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1.
Arterioscler Thromb Vasc Biol ; 41(3): 1105-1123, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33406884

RESUMO

OBJECTIVE: Atherosclerosis predominantly forms in regions of oscillatory shear stress while regions of laminar shear stress are protected. This protection is partly through the endothelium in laminar flow regions expressing an anti-inflammatory and antithrombotic gene expression program. Several molecular pathways transmitting these distinct flow patterns to the endothelium have been defined. Our objective is to define the role of the MEF2 (myocyte enhancer factor 2) family of transcription factors in promoting an atheroprotective endothelium. Approach and Results: Here, we show through endothelial-specific deletion of the 3 MEF2 factors in the endothelium, Mef2a, -c, and -d, that MEF2 is a critical regulator of vascular homeostasis. MEF2 deficiency results in systemic inflammation, hemorrhage, thrombocytopenia, leukocytosis, and rapid lethality. Transcriptome analysis reveals that MEF2 is required for normal regulation of 3 pathways implicated in determining the flow responsiveness of the endothelium. Specifically, MEF2 is required for expression of Klf2 and Klf4, 2 partially redundant factors essential for promoting an anti-inflammatory and antithrombotic endothelium. This critical requirement results in phenotypic similarities between endothelial-specific deletions of Mef2a/c/d and Klf2/4. In addition, MEF2 regulates the expression of Notch family genes, Notch1, Dll1, and Jag1, which also promote an atheroprotective endothelium. In contrast to these atheroprotective pathways, MEF2 deficiency upregulates an atherosclerosis promoting pathway through increasing the amount of TAZ (transcriptional coactivator with PDZ-binding motif). CONCLUSIONS: Our results implicate MEF2 as a critical upstream regulator of several transcription factors responsible for gene expression programs that affect development of atherosclerosis and promote an anti-inflammatory and antithrombotic endothelium. Graphic Abstract: A graphic abstract is available for this article.


Assuntos
Aterosclerose/metabolismo , Endotélio Vascular/metabolismo , Fatores de Transcrição MEF2/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/patologia , Endotélio Vascular/patologia , Feminino , Regulação da Expressão Gênica , Homeostase , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição MEF2/deficiência , Fatores de Transcrição MEF2/genética , Masculino , Camundongos , Camundongos Knockout , Receptores Notch/genética , Transdução de Sinais , Transativadores/metabolismo
2.
Int J Mol Sci ; 21(24)2020 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-33322515

RESUMO

Skeletal myogenesis is a multi-stage process that includes the cell cycle exit, myogenic transcriptional activation, and morphological changes to form multinucleated myofibers. Recent studies have shown that saturated fatty acids (SFA) and miRNAs play crucial roles in myogenesis and muscle homeostasis. Nevertheless, the target molecules and myogenic regulatory mechanisms of miRNAs are largely unknown, particularly when myogenesis is dysregulated by SFA deposition. This study investigated the critical role played by miR-96-5p on the myogenic differentiation in C2C12 myoblasts. Long-chain SFA palmitic acid (PA) significantly reduced FHL1 expression and inhibited the myogenic differentiation of C2C12 myoblasts but induced miR-96-5p expression. The knockdown of FHL1 by siRNA stimulated cell proliferation and inhibited myogenic differentiation of myoblasts. Interestingly, miR-96-5p suppressed FHL1 expression by directly targeting the 3'UTR of FHL1 mRNA. The transfection of an miR-96-5p mimic upregulated the expressions of cell cycle-related genes, such as PCNA, CCNB1, and CCND1, and increased myoblast proliferation. Moreover, the miR-96-5p mimic inhibited the expressions of myogenic factors, such as myoblast determination protein (MyoD), myogenin (MyoG), myocyte enhancer factor 2C (MEF2C), and myosin heavy chain (MyHC), and dramatically impeded differentiation and fusion of myoblasts. Overall, this study highlights the role of miR-96-5p in myogenesis via FHL1 suppression and suggests a novel regulatory mechanism for myogenesis mediated by miRNA in a background of obesity.


Assuntos
Ácido Palmítico/farmacologia , Regiões 3' não Traduzidas/genética , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Imunofluorescência , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Desenvolvimento Muscular/efeitos dos fármacos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteína MyoD/genética , Proteína MyoD/metabolismo , Miogenina/genética , Miogenina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
PLoS One ; 15(12): e0242884, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33315889

