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2.
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
3.
Biochim Biophys Acta Gene Regul Mech ; 1862(10): 194435, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31678627

RESUMO

Cardiovascular development is governed by a complex interplay between inducting signals such as Bmps and Fgfs leading to activation of cardiac specific transcription factors such as Nkx2.5, Mef2c and Srf that orchestrate the initial steps of cardiogenesis. Over the last decade we have witnessed the discovery of novel layers of gene regulation, i.e. post-transcriptional regulation exerted by non-coding RNAs. The function role of small non coding RNAs has been widely demonstrated, e.g. miR-1 knockout display several cardiovascular abnormalities during embryogenesis. More recently long non-coding RNAs have been also reported to modulate gene expression and function in the developing heart, as exemplified by the embryonic lethal phenotypes of Fendrr and Braveheart knock out mice, respectively. In this study, we investigated the differential expression profile during cardiogenesis of previously reported lncRNAs in heart development. Our data revealed that Braveheart, Fendrr, Carmen display a preferential adult expression while Miat, Alien, H19 preferentially display chamber-specific expression at embryonic stages. We also demonstrated that these lncRNAs are differentially regulated by Nkx2.5, Srf and Mef2c, Pitx2 > Wnt > miRNA signaling pathway and angiotensin II and thyroid hormone administration. Importantly isoform-specific expression and distinct nuclear vs cytoplasmic localization of Braveheart, Carmen and Fendrr during chamber morphogenesis is observed, suggesting distinct functional roles of these lncRNAs in atrial and ventricular chambers. Furthermore, we demonstrate by in situ hybridization a dynamic epicardial, myocardial and endocardial expression of H19 during cardiac development. Overall our data support novel roles of these lncRNAs in different temporal and tissue-restricted fashion during cardiogenesis.


Assuntos
Sistema Cardiovascular/crescimento & desenvolvimento , Coração/crescimento & desenvolvimento , RNA Longo não Codificante/genética , Fatores de Transcrição/genética , Animais , Sistema Cardiovascular/metabolismo , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteína Homeobox Nkx-2.5/genética , Proteínas de Homeodomínio/genética , Hibridização In Situ , Fatores de Transcrição MEF2/genética , Camundongos , Camundongos Knockout , MicroRNAs/genética , Fator de Resposta Sérica/genética
4.
PLoS Biol ; 17(10): e3000467, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31589602

RESUMO

Skeletal muscles consist of fibers of differing metabolic activities and contractility, which become remodeled in response to chronic exercise, but the epigenomic basis for muscle identity and adaptation remains poorly understood. Here, we used chromatin immunoprecipitation sequencing of dimethylated histone 3 lysine 4 and acetylated histone 3 lysine 27 as well as transposase-accessible chromatin profiling to dissect cis-regulatory networks across muscle groups. We demonstrate that in vivo enhancers specify muscles in accordance with myofiber composition, show little resemblance to cultured myotube enhancers, and identify glycolytic and oxidative muscle-specific regulators. Moreover, we find that voluntary wheel running and muscle-specific peroxisome proliferator-activated receptor gamma coactivator-1 alpha (Pgc1a) transgenic (mTg) overexpression, which stimulate endurance performance in mice, result in markedly different changes to the epigenome. Exercise predominantly leads to enhancer hypoacetylation, whereas mTg causes hyperacetylation at different sites. Integrative analysis of regulatory regions and gene expression revealed that exercise and mTg are each associated with myocyte enhancer factor (MEF) 2 and estrogen-related receptor (ERR) signaling and transcription of genes promoting oxidative metabolism. However, exercise was additionally associated with regulation by retinoid X receptor (RXR), jun proto-oncogene (JUN), sine oculis homeobox factor (SIX), and other factors. Overall, our work defines the unique enhancer repertoires of skeletal muscles in vivo and reveals that divergent exercise-induced or PGC1α-driven epigenomic programs direct partially convergent transcriptional networks.


