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1.
Nat Commun ; 12(1): 5242, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34475398

RESUMO

Genome-wide association studies (GWAS) have linked single nucleotide polymorphisms (SNPs) at >250 loci in the human genome to type 2 diabetes (T2D) risk. For each locus, identifying the functional variant(s) among multiple SNPs in high linkage disequilibrium is critical to understand molecular mechanisms underlying T2D genetic risk. Using massively parallel reporter assays (MPRA), we test the cis-regulatory effects of SNPs associated with T2D and altered in vivo islet chromatin accessibility in MIN6 ß cells under steady state and pathophysiologic endoplasmic reticulum (ER) stress conditions. We identify 1,982/6,621 (29.9%) SNP-containing elements that activate transcription in MIN6 and 879 SNP alleles that modulate MPRA activity. Multiple T2D-associated SNPs alter the activity of short interspersed nuclear element (SINE)-containing elements that are strongly induced by ER stress. We identify 220 functional variants at 104 T2D association signals, narrowing 54 signals to a single candidate SNP. Together, this study identifies elements driving ß cell steady state and ER stress-responsive transcriptional activation, nominates causal T2D SNPs, and uncovers potential roles for repetitive elements in ß cell transcriptional stress response and T2D genetics.


Assuntos
Diabetes Mellitus Tipo 2/genética , Estresse do Retículo Endoplasmático/genética , Células Secretoras de Insulina/patologia , Polimorfismo de Nucleotídeo Único , Ativação Transcricional/genética , Alelos , Animais , Linhagem Celular , Cromatina/metabolismo , Diabetes Mellitus Tipo 2/patologia , Estudo de Associação Genômica Ampla , Humanos , Camundongos , Locos de Características Quantitativas , Elementos Nucleotídeos Curtos e Dispersos/genética
2.
Nat Commun ; 12(1): 5066, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417456

RESUMO

Prostate cancer (PCa) shows strong dependence on the androgen receptor (AR) pathway. Here, we show that squalene epoxidase (SQLE), an enzyme of the cholesterol biosynthesis pathway, is overexpressed in advanced PCa and its expression correlates with poor survival. SQLE expression is controlled by micro-RNA 205 (miR-205), which is significantly downregulated in advanced PCa. Restoration of miR-205 expression or competitive inhibition of SQLE led to inhibition of de novo cholesterol biosynthesis. Furthermore, SQLE was essential for proliferation of AR-positive PCa cell lines, including abiraterone or enzalutamide resistant derivatives, and blocked transactivation of the AR pathway. Inhibition of SQLE with the FDA approved antifungal drug terbinafine also efficiently blocked orthotopic tumour growth in mice. Finally, terbinafine reduced levels of prostate specific antigen (PSA) in three out of four late-stage PCa patients. These results highlight SQLE as a therapeutic target for the treatment of advanced PCa.


Assuntos
Colesterol/biossíntese , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Esqualeno Mono-Oxigenase/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular , Estudos de Coortes , Simulação por Computador , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Masculino , Camundongos , Camundongos SCID , MicroRNAs/genética , Pessoa de Meia-Idade , Invasividade Neoplásica , Metástase Neoplásica , Estadiamento de Neoplasias , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/patologia , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/metabolismo , Esqualeno Mono-Oxigenase/genética , Esqualeno Mono-Oxigenase/metabolismo , Terbinafina/farmacologia , Ativação Transcricional/genética
3.
FASEB J ; 35(9): e21814, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34369624

