Super enhancers are important regulators of gene expression that often overlap with protein-coding genes. However, it is unclear whether the overlapping protein-coding genes and the RNA derived from them contribute to enhancer activity. Using an erythroid-specific super enhancer that overlaps the Cpox gene as a model, Cpox pre-mRNA is found to have a non-coding function in regulating neighbouring protein-coding genes, eRNA expression and TAD interactions. Depletion of Cpox pre-mRNA leads to accumulation of H3K27me3 and release of p300 from the Cpox locus, activating an intra-TAD enhancer and gene expression. Additionally, a head-to-tail interaction between the TAD boundary genes Cpox and Dcbld2 is identified, facilitated by a novel type of repressive loop anchored by p300 and PRC2/H3K27me3. These results uncover a regulatory role for pre-mRNA transcribed within a super enhancer context and provide insight into head-to-tail inter-gene interaction in the regulation of gene expression and oncogene activation.

Cancer has been characterized as a wound that does not heal. Malignant cells are morphologically distinct from normal proliferating cells but have extensive similarities to tissues undergoing wound healing and/or regeneration. The mechanistic basis of this similarity has, however, remained enigmatic. Here, we show that the genomic region upstream of Myc, which carries more cancer susceptibility in humans than any other genomic region, is required for intestinal regeneration after radiation damage. Failure to regenerate is associated with inefficient Ly6a/Sca1+ stem/progenitor cell mobilization, and almost complete failure to re-establish Lgr5+ cell compartment in the intestinal crypts. The Myc upstream region is also critical for growth of adult intestinal cells in 3D organoid culture. We show that culture conditions recapitulating most aspects of adult normal tissue architecture still reprogram normal cells to proliferate using a mechanism similar to that employed by cancer cells. Our results establish a function for the Myc 2-540 super-enhancer region as the genetic link between tissue regeneration and tumorigenesis, and demonstrates that normal tissue renewal and regeneration of tissues after severe damage are mechanistically distinct.

A growing body of evidence suggests that nuclear bodies, condensates of RNAs and proteins within the nucleus, are assembled through liquid-liquid phase separation. Some nuclear bodies, such as paraspeckles, are scaffolded by a class of RNAs known as architectural RNAs. From a materials science perspective, RNAs are categorized as polymers, which have been extensively studied in soft matter physics. While soft matter physics has the potential to provide significant insights, it is not directly applicable because transcription and other biochemical processes differentiate RNAs from other polymers studied in this field. Therefore, an interdisciplinary research fusing molecular biology and soft matter physics offers a powerful approach to studying nuclear bodies. This review introduces the biophysical insights provided by such interdisciplinary research in the assembly and regulation of nuclear bodies.

Liquid-liquid phase separation (LLPS) is a significant reversible and dynamic process in organisms. Cells form droplets that are distinct from membrane-bound cell organelles by phase separation to keep biochemical processes in order. Nevertheless, the pathological state of LLPS contributes to the progression of a variety of tumor-related pathogenic issues. Sarcoma is one kind of highly malignant tumor characterized by aggressive metastatic potential and resistance to conventional therapeutic agents. Despite the significant clinical relevance, research on phase separation in sarcomas currently faces several major challenges. These include the limited availability of sarcoma samples, insufficient attention from the research community, and the complex genetic heterogeneity of sarcomas. Recently, emerging evidence have elaborated the specific effects and pathways of phase separation on different sarcoma subtypes, including the effect of sarcoma fusion proteins and other physicochemical factors on phase separation. This review aims to summarize the multiple roles of phase separation in sarcoma and novel molecular inhibitors that target phase separation. These insights will broaden the understanding of the mechanisms concerning sarcoma and offer new perspectives for future therapeutic strategies.

The precise regulation of the transcription of genes is essential for normal development and for the maintenance of life. Aberrant gene expression changes drive many human diseases. Despite this, we still do not completely understand how precise gene regulation is controlled in living systems. Enhancers are key regulatory elements that enable cells to specifically activate genes in response to environmental cues, or in a stage or tissue-specific manner. Any model of enhancer activity needs to answer two main questions: (1) how enhancers are able to identify and act on specific genes and (2) how enhancers influence transcription. To address these points, we first outline some of the basic principles that can be established from simpler prokaryotic systems, then discuss recent work on aberrant enhancer activity in leukemia. We argue that highly specific protein-protein interactions are a key driver of enhancer-promoter proximity, allowing enhancer-bound factors to directly act on RNA polymerase and activate transcription.

Maternal-deposited factors initiate zygotic genome activation (ZGA), driving the maternal-to-zygotic transition; however, the coordination between maternal coactivators and transcription factors (TFs) in this process remains unclear. In this study, by profiling the dynamic landscape of p300 during mouse ZGA, we reveal its role in promoting RNA polymerase II (Pol II) pre-configuration at ZGA gene regions and sequentially establishing enhancer activity and regulatory networks. Moreover, p300/CBP-catalyzed acetylation drives Pol II elongation and minor ZGA gene expression by inducing pivotal TFs such as Dux. Remarkably, the supplementation of exogenous Dux rescues ZGA failure and developmental defects caused by the loss of p300/CBP acetylation. DUX functions as a pioneer factor, guiding p300 and Pol II to minor ZGA gene regions and activating them in a manner dependent on the non-catalytic functions of p300/CBP. Together, our findings reveal a mutual dependency between p300/CBP and DUX, highlighting their coordinated role in regulating minor ZGA activation.

