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Heterochromatin is critical for maintaining genome stability, especially in flowering plants, where it relies on a feedback loop involving the H3K9 methyltransferase, KRYPTONITE (KYP), and the DNA methyltransferase CHROMOMETHYLASE3 (CMT3). The H3K9 demethylase INCREASED IN BONSAI METHYLATION 1 (IBM1) counteracts the detrimental consequences of KYP-CMT3 activity in transcribed genes.IBM1expression inArabidopsisis uniquely regulated by methylation of the 7th intron, allowing it to monitor global H3K9me2 levels. We show the methylated intron is prevalent across flowering plants and its underlying sequence exhibits dynamic evolution. We also find extensive genetic and expression variations inKYP,CMT3, andIBM1across flowering plants. We identifyArabidopsisaccessions resembling weakibm1mutants and Brassicaceae species with reducedIBM1expression or deletions. Evolution towards reduced IBM1 activity in some flowering plants could explain the frequent natural occurrence of diminished or lost CMT3 activity and loss of gene body DNA methylation, ascmt3mutants inA.thalianamitigate the deleterious effects of IBM1. 

Abstract Although DNA methylation is an important gene regulatory mechanism in mammals, its function in arthropods remains poorly understood. Studies in eusocial insects have argued for its role in caste development by regulating gene expression and splicing. However, such findings are not always consistent across studies, and have therefore remained controversial. Here we use CRISPR/Cas9 to mutate the maintenance DNA methyltransferase DNMT1 in the clonal raider ant, Ooceraea biroi . Mutants have greatly reduced DNA methylation, but no obvious developmental phenotypes, demonstrating that, unlike mammals, ants can undergo normal development without DNMT1 or DNA methylation. Additionally, we find no evidence of DNA methylation regulating caste development. However, mutants are sterile, whereas in wild-type ants, DNMT1 is localized to the ovaries and maternally provisioned into nascent oocytes. This supports the idea that DNMT1 plays a crucial but unknown role in the insect germline. 

Abstract Epialleles are meiotically heritable variations in expression states that are independent from changes in DNA sequence. Although they are common in plant genomes, their molecular origins are unknown. Here we show, using mutant and experimental populations, that epialleles in Arabidopsis thaliana that result from ectopic hypermethylation are due to feedback regulation of pathways that primarily function to maintain DNA methylation at heterochromatin. Perturbations to maintenance of heterochromatin methylation leads to feedback regulation of DNA methylation in genes. Using single base resolution methylomes from epigenetic recombinant inbred lines (epiRIL), we show that epiallelic variation is abundant in euchromatin, yet, associates with QTL primarily in heterochromatin regions. Mapping three-dimensional chromatin contacts shows that genes that are hotspots for ectopic hypermethylation have increases in contact frequencies with regions possessing H3K9me2. Altogether, these data show that feedback regulation of pathways that have evolved to maintain heterochromatin silencing leads to the origins of spontaneous hypermethylated epialleles. 

The Arabidopsis DEMETER (DME) DNA glycosylase demethylates the maternal genome in the central cell prior to fertilization and is essential for seed viability. DME preferentially targets small transposons that flank coding genes, influencing their expression and initiating plant gene imprinting. DME also targets intergenic and heterochromatic regions, but how it is recruited to these differing chromatin landscapes is unknown. The C-terminal half of DME consists of 3 conserved regions required for catalysis in vitro. We show that this catalytic core guides active demethylation at endogenous targets, rescuing dme developmental and genomic hypermethylation phenotypes. However, without the N terminus, heterochromatin demethylation is significantly impeded, and abundant CG-methylated genic sequences are ectopically demethylated. Comparative analysis revealed that the conserved DME N-terminal domains are present only in flowering plants, whereas the domain architecture of DME-like proteins in nonvascular plants mainly resembles the catalytic core, suggesting that it might represent the ancestral form of the 5mC DNA glycosylase found in plant lineages. We propose a bipartite model for DME protein action and suggest that the DME N terminus was acquired late during land plant evolution to improve specificity and facilitate demethylation at heterochromatin targets.