Assuntos
Epigênese Genética , Histonas/genética , Células Musculares/metabolismo , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Condicionamento Físico Animal , Acetilação , Animais , Reprogramação Celular , Cromatina/química , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Glicólise/genética , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Musculares/citologia , Músculo Esquelético/citologia , Fosforilação Oxidativa , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Receptores Estrogênicos/genética , Receptores Estrogênicos/metabolismo , Receptores X Retinoide/genética , Receptores X Retinoide/metabolismo , Transdução de Sinais
5.
Genes (Basel) ; 10(8)2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31374988

RESUMO

The MEF2 (myocyte enhancer factor 2) family belongs to the MADS-box superfamily of eukaryotic transcription factors. The vertebrate genes compose four distinct subfamilies designated MEF2A, -B, -C, and -D. There are multiple mef2 genes in the common carp (Cyprinus carpio). So far, the embryonic expression patterns of these genes and the evolution of fish mef2 genes have been barely investigated. In this study, we completed the coding information of C. carpio mef2ca2 and mef2d1 genes via gene cloning and presented two mosaic mef2 sequences as evidence for recombination. We also analyzed the phylogenetic relationship and conserved synteny of mef2 genes and proposed a new evolutionary scenario. In our version, MEF2B and the other three vertebrate subfamilies were generated in parallel from the single last ancestor via two rounds of whole genome duplication events that occurred at the dawn of vertebrates. Moreover, we examined the expression patterns of C. carpio mef2 genes during embryogenesis, by using whole-mount in situ hybridization, and found the notochord to be a new expression site for these genes except for mef2ca1&2. Our results thus provide new insights into the evolution and expression of mef2 genes.


Assuntos
Carpas/genética , Evolução Molecular , Proteínas de Peixes/genética , Fatores de Transcrição MEF2/genética , Animais , Carpas/classificação , Proteínas de Peixes/metabolismo , Fatores de Transcrição MEF2/metabolismo , Notocorda/metabolismo , Filogenia , Sintenia
6.
BMC Med Genomics ; 12(1): 116, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375103

RESUMO

BACKGROUND: Balanced structural variants are mostly described in disease with gene disruption or subtle rearrangement at breakpoints. CASE PRESENTATION: Here we report a patient with mild intellectual deficiency who carries a de novo balanced translocation t(3;5). Breakpoints were fully explored by microarray, Array Painting and Sanger sequencing. No gene disruption was found but the chromosome 5 breakpoint was localized 228-kb upstream of the MEF2C gene. The predicted Topologically Associated Domains analysis shows that it contains only the MEF2C gene and a long non-coding RNA LINC01226. RNA studies looking for MEF2C gene expression revealed an overexpression of MEF2C in the lymphoblastoid cell line of the patient. CONCLUSIONS: Pathogenicity of MEF2C overexpression is still unclear as only four patients with mild intellectual deficiency carrying 5q14.3 microduplications containing MEF2C are described in the literature. The microduplications in these individuals also contain other genes expressed in the brain. The patient presented the same phenotype as 5q14.3 microduplication patients. We report the first case of a balanced translocation leading to an overexpression of MEF2C similar to a functional duplication.


Assuntos
Cromatina/metabolismo , Deficiência Intelectual/genética , Criança , Pré-Escolar , Bandeamento Cromossômico , Cromossomos Humanos Par 3/genética , Cromossomos Humanos Par 5/genética , Feminino , Duplicação Gênica , Humanos , Lactente , Recém-Nascido , Fatores de Transcrição MEF2/genética
7.
Zhongguo Gu Shang ; 32(6): 578-581, 2019 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-31277546

