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1.
Int J Mol Sci ; 22(7)2021 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-33804860

RESUMEN

The transcriptional regulator peroxisome proliferator activated receptor gamma coactivator 1A (PGC-1α), encoded by PPARGC1A, has been linked to neurodegenerative diseases. Recently discovered CNS-specific PPARGC1A transcripts are initiated far upstream of the reference promoter, spliced to exon 2 of the reference gene, and are more abundant than reference gene transcripts in post-mortem human brain samples. The proteins translated from the CNS and reference transcripts differ only at their N-terminal regions. To dissect functional differences between CNS-specific isoforms and reference proteins, we used clustered regularly interspaced short palindromic repeats transcriptional activation (CRISPRa) for selective endogenous activation of the CNS or the reference promoters in SH-SY5Y cells. Expression and/or exon usage of the targets was ascertained by RNA sequencing. Compared to controls, more differentially expressed genes were observed after activation of the CNS than the reference gene promoter, while the magnitude of alternative exon usage was comparable between activation of the two promoters. Promoter-selective associations were observed with canonical signaling pathways, mitochondrial and nervous system functions and neurological diseases. The distinct N-terminal as well as the shared downstream regions of PGC-1α isoforms affect the exon usage of numerous genes. Furthermore, associations of risk genes of amyotrophic lateral sclerosis and Parkinson's disease were noted with differentially expressed genes resulting from the activation of the CNS and reference gene promoter, respectively. Thus, CNS-specific isoforms markedly amplify the biological functions of PPARGC1A and CNS-specific isoforms and reference proteins have common, complementary and selective functions relevant for neurodegenerative diseases.


Asunto(s)
Redes Reguladoras de Genes , Enfermedades Neurodegenerativas/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Regiones Promotoras Genéticas , Activación Transcripcional , Línea Celular Tumoral , Exones , Células HEK293 , Humanos , Neuronas/metabolismo , Motivos de Nucleótidos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transcriptoma
2.
Int J Mol Sci ; 22(7)2021 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-33804861

RESUMEN

Cancer-associated fibroblasts (CAF) are attractive therapeutic targets in the tumor microenvironment. The possibility of using CAFs as a source of therapeutic molecules is a challenging approach in gene therapy. This requires transcriptional targeting of transgene expression by cis-regulatory elements (CRE). Little is known about which CREs can provide selective transgene expression in CAFs. We hypothesized that the promoters of FAP, CXCL12, IGFBP2, CTGF, JAG1, SNAI1, and SPARC genes, the expression of whose is increased in CAFs, could be used for transcriptional targeting. Analysis of the transcription of the corresponding genes revealed that unique transcription in model CAFs was characteristic for the CXCL12 and FAP genes. However, none of the promoters in luciferase reporter constructs show selective activity in these fibroblasts. The CTGF, IGFBP2, JAG1, and SPARC promoters can provide higher transgene expression in fibroblasts than in cancer cells, but the nonspecific viral promoters CMV, SV40, and the recently studied universal PCNA promoter have the same features. The patterns of changes in activity of various promoters relative to each other observed for human cell lines were similar to the patterns of activity for the same promoters both in vivo and in vitro in mouse models. Our results reveal restrictions and features for CAF transcriptional targeting.


Asunto(s)
Fibroblastos Asociados al Cáncer/metabolismo , Regiones Promotoras Genéticas , Transgenes , Microambiente Tumoral/genética , Animales , Línea Celular Tumoral , Quimiocina CXCL12/genética , Factor de Crecimiento del Tejido Conjuntivo/genética , Gelatinasas/genética , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína Jagged-1/genética , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Osteonectina/genética , Serina Endopeptidasas/genética , Factores de Transcripción de la Familia Snail/genética , Activación Transcripcional
3.
Int J Mol Sci ; 22(7)2021 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-33810223