Proper development of surface epithelium (SE) is a requisite for the normal development and function of ectodermal appendages; however, the molecular mechanisms underlying SE commitment remain largely unexplored. Here, we developed a KRT8 reporter system and utilized it to identify FOXO4 and SP6 as novel, essential regulators governing SE commitment. We found that the FOXO4-SP6 axis governs SE fate and its abrogation markedly impedes SE fate determination. Mechanistically, FOXO4 regulates SE initiation by shaping the SE chromatin accessibility landscape and regulating the deposition of H3K4me3. SP6, as a novel effector of FOXO4, activates SE-specific genes through modulating the H3K27ac deposition across their super-enhancers. Our work highlights the regulatory function of the FOXO4-SP6 axis in SE development, contributing to an improved understanding of SE fate decisions and providing a research foundation for the therapeutic application of ectodermal dysplasia.

The biological functions of extragenic enhancer RNAs and their impact on disease risk remain relatively underexplored. In this work, we develop in silico models of genetically regulated expression of enhancer RNAs across 49 cell and tissue types, characterizing their degree of genetic control. Leveraging the estimated genetically regulated expression for enhancer RNAs and canonical genes in a large-scale DNA biobank (N > 70,000) and high-resolution Hi-C contact data, we train a deep learning-based model of pairwise three-dimensional chromatin contact frequency for enhancer-enhancer and enhancer-gene pairs in cerebellum and whole blood. Notably, the use of genetically regulated expression of enhancer RNAs provides substantial tissue-specific predictive power, supporting a role for these transcripts in modulating spatial chromatin organization. We identify schizophrenia-associated enhancer RNAs independent of GWAS loci using enhancer RNA-based TWAS and determine the causal effects of these enhancer RNAs using Mendelian randomization. Using enhancer RNA-based TWAS, we generate a comprehensive resource of tissue-specific enhancer associations with complex traits in the UK Biobank. Finally, we show that a substantially greater proportion (63%) of GWAS associations colocalize with causal regulatory variation when enhancer RNAs are included.

Flower formation has been a primary focus in botanical research, leading to the identification of multiple factors regulating flowering over the past 30 years. The MADS transcription factors SEPALLATA3 (SEP3) and APETALA1 (AP1) are essential for floral meristem development and organ identity. In Arabidopsis, SEP3 functions as a central integrator, combining MADS proteins into a tetrameric complex, with its interaction with AP1 playing a key role in sepal and petal formation. This research explores AtSEP3 and AtAP1, with particular emphasis on the Leu residue in the K1 subfunctional domain of AtSEP3, which is necessary for their interaction. A predicted structural model of AP1 was used, followed by protein docking with SEP3, which indicated that Leu residues at positions 115 and 116 are critical binding sites. Mutations at these position were examined through yeast two-hybrid assays and other techniques, identifying Leu 116 as a significant site. Subsequent purification and EMSA analysis revealed that mutations in the leucine zipper of SEP3 decreased its DNA binding ability. Observations of transgenic plants showed that disruption of AtSEP3 and AtAP1 interaction resulted in extended vegetative growth, increased size and number of rosette leaves, and modifications in floral structures. This study offers new insights into the interaction mechanism between AP1 and SEP3 during flowering.

Bidirectional enhancer RNA (eRNA) transcription is a widespread response to environmental signals and glucocorticoids. We investigated whether single nucleotide polymorphisms (SNPs) within dynamically regulated eRNA-transcribing regions contribute to genetic variation in asthma. Through applying multivariate regression modeling with permutation-based significance thresholding to a large clinical cohort, we identified novel associations between asthma and 35 SNPs located in eRNA-transcribing regions implicated in regulating cellular processes relevant to asthma, including rs258760 (mean allele frequency = 0.34, asthma odds ratio = 0.95; P = 5.04E-03). We show that rs258760 disrupts an active aryl hydrocarbon receptor (AHR) response element linked to transcriptional regulation of the glucocorticoid receptor gene by AHR ligands, which are commonly found in combusted air pollution. The role of rs258760 as a protective variant for asthma was independently validated using UK Biobank data. Our findings establish eRNA signatures as a tool for discovery of functional genetic variants and define a novel association between air pollution, glucocorticoid signaling and asthma.

Cellular pliancy refers to the unique disposition of different stages of cellular differentiation to transform when exposed to specific oncogenic insults. This concept highlights a strong interconnection between cellular identity and tumorigenesis, and implies overcoming of epigenetic barriers defining cellular states. Emerging evidence suggests that the cell-type-specific response to intrinsic and extrinsic stresses is modulated by accessibility to certain areas of the genome. Understanding the interplay between epigenetic mechanisms, cellular differentiation, and oncogenic insults is crucial for deciphering the complex nature of tumorigenesis and developing targeted therapies. Hence, cellular pliancy relies on a dynamic cooperation between the cellular identity and the cellular context through epigenetic control, including the reactivation of cellular mechanisms, such as epithelial-to-mesenchymal transition (EMT). Such mechanisms and pathways confer plasticity to the cell allowing it to adapt to a hostile environment in a context of tumor initiation, thus changing its cellular identity. Indeed, growing evidence suggests that cancer is a disease of cell identity crisis, whereby differentiated cells lose their defined identity and gain progenitor characteristics. The loss of cell fate commitment is a central feature of tumorigenesis and appears to be a prerequisite for neoplastic transformation. In this context, EMT-inducing transcription factors (EMT-TFs) cooperate with mitogenic oncoproteins to foster malignant transformation. The aberrant activation of EMT-TFs plays an active role in tumor initiation by alleviating key oncosuppressive mechanisms and by endowing cancer cells with stem cell-like properties, including the ability to self-renew, thus changing the course of tumorigenesis. This highly dynamic phenotypic change occurs concomitantly to major epigenome reorganization, a key component of cell differentiation and cancer cell plasticity regulation. The concept of pliancy was initially proposed to address a fundamental question in cancer biology: why are some cells more likely to become cancerous in response to specific oncogenic events at particular developmental stages? We propose the concept of epipliancy, whereby a difference in epigenetic configuration leads to malignant transformation following an oncogenic insult. Here, we present recent studies furthering our understanding of how the epigenetic landscape may impact the modulation of cellular pliancy during early stages of cancer initiation.

