RESUMEN
In tomato, DNA methylation has an inhibitory effect on fruit ripening. The inhibition of DNA methyltransferase by 5-azacytidine results in premature fruit ripening. Methyl CpG binding domain (MBD) proteins are the readers of DNA methylation marks and help in the recruitment of chromatin-modifying enzymes which affect gene expression. Therefore, we investigate their contribution during fruit development. In this study, we identified and analyzed 18 putative genes of Solanum lycopersicum and Solanum pimpinellifolium encoding MBD proteins. We also identified tomato MBD syntelogs in Capsicum annum and Solanum tuberosum. Sixty-three MBD genes identified from four different species of solanaceae were classified into three groups. An analysis of the conserved domains in these proteins identified additional domains along with MBD motif. The transcript profiling of tomato MBDs in wild-type and two non-ripening mutants, rin and Nr, indicated constructive information regarding their involvement during fruit development. When we performed a stage-specific expression analysis during fruit ripening, a gradual decrease in transcript accumulation in the wild-type fruit was detected. However, a very low expression was observed in the ripening mutants. Furthermore, many ethylene-responsive cis-elements were found in SlMBD gene promoters, and some of them were induced in the presence of exogenous ethylene. Further, we detected the possible role of these MBDs in abiotic stresses. We found that few genes were differentially expressed under various abiotic stress conditions. Our results provide an evidence of the involvement of the tomato MBDs in fruit ripening and abiotic stress responses, which would be helpful in further studies on these genes in tomato fruit ripening.
Asunto(s)
Dominio de Unión a CpG-Metil/genética , Solanum lycopersicum/genética , Capsicum/genética , Metilación de ADN/genética , Etilenos , Frutas/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/genética , Genes de Plantas/genética , Estudio de Asociación del Genoma Completo , Proteínas de Plantas/genética , Regiones Promotoras Genéticas/genética , Solanum tuberosum/genética , Estrés FisiológicoRESUMEN
Methyl-CpG binding protein 2 (MeCP2) is a multifunctional protein that guides neuronal development through its binding to DNA, recognition of sites of methyl-CpG (mCpG) DNA modification, and interaction with other regulatory proteins. Our study explores the relationship between mCpG and hydroxymethyl-CpG (hmCpG) recognition mediated by its mCpG binding domain (MBD) and binding cooperativity mediated by its C-terminal polypeptide. Previous study of the isolated MBD of MeCP2 documented an unusual mechanism by which ion uptake is required for discrimination of mCpG and hmCpG from CpG. MeCP2 binding cooperativity suppresses discrimination of modified DNA and is highly sensitive to both the total ion concentration and the type of counterions. Higher than physiological total ion concentrations completely suppress MeCP2 binding cooperativity, indicating a dominant electrostatic component to the interaction. Substitution of SO4(2-) for Cl(-) at physiological total ion concentrations also suppresses MeCP2 binding cooperativity, This effect is of particular note as the intracellular Cl(-) concentration changes during neuronal development. A related effect is that the protein-stabilizing solutes, TMAO and glutamate, reduce MeCP2 (but not isolated MBD) binding affinity by 2 orders of magnitude without affecting the apparent binding cooperativity. These observations suggest that polypeptide flexibility facilitates DNA binding by MeCP2. Consistent with this view, nuclear magnetic resonance (NMR) analyses show that ions have discrete effects on the structure of MeCP2, both MBD and the C-terminal domains. Notably, anion substitution results in changes in the NMR chemical shifts of residues, including some whose mutation causes the autism spectrum disorder Rett syndrome. Binding cooperativity makes MeCP2 an effective competitor with histone H1 for accessible DNA sites. The relationship between MeCP2 binding specificity and cooperativity is discussed in the context of chromatin binding, neuronal function, and neuronal development.