RESUMEN

DNA can adopt various structures besides the B-form. Among them, cruciform structures are formed on inverted repeat (IR) sequences. While cruciform formable IRs (CFIRs) are sometimes found in regulatory regions of transcription, their function in transcription remains elusive, especially in eukaryotes. We found a cluster of CFIRs within the mouse Pou5f1 enhancer. Here, we demonstrate that this cluster or some member(s) plays an active role in the transcriptional regulation of not only Pou5f1, but also Sox2, Nanog, Klf4 and Esrrb. To clarify in vivo function of the cluster, we performed genome editing using mouse ES cells, in which each of the CFIRs was altered to the corresponding mirror repeat sequence. The alterations reduced the level of the Pou5f1 transcript in the genome-edited cell lines, and elevated those of Sox2, Nanog, Klf4 and Esrrb. Furthermore, transcription of non-coding RNAs (ncRNAs) within the enhancer was also upregulated in the genome-edited cell lines, in a similar manner to Sox2, Nanog, Klf4 and Esrrb. These ncRNAs are hypothesized to control the expression of these four pluripotency genes. The CFIRs present in the Pou5f1 enhancer seem to be important to maintain the integrity of ES cells.


Asunto(s)
Elementos de Facilitación Genéticos , Células Madre Embrionarias de Ratones/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Animales , Línea Celular , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo , Conformación de Ácido Nucleico , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Receptores Estrogénicos/genética , Receptores Estrogénicos/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Activación Transcripcional , Regulación hacia Arriba
4.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-33802993

RESUMEN

Despite advances in the preparation of metal oxide (MO) nanoparticles (NPs) as catalysts for various applications, concerns about the biosafety of these particles remain. In this study, we prepared transition metal-doped cerium oxide (TM@CeO2; TM = Cr, Mn, Fe, Co, or Ni) nanoparticles and investigated the mechanism underlying dopant-dependent toxicity in HaCaT human keratinocytes. We show that doping with Cr or Co but not Fe, Mn, or Ni increased the toxicity of CeO2 NPs in dose- and time-dependent manners and led to apoptotic cell death. Interestingly, while both undoped and transition metal-doped NPs increased intracellular reactive oxygen species (ROS), toxic Cr@CeO2 and Co@CeO2 NPs failed to induce the expression of NRF2 (nuclear factor erythroid 2-related factor 2) as well as its downstream target genes involved in the antioxidant defense system. Moreover, activation of NRF2 transcription was correlated with dynamic changes in H3K4me3 and H3K27me3 at the promoter of NRF2, which was not observed in cells exposed to Cr@CeO2 NPs. Furthermore, exposure to relatively non-toxic Fe@CeO2 NPs, but not the toxic Cr@CeO2 NPs, resulted in increased binding of MLL1 complex, a major histone lysine methylase mediating trimethylation of histone H3 lysine 4, at the NRF2 promoter. Taken together, our findings strongly suggest that failure of cells to respond to oxidative stress is critical for dopant-dependent toxicity of CeO2 NPs and emphasize that careful evaluation of newly developed NPs should be preceded before industrial or biomedical applications.


Asunto(s)
Cerio/toxicidad , Histonas/metabolismo , Factor 2 Relacionado con NF-E2/genética , Nanopartículas/toxicidad , Activación Transcripcional/genética , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Metilación , Factor 2 Relacionado con NF-E2/metabolismo , Nanopartículas/ultraestructura , Regiones Promotoras Genéticas/genética , Especies Reactivas de Oxígeno/metabolismo
5.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-33802672

RESUMEN

Raf kinase inhibitory protein (RKIP), also known as a phosphatidylethanolamine-binding protein 1 (PEBP1), functions as a tumor suppressor and regulates several signaling pathways, including ERK and NF-κκB. RKIP is severely downregulated in human malignant cancers, indicating a functional association with cancer metastasis and poor prognosis. The transcription regulation of RKIP gene in human cancers is not well understood. In this study, we suggested a possible transcription mechanism for the regulation of RKIP in human cancer cells. We found that Metadherin (MTDH) significantly repressed the transcriptional activity of RKIP gene. An analysis of publicly available datasets showed that the knockdown of MTDH in breast and endometrial cancer cell lines induced the expression RKIP. In addition, the results obtained from qRT-PCR and ChIP analyses showed that MTDH considerably inhibited RKIP expression. In addition, the RKIP transcript levels in MTDH-knockdown or MTDH-overexpressing MCF-7 cells were likely correlated to the protein levels, suggesting that MTDH regulates RKIP expression. In conclusion, we suggest that MTDH is a novel factor that controls the RKIP transcription, which is essential for cancer progression.


