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The first magnetic ligand-based electrochemical assay aimed at the determination of BRD4 was developed and validated. BRD4 is an epigenetic regulator of great interest in oncology in relation to its overexpression observed in the pathogenesis of several cancer diseases. BRD4 also represents a major target for the development of innovative treatments aimed at protein inhibition or degradation. Despite the relevance of BRD4 both for diagnostics and therapeutic purposes, current methodologies for its determination are limited to commercial ELISA kits. We present a novel magnetic ligand-based assay for the electrochemical determination of BRD4. The developed assay is based on the use of a small synthetic fragment of the natural protein ligand for BRD4 as receptor, thus exploiting the intrinsic biological protein-protein recognition mechanism. In addition, the assay features the use of magnetic beads as immobilization platforms and peroxidase-conjugated monoclonal anti-BRD4 antibody for the generation of the electrochemical signal. The ligand-based assay shows outstanding performance in terms of rapidity, with results achievable in less than 20 min, no matrix effect when applied to human plasma or cell lysate samples, and excellent specificity. The proposed method exhibits a limit of detection of 2.66 nM and a response range tunable as a function of the amount of immobilized receptor. The developed ligand-based assay was successfully applied to the accurate determination of BRD4 in untreated cell lysates, as proven by the ELISA reference method. The good performance of the proposed bioassay for determination of BRD4 showed potential application of this strategy in convenient point-of-care testing.

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Background: Hepatoblastoma (HB) is the most common pediatric hepatic malignancy. Despite the progress in HB treatment, investigating HB pathomechanisms to optimize stratification and therapies remains a focal point to improve the outcome for high-risk patients. Methods: Here, we pointed to explore the impact of these mechanisms in HB. An observational study was performed on liver samples from a cohort of 17 patients with a diagnosis of HB and two normal liver samples. The in vitro experiments were executed on the Huh6 human HB cell line treated with the FAK inhibitor TAE226. Results: Our results highlight a significant up-regulation of mRNA and protein expression of FAK in livers from HB with respect to normal livers. The increased protein expression of total and Tyr397 phosphorylated FAK (pTyr397FAK) was significantly correlated with the expression of some epigenetic regulators of histone H3 methylation and acetylation. Of note, the expression of pTyr397FAK, N-methyltransferase enzyme (EZH2) and tri-methylation of the H3K27 residue correlated with tumor size and alpha-fetoprotein (AFP) levels. Finally, TAE226 caused a significant reduction of pTyr397FAK, epigenetic regulators, AFP, EPCAM, OCT4, and SOX2, in association with anti-proliferative and pro-apoptotic effects on HB cells. Conclusion: Our results suggest a role of FAK in HB that requires further investigations mainly focused on the exploration of its effective diagnostic and therapeutic translatability.

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To achieve exquisite control over the epigenome, we need a better predictive understanding of how transcription factors, chromatin regulators, and their individual domain's function, both as modular parts and as full proteins. Transcriptional effector domains are one class of protein domains that regulate transcription and chromatin. These effector domains either repress or activate gene expression by interacting with chromatin-modifying enzymes, transcriptional cofactors, and/or general transcriptional machinery. Here, we discuss important design considerations for high-throughput investigations of effector domains, recent advances in discovering new domains in human cells and testing how domain function depends on amino acid sequence. For every effector domain, we would like to know the following: What role does the cell type, signaling state, and targeted context have on activation, silencing, and epigenetic memory? Large-scale measurements of transcriptional activities can help systematically answer these questions and identify general rules for how all these parameters affect effector domain activities. Last, we discuss what steps need to be taken to turn a newly discovered effector domain into a robust, precise epigenome editor. With more carefully considered high-throughput investigations, soon we will have better predictive control over the epigenome.

