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Introduction: Natural plants are valuable resources for exploring new bioactive compounds. Artemisia vulgaris L. is a traditional Chinese medicinal herb that has been historically used for treating multiple diseases. Active compounds isolated and extracted from A. vulgaris L. typically possess immunomodulatory and anti-inflammatory properties. Artemvulactone E (AE) is a new sesquiterpene lactone isolated and extracted from A. vulgaris L. with unclear biological activities. Methods: The immunoregulatory effects of AE on macrophages were assessed by ELISA, RT-qPCR, immunofluorescence, and western blot assay. The effect of AE on lipopolysaccharide (LPS) -relates signaling pathways was examined by western blot assay. In zebrafish models, the larvae were yolk-microinjected with LPS to establish inflammation model and the effect of AE was evaluated by determining the survival rate, heart rate, yolk sac edema size, neutrophils and macrophages infiltration of zebrafish. The interaction between AE and Toll-like receptor 4 (TLR4) was examined by molecular docking and dynamic stimulation. Results: AE reduced the expression and secretion of pro-inflammatory cytokines (TNF-α and IL-6), inflammatory mediators iNOS and COX-2, as well as decreases the production of intracellular NO and ROS in LPS-stimulated macrophages. In addition, AE exerted its anti-inflammatory effect synergistically by inhibiting MAPK/JAK/STAT3-NF-κB signaling pathways. Furthermore, AE enhanced the survival rate and attenuated inflammatory response in zebrafish embryos treated with LPS. Finally, the molecular dynamics results indicate that AE forms stable complexes with LPS receptor TLR4 through the Ser127 residue, thus completely impairing the subsequent activation of MAPK-NF-κB signaling. Conclusion: AE exhibits notable anti-inflammatory activity and represents as a potential agent for treating inflammation-associated diseases.
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Cellular purines, particularly adenosine 5'-triphosphate (ATP), fuel many metabolic reactions, but less is known about the direct effects of pyrimidines on cellular metabolism. We found that pyrimidines, but not purines, maintain pyruvate oxidation and the tricarboxylic citric acid (TCA) cycle by regulating pyruvate dehydrogenase (PDH) activity. PDH activity requires sufficient substrates and cofactors, including thiamine pyrophosphate (TPP). Depletion of cellular pyrimidines decreased TPP synthesis, a reaction carried out by TPP kinase 1 (TPK1), which reportedly uses ATP to phosphorylate thiamine (vitamin B1). We found that uridine 5'-triphosphate (UTP) acts as the preferred substrate for TPK1, enabling cellular TPP synthesis, PDH activity, TCA-cycle activity, lipogenesis, and adipocyte differentiation. Thus, UTP is required for vitamin B1 utilization to maintain pyruvate oxidation and lipogenesis.
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Ciclo do Ácido Cítrico , Lipogênese , Pirimidinas , Complexo Piruvato Desidrogenase , Piruvatos , Trifosfato de Adenosina/metabolismo , Pirimidinas/metabolismo , Piruvatos/metabolismo , Tiamina/metabolismo , Tiamina Pirofosfato/metabolismo , Uridina Trifosfato/metabolismo , Oxirredução , Proteínas Quinases/metabolismo , Humanos , Células HeLa , Complexo Piruvato Desidrogenase/metabolismoRESUMO
Recent studies have identified a previously uncharacterized protein C11orf53 (now named POU2AF2/OCA-T1), which functions as a robust co-activator of POU2F3, the master transcription factor which is critical for both normal and neoplastic tuft cell identity and viability. Here, we demonstrate that POU2AF2 dictates opposing transcriptional regulation at distal enhance elements. Loss of POU2AF2 leads to an inhibition of active enhancer nearby genes, such as tuft cell identity genes, and a derepression of Polycomb-dependent poised enhancer nearby genes, which are critical for cell viability and differentiation. Mechanistically, depletion of POU2AF2 results in a global redistribution of the chromatin occupancy of the SWI/SNF complex, leading to a significant 3D genome structure change and a subsequent transcriptional reprogramming. Our genome-wide CRISPR screen further demonstrates that POU2AF2 depletion or SWI/SNF inhibition leads to a PTEN-dependent cell growth defect, highlighting a potential role of POU2AF2-SWI/SNF axis in small cell lung cancer (SCLC) pathogenesis. Additionally, pharmacological inhibition of SWI/SNF phenocopies POU2AF2 depletion in terms of gene expression alteration and cell viability decrease in SCLC-P subtype cells. Therefore, impeding POU2AF2-mediated transcriptional regulation represents a potential therapeutic approach for human SCLC therapy.
