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Lysine crotonylation (Kcr), a newly discovered post-translational modification, played a crucial role in physiology and disease progression. However, the roles of crotonylation in oocyte meiotic resumption remain elusive. As abnormal cumulus cell development will cause oocyte maturation arrest and female infertility, we report that cumulus cells surrounding human meiotic arrested oocytes showed significantly lower crotonylation, which was associated with decreased EP300 expression and blocked cumulus cell expansion. In cultured human cumulus cells, exogenous crotonylation or EP300 activator promoted cell proliferation and reduced cell apoptosis, whereas EP300 knockdown induced the opposite effect. Transcriptome profiling analysis in human cumulus cells indicated that functions of crotonylation were associated with activation of epidermal growth factor receptor (EGFR) pathway. Importantly, we characterized the Kcr proteomics landscape in cumulus cells by LC-MS/MS analysis, and identified that annexin A2 (ANXA2) was crotonylated in cumulus cells in an EP300-dependent manner. Crotonylation of ANXA2 enhanced the ANXA2-EGFR binding, and then activated the EGFR pathway to affect cumulus cell proliferation and apoptosis. Using mouse oocytes IVM model and EP300 knockout mice, we further confirmed that crotonylation alteration in cumulus cells affected the oocyte maturation. Together, our results indicated that EP300-mediated crotonylation is important for cumulus cells functions and oocyte maturation., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cervical cancer, a common malignancy in women, poses a significant health burden worldwide. In this study, we aimed to investigate the expression, function, and potential mechanisms of NADH: ubiquinone oxidoreductase subunit A8 (NDUFA8) in cervical cancer. The Gene Expression Profiling Interactive Analysis (GEPIA) database and immunohistochemical scoring were used to analyze NDUFA8 expression in cervical cancer tissues and normal tissues. Quantitative real-time PCR and Western blot analyses were performed to assess the expression level of NDUFA8 in cervical cancer cell lines. NDUFA8 knockdown or overexpression experiments were conducted to evaluate its impact on cell proliferation and apoptosis. The mitochondrial respiratory status was analyzed by measuring cellular oxygen consumption, adenosine triphosphate (ATP) levels, and the expression levels of Mitochondrial Complex I activity, and Mitochondrial Complex IV-associated proteins Cytochrome C Oxidase Subunit 5B (COX5B) and COX6C. NDUFA8 exhibited high expression levels in cervical cancer tissues, and these levels were correlated with reduced survival rates. A significant upregulation of NDUFA8 expression was observed in cervical cancer cell lines compared to normal cells. Silencing NDUFA8 hindered cell proliferation, promoted apoptosis, and concurrently suppressed cellular mitochondrial respiration, resulting in decreased levels of available ATP. Conversely, NDUFA8 overexpression induced the opposite effects. Herein, we also found that E1A Binding Protein P300 (EP300) overexpression facilitated Histone H3 Lysine 27 (H3K27) acetylation enrichment, enhancing the activity of the NDUFA8 promoter region. NDUFA8, which is highly expressed in cervical cancer, is regulated by transcriptional control via EP300/H3K27 acetylation. By promoting mitochondrial respiration, NDUFA8 contributes to cervical cancer cell proliferation and apoptosis. These findings provide novel insights into NDUFA8 as a therapeutic target in cervical cancer., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare that are relevant to the content of this article., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The transcriptional activation function of YAP in cancer development has been widely studied. However, the underlying regulatory mechanisms remain largely unknown. In this study, we found that EP300, one histone acetyltransferase, interacted with YAP and was recruited into the phase separated condensates of YAP. Transcriptomic analysis revealed substantial alterations in gene expression upon EP300 depletion, with downregulated genes associated with cancer progression and Hippo-YAP pathway. Notably, disruption of EP300 inhibited the transcriptional activation of YAP and reduced the binding of H3K27ac on YAP target oncogenes in Hippo pathway. Moreover, depletion of EP300 effectively inhibited YAP-driven tumor growth. Taken together, these results indicate that EP300 contributes to lung cancer progression by promoting the oncogenic transcription of YAP through H3K27ac, which suggests that YAP-EP300 axis may be potential therapeutic targets for lung cancer treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Aim: This study investigated the role of lncRNA LINC01232 in ferroptosis of colorectal cancer (CRC). Materials & methods: Real time quantitative polymerase chain reaction or western blot experiments were performed to examine relevant mRNAs and proteins expression. The kit assays evaluated malondialdehyde, iron, Fe 2+ and glutathione levels. ROS levels were verified by flow cytometry. Chromatin immunoprecipitation and RNA immunoprecipitation analysis monitored the correlation among LINC01232 , H3K27ac, p300 and ARNTL2. Results: LINC01232 or ARNTL2 knockdown facilitated erastin-induced ferroptosis. The interaction between LINC01232 and p300 resulted in the enhancement of H3K27ac levels at ARNTL2 promoter to promote ARNTL2 transcriptional activity. ARNTL2 overexpression reversed the promoting effect of LINC01232 knockdown on ferroptosis. Conclusion: LINC01232 inhibited the ferroptosis in CRC by epigenetically upregulating the transcriptional activity of ARNTL2 .

Background: EP300 (E1A binding protein p300) played a significant role in serial diseases such as cancer, neurodegenerative disease. Therefore, it became a significant target., Methods: Targeting EP300 discovery of a novel drug to alleviate these diseases. In this paper, 17 candidate compounds were obtained using a structure-based virtual screening approach, 4449-0460, with an IC 50 of 5.89 ± 2.08 uM, which was identified by the EP300 bioactivity test. 4449-0460 consisted of three rings. The middle benzene ring connected the 5-ethylideneimidazolidine-2,4-dione group and the 3-F-Phenylmethoxy group., Results: Furthermore, the interaction mechanism between 4449-0460 and EP300 was explored by combining molecular dynamics (MD) simulations and binding free energy calculation methods., Conclusion: The binding free energy of EP300 with 4449-0460 was -10.93 kcal/mol, and mainly came from the nonpolar energy term (ΔG nonpolar ). Pro1074, Phe1075, Val1079, Leu1084, and Val1138 were the key residues in EP300/4449-0460 binding with more -1 kcal/mol energy contribution. 4449-0460 was a promising inhibitor targeting EP300, which had implications for the development of drugs for EP300-related diseases.

Helix-loop-helix (HLH) transcription factors (TFs) play a key role in various cellular differentiation and function through the regulation of enhancer activity. E2A, a member of the mammalian E-protein family (class I HLH protein), is well known to play an important role in hematopoiesis, especially in adaptive lymphocyte development. E2A instructs B- and T-cell lineage development through the regulation of enhancer activity for B- or T-cell signature gene expression, including Rag1 and Rag2 (Rag1/2) genes. In this chapter, we mainly focus on the function of E2A in B-cell development and on the roles of E2A in establishing the enhancer landscape through the recruitment of EP300/KAT3B, chromatin remodeling complex, mediator, cohesion, and TET proteins. Finally, we demonstrate how E2A orchestrates the assembly of the Rag1/2 gene super-enhancer (SE) formation by changing the chromatin conformation across the Rag gene locus., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Emerging evidence portray the involvement of epigenomic reprogramming in the onset and progression of several malignancies, including breast cancer. Histone acetyltransferase (HAT) p300 is a critical epigenetic regulator that acts as a transcription co-activator and regulates various cellular processes. p300 is overexpressed in breast cancer and promotes cellular invasion and survival, making it a promising druggable target. In this study, the relevance of p300 in different cancer pathways was established. Virtual screening of the FDA-approved drug library was carried out using molecular docking, and the top 10 potential repurposed drugs were identified. Further, recalculation of binding free energy of drug-p300 complexes was carried out using molecular mechanics Poisson-Boltzmann and surface area (MM-PBSA) method after molecular dynamic simulation. Based on molecular dynamic simulation parameters and binding free energy analysis, two drugs, namely Netarsudil (-305.068 kJ/mol) and Imatinib (-260.457 kJ/mol), were identified as potential repurposed drugs to inhibit the activity of p300. In conclusion, these findings suggest, Netarsudil and Imatinib might be a potential repurposed drug to combat breast cancer via p300 inhibition.Communicated by Ramaswamy H. Sarma.