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The natriuretic peptide system (NPS) is the key driving force of the heart's endocrine function. Recent developments in NPS-targeted therapies have been found promising and effective against cardiovascular diseases, including hypertension. Notably, after discovering crosstalk between NPS and the renin-angiotensin-aldosterone system (RAAS), various combinations such as neprilysin/angiotensin II receptor type 1 AT1 receptor inhibitors and neprilysin/renin inhibitors have been preclinically and clinically tested against various cardiac complications. However, the therapeutic effects of such combinations on the pathophysiology of hypertension are poorly understood. Furthermore, the complicated phenomena underlying NPS regulation and function, particularly in hypertension, are still unexplored. Mounting evidence suggests that numerous regulatory mechanisms modulate the expression of NPS, which can be used as potential targets against hypertension and other cardiovascular diseases. Therefore, this review will specifically focus on epigenetic and other regulators of NPS, identifying prospective regulators that might serve as new therapeutic targets for hypertension. More importantly, it will shed light on recent developments in NPS-targeted therapies, such as M-atrial peptides, and their latest combinations with RAAS modulators, such as S086 and sacubitril-aliskiren. These insights will aid in the development of effective therapies to break the vicious cycle of high blood pressure during hypertension, ultimately addressing the expanding global heart failure pandemic.

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Sarcomas comprise a heterogeneous group of malignant tumors of mesenchymal origin. More than 80 entities are associated with different mesenchymal lineages. Sarcomas with fibroblastic, muscle, bone, vascular, adipocytic, and other characteristics are distinguished. Nearly half of all entities contain specific chromosomal translocations that give rise to fusion proteins. These are mostly pathognomonic, and their detection by various molecular techniques supports histopathologic classification. Moreover, the fusion proteins act as oncogenic drivers, and their blockade represents a promising therapeutic approach. This review summarizes the current knowledge on fusion proteins in sarcoma. We categorize the different fusion proteins into functional classes, including kinases, epigenetic regulators, and transcription factors, and describe their mechanisms of action. Interestingly, while fusion proteins acting as transcription factors are found in all mesenchymal lineages, the others have a more restricted pattern. Most kinase-driven sarcomas belong to the fibroblastic/myofibroblastic lineage. Fusion proteins with an epigenetic function are mainly associated with sarcomas of unclear differentiation, suggesting that epigenetic dysregulation leads to a major change in cell identity. Comparison of mechanisms of action reveals recurrent functional modes, including antagonism of Polycomb activity by fusion proteins with epigenetic activity and recruitment of histone acetyltransferases by fusion transcription factors of the myogenic lineage. Finally, based on their biology, we describe potential approaches to block the activity of fusion proteins for therapeutic intervention. Overall, our work highlights differences as well as similarities in the biology of fusion proteins from different sarcomas and provides the basis for a functional classification.

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The retinoblastoma (RB) transcriptional corepressor 1 (RB1) is a critical tumor suppressor gene, governing diverse cellular processes implicated in cancer biology. Dysregulation or deletion in RB1 contributes to the development and progression of various cancers, making it a prime target for therapeutic intervention. RB1's canonical function in cell cycle control and DNA repair mechanisms underscores its significance in restraining aberrant cell growth and maintaining genomic stability. Understanding the complex interplay between RB1 and cellular pathways is beneficial to fully elucidate its tumor-suppressive role across different cancer types and for therapeutic development. As a result, investigating vulnerabilities arising from RB1 deletion-associated mechanisms offers promising avenues for targeted therapy. Recently, several findings highlighted multiple methods as a promising strategy for combating tumor growth driven by RB1 loss, offering potential clinical benefits in various cancer types. This review summarizes the multifaceted role of RB1 in cancer biology and its implications for targeted therapy.

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KEY MESSAGE: The gametophytic epigenetic regulators, MEA and DME, extend their synergistic role to the sporophytic development by regulating the meristematic activity via restricting the gene expression in the shoot apex. The gametophyte-to-sporophyte transition facilitates the alternation of generations in a plant life cycle. The epigenetic regulators DEMETER (DME) and MEDEA (MEA) synergistically control central cell proliferation and differentiation, ensuring proper gametophyte-to-sporophyte transition in Arabidopsis. Mutant alleles of DME and MEA are female gametophyte lethal, eluding the recovery of recessive homozygotes to examine their role in the sporophyte. Here, we exploited the paternal transmission of these mutant alleles coupled with CENH3-haploid inducer to generate mea-1;dme-2 sporophytes. Strikingly, the simultaneous loss of function of MEA and DME leads to the emergence of ectopic shoot meristems at the apical pole of the plant body axis. DME and MEA are expressed in the developing shoot apex and regulate the expression of various shoot-promoting factors. Chromatin immunoprecipitation (ChIP), DNA methylation, and gene expression analysis revealed several shoot regulators as potential targets of MEA and DME. RNA interference-mediated transcriptional downregulation of shoot-promoting factors STM, CUC2, and PLT5 rescued the twin-plant phenotype to WT in 9-23% of mea-1-/-;dme-2-/- plants. Our findings reveal a previously unrecognized synergistic role of MEA and DME in restricting the meristematic activity at the shoot apex during sporophytic development.

