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.

Multiple myeloma (MM) is a B-cell malignancy accounting for 20 % of all blood-associated cancers. MM patients with a poorer prognosis and high-risk stratification were previously observed to be causally linked to the constitutive activation of non-canonical NF-κB (ncNF-κB) pathway. Consistent with this, the ncNF-κB p52 transcription factor was earlier found to regulate the enhancer landscape of MM to potentiate oncogenic transcription. However, the mechanism by which aberrant p52 expression is involved in coordinating enhancer activity has not been well explored. In this study, we analysed H3K27ac ChIP-seq and ATAC-seq data from MM cell lines and patient samples to screen for putative transcription factors that cooperate with p52 to regulate enhancers activated in MM. We report that ATF4 interacts with p52 and together, this complex mediates the activity of a subset of MM-associated enhancers through the recruitment of histone acetyltransferases (HATs), p300 and CBP (CREB-binding protein). We also identified a ATF4:p52 regulated target gene BACH1 under the regulation of a proximal super-enhancer, which was found to drive oncogenesis in MM by promoting cell cycle progression and proliferation. Together, our findings provide further mechanistic insights into how aberrant enhancer activation observed in MM tumours could lead to disease progression.

Lung cancer remains one of the most prevalent malignancies worldwide. This study investigates the role of histone deacetylase 4 (HDAC4) in mediating chemoresistance in lung adenocarcinoma (LUAD). Super-enhancers (SEs), known to regulate aberrant gene expression, are critical drivers of tumor progression. We identified a specific super-enhancer region associated with HDAC4, referred to as HDAC4-SE. Among its nearby genes, TWIST2 emerged as a key player, strongly linked to chemoresistance and the epithelial-to-mesenchymal transition (EMT). We demonstrated that HDAC4-SE regulates TWIST2 expression, thereby contributing to chemoresistance in LUAD. Through bioinformatics analysis, we identified transcription factors binding to both the promoter of TWIST2 and the activation region of HDAC4-SE, with CCAAT/enhancer-binding protein beta (CEBPB) identified as a central regulator. Chromatin immunoprecipitation (ChIP) assays confirmed that CEBPB binds to both the HDAC4-SE and the TWIST2 promoter. Additionally, our investigation into the involvement of long non-coding RNAs (lncRNAs) revealed that LINC01940 might mediate the regulatory effects of HDAC4-SE on downstream genes. In conclusion, we uncovered a novel HDAC4-SE/LINC01940/CEBPB/TWIST2 signaling pathway that drives chemoresistance and tumor progression in LUAD. This pathway offers promising insights into potential therapeutic targets to overcome chemoresistance in lung cancer.

Gene-environment interactions are pivotal contributors to nitrosamine-induced esophageal carcinogenesis. While genetic mechanisms in esophageal carcinoma (ESCA) are well-defined, epigenetic drivers remain elusive. This study identifies a novel mechanism of epigenetic regulation centered on B-cell lymphoma-2-associated transcription factor 1 (Bclaf1) in nitrosamine-induced (Methylnitronitrosoguanidine, MNNG) esophageal carcinogenesis. In nitrosamine-induced malignant transformation cells (MNNG-M), Bclaf1 expression is progressively increased with malignancy, and elevated Bclaf1 levels are correlated with poor prognosis in ESCA patients. Functionally, Bclaf1 significantly promotes the abnormal proliferation of MNNG-M and ESCA cells in vitro and in vivo. Mechanistically, transposase-accessible chromatin sequencing (ATAC-seq) results suggest that Bclaf1 silencing markedly reduces chromatin accessibility, thereby impairing the synthesis of newly transcribed RNA. Bclaf1 activates RNA polymerase II subunit POLR2A to promote chromatin accessibility through distinct transcriptional and splicing mechanisms. More specifically, cleavage under targets and tagmentation (CUT&Tag) assays revealed Bclaf1/P300/H3K27ac co-recruitment at the POLR2A promoter, driving transcription via the E2/E3 elements of the POLR2A super-enhancer. Additionally, RNA-binding protein immunoprecipitation (RIP) assays demonstrated that the Bclaf1 cofactor, small nuclear ribonucleoprotein polypeptide A (SNRPA), interacts with pre-POLR2A to regulate its splicing. Collectively, our study reveals that Bclaf1 facilitates nitrosamine-induced ESCA by controlling POLR2A transcriptional and splicing activities, providing novel insight for early detection and intervention.

