RESUMEN
Twelve human THAP proteins share the THAP domain, an evolutionary conserved zinc-finger DNA-binding domain. Studies of different THAP proteins have indicated roles in gene transcription, cell proliferation and development. We have analyzed this protein family, focusing on THAP7 and THAP11. We show that human THAP proteins possess differing homo- and heterodimer formation properties and interaction abilities with the transcriptional co-regulator HCF-1. HEK-293 cells lacking THAP7 were viable but proliferated more slowly. In contrast, HEK-293 cells were very sensitive to THAP11 alteration. Nevertheless, HEK-293 cells bearing a THAP11 mutation identified in a patient suffering from cobalamin disorder (THAP11F80L) were viable although proliferated more slowly. Cobalamin disorder is an inborn vitamin deficiency characterized by neurodevelopmental abnormalities, most often owing to biallelic mutations in the MMACHC gene, whose gene product MMACHC is a key enzyme in the cobalamin (vitamin B12) metabolic pathway. We show that THAP11F80L selectively affected promoter binding by THAP11, having more deleterious effects on a subset of THAP11 targets, and resulting in altered patterns of gene expression. In particular, THAP11F80L exhibited a strong effect on association with the MMACHC promoter and led to a decrease in MMACHC gene transcription, suggesting that the THAP11F80L mutation is directly responsible for the observed cobalamin disorder.
Asunto(s)
Oxidorreductasas/genética , Proteínas Represoras/genética , Deficiencia de Vitamina B 12/genética , Vitamina B 12/genética , Línea Celular , Proliferación Celular/genética , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/genética , Células HEK293 , Factor C1 de la Célula Huésped/genética , Humanos , Redes y Vías Metabólicas/genética , Mutación/genética , Regiones Promotoras Genéticas , Unión Proteica/genética , Vitamina B 12/metabolismo , Deficiencia de Vitamina B 12/metabolismo , Deficiencia de Vitamina B 12/patologíaRESUMEN
RNA polymerase II (Pol II) small nuclear RNA (snRNA) promoters and type 3 Pol III promoters have highly similar structures; both contain an interchangeable enhancer and "proximal sequence element" (PSE), which recruits the SNAP complex (SNAPc). The main distinguishing feature is the presence, in the type 3 promoters only, of a TATA box, which determines Pol III specificity. To understand the mechanism by which the absence or presence of a TATA box results in specific Pol recruitment, we examined how SNAPc and general transcription factors required for Pol II or Pol III transcription of SNAPc-dependent genes (i.e., TATA-box-binding protein [TBP], TFIIB, and TFIIA for Pol II transcription and TBP and BRF2 for Pol III transcription) assemble to ensure specific Pol recruitment. TFIIB and BRF2 could each, in a mutually exclusive fashion, be recruited to SNAPc. In contrast, TBP-TFIIB and TBP-BRF2 complexes were not recruited unless a TATA box was present, which allowed selective and efficient recruitment of the TBP-BRF2 complex. Thus, TBP both prevented BRF2 recruitment to Pol II promoters and enhanced BRF2 recruitment to Pol III promoters. On Pol II promoters, TBP recruitment was separate from TFIIB recruitment and enhanced by TFIIA. Our results provide a model for specific Pol recruitment at SNAPc-dependent promoters.
Asunto(s)
Regiones Promotoras Genéticas , ARN Polimerasa III/metabolismo , ARN Polimerasa II/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Células HEK293 , Humanos , Mutación , Unión Proteica , Dominios Proteicos , Transporte de Proteínas , TATA Box/genética , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIIB/metabolismo , Factores de Transcripción/metabolismoRESUMEN
RNA polymerase III (Pol III) is tightly controlled in response to environmental cues, yet a genomic-scale picture of Pol III regulation and the role played by its repressor MAF1 is lacking. Here, we describe genome-wide studies in human fibroblasts that reveal a dynamic and gene-specific adaptation of Pol III recruitment to extracellular signals in an mTORC1-dependent manner. Repression of Pol III recruitment and transcription are tightly linked to MAF1, which selectively localizes at Pol III loci, even under serum-replete conditions, and increasingly targets transcribing Pol III in response to serum starvation. Combining Pol III binding profiles with EU-labeling and high-throughput sequencing of newly synthesized small RNAs, we show that Pol III occupancy closely reflects ongoing transcription. Our results exclude the long-term, unproductive arrest of Pol III on the DNA as a major regulatory mechanism and identify previously uncharacterized, differential coordination in Pol III binding and transcription under different growth conditions.