RESUMO

Alteration in glucose homeostasis during cancer metabolism is an important phenomenon. Though several important transcription factors have been well studied in the context of the regulation of metabolic gene expression, the role of epigenetic readers in this regard remains still elusive. Epigenetic reader protein transcription factor 19 (TCF19) has been recently identified as a novel glucose and insulin-responsive factor that modulates histone posttranslational modifications to regulate glucose homeostasis in hepatocytes. Here we report that TCF19 interacts with a non-histone, well-known tumor suppressor protein 53 (p53) and co-regulates a wide array of metabolic genes. Among these, the p53-responsive carbohydrate metabolic genes Tp53-induced glycolysis and apoptosis regulator (TIGAR) and Cytochrome C Oxidase assembly protein 2 (SCO2), which are the key regulators of glycolysis and oxidative phosphorylation respectively, are under direct regulation of TCF19. Remarkably, TCF19 can form different transcription activation/repression complexes which show substantial overlap with that of p53, depending on glucose-mediated variant stress situations as obtained from IP/MS studies. Interestingly, we observed that TCF19/p53 complexes either have CBP or HDAC1 to epigenetically program the expression of TIGAR and SCO2 genes depending on short-term high glucose or prolonged high glucose conditions. TCF19 or p53 knockdown significantly altered the cellular lactate production and led to increased extracellular acidification rate. Similarly, OCR and cellular ATP production were reduced and mitochondrial membrane potential was compromised upon depletion of TCF19 or p53. Subsequently, through RNA-Seq analysis from patients with hepatocellular carcinoma, we observed that TCF19/p53-mediated metabolic regulation is fundamental for sustenance of cancer cells. Together the study proposes that TCF19/p53 complexes can regulate metabolic gene expression programs responsible for mitochondrial energy homeostasis and stress adaptation.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Mitocôndrias/genética , Chaperonas Moleculares/genética , Monoéster Fosfórico Hidrolases/genética , Fatores de Transcrição/genética , Transcrição Genética/genética , Proteína Supressora de Tumor p53/genética , Adaptação Biológica/genética , Apoptose/genética , Linhagem Celular Tumoral , Metabolismo Energético/genética , Glucose/genética , Células Hep G2 , Homeostase/genética , Humanos , Potencial da Membrana Mitocondrial/genética , Estresse Fisiológico/genética , Ativação Transcricional/genética
4.
FASEB J ; 35(9): e21827, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34383980

RESUMO

Neuron-derived orphan receptor 1, NR4A3 (Nor1)/NR4A3 is an orphan nuclear receptor involved in the transcriptional control of developmental and neurological functions. Oxidative stress-induced conditions are primarily associated with neurological defects in humans, yet the impact on Nor1-mediated transcription of neuronal genes remains with unknown mechanism. Here, we demonstrate that Nor1 is a non-conventional target of SUMO2/3 conjugation at Lys-137 contained in an atypic ψKxSP motif referred to as the pSuM. Nor1 pSuM SUMOylation differs from the canonical process with the obligate phosphorylation of Ser-139 by Ras signaling to create the required negatively charged interface for SUMOylation. Additional phosphorylation at sites flanking the pSuM is also mediated by the coordinated action of protein kinase casein kinase 2 to function as a small ubiquitin-like modifier enhancer, regulating Nor1-mediated transcription and proteasomal degradation. Nor1 responsive genes involved in cell proliferation and metabolism, such as activating transcription factor 3, cyclin D1, CASP8 and FADD-like apoptosis regulator, and enolase 3 were upregulated in response to pSuM disruption in mouse HT-22 hippocampal neuronal cells and human neuroblastoma SH-SY5Y cells. We also identified critical antioxidant genes, such as catalase, superoxide dismutase 1, and microsomal glutathione S-transferase 2, as responsive targets of Nor1 under pSuM regulation. Nor1 SUMOylation impaired gene transcription through less effective Nor1 chromatin binding and reduced enrichment of histone H3K27ac marks to gene promoters. These effects resulted in decreased neuronal cell growth, increased apoptosis, and reduced survival to oxidative stress damage, underlying the role of pSuM-modified Nor1 in redox homeostasis. Our findings uncover a hierarchical post-translational mechanism that dictates Nor1 non-canonical SUMOylation, disrupting Nor1 transcriptional competence, and neuroprotective redox sensitivity.


Assuntos
Sobrevivência Celular/genética , Proteínas de Ligação a DNA/genética , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Sumoilação/genética , Animais , Apoptose/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Quinase do Ponto de Checagem 2/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Hipocampo/metabolismo , Homeostase/genética , Humanos , Camundongos , Neuroblastoma/genética , Neurônios/metabolismo , Oxirredução , Estresse Oxidativo/genética , Fosforilação/genética , Regiões Promotoras Genéticas/genética , Processamento de Proteína Pós-Traducional/genética , Transcrição Genética/genética , Ativação Transcricional/genética , Regulação para Cima/genética
5.
Mol Cell ; 81(16): 3368-3385.e9, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34375583