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.

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.

JMJD1C (Jumonji Domain Containing 1C), a member of the lysine demethylase 3 (KDM3) family, is universally required for the survival of several types of acute myeloid leukemia (AML) cells with different genetic mutations, representing a therapeutic opportunity with broad application. Yet how JMJD1C regulates the leukemic programs of various AML cells is largely unexplored. Here we show that JMJD1C interacts with the master hematopoietic transcription factor RUNX1, which thereby recruits JMJD1C to the genome to facilitate a RUNX1-driven transcriptional program that supports leukemic cell survival. The underlying mechanism hinges on the long N-terminal disordered region of JMJD1C, which harbors two inseparable abilities: condensate formation and direct interaction with RUNX1. This dual capability of JMJD1C may influence enhancer-promoter contacts crucial for the expression of key leukemic genes regulated by RUNX1. Our findings demonstrate a previously unappreciated role for the non-catalytic function of JMJD1C in transcriptional regulation, underlying a mechanism shared by different types of leukemias.

Pulmonary artery endothelial cell (PAEC) dysfunction is a pathological hallmark of pulmonary hypertension (PH). Yet, the roles of long noncoding RNAs (lncRNAs) driven by super-enhancers (SEs) in PAECs are not well understood. In this study, we focused on the PAEC-specific SE-associated lncRNA HCG20 (HLA complex group 20) and to elucidate its role and underlying mechanisms in the progression of PH.

ChIP-qPCR, chromosome conformation capture followed by PCR, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9), and dual-luciferase reporter assays were used to identify dysregulated SE-associated lncRNAs in PAECs and to investigate the pathological role of HCG20. The role of HCG20 in pathological processes was validated in rodent models of PH induced by SU5416/hypoxia, monocrotaline, or hypoxia alone, through adeno-associated virus-mediated endothelial-specific HCG20 overexpression or knockdown of HCG20. RNA pull-down, mass spectrometry, RNA immunoprecipitation, and RNA sequencing were used to elucidate the underlying mechanisms of HCG20-mediated PAEC dysfunction.

We identified the SE-associated lncRNA HCG20 from histone H3 lysine-27 acetylation (H3K27ac) and histone H3 lysine-4 monomethylation (H3K4me1) ChIP-seq data derived from PAECs of patients with PH. A significant upregulation of HCG20 was found in hypoxia-induced human PAECs, lung tissues, and the plasma of patients with PH. Antisense oligonucleotide and CRISPR/Cas9, which, respectively, target HCG20 and its SE, alleviate hypoxia-induced pyroptosis and subsequent endothelial-to-mesenchymal transition. Human pulmonary artery smooth muscle cells internalize human PAEC-derived exosomes containing HCG20, inducing their excessive proliferation. Targeted delivery of HCG20 into the pulmonary vascular endothelium induced pulmonary vasculature remodeling and increased pulmonary artery systolic blood pressure in rodents. Mechanistically, HCG20 directly bound and stabilized the U2AF2 (U2 small nuclear RNA auxiliary factor 2) protein, thereby facilitating its impact on the alternative splicing of EIF2AK2 (eukaryotic translation initiation factor 2 alpha kinase 2). Furthermore, we identified a novel mouse ortholog gene, 4833427F10Rik (named Hcg20), of HCG20 for the first time. Our study demonstrated that specific interference with Hcg20 in the pulmonary vascular intima has been shown to ameliorate hypoxia-induced PH.

Collectively, our data suggest that HCG20, driven by SE, contributes to PAEC dysfunction through U2AF2-mediated alternative splicing of EIF2AK2. Our work underscores the potential of using HCG20 as a novel biomarker and a promising target for the treatment of PH.