RESUMO

OBJECTIVE: To investigate the effect of Xiaozhong Zhitong Ointment(XZZTO) on remodeling and repair of skeletal muscle injury in rats based on the expression mechanism of microRNA. METHODS: The rat gastrocnemius injury model was established by blunt contusion model. The expression of MEF2 gene and protein in gastrocnemius muscle was detected by quantitative PCR at 4, 7, 14 and 21 days after injury with XZZTO. The mechanism of the effect of XZZTO on the muscle remodeling and repair of rat gastrocnemius contusion model was discussed. RESULTS: The expression level of MEF2 in the treatment group was significantly higher than that of the control group and model group, which further confirmed the important role of MEF2 in inducing skeletal muscle remodeling and repair process in the topical drugs. The expression of MEF2 increased at 7 days after injury and remained at a high level until 21 days after injury. Compared with the model group, the peak expression period was about 14 days, and then returned to the general state. CONCLUSIONS: The expression level of MEF2 shows an upward trend. Even 21 days after injury, the expression of MEF2 dose not show a significant downward trend. It can be seen that XZZTO can promote the expression of MEF2. At the same time, XZZTO can regulate the regeneration and repair of skeletal muscle. Therefore, XZZTO can play a regeneration and repair role after skeletal muscle injury through gene regulation.


Assuntos
Fatores de Transcrição MEF2/genética , Músculo Esquelético , Animais , Contusões , Trato Gastrointestinal , Regulação da Expressão Gênica , Pomadas , Proteínas , RNA Mensageiro , Ratos
8.
Biochimie ; 163: 152-162, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31199942

RESUMO

Extra-cellular signal regulated kinase-5 (Erk-5), a transcriptional activator and regulator of endothelial cells (ECs) homeostasis, has been implicated in shear stress-induced endothelial dysfunction (ED), however its role in oxidized low-density lipoprotein (oxLDL)- induced ED during metabolic stress is not known. Herein, regulation and function of Erk-5 in oxLDL-induced EC death, inflammation and dysfunction has been investigated. Primary Human Umbilical Vein Endothelial Cells (pHUVECs) were stimulated with oxLDL. MTT and Trypan blue exclusion assays to assess cell viability, RT-qPCR and Western blotting assays to determine expression of endothelial and inflammatory markers and ED mediators at mRNA and protein levels, respectively were performed. Monocyte adhesion assay was performed to examine monocytes adherence to oxLDL-stimulated pHUVECs. The exposure of oxLDL induced a dose- and time-dependent decrease in pHUVECs viability, which concurred with decreased Erk-5 expression. Further, oxLDL (100 µg/ml) decreased the expression of endothelial markers eNOS and vWF, and increased the expression of ICAM-1, at both mRNA and protein levels. SiRNA-mediated silencing of Erk-5 or its inhibition showed that changes in eNOS, vWF and ICAM-1 expression could be mediated through Erk-5. Furthermore, oxLDL decreased the levels of Erk-5's upstream regulator MEK5 and downstream regulators Mef2c and KLF2, which were similar to their expressions in Erk-5 silenced cells. Fisetin, a phytochemical and bioflavonoid, could reduce the effect of oxLDL in ECs by upregulating the expression of endothelial markers including Erk-5, and downregulating the expression of inflammation markers. These results suggest that Erk-5 could be a critical regulator of oxLDL-induced EC death, inflammation and dysfunction via downregulation of Erk-5/Mef2c-KLF2 signaling pathway, which can be ameliorated by a bioflavonoid, fisetin.


Assuntos
Flavonoides/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Lipoproteínas LDL/toxicidade , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Monócitos/fisiologia , Transdução de Sinais , Aterosclerose/induzido quimicamente , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/prevenção & controle , Células Cultivadas , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Inflamação/prevenção & controle , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Lipoproteínas LDL/farmacologia , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Substâncias Protetoras/farmacologia
9.
Int J Mol Sci ; 20(11)2019 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-31167510