RESUMO

The mechanistic understanding of nascent RNAs in transcriptional control remains limited. Here, by a high sensitivity method methylation-inscribed nascent transcripts sequencing (MINT-seq), we characterized the landscapes of N6-methyladenosine (m6A) on nascent RNAs. We uncover heavy but selective m6A deposition on nascent RNAs produced by transcription regulatory elements, including promoter upstream antisense RNAs and enhancer RNAs (eRNAs), which positively correlates with their length, inclusion of m6A motif, and RNA abundances. m6A-eRNAs mark highly active enhancers, where they recruit nuclear m6A reader YTHDC1 to phase separate into liquid-like condensates, in a manner dependent on its C terminus intrinsically disordered region and arginine residues. The m6A-eRNA/YTHDC1 condensate co-mixes with and facilitates the formation of BRD4 coactivator condensate. Consequently, YTHDC1 depletion diminished BRD4 condensate and its recruitment to enhancers, resulting in inhibited enhancer and gene activation. We propose that chemical modifications of eRNAs together with reader proteins play broad roles in enhancer activation and gene transcriptional control.


Assuntos
Adenosina/análogos & derivados , Proteínas de Ciclo Celular/genética , Proteínas do Tecido Nervoso/genética , Fatores de Processamento de RNA/genética , RNA/genética , Fatores de Transcrição/genética , Adenosina/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Humanos , Metilação , Elementos Reguladores de Transcrição/genética , Ativação Transcricional/genética
6.
Orphanet J Rare Dis ; 16(1): 334, 2021 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-34332615

RESUMO

BACKGROUND: TBX1 (T-box transcription factor 1) is a major candidate gene that likely contributes to the etiology of velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS). Although the haploinsufficiency of TBX1 in both mice and humans results in congenital cardiac malformations, little has been elucidated about its upstream regulation. We aimed to explore the transcriptional regulation and dysregulation of TBX1. METHODS: Different TBX1 promoter reporters were constructed. Luciferase assays and electrophoretic mobility shift assays (EMSAs) were used to identify a cis-regulatory element within the TBX1 promoter region and its trans-acting factor. The expression of proteins was identified by immunohistochemistry and immunofluorescence. Variants in the cis-regulatory element were screened in conotruncal defect (CTD) patients. In vitro functional assays were performed to show the effects of the variants found in CTD patients on the transactivation of TBX1. RESULTS: We identified a cis-regulatory element within intron 1 of TBX1 that was found to be responsive to GATA6 (GATA binding protein 6), a transcription factor crucial for cardiogenesis. The expression patterns of GATA6 and TBX1 overlapped in the pharyngeal arches of human embryos. Transfection experiments and EMSA indicated that GATA6 could activate the transcription of TBX1 by directly binding with its GATA cis-regulatory element in vitro. Furthermore, sequencing analyses of 195 sporadic CTD patients without the 22q11.2 deletion or duplication identified 3 variants (NC_000022.11:g.19756832C > G, NC_000022.11:g.19756845C > T, and NC_000022.11:g. 19756902G > T) in the non-coding cis-regulatory element of TBX1. Luciferase assays showed that all 3 variants led to reduced transcription of TBX1 when incubated with GATA6. CONCLUSIONS: Our findings showed that TBX1 might be a direct transcriptional target of GATA6, and variants in the non-coding cis-regulatory element of TBX1 disrupted GATA6-mediated transactivation.


Assuntos
Síndrome de DiGeorge , Cardiopatias Congênitas , Animais , Síndrome de DiGeorge/genética , Fator de Transcrição GATA6 , Humanos , Camundongos , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional/genética
7.
Int J Mol Sci ; 22(16)2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34445672

RESUMO

In mammalian cells, two cellular organelles, mitochondria and peroxisomes, share the ability to degrade fatty acid chains. Although each organelle harbors its own fatty acid ß-oxidation pathway, a distinct mitochondrial system feeds the oxidative phosphorylation pathway for ATP synthesis. At the same time, the peroxisomal ß-oxidation pathway participates in cellular thermogenesis. A scientific milestone in 1965 helped discover the hepatomegaly effect in rat liver by clofibrate, subsequently identified as a peroxisome proliferator in rodents and an activator of the peroxisomal fatty acid ß-oxidation pathway. These peroxisome proliferators were later identified as activating ligands of Peroxisome Proliferator-Activated Receptor α (PPARα), cloned in 1990. The ligand-activated heterodimer PPARα/RXRα recognizes a DNA sequence, called PPRE (Peroxisome Proliferator Response Element), corresponding to two half-consensus hexanucleotide motifs, AGGTCA, separated by one nucleotide. Accordingly, the assembled complex containing PPRE/PPARα/RXRα/ligands/Coregulators controls the expression of the genes involved in liver peroxisomal fatty acid ß-oxidation. This review mobilizes a considerable number of findings that discuss miscellaneous axes, covering the detailed expression pattern of PPARα in species and tissues, the lessons from several PPARα KO mouse models and the modulation of PPARα function by dietary micronutrients.


