of CBX2 gene., Results: An SE-related prognostic model including CBX2, TPX2, EFNA3, DNASE1L3 and SOCS2 was established and validated. According to this risk model, patients in the high-risk group had a significantly worse prognosis, and their immune cell infiltration was significantly different from that of low-risk group. Moreover, the high-risk group exhibited a significant enrichment of tumor-associated pathological pathways. The SERGs signature can generally be utilized to screen HCC patients who are likely to respond to immunotherapy, as there is a positive correlation between the risk score and the Tumor Immune Dysfunction and Exclusion (TIDE) score. Furthermore, the downregulation of the CBX2 gene expression was found to inhibit HCC cell viability, migration, and cell cycle progression, while simultaneously promoting apoptosis., Conclusions: We developed a novel HCC prognostic model utilizing SERGs, indicating that patients with high-risk score not only face a poorer prognosis but also may exhibit a diminished therapeutic response to immune checkpoint inhibitors (ICIs). This model is designed to tailor personalized treatment strategies to the individual needs of each patient, thereby improving the overall clinical outcomes for HCC patients. Furthermore, CBX2 is a promising candidate for therapeutic intervention in HCC., (© 2024. The Author(s).)

Background: Heat shock transcription factors (HSFs) play crucial roles in the development of malignancies. However, the specific roles of HSFs in hepatocellular carcinoma (HCC) have yet to be fully elucidated., Aims: To explore the involvement of the HSF family, particularly HSF1, in the progression and prognosis of HCC., Materials & Methods: We conducted a thorough analysis of HSF expression and copy number variations across various cancer datasets. Specifically focusing on HSF1, we examined its expression levels and prognostic implications in HCC. In vitro and in vivo experiments were carried out to evaluate the impact of HSF1 on liver cancer cell proliferation. Additionally, we utilized CUT&Tag, H3K27 acetylation enrichment, and RNA sequencing (RNA-seq) to investigate the super-enhancer (SE) regulatory landscapes of HSF1 in liver cancer cell lines., Results: HSF1 expression is elevated in HCC and is linked to poor prognosis in several datasets. HSF1 stimulates liver cancer cell proliferation both in vitro and in vivo, partly through modulation of H3K27ac levels, influencing enhancer distribution. Mechanistically, our findings demonstrate that HSF1 transcriptionally activates MYCN expression by binding to its promoter and SE elements, thereby promoting liver cancer cell proliferation. Moreover, increased MYCN expression was detected in HCC tumors and correlated with unfavorable patient outcomes., Discussion: Our study sheds light on previously unexplored aspects of HSF1 biology, identifying it as a transcription factor capable of shaping the epigenetic landscape in the context of HCC. Given HSF1's potential as an epigenetic regulator, targeting the HSF1-MYCN axis could open up new therapeutic possibilities for HCC treatment., Conclusion: The HSF1-MYCN axis constitutes a transcription-dependent regulatory mechanism that may function as both a prognostic indicator and a promising therapeutic target in liver cancer. Further exploration of this axis could yield valuable insights into novel treatment strategies for HCC., (© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)

Super-enhancers (SEs) play an important role in regulating tumor-specific gene expression. JQ1, a Bromodomain-containing protein 4 (BRD4) inhibitor, exerts antitumor effects by disrupting SE-mediated regulation of gene expression. We investigated the anti-adult T-cell leukemia/lymphoma (ATL) effects of JQ1. JQ1 induced apoptosis and inhibited ATL cell proliferation. JQ1 suppressed RUNX1 expression through the disruption of SE-mediated gene regulation. In the previous reports, it was shown that IC 50 s of AI-10-104 and Ro5-3335, RUNX1 inhibitors were 1-10 µM for lymphoblastic leukemia cell lines carrying RUNX1 mutations. In the present study, we demonstrated that IC 50 s of AI-10-104 and Ro5-3335 were also 1-10 µM or lower for ATL cell lines. Simultaneously, AI-10-104 suppressed MYC proto-oncogene (c-MYC) expression. RUNX1 is a potential therapeutic target for ATL that promotes c-MYC expression. We showed that RUNX1 expression is regulated via SEs in ATL and that RUNX1 may be a novel therapeutic target for ATL.

Our previous studies determined that elevating SOX2 in a wide range of tumor cells leads to a reversible state of tumor growth arrest. Efforts to understand how tumor cell growth is inhibited led to the discovery of a SOX2:MYC axis that is responsible for downregulating c-MYC (MYC) when SOX2 is elevated. Although we had determined that elevating SOX2 downregulates MYC transcription, the mechanism responsible was not determined. Given the challenges of targeting MYC clinically, we set out to identify how elevating SOX2 downregulates MYC transcription. In this study, we focused on the MYC promoter region and an upstream region of the MYC locus that contains a MYC super-enhancer encompassing five MYC enhancers and which is associated with several cancers. Here we report that BRD4 and p300 associate with each of the MYC enhancers in the upstream MYC super-enhancer as well as the MYC promoter region and that elevating SOX2 decreases the recruitment of BRD4 and p300 to these sites. Additionally, we determined that elevating SOX2 leads to increases in the association of SOX2 and H3K27me3 within the MYC super-enhancer and the promoter region of MYC. Importantly, we conclude that the increases in SOX2 within the MYC super-enhancer precipitate a cascade of events that culminates in the repression of MYC transcription. Together, our studies identify a novel molecular mechanism able to regulate MYC transcription in two distinctly different tumor types and provide new mechanistic insights into the molecular interrelationships between two master regulators, SOX2 and MYC, widely involved in multiple cancers., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Gene regulation is crucial for cellular function and homeostasis. It involves diverse mechanisms controlling the production of specific gene products and contributing to tissue-specific variations in gene expression. The dysregulation of genes leads to disease, emphasizing the need to understand these mechanisms. Computational methods have jointly studied transcription factors (TFs), microRNA (miRNA), and messenger RNA (mRNA) to investigate gene regulatory networks. However, there remains a knowledge gap in comprehending gene regulatory networks. On the other hand, super-enhancers (SEs) have been implicated in miRNA biogenesis and function in recent experimental studies, in addition to their pivotal roles in cell identity and disease progression. However, statistical/computational methodologies harnessing the potential of SEs in deciphering gene regulation networks remain notably absent. However, to understand the effect of miRNA on mRNA, existing statistical/computational methods could be updated, or novel methods could be developed by accounting for SEs in the model. In this review, we categorize existing computational methods that utilize TF and miRNA data to understand gene regulatory networks into three broad areas and explore the challenges of integrating enhancers/SEs. The three areas include unraveling indirect regulatory networks, identifying network motifs, and enriching pathway identification by dissecting gene regulators. We hypothesize that addressing these challenges will enhance our understanding of gene regulation, aiding in the identification of therapeutic targets and disease biomarkers. We believe that constructing statistical/computational models that dissect the role of SEs in predicting the effect of miRNA on gene regulation is crucial for tackling these challenges.

