Search

Background & Aims: The incidence of Crohn's disease (CD) continues to increase worldwide. The contribution of CD4 + cell populations remains to be elucidated. We aimed to provide an in-depth transcriptional assessment of CD4 + T cells driving chronic inflammation in CD., Methods: We performed single-cell RNA-sequencing in CD4 + T cells isolated from ileal biopsies of patients with CD compared with healthy individuals. Cells underwent clustering analysis, followed by analysis of gene signaling networks. We overlapped our differentially expressed genes with publicly available microarray data sets and performed functional in vitro studies, including an in vitro suppression assay and organoid systems, to model gene expression changes observed in CD regulatory T (Treg) cells and to test predicted therapeutics., Results: We identified 5 distinct FOXP3 + regulatory Treg subpopulations. Tregs isolated from healthy controls represent the origin of pseudotemporal development into inflammation-associated subtypes. These proinflammatory Tregs displayed a unique responsiveness to tumor necrosis factor-α signaling with impaired suppressive activity in vitro and an elevated cytokine response in an organoid coculture system. As predicted in silico, the histone deacetylase inhibitor vorinostat normalized gene expression patterns, rescuing the suppressive function of FOXP3 + cells in vitro., Conclusions: We identified a novel, proinflammatory FOXP3 + T cell subpopulation in patients with CD and developed a pipeline to specifically target these cells using the US Food and Drug Administration-approved drug vorinostat., (Copyright © 2024 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Enriching the soil with organic matter such as humic and fulvic acid to increase its content available nutrients, improves the chemical properties of the soil and increases plant growth as well as grain yield. In this study, we conducted a field experiment using humic acid (HA), fulvic acid (FA) and recommended dose (RDP) of phosphorus fertilizer to treat Hordeum vulgare seedling, in which four concentrations from HA, FA and RDP (0.0 %, 50 %, 75 % and 100%) under saline soil conditions . Moreover, some agronomic traits (e.g. grain yield, straw yield, spikes weight, plant height, spike length and spike weight) in barley seedling after treated with different concentrations from HA, FA and RDP were determined. As such the beneficial effects of these combinations to improve plant growth, N, P, and K uptake, grain yield, and its components under salinity stress were assessed., Results: The findings showed that the treatments HA + 100% RDP (T1), HA + 75% RDP (T2), FA + 100% RDP (T5), HA + 50% RDP (T3), and FA + 75% RDP (T6), improved number of spikes/plant, 1000-grain weight, grain yield/ha, harvest index, the amount of uptake of nitrogen (N), phosphorous (P) and potassium (K) in straw and grain. The increase for grain yield over the control was 64.69, 56.77, 49.83, 49.17, and 44.22% in the first season, and 64.08, 56.63, 49.19, 48.87, and 43.69% in the second season,. Meanwhile, the increase for grain yield when compared to the recommended dose was 22.30, 16.42, 11.27, 10.78, and 7.11% in the first season, and 22.17, 16.63, 11.08, 10.84, and 6.99% in the second season. Therefore, under salinity conditions the best results were obtained when, in addition to phosphate fertilizer, the soil was treated with humic acid or foliar application the plants with fulvic acid under one of the following treatments: HA + 100% RDP (T1), HA + 75% RDP (T2), FA + 100% RDP (T5), HA + 50% RDP (T3), and FA + 75% RDP (T6)., Conclusions: The result of the use of organic amendments was an increase in the tolerance of barley plant to salinity stress, which was evident from the improvement in the different traits that occurred after the treatment using treatments that included organic amendments (humic acid or fulvic acid)., (© 2024. The Author(s).)

Background: The development of Crohn's disease [CD] involves immune cell signalling pathways regulated by epigenetic modifications. Aberrant DNA methylation has been identified in peripheral blood and bulk intestinal tissue from CD patients. However, the DNA methylome of disease-associated intestinal CD4+ lymphocytes has not been evaluated., Materials and Methods: Genome-wide DNA methylation sequencing was performed from terminal ileum CD4+ cells from 21 CD patients and 12 age- and sex-matched controls. Data were analysed for differentially methylated CpGs [DMCs] and methylated regions [DMRs]. Integration was performed with RNA-sequencing data to evaluate the functional impact of DNA methylation changes on gene expression. DMRs were overlapped with regions of differentially open chromatin [by ATAC-seq] and CCCTC-binding factor [CTCF] binding sites [by ChIP-seq] between peripherally derived Th17 and Treg cells., Results: CD4+ cells in CD patients had significantly increased DNA methylation compared to those from the controls. A total of 119 051 DMCs and 8113 DMRs were detected. While hypermethylated genes were mostly related to cell metabolism and homeostasis, hypomethylated genes were significantly enriched within the Th17 signalling pathway. The differentially enriched ATAC regions in Th17 cells [compared to Tregs] were hypomethylated in CD patients, suggesting heightened Th17 activity. There was significant overlap between hypomethylated DNA regions and CTCF-associated binding sites., Conclusions: The methylome of CD patients shows an overall dominant hypermethylation yet hypomethylation is more concentrated in proinflammatory pathways, including Th17 differentiation. Hypomethylation of Th17-related genes associated with areas of open chromatin and CTCF binding sites constitutes a hallmark of CD-associated intestinal CD4+ cells., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A major hurdle to the application of precision oncology in pancreatic cancer is the lack of molecular stratification approaches and targeted therapy for defined molecular subtypes. In this work, we sought to gain further insight and identify molecular and epigenetic signatures of the Basal-like A pancreatic ductal adenocarcinoma (PDAC) subgroup that can be applied to clinical samples for patient stratification and/or therapy monitoring. We generated and integrated global gene expression and epigenome mapping data from patient-derived xenograft models to identify subtype-specific enhancer regions that were validated in patient-derived samples. In addition, complementary nascent transcription and chromatin topology (HiChIP) analyses revealed a Basal-like A subtype-specific transcribed enhancer program in PDAC characterized by enhancer RNA (eRNA) production that is associated with more frequent chromatin interactions and subtype-specific gene activation. Importantly, we successfully confirmed the validity of eRNA detection as a possible histologic approach for PDAC patient stratification by performing RNA-ISH analyses for subtype-specific eRNAs on pathologic tissue samples. Thus, this study provides proof-of-concept that subtype-specific epigenetic changes relevant for PDAC progression can be detected at a single-cell level in complex, heterogeneous, primary tumor material., Implications: Subtype-specific enhancer activity analysis via detection of eRNAs on a single-cell level in patient material can be used as a potential tool for treatment stratification., (©2023 American Association for Cancer Research.)