Assuntos
Ácidos Graxos/metabolismo , PPAR alfa/metabolismo , Peroxissomos/metabolismo , Acil-CoA Oxidase/metabolismo , Animais , Humanos , Fígado/metabolismo , Oxirredução , Oxirredutases/metabolismo , PPAR alfa/fisiologia , Proliferadores de Peroxissomos , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Ácido Retinoico/metabolismo , Elementos de Resposta/genética , Receptores X de Retinoides/metabolismo , Ativação Transcricional/genética
8.
Nat Immunol ; 22(9): 1093-1106, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34282331

RESUMO

Neutrophils display distinct gene expression patters depending on their developmental stage, activation state and tissue microenvironment. To determine the transcription factor networks that shape these responses in a mouse model, we integrated transcriptional and chromatin analyses of neutrophils during acute inflammation. We showed active chromatin remodeling at two transition stages: bone marrow-to-blood and blood-to-tissue. Analysis of differentially accessible regions revealed distinct sets of putative transcription factors associated with control of neutrophil inflammatory responses. Using ex vivo and in vivo approaches, we confirmed that RUNX1 and KLF6 modulate neutrophil maturation, whereas RELB, IRF5 and JUNB drive neutrophil effector responses and RFX2 and RELB promote survival. Interfering with neutrophil activation by targeting one of these factors, JUNB, reduced pathological inflammation in a mouse model of myocardial infarction. Therefore, our study represents a blueprint for transcriptional control of neutrophil responses in acute inflammation and opens possibilities for stage-specific therapeutic modulation of neutrophil function in disease.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Inflamação/imunologia , Neutrófilos/imunologia , Ativação Transcricional/genética , Animais , Células CHO , Linhagem Celular , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Cricetulus , Feminino , Fatores Reguladores de Interferon/metabolismo , Fator 6 Semelhante a Kruppel/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/patologia , Fatores de Transcrição de Fator Regulador X/metabolismo , Fator de Transcrição RelB/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética/genética
9.
Int J Mol Sci ; 22(14)2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34298922

RESUMO

The coordinated transcription of the genome is the fundamental mechanism in molecular biology. Transcription in eukaryotes is carried out by three main RNA polymerases: Pol I, II, and III. One basic problem is how a decrease in tRNA levels, by downregulating Pol III efficiency, influences the expression pattern of protein-coding genes. The purpose of this study was to determine the mRNA levels in the yeast mutant rpc128-1007 and its overdose suppressors, RBS1 and PRT1. The rpc128-1007 mutant prevents assembly of the Pol III complex and functionally mimics similar mutations in human Pol III, which cause hypomyelinating leukodystrophies. We applied RNAseq followed by the hierarchical clustering of our complete RNA-seq transcriptome and functional analysis of genes from the clusters. mRNA upregulation in rpc128-1007 cells was generally stronger than downregulation. The observed induction of mRNA expression was mostly indirect and resulted from the derepression of general transcription factor Gcn4, differently modulated by suppressor genes. rpc128-1007 mutation, regardless of the presence of suppressors, also resulted in a weak increase in the expression of ribosome biogenesis genes. mRNA genes that were downregulated by the reduction of Pol III assembly comprise the proteasome complex. In summary, our results provide the regulatory links affected by Pol III assembly that contribute differently to cellular fitness.