Hypoxia and dysregulated phase separation can both activate oncogenic transcriptomic profiles. However, whether hypoxia regulates transcription-associated phase separation remains unknown. Here, we find that zinc fingers and homeoboxes 2 (ZHX2) undergoes phase separation in response to hypoxia, promoting their occupancy on chromatin and activating a cluster of oncogene transcription that is enriched by metastatic genes distinct from the targets of hypoxia-inducible factor (HIF) and pathologically relevant to breast cancer. Hypoxia induces ZHX2 phase separation via a proline-rich intrinsically disordered region (IDR), enhancing phosphorylation of ZHX2 at S625 and S628 that incorporates CCCTC-binding factor (CTCF) in condensates to alter chromatin looping, consequently driving metastatic gene transcription and cancer metastasis. Our findings provide significant insight into oncogene activation and suggest a phase-separation-based therapeutic strategy for cancer.

Kaposi's sarcoma-associated herpesvirus (KSHV) genome contains a terminal repeats (TR) sequence. Previous studies demonstrated that KSHV TR functions as a gene enhancer for inducible lytic gene promoters. Gene enhancers anchor bromodomain-containing protein 4 (BRD4) at specific genomic region, where BRD4 interacts flexibly with transcription-related proteins through its intrinsically disordered domain and exerts transcription regulatory function. Here, we generated recombinant KSHV with reduced TR copy numbers and studied BRD4 recruitment and its contributions to the inducible promoter activation. Reducing the TR copy numbers from 21 (TR21) to 5 (TR5) strongly attenuated viral gene expression during de novo infection and impaired reactivation. The EF1α promoter encoded in the KSHV BAC backbone also showed reduced promoter activity, suggesting a global attenuation of transcription activity within TR5 latent episomes. Isolation of reactivating cells confirmed that the reduced inducible gene transcription from TR-shortened DNA template and is mediated by decreased efficacies of BRD4 recruitment to viral gene promoters. Separating the reactivating iSLK cell population from non-responders showed that reactivatable iSLK cells harbored larger LANA nuclear bodies (NBs) compared to non-responders. The cells with larger LANA NBs, either due to prior transcription activation or TR copy number, supported KSHV reactivation more efficiently than those with smaller LANA NBs. With auxin-inducible LANA degradation, we confirmed that LANA is responsible for BRD4 occupancies on latent chromatin. Finally, with purified fluorescence-tagged proteins, we demonstrated that BRD4 is required for LANA to form liquid-liquid phase-separated dots. The inclusion of TR DNA fragments further facilitated the formation of larger BRD4-containing LLPS in the presence of LANA, similar to the "cellular enhancer dot" formed by transcription factor-DNA bindings. These results suggest that LANA binding to TR establishes an enhancer domain for infected KSHV episomes. The strength of this enhancer, regulated by TR length or transcription memory, determines the outcome of KSHV replication.

Gene enhancers are genomic domains that regulate frequency and duration of transcription burst at gene promoters, with BRD4 playing a critical role in their enhancer functions. KSHV latent mini-chromosome also contains an enhancer domain made with multiple copies of 801 bp identical repeat DNA fragments, terminal repeats. Here, we utilized manipulable mini-scale chromatins with convenient inducible KSHV reactivation to systematically examine the association between enhancer strength and the outcome of inducible promoter activation. This study illustrated the amount of BRD4 recruitment at the enhancer associated with frequencies of BRD4 distribution to the inducible promoters during KSHV reactivation and, therefore, KSHV lytic replication. Recruitment of BRD4 to the TR is specifically regulated by KSHV latent protein, LANA. KSHV evolves clever enhancer elements designed to be regulated by the KSHV own latent protein, LANA.

Abnormal metabolism is a characteristic of malignant tumors. Numerous factors play roles in the regulation of tumor metabolism. As epigenetic regulators, enhancers, especially the super-enhancers (SEs), serve as platforms for transcription factors that regulate the expression of metabolism-related enzymes or transporters at the gene level. In this study, we review the effects of enhancer/ SE-driven genes on tumor metabolism and immunity. Enhancers/SEs play regulatory roles in glucose metabolism (glycolysis, gluconeogenesis, tricarboxylic acid (TCA) cycle, pyruvate, and pentose phosphate pathway, lipid metabolism (cholesterol, fatty acid, phosphatide, and sphingolipid), and amino acid metabolism (glutamine, tryptophan, arginine, and cystine). By regulating tumor metabolism, enhancers and SEs can reprogram tumor microenvironment, especially the status of various immune cells. Therefore, interfering enhancers/SEs that regulate the tumor metabolism is likely to enhance the effectiveness of immunotherapy.

