skip to main content

Title: Mechanistic basis for maintenance of CHG DNA methylation in plants

DNA methylation is an evolutionarily conserved epigenetic mechanism essential for transposon silencing and heterochromatin assembly. In plants, DNA methylation widely occurs in the CG, CHG, and CHH (H = A, C, or T) contexts, with the maintenance of CHG methylation mediated by CMT3 chromomethylase. However, how CMT3 interacts with the chromatin environment for faithful maintenance of CHG methylation is unclear. Here we report structure-function characterization of the H3K9me2-directed maintenance of CHG methylation by CMT3 and itsZea maysortholog ZMET2. Base-specific interactions and DNA deformation coordinately underpin the substrate specificity of CMT3 and ZMET2, while a bivalent readout of H3K9me2 and H3K18 allosterically stimulates substrate binding. Disruption of the interaction with DNA or H3K9me2/H3K18 led to loss of CMT3/ZMET2 activity in vitro and impairment of genome-wide CHG methylation in vivo. Together, our study uncovers how the intricate interplay of CMT3, repressive histone marks, and DNA sequence mediates heterochromatic CHG methylation.

; ; ; ; ; ; ;
Publication Date:
Journal Name:
Nature Communications
Nature Publishing Group
Sponsoring Org:
National Science Foundation
More Like this
  1. In plants and mammals, DNA methylation plays a critical role in transcriptional silencing by delineating heterochromatin from transcriptionally active euchromatin. A homeostatic balance between heterochromatin and euchromatin is essential to genomic stability. This is evident in many diseases and mutants for heterochromatin maintenance, which are characterized by global losses of DNA methylation coupled with localized ectopic gains of DNA methylation that alter transcription. Furthermore, we have shown that genome-wide methylation patterns inArabidopsis thalianaare highly stable over generations, with the exception of rare epialleles. However, the extent to which natural variation in the robustness of targeting DNA methylation to heterochromatin exists, and the phenotypic consequences of such variation, remain to be fully explored. Here we describe the finding that heterochromatin and genic DNA methylation are highly variable among 725A. thalianaaccessions. We found that genic DNA methylation is inversely correlated with that in heterochromatin, suggesting that certain methylation pathway(s) may be redirected to genes upon the loss of heterochromatin. This redistribution likely involves a feedback loop involving the DNA methyltransferase, CHROMOMETHYLASE 3 (CMT3), H3K9me2, and histone turnover, as highly expressed, long genes with a high density of CMT3-preferred CWG sites are more likely to be methylated. Importantly, although the presence of CGmore »methylation in genes alone may not affect transcription, genes containing CG methylation are more likely to become methylated at non-CG sites and silenced. These findings are consistent with the hypothesis that natural variation in DNA methylation homeostasis may underlie the evolution of epialleles that alter phenotypes.

    « less
  2. Abstract

    Despite the known critical regulatory functions of microRNAs, histone modifications, and DNA methylation in reprograming plant epigenomes in response to pathogen infection, the molecular mechanisms underlying the tight coordination of these components remain poorly understood. Here, we show how Arabidopsis (Arabidopsis thaliana) miR778 coordinately modulates the root transcriptome, histone methylation, and DNA methylation via post-transcriptional regulation of the H3K9 methyltransferases SU(var)3-9 homolog 5 (SUVH5) and SUVH6 upon infection by the beet cyst nematode Heterodera schachtii. miR778 post-transcriptionally silences SUVH5 and SUVH6 upon nematode infection. Manipulation of the expression of miR778 and its two target genes significantly altered plant susceptibility to H. schachtii. RNA-seq analysis revealed a key role of SUVH5 and SUVH6 in reprograming the transcriptome of Arabidopsis roots upon H. schachtii infection. In addition, chromatin immunoprecipitation (ChIP)-seq analysis established SUVH5 and SUVH6 as the main enzymes mediating H3K9me2 deposition in Arabidopsis roots in response to nematode infection. ChIP-seq analysis also showed that these methyltransferases possess distinct DNA binding preferences in that they are targeting transposable elements under noninfected conditions and protein-coding genes in infected plants. Further analyses indicated that H3K9me2 deposition directed by SUVH5 and SUVH6 contributes to gene expression changes both in roots and in nematode feedingmore »sites and preferentially associates with CG DNA methylation. Together, our results uncovered multi-layered epigenetic regulatory mechanisms coordinated by miR778 during Arabidopsis–H. schachtii interactions.

    « less
  3. Meiotic recombination is a fundamental process that generates genetic diversity and ensures the accurate segregation of homologous chromosomes. While a great deal is known about genetic factors that regulate recombination, relatively little is known about epigenetic factors, such as DNA methylation. In maize, we examined the effects on meiotic recombination of a mutation in a component of the RNA-directed DNA methylation pathway,Mop1(Mediator of paramutation1), as well as a mutation in a component of thetrans-acting small interference RNA biogenesis pathway,Lbl1(Leafbladeless1). MOP1 is of particular interest with respect to recombination because it is responsible for methylation of transposable elements that are immediately adjacent to transcriptionally active genes. In themop1mutant, we found that meiotic recombination is uniformly decreased in pericentromeric regions but is generally increased in gene rich chromosomal arms. This observation was further confirmed by cytogenetic analysis showing that although overall crossover numbers are unchanged, they occur more frequently in chromosomal arms inmop1mutants. Using whole genome bisulfite sequencing, our data show that crossover redistribution is driven by loss of CHH (where H = A, T, or C) methylation within regions near genes. In contrast to what we observed inmop1mutants, no significant changes were observed in the frequency of meiotic recombination inlbl1mutants. Ourmore »data demonstrate that CHH methylation has a significant impact on the overall recombination landscape in maize despite its low frequency relative to CG and CHG methylation.

    « less
  4. 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.
  5. Transcriptional gene silencing (TGS) can serve as an innate immunity against invading DNA viruses throughout Eukaryotes. Geminivirus code for TrAP protein to suppress the TGS pathway. Here, we identified an Arabidopsis H3K9me2 histone methyltransferase, Su(var)3-9 homolog 4/Kryptonite (SUVH4/KYP), as a bona fide cellular target of TrAP. TrAP interacts with the catalytic domain of KYP and inhibits its activity in vitro. TrAP elicits developmental anomalies phenocopying several TGS mutants, reduces the repressive H3K9me2 mark and CHH DNA methylation, and reactivates numerous endogenous KYP-repressed loci in vivo. Moreover, KYP binds to the viral chromatin and controls its methylation to combat virus infection. Notably, kyp mutants support systemic infection of TrAP-deficient Geminivirus. We conclude that TrAP attenuates the TGS of the viral chromatin by inhibiting KYP activity to evade host surveillance. These findings provide new insight on the molecular arms race between host antiviral defense and virus counter defense at an epigenetic level.