RESUMO

Loss of retinal ganglion cells (RGCs) in optic neuropathies results in permanent partial or complete blindness. Myocyte enhancer factor 2 (MEF2) transcription factors have been shown to play a pivotal role in neuronal systems, and in particular MEF2A knockout was shown to enhance RGC survival after optic nerve crush injury. Here we expanded these prior data to study bi-allelic, tri-allelic and heterozygous allele deletion. We observed that deletion of all MEF2A, MEF2C, and MEF2D alleles had no effect on RGC survival during development. Our extended experiments suggest that the majority of the neuroprotective effect was conferred by complete deletion of MEF2A but that MEF2D knockout, although not sufficient to increase RGC survival on its own, increased the positive effect of MEF2A knockout. Conversely, MEF2A over-expression in wildtype mice worsened RGC survival after optic nerve crush. Interestingly, MEF2 transcription factors are regulated by post-translational modification, including by calcineurin-catalyzed dephosphorylation of MEF2A Ser-408 known to increase MEF2A-dependent transactivation in neurons. However, neither phospho-mimetic nor phospho-ablative mutation of MEF2A Ser-408 affected the ability of MEF2A to promote RGC death in vivo after optic nerve injury. Together these findings demonstrate that MEF2 gene expression opposes RGC survival following axon injury in a complex hierarchy, and further support the hypothesis that loss of or interference with MEF2A expression might be beneficial for RGC neuroprotection in diseases such as glaucoma and other optic neuropathies.


Assuntos
Fatores de Transcrição MEF2/metabolismo , Traumatismos do Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/patologia , Células Ganglionares da Retina/patologia , Alelos , Animais , Contagem de Células , Humanos , Fatores de Transcrição MEF2/deficiência , Fatores de Transcrição MEF2/genética , Camundongos , Traumatismos do Nervo Óptico/genética , Mutação Puntual , Transdução de Sinais
5.
Sci Rep ; 10(1): 7734, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32382091

RESUMO

Kruppel-like factor 2 (KLF2) is a positive transcriptional regulator of several endothelial protective molecules, including thrombomodulin (TM), a surface receptor, and endothelial nitric oxide synthase (eNOS), an enzyme that generates nitric oxide (NO). Loss of TM and eNOS causes endothelial dysfunction, which results in suppressed generation of activated protein C (APC) by TM-thrombin complex and in upregulation of intercellular adhesion molecule 1 (ICAM-1). Mechanistic studies revealed that activation of extracellular signal-regulated kinase 5 (ERK5) via upregulation of myocyte enhancer factor 2 (MEF2) induces KLF2 expression. Radiation causes endothelial dysfunction, but no study has investigated radiation's effects on the KLF2 pathway. Because fractionated radiation is routinely used during cancer radiotherapy, we decided to delineate the effects of radiation dose fractionation on the KLF2 signaling cascade at early time points (up to 24 h). We exposed human primary endothelial cells to radiation as a series of fractionated or as a single exposure, with the same total dose delivered to each group. We measured the expression and activity of critical members of the KLF2 pathway at subsequent time points, and determined whether pharmacological upregulation of KLF2 can reverse the radiation effects. Compared to single exposure, fractionated radiation profoundly suppressed KLF2, TM, and eNOS levels, subdued APC generation, declined KLF2 binding ability to TM and eNOS promoters, enhanced ICAM-1 expression, and decreased expression of upstream regulators of KLF2 (ERK5 and MEF2). Pharmacological inhibitors of the mevalonate pathway prevented fractionated-radiation-induced suppression of KLF2, TM, and eNOS expression. Finally, fractionated irradiation to thoracic region more profoundly suppressed KLF2 and enhanced ICAM-1 expression than single exposure in the lung at 24 h. These data clearly indicate that radiation dose fractionation plays a critical role in modulating levels of KLF2, its upstream regulators, and its downstream target molecules in endothelial cells. Our findings will provide important insights for selecting fractionated regimens during radiotherapy and for developing strategies to alleviate radiotherapy-induced toxicity to healthy tissues.


Assuntos
Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Fatores de Transcrição Kruppel-Like/genética , Óxido Nítrico Sintase Tipo III/genética , Trombomodulina/genética , Fracionamento da Dose de Radiação , Relação Dose-Resposta à Radiação , Regulação da Expressão Gênica/efeitos da radiação , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/genética , Fatores de Transcrição MEF2/genética , Proteína Quinase 7 Ativada por Mitógeno/genética , Neoplasias/genética , Neoplasias/patologia , Neoplasias/radioterapia , Radiação , Transdução de Sinais/efeitos da radiação
6.
Proc Natl Acad Sci U S A ; 117(24): 13828-13838, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32461370

RESUMO

Despite its popularity, chromatin immunoprecipitation followed by sequencing (ChIP-seq) remains a tedious (>2 d), manually intensive, low-sensitivity and low-throughput approach. Here, we combine principles of microengineering, surface chemistry, and molecular biology to address the major limitations of standard ChIP-seq. The resulting technology, FloChIP, automates and miniaturizes ChIP in a beadless fashion while facilitating the downstream library preparation process through on-chip chromatin tagmentation. FloChIP is fast (<2 h), has a wide dynamic range (from 106 to 500 cells), is scalable and parallelized, and supports antibody- or sample-multiplexed ChIP on both histone marks and transcription factors. In addition, FloChIP's interconnected design allows for straightforward chromatin reimmunoprecipitation, which allows this technology to also act as a microfluidic sequential ChIP-seq system. Finally, we ran FloChIP for the transcription factor MEF2A in 32 distinct human lymphoblastoid cell lines, providing insights into the main factors driving collaborative DNA binding of MEF2A and into its role in B cell-specific gene regulation. Together, our results validate FloChIP as a flexible and reproducible automated solution for individual or sequential ChIP-seq.