RESUMO

Understanding the molecular mechanisms of skeletal myoblast differentiation is essential for studying muscle developmental biology. In our previous study, we reported that knockdown of myocyte enhancer factor 2A (MEF2A) inhibited myoblast differentiation. Here in this study, we further identified that MEF2A controlled this process through regulating the maternally expressed 3 (MEG3)-iodothyronine deiodinase 3 (DIO3) miRNA mega cluster and protein phosphatase 2A (PP2A) signaling. MEF2A was sufficient to induce MEG3 expression in bovine skeletal myoblasts. A subset of miRNAs in the MEG3-DIO3 miRNA cluster was predicted to target PP2A subunit genes. Consistent with these observations, MEF2A regulated PP2A signaling through its subunit gene protein phosphatase 2 regulatory subunit B, gamma (PPP2R2C) during bovine myoblast differentiation. MiR-758 and miR-543 in the MEG3-DIO3 miRNA cluster were down-regulated in MEF2A-depleted myocytes. Expression of miR-758 and miR-543 promoted myoblast differentiation and repressed PPP2R2C expression. Luciferase activity assay showed that PPP2R2C was post-transcriptionally targeted by miR-758 and miR-543. Taken together, these results reveal that the MEG3-DIO3 miRNAs function at downstream of MEF2A to modulate PP2A signaling in bovine myoblast differentiation.


Assuntos
Iodeto Peroxidase/genética , Fatores de Transcrição MEF2/genética , Família Multigênica , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/metabolismo , Proteína Fosfatase 2/metabolismo , RNA Longo não Codificante/genética , Animais , Bovinos , Diferenciação Celular , Regulação da Expressão Gênica , Modelos Biológicos , Interferência de RNA , Transdução de Sinais
10.
Mol Cell ; 75(1): 26-38.e3, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31130364

RESUMO

Growth factor signaling is initiated at the plasma membrane and propagated through the cytoplasm for eventual relay to intracellular organelles such as lysosomes. The serine/threonine kinase mTOR participates in growth factor signaling as a component of two multi-subunit complexes, mTORC1 and mTORC2. mTORC1 associates with lysosomes, and its activity depends on the positioning of lysosomes within the cytoplasm, although there is no consensus regarding the exact effect of perinuclear versus peripheral distribution. mTORC2 and its substrate kinase AKT have a widespread distribution, but they are thought to act mainly at the plasma membrane. Using cell lines with knockout of components of the lysosome-positioning machinery, we show that perinuclear clustering of lysosomes delays reactivation of not only mTORC1, but also mTORC2 and AKT upon serum replenishment. These experiments demonstrate the existence of pools of mTORC2 and AKT that are sensitive to lysosome positioning.


Assuntos
Núcleo Celular/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Proteínas Proto-Oncogênicas c-akt/genética , Fatores de Ribosilação do ADP/deficiência , Fatores de Ribosilação do ADP/genética , Sistemas CRISPR-Cas , Núcleo Celular/ultraestrutura , Meios de Cultura Livres de Soro , Endossomos/metabolismo , Endossomos/ultraestrutura , Edição de Genes , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Cinesina/deficiência , Cinesina/genética , Lisossomos/ultraestrutura , Fatores de Transcrição MEF2/deficiência , Fatores de Transcrição MEF2/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
11.
Mol Med Rep ; 19(6): 4927-4934, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30957182

RESUMO

Congenital heart disease (CHD) is the most common type of birth defect, and the leading cause of fetal mortality. The long noncoding RNA (lncRNA) uc.457 is differentially expressed in cardiac tissue from patients with a ventricular septal defect; however, its role in cardiac development and CHD remains unknown. In the present study, the role of uc.457 in the differentiation and maturation of cardiomyocytes was investigated. Bioinformatics approaches were employed to analyze putative transcription factor (TF) regulation, histone modifications and the biological functions of uc.457. Subsequently, uc.457 overexpression and small interfering RNA­mediated knockdown were performed to evaluate the functional role of the lncRNA in the dimethyl sulfoxide­induced differentiation of P19 cells into cardiomyocytes. Bioinformatics analyses predicted that uc.457 binds to TFs associated with cardiomyocyte growth and cardiac development. Cell Counting Kit­8 assays demonstrated that uc.457 overexpression inhibited cell proliferation, whereas knockdown of uc.457 enhanced the proliferation of differentiating cardiomyocytes. Additionally, reverse transcription­quantitative polymerase chain reaction and western blot analyses revealed that overexpression of uc.457 suppressed the mRNA and protein expression of histone cell cycle regulation defective homolog A, natriuretic peptide A, cardiac muscle troponin T and myocyte­specific enhancer factor 2C. Collectively, the results indicated that overexpression of uc.457 inhibited the differentiation and proliferation of cardiomyocytes, suggesting that dysregulated uc.457 expression may be associated with CHD.