Asunto(s)
Progresión de la Enfermedad , Proteínas de la Membrana/metabolismo , Neoplasias/genética , Neoplasias/patología , Proteínas de Unión a Fosfatidiletanolamina/genética , Proteínas de Unión al ARN/metabolismo , Transcripción Genética , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de la Membrana/genética , Mutación/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas de Unión al ARN/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Regulación hacia Arriba/genética
6.
Mol Cell ; 81(8): 1666-1681.e6, 2021 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-33823140

RESUMEN

Nuclear speckles are prominent nuclear bodies that contain proteins and RNA involved in gene expression. Although links between nuclear speckles and gene activation are emerging, the mechanisms regulating association of genes with speckles are unclear. We find that speckle association of p53 target genes is driven by the p53 transcription factor. Focusing on p21, a key p53 target, we demonstrate that speckle association boosts expression by elevating nascent RNA amounts. p53-regulated speckle association did not depend on p53 transactivation functions but required an intact proline-rich domain and direct DNA binding, providing mechanisms within p53 for regulating gene-speckle association. Beyond p21, a substantial subset of p53 targets have p53-regulated speckle association. Strikingly, speckle-associating p53 targets are more robustly activated and occupy a distinct niche of p53 biology compared with non-speckle-associating p53 targets. Together, our findings illuminate regulated speckle association as a mechanism used by a transcription factor to boost gene expression.


Asunto(s)
Núcleo Celular/genética , Regulación de la Expresión Génica/genética , Proteínas Nucleares/genética , ARN/genética , Activación Transcripcional/genética , Proteína p53 Supresora de Tumor/genética , ADN/genética , Células HEK293 , Humanos , Cuerpos de Inclusión Intranucleares/genética , Unión Proteica/genética , Factores de Transcripción/genética , Transcripción Genética/genética
7.
Int J Mol Sci ; 22(5)2021 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-33800795

RESUMEN

Plant growth and development are challenged by biotic and abiotic stresses including salinity and heat stresses. For Populus simonii × P. nigra as an important greening and economic tree species in China, increasing soil salinization and global warming have become major environmental challenges. We aim to unravel the molecular mechanisms underlying tree tolerance to salt stress and high temprerature (HT) stress conditions. Transcriptomics revealed that a PsnNAC036 transcription factor (TF) was significantly induced by salt stress in P. simonii × P. nigra. This study focuses on addressing the biological functions of PsnNAC036. The gene was cloned, and its temporal and spatial expression was analyzed under different stresses. PsnNAC036 was significantly upregulated under 150 mM NaCl and 37 °C for 12 h. The result is consistent with the presence of stress responsive cis-elements in the PsnNAC036 promoter. Subcellular localization analysis showed that PsnNAC036 was targeted to the nucleus. Additionally, PsnNAC036 was highly expressed in the leaves and roots. To investigate the core activation region of PsnNAC036 protein and its potential regulatory factors and targets, we conducted trans-activation analysis and the result indicates that the C-terminal region of 191-343 amino acids of the PsnNAC036 was a potent activation domain. Furthermore, overexpression of PsnNAC036 stimulated plant growth and enhanced salinity and HT tolerance. Moreover, 14 stress-related genes upregulated in the transgenic plants under high salt and HT conditions may be potential targets of the PsnNAC036. All the results demonstrate that PsnNAC036 plays an important role in salt and HT stress tolerance.


Asunto(s)
Genes de Plantas , Respuesta al Choque Térmico/genética , Proteínas de Plantas/fisiología , Populus/genética , Estrés Salino/genética , Plantas Tolerantes a la Sal/genética , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Clorofila/biosíntesis , Cruzamientos Genéticos , Regulación de la Expresión Génica de las Plantas , Calor , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Populus/fisiología , Regiones Promotoras Genéticas/genética , Salinidad , Plantas Tolerantes a la Sal/crecimiento & desarrollo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Fracciones Subcelulares/metabolismo , Tabaco/genética , Tabaco/metabolismo , Factores de Transcripción/genética , Activación Transcripcional
8.
Int J Mol Sci ; 22(6)2021 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-33809823