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Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Humanos , Regulação da Expressão Gênica , Fatores de Transcrição/metabolismo , Neoplasias Pulmonares/genética , Diferenciação Celular , Sequências Reguladoras de Ácido NucleicoRESUMO
KEY MESSAGE: One major gene controlling primary root length (PRL) in Gossypium arboreum is identified and this research provides a theoretical basis for root development for cotton. Primary root elongation is an essential process in plant root system structure. Here, we investigated the primary root length (PRL) of 215 diploid cotton (G. arboreum) accessions at 5, 8, 10, 15 days after sowing. A Genome-wide association study was performed for the PRL, resulting in 49 significant SNPs associated with 32 putative candidate genes. The SNP with the strongest signal (Chr07_8047530) could clearly distinguish the PRLs between accessions with two haplotypes. GamurG is the only gene that showed higher relative expression in the long PRL genotypes than the short PRL genotypes, which indicated it was the most likely candidate gene for regulating PRL. Moreover, the GamurG-silenced cotton seedlings showed a shorter PRL, while the GamurG-overexpressed Arabidopsis exhibited a significantly longer PRL. Our findings provide insight into the regulation mechanism of cotton root growth and will facilitate future breeding programs to optimize the root system structure in cotton.
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Estudo de Associação Genômica Ampla , Gossypium , Melhoramento Vegetal , Genótipo , Haplótipos , Regulação da Expressão Gênica de PlantasRESUMO
Cancer genome sequencing consortiums have recently catalogued an abundance of somatic mutations, across a wide range of human cancers, in the chromatin-modifying enzymes that regulate gene expression. Defining the molecular mechanisms underlying the potentially oncogenic functions of these epigenetic mutations could serve as the basis for precision medicine approaches to cancer therapy. MLL4 encoded by the KMT2D gene highly mutated in a large number of human cancers, is a key histone lysine monomethyltransferase within the Complex of Proteins Associated with Set1 (COMPASS) family that regulates gene expression through enhancer function, potentially functioning as a tumor suppressor. We report that the KMT2D mutations which cause MLL4 protein truncation also alter MLL4's subcellular localization, resulting in loss-of-function in the nucleus and gain-of-function in the cytoplasm. We demonstrate that isogenic correction of KMT2D truncation mutation rescues the aberrant localization phenotype and restores multiple regulatory functions of MLL4, including COMPASS integrity/stabilization, histone H3K4 mono-methylation, enhancer activation, and therefore transcriptional regulation. Moreover, isogenic correction diminishes the sensitivity of KMT2D-mutated cancer cells to targeted metabolic inhibition. Using immunohistochemistry, we identified that cytoplasmic MLL4 is unique to the tissue of bladder cancer patients with KMT2D truncation mutations. Using a preclinical carcinogen model of bladder cancer in mouse, we demonstrate that truncated cytoplasmic MLL4 predicts response to targeted metabolic inhibition therapy for bladder cancer and could be developed as a biomarker for KMT2D-mutated cancers. We also highlight the broader potential for prognosis, patient stratification and treatment decision-making based on KMT2D mutation status in MLL4 truncation-relevant diseases, including human cancers and Kabuki Syndrome.