Oxidized low-density lipoprotein (ox-LDL) mediated inflammatory damage, which possibly induces atherosclerosis (AS); however, the role of miRNA in this process has rarely been reported. In this paper, we study the ox-LDL-related endothelial cell damage and changes of macrophages. The bioinformatics method was used to analyze the expression changes of miRNA in AS patients, luciferase assay was used to study the interaction of protein and miRNA, and co-IP and ubiquitination experiments were used to analyze protein interaction. Flow cytometry was used to detect the polarization of macrophages. Database analysis showed that the expression of miR-21-5p was upregulated in AS patients. Luciferase assay showed that miR-21-5p can bind to SKP2 and subsequently influence ubiquitination of EP300. Overexpression of EP300 strengthens the HMGB1-induced acetylation and subsequently mediates the dissociation of HMGB1 from SIRT1, and thus HMGB1 could be secreted outside the cell. The HMGB1 released from endothelial cells can promote macrophage M1 polarization. This study shows that ox-LDL activates the SKP2/EP300 pathway through promoting upregulation of miR-21-5p, thereby acetylating and secreting HMGB1 outside the endothelium, subsequently enhancing macrophage polarization to further stabilize the inflammation situation., (© 2023 Wiley Periodicals LLC.)

The internal exposure dose of bisphenol S (BPS) is increasing since its use as a substitute for BPA. The relationship between BPS and nonalcoholic liver disease (NAFLD) and the underlying mechanism remain unclarified. In this study, we evaluated the correlation of BPS with NAFLD in populations from the Jiangsu Survey and the 2013-2016 National Health Nutrition Examination Survey and unraveled the molecular pathway by which BPS blocked hepatic autophagy, contributing to lipid accumulation. The study found that serum and urine BPS were associated with NAFLD risks in both the Chinese and US populations. For each additional unit of the BPS level, the NAFLD risk increased by 3.163-fold (serum) and 3.979-fold (urine) in the Chinese population. In addition, after BPS exposure at a dose equivalent to human exposure for 20 weeks, mice developed liver lipid accumulation. BPS could trigger PPARα-mediated transcriptional activation of EP300 expression. BPS promoted the translocation of EP300 from the nucleus to the cytoplasm to regulate the acetylation of Raptor and the activation of mTORC1, which in turn induced autophagy blockage and interfered with lipid degradation in hepatocytes. Conversely, knockdown of EP300 reduced Raptor acetylation and ameliorated autophagy blockage. This study demonstrated that EP300 was a key enzyme for the development of BPS-related NAFLD and provided novel evidence that BPS causes NAFLD.

Surgical resection remains the primary approach for treating colorectal cancer, which is among the prevalent types of cancers affecting the digestive system. Tumor-infiltrating lymphocyte (TIL) therapy has emerged as a prominent area of study in the field of tumor immunotherapy in recent times, with the potential to serve as a supplementary treatment for colorectal cancer. For this investigation, we employed single-cell sequencing data to assess the manifestation extent of miR-26a-5p exists in healthy colon tissue, tissue affected by colorectal cancer, and tissue adjacent to the tumor. According to our findings, tumor-infiltrating T lymphocytes express comparatively less miR-26a-5p in comparison to normal T lymphocytes, the role of it in modulating the function of tumor-infiltrating T lymphocytes is suggested. Studies on miR-26a-5p's involvement in tumor-infiltrating T lymphocytes is limited, despite previous evidence indicating its ability to facilitate the development and advancement of cancerous cells. As a result of our experiments, we concluded that miR-26a-5p hindered the PI3K/AKT/mTOR(PAM) signaling pathway, reducing the ability of CD8+ tumor-infiltrating cells eradicate tumors. Using bioinformatics tools, we utilized prediction methods to identify EP300 as the specific gene targeted by miR-26a-5p. Subsequent research understood that downregulation of EP300 counteracted the suppressive impact exerted by miR-26a-5p on the stimulation of PAM signaling pathway, while it also diminishes the viability and cytotoxicity of CD8+ tumor-infiltrating lymphocytes. Therefore, miR-26a-5p emerges as a compelling option for the effective control of TIL therapy.

Melanomas can adopt multiple transcriptional states. Little is known about the epigenetic drivers of these cell states, limiting our ability to regulate melanoma heterogeneity. Here, we identify stress-induced HDAC8 activity as driving melanoma brain metastasis development. Exposure of melanocytes and melanoma cells to multiple stresses increases HDAC8 activation leading to a neural crest-stem cell transcriptional state and an amoeboid, invasive phenotype that increases seeding to the brain. Using ATAC-Seq and ChIP-Seq we show that increased HDAC8 activity alters chromatin structure by increasing H3K27ac and enhancing accessibility at c-Jun binding sites. Functionally, HDAC8 deacetylates the histone acetyltransferase EP300, causing its enzymatic inactivation. This, in turn, increases binding of EP300 to Jun-transcriptional sites and decreases binding to MITF-transcriptional sites. Inhibition of EP300 increases melanoma cell invasion, resistance to stress and increases melanoma brain metastasis development. HDAC8 is identified as a mediator of transcriptional co-factor inactivation and chromatin accessibility that drives brain metastasis., (© 2023. The Author(s).)

Importance: African swine fever (ASF) is an acute, hemorrhagic, and severe porcine infectious disease caused by African swine fever virus (ASFV). ASF outbreaks severely threaten the global pig industries and result in serious economic losses. No safe and efficacious commercial vaccine is currently available except in Vietnam. To date, large gaps in the knowledge concerning viral biological characteristics and immunoevasion strategies have hindered the ASF vaccine design. In this study, we demonstrate that pD129L negatively regulates the type I interferon (IFN) signaling pathway by interfering with the interaction of the transcriptional coactivator p300 and IRF3, thereby inhibiting the induction of type I IFNs. This study reveals a novel immunoevasion strategy employed by ASFV, shedding new light on the intricate mechanisms for ASFV to evade the host immune responses., Competing Interests: The authors declare no conflict of interest.

Background: Histone methyltransferases are crucial regulators in non-small cell lung cancer (NSCLC) development. This study explored the mechanism of histone methyltransferase SET domain containing 1A (SETD1A)-mediated H3K4me2 methylation in NSCLC cell ferroptosis and provides novel targets for NSCLC treatment., Methods: Upon downregulation of SETD1A in NSCLC cell lines, cell proliferation potential, malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) activities, iron content, and SETD1A, long noncoding RNA HOXC cluster antisense RNA 3 (lncRNA HOXC-AS3), E1A binding protein p300 (EP300), glutathione peroxidase 4 (GPX4) expressions were determined via cell counting kit-8, ELISA, iron assay kits, RT-qPCR, and western blot. Enrichment levels of SETD1A and H3K4me3 in the HOXC-AS3 promotor were measured via chromatin immunoprecipitation, and the binding of HOXC-AS3 and EP300 was analyzed via RNA immunoprecipitation. Rescue experiments were performed to confirm their roles in NSCLC cell ferroptosis. Xenograft tumor models were established to validate the role of SETD1A in vivo., Results: SETD1A, H3K4me3, HOXC-AS3, and EP300 were highly-expressed in NSCLC cells. Silencing SETD1A inhibited NSCLC cell proliferation, increased MDA and iron levels, and decreased SOD, GSH, and GPX4 levels. SETD1A downregulation reduced H3K4me3 level, HOXC-AS3 expression, the binding of HOXC-AS3 to EP300, and EP300 stability. Overexpression of HOXC-AS3 or EP300 reversed the promotion of silencing SETD1A on NSCLC cell ferroptosis. Silencing SETD1A reduced tumor volume and weight and positive rate of ki67 and increased ferroptosis through the HOXC-AS3/EP300 axis., Conclusion: SETD1A-mediated H3K4me2 methylation promoted HOXC-AS3 expression, binding of HOXC-AS3 to EP300, and EP300 stability, thereby suppressing NSCLC cell ferroptosis., (© 2023 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Fibrosis is a condition characterized by the excessive accumulation of extracellular matrix proteins in tissues, leading to organ dysfunction and failure. Recent studies have identified EP300, a histone acetyltransferase, as a crucial regulator of the epigenetic changes that contribute to fibrosis. In fact, EP300-mediated acetylation of histones alters global chromatin structure and gene expression, promoting the development and progression of fibrosis. Here, we review the role of EP300-mediated epigenetic regulation in multi-organ fibrosis and its potential as a therapeutic target. We discuss the preclinical evidence that suggests that EP300 inhibition can attenuate fibrosis-related molecular processes, including extracellular matrix deposition, inflammation, and epithelial-to-mesenchymal transition. We also highlight the contributions of small molecule inhibitors and gene therapy approaches targeting EP300 as novel therapies against fibrosis.