Objective: Pancreatic ductal adenocarcinoma (PDAC) displays a remarkable propensity towards therapy resistance. However, molecular epigenetic and transcriptional mechanisms enabling this are poorly understood. In this study, we aimed to identify novel mechanistic approaches to overcome or prevent resistance in PDAC., Design: We used in vitro and in vivo models of resistant PDAC and integrated epigenomic, transcriptomic, nascent RNA and chromatin topology data. We identified a JunD-driven subgroup of enhancers, called interactive hubs (iHUBs), which mediate transcriptional reprogramming and chemoresistance in PDAC., Results: iHUBs display characteristics typical for active enhancers (H3K27ac enrichment) in both therapy sensitive and resistant states but exhibit increased interactions and production of enhancer RNA (eRNA) in the resistant state. Notably, deletion of individual iHUBs was sufficient to decrease transcription of target genes and sensitise resistant cells to chemotherapy. Overlapping motif analysis and transcriptional profiling identified the activator protein 1 (AP1) transcription factor JunD as a master transcription factor of these enhancers. JunD depletion decreased iHUB interaction frequency and transcription of target genes. Moreover, targeting either eRNA production or signaling pathways upstream of iHUB activation using clinically tested small molecule inhibitors decreased eRNA production and interaction frequency, and restored chemotherapy responsiveness in vitro and in vivo. Representative iHUB target genes were found to be more expressed in patients with poor response to chemotherapy compared with responsive patients., Conclusion: Our findings identify an important role for a subgroup of highly connected enhancers (iHUBs) in regulating chemotherapy response and demonstrate targetability in sensitisation to chemotherapy., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Background & Aims: Although T-cell intrinsic expression of G9a has been associated with murine intestinal inflammation, mechanistic insight into the role of this methyltransferase in human T-cell differentiation is ill defined, and manipulation of G9a function for therapeutic use against inflammatory disorders is unexplored., Methods: Human naive T cells were isolated from peripheral blood and differentiated in vitro in the presence of a G9a inhibitor (UNC0642) before being characterized via the transcriptome (RNA sequencing), chromatin accessibility (assay for transposase-accessible chromatin by sequencing), protein expression (cytometry by time of flight, flow cytometry), metabolism (mitochondrial stress test, ultrahigh performance liquid chromatography-tandem mas spectroscopy) and function (T-cell suppression assay). The in vivo role of G9a was assessed using 3 murine models., Results: We discovered that pharmacologic inhibition of G9a enzymatic function in human CD4 T cells led to spontaneous generation of FOXP3 + T cells (G9a-inibitors-T regulatory cells [Tregs]) in vitro that faithfully reproduce human Tregs, functionally and phenotypically. Mechanistically, G9a inhibition altered the transcriptional regulation of genes involved in lipid biosynthesis in T cells, resulting in increased intracellular cholesterol. Metabolomic profiling of G9a-inibitors-Tregs confirmed elevated lipid pathways that support Treg development through oxidative phosphorylation and enhanced lipid membrane composition. Pharmacologic G9a inhibition promoted Treg expansion in vivo upon antigen (gliadin) stimulation and ameliorated acute trinitrobenzene sulfonic acid-induced colitis secondary to tissue-specific Treg development. Finally, Tregs lacking G9a expression (G9a-knockout Tregs) remain functional chronically and can rescue T-cell transfer-induced colitis., Conclusion: G9a inhibition promotes cholesterol metabolism in T cells, favoring a metabolic profile that facilitates Treg development in vitro and in vivo. Our data support the potential use of G9a inhibitors in the treatment of immune-mediated conditions including inflammatory bowel disease., (Copyright © 2023 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Loss of AT-rich interactive domain-containing protein 1A (ARID1A) fosters acinar-to-ductal metaplasia (ADM) and pancreatic carcinogenesis by down-regulating transcription programs controlling acinar cell identity. However, how ARID1A reacts to metaplasia-triggering environmental cues remains elusive. Here, we aimed to elucidate the role of ARID1A in controlling ductal pancreatic gene signatures and deciphering hierarchical signaling cues determining ARID1A-dependent chromatin regulation during acinar cell reprogramming., Methods: Acinar cell explants with differential ARID1A status were subjected to genome-wide expression analyses. The impact of epidermal growth factor receptor (EGFR) signaling, NFATc1 activity, and ARID1A status on acinar reprogramming