Identification of metastasis drivers of triple-negative breast cancer (TNBC) is a multifaceted challenge. Here, we identified TATA-box binding protein associated factor 7 (TAF7) as a candidate to modulate TNBC metastasis. TAF7 exhibited high expression in metastatic TNBC patients, and its elevated expression showed a negative correlation with overall survival in TNBC patients. The knockdown of TAF7 suppressed the migration and invasion of TNBC, suggesting TAF7 plays a role in the metastatic processes. Further, TAF7 was enhancing serum amyloid A1 (SAA1) transcription by binding to a specific motif in the SAA1 gene promoter. The elevated SAA1 in TNBC cells directly increased E-cadherin and N-cadherin phosphorylation thereby regulating cell adhesion. Mechanistically, TAF7 modulated cell invasion, migration, and lung metastasis through an SAA1-dependent manner in vitro and in vivo experiments. Taken together, it is likely that TAF7 could directly act on the SAA1 gene promoter, upregulating SAA1 and consequently promoting TNBC metastasis.

Effective transcriptional activation relies on the spatial interaction between specific DNA elements. DNA interactions have also been observed between DNA viruses and their hosts, with limited understanding of the involved details. Baculovirus is a representative species of DNA virus and has been reported to interact with the host genome in our previous study. However, the biological significance of the baculovirus-host trans-species DNA interaction and its underlying mechanisms remain elusive. Here, using Bombyx mori nucleopolyhedrovirus (BmNPV) as the model virus, we combine epigenome, transcriptome, and biochemical assays to investigate the baculovirus-host DNA interaction. Our data show that BmNPV hijacks the transcriptional regulatory capacity of host super-enhancers (SEs) by physically interacting with these regions on the host genome. This results in the usurpation of the activating capacity of an SE-binding transcription factor GATA by the virus, thereby impairing the SE-induced specific transcriptional activation of the target antiviral genes. Moreover, the hijacked regulatory capacity is spread on BmNPV genome through cis-interaction of viral DNA, leading to enhanced viral gene expression. Overall, our results provide novel insights into the intricate interplay of viruses with host gene expression regulatory networks and broaden the vision in the mechanisms of viral exploitation on cellular machinery.

Human health depends on perplexing defensive cellular responses against microbial pathogens like Viruses. Despite the major effort undertaken, the (epi)genomic mechanisms that human cells utilize to tailor defensive gene expression programs against microbial attacks have remained inadequately understood, mainly due to a significant lack of recording of the in vivo functional cis-regulatory modules (CRMs) of the human genome. Here, we introduce the virus-responsive fate of the human (epi)genome as characterized in naïve and infected cells by functional genomics, computational biology, DNA evolution, and DNA Grammar and Syntax investigations. We discovered that multitudes of novel functional virus-responsive CRMs (vrCRMs) compose typical enhancers (tEs), super-enhancers (SEs), repetitive-DNA enhancers (rDEs), and stand-alone functional genomic stretches that grant human cells regulatory underpinnings for layering basal immunity and eliminating illogical/harmful defensive responses under homeostasis, yet stimulating virus-responsive genes and transposable elements (TEs) upon infection. Moreover, extensive epigenomic reprogramming of previously unknown SE landscapes marks the transition from naïve to antiviral human cell states and involves the functions of the antimicrobial transcription factors (TFs), including interferon response factor 3 (IRF3) and nuclear factor-κB (NF-κB), as well as coactivators and transcriptional apparatus, along with intensive modifications/alterations in histone marks and chromatin accessibility. Considering the polyphyletic evolutionary fingerprints of the composite DNA sequences of the vrCRMs assessed by TFs-STARR-seq, ranging from the animal to microbial kingdoms, the conserved features of antimicrobial TFs and chromatin complexes, and their pluripotent stimulus-induced activation, these findings shed light on how mammalian (epi)genomes evolved their functions to interpret the exogenous stress inflicted and program defensive transcriptional responses against microbial agents. Crucially, many known human short variants, e.g. single-nucleotide polymorphisms (SNPs), insertions, deletions etc., and quantitative trait loci (QTLs) linked to autoimmune diseases, such as multiple sclerosis (MS), systemic lupus erythematosus (SLE), Crohn's disease (CD) etc., were mapped within or vastly proximal (±2.5 kb) to the novel in vivo functional SEs and vrCRMs discovered, thus underscoring the impact of their (mal)functions on human physiology and disease development. Hence, we delved into the virus-responsive fate of the human (epi)genome and illuminated its architecture, function, evolutionary origins, and its significance for cellular homeostasis. These results allow us to chart the "Human hyper-Atlas of virus-infection", an integrated "molecular in silico" encyclopedia situated in the UCSC Genome Browser that benefits our mechanistic understanding of human infectious/(auto)immune diseases development and can facilitate the generation of in vivo preclinical animal models, drug design, and evolution of therapeutic applications.

Ovarian cancer is a gynecological malignancy with the highest recurrence and mortality rates. Although niraparib can effectively affect its progression, the challenge of drug resistance remains. Herein, niraparib-resistant ovarian cancer cell lines were constructed to identify the abnormally activated enhancers and associated target genes via RNA in situ conformation sequencing. Notably, the target gene RAD23A was markedly upregulated in niraparib-resistant cells, and inhibiting RAD23A restored their sensitivity. Additionally, abnormal activation of glycolysis in niraparib-resistant cells induced lactate accumulation, which promoted the lactylation of histone H4K12 lysine residues. Correlation analysis showed that key glycolysis enzymes such as pyruvate kinase M and lactate dehydrogenase A were significantly positively correlated with RAD23A expression in ovarian cancer. Additionally, H4K12la activated the super-enhancer (SE) of niraparib and RAD23A expression via MYC transcription factor, thereby enhancing the DNA damage repair ability and promoting the drug resistance of ovarian cancer cells. Overall, the findings of this study indicate that lactic acid accumulation leads to lactylation of histone H4K12la, thereby upregulating SE-mediated abnormal RAD23A expression and promoting niraparib resistance in ovarian cancer cells, suggesting RAD23A as a potential therapeutic target for niraparib-resistant ovarian cancer.