Assuntos
RNA Longo não Codificante/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Interferência de RNA , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Troponina T/genética , Troponina T/metabolismo
12.
Seizure ; 67: 86-90, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30922778

RESUMO

PURPOSE: MEF2C-related epilepsy has been poorly described in the literature, despite a consistent MEF2C haploinsufficiency phenotype characterized by severe language impairment and motor delay (MIM# 613443). We aimed to delineate the spectrum of electroclinical manifestations of MEF2C-related epilepsy from an illustrative case and literature review. METHODS: A retrospective chart review of our case was performed followed by a literature review on PubMed and OMIM. Publications including patients with MEF2C pathogenic, likely pathogenic variants, or microdeletions without involvement of other genes were selected. RESULTS: The index case is a 2-year-old male with global developmental delay who presented at 7 months with atypical febrile seizures, generalized myoclonias, and focal impaired awareness seizures. Neuroimaging studies were unremarkable and electroencephalograms showed high voltage 200-400uV, 2-2.5 Hz generalized spike-and-waves and polyspikes with alternating frontal predominance, and multifocal spike-and-slow waves. Whole exome sequencing showed an unreported de novo likely pathogenic variant in the MEF2C gene c.236 G > C (p.Arg79Pro). Data from ten additional publications including 22 patients were gathered. From the 23 patients in total, 19 (82%) had seizures. Febrile seizures were most common, but myoclonic, focal-onset and generalized seizures were also reported. Electroencephalogram findings were described in eleven, and nine (82%) showed epileptiform abnormalities. CONCLUSION: MEF2C-related epilepsy may be described as a spectrum of manifestations including febrile seizures, myoclonia, and focal-onset or generalized seizures. Electroencephalogram is consistently abnormal, showing findings such as background slowing, multifocal and generalized epileptiform discharges and polyspikes. It remains unclear whether most patients are responsive or refractory to treatment with anti-epileptic medications.


Assuntos
Epilepsia/genética , Mutação , Encéfalo/fisiopatologia , Pré-Escolar , Epilepsia/fisiopatologia , Epilepsia/psicologia , Epilepsia/terapia , Humanos , Fatores de Transcrição MEF2/genética , Masculino , Fenótipo
13.
BMC Mol Biol ; 20(1): 8, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30885136

RESUMO

BACKGROUND: Myocyte enhancer factor 2A (MEF2A) plays an important role in cell proliferation, differentiation and survival. Functional deletion or mutation in MEF2A predisposes individuals to cardiovascular disease mainly caused by vascular endothelial dysfunction. However, the effect of the inhibition of MEF2A expression on human coronary artery endothelial cells (HCAECs) is unclear. In this study, expression of MEF2A was inhibited by specific small interference RNA (siRNA), and changes in mRNA profiles in response to MEF2A knockdown were analyzed using an Agilent human mRNA array. RESULTS: Silencing of MEF2A in HCAECs accelerated cell senescence and suppressed cell proliferation. Microarray analysis identified 962 differentially expressed genes (DEGs) between the MEF2A knockdown group and the negative control group. Annotation clustering analysis showed that the DEGs were preferentially enriched in gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to proliferation, development, survival, and inflammation. Furthermore, 61 of the 578 downregulated DEGs have at least one potential MEF2A binding site in the proximal promoter and were mostly enriched in the GO terms "reproduction" and "cardiovascular." The protein-protein interaction network analyzed for the downregulated DEGs and the DEGs in the GO terms "cardiovascular" and "aging" revealed that PIK3CG, IL1B, IL8, and PRKCB were included in hot nodes, and the regulation of the longevity-associated gene PIK3CG by MEF2A has been verified at the protein level, suggesting that PIK3CG might play a key role in MEF2A knockdown induced HCAEC senescence. CONCLUSIONS: MEF2A knockdown accelerates HCAEC senescence, and the underlying molecular mechanism may be involved in down-regulation of the genes related with cell proliferation, development, inflammation and survival, in which PIK3CG may play a key role.