processes were characterized by ex vivo ADM assays and transgenic mouse models. EGFR-dependent ARID1A chromatin binding was studied by chromatin immunoprecipitation sequencing analysis and cellular fractionation., Results: EGFR signaling interferes with ARID1A-dependent transcription by inducing genome-wide ARID1A displacement, thereby phenocopying ARID1A loss-of-function mutations and inducing a shift toward ADM permissive ductal transcription programs. Moreover, we show that EGFR signaling is required to push ARID1A-deficient acinar cells toward a metaplastic phenotype. Mechanistically, we identified the transcription factor nuclear factor of activated T cells 1 (NFATc1) as the central regulatory hub mediating both EGFR signaling-induced genomic ARID1A displacement and the induction of ADM-promoting gene signatures in the absence of ARID1A. Consequently, pharmacologic inhibition of NFATc1 or its depletion in transgenic mice not only preserves genome-wide ARID1A occupancy, but also attenuates acinar metaplasia led by ARID1A loss., Conclusions: Our data describe an intimate relationship between environmental signaling and chromatin remodeling in orchestrating cell fate decisions in the pancreas, and illustrate how ARID1A loss influences transcriptional regulation in acinar cell reprogramming., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: H3K27M-mutant diffuse midline glioma (DMG) is a lethal brain tumor that usually occurs in children. Despite advances in our understanding of its underlying biology, efficacious therapies are severely lacking., Methods: We screened a library of drugs either FDA-approved or in clinical trial using a library of patient-derived H3K27M-mutant DMG cell lines with cell viability as the outcome. Results were validated for clinical relevance and mechanistic importance using patient specimens from biopsy and autopsy, patient-derived cell lines, inhibition by gene knockdown and small molecule inhibitors, and patient-derived xenografts., Results: Kinase inhibitors were highly toxic to H3K27M-mutant DMG cells. Within this class, STAT3 inhibitors demonstrated robust cytotoxic activity in vitro. Mechanistic analyses revealed one form of activated STAT3, phospho-tyrosine- 705 STAT3 (pSTAT3), was selectively upregulated in H3K27M-mutant cell lines and clinical specimens. STAT3 inhibition by CRISPR/Cas9 knockout, shRNA or small molecule inhibition reduced cell viability in vitro, and partially restored expression of the polycomb repressive mark H3K27me3, which is classically lost in H3K27M-mutant DMG. Putative STAT3-regulated genes were enriched in an H3K27M-knockout DMG cell line, indicating relative gain of STAT3 signaling in K27M-mutant cells. Treatment of patient-derived intracranial xenografts with WP1066, a STAT3 pathway inhibitor currently in clinical use for pediatric brain tumors, resulted in stasis of tumor growth, and increased overall survival. Finally, pSTAT3(Y705) was detected in circulating plasma extracellular vesicles of patients with H3K27M-mutant DMG., Conclusions: STAT3 is a biologically relevant therapeutic target in H3K27M-mutant DMG. STAT3 inhibition should be considered in future clinical trials., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Cardiovascular diseases are one of the major causes of mortalities worldwide. In the present research, new synthetic derivatives of thiazole were studied using isolated hearts and blood vessels of rats. The heart and thoracic aorta were tested with six new synthesized thiazole acetic acid derivatives (SMVA-10, SMVA-35, SMVA-40, SMVA-41, SMVA-42 and SMVA-60), and the data obtained were statistically analyzed and compared. Isolated rat hearts were used to record the changes in developed tension and heart rate, while thoracic aortas were used to measure the contractile response, before and after treatments. Analysis of the results indicated a significant (p < 0.01) increase in developed tension with the addition of SMVA-35, SMVA-40, SMVA-41 and SMVA-42, which was augmented in the presence of adrenaline without affecting the heart rate. On the other hand, acetylcholine significantly decreased the developed tension, which was significantly reversed (p < 0.01) in the presence of compounds (SMVA-35 and SMVA-60). However, in the presence of SMVA-35 and SMVA-40, acetylcholine-induced bradycardia was significantly (p < 0.01) reduced. Furthermore, only SMVA-42 induced a dose-dependent contractile response in the isolated blood vessel, which was abolished in the presence of prazosin. Therefore, it can be concluded that some of the new synthesized thiazole derivatives exhibited promising results by raising the developed tension without changing the heart rate or blood vessel function, which could be helpful in failing heart conditions. However, more research is required to fully comprehend the function, mechanism and effectiveness of the compounds.