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Targeted therapies, including small molecule inhibitors directed against aberrant kinase signaling and chromatin regulators, are emerging treatment options for high-grade gliomas (HGG). However, when translating these inhibitors into the clinic, their efficacy is generally limited to partial and transient responses. Recent studies in models of high-grade gliomas reveal a convergence of epigenetic regulators and kinase signaling networks that often cooperate to promote malignant properties and drug resistance. This review examines the interplay between five well-characterized groups of chromatin regulators, including the histone deacetylase (HDAC) family, bromodomain and extraterminal (BET)-containing proteins, protein arginine methyltransferase (PRMT) family, Enhancer of zeste homolog 2 (EZH2), and lysine-specific demethylase 1 (LSD1), and various signaling pathways essential for cancer cell growth and progression. These specific epigenetic regulators were chosen for review due to their targetability via pharmacological intervention and clinical relevance. Several studies have demonstrated improved efficacy from the dual inhibition of the epigenetic regulators and signaling kinases. Overall, the interactions between epigenetic regulators and kinase signaling pathways are likely influenced by several factors, including individual glioma subtypes, preexisting mutations, and overlapping/interdependent functions of the chromatin regulators. The insights gained by understanding how the genome and epigenome cooperate in high-grade gliomas will guide the design of future therapeutic strategies that utilize dual inhibition with improved efficacy and overall survival.

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Histone deacetylases (HDACs) are a class of epigenetic enzymes that are closely related to tumorigenesis and suppress the expression of tumor suppressor genes. Whereas the HDACs inhibitors can release DNA into the cytoplasm and trigger innate immunity. However, the high density of chromatin limits DNA damage and release. In this study, suitable nanosized CycNHOH NPs (150 nm) and CypNHOH NPs (85 nm) efficiently accumulate at the tumor site due to the enhanced permeability and retention (EPR) effect. In addition, robust single-linear oxygen generation and good photothermal conversion efficiency under NIR laser irradiation accelerated the DNA damage process. By effectively initiating immune cell death, CypNHOH NPs activated both innate and adaptive immunity by maturing dendritic cells, infiltrating tumors with natural killer cells, and activating cytotoxic T lymphocytes, which offer a fresh perspective for the development of photo-immunotherapy.

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OBJECTIVE: Epigenetic abnormalities have a critical role in breast cancer by regulating gene expression; however, the intricate interrelationships and key roles of approximately 400 epigenetic regulators in breast cancer remain elusive. It is important to decipher the comprehensive epigenetic regulatory network in breast cancer cells to identify master epigenetic regulators and potential therapeutic targets. METHODS: We employed high-throughput sequencing-based high-throughput screening (HTS2) to effectively detect changes in the expression of 2,986 genes following the knockdown of 400 epigenetic regulators. Then, bioinformatics analysis tools were used for the resulting gene expression signatures to investigate the epigenetic regulations in breast cancer. RESULTS: Utilizing these gene expression signatures, we classified the epigenetic regulators into five distinct clusters, each characterized by specific functions. We discovered functional similarities between BAZ2B and SETMAR, as well as CLOCK and CBX3. Moreover, we observed that CLOCK functions in a manner opposite to that of HDAC8 in downstream gene regulation. Notably, we constructed an epigenetic regulatory network based on the gene expression signatures, which revealed 8 distinct modules and identified 10 master epigenetic regulators in breast cancer. CONCLUSIONS: Our work deciphered the extensive regulation among hundreds of epigenetic regulators. The identification of 10 master epigenetic regulators offers promising therapeutic targets for breast cancer treatment.