Tumorigenic drivers of MYCN gene nonamplified neuroblastoma remain largely uncharacterized. Long noncoding RNAs (lncRNAs) regulate tumorigenesis, however, there is little literature on therapeutic targeting of lncRNAs with small molecule compounds. Here PRKCQ-AS1 is identified as the lncRNA most overexpressed in MYCN nonamplified, compared with MYCN-amplified, neuroblastoma cell lines. PRKCQ-AS1 expression is controlled by super-enhancers, and PRKCQ-AS1 RNA bound to MSI2 protein. RNA immunoprecipitation and sequencing identified BMX mRNA as the transcript most significantly disrupted from binding to MSI2 protein, after PRKCQ-AS1 knockdown. PRKCQ-AS1 or MSI2 knockdown reduces, while its overexpression enhances, BMX mRNA stability and expression, ERK protein phosphorylation and MYCN nonamplified neuroblastoma cell proliferation. PRKCQ-AS1 knockdown significantly suppresses neuroblastoma progression in mice. In human neuroblastoma tissues, high levels of PRKCQ-AS1 and MSI2 expression correlate with poor patient outcomes, independent of current prognostic markers. AlphaScreen of a compound library identifies NSC617570 as an efficient inhibitor of PRKCQ-AS1 RNA and MSI2 protein interaction, and NSC617570 reduces BMX expression, ERK protein phosphorylation, neuroblastoma cell proliferation in vitro and tumor progression in mice. The study demonstrates that PRKCQ-AS1 RNA interacts with MSI2 protein to induce neuroblastoma tumorigenesis, and that targeting PRKCQ-AS1 and MSI2 interaction with small molecule compounds is an effective anticancer strategy.

Developing cancer therapies that induce robust death of the malignant cell is critical to prevent relapse. Highly effective strategies, such as immunotherapy, exemplify this observation. Here we provide the structural and molecular underpinnings for an approach that leverages chemical induced proximity to produce specific cell killing of diffuse large B cell lymphoma, the most common non-Hodgkin's lymphoma. We develop KAT-TCIPs (lysine acetyltransferase transcriptional/epigenetic chemical inducers of proximity) that redirect p300 and CBP to activate programmed cell death genes normally repressed by the oncogenic driver, BCL6. Acute treatment rapidly reprograms the epigenome to initiate apoptosis and repress c-MYC. The crystal structure of the chemically induced p300-BCL6 complex reveals how chance interactions between the two proteins can be systematically exploited to produce the exquisite potency and selectivity of KAT-TCIPs. Thus, the malignant function of an oncogenic driver can be co-opted to activate robust cell death, with implications for precision epigenetic therapies.

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.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest solid cancers; thus, identifying more effective therapies is a major unmet need. In this study, we characterized the super-enhancer (SE) landscape of human PDAC to identify drivers of the disease that might be targetable. This analysis revealed MICAL2 as an SE-associated gene in human PDAC, which encodes the flavin monooxygenase enzyme that induces actin depolymerization and indirectly promotes serum response factor transcription by modulating the availability of serum response factor coactivators such as myocardin-related transcription factors (MRTF-A and MRTF-B). MICAL2 was overexpressed in PDAC, and high-MICAL2 expression correlated with poor patient prognosis. Transcriptional analysis revealed that MICAL2 upregulates KRAS and epithelial-mesenchymal transition signaling pathways, contributing to tumor growth and metastasis. In loss- and gain-of-function experiments in human and mouse PDAC cells, MICAL2 promoted both ERK1/2 and AKT activation. Consistent with its role in actin depolymerization and KRAS signaling, loss of MICAL2 also inhibited macropinocytosis. MICAL2, MRTF-A, and MRTF-B influenced PDAC cell proliferation and migration and promoted cell-cycle progression in vitro. Importantly, MICAL2 supported in vivo tumor growth and metastasis. Interestingly, MRTF-B, but not MRTF-A, phenocopied MICAL2-driven phenotypes in vivo. This study highlights the multiple ways in which MICAL2 affects PDAC biology and provides a foundation for future investigations into the potential of targeting MICAL2 for therapeutic intervention. Significance: Characterization of the epigenomic landscape of pancreatic cancer to identify early drivers of tumorigenesis uncovered MICAL2 as a super-enhancer-associated gene critical for tumor progression that represents a potential pharmacologic target.