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Histonas , Neoplasias da Bexiga Urinária , Humanos , Animais , Camundongos , Histonas/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Prognóstico , Histona-Lisina N-Metiltransferase/metabolismo , MutaçãoRESUMO
All eukaryotic cells require a minimal iron threshold to sustain anabolic metabolism. However, the mechanisms by which cells sense iron to regulate anabolic processes are unclear. Here we report a previously undescribed eukaryotic pathway for iron sensing in which molecular iron is required to sustain active histone demethylation and maintain the expression of critical components of the pro-anabolic mTORC1 pathway. Specifically, we identify the iron-binding histone-demethylase KDM3B as an intrinsic iron sensor that regulates mTORC1 activity by demethylating H3K9me2 at enhancers of a high-affinity leucine transporter, LAT3, and RPTOR. By directly suppressing leucine availability and RAPTOR levels, iron deficiency supersedes other nutrient inputs into mTORC1. This process occurs in vivo and is not an indirect effect by canonical iron-utilizing pathways. Because ancestral eukaryotes share homologues of KDMs and mTORC1 core components, this pathway probably pre-dated the emergence of the other kingdom-specific nutrient sensors for mTORC1.
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Histonas , Transdução de Sinais , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Leucina/metabolismo , Histonas/genética , Histonas/metabolismo , Ferro/metabolismo , Proteína Regulatória Associada a mTOR/metabolismo , DesmetilaçãoRESUMO
Epigenetic status-altering mutations in chromatin-modifying enzymes are a feature of human diseases, including many cancers. However, the functional outcomes and cellular dependencies arising from these mutations remain unresolved. In this study, we investigated cellular dependencies, or vulnerabilities, that arise when enhancer function is compromised by loss of the frequently mutated COMPASS family members MLL3 and MLL4. CRISPR dropout screens in MLL3/4-depleted mouse embryonic stem cells (mESCs) revealed synthetic lethality upon suppression of purine and pyrimidine nucleotide synthesis pathways. Consistently, we observed a shift in metabolic activity toward increased purine synthesis in MLL3/4-KO mESCs. These cells also exhibited enhanced sensitivity to the purine synthesis inhibitor lometrexol, which induced a unique gene expression signature. RNA-Seq identified the top MLL3/4 target genes coinciding with suppression of purine metabolism, and tandem mass tag proteomic profiling further confirmed upregulation of purine synthesis in MLL3/4-KO cells. Mechanistically, we demonstrated that compensation by MLL1/COMPASS was underlying these effects. Finally, we demonstrated that tumors with MLL3 and/or MLL4 mutations were highly sensitive to lometrexol in vitro and in vivo, both in culture and in animal models of cancer. Our results depicted a targetable metabolic dependency arising from epigenetic factor deficiency, providing molecular insight to inform therapy for cancers with epigenetic alterations secondary to MLL3/4 COMPASS dysfunction.
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Neoplasias , Proteômica , Humanos , Animais , Camundongos , Histona-Lisina N-Metiltransferase/genética , Mutação , Neoplasias/genética , Epigênese GenéticaRESUMO
Construction of Z-scheme heterojunction catalysts with high-speed charge transfer channels for efficient photocatalytic hydrogen production from water splitting is still a challenge. In this work, a lattice-defect-induced atom migration strategy is proposed to construct an intimate interface. The oxygen vacancies of cubic CeO2 obtained from a Cu2O template are used to induce lattice oxygen migration and form SO bonds with CdS to form a close contact heterojunction with a hollow cube. The hydrogen production efficiency reaches â¼12.6 mmol·g-1·h-1 and maintains a high value over 25 h. A series of photocatalytic tests combined with density functional theory (DFT) calculations show that the close contact heterostructure not only promotes the separation/transfer of photogenerated electron-hole pairs but also regulates the intrinsic catalytic activity of the surface. A large number of oxygen vacancies and SO bonds at the interface participate in charge transfer, which accelerates the migration of photogenerated carriers. The hollow structure improves the ability to capture visible light. Therefore, the synthesis strategy proposed in this work, as well as the in-depth discussion of the interface chemical structure and charge transfer mechanism, provides new theoretical support for the further development of photolytic hydrogen evolution catalysts.