Asunto(s)
ADN/metabolismo , Proteína 2 de Unión a Metil-CpG/metabolismo , ADN/genética , Epigénesis Genética , Histonas/metabolismo , Humanos , Cinética , Dominio de Unión a CpG-Metil/genética , Proteína 2 de Unión a Metil-CpG/química , Proteína 2 de Unión a Metil-CpG/genética , Neurogénesis , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Síndrome de Rett/genética , Síndrome de Rett/metabolismoRESUMEN
DNA methylation, mediated by double-stranded RNA, is a conserved epigenetic phenomenon that protects a genome from transposons, silences unwanted genes and has a paramount function in plant or animal development. Methyl CpG binding domain proteins are members of a class of proteins that bind to methylated DNA. The Arabidopsis thaliana genome encodes 13 methyl CpG binding domain (MBD) proteins, but the molecular/biological functions of most of these proteins are still not clear. In the present study, we identified four proteins that interact with AtMBD6. Interestingly, three of them contain RNA binding domains and are co-localized with AtMBD6 in the nucleus. The interacting partners includes AtRPS2C (a 40S ribosomal protein), AtNTF2 (nuclear transport factor 2) and AtAGO4 (Argonoute 4). The fourth protein that physically interacts with AtMBD6 is a histone-modifying enzyme, histone deacetylase 6 (AtHDA6), which is a known component of the RNA-mediated gene silencing system. Analysis of genomic DNA methylation in the atmbd6, atrps2c and atntf2 mutants, using methylation-sensitive PCR detected decreased DNA methylation at miRNA/siRNA producing loci, pseudogenes and other targets of RNA-directed DNA methylation. Our results indicate that AtMBD6 is involved in RNA-mediated gene silencing and it binds to RNA binding proteins like AtRPS2C, AtAGO4 and AtNTF2. AtMBD6 also interacts with histone deacetylase AtHDA6 that might have a role in chromatin condensation at the targets of RdDM.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas Argonautas/genética , Metilación de ADN/genética , Proteínas de Unión al ADN/genética , Histona Desacetilasas/genética , Proteínas de Transporte de Membrana/genética , Secuencia de Aminoácidos/genética , Arabidopsis/genética , Sitios de Unión , Núcleo Celular/genética , Islas de CpG/genética , Silenciador del Gen , Dominio de Unión a CpG-Metil/genética , Proteínas de Unión al ARN/genéticaRESUMEN
DNA methyltransferase (DNMT) inhibitors are epigenetic drugs used to treat myelodysplastic syndrome. They not only induce DNA demethylation but also have significant cytostatic and cytotoxic effects; however, the relationships between these characteristics have not been established yet due to the lack of a method to induce only DNA demethylation. Herein, we show that a fusion protein comprised of the methyl-CpG-binding domain (MBD) and the catalytic domain of Ten-eleven translocation protein 1 (TET1-CD) globally demethylates and upregulates a number of methylated genes. These upregulated genes frequently contained CpG islands (CGIs) within ± 1000 bp of the transcription start site (TSS). Interestingly, 65% of the genes upregulated fivefold or more by MBD-TET1-CDwt were also reactivated after treatment with a DNMT inhibitor, 5-azacytidine (Aza-CR), suggesting that gene reactivation by both methods primarily shares the same mechanism, DNA demethylation. In order to examine whether DNA demethylation affects the growth of cancer cells, we have established a tetracycline inducible system that can regulate the expression of MBD-TET1-CDwt in a prostate cancer cell line, LNCaP. The induction of MBD-TET1-CDwt demethylated and upregulated glutathione S-transferase pi 1 (GSTP1), one of the hypermethylated genes in prostate cancer. In accordance with the reactivation of methylated genes, induction of MBD-TET1-CDwt extensively suppressed the growth of LNCaP cells through G1/S arrest. These results clearly indicate that TET oxidase activity recruited at methyl-CpG sites through MBD induces reactivation of hypermethylated genes by DNA demethylation and allows us to analyze the effect of only global DNA demethylation in a wide variety of cancer cells.
Asunto(s)
Islas de CpG , Metilación de ADN , Dominio de Unión a CpG-Metil , Oxigenasas de Función Mixta/metabolismo , Dominios y Motivos de Interacción de Proteínas , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Epigénesis Genética , Regulación de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Gutatión-S-Transferasa pi/genética , Gutatión-S-Transferasa pi/metabolismo , Humanos , Dominio de Unión a CpG-Metil/genética , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Regiones Promotoras Genéticas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Sitio de Iniciación de la TranscripciónRESUMEN
DNA methylation is a major epigenetic marker in plants that plays a crucial role in transcriptional and developmental regulation. The DNA methylation 'code' is thought to be 'read' by a set of proteins containing methyl-CpG-binding domain (MBD). However, little is known about MBD genes in common wheat (Triticum aestivum L.). Here, we report the isolation and characterization of TaMBD6 and its homeologues (TaMBD6_A, TaMBD6_B, and TaMBD6_D) in hexaploid wheat. The cDNA was quite different among the three homeologues and InDel mutations were detected in 5'-UTR and coding region. Two types of TRs (tandem repeats) -- TR1 (57 bp) and TR2 (39 bp) -- occurred in the coding region. TaMBD6_B harbored five copies of TR1 and two copies of TR2. In contrast, TaMBD6_A lacked 30 bp between the 2nd and 3rd copy of TR1, while TaMBD6_D was missing two copies of TR1 but had three copies of TR2. TaMBD6_A, TaMBD6_B, and TaMBD6_D encoded 435, 446, and 420 amino acids, respectively. Structural analysis of TaMBD6 protein indicated that each of the three homeologues had an identical MBD domain at the N-terminal, as well as a typical nuclear localization signal. Although genomics analysis showed that two introns were included, the length of the first intron varied from 3100 bp to 3471 bp and their sequences were very different. Expression analysis demonstrated that the transcription level of TaMBD6 began to increase gradually in developing grains at 15 days after pollination while decreasing significantly in endosperm and embryo tissues during germination. Expression of TaMBD6 appeared to be positively correlated with starch metabolism in the endosperm but was negatively correlated with embryo formation and sprouting. We were also interested to learn that TaMBD6 homeologues were differentially expressed in developing wheat plants and that their expression patterns were variously affected by vernalization treatment. Further investigation revealed that TaMBD6 was induced by prolonged chilling, indicating that the protein is potentially involved in regulating the developmental transition from vegetative to reproductive stages. Although the homeologues generally showed similar differential expression patterns, TaMBD6_D and TaMBD6_B contribute more to the processes of grain development and germination while TaMBD6_A is predominant in mature plants.