Deciphering cell identity genes is pivotal to understanding cell differentiation, development, and cell identity dysregulation involving diseases. Here, we introduce SCIG, a machine-learning method to uncover cell identity genes in single cells. In alignment with recent reports that cell identity genes (CIGs) are regulated with unique epigenetic signatures, we found CIGs exhibit distinctive genetic sequence signatures, e.g. unique enrichment patterns of cis-regulatory elements. Using these genetic sequence signatures, along with gene expression information from single-cell RNA-seq data, SCIG uncovers the identity genes of a cell without a need for comparison to other cells. CIG score defined by SCIG surpassed expression value in network analysis to reveal the master transcription factors (TFs) regulating cell identity. Applying SCIG to the human endothelial cell atlas revealed that the tissue microenvironment is a critical supplement to master TFs for cell identity refinement. SCIG is publicly available at https://doi.org/10.5281/zenodo.14726426  , offering a valuable tool for advancing cell differentiation, development, and regenerative medicine research.

HBV integration is considered as the main contributor to hepatocellular carcinoma (HCC). However, whether HBV integrated sequences determine genotype pathogenicity and how to block their function during HCC progression remains unclear.

An in vitro HBV-infected PHH model and liver cancer cell lines were established to confirm the pathogenic potential of HBV-SITEs. The roles of HBV-SITE-1 in HCC development were analyzed using cellular phenotypic assays and molecular biology techniques, including the combined analysis of RNA-seq and ChIP-seq. Animal models were also used to evaluate the therapeutic effect of HBV-miR-2 inhibitors.

We identified nine fragments of HBV Sequences Integrated To Enhancer, termed as "HBV-SITEs". Particularly, a single nucleotide variation (T > G) was embedded at seed sequence of HBV-miR-2 in the highest integrated HBV-SITE-1 between genotypes B and H. Unexpectedly, B-HBV-SITE-1, not H-HBV-SITE-1, could abnormally activate oncogenic genes including TERT and accelerate HCC cell proliferation and migration. Meanwhile, HBV-miR-2 was gradually increased in HBV-infected cells and patient plasma with different HCC stages. Importantly, 227 genes upregulated by HBV, were also activated by HBV-miR-2 through triggering HBV-SITE-1 enhancer. Conversely, enhancer activities were particularly decreased by HBV-miR-2 inhibitors, and further downregulated activated oncogenic genes. Finally, HCC growth was dramatically restrained and HBV-induced transcripts were systematically reduced via injection of HBV-miR-2 inhibitors in animal models.

HBV-SITEs were identified as novel oncogenic elements for HCC, which provides an insightful perspective for the other cancers caused by oncogenic DNA viruses. We demonstrated that the integrated HBV sequence itself acted as oncogenic enhancers and nucleotide variations of HBV genotypes account for particular pathogenic progression, supporting that the viral nucleotide sequences are vital pathogenic substances beyond viral proteins. And modulation of their enhancer activities could be clinically achievable strategy for blocking DNA viruses-related cancer progression in the future.

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.

Lipid storage and epigenetic dysregulation are key features for clear cell renal carcinoma (ccRCC). However, the interplay between fatty acid metabolism and epigenetics in ccRCC remains to be further demonstrated. Here, the landscape of active enhancers is profiled in paired ccRCC samples and identifies 10171 gain variant enhancer loci (VELs) in the tumor tissues. Experimental validation reveals the enhancers targeting FABP5, FABP6, LPCAT1, MET, SEMA5B, SH3GL1, SNX33, and RHBDF2 are oncogenic. Further studies in organoids and animal models prove FABP5 as an oncogene. HIF-2α and ZNF692 transcription factors regulate FABP5 expression through directly binding to its promoter and enhancer. FABP5 is essential for the lipid droplet formation driven by HIFs and critical for H3K27ac and enhancer activity in ccRCC cells. Thus, the study has identified potential targets for drug design and diagnosis and discovered the function of a feedback loop between epigenetics and lipid metabolism regulated by FABP5 in ccRCC.