Glycolysis is a major determinant of pulmonary artery smooth muscle cell (PASMC) proliferation in pulmonary hypertension (PH). Circular RNAs (circRNAs) are powerful regulators of glycolysis in multiple diseases; however, the role of circRNAs in glycolysis in PH has been poorly characterized. The aim of this study was to uncover the regulatory mechanism of a new circRNA, circNAP1L4, in human pulmonary artery smooth muscle cell (HPASMC) proliferation through the host protein NAP1L4 to regulate the super-enhancer-driven glycolysis gene hexokinase II (HK II). CircNAP1L4 was downregulated in hypoxic HPASMCs and plasma of PH patients. Functionally, circNAP1L4 overexpression inhibited glycolysis and proliferation in hypoxic HPASMCs. Mechanistically, circNAP1L4 directly bound to its host protein NAP1L4 and affected the ability of NAP1L4 to move into the nucleus to regulate the epigenomic signals of the super-enhancer of HK II. Intriguingly, circNAP1L4 overexpression inhibited the proliferation but not the migration of human pulmonary arterial endothelial cells (HPAECs) cocultured with HPASMCs. Furthermore, pre-mRNA-processing-splicing Factor 8 (PRP8) was found to regulate the production ratio of circNAP1L4 and linear NAP1L4. In vivo, targeting circNAP1L4 alleviates SU5416 combined with hypoxia (SuHx)-induced PH. Overall, these findings reveal a new circRNA that inhibits PASMC proliferation and serves as a therapeutic target for PH., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Super-enhancer-associated transcription factor networks define cell identity in neuroblastoma (NB). Dysregulation of these transcription factors contributes to the initiation and maintenance of NB by enforcing early developmental identity states. We report that the class I basic helix-loop-helix (bHLH) transcription factor 4 (TCF4; also known as E2-2) is a critical NB dependency gene that significantly contributes to these identity states through heterodimerization with cell-identity-specific bHLH transcription factors. Knockdown of TCF4 significantly induces apoptosis in vitro and inhibits tumorigenicity in vivo. We used genome-wide expression profiling, TCF4 chromatin immunoprecipitation sequencing (ChIP-seq) and TCF4 immunoprecipitation-mass spectrometry to determine the role of TCF4 in NB cells. Our results, along with recent findings in NB for the transcription factors T-box transcription factor TBX2, heart- and neural crest derivatives-expressed protein 2 (HAND2) and twist-related protein 1 (TWIST1), propose a role for TCF4 in regulating forkhead box protein M1 (FOXM1)/transcription factor E2F-driven gene regulatory networks that control cell cycle progression in cooperation with N-myc proto-oncogene protein (MYCN), TBX2, and the TCF4 dimerization partners HAND2 and TWIST1. Collectively, we showed that TCF4 promotes cell proliferation through direct transcriptional regulation of the c-MYC/MYCN oncogenic program that drives high-risk NB. Mechanistically, our data suggest the novel finding that TCF4 acts to support MYC activity by recruiting multiple factors known to regulate MYC function to sites of colocalization between critical NB transcription factors, TCF4 and MYC oncoproteins. Many of the TCF4-recruited factors are druggable, giving insight into potential therapies for high-risk NB. This study identifies a new function for class I bHLH transcription factors (e.g., TCF3, TCF4, and TCF12) that are important in cancer and development., (© 2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: The mesenchymal (MES) subtype of glioblastoma (GBM) is believed to be influenced by both cancer cell-intrinsic alterations and extrinsic cellular interactions, yet the underlying mechanisms remain unexplored., Methods: Identification of microglial heterogeneity by bioinformatics analysis. Transwell migration, invasion assays, and tumor models were used to determine gene function and the role of small molecule inhibitors. RNA sequencing, chromatin immunoprecipitation, and dual-luciferase reporter assays were performed to explore the underlying regulatory mechanisms., Results: We identified the inflammatory microglial subtype of tumor-associated microglia (TAM) and found that its specific gene integrin beta 2 (ITGB2) was highly expressed in TAM of MES GBM tissues. Mechanistically, the activation of ITGB2 in microglia promoted the interaction between the SH2 domain of STAT3 and the cytoplasmic domain of ITGB2, thereby stimulating the JAK1/STAT3/IL-6 signaling feedback to promote the MES transition of GBM cells. Additionally, microglia communicated with GBM cells through the interaction between the receptor ITGB2 on microglia and the ligand ICAM-1 on GBM cells, while an increased secretion of ICAM-1 was induced by the proinflammatory cytokine leukemia inhibitory factor (LIF). Further studies demonstrated that inhibition of cyclin-dependent kinase 7 substantially reduced the recruitment of SNW1 to the super-enhancer of LIF, resulting in transcriptional inhibition of LIF. We identified notoginsenoside R1 as a novel LIF inhibitor that exhibited synergistic effects in combination with temozolomide., Conclusions: Our research reveals that the epigenetic-mediated interaction of GBM cells with TAM drives the MES transition of GBM and provides a novel therapeutic avenue for patients with MES GBM., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Early detection of cancer remains an unmet need in clinical practice, and high diagnostic sensitivity and specificity biomarkers are urgently required. Here, we attempted to identify secreted proteins encoded by super-enhancer (SE)-driven genes as diagnostic biomarkers for esophageal squamous cell carcinoma (ESCC)., Methods: We conducted an integrative analysis of multiple data sets including ChIP-seq data, secretome data, CCLE data and GEO data to screen secreted proteins encoded by SE-driven genes. Using ELISA, we further identified up-regulated secreted proteins through a small size of clinical samples and verified in a multi-centre validation stage (345 in test cohort and 231 in validation cohort). Receiver operating characteristic curves were used to calculate diagnostic accuracy. Artificial intelligence (AI) method named gradient boosting machine (GBM) were applied for model construction to enhance diagnostic accuracy., Results: Serum EFNA1 and MMP13 were identified, and showed significantly higher levels in ESCC patients compared to normal controls. An integrated Five-Biomarker Panel (iFBPanel) established by combining EFNA1, MMP13, carcino-embryonic antigen, Cyfra21-1 and squmaous cell carcinoma antigen had AUCs of 0.881 and 0.880 for ESCC in test and validation cohorts, respectively. Importantly, the iFBPanel also exhibited good performance in detecting early-stage ESCC patients (0.872 and 0.864). Furthermore, the iFBPanel was further empowered by AI technology which showed excellent diagnostic performance in early-stage ESCC (0.927 and 0.907)., Conclusions: Our study suggested that serum EFNA1 and MMP13 could potentially assist ESCC detection, and provided an easy-to-use detection model that might help the diagnosis of early-stage ESCC., Competing Interests: Conflict of interest The author has no potential conflicts of interest to disclose., (© 2023 The Authors. Published by Elsevier B.V. on behalf of Chang Gung University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Objective: Rare study of the non-coding and regulatory regions of the genome limits our ability to decode the mechanisms of fatty liver hemorrhage syndrome (FLHS) in chickens., Methods: Herein, we constructed the high-fat diet-induced FLHS chicken model to investigate the genome-wide active enhancers and transcriptome by H3K27ac target chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-Seq) profiles of normal and FLHS liver tissues. Concurrently, an integrative analysis combining ChIP-seq with RNA-Seq and a comparative analysis with chicken FLHS, rat non-alcoholic fatty liver disease (NAFLD) and human NAFLD at the transcriptome level revealed the enhancer and super enhancer target genes and conservative genes involved in metabolic processes., Results: In total, 56 and 199 peak-genes were identified in upregulated peak-genes positively regulated by H3K27ac (Cor (peak-gene correlation) ≥0.5 and log2(FoldChange) ≥1) (PP) and downregulated peak-genes positively regulated by H3K27ac (Cor (peak-gene correlation) ≥0.5 and log2(FoldChange)≤-1) (PN), respectively; then we screened key regulatory targets mainly distributing in lipid metabolism (PCK1, APOA4, APOA1, INHBE) and apoptosis (KIT, NTRK2) together with MAPK and PPAR signaling pathway in FLHS. Intriguingly, PCK1 was also significantly covered in up-regulated super-enhancers (SEs), which further implied the vital role of PCK1 during the development of FLHS., Conclusion: Together, our studies have identified potential therapeutic biomarkers of PCK1 and elucidated novel insights into the pathogenesis of FLHS, especially for the epigenetic perspective.

