RESUMEN
Estrogen receptor α (ERα) mediates the effects of 17ß-estradiol (E2) in normal mammary gland, and it is a key participant in breast cancer tumor development. ERα transactivation activity is mediated by the synergistic interaction of two domains designated AF1 and AF2. The function of AF2 is to recruit coactivator and corepressor proteins that allow ERα to oscillate between the roles of transcriptional activator and repressor. In contrast, the mechanism responsible for AF-1 transcriptional activity is not completely understood. In this study, we identified tristetraproline (TTP) as a novel ERα-associated protein. TTP expression in MCF7 cells repressed ERα transactivation and reduced MCF7 cell proliferation and the ability of the cells to form tumors in a mouse model. We show that TTP transcriptional activity is mediated through its recruitment to the promoter region of ERα target genes and its interaction with histone deacetylases, in particular with HDAC1. TTP expression attenuates the coactivating activity of SRC-1, suggesting that exchange between TTP and other coactivators may play an important role in fine-tuning ERα transactivation. These results indicate that TTP acts as a bona fide ERα corepressor and suggest that this protein may be a contributing factor in the development of E2-dependent tumors in breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Receptor alfa de Estrógeno/metabolismo , Tristetraprolina/metabolismo , Animales , Neoplasias de la Mama/genética , Proliferación Celular , Proteínas Co-Represoras/metabolismo , Estradiol/metabolismo , Estradiol/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Histona Desacetilasas/metabolismo , Humanos , Células MCF-7 , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Transcripción Genética/fisiologíaRESUMEN
The transcriptional co-repressor C-terminal binding protein (CtBP) interacts with a number of repressor proteins and chromatin modifying enzymes. How the biochemical properties including binding of dinucleotide, oligomerization, and dehydrogenase domains of CtBP1 direct the assembly of a functional co-repressor to influence gene expression is not well understood. In the current study we demonstrate that CtBP1 assembles into a tetramer in a NAD(H)-dependent manner, proceeding through a dimeric intermediate. We find that NAD-dependent oligomerization correlates with NAD(+) binding affinity and that the carboxyl terminus is required for assembly of a dimer of dimers. Mutant CtBP1 proteins that abrogate dinucleotide-binding retain wild type affinity for the PXDLS motif, but do not self-associate either in vitro or in vivo. CtBP1 proteins with mutations in the dehydrogenase domain still retain the ability to self-associate and bind target proteins. Both co-immunoprecipitation and mammalian two-hybrid experiments demonstrate that CtBP1 self-association occurs within the nucleus, and depends on dinucleotide binding. Repression of transcription does not depend on dinucleotide binding or an intact dehydrogenase domain, but rather depends on the amino-terminal domain that recruits PXDLS containing targets. We show that tryptophan 318 (Trp(318)) is a critical residue for tetramer assembly and likely functions as a switch for effective dimerization following NAD(+) binding. These results suggest that dinucleotide binding permits CtBP1 to form an intranuclear homodimer through a Trp(318) switch, creating a nucleation site for multimerization through the C-terminal domain for tetramerization to form an effective repression complex.