Introduction: EP300 is considered to be a cancer suppressor gene that plays a role in tumor development, but some studies have reported that it is not an oral squamous cell carcinoma suppressor gene, because there was neither epigenetic inactivation of the gene nor a mutation resulting in functional impairment. However, there is no relevant study on whether EP300 is the exact carcinogenic effect and its mechanisms of carcinogenic effects in oral squamous cell carcinoma., Methods: Western blot analysis and quantitative real time polymerase chain reaction experiments verified the protein and mRNA expression of EP300 in oral squamous cell carcinoma; The effects of EP300 knockout on glucose consumption and lactic acid production were detected by glycolysis experiments; The relationship between pathway-related proteins and EP300 was verified by bioinformatics analysis and co-immunoprecipitation experiment., Results: Our experimental results confirm that the protein and mRNA of EP300 are highly expressed in oral squamous cell carcinoma, and after knocking out the EP300, the glycolysis ability, invasion, migration, and other biological functions of oral squamous cell carcinoma, are inhibited at the same time. Pathway-related experiments have confirmed that EP300 plays a role in promoting cancer through the transforming growth factor-beta receptor II (TGF-βRII)/EP300/Smad4 cascade pathway., Conclusion: EP300 plays a carcinogenic role in OSCC showed that the TGF-βRII/EP300/Smad4 cascade pathway is involved in oral squamous cell carcinoma., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

As one of the major acetyltransferases in mammalian cells, p300 (also known as EP300) and its highly related protein CBP (also known as CREBBP), collectively termed p300/CBP, is characterized as a key regulator in gene transcription by modulating the acetylation of histones. In recent decades, proteomic analyses have revealed that p300 is also involved in the regulation of various cellular processes by acetylating many non-histone proteins. Among the identified substrates, some are key players involved in different autophagy steps, which together establish p300 as a master regulator of autophagy. Accumulating evidence has shown that p300 activity is controlled by many distinct cellular pathways to regulate autophagy in response to cellular or environmental stimuli. In addition, several small molecules have been shown to regulate autophagy by targeting p300, suggesting that manipulation of p300 activity is sufficient for controlling autophagy. Importantly, dysfunction of p300-regulated autophagy has been implicated in a number of human disorders, such as cancer, aging and neurodegeneration, highlighting p300 as a promising target for the drug development of autophagy-related human disorders. Here, we focus on the roles of p300-mediated protein acetylation in the regulation of autophagy and discuss implications for autophagy-related human disorders., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Background: Bladder cancer (BCa) is the most common malignant tumor of the urinary system, with transitional cell carcinoma (TCC) being the predominant type. EP300 encodes a lysine acetyltransferase that regulates a large subset of genes by acetylating histones and non-histone proteins. We previously identified several bladder cancer-associated mutations in EP300 using high-throughput sequencing; however, the functional consequences of these mutations remain unclear., Methods: Bladder cancer cells T24 and TCC-SUP were infected with shEP300 lentiviruses to generate stable EP300 knockdown cell lines. The expression levels of EP300, p16 and p21 were detected by real-time PCR and western blots. The transcriptional activity of p16 and p21 were detected by dual luciferase assay. Cell proliferation assay, flow cytometric analyses of cell cycle, invasion assay and xenograft tumor model were used to measure the effect of EP300-R1627W mutation in bladder cancer. Immunoprecipitation was used to explore the relationship between EP300-R1627W mutation and p53. Structural analysis was used to detect the structure of EP300-R1627W protein compared to EP300-wt protein., Results: we screened the mutations of EP300 and found that the EP300-R1627W mutation significantly impairs EP300 transactivation activity. Notably, we demonstrated that the R1627W mutation impairs EP300 acetyltransferase activity, potentially by interfering with substrate binding. Finally, we show that EP300-R1627W is more aggressive in growth and invasion in vitro and in vivo compared to cells expressing EP300-wt. We also found that the EP300-R1627W mutation occurs frequently in seven different types of cancers., Conclusion: In summary, our work defines a driver role of EP300-R1627W in bladder cancer development and progression., (© 2023. The Author(s).)

Patients carrying autosomal dominant mutations in the histone/lysine acetyl transferases CBP or EP300 develop a neurodevelopmental disorder: Rubinstein-Taybi syndrome (RSTS). The biological pathways underlying these neurodevelopmental defects remain elusive. Here, we unravel the contribution of a stress-responsive pathway to RSTS. We characterize the structural and functional interaction between CBP/EP300 and heat-shock factor 2 (HSF2), a tuner of brain cortical development and major player in prenatal stress responses in the neocortex: CBP/EP300 acetylates HSF2, leading to the stabilization of the HSF2 protein. Consequently, RSTS patient-derived primary cells show decreased levels of HSF2 and HSF2-dependent alteration in their repertoire of molecular chaperones and stress response. Moreover, we unravel a CBP/EP300-HSF2-N-cadherin cascade that is also active in neurodevelopmental contexts, and show that its deregulation disturbs neuroepithelial integrity in 2D and 3D organoid models of cerebral development, generated from RSTS patient-derived iPSC cells, providing a molecular reading key for this complex pathology., (© 2022. The Author(s).)

Background: RBBP4 activates transcription by histone acetylation, but the partner histone acetyltransferases are unknown. Thus, we investigated the hypothesis that RBBP4 interacts with p300 in a complex in glioblastoma (GBM)., Methods: shRNA silencing of RBBP4 or p300 and RNAseq was used to identify genes co-regulated by RBBP4 and p300 in GBM43 patient-derived xenograft (PDX). RBBP4/p300 complex was demonstrated using proximity ligation assay (PLA) and ChIPseq delineated histone H3 acetylation and RBBP4/p300 complex binding in promoters/enhancers. Temozolomide (TMZ)-induced DNA double strand breaks (DSBs) were evaluated by γ-H2AX and proliferation by CyQuant and live cell monitoring assays. In vivo efficacy was based on survival of mice with orthotopic tumors., Results: shRBBP4 and shp300 downregulated 4768 genes among which 1485 (31%) were commonly downregulated by both shRNAs, while upregulated genes were 2484, including 863 (35%) common genes. The pro-survival genes were the top-ranked among the downregulated genes, including C-MYC. RBBP4/p300 complex was demonstrated in the nucleus, and shRBBP4 or shp300 significantly sensitized GBM cells to TMZ compared to the control shNT in vitro (P < .05). Moreover, TMZ significantly prolonged the survival of mice bearing GBM22-shRBBP4 orthotopic tumors compared with control shNT tumors (median shNT survival 52 days vs. median shRBBP4 319 days; P = .001). CREB-binding protein (CBP)/p300 inhibitor CPI-1612 suppressed H3K27Ac and RBBP4/p300 complex target proteins, including C-MYC, and synergistically sensitized TMZ in vitro. Pharmacodynamic evaluation confirmed brain penetration by CPI-1612 supporting further investigation to evaluate efficacy to sensitize TMZ., Conclusions: RBBP4/p300 complex is present in GBM cells and is a potential therapeutic target., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Angiogenesis is required in many physiological conditions, including bone regeneration, wound healing, and tissue regeneration. Mesenchymal stem cells-derived extracellular matrix (MSCs-ECM) could guide intricate cellular and tissue processes such as homeostasis, healing and regeneration., Methods: The purpose of this study is to explore the effect and mechanism of ECM derived from decellularized Wharton's Jelly-derived mesenchymal stem cells (WJ-MSCs) on endothelial cell viability and angiogenesis. The human umbilical vein endothelial cells (HUVECs) were pretreated with WJ-MSCs ECM for 2d/7d/14d, respectively. After pretreatment, the angiogenesis ability of HUVECs was detected., Results: In this study, we found for the first time that WJ-MSCs ECM could improve the angiogenesis ability of HUVECs with a time-dependent manner in vitro. Mechanically, WJ-MSCs ECM activated the focal adhesion kinase (FAK)/P38 signaling pathway via integrin αVβ3, which further promoted the expression of the cellular (c)-Myc. Further, c-Myc increased histone acetylation levels of the vascular endothelial growth factor (VEGF) promoter by recruiting P300, which ultimately promoting VEGF expression., Conclusions: ECM derived from Wharton's Jelly-derived mesenchymal stem cells promotes angiogenesis via integrin αVβ3/c-Myc/P300/VEGF. This study is expected to provide a new approach to promote angiogenesis in bone and tissue regeneration., (© 2022. The Author(s).)