Assuntos
Senescência Celular/genética , Vasos Coronários/citologia , Células Endoteliais , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Células Endoteliais/citologia , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Inflamação/genética , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/fisiologia
14.
Development ; 146(3)2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30723106

RESUMO

Dynamic organization of chromatin within the three-dimensional nuclear space has been postulated to regulate gene expression and cell fate. Here, we define the genome-wide distribution of nuclear peripheral heterochromatin as a multipotent P19 cell adopts either a neural or a cardiac fate. We demonstrate that H3K9me2-marked nuclear peripheral heterochromatin undergoes lineage-specific reorganization during cell-fate determination. This is associated with spatial repositioning of genomic loci away from the nuclear periphery as shown by 3D immuno-FISH. Locus repositioning is not always associated with transcriptional changes, but a subset of genes is upregulated. Mef2c is specifically repositioned away from the nuclear periphery during early neurogenic differentiation, but not during early cardiogenic differentiation, with associated transcript upregulation. Myocd is specifically repositioned during early cardiogenic differentiation, but not during early neurogenic differentiation, and is transcriptionally upregulated at later stages of cardiac differentiation. We provide experimental evidence for lineage-specific regulation of nuclear architecture during cell-fate determination in a mouse cell line.


Assuntos
Diferenciação Celular , Montagem e Desmontagem da Cromatina , Heterocromatina/metabolismo , Histonas/metabolismo , Células-Tronco Multipotentes/metabolismo , Linhagem Celular , Heterocromatina/genética , Histonas/genética , Humanos , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Miócitos Cardíacos/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Regulação para Cima
15.
Thromb Haemost ; 119(5): 716-725, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30731491

RESUMO

Megakaryopoiesis produces specialized haematopoietic stem cells in the bone marrow that give rise to megakaryocytes which ultimately produce platelets. Defects in megakaryopoiesis can result in altered platelet counts and physiology, leading to dysfunctional haemostasis and thrombosis. Additionally, dysregulated megakaryopoiesis is also associated with myeloid pathologies. Transcription factors play critical roles in cell differentiation by regulating the temporal and spatial patterns of gene expression which ultimately decide cell fate. Several transcription factors have been described as regulating megakaryopoiesis including myocyte enhancer factor 2C (MEF2C); however, the genes regulated by MEF2C that influence megakaryopoiesis have not been reported. Using chromatin immunoprecipitation-sequencing and Gene Ontology data we identified five candidate genes that are bound by MEF2C and regulate megakaryopoiesis: MOV10, AGO3, HDAC1, RBBP5 and WASF2. To study expression of these genes, we silenced MEF2C gene expression in the Meg01 megakaryocytic cell line and in induced pluripotent stem cells by CRISPR/Cas9 editing. We also knocked down MEF2C expression in cord blood-derived haematopoietic stem cells by siRNA. We found that absent or reduced MEF2C expression resulted in defects in megakaryocytic differentiation and reduced levels of the candidate target genes. Luciferase assays confirmed that genomic sequences within the target genes are regulated by MEF2C levels. Finally, we demonstrate that small deletions linked to a platelet count-associated single nucleotide polymorphism alter transcriptional activity, suggesting a mechanism by which genetic variation in MEF2C alters platelet production. These data help elucidate the mechanism behind MEF2C regulation of megakaryopoiesis and genetic variation driving platelet production.