Diabetic nephropathy (DN) is a serious kidney illness characterized by proteinuria, glomerular enlargement, reduced glomerular filtration, and renal fibrosis. DN is the most common cause of end-stage kidney disease, accounting for nearly one-third of all cases of diabetes worldwide. Hyperglycemia is a major factor in the onset and progression of diabetic nephropathy. Many contemporary medicines are derived from plants since they have therapeutic properties and are relatively free of adverse effects. Glycosides, alkaloids, terpenoids, and flavonoids are among the few chemical compounds found in plants that are utilized to treat diabetic nephropathy. The purpose of this review was to consolidate information on the clinical and pharmacological evidence supporting the use of a variety of medicinal plants to treat diabetic nephropathy.

Background & Aims: Biliary disease is associated with a proliferative/fibrogenic ductular reaction (DR). p300 is an epigenetic regulator that acetylates lysine 27 on histone 3 (H3K27ac) and is activated during fibrosis. Long non-coding RNAs (lncRNAs) are aberrantly expressed in cholangiopathies, but little is known about how they recruit epigenetic complexes and regulate DR. We investigated epigenetic complexes, including transcription factors (TFs) and lncRNAs, contributing to p300-mediated transcription during fibrosis., Methods: We evaluated p300 in vivo using tamoxifen-inducible, cholangiocyte-selective, p300 knockout (KO) coupled with bile duct ligation (BDL) and Mdr KO mice treated with SGC-CBP30. Primary cholangiocytes and liver tissue were analyzed for expression of Acta2-as1 lncRNA by qPCR and RNA in situ hybridization. In vitro, we performed RNA-sequencing in human cholangiocytes with a p300 inhibitor. Cholangiocytes were exposed to lipopolysaccharide (LPS) as an injury model. We confirmed formation of a p300/ELK1 complex by immunoprecipitation (IP). RNA IP was used to examine interactions between ACTA2-AS1 and p300. Chromatin IP assays were used to evaluate p300/ELK1 occupancy and p300-mediated H3K27ac. Organoids were generated from ACTA2-AS1-depleted cholangiocytes., Results: BDL-induced DR and fibrosis were reduced in Krt19-Cre ERT /p300 fl/fl mice. Similarly, Mdr KO mice were protected from DR and fibrosis after SGC-CBP30 treatment. In vitro, depletion of ACTA2-AS1 reduced expression of proliferative/fibrogenic markers, reduced LPS-induced cholangiocyte proliferation, and impaired organoid formation. ACTA2-AS1 regulated transcription by facilitating p300/ELK1 binding to the PDGFB promoter after LPS exposure. Correspondingly, LPS-induced H3K27ac was mediated by p300/ELK1 and was reduced in ACTA2-AS1-depleted cholangiocytes., Conclusion: Cholangiocyte-selective p300 KO or p300 inhibition attenuate DR/fibrosis in mice. ACTA2-AS1 influences recruitment of p300/ELK1 to specific promoters to drive H3K27ac and epigenetic activation of proliferative/fibrogenic genes. This suggests that cooperation between epigenetic co-activators and lncRNAs facilitates DR/fibrosis in biliary diseases., Lay Summary: We identified a three-part complex containing an RNA molecule, a transcription factor, and an epigenetic enzyme. The complex is active in injured bile duct cells and contributes to activation of genes involved in proliferation and fibrosis., Competing Interests: Conflict of interest The authors declare no conflicts of interest relevant to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Triple Negative Breast Cancer (TNBC) accounts for 15-20% of all breast cancer cases, yet is responsible for a disproportionately high percentage of breast cancer mortalities. Thus, there is an urgent need to identify novel biomarkers and therapeutic targets based on the molecular events driving TNBC pathobiology. Estrogen receptor beta (ERβ) is known to elicit anti-cancer effects in TNBC, however its mechanisms of action remain elusive. Here, we report the expression profiles of ERβ and its association with clinicopathological features and patient outcomes in the largest cohort of TNBC to date. In this cohort, ERβ was expressed in approximately 18% of TNBCs, and expression of ERβ was associated with favorable clinicopathological features, but correlated with different overall survival outcomes according to menopausal status. Mechanistically, ERβ formed a co-repressor complex involving enhancer of zeste homologue 2/polycomb repressive complex 2 (EZH2/PRC2) that functioned to suppress oncogenic NFκB/RELA (p65) activity. Importantly, p65 was shown to be required for formation of this complex and for ERβ-mediated suppression of TNBC. Our findings indicate that ERβ+ tumors exhibit different characteristics compared to ERβ- tumors and demonstrate that ERβ functions as a molecular switch for EZH2, repurposing it for tumor suppressive activities and repression of oncogenic p65 signaling., (© 2022. The Author(s).)