LncRNAs partake in the biological processes contributing to development of autism spectrum disorder (ASD). The aim of the present study is to investigate the effects of lncRNA maternally expressed gene 3 (MEG3) on viability and apoptosis of hippocampal neurons from ASD rats. Rats with ASD were induced using valproic acid (VPA) with normal saline (NS) as control. We performed microarray analysis on hippocampal tissues of NS rats and ASD rats to screen the differentially expressed lncRNAs. MEG3 loss in rats alleviated the impairment of learning and memory abilities induced by VPA, and promoted neuronal viability and inhibited apoptosis. MEG3 could recruit the transcription factor E1A binding protein p300 (EP300) in the nucleus and promote the cadherin 2 (CDH2) expression. CDH2 depletion in rats ameliorated the impairment of learning and memory capacities in ASD rats. After upregulation of CDH2 in neurons with sh-MEG3, we found diminished viability and increased apoptosis in hippocampal neurons of ASD rats. Taken together, MEG3 supports activation of CDH2 via EP300, thus repressing the viability of hippocampal neurons. Therefore, MEG3 upregulation may be partially responsible for the pathogenesis of ASD., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The rapid transcriptional response to the transcription factor, glucocorticoid receptor (GR), including gene activation or repression, is mediated by the spatial association of genes with multiple GR binding sites (GBSs) over large genomic distances. However, only a minority of the GBSs have independent GR-mediated activating capacity, and GBSs with independent repressive activity were rarely reported. To understand the positive and negative effects of GR we mapped the regulatory environment of its gene targets. We show that the chromatin interaction networks of GR-activated and repressed genes are spatially separated and vary in the features and configuration of their GBS and other non-GBS regulatory elements. The convergence of the KLF4 pathway in GR-activated domains and the STAT6 pathway in GR-repressed domains, impose opposite transcriptional effects to GR, independent of hormone application. Moreover, the ROR and Rev-erb transcription factors serve as positive and negative regulators, respectively, of GR-mediated gene activation. We found that the spatial crosstalk between GBSs and non-GBSs provides a physical platform for sequestering the Ep300 co-activator from non-GR regulatory loci in both GR-activated and -repressed gene compartments. While this allows rapid gene repression, Ep300 recruitment to GBSs is productive specifically in the activated compartments, thus providing the basis for gene induction., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Wnt signaling regulates the balance between stemness and differentiation in multiple tissues and in cancer. RNF43-mutant pancreatic cancers are dependent on Wnt production, and pharmacologic blockade of the pathway, e.g., by PORCN inhibitors, leads to tumor differentiation. However, primary resistance to these inhibitors has been observed. To elucidate potential mechanisms, we performed in vivo CRISPR screens in PORCN inhibitor-sensitive RNF43-mutant pancreatic cancer xenografts. As expected, genes in the Wnt pathway whose loss conferred drug resistance were identified, including APC, AXIN1, and CTNNBIP1. Unexpectedly, the screen also identified the histone acetyltransferase EP300 (p300), but not its paralog, CREBBP (CBP). We found that EP300 is silenced due to genetic alterations in all the existing RNF43-mutant pancreatic cancer cell lines that are resistant to PORCN inhibitors. Mechanistically, loss of EP300 directly downregulated GATA6 expression, thereby silencing the GATA6-regulated differentiation program and leading to a phenotypic transition from the classical subtype to the dedifferentiated basal-like/squamous subtype of pancreatic cancer. EP300 mutation and loss of GATA6 function bypassed the antidifferentiation activity of Wnt signaling, rendering these cancer cells resistant to Wnt inhibition.

Background: Preeclampsia is a pregnancy-related complication that causes maternal and fetal mortality. Despite extensive studies showing the role of hypoxia in preeclampsia progression, the specific mechanism remains unclear. The purpose of this study was to explore the possible mechanism underlying hypoxia in preeclampsia., Methods: Human trophoblast-like JEG-3 cell line was used to investigate the molecular mechanisms underlying hypoxia contribution to preeclampsia and the expression correlation of key molecules was examined in human placental tissues. Methods include JEG-3 cell culture and hypoxia induction, RNA isolation and quantitative real-time PCR, transient transfection and dual-luciferase assay, western blot, immunoprecipitation, immunofluorescence staining, cell proliferation assay, chromatin immunoprecipitation assay, obtainment of human placental tissue sample and immunohistochemistry staining., Results: Hypoxia-Inducible Factor-1α is up-regulated in clinical preeclampsia samples, where Regulator of G Protein Signaling 2 is down-regulated. Mechanistically, Hypoxia-Inducible Factor-1α is induced in response to hypoxia, which up-regulates E1A binding protein P300 expression and thereby forms a Hypoxia-Inducible Factor-1α/E1A binding protein P300 protein-protein complex that binds to the promoter of gene Regulator of G Protein Signaling 2 and subsequently inhibits the transcription of Regulator of G Protein Signaling 2, possibly contributing to the preeclampsia development. In addition, the expression of E1A binding protein P300 is increased in preeclampsia samples, and the expression of Regulator of G Protein Signaling 2 in preeclamptic placentas inversely correlates with the levels of E1A binding protein P300., Conclusion: Our findings may provide novel insights into understanding the molecular pathogenesis of preeclampsia and may be a prognostic biomarker and therapeutic target for preeclampsia., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Aims: Arrhythmogenic cardiomyopathy (ACM) is a primary myocardial disease that typically manifests with cardiac arrhythmias, progressive heart failure, and sudden cardiac death (SCD). ACM is mainly caused by mutations in genes encoding desmosome proteins. Desmosomes are cell-cell adhesion structures and hubs for mechanosensing and mechanotransduction. The objective was to identify the dysregulated molecular and biological pathways in human ACM in the absence of overt heart failure., Methods and Results: Transcriptomes in the right ventricular endomyocardial biopsy samples from three independent individuals carrying truncating mutations in the DSP gene and five control samples were analysed by RNA-Seq (discovery group). These cases presented with cardiac arrhythmias and had a normal right ventricular function. The RNA-Seq analysis identified ∼5000 differentially expressed genes (DEGs), which predicted suppression of the Hippo and canonical WNT pathways, among others. Dysregulated genes and pathways, identified by RNA-Seq, were tested for validation in the right and left ventricular tissues from five independent autopsy-confirmed ACM cases with defined mutations (validation group), who were victims of SCD and had no history of heart failure. Protein levels and nuclear localization of the cWNT and Hippo pathway transcriptional regulators were reduced in the right and left ventricular validation samples. In contrast, levels of acetyltransferase EP300, known to suppress the Hippo and canonical WNT pathways, were increased and its bona fide target TP53 was acetylated. RNA-Seq data identified apical junction, reflective of cell-cell attachment, as the most disrupted biological pathway, which were corroborated by disrupted desmosomes and intermediate filament structures. Moreover, the DEGs also predicted dysregulation of over a dozen canonical signal transduction pathways, including the Tec kinase and integrin signalling pathways. The changes were associated with increased apoptosis and fibro-adipogenesis in the ACM hearts., Conclusion: Altered apical junction structures are associated with activation of the EP300-TP53 and suppression of the Hippo/cWNT pathways in human ACM caused by defined mutations in the absence of an overt heart failure. The findings implicate altered mechanotransduction in the pathogenesis of ACM., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