Biomolecular condensates formed via liquid-liquid phase separation are ubiquitous in cells, especially in the nucleus. While condensates containing one or two kinds of biomolecules have been relatively well characterized, those with more heterogeneous biomolecular components and interactions between biomolecules inside are largely unknown. This study used residue-resolution molecular dynamics simulations to investigate heterogeneous protein assemblies that include four master transcription factors in mammalian embryonic stem cells: Oct4, Sox2, Klf4, and Nanog. Molecular dynamics simulations of the mixture systems showed highly heterogeneous and dynamic behaviors; protein condensates mainly contain Sox2, Klf4, and Nanog, while most Oct4 are dissolved into the dilute phase. The condensate forms loosely interacting clusters where Klf4 is the most abundant, suggesting that Klf4 serves as a scaffold of the condensate, and Sox2 and Nanog are bound to Klf4 for stabilizing the condensate. Oct4 is moderately recruited to the condensate, serving as a client mainly via its interaction with Sox2. This study highlights the importance of intermolecular interaction between different transcription factors on the condensate formations with heterogeneous biomolecular components.

Enhancers are non-coding DNA regions that facilitate gene transcription, with a specialized subset, super-enhancers, known to exert exceptionally strong transcriptional activation effects. Super-enhancers have been implicated in oncogenesis, and their identification is achievable through histone mark chromatin immunoprecipitation followed by sequencing data using existing analytical tools. However, conventional super-enhancer detection methodologies often do not accurately reflect actual gene expression levels, and the large volume of identified super-enhancers complicates comprehensive analysis. To address these limitations, we developed the super-enhancer to gene links (SE-to-gene Links) analysis, a platform named "SEgene" which incorporates the peak-to-gene links approach-a statistical method designed to reveal correlations between genes and peak regions ( https://github.com/hamamoto-lab/SEgene ). This platform enables a targeted evaluation of super-enhancer regions in relation to gene expression, facilitating the identification of super-enhancers that are functionally linked to transcriptional activity. Here, we demonstrate the application of SE-to-gene Links analysis to public datasets, confirming its efficacy in accurately detecting super-enhancers and identifying functionally associated genes. Additionally, SE-to-gene Links analysis identified ERBB2 as a significant gene of interest in the lung adenocarcinoma dataset from the National Cancer Center Japan cohort, suggesting a potential impact across multiple patient samples. Thus, the SE-to-gene Links analysis provides an analytical tool for evaluating super-enhancers as potential therapeutic targets, supporting the identification of clinically significant super-enhancer regions and their functionally associated genes.

Exhausted CD8 T cells (TEX) arising during chronic infections and cancer have reduced functional capacity and limited fate flexibility that prevents optimal disease control and response to immunotherapies. Compared to memory (TMEM) cells, TEX have a unique open chromatin landscape underlying a distinct gene expression program. How TEX transcriptional and epigenetic landscapes are regulated through histone post-translational modifications (hPTMs) remains unclear. Here, we profiled key activating (H3K27ac and H3K4me3) and repressive (H3K27me3 and H3K9me3) histone modifications in naive CD8 T cells (TN), TMEM and TEX. We identified H3K27ac-associated super-enhancers that distinguish TN, TMEM and TEX, along with key transcription factor networks predicted to regulate these different transcriptional landscapes. Promoters of some key genes were poised in TN, but activated in TMEM or TEX whereas other genes poised in TN were repressed in TMEM or TEX, indicating that both repression and activation of poised genes may enforce these distinct cell states. Moreover, narrow peaks of repressive H3K9me3 were associated with increased gene expression in TEX, suggesting an atypical role for this modification. These data indicate that beyond chromatin accessibility, hPTMs differentially regulate specific gene expression programs of TEX compared to TMEM through both activating and repressive pathways.