Free fatty acids like palmitic acid (PA) are elevated in obesity and diabetes and dysregulate monocyte and macrophage functions, contributing to enhanced inflammation in these cardiometabolic diseases. Epigenetic mechanisms regulating enhancer functions play key roles in inflammatory gene expression, but their role in PA-induced monocyte/macrophage dysfunction is unknown. We found that PA treatment altered the epigenetic landscape of enhancers and super-enhancers (SEs) in human monocytes. Integration with RNA-seq data revealed that PA-induced enhancers/SEs correlated with PA-increased expression of inflammatory and immune response genes, while PA-inhibited enhancers correlated with downregulation of phagocytosis and efferocytosis genes. These genes were similarly regulated in macrophages from mouse models of diabetes and accelerated atherosclerosis, human atherosclerosis, and infectious agents. PA-regulated enhancers/SEs harbored SNPs associated with diabetes, obesity, and body mass index indicating disease relevance. We verified increased chromatin interactions between PA-regulated enhancers/SEs and inflammatory gene promoters and reduced interactions at efferocytosis genes. PA-induced gene expression was reduced by inhibitors of BRD4, and NF-κB. PA treatment inhibited phagocytosis and efferocytosis in human macrophages. Together, our findings demonstrate that PA-induced enhancer dynamics at key monocyte/macrophage enhancers/SEs regulate inflammatory and immune genes and responses. Targeting these PA-regulated epigenetic changes could provide novel therapeutic opportunities for cardiometabolic disorders.

Ischemic stroke is a leading cause of disability and mortality, imposing substantial physical, emotional, and economic burdens on patients and society. This study aimed to explore the regulatory effects of super-enhancers (SEs) on gene expression in the context of ischemic stroke and their potential transcriptional regulatory mechanisms.

Super-enhancers were identified via H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) and ROSE software. RNA-sequencing (RNA-seq) was employed to screen for differentially expressed genes. A comparative analysis of ChIP-seq and RNA-seq data initially identified SE target genes, followed by further screening of key core differentially expressed SE target genes via the random forest method. The identified core SE target genes were initially validated through immunofluorescence and immunoblotting techniques. Additionally, potential core transcriptional regulatory circuits were preliminarily screened via the Coltron algorithm.

We identified SE-associated genes in the ischemic stroke model and electroacupuncture-treated groups, revealing 41 genes uniquely regulated by SEs in the electroacupuncture group compared with 367 in the model group. Enrichment analyses revealed that pathways involved in axon guidance, regulation of lipolysis in adipocytes and sphingolipid signaling pathway were significantly enriched in the SE target genes, suggesting that these pathways may be involved in the therapeutic effects of electroacupuncture. Notably, HDAC7 emerged as a key SE-driven gene; its expression was significantly reduced following electroacupuncture treatment, indicating its potential as a therapeutic target. Protein expression analyses confirmed elevated levels of HDAC7 in the model group, which were reduced by electroacupuncture intervention (p < 0.05). Furthermore, core transcriptional regulatory circuitries involving SOX8, FOXK1, and KLF13 were identified, highlighting their roles in the modulation of SE-mediated gene regulation by acupuncture in the ischemic stroke context.

Overall, our findings provide novel insights into the molecular mechanisms by which acupuncture may treat ischemic stroke, identifying key SE target genes and transcriptional circuits as promising targets for future therapeutic strategies. Further research is warranted to validate these findings in clinical settings and explore the translational potential of acupuncture in ischemic stroke treatment.

Spermatogenesis is a unidirectional differentiation process that generates haploid sperm, but how the gene expression program that directs this process is established is largely unknown. Here we determine the high-resolution three-dimensional (3D) chromatin architecture of mouse male germ cells during spermatogenesis and show that CTCF-mediated 3D chromatin dictates the gene expression program required for spermatogenesis. In undifferentiated spermatogonia, CTCF-mediated chromatin interactions between meiosis-specific super-enhancers (SEs) and their target genes precede activation of these SEs on autosomes. These meiotic SEs recruit the master transcription factor A-MYB (MYBL1) in meiotic spermatocytes, which strengthens their 3D contacts and instructs a burst of meiotic gene expression. We also find that at the mitosis-to-meiosis transition, the germline-specific Polycomb protein SCML2 facilitates the resolution of chromatin loops that are specific to mitotic spermatogonia. Moreover, SCML2 and A-MYB help shape the unique 3D chromatin organization of sex chromosomes during meiotic sex chromosome inactivation. We propose that CTCF-mediated 3D chromatin organization regulates epigenetic priming that directs unidirectional differentiation, thereby determining the cellular identity of the male germline.

p300 and CBP are paralogous epigenetic regulators that are considered promising therapeutic targets for cancer treatment. Small molecule p300/CBP inhibitors have so far been unable to differentiate between these closely related proteins, yet selectivity is desirable in order to probe their distinct cellular functions. Additionally, in multiple cancers, loss-of-function CREBBP mutations set up a paralog dependent synthetic lethality with p300, that could be exploited with a selective therapeutic agent. To address this, we developed p300-targeting heterobifunctional degraders that recruit p300 through its HAT domain using the potent spiro-hydantoin-based inhibitor, iP300w. Lead degrader, BT-O2C, demonstrates improved selectivity and a faster onset of action compared to a recently disclosed A 485-based degrader in HAP1 cells and is cytotoxic in CIC::DUX4 sarcoma (CDS) cell lines (IC50 = 152-221 nM), significantly reducing expression of CDS target genes (ETV1, ETV4, ETV5). Taken together, our results demonstrate that BT-O2C represents a useful tool degrader for further exploration of p300 degradation as a therapeutic strategy.

