RESUMEN
Methylation of lysine 4 (K4) within histone H3 has been linked to active transcription and is removed by LSD1 and the JmjC domain-containing proteins by amino-oxidation or hydroxylation, respectively. Here, we describe the deamination catalyzed by Lysyl oxidase-like 2 protein (LOXL2) as an unconventional chemical mechanism for H3K4 modification. Infrared spectroscopy and mass spectrometry analyses demonstrated that recombinant LOXL2 specifically deaminates trimethylated H3K4. Moreover, LOXL2 activity is linked with the transcriptional control of CDH1 gene by regulating H3K4me3 deamination. These results reveal another H3 modification and provide a different mechanism for H3K4 modification.
Asunto(s)
Aminoácido Oxidorreductasas/fisiología , Histonas/metabolismo , Antígenos CD , Cadherinas/genética , Línea Celular Tumoral , Desaminación , Regulación de la Expresión Génica , Humanos , Lisina/metabolismo , MetilaciónRESUMEN
p120-catenin is an E-cadherin-associated protein that modulates E-cadherin function and stability. In response to Wnt3a, p120-catenin is phosphorylated at Ser268 and Ser269, disrupting its interaction with E-cadherin. Here, we describe that Wnt-induced p120-catenin phosphorylation at Ser268 and Ser269 also enhances its binding to the transcriptional factor Kaiso, preventing Kaiso-mediated inhibition of the ß-catenin-Tcf-4 transcriptional complex. Kaiso-mediated repression of this complex is due to its association not only with Tcf-4 but also with ß-catenin. Disruption of Tcf-4-Kaiso and ß-catenin-Kaiso interactions by p120-catenin not only releases Tcf-4 and ß-catenin enabling its mutual association and the formation of the transcriptional complex but also permits Kaiso binding to methylated CpG islands, an interaction that is weakly inhibited by p120-catenin. Consequently, Wnt stimulates Kaiso association to the CDKN2A promoter, which contains CpG sequences, in cells where these sequences are extensively methylated, such as HT-29 M6, an effect accompanied by decreased expression of its gene product. These results indicate that, when released from E-cadherin by Wnt3a-stimulated phosphorylation, p120-catenin controls the activity of the Kaiso transcriptional factor, enhancing its binding to repressed promoters and relieving its inhibition of the ß-catenin-Tcf-4 transcriptional complex.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Cateninas/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Proteína Wnt3A/metabolismo , Cadherinas/metabolismo , Cateninas/genética , Islas de CpG , Genes p16 , Humanos , Metilación , Fosforilación , Regiones Promotoras Genéticas , Unión Proteica , Transducción de Señal/genética , Factor de Transcripción 4 , beta Catenina/metabolismo , Catenina deltaRESUMEN
Snail1 and Zeb1 are E-cadherin-transcriptional repressors induced during epithelial mesenchymal transition (EMT). In this article we have analyzed the factors controlling Zeb1 expression during EMT. In NMuMG cells treated with TGF-ß, Snail1 RNA and protein are induced 1 h after addition of the cytokine preceding Zeb1 up-regulation that requires 6-8 h. Zeb1 gene expression is caused by increased RNA levels but also by enhanced protein stability and is markedly dependent on Snail1 because depletion of this protein prevents Zeb1 protein and RNA up-regulation. In addition to Snail1, depletion of the Twist transcriptional factor retards Zeb1 stimulation by TGF-ß or decreases Zeb1 expression in other cellular models indicating that this factor is also required for Zeb1 expression. Accordingly, Snail1 and Twist cooperate in the induction of Zeb1: co-transfection of both cDNAs is required for the maximal expression of ZEB1 mRNA. Unexpectedly, the expression of Snail1 and Twist shows a mutual dependence although to a different extent; whereas Twist depletion retards Snail1 up-regulation by TGF-ß, Snail1 is necessary for the rapid increase in Twist protein and later up-regulation of Twist1 mRNA induced by the cytokine. Besides this effect on Twist, Snail1 also induces the nuclear translocation of Ets1, another factor required for Zeb1 expression. Both Twist and Ets1 bind to the ZEB1 promoter although to different elements: whereas Ets1 interacts with the proximal promoter, Twist does it with a 700-bp sequence upstream of the transcription start site. These results indicate that Snail1 controls Zeb1 expression at multiple levels and acts cooperatively with Twist in the ZEB1 gene transcription induction.