Assuntos
Plaquetas/fisiologia , Medula Óssea/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Fatores de Transcrição MEF2/genética , Megacariócitos/fisiologia , Elementos Reguladores de Transcrição/genética , Trombopoese/genética , Sistemas CRISPR-Cas , Diferenciação Celular , Linhagem Celular , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica , Ontologia Genética , Humanos , RNA Interferente Pequeno/genética
16.
Biosci Rep ; 39(3)2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30777932

RESUMO

Akirin1 is found to be involved in myoblast differentiation. However, the mechanism by which the Akirin1 gene regulates myoblast differentiation still remains unclear. In the present study, we found that ectopic expression of Akirin1 promoted myoblast differentiation by increasing the expression of myogenic regulatory factor (MRF) 4 (MRF4) and myocyte enhancer factor 2B (MEF2B) mRNA. Additionally, we showed that ectopic Akirin1 induced cell cycle arrest by up-regulating p21 mRNA. To further uncover the mechanism by which Akirin1 promotes myoblast differentiation, we showed that the enhanced Akirin1 increased the mRNA expression of P38α. Importantly, the enhanced MRF4 expression by Akirin1 can be abrogated by treatment of SB203580, a p38 inhibitor. Similarly, we found that enhanced MEF2B expression by Akirin1 can be abrogated by treatment with LY294002, a PI3K inhibitor. Together, our results indicate that Akirin1 promotes myoblast differentiation by acting on the p38 and PI3K pathways and subsequently inducing the expression of myoblast differentiation factors.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Diferenciação Celular/genética , Fatores de Transcrição MEF2/genética , Mioblastos/metabolismo , Fatores de Regulação Miogênica/genética , Proteínas Nucleares/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Cromonas/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Patos , Imidazóis/farmacologia , Fatores de Transcrição MEF2/metabolismo , Morfolinas/farmacologia , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Fatores de Regulação Miogênica/metabolismo , Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Piridinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
17.
Mol Cells ; 42(2): 175-182, 2019 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-30703870

RESUMO

microRNAs regulate a diverse spectrum of cancer biology, including tumorigenesis, metastasis, stemness, and drug resistance. To investigate miRNA-mediated regulation of drug resistance, we characterized the resistant cell lines to 5-fluorouracil by inducing stable expression of miRNAs using lenti-miRNA library. Here, we demonstrate miR-551a as a novel factor regulating cell survival after 5-FU treatment. miR-551a-expressing cells (Hep3B-lenti-miR-551a) were resistant to 5-FU-induced cell death, and after 5-FU treatment, and showed significant increases in cell viability, cell survival, and sphere formation. It was further shown that myocyte-specific factor 2C is the direct target of miR-551a. Our results suggest that miR-551a plays a novel function in regulating 5-FU-induced cell death, and targeting miR-551a might be helpful to sensitize cells to anti-cancer drugs.


Assuntos
Fluoruracila/farmacologia , MicroRNAs/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Sequência de Bases , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , MicroRNAs/genética , Células Tumorais Cultivadas
18.
Genes (Basel) ; 10(2)2019 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-30678091

RESUMO

Genome-wide association studies (GWASes) revealed several single-nucleotide polymorphisms (SNPs) in the human 17q12-21 locus associated with autoimmune diseases. However, follow-up studies are still needed to identify causative SNPs directly mediating autoimmune risk in the locus. We have chosen six SNPs in high linkage disequilibrium with the GWAS hits that showed the strongest evidence of causality according to association pattern and epigenetic data and assessed their functionality in a local genomic context using luciferase reporter system. We found that rs12946510, rs4795397, rs12709365, and rs8067378 influenced the reporter expression level in leukocytic cell lines. The strongest effect visible in three distinct cell types was observed for rs12946510 that is predicted to alter MEF2A/C and FOXO1 binding sites.