RESUMEN

Dehydrins (DHNs) play an important role in abiotic stress tolerance in a large number of plants, but very little is known about the function of DHNs in pepper plants. Here, we isolated a Y1SK2-type DHN gene "CaDHN3" from pepper. To authenticate the function of CaDHN3 in salt and drought stresses, it was overexpressed in Arabidopsis and silenced in pepper through virus-induced gene silencing (VIGS). Sub-cellular localization showed that CaDHN3 was located in the nucleus and cell membrane. It was found that CaDHN3-overexpressed (OE) in Arabidopsis plants showed salt and drought tolerance phenotypic characteristics, i.e., increased the initial rooting length and germination rate, enhanced chlorophyll content, lowered the relative electrolyte leakage (REL) and malondialdehyde (MDA) content than the wild-type (WT) plants. Moreover, a substantial increase in the activities of antioxidant enzymes; including the superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and lower hydrogen peroxide (H2O2) contents and higher O2•- contents in the transgenic Arabidopsis plants. Silencing of CaDHN3 in pepper decreased the salt- and drought-stress tolerance, through a higher REL and MDA content, and there was more accumulation of reactive oxygen species (ROS) in the CaDHN3-silenced pepper plants than the control plants. Based on the yeast two-hybrid (Y2H) screening and Bimolecular Fluorescence Complementation (BiFC) results, we found that CaDHN3 interacts with CaHIRD11 protein in the plasma membrane. Correspondingly, the expressions of four osmotic-related genes were significantly up-regulated in the CaDHN3-overexpressed lines. In brief, our results manifested that CaDHN3 may play an important role in regulating the relative osmotic stress responses in plants through the ROS signaling pathway. The results of this study will provide a basis for further analyses of the function of DHN genes in pepper.


Asunto(s)
Capsicum/fisiología , Sequías , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Especies Reactivas de Oxígeno/metabolismo , Tolerancia a la Sal/genética , Estrés Fisiológico , Adaptación Biológica , Arabidopsis/fisiología , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Fenotipo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Transporte de Proteínas , Activación Transcripcional
9.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-33799739

RESUMEN

The chromatin remodeler SWI/SNF is an important participant in gene activation, functioning predominantly by opening the chromatin structure on promoters and enhancers. Here, we describe its novel mode of action in which SWI/SNF factors mediate the targeted action of an enhancer. We studied the functions of two signature subunits of PBAP subfamily, BAP170 and SAYP, in Drosophila. These subunits were stably tethered to a transgene reporter carrying the hsp70 core promoter. The tethered subunits mediate transcription of the reporter in a pattern that is generated by enhancers close to the insertion site in multiple loci throughout the genome. Both tethered SAYP and BAP170 recruit the whole PBAP complex to the reporter promoter. However, we found that BAP170-dependent transcription is more resistant to the depletion of other PBAP subunits, suggesting that BAP170 may play a more critical role in establishing enhancer-dependent transcription.


Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Elementos de Facilitación Genéticos/genética , Factores de Transcripción/genética , Transcripción Genética , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta/métodos , Humanos , Hibridación in Situ/métodos , Modelos Genéticos , Regiones Promotoras Genéticas/genética , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional
10.
Nat Commun ; 12(1): 2118, 2021 04 09.
Artículo en Inglés | MEDLINE | ID: mdl-33837181

RESUMEN

Hematopoietic stem cells (HSCs) in adult bone marrow (BM) are usually maintained in a state of quiescence. The cellular mechanism coordinating the balance between HSC quiescence and differentiation is not fully understood. Here, we report that galactose-binding lectin-3 (galectin-3; Gal-3) is upregulated by Tie2 or Mpl activation to maintain quiescence. Conditional overexpression of Gal-3 in mouse HSCs under the transcriptional control of Tie2 or Vav1 promoters (Gal-3 Tg) causes cell cycle retardation via induction of p21. Conversely, the cell cycle of long-term repopulating HSCs (LT-HSCs) in Gal-3-deficient (Gal-3-/-) mice is accelerated, resulting in their exhaustion. Mechanistically, Gal-3 regulates p21 transcription by forming a complex with Sp1, thus blocking cell cycle entry. These results demonstrate that Gal-3 is a negative regulator of cell-cycling in HSCs and plays a crucial role in adult hematopoiesis to prevent HSC exhaustion.