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The formation of two-electron chemical bonds requires the alignment of spins. Hence, it is well established for gas-phase reactions that changing a molecule's electronic spin state can dramatically alter its reactivity. For reactions occurring at surfaces, which are of great interest during, among other processes, heterogeneous catalysis, there is an absence of definitive state-to-state experiments capable of observing spin conservation and therefore the role of electronic spin in surface chemistry remains controversial. Here we use an incoming/outgoing correlation ion imaging technique to perform scattering experiments for O(3P) and O(1D) atoms colliding with a graphite surface, in which the initial spin-state distribution is controlled and the final spin states determined. We demonstrate that O(1D) is more reactive with graphite than O(3P). We also identify electronically nonadiabatic pathways whereby incident O(1D) is quenched to O(3P), which departs from the surface. With the help of molecular dynamics simulations carried out on high-dimensional machine-learning-assisted first-principles potential energy surfaces, we obtain a mechanistic understanding for this system: spin-forbidden transitions do occur, but with low probabilities.
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We employ time-slice and velocity map ion imaging methods to explore the quantum-state resolved dynamics in thermal N2O decomposition on Pd(110). We observe two reaction channels: a thermal channel that is ascribed to N2 products initially trapped at surface defects and a hyperthermal channel involving a direct release of N2 to the gas phase from N2O adsorbed on bridge sites oriented along the [001] azimuth. The hyperthermal N2 is highly rotationally excited up to J = 52 (vâ³ = 0) with a large average translational energy of 0.62 eV. Between 35 and 79% of the estimated barrier energy (1.5 eV) released upon dissociation of the transition state (TS) is taken up by the desorbed hyperthermal N2. The observed attributes of the hyperthermal channel are interpreted by post-transition-state classical trajectories on a density functional theory-based high-dimensional potential energy surface. The energy disposal pattern is rationalized by the sudden vector projection model, which attributes to unique features of the TS. Applying detailed balance, we predict that in the reverse Eley-Rideal reaction, both N2 translational and rotational excitation promote N2O formation.
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Obesity is a metabolic disease characterized by excessive fat storage, and the adipogenic differentiation of adipose-derived stromal cells (ADSCs) is closely linked to its occurrence. Growth differentiation factor 11 (GDF11), a well-known molecule in the field of anti-aging, also has great potential in regulating stem cell differentiation. In this study, we found that GDF11 inhibited adipogenic differentiation of human ADSCs in vitro by activating the WNT/ß-catenin and SMAD2/3 pathways while inhibiting the AKT pathway. Moreover, the transcription factor Kruppel-like factor 15 (KLF15) was discovered to be an important downstream factor for GDF11 in inhibiting adipogenesis via the WNT/ß-catenin pathway. Furthermore, AlphaFold2 structure prediction and inhibitor-blocking experiments revealed that ALK5 is a functional receptor of GDF11. Collectively, we demonstrated that GDF11 is a potential target for inhibiting adipogenic differentiation and combating obesity.
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Herpes simplex virus type 1 (HSV-1) infection-associated herpes simplex encephalitis (HSE) is an occasionally but severe neuronal disease that causes behavioral disorder and impairs cognition. Herein, we demonstrate that the consumption of ketogenic diet (KD), a low-carbohydrate high-fat diet, restricts the neurotropic infection of HSV-1 and HSE progression in mice. KD reduced weight loss, neurodegenerative symptoms, virus production and neuroinflammation, resulting in the enhanced survival rate of HSE mice. Notably, depletion of gut microbes by antibiotics attenuated the protective function of KD on HSV-1-related neuroinflammation and HSE development. Therefore, KD represents as an alternative therapeutic strategy to alleviate or prevent HSE via gut microbiota.
