RESUMEN
Interactions between the splicing machinery and RNA polymerase II increase protein-coding gene transcription. Similarly, exons and splicing signals of enhancer-generated long noncoding RNAs (elncRNAs) augment enhancer activity. However, elncRNAs are inefficiently spliced, suggesting that, compared with protein-coding genes, they contain qualitatively different exons with a limited ability to drive splicing. We show here that the inefficiently spliced first exons of elncRNAs as well as promoter-antisense long noncoding RNAs (pa-lncRNAs) in human and mouse cells trigger a transcription termination checkpoint that requires WDR82, an RNA polymerase II-binding protein, and its RNA-binding partner of previously unknown function, ZC3H4. We propose that the first exons of elncRNAs and pa-lncRNAs are an intrinsic component of a regulatory mechanism that, on the one hand, maximizes the activity of these cis-regulatory elements by recruiting the splicing machinery and, on the other, contains elements that suppress pervasive extragenic transcription.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , ARN Polimerasa II/ultraestructura , ARN Largo no Codificante/genética , Transcripción Genética , Empalme Alternativo/genética , Animales , Proteínas Cromosómicas no Histona/ultraestructura , Proteínas de Unión al ADN/ultraestructura , Exones/genética , Humanos , Ratones , Regiones Promotoras Genéticas/genética , ARN Polimerasa II/genética , Empalme del ARN/genética , ARN sin Sentido/genética , ARN sin Sentido/ultraestructura , ARN Largo no Codificante/ultraestructura , ARN Mensajero/genética , Secuencias Reguladoras de Ácidos Nucleicos/genéticaRESUMEN
Upon recruitment to active enhancers and promoters, RNA polymerase II (Pol II) generates short non-coding transcripts of unclear function. The mechanisms that control the length and the amount of ncRNAs generated by cis-regulatory elements are largely unknown. Here, we show that the adaptor protein WDR82 and its associated complexes actively limit such non-coding transcription. WDR82 targets the SET1 H3K4 methyltransferases and the nuclear protein phosphatase 1 (PP1) complexes to the initiating Pol II. WDR82 and PP1 also interact with components of the transcriptional termination and RNA processing machineries. Depletion of WDR82, SET1, or the PP1 subunit required for its nuclear import caused distinct but overlapping transcription termination defects at highly expressed genes and active enhancers and promoters, thus enabling the increased synthesis of unusually long ncRNAs. These data indicate that transcription initiated from cis-regulatory elements is tightly coordinated with termination mechanisms that impose the synthesis of short RNAs.
Asunto(s)
Núcleo Celular/metabolismo , Elementos de Facilitación Genéticos/fisiología , Regiones Promotoras Genéticas/fisiología , ARN Polimerasa II/metabolismo , ARN no Traducido/biosíntesis , Terminación de la Transcripción Genética/fisiología , Transporte Activo de Núcleo Celular/fisiología , Animales , Núcleo Celular/genética , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Ratones , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , ARN Polimerasa II/genética , ARN no Traducido/genéticaRESUMEN
Interleukin-2 (IL-2) is an essential cytokine for T-lymphocyte homeostasis. We have previously reported that all-trans retinoic acid (atRA) enhances the secretion of IL-2 from human peripheral blood T cells in vitro, followed by increased proliferation and inhibition of spontaneous cell death. In this study we used a transgenic IL-2 gene luciferase reporter model to examine the effects of atRA in vivo. In contrast to the observations in human T cells, we found an overall reduction in luciferase-reported IL-2 gene expression in mice treated with atRA. Whole-body luminescence of anti-CD3-treated and non-treated mice was reduced in mice receiving atRA. Accordingly, after 7 hr, IL-2 gene expression was on average 55% lower in the atRA-treated mice compared with the control mice. Furthermore, mice fed a vitamin A-deficient diet had a significantly higher basal level of luciferase activity compared with control mice, demonstrating that vitamin A modulates IL-2 gene expression in vivo. Importantly, the atRA-mediated inhibition of IL-2 gene expression was accompanied by decreased DNA synthesis in murine T cells, suggesting a physiological relevance of the reduced IL-2 gene expression observed in transgenic reporter mice.