Elucidating the regulatory mechanisms underlying the development of different brain regions in humans is essential for understanding advanced cognition and neuropsychiatric disorders. However, the spatiotemporal organization of three-dimensional (3D) chromatin structure and its regulatory functions across different brain regions remain poorly understood. Here, we generated an atlas of high-resolution 3D chromatin structure across six developing human brain regions, including the prefrontal cortex (PFC), primary visual cortex (V1), cerebellum (CB), subcortical corpus striatum (CS), thalamus (TL), and hippocampus (HP), spanning gestational weeks 11-26. We found that the spatial and temporal dynamics of 3D chromatin organization play a key role in regulating brain region development. We also identified H3K27ac-marked super-enhancers as key contributors to shaping brain region-specific 3D chromatin structures and gene expression patterns. Finally, we uncovered hundreds of neuropsychiatric GWAS SNP-linked genes, shedding light on critical molecules in various neuropsychiatric disorders. In summary, our findings provide important insights into the 3D chromatin regulatory mechanisms governing brain region-specific development and can serve as a valuable resource for advancing our understanding of neuropsychiatric disorders.

During chronic infection and tumor progression, CD8+ T cells lose their effector functions and become exhausted. These exhausted CD8+ T cells are heterogeneous and comprised of progenitors that give rise to effector-like or terminally-exhausted cells. The precise cues and mechanisms directing subset formation are incompletely understood. Here, we show that growth factor independent-1 (Gfi1) is dynamically regulated in exhausted CD8+ T cells. During chronic LCMV Clone 13 infection, a previously under-described Ly108+CX3CR1+ subset expresses low levels of Gfi1 while other established subsets have high expression. Ly108+CX3CR1+ cells possess distinct chromatin profiles and represent a transitory subset that develops to effector-like and terminally-exhausted cells, a process dependent on Gfi1. Similarly, Gfi1 in tumor-infiltrating CD8+ T cells is required for the formation of terminally differentiated cells and endogenous as well as anti-CTLA-induced anti-tumor responses. Taken together, Gfi1 is a key regulator of the subset formation of exhausted CD8+ T cells.

The regulation of RNA polymerase II (RNAPII) activity requires orchestrated responses among genomic regulatory sequences and an expansive set of proteins and protein complexes. Despite intense study over five decades, mechanistic insights continue to emerge. Within the past 10 years, live-cell imaging and single-cell transcriptomics experiments have yielded new information about enhancer-promoter communication, transcription factor dynamics, and the kinetics of RNAPII transcription activation. These insights have established RNAPII re-initiation and bursting as a common regulatory phenomenon with widespread implications for gene regulation in health and disease. Here, we summarize regulatory strategies that help control RNAPII bursting in eukaryotic cells, which is defined as short periods of active transcription followed by longer periods of inactivity. We focus on RNAPII re-initiation (i.e., a "burst" of two or more polymerases that initiate from the same promoter), with an emphasis on molecular mechanisms, open questions, and controversies surrounding this distinct regulatory stage.

Triple-negative breast cancer (TNBC) is a malignant type of breast cancer. Owing to the lack of expression of receptors that serve as molecular targets for standard therapy for breast cancer, conventional cytotoxic chemotherapy is the primary treatment option for TNBC. However, TNBC exhibits a high degree of genomic heterogeneity, rendering it resistant to chemotherapy. Therefore, there is an urgent need to identify novel therapeutic targets for the treatment of TNBC. Advances in massively parallel sequencing technology have enabled the identification of cancer cell-specific gene expression patterns and epigenetic alterations that regulate their expression. Cancer cell-specific super-enhancers (SEs) have been identified as effective therapeutic targets for cancer. In this study, we identified the functional roles of epigenetic changes and their regulatory mechanisms in TNBC cells. TNBC cell-specific SEs were formed near several genes that contribute to malignant cancer cell acquisition. We found that the transcription factor OCT-2 (encoded by POU2F2) was responsible for the formation of SEs and the expression of genes encoded in the vicinity of the SE regions. Overexpression of POU2F2 enhances the metastasis of TNBC cells in mice, and its expression is highly correlated to poor prognosis of TNBC patients. Our findings provide a new insight into cancer cell-specific epigenetic changes induced by OCT-2, which trigger the progression of TNBC, and suggest possible candidates that could be targeted for the treatment of TNBC.