Asunto(s)
Transición Epitelial-Mesenquimal/fisiología , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/metabolismo , Proteína 1 Relacionada con Twist/metabolismo , Animales , Células Cultivadas , Inmunoprecipitación de Cromatina , Transición Epitelial-Mesenquimal/genética , Proteínas de Homeodominio/genética , Humanos , Ratones , Unión Proteica/efectos de los fármacos , Proteína Proto-Oncogénica c-ets-1/genética , Proteína Proto-Oncogénica c-ets-1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/farmacología , Proteína 1 Relacionada con Twist/genética , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
Analysis of infrared polarized absorbance spectra and linear dichroism spectra of reconstituted melibiose permease from Escherichia coli shows that the oriented structures correspond mainly to tilted transmembrane alpha-helices, forming an average angle of approximately 26 degrees with the membrane normal in substrate-free medium. Examination of the deconvoluted linear dichroism spectra in H(2)O and D(2)O makes apparent two populations of alpha-helices differing by their tilt angle (helix types I and II). Moreover, the average helical tilt angle significantly varies upon substrate binding: it is increased upon Na(+) binding, whereas it decreases upon subsequent melibiose binding in the presence of Na(+). In contrast, melibiose binding in the presence of H(+) causes virtually no change in the average tilt angle. The data also suggest that the two helix populations change their tilting and H/D exchange level in different ways depending on the bound substrate(s). Notably, cation binding essentially influences type I helices, whereas melibiose binding modifies the tilting of both helix populations.
Asunto(s)
Escherichia coli/enzimología , Melibiosa/metabolismo , Simportadores/química , Simportadores/metabolismo , Dicroismo Circular , Medición de Intercambio de Deuterio , Unión Proteica , Estructura Secundaria de Proteína , Sodio/química , Sodio/farmacología , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
The product of Snail1 gene is a transcriptional repressor of E-cadherin expression and an inductor of the epithelial-mesenchymal transition in several epithelial tumour cell lines. Transcription of Snail1 is induced when epithelial cells are forced to acquire a mesenchymal phenotype. In this work we demonstrate that Snail1 protein limits its own expression: Snail1 binds to an E-box present in its promoter (at -146 with respect to the transcription start) and represses its activity. Therefore, mutation of the E-box increases Snail1 transcription in epithelial and mesenchymal cells. Evidence of binding of ectopic or endogenous Snail1 to its own promoter was obtained by chromatin immunoprecipitation (ChIP) experiments. Studies performed expressing different forms of Snail1 under the control of its own promoter demonstrate that disruption of the regulatory loop increases the cellular levels of Snail protein. These results indicate that expression of Snail1 gene can be regulated by its product and evidence the existence of a fine-tuning feed-back mechanism of regulation of Snail1 transcription.
Asunto(s)
Elementos E-Box , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Factores de Transcripción/genética , Animales , Sitios de Unión , Línea Celular , Regulación hacia Abajo , Homeostasis , Humanos , Ratones , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción de la Familia Snail , Factores de Transcripción/metabolismoRESUMEN
Methylation of histone H3 lysine 4 is linked to active transcription and can be removed by LSD1 or the JmjC domain-containing proteins by amino-oxidation or hydroxylation, respectively. Here we describe that its deamination can be catalyzed by lysyl oxidase-like 2 protein (LOXL2), presenting an unconventional chemical mechanism for H3K4 modification. Infrared spectroscopy and mass spectrometry analyses demonstrated that recombinant LOXL2 specifically deaminates trimethylated H3K4. Moreover, by regulating H3K4me3 deamination, LOXL2 activity is linked with the transcriptional control of the CDH1 gene. These results reveal the existence of further H3 modification as well as a novel mechanism for H3K4me3 demethylation. DATABASE: The GEO accession number for the data referred to this paper is GSE35600.
Asunto(s)
Aminoácido Oxidorreductasas/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Aminoácido Oxidorreductasas/genética , Antígenos CD , Western Blotting , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Metilación , Oxidación-Reducción , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrofotometría InfrarrojaRESUMEN
Notch activation in aortic endothelial cells (ECs) takes place at embryonic stages during cardiac valve formation and induces endothelial-to-mesenchymal transition (EndMT). Using aortic ECs, we show here that active Notch expression promotes EndMT, resulting in downregulation of vascular endothelial cadherin (VE-cadherin) and upregulation of mesenchymal genes such as those for fibronectin and Snail1/2. In these cells, transforming growth factor ß1 exacerbates Notch effects by increasing Snail1 and fibronectin activation. When Notch-downstream pathways were analyzed, we detected an increase in glycogen synthase kinase 3ß (GSK-3ß) phosphorylation and inactivation that facilitates Snail1 nuclear retention and protein stabilization. However, the total activity of Akt was downregulated. The discrepancy between Akt activity and GSK-3ß phosphorylation is explained by a Notch-induced switch in the Akt isoforms, whereby Akt1, the predominant isoform expressed in ECs, is decreased and Akt2 transcription is upregulated. Mechanistically, Akt2 induction requires the stimulation of the ß-catenin/TCF4 transcriptional complex, which activates the Akt2 promoter. Active, phosphorylated Akt2 translocates to the nucleus in Notch-expressing cells, resulting in GSK-3ß inactivation in this compartment. Akt2, but not Akt1, colocalizes in the nucleus with lamin B in the nuclear envelope. In addition to promoting GSK-3ß inactivation, Notch downregulates Forkhead box O1 (FoxO1), another Akt2 nuclear substrate. Moreover, Notch protects ECs from oxidative stress-induced apoptosis through an Akt2- and Snail1-dependent mechanism.
