RESUMEN
BACKGROUND & AIMS: The incidence of Crohn's disease (CD) continues to increase worldwide. The contribution of CD4+ cell populations remains to be elucidated. We aimed to provide an in-depth transcriptional assessment of CD4+ T cells driving chronic inflammation in CD. METHODS: We performed single-cell RNA-sequencing in CD4+ T cells isolated from ileal biopsies of patients with CD compared with healthy individuals. Cells underwent clustering analysis, followed by analysis of gene signaling networks. We overlapped our differentially expressed genes with publicly available microarray data sets and performed functional in vitro studies, including an in vitro suppression assay and organoid systems, to model gene expression changes observed in CD regulatory T (Treg) cells and to test predicted therapeutics. RESULTS: We identified 5 distinct FOXP3+ regulatory Treg subpopulations. Tregs isolated from healthy controls represent the origin of pseudotemporal development into inflammation-associated subtypes. These proinflammatory Tregs displayed a unique responsiveness to tumor necrosis factor-α signaling with impaired suppressive activity in vitro and an elevated cytokine response in an organoid coculture system. As predicted in silico, the histone deacetylase inhibitor vorinostat normalized gene expression patterns, rescuing the suppressive function of FOXP3+ cells in vitro. CONCLUSIONS: We identified a novel, proinflammatory FOXP3+ T cell subpopulation in patients with CD and developed a pipeline to specifically target these cells using the US Food and Drug Administration-approved drug vorinostat.
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Enfermedad de Crohn , Humanos , Enfermedad de Crohn/tratamiento farmacológico , Enfermedad de Crohn/genética , Enfermedad de Crohn/metabolismo , Vorinostat/metabolismo , Linfocitos T Reguladores/metabolismo , Inflamación/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismoRESUMEN
BACKGROUND & AIMS: The highly heterogeneous cellular and molecular makeup of pancreatic ductal adenocarcinoma (PDAC) not only fosters exceptionally aggressive tumor biology, but contradicts the current concept of one-size-fits-all therapeutic strategies to combat PDAC. Therefore, we aimed to exploit the tumor biological implication and therapeutic vulnerabilities of a clinically relevant molecular PDAC subgroup characterized by SMAD4 deficiency and high expression of the nuclear factor of activated T cells (SMAD4-/-/NFATc1High). METHODS: Transcriptomic and clinical data were analyzed to determine the prognostic relevance of SMAD4-/-/NFATc1High cancers. In vitro and in vivo oncogenic transcription factor complex formation was studied by immunoprecipitation, proximity ligation assays, and validated cross model and species. The impact of SMAD4 status on therapeutically targeting canonical KRAS signaling was mechanistically deciphered and corroborated by genome-wide gene expression analysis and genetic perturbation experiments, respectively. Validation of a novel tailored therapeutic option was conducted in patient-derived organoids and cells and transgenic as well as orthotopic PDAC models. RESULTS: Our findings determined the tumor biology of an aggressive and chemotherapy-resistant SMAD4-/-/NFATc1High subgroup. Mechanistically, we identify SMAD4 deficiency as a molecular prerequisite for the formation of an oncogenic NFATc1/SMAD3/cJUN transcription factor complex, which drives the expression of RRM1/2. RRM1/2 replenishes nucleoside pools that directly compete with metabolized gemcitabine for DNA strand incorporation. Disassembly of the NFATc1/SMAD3/cJUN complex by mitogen-activated protein kinase signaling inhibition normalizes RRM1/2 expression and synergizes with gemcitabine treatment in vivo to reduce the proliferative index. CONCLUSIONS: Our results suggest that PDAC characterized by SMAD4 deficiency and oncogenic NFATc1/SMAD3/cJUN complex formation exposes sensitivity to a mitogen-activated protein kinase signaling inhibition and gemcitabine combination therapy.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Gemcitabina , Línea Celular Tumoral , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína smad3/metabolismoRESUMEN