Primary sclerosing cholangitis (PSC) is a chronic fibroinflammatory disease of the biliary tract characterized by cellular senescence and periportal fibrogenesis. Specific disease features that are cell intrinsic and either genetically or epigenetically mediated remain unclear due in part to a lack of appropriate, patient-specific, in vitro models. Recently, our group developed systems to create induced pluripotent stem cell (iPSC)-derived cholangiocytes (iDCs) and biliary epithelial organoids (cholangioids). We use these models to investigate whether PSC cholangiocytes are intrinsically predisposed to cellular senescence. Skin fibroblasts from healthy controls and subjects with PSC were reprogrammed to pluripotency, differentiated to cholangiocytes, and subsequently grown in three-dimensional matrigel-based culture to induce formation of cholangioids. RNA sequencing (RNA-seq) on iDCs showed significant differences in gene expression patterns, including enrichment of pathways associated with cell cycle, senescence, and hepatic fibrosis, that correlate with PSC. These pathways also overlapped with RNA-seq analysis on isolated cholangiocytes from subjects with PSC. Exome sequencing on the subjects with PSC revealed genetic variants of unknown significance in the genes identified in these pathways. Three-dimensional culture revealed smaller size, lack of a central lumen, and increased cellular senescence in PSC-derived cholangioids. Congruent with this, PSC-derived iDCs showed increased secretion of the extracellular matrix molecule fibronectin as well as the inflammatory cytokines interleukin-6, and chemokine (C-C motif) ligand 2. Conditioned media (CM) from PSC-derived iDCs more potently activated hepatic stellate cells compared to control CM. Conclusion: We demonstrated efficient generation of iDCs and cholangioids from patients with PSC that show disease-specific features. PSC cholangiocytes are intrinsically predisposed to cellular senescence. These features are unmasked following biliary differentiation of pluripotent stem cells and have functional consequences in epithelial organoids., (© 2021 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Alcoholic hepatitis (AH) is associated with liver neutrophil infiltration through activated cytokine pathways leading to elevated chemokine expression. Super-enhancers are expansive regulatory elements driving augmented gene expression. Here, we explore the mechanistic role of super-enhancers linking cytokine TNFα with chemokine amplification in AH. RNA-seq and histone modification ChIP-seq of human liver explants show upregulation of multiple CXCL chemokines in AH. Liver sinusoidal endothelial cells (LSEC) are identified as an important source of CXCL expression in human liver, regulated by TNFα/NF-κB signaling. A super-enhancer is identified for multiple CXCL genes by multiple approaches. dCas9-KRAB-mediated epigenome editing or pharmacologic inhibition of Bromodomain and Extraterminal (BET) proteins, transcriptional regulators vital to super-enhancer function, decreases chemokine expression in vitro and decreases neutrophil infiltration in murine models of AH. Our findings highlight the role of super-enhancer in propagating inflammatory signaling by inducing chemokine expression and the therapeutic potential of BET inhibition in AH treatment., (© 2021. The Author(s).)

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

FOXP3+ Tregs are expanded within the inflamed intestine of human Crohn's disease, yet FOXP3-mediated gene repression within these cells is lost. The polycomb repressive complexes play a role in FOXP3 target gene regulation, but deeper mechanistic insight is incomplete. We have now specifically identified the polycomb-repressive complex 1 (PRC1) family member, BMI1 in the regulation of a proinflammatory enhancer network in both human and murine Tregs. Using human Tregs and lamina propria T cells, we inferred PRC1 to regulate Crohn's associated gene networks through assays of chromatin accessibility. Conditional deletion of BMI1 in murine FOXP3+ cells led to systemic inflammation. BMI1-deficient Tregs beared a TH1/TH17-like phenotype as assessed by assays of genome wide transcription, chromatin accessibility and proteomic techniques. Finally, BMI1 mutant FOXP3+ cells did not suppress colitis in the adoptive transfer model of human inflammatory bowel disease. We propose that BMI1 plays an important role in enforcing Treg identity in vitro and in vivo. Loss of Treg identity via genetic or transient BMI1 depletion perturbs the epigenome and converts Tregs into Th1/Th17-like proinflammatory cells, a transition relevant to human Crohn's disease associated CD4+ T cells.

Pancreatic ductal adenocarcinoma (PDAC) displays a dismal prognosis due to late diagnosis and high chemoresistance incidence. For advanced disease stages or patients with comorbidities, treatment options are limited to gemcitabine alone or in combination with other drugs. While gemcitabine resistance has been widely attributed to the levels of one of its targets, RRM1, the molecular consequences of gemcitabine resistance in PDAC remain largely elusive. Here we sought to identify genomic, epigenomic, and transcriptomic events associated with gemcitabine resistance in PDAC and their potential clinical relevance. We found that gemcitabine-resistant cells displayed a coamplification of the adjacent RRM1 and STIM1 genes. Interestingly, RRM1, but not STIM1, was required for gemcitabine resistance, while high STIM1 levels caused an increase in cytosolic calcium concentration. Higher STIM1-dependent calcium influx led to an impaired endoplasmic reticulum stress response and a heightened nuclear factor of activated T-cell activity. Importantly, these findings were confirmed in patient and patient-derived xenograft samples. Taken together, our study uncovers previously unknown biologically relevant molecular properties of gemcitabine-resistant tumors, revealing an undescribed function of STIM1 as a rheostat directing the effects of calcium signaling and controlling epigenetic cell fate determination. It further reveals the potential benefit of targeting STIM1-controlled calcium signaling and its downstream effectors in PDAC. SIGNIFICANCE: Gemcitabine-resistant and some naïve tumors coamplify RRM1 and STIM1 , which elicit gemcitabine resistance and induce a calcium signaling shift, promoting ER stress resistance and activation of NFAT signaling., (©2021 American Association for Cancer Research.)

Pancreatic ductal adenocarcinoma (PDAC) displays a particularly poor prognosis and low survival rate, mainly due to late diagnosis and high incidence of chemotherapy resistance. Genomic aberrations, together with changes in the epigenomic profile, elicit a shift in cellular signaling response and a transcriptional reprograming in pancreatic tumors. This endows them with malignant attributes that enable them to not only overcome chemotherapeutic challenges, but to also attain diverse oncogenic properties. In fact, certain genetic amplifications elicit a rewiring of calcium signaling, which can confer ER stress resistance to tumors while also aberrantly activating known drivers of oncogenic programs such as NFAT. While calcium is a well-known second messenger, the transcriptional programs driven by aberrant calcium signaling remain largely undescribed in pancreatic cancer. In this review, we focus on calcium-dependent signaling and its role in epigenetic programs and transcriptional regulation. We also briefly discuss genetic aberration events, exemplifying how genetic alterations can rewire cellular signaling cascades, including calcium-dependent ones.

