Enhancer-promoter Interactions Research Articles

Endoplasmic reticulum stress related super-enhancers suppress cuproptosis via glycolysis reprogramming in lung adenocarcinoma

The role of copper in tumor progression is thought to be a double-edged sword. Moderate levels of copper promote tumor progression, while excess copper induces a novel form of programmed cell death known as cuproptosis. However, the relationship between lung adenocarcinoma (LUAD) and cuproptosis remains poorly understood. Copper colorimetric assay identified the progression of LUAD simultaneous associated with higher copper accumulation. Single-cell RNA sequencing further identified the activation of unfolded protein response correlates with copper accumulation, particularly the spliced form of XBP1 (XBP1s). XBP1s negatively regulates the protein level of LIPT1 to inhibit LUAD cell death induced by copper-loaded ionophore elesclomol. CUT&Tag-seq and chromosome conformation capture (3 C) experiment showed that XBP1s affect the frequency of MGRN1 promoter-enhancer interactions in various copper environments by forming super-enhancers. Additionally, MGRN1 promotes the ubiquitination and degradation of LIPT1, which in turn supports glycolysis in LUAD cells. In mouse xenograft models, overexpression of XBP1s significantly inhibits the cuproptosis induced by copper ionophores. Co-administration with SEs inhibitor and copper ionophore also markedly reduced tumor volume and growth rate. Our study sheds light on the molecular mechanism by which XBP1s affect the cuproptosis through super-enhancers formation in LUAD and suggested the potential clinical value of copper ionophore as well as a potential biomarker XBP1s for treatment response.

SARE: an optimized method for detection functional regulatory elements in genome

BackgroundMetaheuristic algorithms have been widely employed to solve optimization problems, but their application in analyzing health-related data remains limited. This is primarily due to the complexity of health data, particularly genomic and epigenetic datasets. In genomics, a critical challenge is identifying genomic factors that collaboratively influence disease-associated genes. Enhancer–promoter interactions play a key role in gene regulation, and accurately predicting these elements is essential for developing tailored therapeutic strategies. Despite advances in computational tools, limitations such as fixed-fragment approaches and computational inefficiency hinder the detection of biologically relevant interactions.Materials and methodsHis study introduces the simulation annealing regulatory element (SARE) method, a novel approach based on the simulated annealing algorithm, to identify enhancer–promoter-like interactions from Hi-C (chromosome conformation capture) datasets. The SARE method addresses the limitations of traditional fixed-fragment approaches by detecting variable-length regulatory elements. The Hi-C dataset utilized in this research was derived from mouse embryonic stem cells (ESCs) and processed using advanced filtering techniques to retain valid interactions. A sensitivity analysis was performed to optimize critical parameters, such as the cooling schedule and initial configurations, ensuring robust performance across datasets.ResultsThe proposed method was benchmarked against traditional techniques, including HiCUP and HiC-Pro, using statistical metrics such as precision (0.85), recall (0.78), and F1-score (0.81). SARE demonstrated superior performance, identifying a significantly higher number of interactions with increased biological relevance. Approximately 70% of the detected interactions overlapped with known enhancer–promoter pairs, while the remaining 30% potentially represent novel regulatory mechanisms. Computational efficiency analysis revealed that SARE reduced runtime and memory usage compared to traditional methods, making it suitable for high-throughput applications.ConclusionThe SARE method represents a significant advancement in genomic interaction analysis, offering enhanced sensitivity, efficiency, and biological relevance. By addressing the limitations of traditional tools and identifying both known and novel regulatory elements, SARE provides valuable insights into the mechanisms of gene regulation and chromatin organization. Future studies should focus on expanding the application of SARE to diverse organisms, tissues, and cell types, as well as integrating complementary datasets such as chromatin accessibility and histone modification maps to further validate its findings. Additionally, benchmarking against machine learning-based approaches will establish its position as a robust tool in genomic research.

Deregulated enhancer-promoter communication in cancer through altered nuclear architecture.