Assuntos
Automação/métodos , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Histonas/metabolismo , Fatores de Transcrição MEF2/metabolismo , Automação/instrumentação , Linfócitos B/química , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Sequenciamento de Cromatina por Imunoprecipitação/instrumentação , Histonas/química , Histonas/genética , Humanos , Fatores de Transcrição MEF2/química , Fatores de Transcrição MEF2/genética , Ligação Proteica
7.
Sci Rep ; 10(1): 4050, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-32132660

RESUMO

Skeletal muscle represents the largest pool of body zinc, however, little is known about muscle zinc homeostasis or muscle-specific zinc functions. Zip14 (Slc39a14) was the most highly expressed zinc transporter in skeletal muscle of mice in response to LPS-induced inflammation. We compared metabolic parameters of skeletal muscle from global Zip14 knockout (KO) and wild-type mice (WT). At basal steady state Zip14 KO mice exhibited a phenotype that included muscle wasting and metabolic endotoxemia. Microarray and qPCR analysis of gastrocnemius muscle RNA revealed that ablation of Zip14 produced increased muscle p-Mef2c, Hspb7 and miR-675-5p expression and increased p38 activation. ChIP assays showed enhanced binding of NF-[Formula: see text] to the Mef2c promoter. In contrast, LPS-induced systemic inflammation enhanced Zip14-dependent zinc uptake by muscle, increased expression of Atrogin1 and MuRF1 and markedly reduced MyoD. These signatures of muscle atrophy and cachexia were not influenced by Zip14 ablation, however. LPS-induced miR-675-3p and -5p expression was Zip14-dependent. Collectively, these results with an integrative model are consistent with a Zip14 function in skeletal muscle at steady state that supports myogenesis through suppression of metabolic endotoxemia and that Zip14 ablation coincides with sustained activity of phosphorylated components of signaling pathways including p-Mef2c, which causes Hspb7-dependent muscle wasting.


Assuntos
Proteínas de Transporte de Cátions/deficiência , Endotoxemia , Proteínas de Choque Térmico HSP27/metabolismo , MicroRNAs/metabolismo , Músculo Esquelético/metabolismo , Síndrome de Emaciação , Animais , Proteínas de Transporte de Cátions/metabolismo , Endotoxemia/genética , Endotoxemia/metabolismo , Deleção de Genes , Proteínas de Choque Térmico HSP27/genética , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos , Camundongos Knockout , MicroRNAs/genética , Síndrome de Emaciação/genética , Síndrome de Emaciação/metabolismo
8.
BMC Cardiovasc Disord ; 20(1): 139, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32183703

RESUMO

BACKGROUND: The incidence of CHD is the highest among birth defects and is increasing year to year. CHD seriously harms the health of infants and young children and presents a large economic burden to families and society. The pathogenesis of CHD and preventive measures are the focus of current research. Our research aimed to explore the intervention effect of folic acid on heart abnormalities resulting from sodium arsenic (NaAsO2) exposure during the periconception period. METHODS: Sixty 35-day-old female SD rats were randomly divided into 5 groups with 12 rats in each group. Group A was the control group. The rats were given distilled water and ordinary chow. The rats in group B were given distilled water containing 75 mg/L NaAsO2 and ordinary chow. The rats in groups C, D, and E were given distilled water containing 75 mg/L NaAsO2 and chow containing 0.53 mg/kg, 5.3 mg/kg, and 10.6 mg/kg folic acid, respectively. The general condition of the embryos and the histopathology of the embryonic hearts were examined. The acetylation levels of histone H3K9 in heart tissues and the expression levels of Mef2C (which is related to heart development) were observed. RESULTS: The embryo weight and placental weight of groups B-E were significantly lower than those of group A (P < 0.05). The heart malformation rate of the fetal rats in groups B-E was significantly higher than that of the fetal rats in group A (P < 0.05). We found that the level of H3K9 acetylation in fetal rat cardiomyocytes in groups B-E was significantly higher than that in group A (P < 0.05) and that the level of H3K9 acetylation in groups C-E was lower than that in group B (P < 0.05). The mRNA level of Mef2C in fetal rat cardiomyocytes in group B-E was significantly higher than that in group A (P < 0.05), and the mRNA level of Mef2C in groups C-E was significantly lower than that in group B (P < 0.05). CONCLUSION: Supplementation with folic acid during the periconception period can interfere with the toxic effects of arsenic on the heart. The mechanism may be that lowering the acetylation levels of histone H3K9 in heart tissues leads to decreased expression levels of Mef2C, which may play a protective role in heart development in fetal rats.