Assuntos
RNA Polimerase III/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , RNA Polimerases Dirigidas por DNA/genética , Regulação para Baixo/genética , Regulação Fúngica da Expressão Gênica/genética , Humanos , RNA Polimerase II/genética , RNA de Transferência/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transcrição Genética/genética , Ativação Transcricional/genética , Transcriptoma/genética , Regulação para Cima/genética
10.
Aging (Albany NY) ; 13(12): 16043-16061, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34167089

RESUMO

HMGA1 protein is an architectural transcription factor that has been implicated in the progression of multiple malignant tumors. However, the role of HMGA1 in the growth and metastasis of gastric cancer (GC) has not yet been elucidated. Here, we show that HMGA1 is overexpressed in GC cells and the high expression of HMGA1 was correlated with worse survival in GC patients using a bioinformatics assay. Functionally, HMGA1 affected the EdU incorporation, colony formation, migration and invasion of GC cells by exogenously increasing or decreasing the expression of HMGA1. Mechanistically, HMGA1 directly bound to the SUZ12 and CCDC43 promoter and transactivated its expression in GC cells. Inhibition of SUZ12 and CCDC43 attenuated the proliferation, migration and invasiveness of HMGA1-overexpressing GC cells in vitro. Moreover, both HMGA1 and SUZ12/CCDC43 were highly expressed in cancer cells but not in normal gastric tissues, and their expressions were positively correlated. Finally, a tail vein metastatic assay showed that HMGA1 promoted SUZ12/CCDC43-mediated GC cell metastasis in vivo. Our findings suggest that HMGA1 promotes GC growth and metastasis by transactivating SUZ12 and CCDC43 expression, highlighting HMGA1 as a potential prognostic biomarker in the treatment of GC.


Assuntos
Proteína HMGA1a/metabolismo , Proteínas de Neoplasias/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Fatores de Transcrição/genética , Ativação Transcricional/genética , Animais , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Metástase Neoplásica , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Fatores de Transcrição/metabolismo , Regulação para Cima/genética
11.
Mol Cell ; 81(14): 2875-2886.e5, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34171296

RESUMO

In pathogenic mycobacteria, transcriptional responses to antibiotics result in induced antibiotic resistance. WhiB7 belongs to the Actinobacteria-specific family of Fe-S-containing transcription factors and plays a crucial role in inducible antibiotic resistance in mycobacteria. Here, we present cryoelectron microscopy structures of Mycobacterium tuberculosis transcriptional regulatory complexes comprising RNA polymerase σA-holoenzyme, global regulators CarD and RbpA, and WhiB7, bound to a WhiB7-regulated promoter. The structures reveal how WhiB7 interacts with σA-holoenzyme while simultaneously interacting with an AT-rich sequence element via its AT-hook. Evidently, AT-hooks, rare elements in bacteria yet prevalent in eukaryotes, bind to target AT-rich DNA sequences similarly to the nuclear chromosome binding proteins. Unexpectedly, a subset of particles contained a WhiB7-stabilized closed promoter complex, revealing this intermediate's structure, and we apply kinetic modeling and biochemical assays to rationalize how WhiB7 activates transcription. Altogether, our work presents a comprehensive view of how WhiB7 serves to activate gene expression leading to antibiotic resistance.


Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Fator Intrínseco/genética , Mycobacterium tuberculosis/genética , Fatores de Transcrição/genética , Ativação Transcricional/genética , Antibacterianos/farmacologia , Microscopia Crioeletrônica/métodos , RNA Polimerases Dirigidas por DNA/genética , Regulação Bacteriana da Expressão Gênica/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Fator sigma/genética
12.
J Biol Chem ; 297(1): 100814, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34081964

RESUMO

Nuclear receptors (NRs) activate transcription of target genes in response to binding of ligands to their ligand-binding domains (LBDs). Typically, in vitro assays use either gene expression or the recruitment of coactivators to the isolated LBD of the NR of interest to measure NR activation. However, this approach ignores that NRs function as homo- as well as heterodimers and that the LBD harbors the main dimerization interface. Cofactor recruitment is thereby interconnected with oligomerization status as well as ligand occupation of the partnering LBD through allosteric cross talk. Here we present a modular set of homogeneous time-resolved FRET-based assays through which we investigated the activation of PPARγ in response to ligands and the formation of heterodimers with its obligatory partner RXRα. We introduced mutations into the RXRα LBD that prevent coactivator binding but do not interfere with LBD dimerization or ligand binding. This enabled us to specifically detect PPARγ coactivator recruitment to PPARγ:RXRα heterodimers. We found that the RXRα agonist SR11237 destabilized the RXRα homodimer but promoted formation of the PPARγ:RXRα heterodimer, while being inactive on PPARγ itself. Of interest, incorporation of PPARγ into the heterodimer resulted in a substantial gain in affinity for coactivator CBP-1, even in the absence of ligands. Consequently, SR11237 indirectly promoted coactivator binding to PPARγ by shifting the oligomerization preference of RXRα toward PPARγ:RXRα heterodimer formation. These results emphasize that investigation of ligand-dependent NR activation should take NR dimerization into account. We envision these assays as the necessary assay tool kit for investigating NRs that partner with RXRα.