Asunto(s)
Oxidorreductasas de Alcohol/química , Proteínas de Unión al ADN/química , NAD/metabolismo , Triptófano/química , Secuencias de Aminoácidos , Sitios de Unión , Línea Celular Tumoral , Cromatografía en Gel , Reactivos de Enlaces Cruzados/farmacología , Transferencia Resonante de Energía de Fluorescencia , Regulación Neoplásica de la Expresión Génica , Humanos , Mutagénesis , Nucleótidos/química , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Técnicas del Sistema de Dos HíbridosRESUMEN
Bone has remarkable regenerative capacity, but this ability diminishes during aging. Histone deacetylase inhibitors (HDIs) promote terminal osteoblast differentiation and extracellular matrix production in culture. The epigenetic events altered by HDIs in osteoblasts may hold clues for the development of new anabolic treatments for osteoporosis and other conditions of low bone mass. To assess how HDIs affect the epigenome of committed osteoblasts, MC3T3 cells were treated with suberoylanilide hydroxamic acid (SAHA) and subjected to microarray gene expression profiling and high-throughput ChIP-Seq analysis. As expected, SAHA induced differentiation and matrix calcification of osteoblasts in vitro. ChIP-Seq analysis revealed that SAHA increased histone H4 acetylation genome-wide and in differentially regulated genes, except for the 500 bp upstream of transcriptional start sites. Pathway analysis indicated that SAHA increased the expression of insulin signaling modulators, including Slc9a3r1. SAHA decreased phosphorylation of insulin receptor ß, Akt, and the Akt substrate FoxO1, resulting in FoxO1 stabilization. Thus, SAHA induces genome-wide H4 acetylation and modulates the insulin/Akt/FoxO1 signaling axis, whereas it promotes terminal osteoblast differentiation in vitro.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Histonas/genética , Osteoblastos/citología , Osteoblastos/enzimología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Acetilación/efectos de los fármacos , Animales , Proteína Axina/genética , Diferenciación Celular/genética , Línea Celular , Inmunoprecipitación de Cromatina , Perfilación de la Expresión Génica , Genoma/genética , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Ácidos Hidroxámicos/farmacología , Insulina/metabolismo , Ratones , Osteoblastos/efectos de los fármacos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , VorinostatRESUMEN
Secretory phospholipase A2 group IIa (PLA2g2a) is associated with inflammation, hyperlipidemia, and atherogenesis. Transcription of the PLA2g2a gene is induced by multiple cytokines. Here, we report the surprising observation that thyroid hormone (T3) inhibited PLA2g2a gene expression in human and rat hepatocytes as well as in rat liver. Moreover, T3 reduced the cytokine-mediated induction of PLA2g2a, suggesting that the thyroid status may modulate aspects of the inflammatory response. In an effort to dissect the mechanism of repression by T3, we cloned the PLA2g2a gene and identified a negative T3 response element in the promoter. This T3 receptor (TRß)-binding site differed considerably from consensus T3 stimulatory elements. Using in vitro and in vivo binding assays, we found that TRß bound directly to the PLA2g2a promoter as a heterodimer with the retinoid X receptor. Knockdown of nuclear corepressor or silencing mediator for retinoid and thyroid receptors by siRNA blocked the T3 inhibition of PLA2g2a. Using chromatin immunoprecipitation assays, we showed that nuclear corepressor and silencing mediator for retinoid and thyroid receptors were associated with the PLA2g2a gene in the presence of T3. In contrast with the established role of T3 to promote coactivator association with TRß, our experiments demonstrate a novel inverse recruitment mechanism in which liganded TRß recruits corepressors to inhibit PLA2g2a expression.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Fosfolipasas A2 Grupo II/biosíntesis , Hepatocitos/metabolismo , Proteínas Represoras/metabolismo , Elementos de Respuesta/fisiología , Receptores beta de Hormona Tiroidea/metabolismo , Transcripción Genética/fisiología , Triyodotironina/metabolismo , Animales , Fosfolipasas A2 Grupo II/genética , Células Hep G2 , Hepatocitos/citología , Humanos , Hígado/citología , Hígado/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Proteínas Represoras/genética , Receptores X Retinoide/genética , Receptores X Retinoide/metabolismo , Receptores beta de Hormona Tiroidea/genética , Triyodotironina/genéticaRESUMEN
Histone deacetylase 1 (HDAC1) and HDAC2 are components of corepressor complexes that are involved in chromatin remodeling and regulation of gene expression by regulating dynamic protein acetylation. HDAC1 and -2 form homo- and heterodimers, and their activity is dependent upon dimer formation. Phosphorylation of HDAC1 and/or HDAC2 in interphase cells is required for the formation of HDAC corepressor complexes. In this study, we show that during mitosis, HDAC2 and, to a lesser extent, HDAC1 phosphorylation levels dramatically increase. When HDAC1 and -2 are displaced from the chromosome during metaphase, they dissociate from each other, but each enzyme remains in association with components of the HDAC corepressor complexes Sin3, NuRD, and CoREST as homodimers. Enzyme inhibition studies and mutational analyses demonstrated that protein kinase CK2-catalyzed phosphorylation of HDAC1 and -2 is crucial for the dissociation of these two enzymes. These results suggest that corepressor complexes, including HDAC1 or HDAC2 homodimers, might target different cellular proteins during mitosis.