Objectives: Tobacco hazard is one of the most severe public health issues in the world. It is believed that smoking is the most important factor leading to chronic obstructive pulmonary disease (COPD). Endothelial progenitor cells (EPCs) originate from the bone marrow and can effectively repair vascular endothelial damage and improve vascular endothelial function. Current studies suggest that EPCs senescence and EPCs depletion exist in smoking-related COPD, but the molecular mechanism remains unclear., Methods: Co-immunoprecipitation was used to detect the interaction between USP7 and p300. EPCs from smoking COPD patients were isolated, and the expressions of USP7 and p300 were detected by RT-PCR and Western Blot. Different concentrations of cigarette smoke extract (CSE) and USP7 or p300 inhibitors were used to treat EPCs, then the expression of p53, p53 target genes and aging-related genes were detected. Cell Counting Kit - 8 (CCK8) was used to detect cell proliferation, flow cytometry was used to detect cell cycle distribution, β-galactosidase (β-gal) staining and Lamp1 immunofluorescence was used to detect the proportion of aging cells. COPD mouse models were used to confirm the molecular mechanism., Results: USP7 and p300 interacted with each other, and USP7 affected the protein stability of p300 by regulating the ubiquitination of p300. There existed high expressions of USP7 and p300 proteins in EPCs of smoking COPD patients and COPD mouse model. CSE promoted the high expressions of USP7 and p300 in EPCs. Further studies showed that CSE mediated the USP7/p300-dependent high expression of p53 and activated the expression of p53 target genes especially p21. Activation of p53 - p21 pathway finally inhibited cell activity, led to cell cycle arrest and premature senescence of EPCs., Conclusion: CSE mediated up-regulation of USP7 and p300 activated p53 - p21 pathway was a molecular mechanism that might lead to COPD., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Rheumatoid arthritis (RA) is chronic inflammatory autoimmune disease. The crucial role of long non-coding RNA (lncRNA) in the progression of RA has been highlighted. Hence, this study was designed to explore the specific downstream mechanism of lncRNA nuclear-enriched abundant transcript 1 (NEAT1) in RA. Initially, the expression of NEAT1, p-p65, p300, and IL-18 in clinical tissues and cells was determined. Then, interactions among p65, NEAT1, p300, CBP, and IL-18 were investigated by immunofluorescence staining, dual luciferase reporter gene assay, RT-qPCR assay ChIP assay, and RIP assay followed by the analysis of their effects on RA in vivo and in vitro after expression alteration. The expressions of NEAT1, p-p65, p300, and IL-18 were all upregulated in the synovial tissues from the mice and patients with RA. NEAT1 silencing reduced the infiltration of CD4 + T cells and macrophages in synovial tissues, downregulated expression of blood inflammatory factors, relieved RA severity, and lowered incidence of RA in mice. Further, p-p65 could increase the expression of NEAT1 by binding to the NEAT1 promoter region, NEAT1 could co-locate and interact with p300, thus regulating the expression of IL-18 by regulating histone acetylation modification in IL-18 promoter region. NEAT1 aggravated RA via p300/CBP/IL-18 axis, representing a promising therapeutic target in RA., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Tumour-associated macrophages (TAMs) play an important role in promoting the progression of pancreatic ductal adenocarcinoma (PDAC). Here, we aimed to study the epigenetic mechanisms in regulating pro-tumour M2-polarised TAMs in the PDAC tumour microenvironment., Methods: This study was conducted based on ex vivo TAMs isolated from PDAC tissues and in vitro THP1-derived TAM model. RNA-sequencing (RNA-seq), assay for transposase-accessible chromatin with sequencing and chromatin immunoprecipitation sequencing were performed to investigate gene expression, chromatin accessibility, transcription factor binding sites and histone modifications. Gene knockdown in THP1-derived TAMs was performed with lentivirus, and the impact of THP1-derived TAMs on invasion and metastasis ability of PDAC cells were investigated with in vitro and in vivo functional assays. RNA-chromatin interaction was analysed by chromatin isolation through RNA purification with sequencing. RNA-protein interaction was studied by RNA immunoprecipitation and RNA pull-down., Results: Our data showed that the transcription factor CTCF (CCCTC-binding factor) was highly expressed in TAMs and predicted to be significantly enriched in hyper-accessible chromatin regions when compared to monocytes. High infiltration of CTCF + TAMs was significantly associated with poor prognosis in PDAC patients. Knockdown of CTCF in THP1-derived TAMs led to the down-regulation of specific markers for M2-polarised TAMs, including CD206 and CD163. When THP1-derived TAMs with CTCF knockdown, they showed a decreased ability of invasion and metastasis. Further integrative analysis of multi-omics data revealed that prostaglandin-endoperoxide synthase 2 (PTGS2) and PTGS2 antisense NF-κB1 complex-mediated expression regulator RNA (PACERR) were critical downstream targets of CTCF and positively correlated with each other, which are closely situated on a chromosome. Knockdown of PACERR exhibited a similar phenotype as observed in CTCF knockdown THP1-derived TAMs. Moreover, PACERR could directly bind to CTCF and recruit histone acetyltransferase E1A binding protein p300 to the promoter regions of PACERR and PTGS2, thereby enhancing histone acetylation and gene transcription, promoting the M2 polarization of TAMs in PDAC., Conclusions: Our study demonstrated a novel epigenetic regulation mechanism of promoting pro-tumour M2-polarised TAMs in the PDAC tumour microenvironment., (© 2022 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Acute lymphoblastic leukemia with chromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene (MLL-r ALL) remains an incurable disease. Thus, development of a safe and effective therapeutic agent to treat this disease is crucial to address this unmet medical need. BRD4, a member of the bromodomain and extra-terminal domain (BET) protein family, and cyclic AMP response element binding protein binding protein (CBP) and p300, two paralogous histone acetyltransferases, are all considered cancer drug targets and simultaneous targeting of these proteins may have therapeutic advantages. Here, we demonstrate that a BET/CBP/p300 multi-bromodomain inhibitor, CN470, has anti-tumor activity against MLL-r ALL in vitro and in vivo. CN470, potently inhibited ligand binding to the bromodomains of BRD4, CBP, and p300 and suppressed the growth of MLL-r ALL cell lines and patient-derived cells with MLL rearrangements. CN470 suppressed mRNA and protein expression of MYC and induced apoptosis in MLL-r ALL cells, following a cell cycle arrest in the G1 phase. Moreover, CN470 reduced BRD4 binding to acetylated histone H3. The in vivo effects of CN470 were investigated using SEM Luc/GFP cells expressing luminescent markers in an orthotopic mouse model. Mice administered CN470 daily had prolonged survival compared to the vehicle group. Further, CN470 also showed anti-tumor effects against an MLL-r ALL patient-derived xenograft model. These findings suggest that inhibition of BET/CBP/p300 by the multi-bromodomain inhibitor, CN470, represents a promising therapeutic approach against MLL-r ALL., Competing Interests: Declaration of competing interest N. Imayoshi, K. Tanaka, S.-M. Yang, K. Akahane, Y. Toda, S. Hosogi, T. Inukai, S, Okada, D. J. Maloney, I. Kato and E. Ashihara have no financial conflict of interest to disclose. M. Yoshioka is an employee of ConverGene LLC and holds equity in the company., (Copyright © 2021. Published by Elsevier Inc.)

While current thinking posits that insulin signaling to glucose transporter 4 (GLUT4) exocytic translocation and glucose uptake in skeletal muscle and adipocytes is controlled by phosphorylation-based signaling, many proteins in this pathway are acetylated on lysine residues. However, the importance of acetylation and lysine acetyltransferases to insulin-stimulated glucose uptake is incompletely defined. Here, we demonstrate that combined loss of the acetyltransferases E1A binding protein p300 (p300) and cAMP response element binding protein binding protein (CBP) in mouse skeletal muscle caused a complete loss of insulin-stimulated glucose uptake. Similarly, brief (i.e., 1 hour) pharmacological inhibition of p300/CBP acetyltransferase activity recapitulated this phenotype in human and rodent myotubes, 3T3-L1 adipocytes, and mouse muscle. Mechanistically, these effects were due to p300/CBP-mediated regulation of GLUT4 exocytic translocation and occurred downstream of Akt signaling. Taken together, we highlight a fundamental role for acetylation and p300/CBP in the direct regulation of insulin-stimulated glucose transport in skeletal muscle and adipocytes.

Nuclear-localized epidermal growth factor receptor (EGFR) highly correlates with the malignant progression and may be a promising therapeutic target for breast cancer. However, molecular mechanisms of nuclear EGFR in triple-negative breast cancer (TNBC) have not been fully elucidated. Here, we performed gene-annotation enrichment analysis for the interactors of nuclear EGFR and found that RNA-binding proteins (RBPs) were closely associated with nuclear EGFR. We further demonstrated p54 nrb /NONO, one of the RBPs, significantly interacted with nuclear EGFR. NONO was upregulated in 80 paired TNBC tissues and indicated a poor prognosis. Furthermore, NONO knockout significantly inhibited TNBC proliferation in vitro and in vivo. Mechanistically, NONO increased the stability of nuclear EGFR and recruited CREB binding protein (CBP) and its accompanying E1A binding protein p300, thereby enhancing the transcriptional activity of EGFR. In turn, EGFR positively regulated the affinity of NONO to mRNAs of nuclear EGFR downstream genes. Furthermore, the results indicated that the nuclear EGFR/NONO complex played a critical role in tumorigenesis and chemotherapy resistance. Taken together, our findings indicate that NONO enhances nuclear EGFR-mediated tumorigenesis and may be a potential therapeutic target for TNBC patients with nuclear EGFR expression., (© 2021. The Author(s).)