Ovarian cancer, a significant contributor to cancer-related mortality, exhibits limited responsiveness to hormonal therapies targeting the estrogen receptor (ERα). This study aimed to elucidate the mechanisms behind ERα resistance to the therapeutic drug Fulvestrant (ICI182780 or ICI). Notably, compared to the cytoplasmic version, nuclear ERα was minimally degraded by ICI, suggesting a mechanism for drug resistance via the protective confines of the nuclear substructures. Of these substructures, we identified a 1.3MDa Megacomplex comprising transcription factors ERα, FOXA1, and PITX1 using size exclusion chromatography (SEC) in the ovarian cancer cell line, PEO4. ChIP-seq revealed these factors colocalized at 6,775 genomic positions representing sites of Megacomplex formation. Megacomplex ERα exhibited increased resistance to degradation by ICI compared to cytoplasmic and nuclear ERα. A small molecule inhibitor of active chromatin and super-enhancers, JQ1, in combination with ICI significantly enhanced ERα degradation from Megacomplex as revealed by SEC and ChIP-seq. Interestingly, this combination degraded both the cytoplasmic as well as nuclear ERa. Pathway enrichment analysis showed parallel results for RNA-seq gene sets following Estradiol, ICI, or ICI plus JQ1 treatments as those defined by Megacomplex binding identified through ChIP-seq. Furthermore, similar pathway enrichments were confirmed in mass-spec analysis of the Megacomplex macromolecule fractions after modulation by Estradiol or ICI. These findings implicate Megacomplex in ERα-driven ovarian cancer chromatin regulation. This combined treatment strategy exhibited superior inhibition of cell proliferation and viability. Therefore, by uncovering ERα's resistance within the Megacomplex, the combined ICI plus JQ1 treatment elucidates a novel drug treatment vulnerability., Competing Interests: Declaration of Competing Interest ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (Copyright © 2024. Published by Elsevier B.V.)

Recently, biomolecular condensates formed through liquid-liquid phase separation have been widely reported to regulate key intracellular processes involved in cell biology and pathogenesis. BRD4 is a nuclear protein instrumental to the establishment of phase-separated super-enhancers (SEs) to direct the transcription of important genes. We previously observed that protein droplets of BRD4 became hydrophobic as their size increase, implying an ability of SEs to limit the ionization of water molecules by irradiation. Here, we aim to establish if SEs confer radiation resistance in cancer cells. We established an in vitro DNA damage assay that measures the effect of radicals provoked by the Fenton reaction on DNA integrity. This revealed that DNA damage was markedly reduced when BRD4 underwent phase separation with DNA. Accordingly, co-focal imaging analyses revealed that SE foci and DNA damage foci are mutually exclusive in irradiated cells. Lastly, we observed that the radioresistance of cancer cells was significantly reduced when irradiation was combined with ARV-771, a BRD4 de-stabilizer. Our data revealed the existence of innately radioresistant genomic regions driven by phase separation in cancer cells. The disruption of these phase-separated components enfolding genomic DNA may represent a novel strategy to augment the effects of radiotherapy., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology.)

Lung adenocarcinoma (LUAD) is the leading cause of cancer-related death worldwide, but the underlying molecular mechanisms remain largely unclear. The transcription factor (TF) specificity protein 1 (SP1) plays a crucial role in the development of various cancers, including LUAD. Recent studies have indicated that master TFs may form phase-separated macromolecular condensates to promote super-enhancer (SE) assembly and oncogene expression. In this study, we demonstrated that SP1 undergoes phase separation and that its zinc finger 3 in the DNA-binding domain is essential for this process. Through Cleavage Under Targets & Release Using Nuclease (CUT&RUN) using antibodies against SP1 and H3K27ac, we found a significant correlation between SP1 enrichment and SE elements, identified the regulator of the G protein signaling 20 (RGS20) gene as the most likely target regulated by SP1 through SE mechanisms, and verified this finding using different approaches. The oncogenic activity of SP1 relies on its phase separation ability and RGS20 gene activation, which can be abolished by glycogen synthase kinase J4 (GSK-J4), a demethylase inhibitor. Together, our findings provide evidence that SP1 regulates its target oncogene expression through phase separation and SE mechanisms, thereby promoting LUAD cell progression. This study also revealed an innovative target for LUAD therapies through intervening in SP1-mediated SE formation., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: Presbycusis, also referred to as age-related hearing loss (ARHL), is a condition that results from the cumulative effects of aging on an individual's auditory capabilities. Given the limited understanding of epigenetic mechanisms in ARHL, our research focuses on alterations in chromatin-accessible regions., Methods: We employed assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) in conjunction with unique identifier (UID) mRNA-seq between young and aging cochleae, and conducted integrated analysis as well as motif/TF-gene prediction. Additionally, the essential role of super-enhancers (SEs) in the development of ARHL was identified by comparative analysis to previous research. Meanwhile, an ARHL mouse model and an aging mimic hair cell (HC) model were established with a comprehensive identification of senescence phenotypes to access the role of SEs in ARHL progression., Results: The control cochlear tissue exhibited greater chromatin accessibility than cochlear tissue affected by ARHL. Furthermore, the levels of histone 3 lysine 27 acetylation were significantly depressed in both aging cochlea and aging mimic HEI-OC1 cells, highlighting the essential role of SEs in the development of ARHL. The potential senescence-associated super-enhancers (SASEs) of ARHL were identified, most of which exhibited decreased chromatin accessibility. The majority of genes related to the SASEs showed obvious decreases in mRNA expression level in aging HCs and was noticeably altered following treatment with JQ1 (a commonly used SE inhibitor)., Conclusion: The chromatin accessibility in control cochlear tissue was higher than that in cochlear tissue affected by ARHL. Potential SEs involved in ARHL were identified, which might provide a basis for future therapeutics targeting SASEs related to ARHL., (© 2024. The Author(s).)