Assuntos
Arsenitos , Coração Fetal/efeitos dos fármacos , Ácido Fólico/farmacologia , Cardiopatias Congênitas/prevenção & controle , Compostos de Sódio , Acetilação , Animais , Cardiotoxicidade , Feminino , Coração Fetal/anormalidades , Coração Fetal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Cardiopatias Congênitas/induzido quimicamente , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/metabolismo , Histonas/metabolismo , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Exposição Materna , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Gravidez , Ratos Sprague-Dawley
9.
Am J Psychiatry ; 177(6): 526-536, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32046534

RESUMO

OBJECTIVE: Attention deficit hyperactivity disorder (ADHD) is a common, highly heritable neuropsychiatric disorder. ADHD often co-occurs with intellectual disability, and shared overlapping genetics have been suggested. The aim of this study was to identify novel ADHD genes by investigating whether genes carrying rare mutations linked to intellectual disability contribute to ADHD risk through common genetic variants. Validation and characterization of candidates were performed using Drosophila melanogaster. METHODS: Common genetic variants in a diagnostic gene panel of 396 autosomal intellectual disability genes were tested for association with ADHD risk through gene set and gene-wide analyses, using ADHD meta-analytic data from the Psychiatric Genomics Consortium for discovery (N=19,210) and ADHD data from the Lundbeck Foundation Initiative for Integrative Psychiatric Research for replication (N=37,076). The significant genes were functionally validated and characterized in Drosophila by assessing locomotor activity and sleep upon knockdown of those genes in brain circuits. RESULTS: The intellectual disability gene set was significantly associated with ADHD risk in the discovery and replication data sets. The three genes most consistently associated were MEF2C, ST3GAL3, and TRAPPC9. Performing functional characterization of the two evolutionarily conserved genes in Drosophila melanogaster, the authors found that their knockdown in dopaminergic (dMEF2) and circadian neurons (dTRAPPC9) resulted in increased locomotor activity and reduced sleep, concordant with the human phenotype. CONCLUSIONS: This study reveals that a large set of intellectual disability-related genes contribute to ADHD risk through effects of common alleles. Utilizing this continuity, the authors identified TRAPPC9, MEF2C, and ST3GAL3 as novel ADHD candidate genes. Characterization in Drosophila suggests that TRAPPC9 and MEF2C contribute to ADHD-related behavior through distinct neural substrates.


Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Locomoção/genética , Fatores de Regulação Miogênica/genética , Sialiltransferases/genética , Adulto , Idoso , Animais , Ritmo Circadiano , Neurônios Dopaminérgicos/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Estudo de Associação Genômica Ampla , Humanos , Deficiência Intelectual/genética , Fatores de Transcrição MEF2/genética , Masculino , Pessoa de Meia-Idade , Neurônios/metabolismo , Sono/genética
10.
Nat Commun ; 11(1): 734, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-32024818

RESUMO

The discovery of driver mutations is one of the key motivations for cancer genome sequencing. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2658 cancers across 38 tumour types, we describe DriverPower, a software package that uses mutational burden and functional impact evidence to identify driver mutations in coding and non-coding sites within cancer whole genomes. Using a total of 1373 genomic features derived from public sources, DriverPower's background mutation model explains up to 93% of the regional variance in the mutation rate across multiple tumour types. By incorporating functional impact scores, we are able to further increase the accuracy of driver discovery. Testing across a collection of 2583 cancer genomes from the PCAWG project, DriverPower identifies 217 coding and 95 non-coding driver candidates. Comparing to six published methods used by the PCAWG Drivers and Functional Interpretation Working Group, DriverPower has the highest F1 score for both coding and non-coding driver discovery. This demonstrates that DriverPower is an effective framework for computational driver discovery.


Assuntos
Genômica/métodos , Mutação , Neoplasias/genética , Software , Algoritmos , Genoma Humano , Humanos , Fatores de Transcrição MEF2/genética , Taxa de Mutação , Fator 1 de Elongação de Peptídeos/genética , Receptores Acoplados a Proteínas-G/genética , Sequenciamento Completo do Genoma
11.
Sci Rep ; 10(1): 1204, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31988313

RESUMO

Neurodevelopmental disorders (NDDs) are clinically and genetically extremely heterogeneous with shared phenotypes often associated with genes from the same networks. Mutations in TCF4, MEF2C, UBE3A, ZEB2 or ATRX cause phenotypically overlapping, syndromic forms of NDDs with severe intellectual disability, epilepsy and microcephaly. To characterize potential functional links between these genes/proteins, we screened for genetic interactions in Drosophila melanogaster. We induced ubiquitous or tissue specific knockdown or overexpression of each single orthologous gene (Da, Mef2, Ube3a, Zfh1, XNP) and in pairwise combinations. Subsequently, we assessed parameters such as lethality, wing and eye morphology, neuromuscular junction morphology, bang sensitivity and climbing behaviour in comparison between single and pairwise dosage manipulations. We found most stringent evidence for genetic interaction between Ube3a and Mef2 as simultaneous dosage manipulation in different tissues including glia, wing and eye resulted in multiple phenotype modifications. We subsequently found evidence for physical interaction between UBE3A and MEF2C also in human cells. Systematic pairwise assessment of the Drosophila orthologues of five genes implicated in clinically overlapping, severe NDDs and subsequent confirmation in a human cell line revealed interactions between UBE3A/Ube3a and MEF2C/Mef2, thus contributing to the characterization of the underlying molecular commonalities.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Epistasia Genética/genética , Fatores de Regulação Miogênica/genética , Transtornos do Neurodesenvolvimento/genética , Ubiquitina-Proteína Ligases/genética , Animais , Modelos Animais de Doenças , Olho/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Fatores de Transcrição MEF2/genética , Neuroglia/metabolismo , Fenótipo , RNA Interferente Pequeno , Transfecção , Asas de Animais/metabolismo
12.
EBioMedicine ; 51: 102571, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31911274