Autophagic flux can be quantified based on the accumulation of lipidated LC3B in the presence of late-stage autophagy inhibitors. This method has been widely applied to identify novel compounds that activate autophagy. Here we scrutinize this approach and show that bafilomycin A1 (BafA) but not chloroquine is suitable for flux quantification due to the stimulating effect of chloroquine on non-canonical LC3B-lipidation. Significant autophagic flux increase by rapamycin could only be observed when combining it with BafA concentrations not affecting basal flux, a condition which created a bottleneck, rather than fully blocking autophagosome-lysosome fusion, concomitant with autophagy stimulation. When rapamycin was combined with saturating concentrations of BafA, no significant further increase of LC3B lipidation could be detected over the levels induced by the late-stage inhibitor. The large assay window obtained by this approach enables an effective discrimination of autophagy activators based on their cellular potency. To demonstrate the validity of this approach, we show that a novel inhibitor of the acetyltransferase EP300 activates autophagy in a mTORC1-dependent manner. We propose that the creation of a sensitized background rather than a full block of autophagosome progression is required to quantitatively capture changes in autophagic flux., (© 2022. The Author(s).)

CPVL (carboxypeptidase, vitellogenic-like) is a serine carboxypeptidase that was first characterized in human macrophages. However, the function of CPVL remains unclear in a variety of tumors. The quantitative PCR (qPCR), Western blotting, and IHC assays were utilized to measure the CPVL expression. CPVL was significantly upregulated in glioma cells and tissues compared with normal cells and tissues, respectively. Moreover, high CPVL expression was correlated with advanced clinical grade and poor prognosis. Silencing of CPVL promoted glioma cell apoptosis, and it inhibited cell proliferation and tumorigenicity in vitro and in vivo. Ingenuity Pathway Analysis (IPA) demonstrated that CPVL silencing activated the IFN-γ/STAT1 signaling pathway, thereby inducing glioma cell apoptosis. Mechanistically, immunopurification, mass spectrometry, IP, and glutathione S-transferase (GST) pull-down experiments elucidated that CPVL physically interacts with Bruton's tyrosine kinase (BTK) and downregulates the STAT1 phosphorylation through promoting p300-mediated STAT1 acetylation. Our findings reveal the crucial role of CPVL in promoting the progression of glioma through suppressing STAT1 phosphorylation. CPVL might serve as a potential prognostic biomarker and therapeutic target for the treatment of glioma.

Histone proteins are decorated with a combinatorially and numerically diverse set of biochemical modifications. Here, we describe a versatile and scalable approach which enables efficient characterization of histone modifications without the need for recombinant protein production. As proof-of-concept, we first use this system to rapidly profile the histone H3 and H4 residue writing specificities of the human histone acetyltransferase, p300. Subsequently, a large panel of commercially available anti-acetylation antibodies are screened for their specificities, identifying many suitable and unsuitable reagents. Furthermore, this approach enables efficient mapping of the large binary crosstalk space between acetylated residues on histones H3 and H4 and uncovers residue interdependencies affecting p300 activity. These results show that using yeast surface display to study histone modifications is a useful tool that can advance our understanding of chromatin biology by enabling efficient interrogation of the complexity of epigenome modifications., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Inhibitors of bromodomain and extra-terminal motif (BET) proteins are emerging epigenetic therapeutics that suppress gene expressions that drive cancer and inflammation. The present study examined anti-inflammatory effects of a quinazoline-based BET inhibitor, CN210, in a murine ileitis model. CN210 was given orally 30 min before and 24 h after a subcutaneous administration of indomethacin. Macroscopic and histological evidences of ileitis, mucosal myeloperoxidase (MPO) activity and cytokine expressions were evaluated 48 h after the indomethacin administration. To further characterize the anti-inflammatory pathways modulated by CN210, its effects on RAW264 cells treated with lipopolysaccharide (LPS) were investigated. Competitive ligand binding and docking studies of CN210 to CREB-binding protein (CBP) and p300 were also performed. Oral administration of CN210 significantly reduced the severity of ileitis, normalized both proinflammatory MPO activity and concomitant cytokine expressions induced by indomethacin administration. Furthermore, CN210 attenuated the expression of cytokines and reversed the activation of nuclear factor κB (NF-κB) and mitogen-activated protein kinases (MAPK) induced by LPS. Competitive ligand binding assays showed that CN210 bound to the bromodomains of two paralogous histone acetyltransferases, CBP and p300, in addition to the bromodomains of BET proteins. Docking studies of CN210 to the bromodomains of CBP and p300 showed a similarity to the binding mode of SGC-CBP30, a specific CBP/p300 inhibitor. CN210 ameliorates indomethacin-induced ileitis by inhibiting the expression of inflammatory cytokines through the attenuation of NF-κB and MAPK pathways. CN210 thus represents a new mode of therapy for non-steroidal anti-inflammatory drug-induced ileitis and inflammatory bowel disease., (© 2021 Wiley Periodicals LLC.)

We demonstrate for the first time the simultaneous measurement of the acetyltransferase (HAT) and crotonyltransferase (HCT) activities of histone acetylation writer p300 by integrating antibody-based fluorescence labeling with single molecule detection. This methods exhibits good specificity and high sensitivity. Moreover, it can accurately evaluate the kinetic parameters of both the HAT and HCT activities of p300 and screen inhibitors.

Despite radiation forming the curative backbone of over 50% of malignancies, there are no genomically-driven radiosensitizers for clinical use. Herein we perform in vivo shRNA screening to identify targets generally associated with radiation response as well as those exhibiting a genomic dependency. This identifies the histone acetyltransferases CREBBP/EP300 as a target for radiosensitization in combination with radiation in cognate mutant tumors. Further in vitro and in vivo studies confirm this phenomenon to be due to repression of homologous recombination following DNA damage and reproducible using chemical inhibition of histone acetyltransferase (HAT), but not bromodomain function. Selected mutations in CREBBP lead to a hyperacetylated state that increases CBP and BRCA1 acetylation, representing a gain of function targeted by HAT inhibition. Additionally, mutations in CREBBP/EP300 are associated with recurrence following radiation in squamous cell carcinoma cohorts. These findings provide both a mechanism of resistance and the potential for genomically-driven treatment., (© 2021. The Author(s).)

The ARID1A subunit of SWI/SNF chromatin remodeling complexes is a potent tumor suppressor. Here, a degron is applied to detect rapid loss of chromatin accessibility at thousands of loci where ARID1A acts to generate accessible minidomains of nucleosomes. Loss of ARID1A also results in the redistribution of the coactivator EP300. Co-incident EP300 dissociation and lost chromatin accessibility at enhancer elements are highly enriched adjacent to rapidly downregulated genes. In contrast, sites of gained EP300 occupancy are linked to genes that are transcriptionally upregulated. These chromatin changes are associated with a small number of genes that are differentially expressed in the first hours following loss of ARID1A. Indirect or adaptive changes dominate the transcriptome following growth for days after loss of ARID1A and result in strong engagement with cancer pathways. The identification of this hierarchy suggests sites for intervention in ARID1A-driven diseases., Competing Interests: Declaration of interests The others declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Histone acetyltransferases (HATs) play a crucial role in post-translational modification. Among them, overexpression, mutation, or hyperfunction of EP300/CBP has been associated with various cancers. In this study, we identified the novel compound 2-chloro-5-[5-[(E)-[1-(3-chlorophenyl)-3-methyl-5-oxo-pyrazol-4-ylidene]methyl]-2-furyl]benzoic acid (1) as an EP300 HAT inhibitor via virtual screening. Further research has been focused on the design, synthesis, and in vitro biological evaluation of virtual hit derivatives. The studies revealed that 4-pyridone-3-carboxylic acid derivatives exhibited bioisosterism of benzoic acid. Replacement proved effective, providing compounds with similar EP300 HAT-inhibitory activity and improved cell growth-inhibitory activity compared to the benzoic acid analogs. Through these studies, we identified a potent and selective EP300/CBP HAT inhibitor., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Cervical cancer is a serious gynecological cancer and one of the primary causes of mortality in female patients with cancer. Despite advances in cancer research, the molecular mechanism underlying cancer remains poorly understood. High levels of MIR9‑3 host gene (HG) are associated with the occurrence and development of cervical cancer. However, the specific role of MIR9‑3HG during the development of cervical cancer is unclear. In the present study, the expression of MIR9‑3HG was silenced in C33A and SiHa cervical cancer cell lines. Proliferation and apoptosis were measured in these cells using 5‑ethynyl‑2'‑deoxyuridine assay and flow cytometry, respectively. In addition, targeting microRNAs (miRs) of MIR9‑3HG and mRNAs of miR‑498 were predicted using public databases. The predicted interactions between these molecules were validated using RNA immunoprecipitation, RNA pull‑down and luciferase reporter assays. Lastly, C33A cells transfected with short hairpin MIR‑3HG alone or in combination with miR‑498 inhibitor or PC‑EP300 were subcutaneously injected into mice. The levels of miR‑498, EP300 and Ki67 in tumor tissue were measured via reverse transcription‑quantitative PCR or western blotting. MIR9‑3HG knockdown inhibited the proliferation of cervical cancer cells, whilst promoting apoptosis. MIR9‑3HG sponged miR‑498 and inhibited its expression. Additionally, miR‑498 interacted with EP300 and inhibited its expression. Transfection with miR‑498 inhibitor significantly decreased apoptosis levels; this effect was abolished following EP300 silencing in vitro . In vivo , both miR‑498 inhibition and EP300 overexpression reversed the inhibition of tumor growth mediated by MIR‑3HG knockdown. MIR9‑3HG promoted the proliferation cervical cancer cells via EP300 and miR‑498. These in vitro and in vivo findings demonstrate the regulatory role of the MIR9‑3HG/miR‑498/EP300 axis in cervical cancer cell growth. Thus, the present study identified novel molecular targets for the diagnosis and treatment of cervical cancer and provided new insight into the pathogenesis of cervical cancer.