Asunto(s)
Células Madre Adultas/fisiología , Ciclo Celular/fisiología , Galectina 3/metabolismo , Hematopoyesis/genética , Células Madre Hematopoyéticas/fisiología , Animales , Diferenciación Celular/genética , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Femenino , Galectina 3/genética , Ratones , Ratones Noqueados , Modelos Animales , Receptor TIE-2/metabolismo , Receptores de Trombopoyetina/metabolismo , Factor de Transcripción Sp1/metabolismo , Activación Transcripcional , Regulación hacia Arriba
11.
Nat Commun ; 12(1): 1375, 2021 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-33654095

RESUMEN

Cellular adaptation to hypoxia is a hallmark of cancer, but the relative contribution of hypoxia-inducible factors (HIFs) versus other oxygen sensors to tumorigenesis is unclear. We employ a multi-omics pipeline including measurements of nascent RNA to characterize transcriptional changes upon acute hypoxia. We identify an immediate early transcriptional response that is strongly dependent on HIF1A and the kinase activity of its cofactor CDK8, includes indirect repression of MYC targets, and is highly conserved across cancer types. HIF1A drives this acute response via conserved high-occupancy enhancers. Genetic screen data indicates that, in normoxia, HIF1A displays strong cell-autonomous tumor suppressive effects through a gene module mediating mTOR inhibition. Conversely, in advanced malignancies, expression of a module of HIF1A targets involved in collagen remodeling is associated with poor prognosis across diverse cancer types. In this work, we provide a valuable resource for investigating context-dependent roles of HIF1A and its targets in cancer biology.


Asunto(s)
Redes Reguladoras de Genes , Genes Supresores de Tumor , Genómica , Hipoxia/genética , Oncogenes , Línea Celular Tumoral , Supervivencia Celular , Quinasa 8 Dependiente de Ciclina/metabolismo , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Genoma Humano , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias/genética , Neoplasias/patología , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Transcripción Genética , Activación Transcripcional/genética , Regulación hacia Arriba/genética
12.
Nat Commun ; 12(1): 1859, 2021 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-33767140

RESUMEN

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoproteins initiates co-transcriptionally and requires the action of the assembly machinery including the Hsp90/R2TP complex, the Rsa1p:Hit1p heterodimer and the Bcd1 protein. We present genetic interactions between the Rsa1p-encoding gene and genes involved in chromatin organization including RTT106 that codes for the H3-H4 histone chaperone Rtt106p controlling H3K56ac deposition. We show that Bcd1p binds Rtt106p and controls its transcription-dependent recruitment by reducing its association with RNA polymerase II, modulating H3K56ac levels at gene body. We reveal the 3D structures of the free and Rtt106p-bound forms of Bcd1p using nuclear magnetic resonance and X-ray crystallography. The interaction is also studied by a combination of biophysical and proteomic techniques. Bcd1p interacts with a region that is distinct from the interaction interface between the histone chaperone and histone H3. Our results are evidence for a protein interaction interface for Rtt106p that controls its transcription-associated activity.


Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Chaperonas Moleculares/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Activación Transcripcional/fisiología , Proliferación Celular/fisiología , Cromatina/genética , Cristalografía por Rayos X , Histonas/metabolismo , Resonancia Magnética Nuclear Biomolecular , ARN Polimerasa II/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/genética , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transcripción Genética/genética
13.
Nat Commun ; 12(1): 1881, 2021 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-33767157

RESUMEN

To achieve the very high oncoprotein levels required to drive the malignant state cancer cells utilise the ubiquitin proteasome system to upregulate transcription factor levels. Here our analyses identify ALYREF, expressed from the most common genetic copy number variation in neuroblastoma, chromosome 17q21-ter gain as a key regulator of MYCN protein turnover. We show strong co-operativity between ALYREF and MYCN from transgenic models of neuroblastoma in vitro and in vivo. The two proteins form a nuclear coactivator complex which stimulates transcription of the ubiquitin specific peptidase 3, USP3. We show that increased USP3 levels reduce K-48- and K-63-linked ubiquitination of MYCN, thus driving up MYCN protein stability. In the MYCN-ALYREF-USP3 signal, ALYREF is required for MYCN effects on the malignant phenotype and that of USP3 on MYCN stability. This data defines a MYCN oncoprotein dependency state which provides a rationale for future pharmacological studies.