Snail is a zinc finger transcription factor encoded by the SNAI1 gene and triggers a cellular process termed epithelial-mesenchymal transition (EMT) upon its increased expression and/or functional activation. Snail expression and activity are regulated by various extracellular stimuli, including cytokines and environmental factors. Transforming growth factor-β (TGF-β) is a Snail inducer that functions via Smad3-mediated transcriptional activation. In the present study, we identified a distal enhancer that modulates TGF-β-induced SNAI1 expression. ChIP-seq and Hi-C analyses showed that the enhancer is located 46 kb downstream of the SNAI1 gene; in TGF-β-stimulated cells, it associates with Smad3 and interacts with the SNAI1 proximal promoter. Inhibiting the activity of the enhancer using CRISPRi attenuated TGF-β-induced SNAI1 expression, stress fiber formation, and cell motility enhancement, suggesting that the enhancer mediates TGF-β-induced EMT. The enhancer contains a Smad-binding CAGA motif and an activator protein-1 (AP-1) binding motif that function in transcriptional activation. Ras-responsive element binding protein 1 (RREB1), a transcription factor required for TGF-β-induced Snail expression, regulated the basal activity of the enhancer but not its inducibility by TGF-β. In contrast to the enhancer, the association of Smad3 with the proximal promoter was not evident. These findings suggest that the proximal promoter and the distal enhancer respond to distinct signaling cues, integrate them, and cooperatively function to drive SNAI1 expression.

Super-enhancers (SEs) are critical regulators of tumorigenesis and represent promising targets for bromodomain and extra-terminal domain inhibitors (BETi). However, clinical studies across various solid tumors, including triple-negative breast cancer (TNBC), have demonstrated limited BETi efficacy. This study aims to investigate SE heterogeneity in TNBC and its influence on BETi effectiveness, with the goal of advancing BETi precision treatment strategies and enhancing therapeutic efficacy.

We conducted a comprehensive analysis of H3K27ac ChIP-Seq data from TNBC cell lines and clinical samples, integrating multiple bulk RNA-Seq, scRNA-Seq, and stRNA-Seq datasets to characterize the SE landscape and heterogeneity in TNBC. Utilizing various bioinformatics algorithms, CERES scoring, and clinical prognostic data on transcription factors (TFs), we identified core transcriptional regulatory circuits (CRCs) composed of TNBC-specific SEs and master regulators, characterizing different TNBC subtypes. The biological significance of CRCs in these different TNBC subtypes and their influence on BETi sensitivity were evaluated using in vitro and in vivo models.

Our findings revealed a distinct SE landscape in TNBC compared to non-TNBC and normal breast epithelium, allowing TNBC to be classified into distinct subtypes based on TNBC-specific SEs. Importantly, we identified a high-risk mesenchymal development subtype, validated across cell lines and transcriptomic analyses, primarily driven by a CRC consisting of the master regulator VAX2 and a TNBC-specific SE. This SE-VAX2 CRC is essential for sustaining the malignant traits of this subtype and increasing its sensitivity to BETi.

Our research clarifies the heterogeneity of SEs in TNBC and identifies a high-risk mesenchymal development subtype driven by the SE-VAX2 CRC. The subtype shows more sensitivity to BETi, supporting its precision application in TNBC.

Organisms have evolved various protective mechanisms to survive in complex and dynamic environments. Phase separation is a ubiquitous mechanism in plants, animals, and microorganisms. It facilitates the aggregation of biomolecules through weak interactions, forming membrane-less organelles that help organisms respond effectively to stress signals. These biomolecular condensates include DNA, RNA, and proteins. Proteins are categorized into scaffold and client proteins, whose coordinated actions ensure the compartmentalization of cellular signals, thereby regulating various biological processes. Studies indicate that, in response to stress, phase separation modulates gene expression, signal transduction, cytoskeleton dynamics, and protein homeostasis, ensuring the precise spatiotemporal control of cellular functions. These insights underscore the crucial role of phase separation in maintaining cellular integrity and adaptability.