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Keratinocytes are one of the primary cells affected by psoriasis inflammation. Our study aimed to delve deeper into their morphology, transcriptome, and epigenome changes in response to psoriasis-like inflammation. We created a novel cytokine mixture to mimic mild and severe psoriasis-like inflammatory conditions in cultured keratinocytes. Upon induction of inflammation, we observed that the keratinocytes exhibited a mesenchymal-like phenotype, further confirmed by increased VIM mRNA expression and results obtained from confocal microscopy. We performed RNA sequencing to achieve a more global view, revealing 858 and 6987 DEGs in mildly and severely inflamed keratinocytes, respectively. Surprisingly, we found that the transcriptome of mildly inflamed keratinocytes more closely mimicked that of the psoriatic epidermis transcriptome than the severely inflamed keratinocytes. Genes involved in the IL-17 pathway were a major contributor to the similarities of the transcriptomes between mildly inflamed KCs and psoriatic epidermis. Mild and severe inflammation led to the gene regulation of epigenetic modifiers such as HATs, HDACs, DNMTs, and TETs. Immunofluorescence staining revealed distinct 5-hmC patterns in inflamed versus control keratinocytes, and consistently low 5-mC intensity in both groups. However, the global DNA methylation assay detected a tendency of decreased 5-mC levels in inflamed keratinocytes versus controls. This study emphasizes how inflammation severity affects the transcriptomic similarity of keratinocytes to psoriatic epidermis and proves dynamic epigenetic regulation and adaptive morphological changes in inflamed keratinocytes.

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Mammalian SWI/SNF chromatin remodeling complexes move and evict nucleosomes at gene promoters and enhancers to modulate DNA access. Although SWI/SNF subunits are commonly mutated in disease, therapeutic options are limited by our inability to predict SWI/SNF gene targets and conflicting studies on functional significance. Here, we leverage a fast-acting inhibitor of SWI/SNF remodeling to elucidate direct targets and effects of SWI/SNF. Blocking SWI/SNF activity causes a rapid and global loss of chromatin accessibility and transcription. Whereas repression persists at most enhancers, we uncover a compensatory role for the EP400/TIP60 remodeler, which reestablishes accessibility at most promoters during prolonged loss of SWI/SNF. Indeed, we observe synthetic lethality between EP400 and SWI/SNF in cancer cell lines and human cancer patient data. Our data define a set of molecular genomic features that accurately predict gene sensitivity to SWI/SNF inhibition in diverse cancer cell lines, thereby improving the therapeutic potential of SWI/SNF inhibitors.

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To effectively adapt to changing environments, plants must maintain a delicate balance between growth and resistance or tolerance to various stresses. Nitrate, a significant inorganic nitrogen source in soils, not only acts as an essential nutrient but also functions as a critical signaling molecule that regulates multiple aspects of plant growth and development. In recent years, substantial advancements have been made in understanding nitrate sensing, calcium-dependent nitrate signal transmission, and nitrate-induced transcriptional cascades. Mounting evidence suggests that the primary response to nitrate is influenced by environmental conditions, while nitrate availability plays a pivotal role in stress tolerance responses. Therefore, this review aims to provide an overview of the transcriptional and post-transcriptional regulation of key components in the nitrate signaling pathway, namely, NRT1.1, NLP7, and CIPK23, under abiotic stresses. Additionally, we discuss the specificity of nitrate sensing and signaling as well as the involvement of epigenetic regulators. A comprehensive understanding of the integration between nitrate signaling transduction and abiotic stress responses is crucial for developing future crops with enhanced nitrogen-use efficiency and heightened resilience.