Asunto(s)
Carcinogénesis/patología , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuroblastoma/patología , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Cromosomas Humanos Par 17/genética , Variaciones en el Número de Copia de ADN/genética , Células HEK293 , Humanos , Ratones , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/genética , Proteínas Nucleares/genética , Pronóstico , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Factores de Transcripción/genética , Transcripción Genética/genética , Activación Transcripcional/genética , Proteasas Ubiquitina-Específicas/genética , Ubiquitinación/fisiología
14.
Sci Rep ; 11(1): 6811, 2021 03 24.
Artículo en Inglés | MEDLINE | ID: mdl-33762651

RESUMEN

High rate of cardiovascular disease (CVD) has been reported among patients with coronavirus disease 2019 (COVID-19). Importantly, CVD, as one of the comorbidities, could also increase the risks of the severity of COVID-19. Here we identified phospholipase A2 group VII (PLA2G7), a well-studied CVD biomarker, as a hub gene in COVID-19 though an integrated hypothesis-free genomic analysis on nasal swabs (n = 486) from patients with COVID-19. PLA2G7 was further found to be predominantly expressed by proinflammatory macrophages in lungs emerging with progression of COVID-19. In the validation stage, RNA level of PLA2G7 was identified in nasal swabs from both COVID-19 and pneumonia patients, other than health individuals. The positive rate of PLA2G7 were correlated with not only viral loads but also severity of pneumonia in non-COVID-19 patients. Serum protein levels of PLA2G7 were found to be elevated and beyond the normal limit in COVID-19 patients, especially among those re-positive patients. We identified and validated PLA2G7, a biomarker for CVD, was abnormally enhanced in COVID-19 at both nucleotide and protein aspects. These findings provided indications into the prevalence of cardiovascular involvements seen in patients with COVID-19. PLA2G7 could be a potential prognostic and therapeutic target in COVID-19.


Asunto(s)
1-Alquil-2-acetilglicerofosfocolina Esterasa/metabolismo , Enfermedades Cardiovasculares/metabolismo , Macrófagos/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterasa/sangre , 1-Alquil-2-acetilglicerofosfocolina Esterasa/genética , Biomarcadores/metabolismo , /inmunología , Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/virología , China/epidemiología , Minería de Datos/métodos , Humanos , Macrófagos/inmunología , Macrófagos/patología , Polimorfismo de Nucleótido Simple , Activación Transcripcional , Regulación hacia Arriba
15.
Life Sci ; 273: 119292, 2021 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-33667516

RESUMEN

Delta opioids are thought to relieve ischemic injury and have tissue-protective properties. However, the detailed mechanisms of delta opioids have not been well identified. Receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor (EGFR), have been shown to mediate downstream signals of δ opioid receptor (δOR) activation through the metalloproteinase (MMP)-dependent EGF-like growth factor (HB-EGF) excretion pathway, which is called transactivation. In this study, to investigate the role of EGFR in δOR-induced anti-ischemic effects in the brain, we applied the middle cerebral artery occlusion (MCAO) model followed by reperfusion to mimic ischemic stroke injury in rats. Pre-treatment with the δOR agonist [D-ala2, D-leu5] enkephalin (DADLE) improved the neurologic deficits and the decreased infarct volume caused by cerebral ischemia/reperfusion injury, which were blocked by the EGFR inhibitor AG1478 and the MMP inhibitor GM6001, respectively. Further results indicated that DADLE activated EGFR, Akt and ERK1/2 and upregulated EGFR expression in the hippocampus in a time-dependent manner, which were inhibited by AG1478 and GM6001. The enzyme-linked immunosorbent assay (ELISA) results showed that δOR activation led to an increase in HB-EGF release, but HB-EGF in tissue was downregulated at the mRNA and protein levels. Moreover, this protective action caused by δOR agonists may involve attenuated hippocampal cellular apoptosis. Overall, these results demonstrate that MMP-mediated transactivation of EGFR is essential for δOR agonist-induced MCAO/reperfusion injury relief. These findings provide a potential molecular mechanism for the neuroprotective property of δOR and may add new insight into mitigating or preventing injury.