Over the past two decades, cell-to-cell heterogeneity has garnered increasing attention due to its critical role in both developmental and pathological processes. This growing interest has been driven, in part, by the advancements in live-cell and single-molecule imaging techniques. These techniques have provided mechanistic insights into how processes, transcription in particular, contribute to gene expression noise and, ultimately, cell-to-cell heterogeneity. More recently, however, research has expanded to explore how downstream steps in the central dogma influence gene expression noise. In this review, we mostly examine the impact of transcriptional processes on the generation of gene expression noise but also discuss how post-transcriptional mechanisms modulate noise and its propagation to the protein level. This evaluation emphasizes the need for further investigation into how processes beyond transcription shape gene expression noise, highlighting unanswered questions that remain in the field.

Addiction to oncogene-rewired transcriptional networks is a therapeutic vulnerability in cancer cells, underscoring a need to better understand mechanisms that relay oncogene signals to the transcriptional machinery. Here, using human and mouse T cell acute lymphoblastic leukemia (T-ALL) models, we identify an essential requirement for the endosomal sorting complex required for transport protein CHMP5 in T-ALL epigenetic and transcriptional programming. CHMP5 is highly expressed in T-ALL cells where it mediates recruitment of the coactivator BRD4 and the histone acetyl transferase p300 to enhancers and super-enhancers that enable transcription of T-ALL genes. Consequently, CHMP5 depletion causes severe downregulation of critical T-ALL genes, mitigates chemoresistance and impairs T-ALL initiation by oncogenic NOTCH1 in vivo. Altogether, our findings uncover a non-oncogene dependency on CHMP5 that enables T-ALL initiation and maintenance.

Cyclin-dependent kinases (CDKs) are pivotal in regulating cell cycle progression and transcription, making them crucial targets in cancer research. The two types of CDKs that regulate different biological activities are transcription-associated CDKs (e.g., CDK7, 8, 9, 12, and 13) and cell cycle-associated CDKs (e.g., CDK1, 2, 4, and 6). One characteristic of cancer is the dysregulation of CDK activity, which results in unchecked cell division and tumor expansion. Targeting transcriptional CDKs, which control RNA polymerase II activity and gene expression essential for cancer cell survival, has shown promise as a therapeutic approach in recent research. While research into selective inhibitors for transcriptional CDKs is ongoing, inhibitors that target CDK4/6, such as palbociclib and ribociclib, have demonstrated encouraging outcomes in treating breast cancer. CDK7, CDK8, and CDK9 are desirable targets for therapy since they have shown oncogenic roles in a variety of cancer types, such as colorectal, ovarian, and breast malignancies. Even with significant advancements, creating selective inhibitors with negligible off-target effects is still difficult. This review highlights the need for more research to optimize therapeutic strategies and improve patient outcomes by giving a thorough overview of the non-transcriptional roles of CDKs in cancer biology, their therapeutic potential, and the difficulties in targeting these kinases for cancer treatment.

Mast cells (MCs) play a critical role in allergic inflammation, anaphylaxis, and chronic inflammatory diseases such as asthma, COPD, and osteoarthritis. Dysregulated MC activation can lead to MC activation syndrome (MACS), which is observed in patients with long COVID. MCs express the high-affinity receptor for IgE and, upon activation, release mediators and cytokines that trigger anaphylactic shock and promote allergic inflammation. They also interact with epithelial and nerve cells, which are crucial in forming a complex network of cell-cell and gene-gene interactions driving chronic inflammation that can confer resistance to treatment. In this review, in the context of the literature, we focus on experiments conducted in our laboratory investigating how transcription factors and enhancers regulate genes critical in mouse MC differentiation and function related to human lung inflammation.

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.

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.