Enhancers are critical regulators of gene expression. Structural variations in cancer genomes can lead to enhancer hijacking, where oncogenes are activated by mistargeted enhancer activity. Novel enhancer-promoter interactions may also arise through chromosomal rearrangements that create extrachromosomal DNA elements. Additionally, fusion proteins and other mutation-induced alterations in protein properties can lead to the aberrant assembly of proteins into large complexes on the size scale of 0.1-1 μm termed onco-condensates. Transcription factors and co-activators accumulate with cis-regulatory elements in these structures, driving oncogenic programs. Here, we review current evidence of how altered genome architecture and macromolecular assembly result in deregulated enhancer-promoter communication. We discuss emerging strategies to exploit these mechanisms for clinical applications.

The bxd PRE from Bithorax Complex of Drosophila melanogaster Has Weak Insulator Activity.

The autonomous functioning of regulatory domains in the Bithorax complex (BX-C) of Drosophila melanogaster is maintained by boundaries (insulators) that prevent inappropriate enhancer-promoter interactions. Polycomb response elements (PREs) maintain epigenetic memory within regulatory domains and are often located near insulators, enhancing their blocking activity. The bxdPRE is a well-characterized silencer that regulates the bxd/pbx domain of the Ubx gene in the first abdominal segment. We used a boundary replacement strategy to assess the insulator activity of the bxdPRE. Substituting the bxdPRE for the Fab-7 boundary, which separates the Abd-B regulatory domains in the BX-C, revealed that the bxdPRE has weak insulator activity.

A specific pluripotency-associated eRNA controls Nanog locus by shaping the epigenetic landscape and stabilizing enhancer-promoter interaction.

Despite a plethora of studies exploringthe transcriptional regulation of the Nanog gene, the role of theenhancer RNAs (eRNAs) derived from Nanog-interacting super-enhancers (SEs) remains under-investigated. In the present study, we examined the functional role of the eRNAs transcribed from the -5kb NanogSEin mouse embryonic stem cells(mESCs)and found that an eRNA, here defined as -5KNAR, was essential to maintain the Nanog locus in an epigenetically active configuration, thereby ensuring pluripotency. We found that the here identified -5KNAR functionally interacts with the RAD21 protein, suggesting a role in stabilizing a cohesin complex at the Nanog locus, ensuring the generation and maintenance of an enhancer-promoter loop. Silencing of -5KNAR caused a cascade of events,including the generation of a DNA methylation wave (likely spreading from a single methylated CpG site), substantial chromatin remodeling, and loss of the enhancer-promoter loop, inducing Nanog silencing and mESCdifferentiation. Under these conditions, exogenous re-expression of Nanog was unable to restore either the endogenous Nanog expression or the enhancer-promoter interaction,suggesting that, at hierarchical level, the expression of the -5KNAR plays a prominent role in maintaining the pluripotency in mESCs.

Nuclear actin-dependent Meg3 expression suppresses metabolic genes by affecting the chromatin architecture at sites of elevated H3K27 acetylation levels.

Nuclear actin mediates enhancer-dependent transcriptional regulation at compartment level, playing critical roles in 3D genome organization. In β-actin depleted cells, H3K27 acetylation is enhanced, directly affecting enhancer-dependent transcriptional regulation and gene expression changes during compartment-switching. Here, we report these mechanisms are influenced by the long non-coding RNA (lncRNA) Meg3. Bulk RNA-seq analysis and qPCR on wild-type (WT), heterozygous (HET), and β-actin knockout (KO) mouse embryonic fibroblasts (MEFs) show that β-actin depletion significantly alters expression of several lncRNAs, including Meg3. Results from ChIRP-seq, ChIRP-MS, and fRIP-qPCR revealed that in β-actin KO cells, Meg3 becomes enriched and binds to H3K27 acetylation marks within gene regulatory regions. By integrating RNA-seq, H3K27 acetylation ChIP-seq, ATAC-seq, and HiC-seq data through activity by contact (ABC) analysis, we discovered Meg3 binding disrupts promoter-enhancer interactions in β-actin KO cells. These results, combined with metabolomics in WT, HET, and β-actin KO MEFs, show Meg3 binding to regulatory regions at sites of increased H3K27 acetylation impairs the expression of genes involved in the synthesis of chondroitin, heparan, dermatan sulfate, and phospholipases. We propose that in β-actin KO cells Meg3 binds to H3K27 acetylation levels. This interferes with promoter-enhancer interactions, disrupts genome organization, and downregulates gene expression and key metabolic pathways.