Podocyte injury is the hallmark of proteinuric glomerular diseases. Notch3 is neo-activated simultaneously in damaged podocytes and podocyte's progenitor cells of FSGS, indicating a unique role of Notch3. We previously showed that activation of cAMP-PKA pathway alleviated podocyte injury possibly via inhibiting Notch3 expression. However, the mechanisms are unknown. In the present study, Notch3 signaling was significantly activated in ADR-induced podocytes in vitro and in PAN nephrosis rats and patients with idiopathic FSGS in vivo, concomitantly with podocyte dedifferentiation. In cultured podocytes, pCPT-cAMP, a selective cAMP-PKA activator, dramatically blocked ADR-induced activation of Notch3 signaling as well as inhibition of cAMP-PKA pathway, thus alleviating the decreased cell viability and podocyte dedifferentiation. Bioinformatics analysis revealed EP300/CBP, a transcriptional co-activator, as a central hub for the crosstalk between these two signaling pathways. Additionally, CREB/KLF15 in cAMP-PKA pathway competed with RBP-J the major transcriptional factor of Notch3 signaling for binding to EP300/CBP. EP300/CBP siRNA significantly inhibited these two signaling transduction pathways and disrupted the interactions between the above major transcriptional factors. These data indicate a crucial role of EP300/CBP in regulating the crosstalk between cAMP-PKA pathway and Notch3 signaling and modulating the phenotypic change of podocytes, and enrich the reno-protective mechanisms of cAMP-PKA pathway., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Hepatocellular carcinoma (HCC) is a leading cause of cancer related deaths worldwide. It was suggested that albendazole (ABZ) is a powerful inhibitor of several carcinoma types. However, the bioavailability of ABZ is very poor. Additionally, the mechanisms underlying the antitumor effects of ABZ may go beyond its tubulin-inhibiting activity. Therefore, we aimed to examine the effects of ABZ suspension (i.p. and p.o.) and ABZ-loaded cubosomes (LC) on the diethylnitrosamine-induced HCC in mice. ABZ-loaded nanoparticles exhibited a mean particle size of 48.17 ± 0.65 nm and entrapped 93.26 ± 2.48% of ABZ. The in vivo absorption study confirmed a two-fold improvement in the relative bioavailability compared with aqueous ABZ suspension. Furthermore, the oral administration of ABZ cubosomal dispersion demonstrated regression of tumor production rates that was comparable with ABZ (i.p.). ABZ relieved oxidative stress, improved liver function, and decreased necroinflammation score. The antiangiogenic activity was evident as ABZ effectively downregulated tissue expression of CD34, mRNA expression of CD309 and VEGF at the protein expression level. Besides, lower levels of MMP-9 and CXCR4 indicated antimetastatic activity. ABZ showed a considerable level of apoptotic activity as indicated by increased mRNA expression level of p53 and the increased Bax/BCL-2 ratio and active caspase-3. Additionally, Ki-67 expression levels were downregulated showing an antiproliferative potential. These protective effects contributed to increasing survival rate of diethylnitrosamine-treated mice. These effects found to be mediated via interrupting ERK1/2-HIF-1α-p300/CREB interactions. Therefore, our findings revealed that disrupting ERK1/2-HIF-1α-p300/CREB interplay might create a novel therapeutic target for the management of HCC., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Homeostasis of the hematopoietic system is achieved in a hierarchy, with hematopoietic stem cells at the pinnacle. Because only hematopoietic stem cells (HSCs) can self-renew, the size of the hematopoietic system is strictly controlled. In hematopoietic reconstitution experiments, 1 HSC can reconstitute the entire hematopoietic system, whereas 50 multipotent progenitors cannot. This indicates that only HSCs self-renew, whereas non-HSC hematopoietic progenitors are programmed to differentiate or senesce. Oncogenic mutations of the mixed lineage leukemia gene (MLL) overcome this "programmed differentiation" by conferring the self-renewing ability to non-HSC hematopoietic progenitors. In leukemia, mutated MLL proteins constitutively activate a broad range of previously transcribed CpG-rich promoters by an MLL-mediated transcriptional activation system. This system promotes self-renewal by replicating an expression profile similar to that of the mother cell in its daughter cells. In this transcriptional activation system, MLL binds to unmethylated CpG-rich promoters and recruits RNA polymerase II. MLL recruits p300/CBP through its transcriptional activation domain, which acetylates histone H3 at lysines 9, 18, and 27. The AF4 family/ENL family/P-TEFb complex (AEP) binds to acetylated H3K9/18/27 to activate transcription. Gene rearrangements of MLL with AEP- or CBP/p300-complex components generate constitutively active transcriptional machinery of this transcriptional activation system, which causes aberrant self-renewal of leukemia stem cells. Inhibitors of the components of this system effectively decrease their leukemogenic potential., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Introduction: Heart failure is the final pathway for a wide spectrum of myocardial stress, including hypertension and myocardial infarction. However, the potential effects of metformin on cardiac hypertrophy are still unclear., Purpose: The purpose of this study was to investigate whether metformin leads to suppression of hypertrophic responses in cardiomyocytes., Methods and Results: To investigate whether metformin inhibited p300-histone acetyltransferase (HAT), we performed an in vitro HAT assay. Metformin directly inhibited p300-mediated acetylation of histone-H3K9. To examine the effects of metformin on hypertrophic responses, cardiomyocytes prepared from neonatal rats were treated with metformin and stimulated with saline or phenylephrine (PE), a α 1 -adrenergic agonist for 48 h. PE stimulus showed an increase in cell size, myofibrillar organization, expression of the endogenous atrial natriuretic factor and brain natriuretic peptide genes, and acetylation of histone-H3K9 compared with saline-treated cells. These PE-induced changes were inhibited by metformin. Next, to examine the effect of metformin on p300-mediated hypertrophy, cardiomyocytes were transfected with expression vector of p300. Metformin significantly suppressed p300-induced hypertrophic responses and acetylation of histone-H3K9., Conclusions: The study demonstrates that metformin can suppress PE-induced and p300-mediated hypertrophic responses. Metformin may be useful for the treatment of patients with diabetes and heart failure., Competing Interests: Declaration of competing interest None., (Copyright © 2021 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