Transcriptional dysregulation is a hallmark of cancer initiation and progression, driven by genetic and epigenetic alterations. Enhancer reprogramming has emerged as a pivotal driver of carcinogenesis, with cancer cells often relying on aberrant transcriptional programs. The advent of high-throughput sequencing technologies has provided critical insights into enhancer reprogramming events and their role in malignancy. While targeting enhancers presents a promising therapeutic strategy, significant challenges remain. These include the off-target effects of enhancer-targeting technologies, the complexity and redundancy of enhancer networks, and the dynamic nature of enhancer reprogramming, which may contribute to therapeutic resistance. This review comprehensively encapsulates the structural attributes of enhancers, delineates the mechanisms underlying their dysregulation in malignant transformation, and evaluates the therapeutic opportunities and limitations associated with targeting enhancers in cancer.

Glioblastoma is the most common primary malignant brain tumor in adults, with a median survival of just over 1 year. The failure of available treatments to achieve remission in patients with glioblastoma (GBM) has been attributed to the presence of cancer stem cells (CSCs), which are thought to play a central role in tumor development and progression and serve as a treatment-resistant cell repository capable of driving tumor recurrence. In fact, the property of "stemness" itself may be responsible for treatment resistance. In this study, we identify a novel long noncoding RNA (lncRNA), cancer stem cell-associated distal enhancer of SOX2 (CASCADES), that functions as an epigenetic regulator in glioma CSCs (GSCs). CASCADES is expressed in isocitrate dehydrogenase (IDH)-wild-type GBM and is significantly enriched in GSCs. Knockdown of CASCADES in GSCs results in differentiation towards a neuronal lineage in a cell- and cancer-specific manner. Bioinformatics analysis reveals that CASCADES functions as a super-enhancer-associated lncRNA epigenetic regulator of SOX2. Our findings identify CASCADES as a critical regulator of stemness in GSCs that represents a novel epigenetic and therapeutic target for disrupting the CSC compartment in glioblastoma.

The spatial conformation of chromosomes and genomes of single cells is relevant to cellular function and useful for elucidating the mechanism underlying gene expression and genome methylation. The chromosomal contacts (i.e. chromosomal regions in spatial proximity) entailing the three-dimensional (3D) structure of the genome of a single cell can be obtained by single-cell chromosome conformation capture techniques, such as single-cell Hi-C (ScHi-C). However, due to the sparsity of chromosomal contacts in ScHi-C data, it is still challenging for traditional 3D conformation optimization methods to reconstruct the 3D chromosome structures from ScHi-C data. Here, we present a machine learning-based method based on a novel SO(3)-equivariant graph neural network (HiCEGNN) to reconstruct 3D structures of chromosomes of single cells from ScHi-C data. HiCEGNN consistently outperforms both the traditional optimization methods and the only other deep learning method across diverse cells, different structural resolutions, and different noise levels of the data. Moreover, HiCEGNN is robust against the noise in the ScHi-C data.

3D organization of the genome plays a critical role in regulating gene expression. How 3D-genome organization differs among different cell types and relates to cell type-dependent transcriptional regulation remains unclear. Here, we used genome-scale DNA and RNA imaging to investigate 3D-genome organization in transcriptionally distinct cell types in the mouse cerebral cortex. We uncovered a wide spectrum of differences in the nuclear architecture and 3D-genome organization among different cell types, ranging from the size of the cell nucleus to higher-order chromosome structures and radial positioning of chromatin loci within the nucleus. These cell type-dependent variations in nuclear architecture and chromatin organization exhibit strong correlations with both the total transcriptional activity of the cell and transcriptional regulation of cell type-specific marker genes. Moreover, we found that the methylated DNA binding protein MeCP2 promotes active-inactive chromatin segregation and regulates transcription in a nuclear radial position-dependent manner that is highly correlated with its function in modulating active-inactive chromatin compartmentalization.

Rhabdomyosarcoma (RMS) is a common soft tissue sarcoma primarily affecting children and young adults. This disease is more prevalent in children under 15, with two main types: embryonal Rhabdomyosarcoma (eRMS), which has a better prognosis, and alveolar Rhabdomyosarcoma (aRMS), which is more aggressive and associated with specific genetic alterations. The PI3K-Akt-mTOR pathway is often hyperactivated in RMS, contributing to cell proliferation, survival, and resistance to therapies. The presence of phosphorylated components of this pathway correlates with poor survival outcomes. Here, we discuss various therapeutic approaches targeting the PI3K-Akt-mTOR pathway. These include the use of specific inhibitors (e.g., PI3K inhibitors, Akt inhibitors) and combination therapies that may enhance treatment efficacy. Dietary supplements like curcumin and repurposed drugs such as chloroquine are also mentioned for their potential to induce apoptosis in RMS cells. We also emphasize the need for innovative strategies to improve survival rates, which have remained stagnant over the years. Targeting super-enhancers and transcription factors associated with RMS may provide new therapeutic avenues. Overall, this review underscores the critical role of the PI3K-Akt-mTOR pathway in RMS and the potential for targeted therapies to improve patient outcomes.

Small cell carcinoma of the ovary-hypercalcemic type (SCCOHT) is a rare ovarian cancer affecting young females and is driven by the loss of both SWI/SNF ATPases SMARCA4 and SMARCA2. As loss of SWI/SNF alters enhancers, we hypothesized that super-enhancers, which regulate oncogene expression in cancer, are disparately impacted by SWI/SNF loss. We discovered differences between SWI/SNF occupancy at enhancers vs. super-enhancers. SCCOHT super-enhancer target genes were enriched in developmental processes, most notably nervous system development. This may further support neuronal cell-of-origin previously proposed. We found high sensitivity of SCCOHT cell lines to triptolide. Triptolide inhibits expression of many super-enhancer-associated genes, including oncogenes. SALL4 expression is decreased by triptolide and is highly expressed in SCCOHT tumors. In patient-derived xenograft models, triptolide and prodrug minnelide effectively inhibit tumor growth. These results reveal unique features of super-enhancers in SCCOHT, which may be one mechanism through which triptolide has high activity in these tumors.