RESUMO

BACKGROUND: A pathophysiological link exists between dysregulation of MEF2C transcription factors and heart failure (HF), but the underlying mechanisms remain elusive. Alternative splicing of MEF2C exons α, ß and γ provides transcript diversity with gene activation or repression functionalities. METHODS: Neonatal and adult rat ventricular myocytes were used to overexpress MEF2C splicing variants γ+ (repressor) or γ-, or the inactive MEF2Cγ+23/24 (K23T/R24L). Phenotypic alterations in cardiomyocytes were determined by confocal and electron microscopy, flow cytometry and DNA microarray. We used transgenic mice with cardiac-specific overexpression of MEF2Cγ+ or MEF2Cγ- to explore the impact of MEF2C variants in cardiac phenotype. Samples of non-infarcted areas of the left ventricle from patients and mouse model of myocardial infarction were used to detect the expression of MEF2Cγ+ in failing hearts. FINDINGS: We demonstrate a previously unrealized upregulation of the transrepressor MEF2Cγ+ isoform in human and mouse failing hearts. We show that adenovirus-mediated overexpression of MEF2Cγ+ downregulates multiple MEF2-target genes, and drives incomplete cell-cycle reentry, partial dedifferentiation and apoptosis in the neonatal and adult rat. None of these changes was observed in cardiomyocytes overexpressing MEF2Cγ-. Transgenic mice overexpressing MEF2Cγ+, but not the MEF2Cγ-, developed dilated cardiomyopathy, correlated to cell-cycle reentry and apoptosis of cardiomyocytes. INTERPRETATION: Our results provide a mechanistic link between MEF2Cγ+ and deleterious abnormalities in cardiomyocytes, supporting the notion that splicing dysregulation in MEF2C towards the selection of the MEF2Cγ+ variant contributes to the pathogenesis of HF by promoting cardiomyocyte dropout. FUNDING: São Paulo Research Foundation (FAPESP); Brazilian National Research Council (CNPq).


Assuntos
Ciclo Celular/genética , Regulação da Expressão Gênica , Predisposição Genética para Doença , Variação Genética , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Processamento Alternativo , Animais , Apoptose/genética , Modelos Animais de Doenças , Estudos de Associação Genética , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/terapia , Humanos , Fatores de Transcrição MEF2/genética , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Ratos
13.
Biomed Pharmacother ; 122: 109557, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31918265

RESUMO

Prostate cancer (PCa) is a destructive malignancy with a bad prognosis. LncRNA VPS9D1-AS1 has recently been delineated as an oncogene in some kinds of tumor, whereas, the function of VPS9D1-AS1 in PCa remains to be clarified. In this study, we researched its underlying role in PCa. The expression of VPS9D1-AS1 was conspicuously upregulated in PCa tissues and cells. And absence of VPS9D1-AS1 inhibited cell proliferation, migration and invasion, and promoted cell apoptosis in PCa. In addition, VPS9D1-AS1 overexpression led to opposite results. Furthermore, VPS9D1-AS1/MEF2D could sponge with miR-4739. VPS9D1-AS1/MEF2D and miR-4739 were inversely correlated in tumor cells. And the expression of miR-4739 is markedly downregulated in PCa, meanwhile, that of MEF2D exhibited the opposite tendency. However, MEF2D was positively regulated by VPS9D1-AS1. Moreover, MEF2D upregulation offset the suppressive effects of VPS9D1-AS1 deficiency on cell proliferation, migration and invasion in PCa. Additionally, ZEB1 contained the binding sites of VPS9D1-AS1 promoter, and there existed positive relation between them. Taken together, above results illustrated that ZEB1 activated-VPS9D1-AS1 promotes the tumorigenesis and progression of PCa by sponging miR-4739 to upregulate MEF2D, which offering a new useful reference for studying the development process of PCa.