Super-enhancers (SEs) are expansive regions of genomic DNA that regulate the expression of genes involved in cell identity and cell fate. We recently identified developmental stage- and cell type-specific modules within the murine Vsx2 SE. Here, we show that the human VSX2 SE modules have similar developmental stage- and cell type-specific activity in reporter gene assays. By inserting the human sequence of one VSX2 SE module into a mouse with microphthalmia, eye size was rescued. To understand the function of these SE modules during human retinal development, we deleted individual modules in human embryonic stem cells and generated retinal organoids. Deleting one module results in small organoids, recapitulating the small-eyed phenotype of mice with microphthalmia, while deletion of the other module led to disruptions in bipolar neuron development. This prototypical SE serves as a model for understanding developmental stage- and cell type-specific effects of neurogenic transcription factors with complex expression patterns. Moreover, by elucidating the gene regulatory mechanisms, we can begin to examine how dysregulation of these mechanisms contributes to phenotypic diversity and disease., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Immune checkpoints inhibitors are effective but it needs more precise biomarkers for patient selection. We explored the biological significance of LINC00862 in pan-cancer by bioinformatics. And we studied its regulatory mechanisms using chromatin immunoprecipitation and RNA immunoprecipitation assays etc. TCGA and single-cell sequencing data analysis indicated that LINC00862 was overexpressed in the majority of tumor and stromal cells, which was related with poor prognosis. LINC00862 expression was related with immune cell infiltration and immune checkpoints expression, and had a high predictive value for immunotherapy efficacy. Mechanistically, LINC00862 competitively bound to miR-29c-3p to unleash SIRT1's tumor-promoting function. SIRT1 inhibitor-EX527 were screened by virtual screening and verified by in vitro and vivo assays. Notably, acetyltransferase P300-mediated super-enhancer activity stimulated LINC00862 transcription. Collectively, LINC00862 could be a diagnostic and prognostic biomarker. LINC00862 could also be a predictive biomarker for immunotherapy efficacy. Super-enhancer activity is the driver for LINC00862 overexpression in cervical cancer and gastric cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Nasopharyngeal carcinoma (NPC), primarily found in the southern region of China, is a malignant tumor known for its highly metastatic characteristics. The high mortality rates caused by the distant metastasis and disease recurrence remain unsolved clinical problems. In clinic, the berberine (BBR) compound has widely been in NPC therapy to decrease metastasis and disease recurrence, and BBR was documented as a main component with multiple anti-NPC effects. However, the mechanism by which BBR inhibits the growth and metastasis of nasopharyngeal carcinoma remains elusive. Herein, we show that BBR effectively inhibits the growth, metastasis, and invasion of NPC via inducing a specific super enhancer (SE). From a mechanistic perspective, the RNA sequencing (RNA-seq) results suggest that the RAS-RAF1-MEK1/2-ERK1/2 signaling pathway, activated by the epidermal growth factor receptor (EGFR), plays a significant role in BBR-induced autophagy in NPC. Blockading of autophagy markedly attenuated the effect of BBR-mediated NPC cell growth and metastasis inhibition. Notably, BBR increased the expression of EGFR by transcription, and knockout of EGFR significantly inhibited BBR-induced microtubule associated protein 1 light chain 3 (LC3)-II increase and p62 inhibition, proposing that EGFR plays a pivotal role in BBR-induced autophagy in NPC. Chromatin immunoprecipitation sequencing (ChIP-seq) results found that a specific SE existed only in NPC cells treated with BBR. This SE knockdown markedly repressed the expression of EGFR and phosphorylated EGFR (EGFR-p) and reversed the inhibition of BBR on NPC proliferation, metastasis, and invasion. Furthermore, BBR-specific SE may trigger autophagy by enhancing EGFR gene transcription, thereby upregulating the RAS-RAF1-MEK1/2-ERK1/2 signaling pathway. In addition, in vivo BBR effectively inhibited NPC cells growth and metastasis, following an increase LC3 and EGFR and a decrease p62. Collectively, this study identifies a novel BBR-special SE and established a new epigenetic paradigm, by which BBR regulates autophagy, inhibits proliferation, metastasis, and invasion. It provides a rationale for BBR application as the treatment regime in NPC therapy in future., (© 2024. The Author(s).)

Background: Tongue squamous cell carcinoma (TSCC) exhibits an aggressive biological behavior of lymph node and distant metastasis, which contributes to poorer prognosis and results in tongue function loss or death. In addition to known regulators and pathways of cell migration in TSCC, it is important to uncover pivotal switches governing tumor metastasis., Methods: Cancer cell migration-associated transcriptional and epigenetic characteristics were profiled in TSCC, and the specific super-enhancers (SEs) were identified. Molecular function and mechanism studies were used to investigate the pivotal switches in TSCC metastasis., Results: Ameboidal-type cell migration-related genes accompanied by transcriptional and epigenetic activity were enriched in TSCC. Meanwhile, the higher-ranked SE-related genes showed significant differences between 43 paired tumor and normal samples from the TCGA TSCC cohort. In addition, key motifs were detected in SE regions, and transcription factor-related expression levels were significantly associated with TSCC survival status. Notably, BATF and ATF3 regulated the expression of ameboidal-type cell migration-related MMP14 by switching the interaction with the SE region., Conclusion: SEs and related key motifs transcriptional regulate tumor metastasis-associated MMP14 and might be potential therapeutic targets for TSCC., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Super-enhancers play prominent roles in driving robust pathological gene expression, but they are hidden in human genome at noncoding regions, making them difficult to explore. Leukemia inhibitory factor (LIF) is a multifunctional cytokine crucially involved in acute respiratory distress syndrome (ARDS) and lung cancer progression. However, the mechanisms governing LIF regulation in disease contexts remain largely unexplored. In this study, we observed elevated levels of LIF in the bronchoalveolar lavage fluid (BALF) of patients with sepsis-related ARDS compared to those with nonsepsis-related ARDS. Furthermore, both basal and LPS-induced LIF expression were under the control of super-enhancers. Through analysis of H3K27Ac ChIP-seq data, we pinpointed three potential super-enhancers (LIF-SE1, LIF-SE2, and LIF-SE3) located proximal to the LIF gene in cells. Notably, genetic deletion of any of these three super-enhancers using CRISPR-Cas9 technology led to a significant reduction in LIF expression. Moreover, in cells lacking these super-enhancers, both cell growth and invasion capabilities were substantially impaired. Our findings highlight the critical role of three specific super-enhancers in regulating LIF expression and offer new insights into the transcriptional regulation of LIF in ARDS and lung cancer., (© 2024 John Wiley & Sons Ltd.)