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OBJECTIVES: To investigate the role of DNA methylation regulators in the prognosis of clear cell renal cell carcinoma (ccRCC) and to construct a DNA methylation regulator-based signature for predicting patient outcome. METHODS: Data from the TCGA dataset were downloaded and analyzed to identify differentially expressed DNA methylation regulators and their interaction as well as correlation. Consensus clustering was used to establish groups of ccRCC with distinct clinical outcomes. A prognostic signature based on two sets of DNA methylation regulators was established and validated in an independent cohort. RESULTS: Our analysis revealed that the expression levels of DNMT3B, MBD1, SMUG1, DNMT1, DNMT3A, TDG, TET3, MBD2, UHRF2, MBD3, UHRF1, and TET2 were significantly upregulated in ccRCC samples, while UNG, ZBTB4, TET1, ZBTB38, and MECP2 were markedly downregulated. UHRF1 was identified as a hub gene in the DNA methylation regulator interaction network. Significant differences were found regarding overall survival, gender, tumor status, and grade between ccRCC patients in the two risk groups. The prognostic signature, based on two sets of DNA methylation regulators, was an independent prognostic indicator, and these findings were validated in an external, independent cohort. CONCLUSIONS: The study provides evidence that DNA methylation regulators play a significant role in the prognosis of ccRCC and the developed DNA methylation regulator-based signature could effectively predict patient outcome.

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Attenuating the Taxol productivity of fungi with the subculturing and storage under axenic conditions is the challenge that halts the feasibility of fungi to be an industrial platform for Taxol production. This successive weakening of Taxol productivity by fungi could be attributed to the epigenetic down-regulation and molecular silencing of most of the gene clusters encoding Taxol biosynthetic enzymes. Thus, exploring the epigenetic regulating mechanisms controlling the molecular machinery of Taxol biosynthesis could be an alternative prospective technology to conquer the lower accessibility of Taxol by the potent fungi. The current review focuses on discussing the different molecular approaches, epigenetic regulators, transcriptional factors, metabolic manipulators, microbial communications and microbial cross-talking approaches on restoring and enhancing the Taxol biosynthetic potency of fungi to be industrial platform for Taxol production.

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MicroRNAs are critical epigenetic regulators that can be used as diagnostic, prognostic, and therapeutic biomarkers for the treatment of various diseases, including gastrointestinal cancers, among a variety of cellular and molecular biomarkers. MiRNAs have also shown oncogenic or tumor suppressor roles in tumor tissue and other cell types. Studies showed that the dysregulation of miR-28 is involved in cell growth and metastasis of gastrointestinal cancers. MiR-28 plays a key role in controlling the physiological processes of cancer cells including growth and proliferation, migration, invasion, apoptosis, and metastasis. Therefore, miR-28 expression patterns can be used to distinguish patient subgroups. Based on the previous studies, miR-28 expression can be a suitable biomarker to detect tumor size and predict histological grade metastasis. In this review, we summarize the inhibitory effects of miR-28 as a metastasis suppressor in gastrointestinal cancers. miR-28 plays a role as a tumor suppressor in gastrointestinal cancers by regulating cancer cell growth, cell differentiation, angiogenesis, and metastasis. As a result, using it as a prognostic, diagnostic, and therapeutic biomarker in the treatment of gastrointestinal cancers can be a way to solve the problems in this field.

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Viability CRISPR screens have proven indispensable in parsing genome function. However, their application in new, more physiologically relevant culturing systems like patient-derived organoids (PDOs) has been much slower. To probe epigenetic contribution to gastric cancer (GC), the third leading cause of cancer-related deaths worldwide, the first negative selection CRISPR screen in GC PDOs that faithfully preserve primary tumor characteristics is performed. Extensive quality control measurements showing feasibility of CRISPR screens in primary organoid culture are provided. The screen reveals the histone lysine demethylase-1A (KDM1A) to constitute a GC vulnerability. Both genetic and pharmacological inhibition of KDM1A cause organoid growth retardation. Further, it is shown that most of KDM1A cancer-supporting functions center on repression of N-myc downstream regulates gene-1 (NDRG1). De-repression of NDRG1 by KDM1A inhibitors (KDM1Ai) causes inhibition of Wnt signaling and a strong G1 cell cycle arrest. Finally, by profiling 20 GC PDOs, it is shown that NDRG1 upregulation predicts KDM1Ai response with 100% sensitivity and 82% specificity in the tested cohort. Thus, this work pioneers the use of negative selection CRISPR screens in patient-derived organoids, identifies a marker of KDM1Ai response, and accordingly a cohort of patients who may benefit from such therapy.