USP10 stabilizes BAZ1A to drive tumor stemness via an epigenetic mechanism in head and neck squamous cell carcinoma

Aberrant epigenetic remodeling events occurred in head and neck squamous cell carcinoma (HNSCC) contribute to tumor stemness and chemotherapy resistance, yet little is known. In this study, we identified that ubiquitin-specific peptidase 10 (USP10) is up-regulated in HNSCC tissues, and high USP10 is associated with poor prognosis of patients. Functionally, USP10 serving as an oncogene potentiates the proliferation and metastasis of HNSCC cells in vitro and in vivo. Mechanistically, USP10 physically interacts with, deubiquitinate, and stabilizes BAZ1A proteins. In addition, BAZ1A complexes with SOX2 to drive the enhancer-promoter interaction and facilitate the recruitment of BRD4, thereby activating the expressions of cancer stem cells (CSCs)-related signature. Therefore, we found that USP10 relied on BAZ1A to enhance HNSCC stemness, progression, and chemotherapy resistance. The pharmacology research implicated that BAZ1A-IN-1, one specific BAZ1A inhibitor, could effectively inhibit HNSCC stemness, distal metastasis, and cisplatin resistance. Together, our study revealed a novel USP10/BAZ1A/stemness axis and one significant therapeutic target for USP10-driven HNSCC.

PC-SAC: Amethod for high-resolution 3D genome reconstruction from low-resolution Hi-C data.

The three-dimensional (3D) organization of the genome is crucial for gene regulation, with disruptions linked to various diseases. High-throughput Chromosome Conformation Capture (Hi-C) and related technologies have advanced our understanding of 3Dgenome organization by mapping interactions between distalgenomic regions. However, capturing enhancer-promoter interactions at high resolution remains challenging due to the high sequencing depth required. We introduce pC-SAC (probabilistically Constrained Self-Avoiding Chromatin), a novel computational method for producing accurate high-resolution Hi-C matrices from low-resolution data. pC-SAC uses adaptive importance sampling with sequential Monte Carlo to generate ensembles of 3D chromatin chains that satisfy physical constraints derived from low-resolution Hi-C data. Our method achieves over 95% accuracy in reconstructing high-resolution chromatin maps and identifies novel interactions enriched with candidate cis-regulatory elements (cCREs) and expression quantitative trait loci (eQTLs). Benchmarking against state-of-the-art deep learning models demonstrates pC-SAC'sperformance in both short- and long-range interaction reconstruction. pC-SAC offers a cost-effective solution for enhancing the resolution of Hi-C data, thus enabling deeper insights into 3D genome organization and its role in gene regulation and disease. Our tool can be found at https://github.com/G2Lab/pCSAC.

Targeting processive transcription for Myc-driven circuitry in medulloblastoma.