Assuntos
Doenças Autoimunes/genética , Cromossomos Humanos Par 17/genética , Polimorfismo de Nucleotídeo Único , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Humanos , Células Jurkat , Desequilíbrio de Ligação , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Locos de Características Quantitativas
19.
BMC Mol Biol ; 20(1): 1, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30602381

RESUMO

BACKGROUND: MicroRNAs (miRNAs) are small noncoding RNAs of approximately 22 nucleotides, highly conserved among species, which modulate gene expression by cleaving messenger RNA target or inhibiting translation. MiRNAs are involved in the regulation of many processes including cell proliferation, differentiation, neurogenesis, angiogenesis, and apoptosis. Beef tenderness is an organoleptic characteristic of great influence in the acceptance of meat by consumers. Previous studies have shown that collagen level, marbling, apoptosis and proteolysis are among the many factors that affect beef tenderness. Considering that miRNAs can modulate gene expression, this study was designed to identify differentially expressed miRNAs that could be modulating biological processes involved with beef tenderness. RESULTS: Deep sequence analysis of miRNA libraries from longissimus thoracis muscle allowed the identification of 42 novel and 308 known miRNAs. Among the known miRNAs, seven were specifically expressed in skeletal muscle. Differential expression analysis between animals with high (H) and low (L) estimated breeding values for shear force (EBVSF) revealed bta-mir-182 and bta-mir-183 are up-regulated (q value < 0.05) in animals with L EBVSF, and bta-mir-338 is up-regulated in animals with H EBVSF. The number of bovine predicted targets for bta-mir-182, bta-mir-183 and bta-mir-338 were 811, 281 and 222, respectively, which correspond to 1204 unique target genes. Among these, four of them, MEF2C, MAP3K2, MTDH and TNRC6B were common targets of the three differentially expressed miRNAs. The functional analysis identified important pathways related to tenderness such as apoptosis and the calpain-calpastatin system. CONCLUSION: The results obtained indicate the importance of miRNAs in the regulatory mechanisms that influence muscle proteolysis and meat tenderness and contribute to our better understanding of the role of miRNAs in biological processes associated with beef tenderness.


Assuntos
Cruzamento , Bovinos/genética , MicroRNAs/metabolismo , Músculo Esquelético/metabolismo , Carne Vermelha , Animais , Apoptose , Proteínas de Ligação ao Cálcio/metabolismo , MAP Quinase Quinase Quinase 2/genética , MAP Quinase Quinase Quinase 2/metabolismo , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Manitol Desidrogenases/genética , Manitol Desidrogenases/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
20.
Genes Brain Behav ; 18(3): e12519, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30221471

RESUMO

Sensation seeking is a multifaceted, heritable trait which predicts the development of substance use and abuse in humans; similar phenomena have been observed in rodents. Genetic correlations among sensation seeking and substance use indicate shared biological mechanisms, but the genes and networks underlying these relationships remain elusive. Here, we used a systems genetics approach in the BXD recombinant inbred mouse panel to identify shared genetic mechanisms underlying substance use and preference for sensory stimuli, an intermediate phenotype of sensation seeking. Using the operant sensation seeking (OSS) paradigm, we quantified preference for sensory stimuli in 120 male and 127 female mice from 62 BXD strains and the C57BL/6J and DBA/2J founder strains. We used relative preference for the active and inactive levers to dissociate preference for sensory stimuli from locomotion and exploration phenotypes. We identified genomic regions on chromosome 4 (155.236-155.742 Mb) and chromosome 13 (72.969-89.423 Mb) associated with distinct behavioral components of OSS. Using publicly available behavioral data and mRNA expression data from brain regions involved in reward processing, we identified (a) genes within these behavioral QTL exhibiting genome-wide significant cis-eQTL and (b) genetic correlations among OSS phenotypes, ethanol phenotypes and mRNA expression. From these analyses, we nominated positional candidates for behavioral QTL associated with distinct OSS phenotypes including Gnb1 and Mef2c. Genetic covariation of Gnb1 expression, preference for sensory stimuli and multiple ethanol phenotypes suggest that heritable variation in Gnb1 expression in reward circuitry partially underlies the widely reported relationship between sensation seeking and substance use.


Assuntos
Consumo de Bebidas Alcoólicas/genética , Locos de Características Quantitativas , Sensação , Animais , Cromossomos/genética , Condicionamento Operante , Comportamento de Procura de Droga , Feminino , Proteínas Heterotriméricas de Ligação ao GTP/genética , Fatores de Transcrição MEF2/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Recompensa , Biologia de Sistemas
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