Assuntos
MicroRNAs/metabolismo , Neoplasias da Próstata/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Apoptose , Carcinogênese , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Masculino , MicroRNAs/genética , Neoplasias da Próstata/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética
15.
Circ Res ; 126(1): 6-24, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31730408

RESUMO

RATIONALE: Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is evolving rapidly. Recently, second-generation CRISPR/Cas9 activation systems based on nuclease inactive dead (d)Cas9 fused to transcriptional transactivation domains were developed for directing specific guide (g)RNAs to regulatory regions of any gene of interest, to enhance transcription. The application of dCas9 to activate cardiomyocyte transcription in targeted genomic loci in vivo has not been demonstrated so far. OBJECTIVE: We aimed to develop a mouse model for cardiomyocyte-specific, CRISPR-mediated transcriptional modulation, and to demonstrate its versatility by targeting Mef2d and Klf15 loci (2 well-characterized genes implicated in cardiac hypertrophy and homeostasis) for enhanced transcription. METHODS AND RESULTS: A mouse model expressing dCas9 with the VPR transcriptional transactivation domains under the control of the Myh (myosin heavy chain) 6 promoter was generated. These mice innocuously expressed dCas9 exclusively in cardiomyocytes. For initial proof-of-concept, we selected Mef2d, which when overexpressed, led to hypertrophy and heart failure, and Klf15, which is lowly expressed in the neonatal heart. The most effective gRNAs were first identified in fibroblast (C3H/10T1/2) and myoblast (C2C12) cell lines. Using an improved triple gRNA expression system (TRISPR [triple gRNA expression construct]), up to 3 different gRNAs were transduced simultaneously to identify optimal conditions for transcriptional activation. For in vivo delivery of the validated gRNA combinations, we employed systemic administration via adeno-associated virus serotype 9. On gRNA delivery targeting Mef2d expression, we recapitulated the anticipated cardiac hypertrophy phenotype. Using gRNA targeting Klf15, we could enhance its transcription significantly, although Klf15 is physiologically silenced at that time point. We further confirmed specific and robust dCas9VPR on-target effects. CONCLUSIONS: The developed mouse model permits enhancement of gene expression by using endogenous regulatory genomic elements. Proof-of-concept in 2 independent genomic loci suggests versatile applications in controlling transcription in cardiomyocytes of the postnatal heart.


Assuntos
Sistemas CRISPR-Cas , Regulação da Expressão Gênica , Miocárdio/metabolismo , Ativação Transcricional , Animais , Linhagem Celular , Dependovirus/genética , Fibroblastos/metabolismo , Regulação da Expressão Gênica/genética , Genes Sintéticos , Vetores Genéticos/genética , Coração/crescimento & desenvolvimento , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição MEF2/biossíntese , Fatores de Transcrição MEF2/genética , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/genética , Regiões Promotoras Genéticas , Domínios Proteicos , RNA Polimerase III/genética , RNA Guia/genética
16.
Nucleic Acids Res ; 48(2): 646-664, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31754707

RESUMO

Transcriptional networks supervising class IIa HDAC expression are poorly defined. Here we demonstrate that MEF2D is the key factor controlling HDAC9 transcription. This control, which is part of a negative feed-back loop during muscle differentiation, is hijacked in cancer. In leiomyosarcomas the MEF2D/HDAC9 vicious circuit sustains proliferation and cell survival, through the repression of the death receptor FAS. Comprehensive genome-wide studies demonstrate that HDAC4 and HDAC9 control different genetic programs and show both specific and common genomic binding sites. Although the number of MEF2-target genes commonly regulated is similar, only HDAC4 represses many additional genes that are not MEF2D targets. As expected, HDAC4-/- and HDAC9-/- cells increase H3K27ac levels around the TSS of the respective repressed genes. However, these genes rarely show binding of the HDACs at their promoters. Frequently HDAC4 and HDAC9 bind intergenic regions. We demonstrate that these regions, recognized by MEF2D/HDAC4/HDAC9 repressive complexes, show the features of active enhancers. In these regions HDAC4 and HDAC9 can differentially influence H3K27 acetylation. Our studies describe new layers of class IIa HDACs regulation, including a dominant positional effect, and can contribute to explain the pleiotropic actions of MEF2 TFs.


Assuntos
Histona Desacetilases/genética , Leiomiossarcoma/genética , Proteínas Repressoras/genética , Acetilação , Diferenciação Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Elementos Facilitadores Genéticos/genética , Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Leiomiossarcoma/patologia , Fatores de Transcrição MEF2/genética
17.
Gastroenterology ; 158(3): 664-678.e24, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31678303

