RESUMEN
The distribution of the transcription machinery among different sub-nuclear domains raises the question on how the architecture of the nucleus modulates the transcriptional response. Here, we used fluorescence fluctuation analyses to quantitatively explore the organization of the glucocorticoid receptor (GR) in the interphase nucleus of living cells. We found that this ligand-activated transcription factor diffuses within the nucleus and dynamically interacts with bodies enriched in the coregulator NCoA-2, DNA-dependent foci and chromatin targets. The distribution of the receptor among the nuclear compartments depends on NCoA-2 and the conformation of the receptor as assessed with synthetic ligands and GR mutants with impaired transcriptional abilities. Our results suggest that the partition of the receptor in different nuclear reservoirs ultimately regulates the concentration of receptor available for the interaction with specific targets, and thus has an impact on transcription regulation.
Asunto(s)
Núcleo Celular/metabolismo , Cromatina/metabolismo , Regulación de la Expresión Génica , Coactivador 2 del Receptor Nuclear/metabolismo , Receptores de Glucocorticoides/metabolismo , Transcripción Genética , Sitios de Unión , Núcleo Celular/genética , Células Cultivadas , Cromatina/genética , Humanos , Coactivador 2 del Receptor Nuclear/genética , Regiones Promotoras Genéticas , Unión Proteica , Receptores de Glucocorticoides/genética , Activación TranscripcionalRESUMEN
Thyroid hormones (TH) regulate mammary function. Hypothyroidism (HypoT) has deleterious effects on lactation, litter growth and survival. We analyzed the effect of chronic 6-propyl-2-thiouracil (PTU)-induced HypoT in the expression of nuclear receptors, co-regulators and oxytocin receptor (OTR) on lactation (L) days 2, 7 and 14. TH receptors (TRs) were increased on L7 at mRNA and protein levels, except TRα protein, that fell on L14. HypoT decreased TRα2 mRNA on L7 and TRα1 protein on L2, while TRß1 protein increased on L14. HypoT increased estrogen receptor ß (ERß) mRNA on L7 but decreased its protein levels on L14. Progesterone receptor A (PRA) mRNA decreased from L2 to L14 while PRB increased, and at protein levels PRA levels showed a nadir on L7, while PRB peaked. HypoT decreased PRA mRNA and protein and increased PRB mRNA at L14. Nuclear receptor co-activator (NCOA) 1 and RXRα mRNA showed an opposite pattern to the TRs, while NCOA2 increased at L14; HypoT blocked the variations in NCOA1 and NCOA2. HypoT increased NCOR1 on L2 and decreased OTR at L2 and circulating estradiol and NCOR2 at L14. In controls the most notable changes occurred on L7, suggesting it is a key inflection point in mammary metabolism. The low levels of TRα1, NCOA1 and OTR, and increased NCOR1 produced by HypoT on L2 may hinder the mammary ability to achieve normal milk synthesis and ejection, leading to defective lactation. Later on, altered ER and PR expression may impair further mammary function.
Asunto(s)
Expresión Génica , Hipotiroidismo/metabolismo , Lactancia , Receptores de Progesterona/metabolismo , Animales , Femenino , Hipotiroidismo/inducido químicamente , Glándulas Mamarias Animales/metabolismo , Co-Represor 1 de Receptor Nuclear/genética , Co-Represor 1 de Receptor Nuclear/metabolismo , Coactivador 1 de Receptor Nuclear/genética , Coactivador 1 de Receptor Nuclear/metabolismo , Coactivador 2 del Receptor Nuclear/genética , Coactivador 2 del Receptor Nuclear/metabolismo , Propiltiouracilo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas Wistar , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Receptores de Oxitocina/genética , Receptores de Oxitocina/metabolismo , Receptores de Progesterona/genética , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismo , Receptor alfa X Retinoide/genética , Receptor alfa X Retinoide/metabolismoRESUMEN
Reproductive traits are an important component of the economic selection index for beef cattle in the tropics. Phenotypic expression of these traits occurs late because they are measured when the animals reach reproductive age. Association studies using high-density markers have been conducted to identify genes that influence certain traits. The identification of causal mutations in these genes permits the inclusion of these single nucleotide polymorphisms (SNPs) in customised DNA chips to increase efficiency and validity. Therefore, the aim of the present study was to detect causal mutations in the TOX and NCOA2 genes, previously identified by genome-wide association studies of zebu cattle. DNA was extracted from 385 Nellore females and polymorphisms were investigated by polymerase chain reaction sequencing. Five polymorphisms were detected in the NCOA2 gene and four in the TOX gene that were associated with reproductive traits. Analysis of variance showed that SNP 1718 in the NCOA2 gene was significant for early pregnancy probability (P=0.02) and age at first calving (P=0.03), and SNP 2038 in the same gene was significant for days to calving (P=0.03). Studies investigating polymorphisms in other regions of the gene and in other genes should be conducted to identify causal mutations.
Asunto(s)
Proteínas del Grupo de Alta Movilidad/genética , Coactivador 2 del Receptor Nuclear/genética , Polimorfismo de Nucleótido Simple , Reproducción/genética , Alelos , Animales , Bovinos , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Genotipo , Mutación , Fenotipo , EmbarazoRESUMEN
The antagonism exerted by melatonin on the glucocorticoid response has been well established, being strongly dependent on the cellular context. Previously, we found that melatonin inhibits glucocorticoid receptor (GR) dissociation from the chaperone hetero-complex and nuclear translocation on mouse thymocytes. Here, by performing confocal fluorescence microscopy and the Number and Brightness assay we show that in newborn hamster kidney cells (BHK21) melatonin neither affects GR nuclear translocation nor GR homodimerization. Instead, co-immunoprecipitation studies suggest that physiological concentrations of melatonin impair GR interaction with the transcriptional intermediary factor 2 (TIF2). This melatonin effect was not blocked by the MT(1)/MT(2) receptor antagonist luzindole. Curiously, luzindole behaved as an antiglucocorticoid per se by impairing the glucocorticoid-dependent MMTV-driven gene expression affecting neither GR translocation nor GR-TIF2 interaction.