Super enhancers (SEs) are genomic regions comprising multiple closely spaced enhancers, typically occupied by a high density of cell-type-specific master transcription factors (TFs) and frequently enriched in key oncogenes in various tumors, including neuroblastoma (NB), one of the most prevalent malignant solid tumors in children originating from the neural crest. Cyclin-dependent kinase 5 regulatory subunit-associated protein 3 (CDK5RAP3) is a newly identified super-enhancer-driven gene regulated by master TFs in NB; however, its function in NB remains unclear. Through an integrated study of publicly available datasets and microarrays, we observed a significantly elevated CDK5RAP3 expression level in NB, associated with poor patient prognosis. Further research demonstrated that CDK5RAP3 promotes the growth of NB cells, both in vitro and in vivo. Mechanistically, defective CDK5RAP3 interfered with the UFMylation system, thereby triggering endoplasmic reticulum (ER) phagy. Additionally, we provide evidence that CDK5RAP3 maintains the stability of MEIS2, a master TF in NB, and in turn, contributes to the high expression of CDK5RAP3. Overall, our findings shed light on the molecular mechanisms by which CDK5RAP3 promotes tumor progression and suggest that its inhibition may represent a novel therapeutic strategy for NB., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Liquid-liquid phase separation, a novel biochemical phenomenon, has been increasingly studied for its medical applications. It underlies the formation of membrane-less organelles and is involved in many cellular and biological processes. During transcriptional regulation, dynamic condensates are formed through interactions between transcriptional elements, such as transcription factors, coactivators, and mediators. Cancer is a disease characterized by uncontrolled cell proliferation, but the precise mechanisms underlying tumorigenesis often remain to be elucidated. Emerging evidence has linked abnormal transcriptional condensates to several diseases, especially cancer, implying that phase separation plays an important role in tumorigenesis. Condensates formed by phase separation may have an effect on gene transcription in tumors. In the present review, we focus on the correlation between phase separation and transcriptional regulation, as well as how this phenomenon contributes to cancer development.