RESUMO

BACKGROUND & AIMS: Immune checkpoint inhibitors have some efficacy in the treatment of hepatocellular carcinoma (HCC). Programmed cell death 1 ligand 1 (PD-L1), expressed on some cancer cells, binds to the receptor programmed cell death 1 (PDCD1, also called PD1) on T cells to prevent their proliferation and reduce the antigen-tumor immune response. Immune cells that infiltrate some types of HCCs secrete interferon gamma (IFNG). Some HCC cells express myocyte enhancer factor 2D (MEF2D), which has been associated with shorter survival times of patients. We studied whether HCC cell expression of MEF2D regulates expression of PD-L1 in response to IFNG. METHODS: We analyzed immune cells from 20 fresh HCC tissues by flow cytometry. We analyzed 225 fixed HCC tissues (from 2 cohorts) from patients in China by immunohistochemistry and obtained survival data. We created mice with liver-specific knockout of MEF2D (MEF2DLPC-KO mice). We knocked out or knocked down MEF2D, E1A binding protein p300 (p300), or sirtuin 7 (SIRT7) in SMMC-7721, Huh7, H22, and Hepa1-6 HCC cell lines, some incubated with IFNG. We analyzed liver tissues from mice and cell lines by RNA sequencing, immunoblot, dual luciferase reporter, and chromatin precipitation assays. MEF2D protein acetylation and proteins that interact with MEF2D were identified by coimmunoprecipitation and pull-down assays. H22 cells, with MEF2D knockout or without (controls), were transplanted into BALB/c mice, and some mice were given antibodies to deplete T cells. Mice bearing orthotopic tumors grown from HCC cells, with or without knockout of SIRT7, were given injections of an antibody against PD1. Growth of tumors was measured, and tumors were analyzed by immunohistochemistry and flow cytometry. RESULTS: In human HCC specimens, we found an inverse correlation between level of MEF2D and numbers of CD4+ and CD8+ T cells; level of MEF2D correlated with percentages of PD1-positive or TIM3-positive CD8+ T cells. Knockout of MEF2D from H22 cells reduced their growth as allograft tumors in immune-competent mice but not in immune-deficient mice or mice with depletion of CD8+ T cells. When MEF2D-knockout cells were injected into immune-competent mice, they formed smaller tumors that had increased infiltration and activation of T cells compared with control HCC cells. In human and mouse HCC cells, MEF2D knockdown or knockout reduced expression of PD-L1. MEF2D bound the promoter region of the CD274 gene (encodes PD-L1) and activated its transcription. Overexpression of p300 in HCC cells, or knockout of SIRT7, promoted acetylation of MEF2D and increased its binding, along with acetylated histones, to the promoter region of CD274. Exposure of HCC cells to IFNG induced expression of p300 and its binding MEF2D, which reduced the interaction between MEF2D and SIRT7. MEF2D-induced expression of PD-L1 upon IFNG exposure was independent of interferon-regulatory factors 1 or 9. In HCC cells not exposed to IFNG, SIRT7 formed a complex with MEF2D that attenuated expression of PD-L1. Knockout of SIRT7 reduced proliferation of HCC cells and growth of tumors in immune-deficient mice. Compared with allograft tumors grown from control HCC cells, in immune-competent mice, tumors grown from SIRT7-knockout HCC cells expressed higher levels of PD-L1 and had reduced infiltration and activation of T cells. In immune-competent mice given antibodies to PD1, allograft tumors grew more slowly from SIRT7-knockout HCC cells than from control HCC cells. CONCLUSIONS: Expression of MEF2D by HCC cells increases their expression of PD-L1, which prevents CD8+ T-cell-mediated antitumor immunity. When HCC cells are exposed to IFNG, p300 acetylates MEF2D, causing it to bind the CD274 gene promoter and up-regulate PD-L1 expression. In addition to promoting HCC cell proliferation, SIRT7 reduced acetylation of MEF2D and expression of PD-L1 in HCC cells not exposed to IFNG. Strategies to manipulate this pathway might increase the efficacy of immune therapies for HCC.


Assuntos
Antígeno B7-H1/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Sirtuínas/genética , Adolescente , Adulto , Idoso , Animais , Contagem de Linfócito CD4 , Linfócitos T CD8-Positivos/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Feminino , Técnicas de Inativação de Genes , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Humanos , Imunocompetência , Interferon gama/farmacologia , Neoplasias Hepáticas/patologia , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Transplante de Neoplasias , Receptor de Morte Celular Programada 1/metabolismo , Sirtuínas/metabolismo , Transcrição Genética/efeitos dos fármacos , Transcrição Genética/genética , Adulto Jovem
18.
Blood ; 135(1): 56-70, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31697837

RESUMO

Lineage-defining transcription factors (TFs) are compelling targets for leukemia therapy, yet they are among the most challenging proteins to modulate directly with small molecules. We previously used CRISPR screening to identify a salt-inducible kinase 3 (SIK3) requirement for the growth of acute myeloid leukemia (AML) cell lines that overexpress the lineage TF myocyte enhancer factor (MEF2C). In this context, SIK3 maintains MEF2C function by directly phosphorylating histone deacetylase 4 (HDAC4), a repressive cofactor of MEF2C. In this study, we evaluated whether inhibition of SIK3 with the tool compound YKL-05-099 can suppress MEF2C function and attenuate disease progression in animal models of AML. Genetic targeting of SIK3 or MEF2C selectively suppressed the growth of transformed hematopoietic cells under in vitro and in vivo conditions. Similar phenotypes were obtained when cells were exposed to YKL-05-099, which caused cell-cycle arrest and apoptosis in MEF2C-expressing AML cell lines. An epigenomic analysis revealed that YKL-05-099 rapidly suppressed MEF2C function by altering the phosphorylation state and nuclear localization of HDAC4. Using a gatekeeper allele of SIK3, we found that the antiproliferative effects of YKL-05-099 occurred through on-target inhibition of SIK3 kinase activity. Based on these findings, we treated 2 different mouse models of MLL-AF9 AML with YKL-05-099, which attenuated disease progression in vivo and extended animal survival at well-tolerated doses. These findings validate SIK3 as a therapeutic target in MEF2C-addicted AML and provide a rationale for developing druglike inhibitors of SIK3 for definitive preclinical investigation and for studies in human patients.