Asunto(s)
Muerte Celular , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Notch/metabolismo , Factores de Transcripción/genética , Animales , Aorta/citología , Línea Celular , Células Endoteliales/citología , Células Endoteliales/metabolismo , Transición Epitelial-Mesenquimal , Regulación de la Expresión Génica , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Células HEK293 , Humanos , Ratones , Estrés Oxidativo , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-akt/análisis , Proteínas Proto-Oncogénicas c-akt/genética , Factores de Transcripción de la Familia Snail , Porcinos , Factores de Transcripción/análisis , Factores de Transcripción/metabolismo , Regulación hacia Arriba , beta Catenina/metabolismoRESUMEN
The product of the Snail1 gene is a transcriptional repressor required for triggering the epithelial-to-mesenchymal transition. Furthermore, ectopic expression of Snail1 in epithelial cells promotes resistance to apoptosis. In this study, we demonstrate that this resistance to gamma radiation-induced apoptosis caused by Snail1 is associated with the inhibition of PTEN phosphatase. In MDCK cells, mRNA levels of the p53 target gene PTEN are induced after gamma radiation; the transfection of Snail1 prevents this up-regulation. Decreased mRNA levels of PTEN were also detected in RWP-1 cells after the ectopic expression of this transcriptional factor. Snail1 represses and associates to the PTEN promoter as detected both by the electrophoretic mobility shift assay and chromatin immunoprecipitation experiments performed with either endogenous or ectopic Snail1. The binding of Snail1 to the PTEN promoter increases after gamma radiation, correlating with the stabilization of Snail1 protein, and prevents the association of p53 to the PTEN promoter. These results stress the critical role of Snail1 in the control of apoptosis and demonstrate the regulation of PTEN phosphatase by this transcriptional repressor.
Asunto(s)
Apoptosis/efectos de la radiación , Rayos gamma , Regulación de la Expresión Génica , Fosfohidrolasa PTEN/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Daño del ADN , ADN Complementario , Perros , Fase G2 , Genes Reporteros , Humanos , Luciferasas de Luciérnaga/análisis , Luciferasas de Luciérnaga/metabolismo , Luciferasas de Renilla/análisis , Luciferasas de Renilla/metabolismo , Sustancias Luminiscentes/metabolismo , Neoplasias Pancreáticas/patología , Regiones Promotoras Genéticas , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Puromicina/farmacología , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Selección Genética , Factores de Transcripción de la Familia Snail , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/farmacología , TransfecciónRESUMEN
The transcriptional factor Snail1 is a repressor of E-cadherin (CDH1) gene expression essential for triggering epithelial-mesenchymal transition. Snail1 represses CDH1, directly binding its promoter and inducing the synthesis of the Zeb1 repressor. In this article, we show that repression of CDH1 by Snail1, but not by Zeb1, is dependent on the activity of Polycomb repressive complex 2 (PRC2). Embryonic stem (ES) cells null for Suz12, one of the components of PRC2, show higher levels of Cdh1 mRNA than control ES cells. In tumor cells, interference of PRC2 activity prevents the ability of Snail1 to downregulate CDH1 and partially derepresses CDH1. Chromatin immunoprecipitation assays demonstrated that Snail1 increases the binding of Suz12 to the CDH1 promoter and the trimethylation of lysine 27 in histone H3. Moreover, Snail1 interacts with Suz12 and Ezh2, as shown by coimmunoprecipitation experiments. In conclusion, these results demonstrate that Snail1 recruits PRC2 to the CDH1 promoter and requires the activity of this complex to repress E-cadherin expression.
Asunto(s)
Cadherinas/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Cadherinas/genética , Línea Celular , Línea Celular Tumoral , Regulación hacia Abajo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Regiones Promotoras Genéticas/genética , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción de la Familia Snail , Factores de Transcripción/químicaRESUMEN
The accessibility of Escherichia coli melibiose permease to aqueous solvent was studied following hydrogen-deuterium exchange kinetics monitored by attenuated total reflection-Fourier transform infrared spectroscopy under four distinct conditions where MelB forms different complexes with its substrates (H(+), Na(+), melibiose). Analysis of the amide II band upon (2)H(2)O exposure discloses a significant sugar protection of the protein against aqueous solvent, resulting in an 8% less exchange of the corresponding H(+)*melibiose*MelB complex compared with the protein in the absence of sugar. Investigation of the amide I exchange reveals clear substrate effects on beta-sheet accessibility, with the complex H(+)*melibiose*MelB being the most protected state against exchange, followed by Na(+)*melibiose*MelB. Although of smaller magnitude, similar changes in alpha-helices plus non-ordered structures are detected. Finally, no differences are observed when analyzing reverse turn structures. The results suggest that sugar binding induces a remarkable compactness of the carrier's structure, affecting mainly beta-sheet domains of the transporter, which, according to secondary structure predictions, may include cytoplasmic loops 4-5 and 10-11. A possible catalytic role of these two loops in the functioning of MelB is hypothesized.