Super-enhancers (SEs) exerted a crucial role in regulating the transcription of oncogenes across various malignancies while the roles of SEs driven genes and the core regulatory elements remain elusive in LUAD. In this study, cancer-specific-SE-genes of lung adenocarcinoma (LUAD) were profiled through H3K27ac ChIP-seq data of cancer cell lines and normal lung tissues, which enriched in in biological processes and pathways integral to the pathophysiology of LUAD. Based on this study, LUAD cells were susceptible to SEs inhibitors, with a reduction of cell proliferation as well as an elevation of apoptosis upon JQ1 or THZ1 intervention. Moreover, the integration of SEs landscapes, CRISPRi, ChIP-PCR, Hi-C data analysis and dual-luciferase reporter assays revealed that myeloma overexpressed gene (MYEOV) was aberrantly overexpressed in LUAD via transcriptional activation by the core SE elements. Functionally, the knockdown of MYEOV undermined cell proliferation in vitro and tumor growth in vivo. In addition, the knockdown of MYEOV generated a prominent ferroptotic phenotype, characterized by elevation of intracellular ferrous iron, reactive oxygen species and lipid peroxidation, together with alteration in marker proteins (SLC7A11, GPX4, FTH1, and ACSL4). Instead, the overexpression of MYEOV accelerated cell proliferation and abrogated ferroptosis. Clinically, the overexpression of MYEOV was observed in LUAD tissues indicating a poor prognosis in patients with LUAD. Mechanistically, SMPD1-induced autophagic degradation of GPX4 assumed a crucial role in the process of ferroptosis triggered by MYEOV knockdown. Serving as an oncogene repressing ferroptosis, promoting proliferation as well as shortening survival in LUAD, SEs-mediated activation of MYEOV might distinguish as a promising therapeutic target., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Xianhe Xie reports financial support was provided by Department of Science and Technology of Fujian Province, China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Hypoxia and Ferroptosis are associated with the malignant behaviour of cervical cancer. Endothelial PAS domain-containing protein 1 (EPAS1) contributes to the progression of cervical cancer. EPAS1 plays important roles in hypoxia and ferroptosis. Using the GEO dataset, machine-learning algorithms were used to screen for hypoxia- and ferroptosis-related genes (HFRGs) in cervical cancer. EPAS1 was identified as the hub gene. qPCR and WB were used to investigate the expression of EPAS1 in normal and cervical cancer tissues. The proliferation, invasion and migration of EPAS1 cells in HeLa and SiHa cell lines were detected using CCK8, transwell and wound healing assays, respectively. Apoptosis was detected by flow cytometry. A dual-luciferase assay was used to analyse the MALAT1-miR-182-5P-EPAS1 mRNA axis and core promoter elements of the super-enhancer. EPAS1 was significantly overexpressed in cervical cancer tissues. EPAS1 could increase the proliferation, invasion, migration of HeLa and SiHa cells and reduce the apoptosis of HeLa and SiHa cell. According to the double-luciferase assay, EPAS1 expression was regulated by the MALAT1-Mir-182-5p-EPAS1 mRNA axis. EPAS1 is associated with super-enhancers. Double-luciferase assay showed that the core elements of the super-enhancer were E1 and E3. EPAS1, an HFRG, is significantly overexpressed in cervical cancer. EPAS1 promotes malignant behaviour of cervical cancer cells. EPAS1 expression is regulated by super-enhancers and the MALAT1-miR-182-5P- EPAS1 mRNA axis. EPAS1 may be a target for the diagnosis and treatment of cervical cancer., (© 2024 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Variants at the SLC30A8 locus are associated with type 2 diabetes (T2D) risk. The lead variant, rs13266634, encodes an amino acid change, Arg325Trp (R325W), at the C-terminus of the secretory granule-enriched zinc transporter, ZnT8. Although this protein-coding variant was previously thought to be the sole driver of T2D risk at this locus, recent studies have provided evidence for lowered expression of SLC30A8 mRNA in protective allele carriers. In the present study, we examined multiple variants that influence SLC30A8 allele-specific expression. Epigenomic mapping has previously identified an islet-selective enhancer cluster at the SLC30A8 locus, hosting multiple T2D risk and cASE associations, which is spatially associated with the SLC30A8 promoter and additional neighboring genes. Here, we show that deletion of variant-bearing enhancer regions using CRISPR-Cas9 in human-derived EndoC-βH3 cells lowers the expression of SLC30A8 and several neighboring genes and improves glucose-stimulated insulin secretion. While downregulation of SLC30A8 had no effect on beta cell survival, loss of UTP23, RAD21, or MED30 markedly reduced cell viability. Although eQTL or cASE analyses in human islets did not support the association between these additional genes and diabetes risk, the transcriptional regulator JQ1 lowered the expression of multiple genes at the SLC30A8 locus and enhanced stimulated insulin secretion., (© 2024 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Master transcription factors such as TP63 establish super-enhancers (SEs) to drive core transcriptional networks in cancer cells, yet the spatiotemporal regulation of SEs within the nucleus remains unknown. The nuclear pore complex (NPC) may tether SEs to the nuclear pore where RNA export rates are maximal. Here, we report that NUP153, a component of the NPC, anchors SEs to the NPC and enhances TP63 expression by maximizing mRNA export. This anchoring is mediated through protein-protein interaction between the intrinsically disordered regions (IDRs) of NUP153 and the coactivator BRD4. Silencing of NUP153 excludes SEs from the nuclear periphery, decreases TP63 expression, impairs cellular growth, and induces epidermal differentiation of squamous cell carcinoma. Overall, this work reveals the critical roles of NUP153 IDRs in the regulation of SE localization, thus providing insights into a new layer of gene regulation at the epigenomic and spatial level., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Histone H4 asymmetrically dimethylated at arginine 3 (H4R3me2a) is an active histone mark catalyzed by protein arginine methyltransferase 1 (PRMT1), a major arginine methyltransferase in vertebrates catalyzing asymmetric dimethylation of arginine. H4R3me2a stimulates the activity of lysine acetyltransferases such as CBP/p300, which catalyze the acetylation of H3K27, a mark of active enhancers, super-enhancers, and promoters. There are a few studies on the genomic location of H4R3me2a. In chicken polychromatic erythrocytes, H4R3me2a is found in introns and intergenic regions and binds to the globin locus control region (a super-enhancer) and globin regulatory regions. In this report, we analyzed chromatin immunoprecipitation sequencing data for the genomic location of H4R3me2a in the breast cancer cell line MCF7. As in avian cells, MCF7 H4R3me2a is present in intronic and intergenic regions. Nucleosomes with H4R3me2a and H3K27ac next to nucleosome-free regions are found at super-enhancers, enhancers, and promoter regions of expressed genes. Genes with critical roles in breast cancer cells have broad domains of nucleosomes with H4R3me2a, H3K27ac, and H3K4me3. Our results are consistent with PRMT1-mediated H4R3me2a playing a key role in the chromatin organization of regulatory regions of vertebrate genomes., Competing Interests: The authors declare that they have no conflicts of interest.

One major obstacle in the drug treatment of pancreatic ductal adenocarcinoma (PDAC) is its highly fibrotic tumor microenvironment, which is replete with activated pancreatic stellate cells (a-PSCs). These a-PSCs generate abundant extracellular matrix and secrete various cytokines to form biophysical and biochemical barriers, impeding drug access to tumor tissues. Therefore, it is imperative to develop a strategy for reversing PSC activation and thereby removing the barriers to facilitate PDAC drug treatment. Herein, by integrating chromatin immunoprecipitation (ChIP)-seq, Assays for Transposase-Accessible Chromatin (ATAC)-seq, and RNA-seq techniques, this work reveals that super-enhancers (SEs) promote the expression of various genes involved in PSC activation. Disruption of SE-associated transcription with JQ1 reverses the activated phenotype of a-PSCs and decreases stromal fibrosis in both orthotopic and patient-derived xenograft (PDX) models. More importantly, disruption of SEs by JQ1 treatments promotes vascularization, facilitates drug delivery, and alters the immune landscape in PDAC, thereby improving the efficacies of both chemotherapy (with gemcitabine) and immunotherapy (with IL-12). In summary, this study not only elucidates the contribution of SEs of a-PSCs in shaping the PDAC tumor microenvironment but also highlights that targeting SEs in a-PSCs may become a gate-opening strategy that benefits PDAC drug therapy by removing stromal barriers., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Class switch recombination (CSR) plays an important role in adaptive immune response by enabling mature B cells to replace the initial IgM by another antibody class (IgG, IgE or IgA). CSR is preceded by transcription of the IgH constant genes and is controlled by the super-enhancer 3' regulatory region (3'RR) in an activation-specific manner. The 3'RR is composed of four enhancers (hs3a, hs1-2, hs3b and hs4). In mature B cells, 3'RR activity correlates with transcription of its enhancers. CSR can also occur in primary developing B cells though at low frequency, but in contrast to mature B cells, the transcriptional elements that regulate the process in developing B cells are ill-known. In particular, the role of the 3'RR in the control of constant genes' transcription and CSR has not been addressed. Here, by using a mouse line devoid of the 3'RR and a culture system that highly enriches in pro-B cells, we show that the 3'RR activity is indeed required for switch transcription and CSR, though its effect varies in an isotype-specific manner and correlates with transcription of hs4 enhancer only., (© 2024. The Author(s).)