Background & Aims: Transforming growth factor β (TGFβ) upregulates cholangiocyte-derived signals that activate myofibroblasts and promote fibrosis. Using epigenomic and transcriptomic approaches, we sought to distinguish the epigenetic activation mechanisms downstream of TGFβ that mediate transcription of fibrogenic signals., Methods: Chromatin immunoprecipitation (ChIP)-seq and RNA-seq were performed to assess histone modifications and transcriptional changes following TGFβ stimulation. Histone modifications and acetyltransferase occupancy were confirmed using ChIP assays. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) was used to investigate changes in chromatin accessibility. Cholangiocyte cell lines and primary cholangiocytes were used for in vitro studies. Mdr2 -/- and 3,5-diethoxycarboncyl-1,4-dihydrocollidine (DDC)-fed mice were used as animal models., Results: TGFβ stimulation caused widespread changes in histone 3 lysine 27 acetylation (H3K27ac), and was associated with global TGFβ-mediated transcription. In contrast, H3K9ac was gained in a smaller group of chromatin sites and was associated with fibrosis pathways. These pathways included overexpression of hepatic stellate cell (HSC) activators such as fibronectin 1 (FN1) and SERPINE1. The promoters of these genes showed H3K9ac enrichment following TGFβ. Of the acetyltransferases responsible for H3K9ac, cholangiocytes predominantly express Lysine Acetyltransferases 2A (KAT2A). Small interfering RNA knockdown of KAT2A or H3K9ac inhibition prevented the TGFβ-mediated increase in FN1 and SERPINE1. SMAD3 ChIP-seq and ATAC-seq suggested that TGFβ-mediated H3K9ac occurs through SMAD signaling, which was confirmed using colocalization and genetic knockdown studies. Pharmacologic inhibition or cholangiocyte-selective deletion of Kat2a was protective in mouse models of biliary fibrosis., Conclusions: Cholangiocyte expression of HSC-activating signals occurs through SMAD-dependent, KAT2A-mediated, H3K9ac, and can be targeted to prevent biliary fibrosis., (Copyright © 2021 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) is resistant to virtually all chemo- and targeted therapeutic approaches. Epigenetic regulators represent a novel class of drug targets. Among them, BET and HDAC proteins are central regulators of chromatin structure and transcription, and preclinical evidence suggests effectiveness of combined BET and HDAC inhibition in PDAC. Here, we describe that TW9, a newly generated adduct of the BET inhibitor (+)-JQ1 and class I HDAC inhibitor CI994, is a potent dual inhibitor simultaneously targeting BET and HDAC proteins. TW9 has a similar affinity to BRD4 bromodomains as (+)-JQ1 and shares a conserved binding mode, but is significantly more active in inhibiting HDAC1 compared to the parental HDAC inhibitor CI994. TW9 was more potent in inhibiting tumor cell proliferation compared to (+)-JQ1, CI994 alone or combined treatment of both inhibitors. Sequential administration of gemcitabine and TW9 showed additional synergistic antitumor effects. Microarray analysis revealed that dysregulation of a FOSL1-directed transcriptional program contributed to the antitumor effects of TW9. Our results demonstrate the potential of a dual chromatin-targeting strategy in the treatment of PDAC and provide a rationale for further development of multitarget inhibitors., (© 2020 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Epigenetic alterations play a central role in cancer development and progression. The acetylation of histone 3 at lysine 27 (H3K27ac) specifically marks active genes. While chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-seq) analyses are commonly performed in cell lines, only limited data are available from primary tumors. We therefore examined whether cancer-specific alterations in H3K27ac occupancy can be identified in primary rectal cancer. Tissue samples from primary rectal cancer and matched mucosa were obtained. ChIP-seq for H3K27ac was performed and differentially occupied regions were identified. The expression of selected genes displaying differential occupancy between tumor and mucosa were examined in gene expression data from an independent patient cohort. Differential expression of four proteins was further examined by immunohistochemistry. ChIP-seq for H3K27ac in primary rectal cancer and matched mucosa was successfully performed and revealed differential binding on 44 regions. This led to the identification of genes with increased H3K27ac, i.e., RIPK2 , FOXQ1 , KRT23 , and EPHX4 , which were also highly upregulated in primary rectal cancer in an independent dataset. The increased expression of these four proteins was confirmed by immunohistochemistry. This study demonstrates the feasibility of ChIP-seq-based epigenome mapping of primary rectal cancer and confirms the value of H3K27ac occupancy to predict gene expression differences.