Brain injuries can result from accidents, warfare, sports injuries, or brain diseases. Identifying regeneration-associated genes (RAGs) during epigenome remodeling upon brain injury could have a significant impact on reducing neuronal death and subsequent neurodegeneration for patients with brain injury. We previously identified several WNT genes as RAGs involved in the neurite regrowth of injured cortical neurons. Among them, the expression of the Wnt8a gene increased most significantly during neurite regrowth, indicating its potential to promote neuronal regeneration. In this study, we investigated the regulatory mechanism of Wnt8a transcription. An algorithm was developed to predict the novel enhancer regions of candidate genes. By combining active enhancer marks, histone H3 lysine 27 acetylation (H3K27ac), and histone H3 lysine 4 mono-methylation (H3K4me1), we identified a candidate enhancer region for Wnt8a located 1.7 Mb upstream and 0.1 Mb downstream of the Wnt8a gene. This region was organized into enhancers (Ens) 1-15. Enhancer RNA expression from the predicted En1-15 regions, DNA topological dynamics, and the activity of predicted enhancers were analyzed to validate the candidate active enhancers. Our findings showed that the En8, 9, 10, 14, and 15 regions expressed higher eRNAs during neurite regrowth. Notably, the En8-2 and En14-2 subregions showed significantly up-regulated H3K4me1 modification during neurite regrowth. Using chromatin conformation capture assays and enhancer-reporter assays, we delineated that the molecular regulation of Wnt8a transcription during neurite regrowth occurs through looped En8-promoter interplay.

Breast cancer (BC) is the most common malignancy with a poor prognosis. Radiotherapy is one of the leading traditional treatments for BC. However, radiotherapy-associated secondary diseases are severe issues for the treatment of BC. The present study integrated multi-omics data to investigate the molecular and epigenetic mechanisms involved in post-radiation BC. The differences in the expression of radiation-associated genes between post-radiation and pre-radiation BC samples were determined. Enrichment analysis revealed that these radiation-associated genes involved diverse biological functions and pathways in BC. Combining epigenetic data, we identified radiation-associated genes whose transcriptional changes might be associated with aberrant methylation. Then, we identified potential therapeutic targets and chemical drugs for post-radiation BC patient treatment by constructing a drug-target association network. Specifically, four radiation-associated genes (CD248, CCDC80, GADD45B, and MMP2) whose increased expression might be regulated by hypomethylation of the corresponding enhancer region were found to have excellent diagnostic effects and clinical prognostic value. Finally, we further used independent samples to verify CD248 expression and established a simple epigenetic regulatory model. In summary, this study provides novel insights for understanding the regulation of target genes mediated by DNA methylation and developing potential biomarkers for radiation-associated secondary diseases in BC.

Cis-regulatory elements (CREs), such as enhancers and promoters, are fundamental regulators of gene expression and, across different cell types, the MYC locus utilizes a diverse regulatory architecture driven by multiple CREs. To better understand differences in CRE function, we perform pooled CRISPR inhibition (CRISPRi) screens to comprehensively probe the 2.8 Mb topologically-associated domain containing MYC in 6 human cancer cell lines with nucleotide resolution. We map 32 CREs where inhibition leads to changes in cell growth, including 8 that overlap previously identified enhancers. Targeting specific CREs decreases MYC expression by as much as 60%, and cell growth by as much as 50%. Using 3-D enhancer contact mapping, we find that these CREs almost always contact MYC but less than 10% of total MYC contacts impact growth when silenced, highlighting the utility of our approach to identify phenotypically-relevant CREs. We also detect an enrichment of lineage-specific transcription factors (TFs) at MYC CREs and, for some of these TFs, find a strong, tumor-specific correlation between TF and MYC expression not found in normal tissue. Taken together, these CREs represent systematically identified, functional regulatory regions and demonstrate how the same region of the human genome can give rise to complex, tissue-specific gene regulation.

Protein intrinsically disordered regions (IDRs) are critical gene-regulatory components and aberrant fusions between IDRs and DNA-binding/chromatin-associating domains cause diverse human cancers. Despite this importance, how IDRs influence gene expression, and how aberrant IDR fusion proteins provoke oncogenesis, remains incompletely understood. Here we develop a series of synthetic dCas9-IDR fusions to establish that locus-specific recruitment of IDRs can be sufficient to stimulate endogenous gene expression. Using dCas9 fused to the paradigmatic leukemogenic NUP98 IDR, we also demonstrate that IDRs can activate transcription via localized biomolecular condensation and in a manner that is dependent upon overall IDR concentration, local binding density, and amino acid composition. To better clarify the oncogenic role of IDRs, we construct clinically observed NUP98 IDR fusions and show that, while generally non-overlapping, oncogenic NUP98-IDR fusions convergently drive a core leukemogenic gene expression program in donor-derived human hematopoietic stem cells. Interestingly, we find that this leukemic program arises through differing mechanistic routes based upon IDR fusion partner; either distributed intragenic binding and intrachromosomal looping, or dense binding at promoters. Altogether, our studies clarify the gene-regulatory roles of IDRs and, for the NUP98 IDR, connect this capacity to pathological cellular programs, creating potential opportunities for generalized and mechanistically tailored therapies.