RESUMEN
Proper regulation of epigenetic states of chromatin is crucial to achieve tissue-specific gene expression during embryogenesis. The lung-specific gene products, surfactant proteins B (SP-B) and C (SP-C), are synthesized in alveolar epithelial cells and prevent alveolar collapse. Epigenetic regulation of these surfactant proteins, however, remains unknown. Here we report that MCRIP1, a regulator of the CtBP transcriptional co-repressor, promotes the expression of SP-B and SP-C by preventing CtBP-mediated epigenetic gene silencing. Homozygous deficiency of Mcrip1 in mice causes fatal respiratory distress due to abnormal transcriptional repression of these surfactant proteins. We found that MCRIP1 interferes with interactions of CtBP with the lung-enriched transcriptional repressors, Foxp1 and Foxp2, thereby preventing the recruitment of the CtBP co-repressor complex to the SP-B and SP-C promoters and maintaining them in an active chromatin state. Our findings reveal a molecular mechanism by which cells prevent inadvertent gene silencing to ensure tissue-specific gene expression during organogenesis.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Proteínas Co-Represoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Pulmón/metabolismo , Proteína B Asociada a Surfactante Pulmonar/metabolismo , Proteína C Asociada a Surfactante Pulmonar/metabolismo , Animales , Línea Celular Tumoral , Epitelio/crecimiento & desarrollo , Epitelio/metabolismo , Epitelio/patología , Factores de Transcripción Forkhead/metabolismo , Expresión Génica , Silenciador del Gen , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Pulmón/crecimiento & desarrollo , Pulmón/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Represoras/metabolismo , Insuficiencia Respiratoria/metabolismo , Insuficiencia Respiratoria/patologíaRESUMEN
The ERK pathway not only upregulates growth-promoting genes, but also downregulates anti-proliferative and tumor-suppressive genes. In particular, ERK signaling contributes to repression of the E-cadherin gene during epithelial-mesenchymal transition (EMT). The CtBP transcriptional co-repressor is also involved in gene silencing of E-cadherin. However, the functional relationship between ERK signaling and CtBP is unknown. Here, we identified an ERK substrate, designated MCRIP1, which bridges ERK signaling and CtBP-mediated gene silencing. CtBP is recruited to promoter elements of target genes by interacting with the DNA-binding transcriptional repressor ZEB1. We found that MCRIP1 binds to CtBP, thereby competitively inhibiting CtBP-ZEB1 interaction. When phosphorylated by ERK, MCRIP1 dissociates from CtBP, allowing CtBP to interact with ZEB1. In this manner, the CtBP co-repressor complex is recruited to, and silences, the E-cadherin promoter by inducing chromatin modifications. Our findings reveal a molecular mechanism underlying ERK-induced epigenetic gene silencing during EMT and its dysregulation in cancer.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Cadherinas/genética , Proteínas de Unión al ADN/genética , Epigénesis Genética , Transición Epitelial-Mesenquimal/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Oxidorreductasas de Alcohol/metabolismo , Secuencia de Aminoácidos , Cadherinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Silenciador del Gen , Células HEK293 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Datos de Secuencia Molecular , Fosforilación , Plásmidos/química , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Técnicas del Sistema de Dos Híbridos , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
Hypoxia-inducible factor 1 (HIF-1) plays a key role in the cellular adaptation to hypoxia. Although HIF-1 is usually strongly suppressed by posttranslational mechanisms during normoxia, HIF-1 is active and enhances tumorigenicity in malignant tumor cells that express the membrane protease MT1-MMP. The cytoplasmic tail of MT1-MMP, which can bind a HIF-1 suppressor protein called factor inhibiting HIF-1 (FIH-1), promotes inhibition of FIH-1 by Mint3 during normoxia. To explore possible links between HIF-1 activation by MT1-MMP/Mint3 and tumor growth signals, we surveyed a panel of 252 signaling inhibitors. The mTOR inhibitor rapamycin was identified as a possible modulator, and it inhibited the mTOR-dependent phosphorylation of Mint3 that is required for FIH-1 inhibition. A mutant Mint3 protein that cannot be phosphorylated exhibited a reduced ability to inhibit FIH-1 and promoted tumor formation in mice. These data suggest a novel molecular link between the important hub proteins MT1-MMP and mTOR that contributes to tumor malignancy.
Asunto(s)
Factor 1 Inducible por Hipoxia/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Antibióticos Antineoplásicos/farmacología , Carbazoles/farmacología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Factor 1 Inducible por Hipoxia/genética , Immunoblotting , Metaloproteinasa 14 de la Matriz/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Mutación , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Fosforilación/efectos de los fármacos , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/genética , Trasplante HeterólogoRESUMEN
Oxygen is a vital requirement for multi-cellular organisms to generate energy and cells have developed multiple compensatory mechanisms to adapt to stressful hypoxic conditions. Such adaptive mechanisms are intricately interconnected with other signaling pathways that regulate cellular functions such as cell growth. However, our understanding of the overall system governing the cellular response to the availability of oxygen remains limited. To identify new genes involved in the response to hypoxic stress, we have performed a genome-wide gene knockdown analysis in human lung carcinoma PC8 cells using an shRNA library carried by a lentiviral vector. The knockdown analysis was performed under both normoxic and hypoxic conditions to identify shRNA sequences enriched or lost in the resulting selected cell populations. Consequently, we identified 56 candidate genes that might contribute to the cellular response to hypoxia. Subsequent individual knockdown of each gene demonstrated that 13 of these have a significant effect upon oxygen-sensitive cell growth. The identification of BCL2L1, which encodes a Bcl-2 family protein that plays a role in cell survival by preventing apoptosis, validates the successful design of our screen. The other selected genes have not previously been directly implicated in the cellular response to hypoxia. Interestingly, hypoxia did not directly enhance the expression of any of the identified genes, suggesting that we have identified a new class of genes that have been missed by conventional gene expression analyses to identify hypoxia response genes. Thus, our genetic screening method using a genome-wide shRNA library and the newly-identified genes represent useful tools to analyze the cellular systems that respond to hypoxic stress.