RESUMEN
DNA double-strand breaks (DSBs) represent one of the most threatening lesions to the integrity of genomes. In yeast Saccharomyces cerevisiae, NuA4, a histone acetylation complex, is recruited to DSBs, wherein it acetylates histones H2A and H4, presumably relaxing the chromatin and allowing access to repair proteins. Two subunits of NuA4, Yng2 and Eaf3, can interact in vitro with methylated H3K4 and H3K36 via their plant homeodomain (PHD) and chromodomain. However, the roles of the two domains and how they interact in a combinatorial fashion are still poorly characterized. In this study, we generated mutations in the PHD and chromodomain that disrupt their interaction with methylated H3K4 and H3K36. We demonstrate that the combined mutations in both the PHD and chromodomain impair the NuA4 recruitment, reduce H4K12 acetylation at the DSB site, and confer sensitivity to bleomycin that induces DSBs. In addition, the double mutant cells are defective in DSB repair as judged by Southern blot and exhibit prolonged activation of phospho-S129 of H2A. Cells harboring the H3K4R, H3K4R, K36R, or set1Δ set2Δ mutant that disrupts H3K4 and H3K36 methylation also show very similar phenotypes to the PHD and chromodomain double mutant. Our results suggest that multivalent interactions between the PHD, chromodomain, and methylated H3K4 and H3K36 act in a combinatorial manner to recruit NuA4 and regulate the NuA4 activity at the DSB site.
Asunto(s)
ADN de Hongos/metabolismo , Histona Acetiltransferasas/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Acetilación , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Sitios de Unión , Bleomicina/farmacología , Cromatina/metabolismo , Roturas del ADN de Doble Cadena , Farmacorresistencia Fúngica/genética , Histona Acetiltransferasas/química , Histona Acetiltransferasas/genética , Histonas/metabolismo , Metilación , Mutación , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The phytotoxic effects of copper (Cu) and cadmium (Cd) on plant growth are well documented. However, Cu and Cd toxicity targets and the cellular systems contributing to acquisition of tolerance are not fully understood at the molecular level. We aimed to identify genes and pathways that discriminate the actions of Cu and Cd in rice roots (Oryza sativa L. cv. TN67). The transcripts of 1,450 and 1,172 genes were regulated after Cu and Cd treatments, respectively. We identified 882 genes specifically respond to Cu treatment, and 604 unique genes as Cd-responsive by comparison of expression profiles of these two regulated gene groups. Gene ontology analysis for 538 genes involved in primary metabolism, oxidation reduction and response to stimulus was changed in response to both metals. In the individual aspect, Cu specifically altered levels of genes involved in vesicle trafficking transport, fatty acid metabolism and cellular component biogenesis. Cd-regulated genes related to unfolded protein binding and sulfate assimilation. To further characterize the functions of vesicle trafficking transport under Cu stress, interference of excytosis in root tissues was conducted by inhibitors and silencing of Exo70 genes. It was demonstrated that vesicle-trafficking is required for mediation of Cu-induced reactive oxygen species (ROS) production in root tissues. These results may provide new insights into understanding the molecular basis of the early metal stress response in plants.
Asunto(s)
Cadmio/toxicidad , Cobre/toxicidad , Oryza/efectos de los fármacos , Oryza/genética , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genética , Transcriptoma/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Genes de Plantas/genética , Anotación de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Oryza/crecimiento & desarrollo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Virus de Plantas/efectos de los fármacos , Virus de Plantas/genética , Especies Reactivas de Oxígeno/metabolismo , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Plantones/efectos de los fármacos , Plantones/genética , Plantones/crecimiento & desarrollo , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Nicotiana/efectos de los fármacos , Nicotiana/genética , Nicotiana/virología , Transcriptoma/efectos de los fármacos , Vesículas Transportadoras/efectos de los fármacos , Vesículas Transportadoras/metabolismoRESUMEN
Mercury (Hg) is a serious environmental pollution threats to the planet. Accumulation of Hg in plants disrupts many cellular-level functions and inhibits growth and development, but the mechanism is not fully understood. We investigated cellular, biochemical and proteomic changes in rice roots under Hg stress. Root growth rate was decreased and Hg, reactive oxygen species (ROS), and malondialdehyde (MDA) content and lipoxygenase activity were increased significantly with increasing Hg concentration in roots. We revealed a time-dependent alteration in total glutathione content and enzymatic activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POD) during Hg stress. 2-D electrophoresis revealed differential expression of 25 spots with Hg treatment of roots: 14 spots were upregulated and 11 spots downregulated. These differentially expressed proteins were identified by ESI-MS/MS to be involved in cellular functions including redox and hormone homeostasis, chaperone activity, metabolism, and transcription regulation. These results may provide new insights into the molecular basis of the Hg stress response in plants.