BACKGROUND & AIMS: Although T-cell intrinsic expression of G9a has been associated with murine intestinal inflammation, mechanistic insight into the role of this methyltransferase in human T-cell differentiation is ill defined, and manipulation of G9a function for therapeutic use against inflammatory disorders is unexplored. METHODS: Human naive T cells were isolated from peripheral blood and differentiated in vitro in the presence of a G9a inhibitor (UNC0642) before being characterized via the transcriptome (RNA sequencing), chromatin accessibility (assay for transposase-accessible chromatin by sequencing), protein expression (cytometry by time of flight, flow cytometry), metabolism (mitochondrial stress test, ultrahigh performance liquid chromatography-tandem mas spectroscopy) and function (T-cell suppression assay). The in vivo role of G9a was assessed using 3 murine models. RESULTS: We discovered that pharmacologic inhibition of G9a enzymatic function in human CD4 T cells led to spontaneous generation of FOXP3+ T cells (G9a-inibitors-T regulatory cells [Tregs]) in vitro that faithfully reproduce human Tregs, functionally and phenotypically. Mechanistically, G9a inhibition altered the transcriptional regulation of genes involved in lipid biosynthesis in T cells, resulting in increased intracellular cholesterol. Metabolomic profiling of G9a-inibitors-Tregs confirmed elevated lipid pathways that support Treg development through oxidative phosphorylation and enhanced lipid membrane composition. Pharmacologic G9a inhibition promoted Treg expansion in vivo upon antigen (gliadin) stimulation and ameliorated acute trinitrobenzene sulfonic acid-induced colitis secondary to tissue-specific Treg development. Finally, Tregs lacking G9a expression (G9a-knockout Tregs) remain functional chronically and can rescue T-cell transfer-induced colitis. CONCLUSION: G9a inhibition promotes cholesterol metabolism in T cells, favoring a metabolic profile that facilitates Treg development in vitro and in vivo. Our data support the potential use of G9a inhibitors in the treatment of immune-mediated conditions including inflammatory bowel disease.
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Linfocitos T CD4-Positivos , Colitis , Ratones , Humanos , Animales , Metabolismo de los Lípidos , Linfocitos T Reguladores/metabolismo , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colitis/genética , Cromatina , Inflamación , Colesterol , Lípidos , Factores de Transcripción Forkhead/metabolismoRESUMEN
BACKGROUND: Breast cancer (BC) is the most frequent tumor entity in women worldwide with a high chance of therapeutic response in early- and non-metastatic disease stages. Among all BC subtypes, triple-negative BC (TNBC) is the most challenging cancer subtype lacking effective molecular targets due to the particular enrichment of cancer stem cells (CSCs), frequently leading to a chemoresistant phenotype and metastasis. The Ubiquitin Specific Peptidase 22 (USP22) is a deubiquitinase that has been frequently associated with a CSC-promoting function and intimately implicated in resistance to conventional therapies, tumor relapse, metastasis and overall poor survival in a broad range of cancer entities, including BC. To date, though, the role of USP22 in TNBC has been only superficially addressed. METHODS: The current study utilized the MMTV-cre, Usp22fl/fl transgenic mouse model to study the involvement of USP22 in the stem cell-like properties of the growing mammary tissue. Additionally, we combined high-throughput transcriptomic analyses with publicly available patient transcriptomic data and utilized TNBC culture models to decipher the functional role of USP22 in the CSC characteristics of this disease. RESULTS: Interestingly, we identified that USP22 promotes CSC properties and drug tolerance by supporting the oxidative phosphorylation program, known to be largely responsible for the poor response to conventional therapies in this particularly aggressive BC subtype. CONCLUSIONS: This study suggests a novel tumor-supportive role of USP22 in sustaining cellular respiration to facilitate the drug-tolerant behavior of HER2+-BC and TNBC cells. Therefore, we posit USP22 as a promising therapeutic target to optimize standard therapies and combat the aggressiveness of these malignancies. Video Abstract.
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Neoplasias de la Mama Triple Negativas , Animales , Femenino , Humanos , Ratones , Línea Celular Tumoral , Respiración de la Célula , Modelos Animales de Enfermedad , Recurrencia Local de Neoplasia , Neoplasias de la Mama Triple Negativas/patología , Ubiquitina TiolesterasaRESUMEN
The MDM2 oncoprotein ubiquitinates and antagonizes p53 but may also carry out p53-independent functions. Here we report that MDM2 is required for the efficient generation of induced pluripotent stem cells (iPSCs) from murine embryonic fibroblasts, in the absence of p53. Similarly, MDM2 depletion in the context of p53 deficiency also promoted the differentiation of human mesenchymal stem cells and diminished clonogenic survival of cancer cells. Most of the MDM2-controlled genes also responded to the inactivation of the Polycomb Repressor Complex 2 (PRC2) and its catalytic component EZH2. MDM2 physically associated with EZH2 on chromatin, enhancing the trimethylation of histone 3 at lysine 27 and the ubiquitination of histone 2A at lysine 119 (H2AK119) at its target genes. Removing MDM2 simultaneously with the H2AK119 E3 ligase Ring1B/RNF2 further induced these genes and synthetically arrested cell proliferation. In conclusion, MDM2 supports the Polycomb-mediated repression of lineage-specific genes, independent of p53.