Asunto(s)
Isquemia Encefálica/prevención & control , Leucina Encefalina-2-Alanina/farmacología , Receptores ErbB/metabolismo , Infarto de la Arteria Cerebral Media/complicaciones , Receptores Opioides delta/agonistas , Daño por Reperfusión/prevención & control , Activación Transcripcional , Animales , Apoptosis , Isquemia Encefálica/etiología , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Receptores ErbB/genética , Masculino , Fosforilación , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/etiología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología
16.
Eur J Med Chem ; 216: 113285, 2021 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-33662676

RESUMEN

The development of resistance poses a serious problem in the therapy of cancer due to the necessity of a multiple-drug and unlimited treatment of affected patients. In chronic myeloid leukemia (CML), the introduction of imatinib has revolutionized the therapy. The persistence of an untreatable cancer stem cell pool and other resistance-causing factors, however, also impede the cure of this malignancy. New therapeutic approaches are therefore essential to overcome current treatment drawbacks. In this regard, an intervention in the STAT5 signaling pathway can significantly improve drug response, as this central signaling node induces the formation of highly resistant CML cells. In the present study, we continued the design of efficient chemosensitizers derived from the partial PPARγ agonist telmisartan. The developed 2-carbonitriles or 2-carboxymethyl esters showed improved potency in sensitizing K562-resistant cells to imatinib treatment, even at concentrations, which are considered patient-relevant. At 5 µM, for instance, 2d sensitized the cells in such a manner that the resistance was fully overcome and the recovered efficacy of imatinib resulted in >76% cell death. Importantly, all compounds were non-cytotoxic per se. A transactivation experiment showed that only the carbonitriles are partial agonists of PPARγ, which does not seem to be involved in the mode of action. Yet, immunoassays revealed a suppression of the STAT5 phosphorylation status by co-application of the most active derivatives with imatinib. This mechanism consequently resulted in reduced cell proliferation and induction of cell death in resistant CML cells.


Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Nitrilos/química , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Diseño de Fármacos , Regulación de la Expresión Génica , Humanos , Mesilato de Imatinib/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Nitrilos/farmacología , PPAR gamma/genética , PPAR gamma/metabolismo , Fosforilación/efectos de los fármacos , Factor de Transcripción STAT5/antagonistas & inhibidores , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Telmisartán/química , Telmisartán/farmacología , Activación Transcripcional/efectos de los fármacos , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo
17.
APMIS ; 129(5): 241-253, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33683784

RESUMEN

Human endogenous retroviruses (HERVs) are remnants of ancient retroviral germline infections. Most HERV sequences are silenced in somatic cells, but interest is emerging on the involvement of HERV derived transcripts and proteins in human physiology and disease. A HERV-W encoded protein, syncytin-1, has been co-opted into fetal physiology, where it plays a role in trophoblast formation. Altered HERV transcription and expression of HERV derived proteins are associated with various cancer types and neurological diseases such as multiple sclerosis (MS). The implication of HERVs as potential mediators of both health and disease suggests important roles of regulatory mechanisms and alterations of these in physiological and pathological processes. The regulation of HERV sequences is mediated by a wide variety of mechanisms, and the focus of this review is on selected aspects of these, including epigenetic mechanisms such as CpG methylation and histone modifications of the HP1-H3K9me axis, viral transactivation events, and regulatory perspectives of transient stimuli in the microenvironment. Increasing knowledge of the regulation of HERV sequences will not only contribute to the understanding of complex pathogeneses, but also may pinpoint potential targets for better diagnosis and treatment in complex diseases as MS.