The precursor of sterol regulatory element-binding protein-2 (SREBP2) is a membrane-bound transcription factor regulating cholesterol biosynthesis. Under cholesterol-deficient conditions, mature SREBP2 is released from membrane-bound precursors through proteolytic cleavage and enters the nucleus. However, regulation of the transcriptional activity of nuclear SREBP2 (nSREBP2) is poorly understood. In the present study, we reported that nSREBP2 forms nuclear condensates through its amino-terminal, intrinsically disordered region (IDR) and works together with transcription coactivators, partly on superenhancers, for the transcriptional activation of SREBP2 target genes. Substitution of a conserved phenylalanine by alanine within the IDR abolishes the formation of nSREBP2 condensates and reduces its transcriptional activity. This can be effectively rescued by fusion with a phase separation driving FUS-IDR. Knock-in of the phenylalanine-to-alanine substitution in male mice compromises feeding-induced nSREBP2 activity and lowers hepatic and circulating cholesterol levels, underscoring the functional significance of nSREBP2 condensates. Together, the present study reveals that nuclear condensates driven by nSREBP2 N-terminal IDR facilitate the efficient activation of lipogenic genes and play an important role in cholesterol homeostasis.

FMS-like tyrosine kinase-3 (FLT3), a class 3 receptor tyrosine kinase, can be activated by mutations of internal tandem duplication (FLT3-ITD) or point mutations in the tyrosine kinase domain (FLT3-TKD), leading to constitutive activation of downstream signaling cascades, including the JAK/STAT5, PI3K/AKT/mTOR and RAS/MAPK pathways, which promote the progression of leukemic cells. Despite the initial promise of FLT3 inhibitors, the discouraging outcomes in the treatment of FLT3-ITD-positive acute myeloid leukemia (AML) promote the pursuit of more potent and enduring therapeutic approaches. The histone acetyltransferase complex comprising the E1A binding protein P300 and its paralog CREB-binding protein (p300/CBP) is a promising therapeutic target, but the development of effective p300/CBP inhibitors faces challenges due to inherent resistance and low efficacy, often exacerbated by the absence of reliable clinical biomarkers for patient stratification. In this study we investigated the role of p300/CBP in FLT3-ITD AML and evaluated the therapeutic potential of targeting p300/CBP alone or in combination with FLT3 inhibitors. We showed that high expression of p300 was significantly associated with poor prognosis in AML patients and positively correlated with FLT3 expression. We unveiled that the p300/CBP inhibitors A485 or CCS1477 dose-dependently downregulated FLT3 transcription via abrogation of histone acetylation in FLT3-ITD AML cells; in contrast, the FLT3 inhibitor quizartinib reduced the level of H3K27Ac. Concurrent inhibition of p300/CBP and FLT3 enhanced the suppression of FLT3 signaling and H3K27 acetylation, concomitantly reducing the phosphorylation of STAT5, AKT, ERK and the expression of c-Myc, thereby leading to synergistic antileukemic effects both in vitro and in vivo. Moreover, we found that p300/CBP-associated transcripts were highly expressed in quizartinib-resistant AML cells with FLT3-TKD mutation. Targeting p300/CBP with A485 or CCS1477 retained the efficacy of quizartinib, suggesting marked synergy when combined with p300/CBP inhibitors in quizartinib-resistant AML models, as well as primary FLT3-ITD+ AML samples. These results demonstrate a potential therapeutic strategy of combining p300/CBP and FLT3 inhibitors to treat FLT3-ITD and FLT3-TKD AML.

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.

Epithelial ovarian cancer (EOC) genetics research has been focused on germline or somatic mutations independently. Emerging evidence suggests that the somatic mutational landscape can be shaped by the germline genetic background. In this study, we aim to unravel the role of somatic alterations within EOC germline susceptibility regions by incorporating functional annotations. We investigate somatic events, including mutational signatures, point mutations, copy number alterations, and transcription factor binding disruptions, within 33 EOC germline susceptibility regions. Our analysis identifies significant associations between candidate germline susceptibility genes and somatic mutational signatures known to be key risk factors for EOC, such as mismatch repair deficiency, age-related mutagenesis, and homologous recombination deficiency. In addition, we find somatic point mutations and copy number alterations are significantly enriched in histotype-specific active enhancers and promoters within EOC risk loci. Furthermore, we examine the impact of germline variants and somatic mutations on transcription factor binding sites, identifying cancer developmental transcription factor motifs frequently affected by both types of mutations. Overall, our study highlights the importance of integrating germline and somatic mutations with regulatory and epigenomic data to gain insights into the genetic basis of EOC.