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Ensamble y Desensamble de Cromatina , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Células Madre Neoplásicas/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Supervivencia Celular , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Histonas/metabolismo , Humanos , Células MCF-7 , Metilación , Ratones , Osteogénesis , Fenotipo , Complejo Represivo Polycomb 1/metabolismo , Complejo Represivo Polycomb 2/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Interferencia de ARN , Transducción de Señal , Factores de Tiempo , Transfección , Proteína p53 Supresora de Tumor/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) displays a remarkable propensity towards therapy resistance. However, molecular epigenetic and transcriptional mechanisms enabling this are poorly understood. In this study, we aimed to identify novel mechanistic approaches to overcome or prevent resistance in PDAC. DESIGN: We used in vitro and in vivo models of resistant PDAC and integrated epigenomic, transcriptomic, nascent RNA and chromatin topology data. We identified a JunD-driven subgroup of enhancers, called interactive hubs (iHUBs), which mediate transcriptional reprogramming and chemoresistance in PDAC. RESULTS: iHUBs display characteristics typical for active enhancers (H3K27ac enrichment) in both therapy sensitive and resistant states but exhibit increased interactions and production of enhancer RNA (eRNA) in the resistant state. Notably, deletion of individual iHUBs was sufficient to decrease transcription of target genes and sensitise resistant cells to chemotherapy. Overlapping motif analysis and transcriptional profiling identified the activator protein 1 (AP1) transcription factor JunD as a master transcription factor of these enhancers. JunD depletion decreased iHUB interaction frequency and transcription of target genes. Moreover, targeting either eRNA production or signaling pathways upstream of iHUB activation using clinically tested small molecule inhibitors decreased eRNA production and interaction frequency, and restored chemotherapy responsiveness in vitro and in vivo. Representative iHUB target genes were found to be more expressed in patients with poor response to chemotherapy compared with responsive patients. CONCLUSION: Our findings identify an important role for a subgroup of highly connected enhancers (iHUBs) in regulating chemotherapy response and demonstrate targetability in sensitisation to chemotherapy.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Factores de Transcripción/genética , ARN , Elementos de Facilitación Genéticos/genética , Regulación Neoplásica de la Expresión Génica , Línea Celular Tumoral , Neoplasias PancreáticasRESUMEN
BACKGROUND & AIMS: Transforming growth factor ß (TGFß) upregulates cholangiocyte-derived signals that activate myofibroblasts and promote fibrosis. Using epigenomic and transcriptomic approaches, we sought to distinguish the epigenetic activation mechanisms downstream of TGFß that mediate transcription of fibrogenic signals. METHODS: Chromatin immunoprecipitation (ChIP)-seq and RNA-seq were performed to assess histone modifications and transcriptional changes following TGFß stimulation. Histone modifications and acetyltransferase occupancy were confirmed using ChIP assays. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) was used to investigate changes in chromatin accessibility. Cholangiocyte cell lines and primary cholangiocytes were used for in vitro studies. Mdr2-/- and 3,5-diethoxycarboncyl-1,4-dihydrocollidine (DDC)-fed mice were used as animal models. RESULTS: TGFß stimulation caused widespread changes in histone 3 lysine 27 acetylation (H3K27ac), and was associated with global TGFß-mediated transcription. In contrast, H3K9ac was gained in a smaller group of chromatin sites and was associated with fibrosis pathways. These pathways included overexpression of hepatic stellate cell (HSC) activators such as fibronectin 1 (FN1) and SERPINE1. The promoters of these genes showed H3K9ac enrichment following TGFß. Of the acetyltransferases responsible for H3K9ac, cholangiocytes predominantly express Lysine Acetyltransferases 2A (KAT2A). Small interfering RNA knockdown of KAT2A or H3K9ac inhibition prevented the TGFß-mediated increase in FN1 and SERPINE1. SMAD3 ChIP-seq and ATAC-seq suggested that TGFß-mediated H3K9ac occurs through SMAD signaling, which was confirmed using colocalization and genetic knockdown studies. Pharmacologic inhibition or cholangiocyte-selective deletion of Kat2a was protective in mouse models of biliary fibrosis. CONCLUSIONS: Cholangiocyte expression of HSC-activating signals occurs through SMAD-dependent, KAT2A-mediated, H3K9ac, and can be targeted to prevent biliary fibrosis.