Asunto(s)
Retrovirus Endógenos/genética , Desarrollo Fetal/genética , Regulación Viral de la Expresión Génica , Neoplasias/patología , Enfermedades del Sistema Nervioso/patología , Retrovirus Endógenos/patogenicidad , Epigénesis Genética , Productos del Gen env/fisiología , Humanos , Inmunidad/genética , Inmunidad/inmunología , Neoplasias/genética , Neoplasias/inmunología , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/inmunología , Proteínas Gestacionales/fisiología , Activación Transcripcional
18.
Mol Cell ; 81(8): 1651-1665.e4, 2021 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-33705711

RESUMEN

Enhancers harbor binding motifs that recruit transcription factors (TFs) for gene activation. While cooperative binding of TFs at enhancers is known to be critical for transcriptional activation of a handful of developmental enhancers, the extent of TF cooperativity genome-wide is unknown. Here, we couple high-resolution nuclease footprinting with single-molecule methylation profiling to characterize TF cooperativity at active enhancers in the Drosophila genome. Enrichment of short micrococcal nuclease (MNase)-protected DNA segments indicates that the majority of enhancers harbor two or more TF-binding sites, and we uncover protected fragments that correspond to co-bound sites in thousands of enhancers. From the analysis of co-binding, we find that cooperativity dominates TF binding in vivo at the majority of active enhancers. Cooperativity is highest between sites spaced 50 bp apart, indicating that cooperativity occurs without apparent protein-protein interactions. Our findings suggest nucleosomes promoting cooperativity because co-binding may effectively clear nucleosomes and promote enhancer function.


Asunto(s)
Elementos de Facilitación Genéticos/genética , Unión Proteica/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Sitios de Unión/genética , Línea Celular , Drosophila/genética , Drosophila/metabolismo , Genoma/genética , Nucleasa Microcócica/genética , Nucleosomas/genética , Nucleosomas/metabolismo , Mapas de Interacción de Proteínas/genética , Activación Transcripcional/genética
19.
Mol Cell ; 81(8): 1732-1748.e8, 2021 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-33730542

RESUMEN

During self-renewal, cell-type-defining features are drastically perturbed in mitosis and must be faithfully reestablished upon G1 entry, a process that remains largely elusive. Here, we characterized at a genome-wide scale the dynamic transcriptional and architectural resetting of mouse pluripotent stem cells (PSCs) upon mitotic exit. We captured distinct waves of transcriptional reactivation with rapid induction of stem cell genes and transient activation of lineage-specific genes. Topological reorganization at different hierarchical levels also occurred in an asynchronous manner and showed partial coordination with transcriptional resetting. Globally, rapid transcriptional and architectural resetting associated with mitotic retention of H3K27 acetylation, supporting a bookmarking function. Indeed, mitotic depletion of H3K27ac impaired the early reactivation of bookmarked, stem-cell-associated genes. However, 3D chromatin reorganization remained largely unaffected, suggesting that these processes are driven by distinct forces upon mitotic exit. This study uncovers principles and mediators of PSC molecular resetting during self-renewal.


Asunto(s)
Cromatina/genética , Código de Histonas/genética , Histonas/genética , Mitosis/genética , Células Madre Pluripotentes/fisiología , Acetilación , Animales , Línea Celular , Drosophila/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Transcripción Genética/genética , Activación Transcripcional/genética
20.
Mol Cell ; 81(8): 1682-1697.e7, 2021 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-33651988

RESUMEN

The coactivator p300/CREB-binding protein (CBP) regulates genes by facilitating the assembly of transcriptional machinery and by acetylating histones and other factors. However, it remains mostly unclear how both functions of p300 are dynamically coordinated during gene control. Here, we showed that p300 can orchestrate two functions through the formation of dynamic clusters with certain transcription factors (TFs), which is mediated by the interactions between a TF's transactivation domain (TAD) and the intrinsically disordered regions of p300. Co-condensation can enable spatially defined, all-or-none activation of p300's catalytic activity, priming the recruitment of coactivators, including Brd4. We showed that co-condensation can modulate transcriptional initiation rate and burst duration of target genes, underlying nonlinear gene regulatory functions. Such modulation is consistent with how p300 might shape gene bursting kinetics globally. Altogether, these results suggest an intriguing gene regulation mechanism, in which TF and p300 co-condensation contributes to transcriptional bursting regulation and cooperative gene control.


Asunto(s)
Proteína p300 Asociada a E1A/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Activación Transcripcional/genética , Acetilación , Animales , Células CHO , Proteína de Unión a CREB/metabolismo , Línea Celular , Cricetulus , Regulación de la Expresión Génica/genética , Células HEK293 , Histonas/metabolismo , Humanos , Cinética , Ratones , Transactivadores/metabolismo
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