Assuntos
Compostos de Anilina/farmacologia , Leucemia Mieloide Aguda/prevenção & controle , Fatores de Transcrição MEF2/metabolismo , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Pirimidinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Apoptose , Ciclo Celular , Proliferação de Células , Feminino , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Fatores de Transcrição MEF2/genética , Camundongos , Camundongos Endogâmicos C57BL , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Biochim Biophys Acta Mol Cell Res ; 1867(2): 118610, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31738957

RESUMO

OBJECTIVE: In muscle cells, the peroxisome proliferator-activated receptor γ co-activator 1 (PGC-1) signaling network, which has been shown to be disturbed in the skeletal muscle in several chronic diseases, tightly controls mitochondrial biogenesis and oxidative substrate metabolism. Previously, we showed that inactivation of glycogen synthase kinase (GSK)-3ß potently increased Pgc-1α abundance and oxidative metabolism in skeletal muscle cells. The current study aims to unravel the molecular mechanism driving the increase in Pgc-1α mediated by GSK-3ß inactivation. METHODS: GSK-3ß was inactivated genetically or pharmacologically in C2C12 myotubes and the requirement of transcription factors known to be involved in Pgc-1α transcription for increases in Pgc-1α abundance mediated by inactivation of GSK-3ß was examined. RESULTS: Enhanced PGC-1α promoter activation after GSK-3ß inhibition suggested a transcriptionally-controlled mechanism. While myocyte enhancer factor (MEF)2 transcriptional activity was unaltered, GSK-3ß inactivation increased the abundance and activity of the transcription factors estrogen-related receptor (ERR)α and ERRγ. Pharmacological inhibition or knock-down of ERRα and ERRγ however failed to prevent increases in Pgc-1α mRNA mediated by GSK-3ß inactivation. Interestingly, GSK-3ß inactivation activated transcription factor EB (TFEB), evidenced by decreased phosphorylation and enhanced nuclear localization of the TFEB protein. Moreover, knock-down of TFEB completely prevented increases in Pgc-1α gene expression, PGC-1α promoter activity and PGC-1α protein abundance induced by GSK-3ß inactivation. Furthermore, mutation of a specific TFEB binding site on the PGC-1α promoter blocked promoter activation upon inhibition of GSK-3ß. CONCLUSIONS: In skeletal muscle, GSK-3ß inactivation causes dephosphorylation and nuclear translocation of TFEB resulting in TFEB-dependent induction of Pgc-1α expression.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/genética , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Fosforilação , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Estrogênicos/antagonistas & inibidores , Receptores Estrogênicos/genética , Receptores Estrogênicos/metabolismo , Transdução de Sinais , Ativação Transcricional , Regulação para Cima
20.
PLoS Genet ; 15(12): e1008507, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31790396

RESUMO

Deleterious genetic mutations allow developmental biologists to understand how genes control development. However, not all loss of function genetic mutants develop phenotypic changes. Many deleterious mutations only produce a phenotype in a subset of mutant individuals, a phenomenon known as incomplete penetrance. Incomplete penetrance can confound analyses of gene function and our understanding of this widespread phenomenon remains inadequate. To better understand what controls penetrance, we capitalized on the zebrafish mef2ca mutant which produces craniofacial phenotypes with variable penetrance. Starting with a characterized mef2ca loss of function mutant allele, we used classical selective breeding methods to generate zebrafish strains in which mutant-associated phenotypes consistently appear with low or high penetrance. Strikingly, our selective breeding for low penetrance converted the mef2ca mutant allele behavior from homozygous lethal to homozygous viable. Meanwhile, selective breeding for high penetrance converted the mef2ca mutant allele from fully recessive to partially dominant. Comparing the selectively-bred low- and high-penetrance strains revealed that the strains initially respond similarly to the mutation, but then gene expression differences between strains emerge during development. Thus, altered temporal genetic circuitry can manifest through selective pressure to modify mutant penetrance. Specifically, we demonstrate differences in Notch signaling between strains, and further show that experimental manipulation of the Notch pathway phenocopies penetrance changes occurring through selective breeding. This study provides evidence that penetrance is inherited as a liability-threshold trait. Our finding that vertebrate animals can overcome a deleterious mutation by tuning genetic circuitry complements other reported mechanisms of overcoming deleterious mutations such as transcriptional adaptation of compensatory genes, alternative mRNA splicing, and maternal deposition of wild-type transcripts, which are not observed in our system. The selective breeding approach and the resultant genetic circuitry change we uncovered advances and expands our current understanding of genetic and developmental resilience.


Assuntos
Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Receptores Notch/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais , Epistasia Genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Mutação com Perda de Função , Masculino , Ossificação Heterotópica/genética , Penetrância , Fenótipo , Seleção Artificial , Transdução de Sinais , Fatores de Transcrição/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética
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