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Conductos Biliares/patología , Epigénesis Genética/genética , Histonas/metabolismo , Cirrosis Hepática Biliar/genética , Factor de Crecimiento Transformador beta/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Acetilación/efectos de los fármacos , Animales , Conductos Biliares/citología , Conductos Biliares/efectos de los fármacos , Línea Celular , Secuenciación de Inmunoprecipitación de Cromatina , Modelos Animales de Enfermedad , Epigénesis Genética/efectos de los fármacos , Epigenómica , Técnicas de Silenciamiento del Gen , Histona Acetiltransferasas/antagonistas & inhibidores , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Humanos , Cirrosis Hepática Biliar/inducido químicamente , Cirrosis Hepática Biliar/tratamiento farmacológico , Cirrosis Hepática Biliar/patología , Ratones , Ratones Noqueados , Miofibroblastos/patología , Cultivo Primario de Células , Piridinas/administración & dosificación , Piridinas/toxicidad , RNA-Seq , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Schwann cells are the nerve ensheathing cells of the peripheral nervous system. Absence, loss and malfunction of Schwann cells or their myelin sheaths lead to peripheral neuropathies such as Charcot-Marie-Tooth disease in humans. During Schwann cell development and myelination chromatin is dramatically modified. However, impact and functional relevance of these modifications are poorly understood. Here, we analyzed histone H2B monoubiquitination as one such chromatin modification by conditionally deleting the Rnf40 subunit of the responsible E3 ligase in mice. Rnf40-deficient Schwann cells were arrested immediately before myelination or generated abnormally thin, unstable myelin, resulting in a peripheral neuropathy characterized by hypomyelination and progressive axonal degeneration. By combining sequencing techniques with functional studies we show that H2B monoubiquitination does not influence global gene expression patterns, but instead ensures selective high expression of myelin and lipid biosynthesis genes and proper repression of immaturity genes. This requires the specific recruitment of the Rnf40-containing E3 ligase by Egr2, the central transcriptional regulator of peripheral myelination, to its target genes. Our study identifies histone ubiquitination as essential for Schwann cell myelination and unravels new disease-relevant links between chromatin modifications and transcription factors in the underlying regulatory network.
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Proteína 2 de la Respuesta de Crecimiento Precoz/fisiología , Neuropatía Hereditaria Motora y Sensorial/metabolismo , Histonas/metabolismo , Sistema Nervioso Periférico/metabolismo , Células de Schwann/metabolismo , Animales , Línea Celular Tumoral , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Sistema Nervioso Periférico/patología , Ratas , Células de Schwann/patología , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Ubiquitin (ub) is a small, highly conserved protein widely expressed in eukaryotic cells. Ubiquitination is a post-translational modification catalyzed by enzymes that activate, conjugate, and ligate ub to proteins. Substrates can be modified either by addition of a single ubiquitin molecule (monoubiquitination), or by conjugation of several ubs (polyubiquitination). Monoubiquitination acts as a signaling mark to control diverse biological processes. The cellular and spatial distribution of ub is determined by the opposing activities of ub ligase enzymes, and deubiquitinases (DUBs), which remove ub from proteins to generate free ub. In mammalian cells, 1-2% of total histone H2B is monoubiquitinated. The SAGA (Spt Ada Gcn5 Acetyl-transferase) is a transcriptional coactivator and its DUB module removes ub from H2Bub1. The mammalian SAGA DUB module has four subunits, ATXN7, ATXN7L3, USP22, and ENY2. Atxn7l3-/- mouse embryos, lacking DUB activity, have a five-fold increase in H2Bub1 retention, and die at mid-gestation. Interestingly, embryos lacking the ub encoding gene, Ubc, have a similar phenotype. Here we provide a current overview of data suggesting that H2Bub1 retention on the chromatin in Atxn7l3-/- embryos may lead to an imbalance in free ub distribution. Thus, we speculate that ATXN7L3-containing DUBs impact the free cellular ub pool during development.