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Dieta Cetogênica , Encefalite por Herpes Simples , Microbioma Gastrointestinal , Herpes Simples , Herpesvirus Humano 1 , Animais , Camundongos , Encefalite por Herpes Simples/diagnóstico , Encefalite por Herpes Simples/tratamento farmacológico , Doenças Neuroinflamatórias , Herpes Simples/tratamento farmacológicoRESUMO
Macrophage infiltration is one of the main pathological features of ulcerative colitis (UC) and ferroptosis is a type of nonapoptotic cell death, connecting oxidative stress and inflammation. However, whether ferroptosis occurs in the colon macrophages of UC mice and whether targeting macrophage ferroptosis is an effective approach for UC treatment remain unclear. The present study revealed that macrophage lipid peroxidation was observed in the colon of UC mice. Subsequently, we screened several main components of essential oil from Artemisia argyi and found that ß-caryophyllene (BCP) had a good inhibitory effect on macrophage lipid peroxidation. Additionally, ferroptotic macrophages were found to increase the mRNA expression of tumor necrosis factor alpha (Tnf-α) and prostaglandin-endoperoxide synthase 2 (Ptgs2), while BCP can reverse the effects of inflammation activated by ferroptosis. Further molecular mechanism studies revealed that BCP activated the type 2 cannabinoid receptor (CB2R) to inhibit macrophage ferroptosis and its induced inflammatory response both in vivo and in vitro. Taken together, BCP potentially ameliorated experimental colitis inflammation by inhibiting macrophage ferroptosis. These results revealed that macrophage ferroptosis is a potential therapeutic target for UC and identified a novel mechanism of BCP in ameliorating experimental colitis.
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Colite Ulcerativa , Colite , Ferroptose , Camundongos , Animais , Colite/induzido quimicamente , Colite/tratamento farmacológico , Sesquiterpenos Policíclicos/farmacologia , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/patologia , Inflamação/tratamento farmacológico , Sulfato de DextranaRESUMO
An imbalance in the activities of the Polycomb and Trithorax complexes underlies numerous human pathologies, including cancer. The BRCA1 associated protein-1 (BAP1) deubiquitinase negatively regulates Polycomb activity and recruits the Trithorax histone H3K4 methyltransferase, mixed-lineage leukemia protein 3 (MLL3) within Complex Proteins Associated with Set1 (COMPASS), to the enhancers of tumor suppressor genes. We previously demonstrated that the BAP1-MLL3 pathway is mutated in several cancers, yet how BAP1 recruits MLL3 to its target loci remains an important unanswered question. We demonstrate that the ASXL2 subunit of the BAP1 complex mediates a direct interaction with MLL3/COMPASS. ASXL2 loss results in decreased MLL3 occupancy at enhancers and reduced BAP1-MLL3 target gene expression. Interaction between ASXL2 and MLL3 is negatively regulated by protein arginine methyltransferase 4 (PRMT4/CARM1), which methylates ASXL2 at R639/R641. ASXL2 methylation blocks binding to MLL3 and impairs the expression of MLL3/COMPASS-dependent genes. This previously unidentified transcriptional repressive function of CARM1 provides insight into the BAP1/MLL3-COMPASS axis and reveals a potential cancer therapeutic target.
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Small cell lung cancer (SCLC), accounting for around 13% of all lung cancers, often results in rapid tumor growth, early metastasis, and acquired therapeutic resistance. The POU class 2 homeobox 3 (POU2F3) is a master regulator of tuft cell identity and defines the SCLC-P subtype that lacks the neuroendocrine markers. Here, we have identified a previously uncharacterized protein, C11orf53, which is coexpressed with POU2F3 in both SCLC cell lines and patient samples. Mechanistically, C11orf53 directly interacts with POU2F3 and is recruited to chromatin by POU2F3. Depletion of C11orf53 reduced enhancer H3K27ac levels and chromatin accessibility, resulting in a reduction of POU2F3-dependent gene expression. On the basis of the molecular function of C11orf53, we renamed it as "POU Class 2 Homeobox Associating Factor 2" (POU2AF2). In summary, our study has identified a new coactivator of POU2F3 and sheds light on the therapeutic potential of targeting POU2AF2/POU2F3 heterodimer in human SCLC.