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Cancer is amongst the deadliest and most disruptive disorders, having a much higher death rate than other diseases worldwide. Human cancer rates continue to rise, thereby posing the most significant concerns for medical health professionals. In the last two decades, researchers have gone past several milestones in tackling cancer while gaining insight into the role of apoptosis in cancer or targeting various biomarker tools for prognosis and diagnosis. Apoptosis which is still a topic full of complexities, can be controlled considerably by B-cell lymphoma 2 (BCL-2) and its family members. Therefore, targeting proteins of this family to prevent tumorigenesis, is essential to focus on the pharmacological features of the anti-apoptotic and pro-apoptotic members, which will help to develop and manage this disorder. This review deals with the advancements of various epigenetic regulators to target BCL-2 family proteins, including the mechanism of several microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). Similarly, a rise in natural and synthetic molecules' research over the last two decades has allowed us to acquire insights into understanding and managing the transcriptional alterations that have led to apoptosis and treating various neoplastic diseases. Furthermore, several inhibitors targeting anti-apoptotic proteins and inducers or activators targeting pro-apoptotic proteins in preclinical and clinical stages have been summarized. Overall, agonistic and antagonistic mechanisms of BCL-2 family proteins conciliated by epigenetic regulators, natural and synthetic agents have proven to be an excellent choice in developing cancer therapeutics.

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Dysregulation of the oncogenic transcription factor HOXA9 is a prominent feature for most aggressive acute myeloid leukemia cases and a strong indicator of poor prognosis in patients. Leukemia subtypes with hallmark overexpression of HOXA9 include those carrying MLL gene rearrangements, NPM1c mutations, and other genetic alternations. A growing body of evidence indicates that HOXA9 dysregulation is both sufficient and necessary for leukemic transformation. The HOXA9 mRNA and protein regulation includes multilayered controls by transcription factors (such as CDX2/4 and USF2/1), epigenetic factors (such as MLL-menin-LEDGF, DOT1L, ENL, HBO1, NPM1c-XPO1, and polycomb proteins), microRNAs (such as miR-126 and miR-196b), long noncoding RNAs (such as HOTTIP), three-dimensional chromatin interactions, and post-translational protein modifications. Recently, insights into the dynamic regulation of HOXA9 have led to an advanced understanding of the HOXA9 regulome and provided new cancer therapeutic opportunities, including developing inhibitors targeting DOT1L, menin, and ENL proteins. This review summarizes recent advances in understanding the molecular mechanisms controlling HOXA9 regulation and the pharmacological approaches that target HOXA9 regulators to treat HOXA9-driven acute myeloid leukemia.

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Plasminogen activator inhibitor-1 (PAI-1) is a specific and rapid-acting inhibitor of endogenous plasminogen activators (uPA and tPA). The global PAI-1 knockout mice (PAI-1KO) develop age-dependent cardiac-selective fibrosis, and young global PAI-1KO mice exhibit augmented susceptibility to developing cardiac fibrosis in response to hypertension. Here, we tested the hypothesis that cardiomyocyte PAI-1 is necessary to provide cardioprotective effects in a left ventricular pressure overload-induced murine model of cardiac hypertrophy and fibrosis using cardiomyocyte-specific PAI-1 knockout (cmPAI-1KO) mice. The results revealed that cmPAI-1KO mice display significantly worse cardiac fibrosis than controls. To investigate the molecular mechanisms responsible for these effects, genome-wide cardiac transcriptome analysis was performed. Loss of cardiomyocyte PAI-1 led to differential expression of 978 genes compared to controls in response to left ventricular pressure overload. Pathway enrichment analysis identified the inflammatory response, cell substrate adhesion, regulation of cytokine production, leukocyte migration, extracellular matrix organization, and cytokine-mediated signaling pathways as being significantly upregulated in cmPAI-1KO hearts. Conversely, specific epigenetic repressors, cation transmembrane transport, muscle system processes, and nitric oxide signaling were significantly downregulated in cmPAI-1KO hearts compared to control hearts in response to left ventricular pressure overload. Collectively, the present study provides strong evidence of the impact of cardiomyocyte PAI-1 in regulation of the transcriptome network involved in the cardiac stress response. In response to stress, the deregulatory impact of cardiomyocyte PAI-1 loss on the cardiac transcriptome may be the underlying cause of cardiac-selective accelerated fibrogenesis in global PAI-1-deficient mice.

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