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Histonas , Ubiquitina , Animales , Desarrollo Embrionario/genética , Histonas/genética , Histonas/metabolismo , Mamíferos/metabolismo , Ratones , Procesamiento Proteico-Postraduccional , Ubiquitina/genética , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Molecular subtyping of cancer offers tremendous promise for the optimization of a precision oncology approach to anticancer therapy. Recent advances in pancreatic cancer research uncovered various molecular subtypes with tumors expressing a squamous/basal-like gene expression signature displaying a worse prognosis. Through unbiased epigenome mapping, we identified deltaNp63 as a major driver of a gene signature in pancreatic cancer cell lines, which we report to faithfully represent the highly aggressive pancreatic squamous subtype observed in vivo, and display the specific epigenetic marking of genes associated with decreased survival. Importantly, depletion of deltaNp63 in these systems significantly decreased cell proliferation and gene expression patterns associated with a squamous subtype and transcriptionally mimicked a subtype switch. Using genomic localization data of deltaNp63 in pancreatic cancer cell lines coupled with epigenome mapping data from patient-derived xenografts, we uncovered that deltaNp63 mainly exerts its effects by activating subtype-specific super enhancers. Furthermore, we identified a group of 45 subtype-specific super enhancers that are associated with poorer prognosis and are highly dependent on deltaNp63. Genes associated with these enhancers included a network of transcription factors, including HIF1A, BHLHE40, and RXRA, which form a highly intertwined transcriptional regulatory network with deltaNp63 to further activate downstream genes associated with poor survival.
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Neoplasias Pancreáticas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Carcinoma de Células Escamosas/genética , Línea Celular Tumoral , Proliferación Celular/genética , Metilación de ADN/genética , Elementos de Facilitación Genéticos/genética , Epigénesis Genética/genética , Epigenómica/métodos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Neoplasias Pancreáticas/metabolismo , Medicina de Precisión , Pronóstico , Secuencias Reguladoras de Ácidos Nucleicos/genéticaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is resistant to virtually all chemo- and targeted therapeutic approaches. Epigenetic regulators represent a novel class of drug targets. Among them, BET and HDAC proteins are central regulators of chromatin structure and transcription, and preclinical evidence suggests effectiveness of combined BET and HDAC inhibition in PDAC. Here, we describe that TW9, a newly generated adduct of the BET inhibitor (+)-JQ1 and class I HDAC inhibitor CI994, is a potent dual inhibitor simultaneously targeting BET and HDAC proteins. TW9 has a similar affinity to BRD4 bromodomains as (+)-JQ1 and shares a conserved binding mode, but is significantly more active in inhibiting HDAC1 compared to the parental HDAC inhibitor CI994. TW9 was more potent in inhibiting tumor cell proliferation compared to (+)-JQ1, CI994 alone or combined treatment of both inhibitors. Sequential administration of gemcitabine and TW9 showed additional synergistic antitumor effects. Microarray analysis revealed that dysregulation of a FOSL1-directed transcriptional program contributed to the antitumor effects of TW9. Our results demonstrate the potential of a dual chromatin-targeting strategy in the treatment of PDAC and provide a rationale for further development of multitarget inhibitors.
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Antineoplásicos/farmacología , Azepinas/química , Carcinoma Ductal Pancreático/genética , Inhibidores de Histona Desacetilasas/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas c-fos/genética , Triazoles/química , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Sinergismo Farmacológico , Epigénesis Genética/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Histona Desacetilasa 1/antagonistas & inhibidores , Humanos , Neoplasias Pancreáticas/metabolismo , Dominios Proteicos/efectos de los fármacos , Factores de Transcripción/química , Factores de Transcripción/metabolismo , GemcitabinaRESUMEN
Extensive changes in posttranslational histone modifications accompany the rewiring of the transcriptional program during stem cell differentiation. However, the mechanisms controlling the changes in specific chromatin modifications and their function during differentiation remain only poorly understood. We show that histone H2B monoubiquitination (H2Bub1) significantly increases during differentiation of human mesenchymal stem cells (hMSCs) and various lineage-committed precursor cells and in diverse organisms. Furthermore, the H2B ubiquitin ligase RNF40 is required for the induction of differentiation markers and transcriptional reprogramming of hMSCs. This function is dependent upon CDK9 and the WAC adaptor protein, which are required for H2B monoubiquitination. Finally, we show that RNF40 is required for the resolution of the H3K4me3/H3K27me3 bivalent poised state on lineage-specific genes during the transition from an inactive to an active chromatin conformation. Thus, these data indicate that H2Bub1 is required for maintaining multipotency of hMSCs and plays a central role in controlling stem cell differentiation.
