Project description:Paramutation is an exception among eukaryotes, in which epigenetic information is conserved through mitosis and meiosis. It has been studied for over 70 years in maize, but the mechanisms involved are largely unknown. All previously described actors of paramutation encoding components of the RNA-dependent DNA-methylation pathway (RdDM) are involved in the biogenesis of 24nt small RNAs. However, no actors of paramutation have been identified in the effector complex of RdDM. Through a combination of reverse genetics, immunolocalization and immunoprecipitation (siRNA-IP) we found that ARGONAUTE104 (AGO104), AGO105 and AGO119 are members of the RdDM effector complex in maize and bind siRNAs produced from the tandem repeats required for paramutation at the b1 locus. We also showed that AGO104 is an effector of the b1 paramutation in maize.
Project description:By using DNA methylation of introgression lines as a marker of past paramutation, we characterise in details the paramutation of the \textit{H06} locus in crosses between Solanum lycopersicum and a range of tomato relatives and cultivars. Paramutation of H06 depends on the timing of sRNA production and conforms to the RNA-directed model of paramutation. By scanning the methylomes of tomato introgression lines for shared regions of differential methylation, thousands of candidate regions for paramutation are identified. Analysing the segregation of the methylation patterns for a subset of these regions shows variations in the penetrance of paramutation-like interactions.
Project description:By using DNA methylation of introgression lines as a marker of past paramutation, we characterise in details the paramutation of the \textit{H06} locus in crosses between Solanum lycopersicum and a range of tomato relatives and cultivars. Paramutation of H06 depends on the timing of sRNA production and conforms to the RNA-directed model of paramutation. By scanning the methylomes of tomato introgression lines for shared regions of differential methylation, thousands of candidate regions for paramutation are identified. Analysing the segregation of the methylation patterns for a subset of these regions shows variations in the penetrance of paramutation-like interactions.
Project description:This research identifies a novel protein required for paramutation at the maize purple plant1 locus. This 'required to maintain repression2' (RMR2) protein represents the founding member of a plant-specific clade of hypothetical proteins. We show that RMR2 is required for transcriptional repression at the Pl1-Rhoades haplotype, for accumulation of 24 nt RNA species, and for maintenance of a 5-methylcytosine pattern distinct from that maintained by RNA polymerase IV. Genetic tests indicate that RMR2 is not required for paramutation occurring at the red1 locus. These results distinguish the paramutation-type mechanisms operating at specific haplotypes. The RMR2 clade of proteins provides a new entry point for understanding the diversity of epigenomic control operating in higher plants.
Project description:This research identifies a novel protein required for paramutation at the maize purple plant1 locus. This 'required to maintain repression2' (RMR2) protein represents the founding member of a plant-specific clade of hypothetical proteins. We show that RMR2 is required for transcriptional repression at the Pl1-Rhoades haplotype, for accumulation of 24 nt RNA species, and for maintenance of a 5-methylcytosine pattern distinct from that maintained by RNA polymerase IV. Genetic tests indicate that RMR2 is not required for paramutation occurring at the red1 locus. These results distinguish the paramutation-type mechanisms operating at specific haplotypes. The RMR2 clade of proteins provides a new entry point for understanding the diversity of epigenomic control operating in higher plants. Examination of small RNAs using Illumina's sequencing-by-synthesis (SBS) platform to deep sequence small RNA libraries made from the 4-cm cobs of rmr2 mutant and non-mutant siblings.
Project description:Methylation of chromosomal DNA in animals and plants is a fundamental mechanism of epigenetic regulation, and the maize genome, with its diverse complement of transposons and repeats, is a paradigm for transgenerational mechanisms such as paramutation and imprinting. We have determined the genome-wide cytosine methylation map of two maize inbred lines, B73 and Mo17, at high coverage and at single nucleotide resolution. Transposon methylation is highest in CG (65%) and CHG (50%) contexts (where H = A, C or T), while methylation in CHH (5%) contexts is guided by 24nt small interfering RNA (siRNA), and not by 21-22nt siRNA. We have found that CG (8%) methylation seems to deter insertion of Mutator transposons into exons, while CHH and CHG methylation at splice donor and acceptor sites strongly inhibits RNA splicing. Methylation differences between parents are inherited in recombinant inbred lines, but methylation switches, guided by siRNA, are widespread and persist for up to 8 generations. These differences influence splicing, and recurrent switching suggest that paramutation is much more common than previously supposed, and may contribute to heterosis. Our results provide a comprehensive high resolution resource for maize genome methylation, as well as a map of recurrent transgenerational epigenetic shifts (paramutation) in the two most commonly used inbred maize lines.
Project description:Paramutations represent directed and meiotically-heritable changes in gene regulation leading to apparent violations of Mendelian inheritance. Although the mechanism and evolutionary importance of paramutation behaviors remain largely unknown, genetic screens in maize (Zea mays) identify five components affecting 24 nucleotide RNA biogenesis as required to maintain repression of a paramutant purple plant1 (pl1) allele. Currently, the RNA polymerase IV largest subunit represents the only component also specifying proper development. Here we identify a chromodomain helicase DNA-binding 3 (CHD3) protein orthologous to Arabidopsis (Arabidopsis thaliana) PICKLE as a second component maintaining both pl1 paramutation, and normal somatic development. In addition, genetic tests show this protein contributes to proper male gametophyte function. The similar mutant phenotypes documented in Arabidopsis and maize implicate some evolutionarily-conserved gene regulation while developmental defects associated with the two paramutation mutants are largely distinct. Our results show that a CHD3 protein responsible for normal plant ontogeny and sperm transmission also maintains meiotically-heritable epigenetic regulatory variation for specific alleles. This finding implicates an intersection of RNA polymerase IV function and nucleosome positioning in the paramutation process.
Project description:Methylation of chromosomal DNA in animals and plants is a fundamental mechanism of epigenetic regulation, and the maize genome, with its diverse complement of transposons and repeats, is a paradigm for transgenerational mechanisms such as paramutation and imprinting. We have determined the genome-wide cytosine methylation map of two maize inbred lines, B73 and Mo17, at high coverage and at single nucleotide resolution. Transposon methylation is highest in CG (65%) and CHG (50%) contexts (where H = A, C or T), while methylation in CHH (5%) contexts is guided by 24nt small interfering RNA (siRNA), and not by 21-22nt siRNA. We have found that CG (8%) methylation seems to deter insertion of Mutator transposons into exons, while CHH and CHG methylation at splice donor and acceptor sites strongly inhibits RNA splicing. Methylation differences between parents are inherited in recombinant inbred lines, but methylation switches, guided by siRNA, are widespread and persist for up to 8 generations. These differences influence splicing, and recurrent switching suggest that paramutation is much more common than previously supposed, and may contribute to heterosis. Our results provide a comprehensive high resolution resource for maize genome methylation, as well as a map of recurrent transgenerational epigenetic shifts (paramutation) in the two most commonly used inbred maize lines. Genome-wide cytosine methylation map in 2 maize strains by bisulfite sequencing, and RNA and small RNA profiles in the same tissue using Illumina platform.