RESUMO
Short- and long-scales intra- and inter-chromosomal interactions are linked to gene transcription, but the molecular events underlying these structures and how they affect cell fate decision during embryonic development are poorly understood. One of the first embryonic cell fate decisions (that is, mesendoderm determination) is driven by the POU factor OCT4, acting in concert with the high-mobility group genes Sox-2 and Sox-17. Here we report a chromatin-remodelling mechanism and enhancer function that mediate cell fate switching. OCT4 alters the higher-order chromatin structure at both Sox-2 and Sox-17 loci. OCT4 titrates out cohesin and switches the Sox-17 enhancer from a locked (within an inter-chromosomal Sox-2 enhancer/CCCTC-binding factor CTCF/cohesin loop) to an active (within an intra-chromosomal Sox-17 promoter/enhancer/cohesin loop) state. SALL4 concomitantly mobilizes the polycomb complexes at the Soxs loci. Thus, OCT4/SALL4-driven cohesin- and polycombs-mediated changes in higher-order chromatin structure mediate instruction of early cell fate in embryonic cells.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Embrião de Mamíferos/metabolismo , Coração/embriologia , Células-Tronco Embrionárias Humanas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOX/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fator de Ligação a CCCTC , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Humanos , Camundongos , Proteínas de Neoplasias , Células-Tronco Pluripotentes , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXF/genética , Fatores de Transcrição SOXF/metabolismo , Fatores de Transcrição/metabolismo , CoesinasRESUMO
RATIONALE: The sympathetic nervous system plays a fundamental role in the regulation of myocardial function. During chronic pressure overload, overactivation of the sympathetic nervous system induces the release of catecholamines, which activate ß-adrenergic receptors in cardiomyocytes and lead to increased heart rate and cardiac contractility. However, chronic stimulation of ß-adrenergic receptors leads to impaired cardiac function, and ß-blockers are widely used as therapeutic agents for the treatment of cardiac disease. MicroRNA-133 (miR-133) is highly expressed in the myocardium and is involved in controlling cardiac function through regulation of messenger RNA translation/stability. OBJECTIVE: To determine whether miR-133 affects ß-adrenergic receptor signaling during progression to heart failure. METHODS AND RESULTS: Based on bioinformatic analysis, ß1-adrenergic receptor (ß1AR) and other components of the ß1AR signal transduction cascade, including adenylate cyclase VI and the catalytic subunit of the cAMP-dependent protein kinase A, were predicted as direct targets of miR-133 and subsequently validated by experimental studies. Consistently, cAMP accumulation and activation of downstream targets were repressed by miR-133 overexpression in both neonatal and adult cardiomyocytes following selective ß1AR stimulation. Furthermore, gain-of-function and loss-of-function studies of miR-133 revealed its role in counteracting the deleterious apoptotic effects caused by chronic ß1AR stimulation. This was confirmed in vivo using a novel cardiac-specific TetON-miR-133 inducible transgenic mouse model. When subjected to transaortic constriction, TetON-miR-133 inducible transgenic mice maintained cardiac performance and showed attenuated apoptosis and reduced fibrosis compared with control mice. CONCLUSIONS: miR-133 controls multiple components of the ß1AR transduction cascade and is cardioprotective during heart failure.
Assuntos
AMP Cíclico/fisiologia , MicroRNAs/fisiologia , Miócitos Cardíacos/fisiologia , Receptores Adrenérgicos beta 1/fisiologia , Sistemas do Segundo Mensageiro/fisiologia , Regiões 3' não Traduzidas/fisiologia , Adenilil Ciclases/fisiologia , Animais , Apoptose , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Progressão da Doença , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Masculino , Metoprolol/farmacologia , Metoprolol/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/genéticaRESUMO
MicroRNAs play an important role in myocardial diseases. MiR-133a regulates cardiac hypertrophy, while miR-29b is involved in cardiac fibrosis. The aim of this study was to evaluate whether miR-133a and miR-29b play a role in myocardial fibrosis caused by Angiotensin II (Ang II)-dependent hypertension. Sprague-Dawley rats were treated for 4 weeks with Ang II (200 ng/kg/min) or Ang II + irbesartan (50 mg/kg/day in drinking water), or saline by osmotic minipumps. At the end of the experimental period, cardiac miR-133a and miR-29b expression was measured by real-time PCR, and myocardial fibrosis was evaluated by morphometric analysis. A computer-based prediction algorithm led to the identification of collagen 1a1 (Col1A1) as a putative target of miR-133a. A reporter plasmid bearing the 3'-untranslated regions (UTRs) of Col1A1 mRNA was constructed and luciferase assay was performed. MiR-133a suppressed the activity of luciferase when the reporter gene was linked to a 3'-UTR segment of Col1A1 (P < 0.01). Mutation of miR-133a binding sites in the 3'-UTR of Col1A1 mRNA abolished miR-133a-mediated repression of reporter gene activity, showing that Col1A1 is a real target of miR-133a. In vivo, Ang II caused an increase in systolic blood pressure (P < 0.0001, tail cuff) and myocardial fibrosis in presence of a decrease in miR-133a (P < 0.01) and miR-29b (P < 0.01), and an increase in Col1A1 expression (P < 0.01). These effects were abolished by Ang II administration + irbesartan. These data demonstrate a relationship between miR-133a and Col1A1, suggesting that myocardial fibrosis occurring in Ang II-dependent hypertension is regulated by the down-regulation of miR-133a and miR-29b through the modulation of Col1A1 expression.