Precise spatio-temporal expression of genes in organogenesis is regulated by the coordinated interplay of DNA elements such as promoter and enhancers present in the regulatory region of a given locus. POU1F1 transcription factor plays a crucial role in the development of somatotrophs, lactotrophs and thyrotrophs in the anterior pituitary gland, and in maintaining high expression of growth hormone, prolactin and TSH. In mouse, expression of POU1F1 is controlled by a region fenced by two CTCF sites, containing 5 upstream enhancer elements, designated E-A (5' to 3'). Elements C, B and A correspond to elements shown previously to play a role in pituitary development and hormonal expression; functional roles for elements E and D have not been reported. We performed comparative sequence analysis of this regulatory region and discovered that three elements, B, C and E, are present in all vertebrate groups except Agnatha. One very long (>2 kb) element (A) is unique to mammals suggesting a specific change in regulation of the gene in this group. Using DNA accessibility assay (ATAC-seq) we showed that conserved elements in anterior pituitary of four non-mammals are open, suggesting functionality as regulatory elements. We showed that, in many non-mammalian vertebrates, an additional upstream exon closely follows element E, leading to alternatively spliced transcripts. Here, element E functions as an alternative promoter, but in mammals this feature is lost, suggesting conversion of alternative promoter to enhancer. Our work shows that regulation of POU1F1 changed markedly during the course of vertebrate evolution, use of a low number of enhancer elements combined with alternative promoters in non-mammalian vertebrates being replaced by use of a unique combination of regulatory units in mammals. Most importantly, our work suggests that evolutionary conversion of alternate promoter to enhancer could be one of the evolutionary mechanisms of enhancer birth., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024 Published by Elsevier Ltd.)

As one of the first identified oncogenic microRNAs, the precise details concerning the transcriptional regulation and function of microRNA-21 (miR-21) are still not completely established. The miR-21 gene is situated on chromosome 17q23.2, positioned at the 3'-UTR of the gene that encodes vacuole membrane protein-1 (VMP1). In this current study, we presented evidence indicating that miR-21 possesses its own gene promoter, which can be found in the intron 10 of the VMP1 gene. Chromatin immunoprecipitation followed by global DNA sequencing (ChIP-seq) revealed the presence of a broad H3K4me3 peak spanning the entire gene body of the primary miR-21 and the existence of super-enhancer clusters in the close proximity to both the miR-21 gene promoter and the transcription termination site in arsenic (As 3+ )-induced cancer stem-like cells (CSCs) and human induced pluripotent stem cells (hiPSCs). In non-transformed human bronchial epithelial cells (BEAS-2B), As 3+ treatment enhanced Nrf2 binding to both the host gene VMP1 of miR-21 and the miR-21 gene. Knockout of Nrf2 inhibited both the basal and As 3+ -induced expressions of miR-21. Furthermore, the As 3+ -enhanced Nrf2 peaks in ChIP-seq fully overlap with these super-enhancers enriched with H3K4me1 and H3K27ac in the miR-21 gene, suggesting that Nrf2 may coordinate with other transcription factors through the super-enhancers to regulate the expression of miR-21 in cellular response to As 3+ . These findings demonstrate the unique genetic and epigenetic characteristics of miR-21 and may provide insights into understanding the novel mechanisms linking environmental As 3+ exposure and human cancers., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Fei Chen reports financial support and administrative support were provided by National Institutes of Health. Fei Chen reports a relationship with National Institutes of Health that includes: funding grants., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Metabolic reprogramming is an essential hallmark of tumors, and metabolic abnormalities are strongly associated with the malignant phenotype of tumor cells. This is closely related to transcriptional dysregulation. Super-enhancers are extremely active cis-regulatory regions in the genome, and can amalgamate a complex set of transcriptional regulatory components that are crucial for establishing tumor cell identity, promoting tumorigenesis, and enhancing aggressiveness. In addition, alterations in metabolic signaling pathways are often accompanied by changes in super-enhancers. Presently, there is a surge in interest in the potential pathogenesis of various tumors through the transcriptional regulation of super-enhancers and oncogenic mutations in super-enhancers. In this review, we summarize the functions of super-enhancers, oncogenic signaling pathways, and tumor metabolic reprogramming. In particular, we focus on the role of the super-enhancer in tumor metabolism and its impact on metabolic reprogramming. This review also discusses the prospects and directions in the field of super-enhancer and metabolic reprogramming., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Engrailed 2 (EN2) is a homeodomain-containing protein that is dysregulated in many types of cancer. However, the role of EN2 in non-small cell lung cancer (NSCLC) and the mechanism underlying its biological function are largely unclear. Here, we showed that EN2 played an oncogenic function in NSCLC and greatly enhanced the malignant phenotype of NSCLC cells. Meanwhile, EN2 was able to boost the expression of a well-studied oncogenic Tenascin-C (TNC) gene, which in turn activated the AKT signaling pathway. Interestingly, we found that EN2 directly bound to the super-enhancer (SE) region in the TNC locus. The histone marker H3K27ac was also enriched in the region, indicating the activation of the SE. Treatment of the cells with JQ1, an inhibitor of SE activity, abrogated the effect of EN2 on the expression of TNC and phosphorylation of AKT-Ser473. Collectively, our work unveils a novel mode of EN2 function, in which EN2 governs the SE in the TNC locus, consequently activating the oncogenic TNC-AKT axis in NSCLC., (© 2023 Wiley Periodicals LLC.)

Genetic variants in super-enhancers (SEs) are increasingly implicated as a disease risk-driving mechanism. Previous studies have reported an associations between benzo[a]pyrene (BaP) exposure and some malignant tumor risk. Currently, it is unclear whether BaP is involved in the effect of genetic variants in SEs on prostate cancer risk, nor the associated intrinsic molecular mechanisms. In the current study, by using logistic regression analysis, we found that rs5750581T>C in 22q-SE was significantly associated with prostate cancer risk (odds ratio = 1.26, P = 7.61 × 10 -5 ). We also have found that the rs6001092T>G, in a high linkage disequilibrium with rs5750581T>C ( r 2 = 0.98), is located in a regulatory aryl hydrocarbon receptor (AhR) motif and may interact with the FAM227A promoter in further bioinformatics analysis. We then performed a series of functional and BaP acute exposure experiments to assess biological function of the genetic variant and the target gene. Biologically, the rs6001092-G allele strengthened the transcription factor binding affinity to AhR, thereby upregulating FAM227A , especially upon exposure to BaP, which induced the malignant phenotypes of prostate cancer. The current study highlights that AhR acts as an environmental sensor of BaP and is involved in the SE-mediated prostate cancer risk, which may provide new insights into the etiology of prostate cancer associated with the inherited SE variants under environmental carcinogen stressors.

Mutations in the SWI/SNF chromatin remodeling complex occur in ~20% of cancers. In rhabdoid tumors defined by loss of the SWI/SNF subunit SMARCB1 , dysregulation of enhancer-mediated gene expression is pivotal in driving oncogenesis. Enhancer dysregulation in this setting is tied to retention of the SWI/SNF ATPase BRG1-which becomes essential in the absence of SMARCB1 -but precisely how BRG1 contributes to this process remains unknown. To characterize how BRG1 participates in chromatin remodeling and gene expression in SMARCB1 -deficient cells, we performed a genome-wide characterization of the impact of BRG1 depletion in multiple rhabdoid tumor cell lines. We find that although BRG1-regulated open chromatin sites are distinct at the locus level, the biological characteristics of the loci are very similar, converging on a set of thematically related genes and pointing to the involvement of the AP-1 transcription factor. The open chromatin sites regulated by BRG1 colocalize with histone-marked enhancers and intriguingly include almost all super-enhancers, revealing that BRG1 plays a critical role in maintaining super-enhancer function in this setting. These studies can explain the essentiality of BRG1 to rhabdoid tumor cell identity and survival and implicate the involvement of AP-1 as a critical downstream effector of rhabdoid tumor cell transcriptional programs.