Assuntos
Proteína de Ligação a CREB/metabolismo , PPAR gama/metabolismo , Multimerização Proteica , Receptor X Retinoide alfa/metabolismo , Benzoatos/farmacologia , Células HEK293 , Humanos , Ligantes , Mutação/genética , Coativador 1 de Receptor Nuclear/metabolismo , PPAR gama/agonistas , PPAR gama/química , Domínios Proteicos , Multimerização Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Reprodutibilidade dos Testes , Receptor X Retinoide alfa/química , Receptor X Retinoide alfa/genética , Retinoides/farmacologia , Rosiglitazona/farmacologia , Ativação Transcricional/genética
13.
Int J Mol Sci ; 22(9)2021 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066536

RESUMO

Plant food production is severely affected by fungi; to cope with this problem, farmers use synthetic fungicides. However, the need to reduce fungicide application has led to a search for alternatives, such as biostimulants. Rare-earth elements (REEs) are widely used as biostimulants, but their mode of action and their potential as an alternative to synthetic fungicides have not been fully studied. Here, the biostimulant effect of gadolinium (Gd) is explored using the plant-pathosystem Arabidopsis thaliana-Botrytis cinerea. We determine that Gd induces local, systemic, and long-lasting plant defense responses to B. cinerea, without affecting fungal development. The physiological changes induced by Gd have been related to its structural resemblance to calcium. However, our results show that the calcium-induced defense response is not sufficient to protect plants against B. cinerea, compared to Gd. Furthermore, a genome-wide transcriptomic analysis shows that Gd induces plant defenses and modifies early and late defense responses. However, the resistance to B. cinerea is dependent on JA/ET-induced responses. These data support the conclusion that Gd can be used as a biocontrol agent for B. cinerea. These results are a valuable tool to uncover the molecular mechanisms induced by REEs.


Assuntos
Arabidopsis/imunologia , Arabidopsis/microbiologia , Botrytis/fisiologia , Ciclopentanos/metabolismo , Etilenos/metabolismo , Gadolínio/farmacologia , Oxilipinas/metabolismo , Substâncias Protetoras/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Botrytis/efeitos dos fármacos , Botrytis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo , Ácido Salicílico/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética
14.
Mol Cell Biol ; 41(8): e0005921, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34031216

RESUMO

Cervical cancer (CC) is a common gynecological malignancy with high morbidity and mortality. Mounting evidence has highlighted that long noncoding RNAs are essential regulators in cancer development. Long intergenic non-protein-coding RNA 997 (LINC00997) was identified for study due to its high expression in CC tissues. The aim of the study was to investigate the function and mechanism of LINC00997 in CC. Reverse transcription-quantitative PCR (RT-qPCR) revealed that LINC00997 RNA expression was also increased in CC cells and LINC00997 copy number was upregulated in CC tissues. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), colony formation, and Transwell assays as well as transmission electron microscopy observation exhibited that LINC00997 depletion inhibited CC cell proliferation, migration, invasion, and autophagy. The relationship between LINC00997 and its downstream genes was confirmed by RNA pulldown, luciferase reporter, and RNA-binding protein immunoprecipitation assays. Mechanistically, LINC00997 upregulated the expression of cullin 2 (CUL2) by interacting with microRNA 574-3p (miR-574-3p). Moreover, Western blot analysis was employed to detect the protein levels of mitogen-activated protein kinase (MAPK) pathway-associated factors in CC cells. LINC00997 activated the MAPK signaling by increasing CUL2 expression, thus promoting malignant phenotypes of CC cells. In conclusion, the LINC00997/miR-574-3p/CUL2 axis contributes to CC cell proliferation, migration, invasion, and autophagy via the activation of MAPK signaling.