Background: Colorectal cancer (CRC) is the fourth leading cause of cancer-related deaths worldwide, and deciphering underlying molecular mechanism is essential. The loss of monoubiquitinated histone H2B (H2Bub1) was correlated with poor prognosis of CRC patients and, accordingly, H2Bub1 was suggested as a tumor-suppressive mark. Surprisingly, our previous work revealed that the H2B ubiquitin ligase RING finger protein 40 (RNF40) might exert tumor-promoting functions. Here, we investigated the effect of RNF40 loss on tumorigenic features of CRC cells and their survival in vitro., Methods: We evaluated the effects of RNF40 depletion in several human CRC cell lines in vitro. To evaluate cell cycle progression, cells were stained with propidium iodide and analyzed by flow cytometry. In addition, to assess apoptosis rates, caspase 3/7 activity was assessed in a Celigo® S-based measurement and, additionally, an Annexin V assay was performed. Genomic occupancy of H2Bub1, H3K79me3, and H3K27ac was determined by chromatin immunoprecipitation. Transcriptome-wide effects of RNF40 loss were evaluated based on mRNA-seq results, qRT-PCR, and Western blot. To rescue apoptosis-related effects, cells were treated with Z-VAD-FMK., Results: Human CRC cell lines displayed decreased cell numbers in vitro after RNF40 depletion. While the differences in confluence were not mediated by changes in cell cycle progression, we discovered highly increased apoptosis rates after RNF40 knockdown due to elevated caspase 3/7 activity. This effect can be explained by reduced mRNA levels of anti-apoptotic and upregulation of pro-apoptotic BCL2 family members. Moreover, the direct occupancy of the RNF40-mediated H2B monoubiquitination was observed in the transcribed region of anti-apoptotic genes. Caspase inhibition by Z-VAD-FMK treatment rescued apoptosis in RNF40-depleted cells. However, knockdown cells still displayed decreased tumorigenic features despite the absence of apoptosis., Conclusions: Our findings reveal that RNF40 is essential for maintaining tumorigenic features of CRC cells in vitro by controlling the expression of genes encoding central apoptotic regulators.

Background: ARID1A (AT-rich interactive domain-containing protein 1A) is a subunit of the BAF chromatin remodeling complex and plays roles in transcriptional regulation and DNA damage response. Mutations in ARID1A that lead to inactivation or loss of expression are frequent and widespread across many cancer types including colorectal cancer (CRC). A tumor suppressor role of ARID1A has been established in a number of tumor types including CRC where the genetic inactivation of Arid1a alone led to the formation of invasive colorectal adenocarcinomas in mice. Mechanistically, ARID1A has been described to largely function through the regulation of enhancer activity., Methods: To mimic ARID1A-deficient colorectal cancer, we used CRISPR/Cas9-mediated gene editing to inactivate the ARID1A gene in established colorectal cancer cell lines. We integrated gene expression analyses with genome-wide ARID1A occupancy and epigenomic mapping data to decipher ARID1A-dependent transcriptional regulatory mechanisms., Results: Interestingly, we found that CRC cell lines harboring KRAS mutations are critically dependent on ARID1A function. In the absence of ARID1A, proliferation of these cell lines is severely impaired, suggesting an essential role for ARID1A in this context. Mechanistically, we showed that ARID1A acts as a co-factor at enhancers occupied by AP1 transcription factors acting downstream of the MEK/ERK pathway. Consistently, loss of ARID1A led to a disruption of KRAS/AP1-dependent enhancer activity, accompanied by a downregulation of expression of the associated target genes., Conclusions: We identify a previously unknown context-dependent tumor-supporting function of ARID1A in CRC downstream of KRAS signaling. Upon the loss of ARID1A in KRAS-mutated cells, enhancers that are co-occupied by ARID1A and the AP1 transcription factors become inactive, thereby leading to decreased target gene expression. Thus, targeting of the BAF complex in KRAS-mutated CRC may offer a unique, previously unknown, context-dependent therapeutic option in CRC.

Tight regulation of gene transcription is essential for normal development, tissue homeostasis, and disease-free survival. Enhancers are distal regulatory elements in the genome that provide specificity to gene expression programs and are frequently misregulated in cancer. Recent studies examined various enhancer-driven malignant dependencies and identified different approaches to specifically target these programs. In this review, we describe numerous features that make enhancers good transcriptional targets in cancer therapy and discuss different approaches to overcome enhancer perturbation. Interestingly, a number of approved therapeutic agents, such as cyclosporine, steroid hormones, and thiazolidinediones, actually function by affecting enhancer landscapes by directly targeting very specific transcription factor programs. More recently, a broader approach to targeting deregulated enhancer programs has been achieved via Bromodomain and Extraterminal (BET) inhibition or perturbation of transcription-related cyclin-dependent kinases (CDK). One challenge to enhancer-targeted therapy is proper patient stratification. We suggest that monitoring of enhancer RNA (eRNA) expression may serve as a unique biomarker of enhancer activity that can help to predict and monitor responsiveness to enhancer-targeted therapies. A more thorough investigation of cancer-specific enhancers and the underlying mechanisms of deregulation will pave the road for an effective utilization of enhancer modulators in a precision oncology approach to cancer treatment.