Medulloblastoma is the most common malignant brain tumor of childhood. The highest-risk tumors are driven by recurrent Myc amplifications (Myc-MB) and experience poorer outcomes despite intensive multimodal therapy. The Myc transcription factor defines core regulatory circuitry for these tumors and acts to broadly amplify downstream pro-survival transcriptional programs. Therapeutic targeting of Myc directly has proven elusive, but inhibiting transcriptional cofactors may present an indirect means of drugging the oncogenic transcriptional circuitry sustaining Myc-MB. Independent CRISPR-Cas9 screens were pooled to identify conserved dependencies in Myc-MB. We performed chromatin conformation capture (Hi-C) from primary patient Myc-MB samples to map enhancer-promoter interactions. We then treated in vitro and xenograft models with CDK9/7 inhibitors to evaluate effect on Myc-driven programs and tumor growth. Eight CRISPR-Cas9 screens performed across three independent labs identify CDK9 as a conserved dependency in Myc-MB. Myc-MB cells are susceptible to CDK9 inhibition, which is synergistic with concurrent inhibition of CDK7. Inhibition of transcriptional CDKs disrupts enhancer-promoter activity in Myc-MB and downregulates Myc-driven transcriptional programs, exerting potent anti-tumor effect. Our findings identify CDK9 inhibition as a translationally promising strategy for the treatment of Myc-MB. CDK9 is an intrinsic dependency in Myc-driven medulloblastomaDual CDK9/7 inhibition disrupts Myc-driven transcriptional circuitryCDK9 inhibitors should be developed as pharmaceutical agents for Myc-MB. Medulloblastoma is the most common malignant brain tumor of childhood, and outcomes for high-risk subgroups remain unsatisfactory despite intensive multimodal therapy. In this study, we pool multiple independent CRISPR-Cas9 screens to identify transcriptional cofactors such as CDK9 as conserved dependencies in Myc-MB. Using Hi-C from primary patient samples, we map Myc enhancer-promoter interactions and show that they can be disrupted using inhibition of transcriptional CDKs. CDK9 inhibitor treatment depletes Myc-driven transcriptional programs, leading to potent anti-tumor effect in vitro and prolongation of xenograft survival in vivo . With a large number of CDK9 inhibitory compounds now in clinical development, this study highlights the opportunity for clinical translation of these for children diagnosed with Myc-MB.

Enhancer transcription profiling reveals an enhancer RNA-driven ferroptosis and new therapeutic opportunities in prostate cancer

Enhancer RNAs (eRNAs), a subclass of non-coding RNAs transcribed from enhancer regions, have emerged as critical regulators of gene expression; however, their functional roles in prostate cancer remain largely unexplored. In this study, we performed integrated chromatin accessibility and transcriptomic analyses using ATAC-seq and RNA-seq on twenty pairs of prostate cancer and matched benign tissues. By incorporating chromatin immunoprecipitation sequencing data, we identified a subset of differentially expressed eRNAs significantly associated with genes involved in prostate development and oncogenic signaling pathways. Among these, lactotransferrin-eRNA (LTFe) was markedly downregulated in prostate cancer tissues, with functional analyses revealing its tumor-suppressive role. Mechanistically, LTFe promotes the transcription of its target gene, lactotransferrin (LTF), by interacting with heterogeneous nuclear ribonucleoprotein F (HNRNPF) and facilitating enhancer-promoter chromatin interactions. Furthermore, we demonstrate that the LTFe-LTF axis facilitates ferroptosis by modulating iron transport. Notably, androgen receptor (AR) signaling disrupts LTFe-associated chromatin looping, leading to ferroptosis resistance. Therapeutically, co- administration of the AR inhibitor enzalutamide and the ferroptosis inducer RSL3 significantly suppressed tumor growth, offering a promising strategy for castration-resistant prostate cancer. Collectively, this study provides novel insights into the mechanistic role of eRNAs in prostate cancer, highlighting the LTFe-LTF axis as a critical epigenetic regulator and potential therapeutic target for improved treatment outcomes.

Distance matters: How protein regulators facilitate enhancer-promoter interactions and transcription.

Distance matters: How protein regulators facilitate enhancer-promoter interactions and transcription.

EPIPDLF: a pre-trained deep learning framework for predicting enhancer-promoter interactions.

Enhancers and promoters, as regulatory DNA elements, play pivotal roles in gene expression, homeostasis, and disease development across various biological processes. With advancing research, it has been uncovered that distal enhancers may engage with nearby promoters to modulate the expression of target genes. This discovery holds significant implications for deepening our comprehension of various biological mechanisms. In recent years, numerous high-throughput wet-lab techniques have been created to detect possible interactions between enhancers and promoters. However, these experimental methods are often time-intensive and costly. To tackle this issue, we have created an innovative deep learning approach, EPIPDLF, which utilizes advanced deep learning techniques to predict EPIs based solely on genomic sequences in an interpretable manner. Comparative evaluations across six benchmark datasets demonstrate that EPIPDLF consistently exhibits superior performance in EPI prediction. Additionally, by incorporating interpretable analysis mechanisms, our model enables the elucidation of learned features, aiding in the identification and biological analysis of important sequences. The source code and data are available at: https://github.com/xzc196/EPIPDLF.