Asunto(s)
Complejos Multiproteicos/metabolismo , ARN Polimerasa III/metabolismo , Proteínas Represoras/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Transcripción Genética/fisiología , Línea Celular , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos/genética , ARN Polimerasa III/genética , Proteínas Represoras/genética , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Cyclosporin A (CsA) generates superoxide in smooth muscle cells. Our earlier studies have demonstrated that the increase in the vasopressin type 1 receptor induced in vascular smooth muscle cells in the presence of CsA is probably due to superoxide (Krauskopf et al., J Biol Chem 278, 41685-41690, 2003). This increase in vasopressin receptor is likely at the base of increased vascular responsiveness to vasoconstrictor hormones and hypertension induced by CsA. Here, we demonstrate that CsA produces superoxide. In addition, our data show that superoxide generation does not originate from the major cellular superoxide generating systems NAD(P)H oxidase or xanthine oxidase. Our results suggest that the side effects of CsA could be diminished with the help of SOD mimetic drugs.
Asunto(s)
Ciclosporina/farmacología , Inmunosupresores/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , Superóxidos/metabolismo , Animales , Aorta/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Etidio/análogos & derivados , Fluoresceínas , Depuradores de Radicales Libres/farmacología , Masculino , Miocitos del Músculo Liso/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/antagonistas & inhibidores , Superóxido Dismutasa/genéticaRESUMEN
Basal calcium leak into smooth muscle was identified 30 years ago yet remains poorly understood. We characterized this leak measuring 45Ca2+ uptake into cultured rat aortic smooth muscle cells. Wash solution (0 degrees C) containing lanthanum (3 mM) removed extracellular tracer and increased cellular 45Ca2+ retention more effectively than EGTA (0.2 mM). Basal Ca2+ entry was 1.45 x 10(9) Ca2+ x cell(-1) x min(-1). This translated to approximately 250 micromol(-1) x min(-1) given cell volumes of 4-15 pl as determined by 3-D image reconstruction. Gadolinium (100 microM) blocked 80% of the leak and exhibited a biphasic concentration-response relation (IC50s=1 microM and 2 mM). Organic ion channel blockers also inhibited approximately 80% of the leak; 45% by nifedipine (10 microM), 7% was exclusively blocked by SKF 96365 (1-[b-[3-(4-Methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole) (50 microM) and 23% was exclusively sensitive to 2-aminoethoxy-diphenylborate (2-APB, 75 microM). Reverse transcriptase polymerase chain reaction revealed TrpC1, 4 and 6 mRNA, and we propose that 2-APB may selectively block TrpC4-containing channels. We conclude that basal Ca2+ entry is mainly due to a basal open probability of excitable Ca2+ -channels.
Asunto(s)
Calcio/metabolismo , Músculo Liso Vascular/metabolismo , Animales , Compuestos de Boro/farmacología , Calcio/farmacocinética , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/genética , Radioisótopos de Calcio , Relación Dosis-Respuesta a Droga , Ácido Egtácico/farmacología , Gadolinio/farmacología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Imidazoles/farmacología , Lantano/farmacología , Masculino , Proteínas de la Membrana/genética , Microscopía Confocal , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Nifedipino/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas WKY , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína 25 Asociada a Sinaptosomas , Canales Catiónicos TRPCRESUMEN
Based on our previous results, we investigated whether cyclosporin A (CsA)-induced vasopressin type 1A receptor up-regulation was mediated by free radicals. We report that CsA analogues with different affinities for cyclophilin and calcineurin were able to up-regulate vasopressin type 1A receptor and to generate free radicals in smooth muscle cells independently of calcineurin. Further, we demonstrate that the antioxidant N-acetyl-L-cysteine blocked the increase in vasopressin type 1A receptor mRNA and protein levels induced by CsA and that low concentrations of prooxidants were able to directly increase vasopressin type 1A receptor mRNA and protein levels. In addition, short exposure to CsA or pro-oxidants was sufficient to significantly increase vasopressin type 1A receptor mRNA and protein levels. Using cell-permeable forms of superoxide dismutase and catalase, we finally show that superoxide mediates the CsA-induced effects on vasopressin type 1A receptor. These results provide strong evidence that CsA-induced superoxide generation is causally involved in vasopressin type 1A receptor expression and demonstrate for the first time that low physiological concentrations of radicals, most probably superoxide, are able to directly affect cellular signaling to increase vasopressin type 1A receptor expression in rat aortic smooth muscle cells.