Functional characterization of Methylated DNA binding proteins in gene silencing

Abstract

The 5-methyl cytosine present in genomic DNA is one of the epigenetic modifications that determines the fate of gene expression. The methylome and transcriptome data from different organisms suggest an inverse relation between DNA methylation and gene expression. Different studies in plants and animals also indicate the interaction of a lot of histone modifying proteins with cytosine methylation. The association of DNA methylation with histone deacetylation and histone methylation is well characterized in plants and animals. A combined effect of these processes leads to chromatin condensation and gene silencing. In higher organisms, the cytosine methylation is read and interpreted by a class of proteins called methyl CpG binding domain (MBD) proteins. Another interesting phenomenon associated with genomic DNA methylation is RNA directed DNA Methylation (RdDM), a process involving RNA molecules directing the methylation of its loci in the genomic DNA. This area of regulation is poorly understood. In the present work four genes encoding MBD proteins from Arabidopsis were characterized. Transgenic plants over-expressing AtMBD4, AtMBD6, AtMBD10 and AtMBD11 were generated. Over-expression of AtMBD4 and AtMBD10 in Arabidopsis led to hypomethylation of genomic DNA which are highly methylated in wild type plants. These genes encoding MBD proteins were further characterized using mutants and knockdown lines. The down-regulation of these genes in Arabidopsis showed abnormal phenotype in root, leaf and flower. These abnormal phenotypes suggest their importance during the process of development. Transcriptome analysis identified many genes affected due to mutation in AtMBD genes. Transcriptome analysis of atmbd4 mutant suggested its physiological and molecular importance in nutrient starvation especially phosphate starvation. Genes involved in biotic stress response were up regulated in the atmbd6 mutant.