Assuntos
Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias do Colo do Útero/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Humanos , Proteínas Quinases Ativadas por Mitógeno/genética , RNA Longo não Codificante/genética , Ativação Transcricional/genética , Neoplasias do Colo do Útero/metabolismo
15.
Nat Commun ; 12(1): 2770, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33986266

RESUMO

CRISPR-based transcriptional activation is a powerful tool for functional gene interrogation; however, delivery difficulties have limited its applications in vivo. Here, we created a mouse model expressing all components of the CRISPR-Cas9 guide RNA-directed Synergistic Activation Mediator (SAM) from a single transcript that is capable of activating target genes in a tissue-specific manner. We optimized Lipid Nanoparticles and Adeno-Associated Virus guide RNA delivery approaches to achieve expression modulation of one or more genes in vivo. We utilized the SAM mouse model to generate a hypercholesteremia disease state that we could bidirectionally modulate with various guide RNAs. Additionally, we applied SAM to optimize gene expression in a humanized Transthyretin mouse model to recapitulate human expression levels. These results demonstrate that the SAM gene activation platform can facilitate in vivo research and drug discovery.


Assuntos
Sistemas CRISPR-Cas/genética , Hipercolesterolemia/genética , Lipossomos/farmacologia , Pré-Albumina/metabolismo , Ativação Transcricional/genética , Animais , Linhagem Celular , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Engenharia Genética/métodos , Células HEK293 , Humanos , Hipercolesterolemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nanopartículas , Pré-Albumina/genética , RNA Guia/genética , RNA Guia/metabolismo
16.
Nat Commun ; 12(1): 3086, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34035267

RESUMO

Meis1 and Meis2 are homeodomain transcription factors that regulate organogenesis through cooperation with Hox proteins. Elimination of Meis genes after limb induction has shown their role in limb proximo-distal patterning; however, limb development in the complete absence of Meis function has not been studied. Here, we report that Meis1/2 inactivation in the lateral plate mesoderm of mouse embryos leads to limb agenesis. Meis and Tbx factors converge in this function, extensively co-binding with Tbx to genomic sites and co-regulating enhancers of Fgf10, a critical factor in limb initiation. Limbs with three deleted Meis alleles show proximal-specific skeletal hypoplasia and agenesis of posterior skeletal elements. This failure in posterior specification results from an early role of Meis factors in establishing the limb antero-posterior prepattern required for Shh activation. Our results demonstrate roles for Meis transcription factors in early limb development and identify their involvement in previously undescribed interaction networks that regulate organogenesis.


Assuntos
Padronização Corporal/genética , Proteínas de Homeodomínio/genética , Botões de Extremidades/metabolismo , Proteína Meis1/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator 10 de Crescimento de Fibroblastos/genética , Fator 10 de Crescimento de Fibroblastos/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/metabolismo , Botões de Extremidades/embriologia , Camundongos Knockout , Camundongos Transgênicos , Modelos Genéticos , Proteína Meis1/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética
17.
PLoS Genet ; 17(4): e1009510, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33826618

RESUMO

The R2R3-MYB transcription factor MYB46 functions as a master switch for secondary cell wall biosynthesis, ensuring the exquisite expression of the secondary wall biosynthetic genes in the tissues where secondary walls are critical for growth and development. At the same time, suppression of its function is needed when/where formation of secondary walls is not desirable. Little is known about how this opposing control of secondary cell wall formation is achieved. We used both transient and transgenic expression of MYB46 and mitogen-activated protein kinase 6 (MPK6) to investigate the molecular mechanism of the post-translational regulation of MYB46. We show that MYB46 is phosphorylated by MPK6, leading to site specific phosphorylation-dependent degradation of MYB46 by the ubiquitin-mediated proteasome pathway. In addition, the MPK6-mediated MYB46 phosphorylation was found to regulate in planta secondary wall forming function of MYB46. Furthermore, we provide experimental evidences that MYB83, a paralog of MYB46, is not regulated by MPK6. The coupling of MPK signaling to MYB46 function provides insights into the tissue- and/or condition-specific activity of MYB46 for secondary wall biosynthesis.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Parede Celular/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Fatores de Transcrição/genética , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/genética , Especificidade de Órgãos/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Regiões Promotoras Genéticas/genética , Estabilidade Proteica , Ativação Transcricional/genética
18.
Mol Genet Metab ; 133(2): 137-147, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33795191