Asunto(s)
Interleucina-2/biosíntesis , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Tretinoina/farmacología , Animales , Benzoatos/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cromanos/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-2/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Transgénicos , FN-kappa B/metabolismo , Receptores de Ácido Retinoico/antagonistas & inhibidores , Receptores de Ácido Retinoico/inmunología , Bazo/inmunología , Linfocitos T/inmunología , Deficiencia de Vitamina A/inmunologíaAsunto(s)
Proteínas/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal , Proteína de Dominio de Muerte Asociada a Receptor de TNF/metabolismo , Factor 1 Asociado a Receptor de TNF/metabolismo , Animales , Humanos , Proteínas/química , Proteínas/genética , Factor 1 Asociado a Receptor de TNF/inmunologíaRESUMEN
A wide range of environmental stress and human disorders involves inappropriate regulation of NF-kappaB, including cancers and numerous inflammatory conditions. We have developed transgenic mice that express luciferase under the control of NF-kappaB, enabling real-time non-invasive imaging of NF-kappaB activity in intact animals. We show that, in the absence of stimulation, strong, intrinsic luminescence is evident in lymph nodes in the neck region, thymus, and Peyer's patches. Treating mice with stressors, such as TNF-alpha, IL-1alpha, or lipopolysaccharide (LPS) increases the luminescence in a tissue-specific manner, with the strongest activity observable in the skin, lungs, spleen, Peyer's patches, and the wall of the small intestine. Liver, kidney, heart, muscle, and adipose tissue exhibit less intense activities. Exposure of the skin to a low dose of UV-B radiation increases luminescence in the exposed areas. In ocular experiments, LPS- and TNF-alpha injected NF-kappaB-luciferase transgenic mice exhibit a 20-40-fold increase in lens NF-kappaB activity, similar to other LPS- and TNF-alpha-responsive organs. Peak NF-kappaB activity occurs 6h after injection of TNF-alpha and 12h after injection of LPS. Peak activities occur, respectively, 3 and 6h later than that in other tissues. Mice exposed to 360J/m(2) of UV-B exhibit a 16-fold increase in NF-kappaB activity 6h after exposure, characteristically similar to TNF-alpha-exposed mice. Thus, in NF-kappaB-luciferase transgenic mice, NF-kappaB activity also occurs in lens epithelial tissue and is activated when the intact mouse is exposed to classical stressors. Furthermore, as revealed by real-time non-invasive imaging, induction of chronic inflammation resembling rheumatoid arthritis produces strong NF-kappaB activity in the affected joints. Finally, we have used the model to demonstrate NF-kappaB regulation by manipulating the Vitamin A status in mice. NF-kappaB activity is elevated in mice fed a Vitamin A deficient (VAD) diet, and suppressed by surplus doses of retinoic acid (RA). We thus demonstrate the development and use of a versatile model for monitoring NF-kappaB activation both in tissue homogenates and in intact animals after the use of classical activators, during disease progression and after dietary intervention.
Asunto(s)
FN-kappa B/metabolismo , Animales , Diagnóstico por Imagen , Regulación de la Expresión Génica , Genes Reporteros , Cristalino/química , Luciferasas/análisis , Mediciones Luminiscentes , Ratones , Ratones Transgénicos , Vitamina A/sangreRESUMEN
Our study aimed to investigate, in vivo, the relationship between vitamin A status and NF-kappaB activity, a transcription factor central in regulating inflammatory and immune responses. We used a novel transgenic murine NF-kappaB-luciferase reporter model that enabled molecular imaging of NF-kappaB activity in live mice via an intensified image-capture apparatus. Whole-body luminescence, which reflects overall NF-kappaB activity, was elevated 2.2-fold in vitamin A-deficient (VAD) mice compared with control mice. Specifically, NF-kappaB activity in VAD mice was increased 1.8-fold in the lymph nodes and 1.4-fold in the thymus and, NF-kappaB induction in UVB radiation-exposed skin was also enhanced in VAD mice compared with control mice. The administration of all-trans retinoic acid to VAD mice resulted in a transient reduction in NF-kappaB activity and, conversely, a single dose of the RAR-pan-antagonist, AGN 194310, administered to control mice, led to a marked, transient induction of whole-body luminescence. Our results suggest that vitamin A status, and vitamin A itself, affects NF-kappaB activity in vivo and that the elevated NF-kappaB activity in VAD may be a mechanism underlying some of the features of VAD syndrome.