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Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Linhagem Celular Tumoral , Cromatina/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Fatores de Transcrição de Octâmero/genética , Fatores de Transcrição de Octâmero/metabolismo , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologiaRESUMO
BACKGROUND: BRCA1-associated protein 1 (BAP1) is an ubiquitin carboxy-terminal hydrolase, which forms a multi-protein complex with different epigenetic factors, such as ASXL1-3 and FOXK1/2. At the chromatin level, BAP1 catalyzes the removal of mono-ubiquitination on histone H2AK119 in collaboration with other subunits within the complex and functions as a transcriptional activator in mammalian cells. However, the crosstalk between different subunits and how these subunits impact BAP1's function remains unclear. RESULTS: We report the identification of the methyl-CpG-binding domain proteins 5 and 6 (MBD5 and MBD6) that bind to the C-terminal PHD fingers of the large scaffold subunits ASXL1-3 and stabilize the BAP1 complex at the chromatin. We further identify a novel Drosophila protein, the six-banded (SBA), as an ortholog of human MBD5 and MBD6, and demonstrate that the core modules of the BAP1 complex is structurally and functionally conserved from Drosophila (Calypso/ASX/SBA) to human cells (BAP1/ASXL/MBD). Dysfunction of the BAP1 complex induced by the misregulation/mutations in its subunit(s) are frequent in many human cancers. In BAP1-dependent human cancers, such as small cell lung cancer (SCLC), MBD6 tends to be a part of the predominant complex formed. Therefore, depletion of MBD6 leads to a global loss of BAP1 occupancy at the chromatin, resulting in a reduction of BAP1-dependent gene expression and tumor growth in vitro and in vivo. CONCLUSIONS: We characterize MBD5 and MBD6 as important regulators of the BAP1 complex and maintain its transcriptional landscape, shedding light on the therapeutic potential of targeting MBD5 and MBD6 in BAP1-dependent human cancers.
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Proteínas de Drosophila , Neoplasias , Animais , Cromatina , Proteínas de Ligação a DNA/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fatores de Transcrição Forkhead/genética , Histonas/metabolismo , Humanos , Mamíferos/genética , Neoplasias/genética , Proteínas Repressoras/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismoRESUMO
Small cell lung cancer (SCLC) is an aggressive disease, with patients diagnosed with either early-stage, limited stage, or extensive stage of SCLC tumor progression. Discovering and targeting the functional biomarkers for SCLC will be crucial in understanding the molecular basis underlying SCLC tumorigenesis to better assist in improving clinical treatment. Emerging studies have demonstrated that dysregulations in BAP1 histone H2A deubiquitinase complex are collectively associated with pathogenesis in human SCLC. Here, we investigated the function of the oncogenic BAP1/ASXL3/BRD4 epigenetic axis in SCLC by developing a next-generation BAP1 inhibitor, iBAP-II, and focusing on the epigenetic balance established between BAP1 and non-canonical PRC1 complexes in regulating SCLC-specific transcriptional programming. We further demonstrated that pharmacologic inhibition of BAP1's catalytic activity disrupted BAP1/ASXL3/BRD4 epigenetic axis by inducing protein degradation of the ASXL3 scaffold protein, which bridges BRD4 and BAP1 at active enhancers. Furthermore, treatment of iBAP-II represses neuroendocrine lineage-specific ASCL1/MYCL/E2F signaling in SCLC cell lines, and dramatically inhibits SCLC cell viability and tumor growth in vivo. In summary, this study has provided mechanistic insight into the oncogenic function of BAP1 in SCLC and highlighted the potential of targeting BAP1's activity as a novel SCLC therapy.
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Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteínas de Ciclo Celular , Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Fatores de Transcrição , Proteínas Supressoras de Tumor , Ubiquitina Tiolesterase , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinogênese , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas Nucleares/metabolismo , Oncogenes , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismoRESUMO
Antibodies offer a powerful means to interrogate specific proteins in a complex milieu. However, antibody availability and reliability can be problematic, whereas epitope tagging can be impractical in many cases. To address these limitations, the Protein Capture Reagents Program (PCRP) generated over a thousand renewable monoclonal antibodies (mAbs) against human presumptive chromatin proteins. However, these reagents have not been widely field-tested. We therefore performed a screen to test their ability to enrich genomic regions via chromatin immunoprecipitation (ChIP) and a variety of orthogonal assays. Eight hundred eighty-seven unique antibodies against 681 unique human transcription factors (TFs) were assayed by ultra-high-resolution ChIP-exo/seq, generating approximately 1200 ChIP-exo data sets, primarily in a single pass in one cell type (K562). Subsets of PCRP mAbs were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and protein binding microarray (PBM) experiments. About 5% of the tested antibodies displayed high-confidence target (i.e., cognate antigen) enrichment across at least one assay and are strong candidates for additional validation. An additional 34% produced ChIP-exo data that were distinct from background and thus warrant further testing. The remaining 61% were not substantially different from background, and likely require consideration of a much broader survey of cell types and/or assay optimizations. We show and discuss the metrics and challenges to antibody validation in chromatin-based assays.