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Diferenciación Celular/genética , Histonas/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Multipotentes/citología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/fisiología , Línea Celular , Ensamble y Desensamble de Cromatina , Quinasa 9 Dependiente de la Ciclina/genética , Quinasa 9 Dependiente de la Ciclina/fisiología , Humanos , Células Madre Mesenquimatosas/metabolismo , Células Madre Multipotentes/metabolismo , Procesamiento Proteico-Postraduccional , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , UbiquitinaciónRESUMEN
Chromatin remodelling precedes transcriptional and structural changes in heart failure. A body of work suggests roles for the developmental Wnt signalling pathway in cardiac remodelling. Hitherto, there is no evidence supporting a direct role of Wnt nuclear components in regulating chromatin landscapes in this process. We show that transcriptionally active, nuclear, phosphorylated(p)Ser675-ß-catenin and TCF7L2 are upregulated in diseased murine and human cardiac ventricles. We report that inducible cardiomyocytes (CM)-specific pSer675-ß-catenin accumulation mimics the disease situation by triggering TCF7L2 expression. This enhances active chromatin, characterized by increased H3K27ac and TCF7L2 occupancies to cardiac developmental and remodelling genes in vivo. Accordingly, transcriptomic analysis of ß-catenin stabilized hearts shows a strong recapitulation of cardiac developmental processes like cell cycling and cytoskeletal remodelling. Mechanistically, TCF7L2 co-occupies distal genomic regions with cardiac transcription factors NKX2-5 and GATA4 in stabilized-ß-catenin hearts. Validation assays revealed a previously unrecognized function of GATA4 as a cardiac repressor of the TCF7L2/ß-catenin complex in vivo, thereby defining a transcriptional switch controlling disease progression. Conversely, preventing ß-catenin activation post-pressure-overload results in a downregulation of these novel TCF7L2-targets and rescues cardiac function. Thus, we present a novel role for TCF7L2/ß-catenin in CMs-specific chromatin modulation, which could be exploited for manipulating the ubiquitous Wnt pathway.
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Cromatina/genética , Factor de Transcripción GATA4/genética , Insuficiencia Cardíaca/genética , Proteína 2 Similar al Factor de Transcripción 7/genética , beta Catenina/genética , Adulto , Animales , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/genética , Progresión de la Enfermedad , Factor de Transcripción GATA4/metabolismo , Perfilación de la Expresión Génica , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Humanos , Ratones Noqueados , Ratones Transgénicos , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/metabolismo , Unión Proteica , Proteína 2 Similar al Factor de Transcripción 7/metabolismo , Vía de Señalización Wnt/genética , beta Catenina/metabolismoRESUMEN
The coordinated temporal and spatial activation of gene expression is essential for proper stem cell differentiation. The Chromodomain Helicase DNA-binding protein 1 (CHD1) is a chromatin remodeler closely associated with transcription and nucleosome turnover downstream of the transcriptional start site (TSS). In this study, we show that CHD1 is required for the induction of osteoblast-specific gene expression, extracellular-matrix mineralization and ectopic bone formation in vivo. Genome-wide occupancy analyses revealed increased CHD1 occupancy around the TSS of differentiation-activated genes. Furthermore, we observed that CHD1-dependent genes are mainly induced during osteoblast differentiation and are characterized by higher levels of CHD1 occupancy around the TSS. Interestingly, CHD1 depletion resulted in increased pausing of RNA Polymerase II (RNAPII) and decreased H2A.Z occupancy close to the TSS, but not at enhancer regions. These findings reveal a novel role for CHD1 during osteoblast differentiation and provide further insights into the intricacies of epigenetic regulatory mechanisms controlling cell fate determination.
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Diferenciación Celular/fisiología , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Diferenciación Celular/genética , Células Cultivadas , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/genética , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Histonas/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , ARN Polimerasa II/metabolismo , ARN Interferente Pequeño/genética , Sitio de Iniciación de la TranscripciónRESUMEN
Bromodomain-containing protein 4 (BRD4) is a member of the bromo- and extraterminal (BET) domain-containing family of epigenetic readers which is under intensive investigation as a target for anti-tumor therapy. BRD4 plays a central role in promoting the expression of select subsets of genes including many driven by oncogenic transcription factors and signaling pathways. However, the role of BRD4 and the effects of BET inhibitors in non-transformed cells remain mostly unclear. We demonstrate that BRD4 is required for the maintenance of a basal epithelial phenotype by regulating the expression of epithelial-specific genes including TP63 and Grainy Head-like transcription factor-3 (GRHL3) in non-transformed basal-like mammary epithelial cells. Moreover, BRD4 occupancy correlates with enhancer activity and enhancer RNA (eRNA) transcription. Motif analyses of cell context-specific BRD4-enriched regions predicted the involvement of FOXO transcription factors. Consistently, activation of FOXO1 function via inhibition of EGFR-AKT signaling promoted the expression of TP63 and GRHL3. Moreover, activation of Src kinase signaling and FOXO1 inhibition decreased the expression of FOXO/BRD4 target genes. Together, our findings support a function for BRD4 in promoting basal mammary cell epithelial differentiation, at least in part, by regulating FOXO factor function on enhancers to activate TP63 and GRHL3 expression.