Liquid condensates: a new barrier to loop extrusion?

Liquid–liquid phase separation (LLPS), driven by dynamic, low-affinity multivalent interactions of proteins and RNA, results in the formation of macromolecular condensates on chromatin. These structures are likely to provide high local concentrations of effector factors responsible for various processes including transcriptional regulation and DNA repair. In particular, enhancers, super-enhancers, and promoters serve as platforms for condensate assembly. In the current paradigm, enhancer-promoter (EP) interaction could be interpreted as a result of enhancer- and promoter-based condensate contact/fusion. There is increasing evidence that the spatial juxtaposition of enhancers and promoters could be provided by loop extrusion (LE) by SMC complexes. Here, we propose that condensates may act as barriers to LE, thereby contributing to various nuclear processes including spatial contacts between regulatory genomic elements.

Pioneer factor GATA6 promotes colorectal cancer through 3D genome regulation.

Colorectal cancer (CRC) is one of the most lethal and prevalent malignancies. While the overexpression of pioneer factor GATA6 in CRC has been linked with metastasis, its role in genome-wide gene expression dysregulation remains unclear. Through studies of primary human CRC tissues and analysis of the TCGA data, we found that GATA6 preferentially binds at CRC-specific active enhancers, with enrichment at enhancer-promoter loop anchors. GATA6 protein also physically interacts with CTCF, suggesting its critical role in 3D genome organization. The ablation of GATA6 through AID and CRISPR systems severely impaired cancer cell clonogenicity and proliferation. Mechanistically, GATA6 knockout induced global loss of CRC-specific open chromatins and extensive alterations of critical enhancer-promoter interactions for CRC oncogenes. Last, we showed that GATA6 knockout greatly reduced tumor growth and improved survival in mice. Together, we revealed a previously unidentified mechanism by which GATA6 contributes to the pathogenesis of colorectal cancer.

JMJD6 Rewires ATF4-Dependent Glutathione Metabolism to Confer Ferroptosis Resistance in SPOP-Mutated Prostate Cancer.

Ferroptosis inducers have shown therapeutic potential in prostate cancer (PCa), but tumor heterogeneity poses a barrier to their efficacy. Distinguishing the regulators orchestrating metabolic crosstalk between cancer cells could shed light on therapeutic strategies to more robustly activate ferroptosis. Here, we found that aberrant accumulation of jumonji domain containing 6 (JMJD6) proteins correlated with poorer prognosis of PCa patients. Mechanistically, PCa-associated speckle type BTB/POZ protein (SPOP) mutants impaired the proteasomal degradation of JMJD6 proteins. Elevated JMJD6 and ATF4 coordinated enhancer-promoter loop interactions to stimulate the glutathione biosynthesis pathway. Independent of androgen receptor, JMJD6 recruited mediator subunits (Med1/14) to assemble de novo enhancers mapping to pivotal genes associated with glutathione metabolism, including SLC7A11, GCLM, ME1, and others. SPOP mutations thus induced intrinsic resistance to ferroptosis, dependent on enhanced JMJD6-ATF4 activity. Consequently, targeting JMJD6 rendered SPOP-mutated PCa selectively sensitive to ferroptosis. The JMJD6 antagonist SKLB325 synergized with erastin in multiple pre-clinical PCa models. Together, this study identifies JMJD6 as a druggable vulnerability in SPOP-mutated PCa to increase sensitivity to ferroptosis inducers.

Exploring the interplay between enhancer-promoter interactions and transcription.

Exploring the interplay between enhancer-promoter interactions and transcription.

Human herpesvirus-associated transposable element activation in human aging brains with Alzheimer's disease.