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Sequenciamento de Cromatina por Imunoprecipitação , Fatores de Transcrição , Sítios de Ligação , Imunoprecipitação da Cromatina , Humanos , Indicadores e Reagentes , Reprodutibilidade dos Testes , Fatores de Transcrição/metabolismoRESUMO
Abnormalities in genetic and epigenetic modifications can lead to drastic changes in gene expression profiles that are associated with various cancer types. Small cell lung cancer (SCLC) is an aggressive and deadly form of lung cancer with limited effective therapies currently available. By utilizing a genome-wide CRISPR-Cas9 dropout screen in SCLC cells, we identified paired box protein 9 (PAX9) as an essential factor that is overexpressed in human malignant SCLC tumor samples and is transcriptionally driven by the BAP1/ASXL3/BRD4 epigenetic axis. Genome-wide studies revealed that PAX9 occupies distal enhancer elements and represses gene expression by restricting enhancer activity. In multiple SCLC cell lines, genetic depletion of PAX9 led to significant induction of a primed-active enhancer transition, resulting in increased expression of a large number of neural differentiation and tumor-suppressive genes. Mechanistically, PAX9 interacted and cofunctioned with the nucleosome remodeling and deacetylase (NuRD) complex at enhancers to repress nearby gene expression, which was reversed by pharmacologic HDAC inhibition. Overall, this study provides mechanistic insight into the oncogenic function of the PAX9/NuRD complex epigenetic axis in human SCLC and suggests that reactivation of primed enhancers may have potential therapeutic efficacy in treating SCLC expressing high levels of PAX9. SIGNIFICANCE: A genome-wide screen in small cell lung cancer reveals PAX9/NuRD-mediated epigenetic enhancer silencing and tumor progression, supporting the development of novel personalized therapeutic approaches targeting the PAX9-regulated network.
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Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Neoplasias/metabolismo , Fator de Transcrição PAX9/metabolismo , Animais , Sistemas CRISPR-Cas , Diferenciação Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla/métodos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos , Modelos Biológicos , Neoplasias/patologia , Fator de Transcrição PAX9/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Carcinoma de Pequenas Células do Pulmão/genética , Fatores de Transcrição/genéticaRESUMO
Soil salinization is a vital factor that restricts the efficient and sustainable development of global agriculture. Studies enlightened that the C2H2 zinc finger proteins (C2H2-ZFP) were involved in regulating the stress response in plants. However, knowledge of the C2H2-ZFP subfamily C1 (ZAT; Zinc finger of Arabidopsis thaliana) in cotton is still a mystery. In this study, 47, 45, 94, and 88 ZAT genes were obtained from diploid A2, D5 and tetraploid AD1, AD2 cotton genomes, respectively. The function of hybridization and allopolyploidy in the evolutionary linkage of allotetraploid cotton was explained by the family of ZAT gene in 4 species. Duplication of gene activities indicates that the family of ZAT gene of cotton evolution was under strong purifying selection. The integration of previous transcriptome data related to NaCl stress, strongly suggests the GhZAT34 and GhZAT79 may interact with salt resistance in upland cotton. The expression level of certain ZAT genes, higher seed germination rate of transgenic Arabidopsis and gene- silenced cotton revealed that both genes were involved in the salt tolerance of upland cotton. This study may pave the substantial understandings into the role of ZATs genes in plants as well as suggest appropriate candidate genes for breeding of cotton varieties against salinity tolerance.