Asunto(s)
Mama/metabolismo , Proteínas de Unión al ADN/genética , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genética , Mama/citología , Proteínas de Ciclo Celular , Línea Celular , Proteínas de Unión al ADN/biosíntesis , Elementos de Facilitación Genéticos , Factores de Transcripción Forkhead/metabolismo , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , ARN Polimerasa II/metabolismo , Transducción de Señal , Factores de Transcripción/biosíntesis , Transcripción Genética , Proteínas Supresoras de Tumor/biosíntesisRESUMEN
Proper temporal epigenetic regulation of gene expression is essential for cell fate determination and tissue development. The Bromodomain-containing Protein-4 (BRD4) was previously shown to control the transcription of defined subsets of genes in various cell systems. In this study we examined the role of BRD4 in promoting lineage-specific gene expression and show that BRD4 is essential for osteoblast differentiation. Genome-wide analyses demonstrate that BRD4 is recruited to the transcriptional start site of differentiation-induced genes. Unexpectedly, while promoter-proximal BRD4 occupancy correlated with gene expression, genes which displayed moderate expression and promoter-proximal BRD4 occupancy were most highly regulated and sensitive to BRD4 inhibition. Therefore, we examined distal BRD4 occupancy and uncovered a specific co-localization of BRD4 with the transcription factors C/EBPb, TEAD1, FOSL2 and JUND at putative osteoblast-specific enhancers. These findings reveal the intricacies of lineage specification and provide new insight into the context-dependent functions of BRD4.
Asunto(s)
Linaje de la Célula/genética , Epigénesis Genética , Células Epiteliales/metabolismo , Células Madre Mesenquimatosas/metabolismo , Proteínas Nucleares/genética , Osteoblastos/metabolismo , Osteocitos/metabolismo , Factores de Transcripción/genética , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteínas de Ciclo Celular , Diferenciación Celular , Línea Celular , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/citología , Antígeno 2 Relacionado con Fos/genética , Antígeno 2 Relacionado con Fos/metabolismo , Perfilación de la Expresión Génica , Humanos , Células Madre Mesenquimatosas/citología , Proteínas Nucleares/metabolismo , Especificidad de Órganos , Osteoblastos/citología , Osteocitos/citología , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Transducción de Señal , Factores de Transcripción de Dominio TEA , Factores de Transcripción/metabolismo , Sitio de Iniciación de la TranscripciónRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a particularly dismal prognosis. Histone deacetylases (HDAC) are epigenetic modulators whose activity is frequently deregulated in various cancers including PDAC. In particular, class-I HDACs (HDAC 1, 2, 3 and 8) have been shown to play an important role in PDAC. In this study, we investigated the effects of the class I-specific HDAC inhibitor (HDACi) 4SC-202 in multiple PDAC cell lines in promoting tumor cell differentiation. We show that 4SC-202 negatively affects TGFß signaling and inhibits TGFß-induced epithelial-to-mesenchymal transition (EMT). Moreover, 4SC-202 markedly induced p21 (CDKN1A) expression and significantly attenuated cell proliferation. Mechanistically, genome-wide studies revealed that 4SC-202-induced genes were enriched for Bromodomain-containing Protein-4 (BRD4) and MYC occupancy. BRD4, a well-characterized acetyllysine reader, has been shown to play a major role in regulating transcription of selected subsets of genes. Importantly, BRD4 and MYC are essential for the expression of a subgroup of genes induced by class-I HDACi. Taken together, our study uncovers a previously unknown role of BRD4 and MYC in eliciting the HDACi-mediated induction of a subset of genes and provides molecular insight into the mechanisms of HDACi action in PDAC.