Human herpesvirus (HHV) has been linked to Alzheimer's disease (AD), but the underlying mechanisms remain unknown. We leveraged functional genomics data from Religious Orders Study or the Rush Memory and Aging Project (ROS/MAP) and Mount Sinai Brain Bank (MSBB) brain biobanks and single-cell RNA-sequencing data from HHV-infected forebrain organoids to investigate HHV-infection-associated transposable element (TE) dysregulation underlying AD etiologies. We identified widespread TE dysregulation in HHV-positive human AD brains, including an astrocyte-specific upregulation of LINE1 subfamily TEs in HHV-positive human AD brains. We further pinpointed astrocyte-specific LINE1 upregulation that could potentially regulate target gene NEAT1 expression via long-range enhancer-promoter chromatin interactions. This LINE1 dysregulation can be partially reversed by the usage of anti-HHV drugs (valacyclovir and acyclovir) in a virus-infected human brain organoid model. Finally, we demonstrated that valacyclovir rescued tau-associated neuropathology and alleviated LINE1 activation in an experimental tau aggregation model. Our analysis provides associations linking molecular, clinical, and neuropathological AD features with HHV infection, which warrants future clinical validation. Via analysis of bulk RNA-seq data in two large-scale human brain biobanks, ROS/MAP (n=109 pathologically confirmed AD and n=44 cognitively healthy controls) and MSBB (n=284 AD and n=150 cognitively healthy controls), we identified widespread TE activation in HHV-positive human AD brains and significantly positive associations of HHV RNA abundance with APOE4 genotype, Braak staging score, and CERAD score. We identified cell type-specific LINE1 upregulation in both microglia and astrocytes of human AD brains via long-range enhancer-promoter chromatin interactions on lncRNA nuclear enriched abundant transcript 1 (NEAT1). We determined that usage of valacyclovir and acyclovir was significantly associated with reduced incidence of AD in a large real-world patient database. Using the HEK293 tau P301S model and U2OS mt-Keima cell model, we determined that valacyclovir treatment rescued tau-associated neuropathology and alleviated activation of LINE1 with increased cellular autophagy-level mechanistically supported clinical benefits of valacyclovir in real-world patient data.

Multivalent nucleosome scaffolding by bromodomain and extraterminal domain tandem bromodomains.

Promoter-promoter and enhancer-promoter interactions are enriched in histone acetylation and central to chromatin organization in active genetic regions. Bromodomains are epigenetic 'readers' that recognize and bind histone acetylation. Bromodomains often exist in tandem or with other reader domains. Cellular knockdown of the bromodomain and extraterminal domain (BET) protein family disrupts chromatin organization, but the mechanisms through which BET proteins preserve chromatin structure are largely unknown. We hypothesize that BET proteins maintain overall chromatin structure by employing their tandem bromodomains to multivalently scaffold acetylated nucleosomes in an intra- or internucleosomal manner. To test this hypothesis biophysically, we used small-angle X-ray scattering, electron paramagnetic resonance, and Rosetta protein modeling to show that a disordered linker separates BET tandem bromodomain acetylation binding sites by 15-157 Å. Most of these modeled distances are sufficient to span the length of a nucleosome (>57 Å). Focusing on the BET family member BRD4, we employed bioluminescence resonance energy transfer and isothermal titration calorimetry to show that BRD4 bromodomain binding of multiple acetylation sites on a histone tail does not increase BRD4-histone tail affinity, suggesting that BET bromodomain intranucleosome binding is not biologically relevant. Using sucrose gradients and amplified luminescent proximity homogeneous (AlphaScreen) assays, we provide the first direct biophysical evidence that BET bromodomains can scaffold multiple acetylated nucleosomes. Taken together, our results demonstrate that BET bromodomains are capable of multivalent internucleosome scaffolding in vitro. The knowledge gained provides implications for how BET bromodomain-mediated acetylated internucleosome scaffolding may maintain cellular chromatin interactions in active genetic regions.

Analytical results for chromatin polymer models with enhancer-promoter interactions

Analytical results for chromatin polymer models with enhancer-promoter interactions

Glucocorticoid receptor suppresses GATA6-mediated RNA polymerase II pause release to modulate classical subtype identity in pancreatic cancer

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer with a 5-year survival rate of 12%. It has two major molecular subtypes: classical and basal, regulated by the master transcription...