Background: A super-enhancer (SE) is a huge cluster of multiple enhancers that control the key genes for cell identity and function. The rise of advanced chromatin immunoprecipitation sequencing (ChIP-seq) technology such as Cleavage Under Targets and Tagmentation (CUT&Tag) allows more SEs to be discovered. However, SE studies in Luchuan and Duroc pigs are very rare in animal husbandry., Results: We used the CUT&Tag technique to identify 145 and 378 SEs from the adipose tissues of Luchuan and Duroc pigs, respectively. There were significant differences in the peak coverage ratio of SE peaks in the gene promoter region between the two breeds. Not only that, peak signals at the start and end point of the SE peak profile showed obvious spikes. The proximal target genes of SE were highly expressed compared with the background genes and the typical enhancer target genes. Subsequently, in conjoint analysis with high-throughput chromosome conformation capture sequencing (Hi-C seq) data, we predicted the remote regulatory genes of SE and found that their expression level was related to the distance of SE extended to the loop's anchor, but not the length of loops. According to our prediction model, SEs can maintain promoter accessibility of partial remote target genes through loop domains. Finally, a batch of SEs closely related to fat metabolism traits were obtained by performing a coalition analysis of quantitative trait loci and SE data., Conclusions: This work enabled us to obtain hundreds of SEs from Luchuan and Duroc pigs. Our model provides a new method for predicting the SE remote target genes based on loop domains, and to further explore the potential role of super-enhancer in the regulation of fat metabolism., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Background: Risk stratification and personalized care are crucial in managing osteosarcoma due to its complexity and heterogeneity. However, current prognostic prediction using clinical variables has limited accuracy. Thus, this study aimed to explore potential molecular biomarkers to improve prognostic assessment., Methods: High-throughput inhibitor screening of 150 compounds with broad targeting properties was performed and indicated a direction towards super-enhancers (SEs). Bulk RNA-seq, scRNA-seq, and immunohistochemistry (IHC) were used to investigate SE-associated gene expression profiles in osteosarcoma cells and patient tissue specimens. Data of 212 osteosarcoma patients who received standard treatment were collected and randomized into training and validation groups for retrospective analysis. Prognostic signatures and nomograms for overall survival (OS) and lung metastasis-free survival (LMFS) were developed using Cox regression analyses. The discriminatory power, calibration, and clinical value of nomograms were evaluated., Results: High-throughput inhibitor screening showed that SEs significantly contribute to the oncogenic transcriptional output in osteosarcoma. Based on this finding, focus was given to 10 SE-associated genes with distinct characteristics and potential oncogenic function. With multi-omics approaches, the hyperexpression of these genes was observed in tumor cell subclusters of patient specimens, which were consistently correlated with poor outcomes and rapid metastasis, and the majority of these identified SE-associated genes were confirmed as independent risk factors for poor outcomes. Two molecular signatures were then developed to predict survival and occurrence of lung metastasis: the SE-derived OS-signature (comprising LACTB, CEP55, SRSF3, TCF7L2, and FOXP1) and the SE-derived LMFS-signature (comprising SRSF3, TCF7L2, FOXP1, and APOLD1). Both signatures significantly improved prognostic accuracy beyond conventional clinical factors., Conclusions: Oncogenic transcription driven by SEs exhibit strong associations with osteosarcoma outcomes. The SE-derived signatures developed in this study hold promise as prognostic biomarkers for predicting OS and LMFS in patients undergoing standard treatments. Integrative prognostic models that combine conventional clinical factors with these SE-derived signatures demonstrate substantially improved accuracy, and have the potential to facilitate patient counseling and individualized management., (© 2024. The Author(s).)

Enhancers are distal DNA elements believed to loop and contact promoters to control gene expression. Recently, we found diffraction-sized transcriptional condensates at genes controlled by clusters of enhancers (super-enhancers). However, a direct function of endogenous condensates in controlling gene expression remains elusive. Here, we develop live-cell super-resolution and multi-color 3D-imaging approaches to investigate putative roles of endogenous condensates in the regulation of super-enhancer controlled gene Sox2. In contrast to enhancer distance, we find instead that the condensate's positional dynamics are a better predictor of gene expression. A basal gene bursting occurs when the condensate is far (>1 μm), but burst size and frequency are enhanced when the condensate moves in proximity (<1 μm). Perturbations of cohesin and local DNA elements do not prevent basal bursting but affect the condensate and its burst enhancement. We propose a three-way kissing model whereby the condensate interacts transiently with gene locus and regulatory DNA elements to control gene bursting., Competing Interests: Declaration of interests I.I.C. is on the Advisory Board at Cell., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Liquid-liquid phase separation, a novel biochemical phenomenon, has been increasingly studied for its medical applications. It underlies the formation of membrane-less organelles and is involved in many cellular and biological processes. During transcriptional regulation, dynamic condensates are formed through interactions between transcriptional elements, such as transcription factors, coactivators, and mediators. Cancer is a disease characterized by uncontrolled cell proliferation, but the precise mechanisms underlying tumorigenesis often remain to be elucidated. Emerging evidence has linked abnormal transcriptional condensates to several diseases, especially cancer, implying that phase separation plays an important role in tumorigenesis. Condensates formed by phase separation may have an effect on gene transcription in tumors. In the present review, we focus on the correlation between phase separation and transcriptional regulation, as well as how this phenomenon contributes to cancer development.

Gastric cancer (GC) is the fifth most prevalent malignancy and the third leading cause of cancer-related mortality globally. Despite intensive efforts to enhance the efficiencies of various therapeutics (chemotherapy, surgical interventions, molecular-targeted therapies, immunotherapies), the prognosis for patients with GC remains poor. This might be predominantly due to the limited understanding of the complicated etiology of GC. Importantly, epigenetic modifications and alterations are crucial during GC development. Super-enhancers (SEs) are a large cluster of adjacent enhancers that greatly activate transcription. SEs sustain cell-specific identity by enhancing the transcription of specific oncogenes. In this review, we systematically summarize how SEs are involved in GC development, including the SE landscape in GC, the SE target genes in GC, and the interventions related to SE functions for treating GC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)