RESUMO

Tristetraprolin (TTP) is a nucleocytoplasmic 326 amino acid protein whose sequence is characterized by possessing two CCCH-type zinc finger domains. In the cytoplasm TTP function is to promote the degradation of mRNAs that contain adenylate/uridylate-rich elements (AREs). Mechanistically, TTP promotes the recruitment of poly(A)-specific deadenylases and exoribonucleases. By reducing the half-life of about 10% of all the transcripts in the cell TTP has been shown to participate in multiple cell processes that include regulation of gene expression, cell proliferation, metabolic homeostasis and control of inflammation and immune responses. However, beyond its role in mRNA decay, in the cell nucleus TTP acts as a transcriptional coregulator by interacting with chromatin modifying enzymes. TTP has been shown to repress the transactivation of NF-κB and estrogen receptor suggesting the possibility that it participates in the transcriptional regulation of hundreds of genes in human cells and its possible involvement in breast cancer progression. In this review, we discuss the cytoplasmic and nuclear functions of TTP and the effect of the dysregulation of its protein levels in the development of human diseases. We suggest that TTP be classified as a moonlighting tumor supressor protein that regulates gene expression through two different mechanims; the decay of ARE-mRNAs and a transcriptional coregulatory function.


Assuntos
Citosol/metabolismo , RNA Mensageiro/metabolismo , Ativação Transcricional/genética , Tristetraprolina/genética , Proliferação de Células/genética , Regulação da Expressão Gênica/genética , Humanos , Inflamação/genética , Inflamação/patologia , Estabilidade de RNA/genética , RNA Mensageiro/genética , Tristetraprolina/metabolismo , Dedos de Zinco/genética
19.
Mol Cell ; 81(8): 1666-1681.e6, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33823140

RESUMO

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.


Assuntos
Núcleo Celular/genética , Regulação da Expressão Gênica/genética , Proteínas Nucleares/genética , RNA/genética , Ativação Transcricional/genética , Proteína Supressora de Tumor p53/genética , DNA/genética , Células HEK293 , Humanos , Corpos de Inclusão Intranuclear/genética , Ligação Proteica/genética , Fatores de Transcrição/genética , Transcrição Genética/genética
20.
Genes (Basel) ; 12(3)2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33809104

RESUMO

Atrial fibrillation (AF) represents the most common type of clinical cardiac arrhythmia and substantially increases the risks of cerebral stroke, heart failure and death. Accumulating evidence has convincingly demonstrated the strong genetic basis of AF, and an increasing number of pathogenic variations in over 50 genes have been causally linked to AF. Nevertheless, AF is of pronounced genetic heterogeneity, and the genetic determinants underpinning AF in most patients remain obscure. In the current investigation, a Chinese pedigree with AF as well as ventricular arrhythmias and hypertrophic cardiomyopathy was recruited. Whole exome sequencing and bioinformatic analysis of the available family members were conducted, and a novel heterozygous variation in the KLF15 gene (encoding Krüppel-like factor 15, a transcription factor critical for cardiac electrophysiology and structural remodeling), NM_014079.4: c.685A>T; p.(Lys229*), was identified. The variation was verified by Sanger sequencing and segregated with autosomal dominant AF in the family with complete penetrance. The variation was absent from 300 unrelated healthy subjects used as controls. In functional assays using a dual-luciferase assay system, mutant KLF15 showed neither transcriptional activation of the KChIP2 promoter nor transcriptional inhibition of the CTGF promoter, alone or in the presence of TGFB1, a key player in the pathogenesis of arrhythmias and cardiomyopathies. The findings indicate KLF15 as a new causative gene responsible for AF as well as ventricular arrhythmias and hypertrophic cardiomyopathy, and they provide novel insight into the molecular mechanisms underlying cardiac arrhythmias and hypertrophic cardiomyopathy.


Assuntos
Arritmias Cardíacas/genética , Fibrilação Atrial/genética , Cardiomiopatias/genética , Predisposição Genética para Doença/genética , Fatores de Transcrição Kruppel-Like/genética , Mutação/genética , Adolescente , Adulto , Idoso , Animais , Grupo com Ancestrais do Continente Asiático/genética , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Células HeLa , Heterozigoto , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Células NIH 3T3 , Linhagem , Regiões Promotoras Genéticas/genética , Ativação Transcricional/genética , Adulto Jovem
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