Background: Cytosolic 5'-nucleotidase 1A (NT5C1A) dephosphorylates non-cyclic nucleoside monophosphates to produce nucleosides and inorganic phosphates. Here, we investigate NT5C1A expression in pancreatic ductal adenocarcinoma (PDAC) and its impact on gemcitabine metabolism and therapeutic efficacy., Methods: NT5C1A expression was determined by semiquantitative immunohistochemistry using tissue microarrays. Gemcitabine metabolites and response were assessed in several human and murine PDAC cell lines using crystal violet assays, Western blot, viability assays, and liquid chromatography tandem mass-spectrometry (LC-MS/MS)., Findings: NT5C1A was strongly expressed in tumor cells of a large subgroup of resected PDAC patients in two independent patient cohorts (44-56% score 2 and 8-26% score 3, n = 414). In contrast, NT5C1A was expressed at very low levels in the tumor stroma, and neither stromal nor tumoral expression was a prognostic marker for postoperative survival. In vitro, NT5C1A overexpression increased gemcitabine resistance by reducing apoptosis levels and significantly decreased intracellular amounts of cytotoxic dFdCTP in +NT5C1A tumor cells. Co-culture experiments with conditioned media from +NT5C1A PSCs improved gemcitabine efficacy in tumor cells. In vivo, therapeutic efficacy of gemcitabine was significantly decreased and serum levels of the inactive gemcitabine metabolite dFdU significantly increased in mice bearing NT5C1A overexpressing tumors., Interpretation: NT5C1A is robustly expressed in tumor cells of resected PDAC patients. Moreover, NT5C1A mediates gemcitabine resistance by decreasing the amount of intracellular dFdCTP, leading to reduced tumor cell apoptosis and larger pancreatic tumors in mice. Further studies should clarify the role of NT5C1A as novel predictor for gemcitabine treatment response in patients with PDAC., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Molecular subtyping of cancer offers tremendous promise for the optimization of a precision oncology approach to anticancer therapy. Recent advances in pancreatic cancer research uncovered various molecular subtypes with tumors expressing a squamous/basal-like gene expression signature displaying a worse prognosis. Through unbiased epigenome mapping, we identified deltaNp63 as a major driver of a gene signature in pancreatic cancer cell lines, which we report to faithfully represent the highly aggressive pancreatic squamous subtype observed in vivo, and display the specific epigenetic marking of genes associated with decreased survival. Importantly, depletion of deltaNp63 in these systems significantly decreased cell proliferation and gene expression patterns associated with a squamous subtype and transcriptionally mimicked a subtype switch. Using genomic localization data of deltaNp63 in pancreatic cancer cell lines coupled with epigenome mapping data from patient-derived xenografts, we uncovered that deltaNp63 mainly exerts its effects by activating subtype-specific super enhancers. Furthermore, we identified a group of 45 subtype-specific super enhancers that are associated with poorer prognosis and are highly dependent on deltaNp63. Genes associated with these enhancers included a network of transcription factors, including HIF1A, BHLHE40, and RXRA, which form a highly intertwined transcriptional regulatory network with deltaNp63 to further activate downstream genes associated with poor survival., Competing Interests: The authors declare no conflict of interest.

Significant attention has recently been given to a class of enhancers termed "super enhancers", while implying that "typical enhancers" are less important. In this report, we examine criteria for identification of super enhancers and address the need to evaluate the differences between BRD4-occupied "typical" and "super" enhancers.

Bromodomain-containing protein 4 (BRD4) is a member of the bromo- and extraterminal (BET) domain-containing family of epigenetic readers which is under intensive investigation as a target for anti-tumor therapy. BRD4 plays a central role in promoting the expression of select subsets of genes including many driven by oncogenic transcription factors and signaling pathways. However, the role of BRD4 and the effects of BET inhibitors in non-transformed cells remain mostly unclear. We demonstrate that BRD4 is required for the maintenance of a basal epithelial phenotype by regulating the expression of epithelial-specific genes including TP63 and Grainy Head-like transcription factor-3 (GRHL3) in non-transformed basal-like mammary epithelial cells. Moreover, BRD4 occupancy correlates with enhancer activity and enhancer RNA (eRNA) transcription. Motif analyses of cell context-specific BRD4-enriched regions predicted the involvement of FOXO transcription factors. Consistently, activation of FOXO1 function via inhibition of EGFR-AKT signaling promoted the expression of TP63 and GRHL3. Moreover, activation of Src kinase signaling and FOXO1 inhibition decreased the expression of FOXO/BRD4 target genes. Together, our findings support a function for BRD4 in promoting basal mammary cell epithelial differentiation, at least in part, by regulating FOXO factor function on enhancers to activate TP63 and GRHL3 expression., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Hypoxia plays a significant role in tumor progression and aggressiveness and implicated in resistance to radiotherapy and chemotherapy. This study aims to characterize the changes in gene expression associated with chronic hypoxia in MCF7 breast cancer cell line and identify a possible biomarker for hypoxia in breast cancer. Breast cancer cells (MCF7) were exposed to 8-hour hypoxic episodes (<1% oxygen) three times a week for a total of 38 episodes. Gene expression changes were profiled using RT- PCR array after 19 and 38 episodes of hypoxia and compared to normoxic cells. Chemoresistance of hypoxic cells toward doxorubicin was assessed using MTT cell proliferation assay. Marked gene expression changes were indentified after 19 and 38 episodes of hypoxia. Only few changes were common in both stages with most genes rebounding at the level of 38 episodes. A notable gene (HNF4A) has been up-regulated by 2 folds after 19 hypoxic shots and further up-regulated by 6.43 folds after 38 hypoxic shots. The half maximal inhibitory concentration (IC50) of doxorubicin in MCF7 cells has increased in a trend proportional to the number of hypoxic episodes then totally rebounded after incubation under normoxia for 3 weeks. This study provides evidence that exposing cells to prolonged periods of hypoxia (weeks) results in different expression changes than those induced by short-term hypoxia (less than 72h)., (Copyright © 2014 Elsevier Inc. All rights reserved.)