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Inhibidores de Histona Desacetilasas/farmacología , Proteínas Nucleares/fisiología , Neoplasias Pancreáticas/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-myc/fisiología , Factores de Transcripción/fisiología , Animales , Benzamidas/farmacología , Carcinoma Ductal Pancreático/patología , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Transición Epitelial-Mesenquimal , Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Histona Desacetilasa 1/metabolismo , Humanos , Ratones Desnudos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Fenotipo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND & AIMS: The ability of exocrine pancreatic cells to change the cellular phenotype is required for tissue regeneration upon injury, but also contributes to their malignant transformation and tumor progression. We investigated context-dependent signaling and transcription mechanisms that determine pancreatic cell fate decisions toward regeneration and malignancy. In particular, we studied the function and regulation of the inflammatory transcription factor nuclear factor of activated T cells 1 (NFATC1) in pancreatic cell plasticity and tissue adaptation. METHODS: We analyzed cell plasticity during pancreatic regeneration and transformation in mice with pancreas-specific expression of a constitutively active form of NFATC1, or depletion of enhancer of zeste 2 homologue 2 (EZH2), in the context of wild-type or constitutively activate Kras, respectively. Acute and chronic pancreatitis were induced by intraperitoneal injection of caerulein. EZH2-dependent regulation of NFATC1 expression was studied in mouse in human pancreatic tissue and cells by immunohistochemistry, immunoblotting, and quantitative reverse transcription polymerase chain reaction. We used genetic and pharmacologic approaches of EZH2 and NFATC1 inhibition to study the consequences of pathway disruption on pancreatic morphology and function. Epigenetic modifications on the NFATC1 gene were investigated by chromatin immunoprecipitation assays. RESULTS: NFATC1 was rapidly and transiently induced in early adaptation to acinar cell injury in human samples and in mice, where it promoted acinar cell transdifferentiation and blocked proliferation of metaplastic pancreatic cells. However, in late stages of regeneration, Nfatc1 was epigenetically silenced by EZH2-dependent histone methylation, to enable acinar cell redifferentiation and prevent organ atrophy and exocrine insufficiency. In contrast, oncogenic activation of KRAS signaling in pancreatic ductal adenocarcinoma cells reversed the EZH2-dependent effects on the NFATC1 gene and was required for EZH2-mediated transcriptional activation of NFATC1. CONCLUSIONS: In studies of human and mouse pancreatic cells and tissue, we identified context-specific epigenetic regulation of NFATc1 activity as an important mechanism of pancreatic cell plasticity. Inhibitors of EZH2 might therefore interfere with oncogenic activity of NFATC1 and be used in treatment of pancreatic ductal adenocarcinoma.
Asunto(s)
Carcinoma Ductal Pancreático/genética , Plasticidad de la Célula/genética , Transformación Celular Neoplásica/genética , Proteína Potenciadora del Homólogo Zeste 2/genética , Regulación de la Expresión Génica , Factores de Transcripción NFATC/genética , Neoplasias Pancreáticas/genética , Regeneración/genética , Células Acinares/fisiología , Animales , Carcinoma Ductal Pancreático/química , Proliferación Celular/genética , Transdiferenciación Celular/genética , Ceruletida , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Proteína Potenciadora del Homólogo Zeste 2/análisis , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Silenciador del Gen , Histonas/metabolismo , Humanos , Metilación , Ratones , Factores de Transcripción NFATC/análisis , Factores de Transcripción NFATC/metabolismo , Páncreas/fisiología , Neoplasias Pancreáticas/química , Pancreatitis Crónica/inducido químicamente , Pancreatitis Crónica/fisiopatología , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transducción de Señal/genética , Transcripción GenéticaRESUMEN
Although inhibitors of bromodomain and extra terminal domain (BET) proteins show promising clinical activity in different hematologic malignancies, a systematic analysis of the consequences of pharmacological BET inhibition on healthy hematopoietic (stem) cells is urgently needed. We found that JQ1 treatment decreases the numbers of pre-, immature and mature B cells while numbers of early pro-B cells remain constant. In addition, JQ1 treatment increases apoptosis in T cells, all together leading to reduced cellularity in thymus, bone marrow and spleen. Furthermore, JQ1 induces proliferation of long-term hematopoietic stem cells, thereby increasing stem cell numbers. Due to increased numbers, JQ1-treated hematopoietic stem cells engrafted better after stem cell transplantation and repopulated the hematopoietic system significantly faster after sublethal myeloablation. As quantity and functionality of hematopoietic stem cells determine the duration of life-threatening myelosuppression, BET inhibition might benefit patients in myelosuppressive conditions.