To clarify the mechanism of semicarbazide-modified α-lactalbumin (SEM-LA)-mediated cytotoxicity, we investigated its effect on human U937 leukemia cells and MCF-7 breast cancer cells in the current study. SEM-LA induced apoptosis in U937 cells, which showed increased NOX4 expression, procaspase-8 degradation, and t-Bid production. FADD depletion inhibited SEM-LA-elicited caspase-8 activation, t-Bid production, and cell death, indicating that SEM-LA activated death receptor-mediated apoptosis in U937 cells. SEM-LA stimulated Ca 2+ -mediated Akt activation, which in turn increased Sp1- and p300-mediated NOX4 transcription. The upregulation of NOX4 expression promoted ROS-mediated p38 MAPK phosphorylation, leading to protein phosphatase 2A (PP2A)-regulated tristetraprolin (TTP) degradation. Remarkably, TTP downregulation increased the stability of TNF-α mRNA, resulting in the upregulation of TNF-α protein expression. Abolishment of Ca 2+ -NOX4-ROS axis-mediated p38 MAPK activation attenuated SEM-LA-induced TNF-α upregulation and protected U937 cells from SEM-LA-mediated cytotoxicity. The restoration of TTP expression alleviated the effect of TNF-α upregulation and cell death induced by SEM-LA. Altogether, the data in this study demonstrate that SEM-LA activates TNF-α-mediated apoptosis in U937 cells through the NOX4/p38 MAPK/PP2A axis. We think that a similar pathway can also explain the death of MCF-7 human breast cancer cells after SEM-LA treatment., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The protein-protein interaction between the KIX motif of the transcriptional coactivator CBP/p300 and the transcriptional activator Myb is a high-value target due to its established role in certain acute myeloid leukemias (AML) and potential contributions to other cancers. However, the CBP/p300 KIX domain has multiple binding sites, several structural homologues, many binding partners, and substantial conformational plasticity, making it challenging to specifically target using small-molecule inhibitors. Here, we report a picomolar dual-site inhibitor (MybLL-tide) of the Myb-CBP/p300 KIX interaction. MybLL-tide has higher affinity for CBP/p300 KIX than any previously reported compounds while also possessing 5600-fold selectivity for the CBP/p300 KIX domain over other coactivator domains. MybLL-tide blocks the association of CBP and p300 with Myb in the context of the proteome, leading to inhibition of key Myb·KIX-dependent genes in AML cells. These results show that MybLL-tide is an effective, modifiable tool to selectively target the KIX domain and assess transcriptional effects in AML cells and potentially other cancers featuring aberrant Myb behavior. Additionally, the dual-site design has applicability to the other challenging coactivators that bear multiple binding surfaces.

Actin cytoskeletal reorganization plays an important role in regulating smooth muscle contraction, which is essential for the modulation of various physiological functions including airway tone. The adapter protein Abi1 (Abelson interactor 1) participates in the control of smooth muscle contraction. The mechanisms by which Abi1 coordinates smooth muscle function are not fully understood. Here, we found that contractile stimulation elicited Abi1 acetylation in human airway smooth muscle (HASM) cells. Mutagenesis analysis identified lysine-416 (K416) as a major acetylation site. Replacement of K416 with Q (glutamine) enhanced the interaction of Abi1 with neuronal Wiskott-Aldrich syndrome protein (N-WASP), an important actin-regulatory protein. Moreover, the expression of K416Q Abi1 promoted actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19 and vimentin phosphorylation at Ser-56. Furthermore, p300 is a lysine acetyltransferase that catalyzes acetylation of histone and non-histone proteins in various cell types. Here, we discovered that a portion of p300 was localized in the cytoplasm of HASM cells. Knockdown of p300 reduced the agonist-induced Abi1 acetylation in HASM cells and in mouse airway smooth muscle tissues. Smooth muscle conditional knockout of p300 inhibited actin polymerization and the contraction of airway smooth muscle tissues without affecting myosin light chain phosphorylation and vimentin phosphorylation. Together, our results suggest that contractile stimulation induces Abi1 acetylation via p300 in smooth muscle. Acetylation at K416 promotes the coupling of Abi1 with N-WASP, which facilitates actin polymerization and smooth muscle contraction. This is a novel acetylation-dependent regulation of the actin cytoskeleton in smooth muscle., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Stresses, such as neurohumoral activation, induced pathological cardiac hypertrophy is the main risk factor for heart failure. The ubiquitin-proteasome system (UPS) plays a key role in maintaining protein homeostasis and cardiac function. However, research on the role and mechanism of deubiquitinating enzymes (DUBs) in cardiac hypertrophy is limited. Here, we observe that the deubiquitinating enzyme ubiquitin-specific protease 12(USP12) is upregulated in Ang II-induced hypertrophic hearts and primary neonatal rat cardiomyocytes (NRCMs). Inhibition of USP12 ameliorate Ang II-induced myocardial hypertrophy, while overexpression of USP12 have the opposite effect. USP12 deficiency also significantly attenuate the phenotype of Ang II-induced cardiac hypertrophy in vivo. Moreover, we demonstrate that USP12 aggravate Ang II-induced cardiac hypertrophy by enhancing METTL3, a methyltransferase which catalyze N 6 -methyladenosine (m 6 A) modification on messenger RNA and acts as a harmful factor in pathological cardiac hypertrophy. Upregulation of METTL3 reverse the reduction of myocardial hypertrophy induced by USP12 silencing in NRCMs. In contrast, knockdown of METTL3 attenuate the aggravation of myocardial hypertrophy in USP12-overexpressing NRCMs. Furthermore, we discover that USP12 promote the expression of METTL3 via upregulating p300. Mechanistically, USP12 binds and stabilizes p300, thereby activating the transcription of its downstream gene METTL3. Finally, our data show that USP12 is partially dependent on the stabilization of p300 to activate METTL3 expression and promote myocardial hypertrophy. Taken together, our results demonstrate that USP12 acts as a pro-hypertrophic deubiquitinating enzyme via enhancing p300/METTL3 axis, indicating that targeting USP12 could be a potential treatment strategy for pathological cardiac hypertrophy., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cytochrome P450 family 1 (CYP1) enzymes contribute both to metabolism of xenobiotics and to the control of endogenous levels of ligands of the aryl hydrocarbon receptor (AhR). Their activities, similar to other CYPs, can be altered in tumor tissues. Here, we examined a possible role of proliferative/survival pathways signaling, which is often deregulated in tumor cells, and possible links with p300 histone acetyltransferase (a transcriptional co-activator) in the control of CYP1 expression, focusing particularly on CYP1A1. Using cell models derived from human liver, we observed that the induction of CYP1A1 expression, as well as other CYP1 enzymes, was reduced in exponentially growing cells, as compared with their non-dividing counterparts. The siRNA-mediated inhibition of proliferation/pro-survival signaling pathway effectors (such as β-catenin and/or Hippo pathway effectors YAP/TAZ) increased the AhR ligand-induced CYP1A1 mRNA levels in liver HepaRG cells, and/or in colon carcinoma HCT-116 cells. The activation of proliferative Wnt/β-catenin signaling in HCT-116 cells reduced both the induction of CYP1 enzymes and the binding of p300 to the promoter of CYP1A1 or CYP1B1 genes. These results seem to indicate that aberrant proliferative signaling in tumor cells could suppress induction of CYP1A1 (or other CYP1 enzymes) via competition for p300 binding. This mechanism could be involved in modulation of the metabolism of both endogenous and exogenous substrates of CYP1A1 (and other CYP1 enzymes), with possible further consequences for alterations of the AhR signaling in tumor cells, or additional functional roles of CYP1 enzymes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The function of the transcription factor, cAMP response element-binding protein (CREB), is activated through S133 phosphorylation by PKA and others. Regarding its inactivation, it is not well defined. cAMP response element-binding protein plays an essential role in promoting cell proliferation, neuronal survival and the synaptic plasticity associated with long-term memory. Our recent studies have shown that CREB is an important player in mediating stress response. Here, we have demonstrated that CREB regulates aging process through suppression of αB-crystallin and activation of the p300-p53-Bak/Bax signaling axis. First, we determined that two specific protein phosphatases, PP-1β and PP-2Aα, can inactivate CREB through S133 dephosphorylation. Subsequently, we demonstrated that cells expressing the S133A-CREB, a mutant mimicking constant dephosphorylation at S133, suppress CREB functions in aging control and stress response. Mechanistically, S133A-CREB not only significantly suppresses CREB control of αB-crystallin gene, but also represses CREB-mediated activation of p53 acetylation and downstream Bak/Bax genes. cAMP response element-binding protein suppression of αB-crystallin and its activation of p53 acetylation are major molecular events observed in human cataractous lenses of different age groups. Together, our results demonstrate that PP-1β and PP-2Aα modulate CREB functions in aging control and stress response through de-regulation of αB-crystallin gene and p300-p53-Bax/Bak signaling axis, which regulates human cataractogenesis in the aging lens., (© 2021 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)