Correction: An AP endonuclease functions in active DNA demethylation and gene imprinting in Arabidopsis.
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ABSTRACT:
SUBMITTER: PLOS Genetics Staff
PROVIDER: S-EPMC4411094 | biostudies-literature | 2015 Apr
REPOSITORIES: biostudies-literature
Publications
Correction: An AP endonuclease functions in active DNA demethylation and gene imprinting in Arabidopsis.
PLoS genetics 20150427 4
[This corrects the article DOI: 10.1371/journal.pgen.1004905.]. ...[more]
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Project description:Active DNA demethylation in plants occurs through base excision repair, beginning with removal of methylated cytosine by the ROS1/DME subfamily of 5-methylcytosine DNA glycosylases. Active DNA demethylation in animals requires the DNA glycosylase TDG or MBD4, which functions after oxidation or deamination of 5-methylcytosine, respectively. However, little is known about the steps following DNA glycosylase action in the active DNA demethylation pathways in plants and animals. We show here that the Arabidopsis APE1L protein has apurinic/apyrimidinic endonuclease activities and functions downstream of ROS1 and DME. APE1L and ROS1 interact in vitro and co-localize in vivo. Whole genome bisulfite sequencing of ape1l mutant plants revealed widespread alterations in DNA methylation. We show that the ape1l/zdp double mutant displays embryonic lethality. Notably, the ape1l+/-zdp-/- mutant shows a maternal-effect lethality phenotype. APE1L and the DNA phosphatase ZDP are required for FWA and MEA gene imprinting in the endosperm and are important for seed development. Thus, APE1L is a new component of the active DNA demethylation pathway and, together with ZDP, regulates gene imprinting in Arabidopsis.
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Project description:DNA methylation is an important epigenetic mark established by the combined actions of methylation and demethylation reactions. Plants use a base excision repair pathway for active DNA demethylation. After 5-methylcytosine removal, the Arabidopsis DNA glycosylase/lyase ROS1 incises the DNA backbone and part of the product has a single-nucleotide gap flanked by 3'- and 5'-phosphate termini. Here we show that the DNA phosphatase ZDP removes the blocking 3' phosphate, allowing subsequent DNA polymerization and ligation steps needed to complete the repair reactions. ZDP and ROS1 interact in vitro and colocalize in vivo in nucleoplasmic foci. Extracts from zdp mutant plants are unable to complete DNA demethylation in vitro, and the mutations cause DNA hypermethylation and transcriptional silencing of a reporter gene. Genome-wide methylation analysis in zdp mutant plants identified hundreds of hypermethylated endogenous loci. Our results show that ZDP functions downstream of ROS1 in one branch of the active DNA demethylation pathway.
| S-EPMC3278721 | biostudies-literature
Project description:DNA methylation is a primary epigenetic modification regulating gene expression and chromatin structure in many eukaryotes. Plants have a unique DNA demethylation system in that 5-methylcytosine (5mC) is directly removed by DNA demethylases, such as DME/ROS1 family proteins, but little is known about the downstream events. During 5mC excision, DME produces 3'-phosphor-α, β-unsaturated aldehyde and 3'-phosphate by successive β- and δ-eliminations, respectively. The kinetic studies revealed that these 3'-blocking lesions persist for a significant amount of time and at least two different enzyme activities are required to immediately process them. We demonstrate that Arabidopsis AP endonucleases APE1L, APE2 and ARP have distinct functions to process such harmful lesions to allow nucleotide extension. DME expression is toxic to E. coli due to excessive 5mC excision, but expression of APE1L or ARP significantly reduces DME-induced cytotoxicity. Finally, we propose a model of base excision repair and DNA demethylation pathway unique to plants.
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Project description:Active DNA demethylation is an important part of epigenetic regulation in plants and animals. How active DNA demethylation is regulated and its relationship with histone modification patterns are unclear. Here, we report the discovery of IDM1, a regulator of DNA demethylation in Arabidopsis. IDM1 is required for preventing DNA hypermethylation of highly homologous multicopy genes and other repetitive sequences that are normally targeted for active DNA demethylation by Repressor of Silencing 1 and related 5-methylcytosine DNA glycosylases. IDM1 binds methylated DNA at chromatin sites lacking histone H3K4 di- or trimethylation and acetylates H3 to create a chromatin environment permissible for 5-methylcytosine DNA glycosylases to function. Our study reveals how some genes are indicated by multiple epigenetic marks for active DNA demethylation and protection from silencing.
| S-EPMC3575687 | biostudies-literature
Project description:De novo DNA methylation through the RNA-directed DNA methylation (RdDM) pathway and active DNA demethylation play important roles in controlling genome-wide DNA methylation patterns in plants. Little is known about how cells manage the balance between DNA methylation and active demethylation activities. Here, we report the identification of a unique RdDM target sequence, where DNA methylation is required for maintaining proper active DNA demethylation of the Arabidopsis genome. In a genetic screen for cellular antisilencing factors, we isolated several REPRESSOR OF SILENCING 1 (ros1) mutant alleles, as well as many RdDM mutants, which showed drastically reduced ROS1 gene expression and, consequently, transcriptional silencing of two reporter genes. A helitron transposon element (TE) in the ROS1 gene promoter negatively controls ROS1 expression, whereas DNA methylation of an RdDM target sequence between ROS1 5' UTR and the promoter TE region antagonizes this helitron TE in regulating ROS1 expression. This RdDM target sequence is also targeted by ROS1, and defective DNA demethylation in loss-of-function ros1 mutant alleles causes DNA hypermethylation of this sequence and concomitantly causes increased ROS1 expression. Our results suggest that this sequence in the ROS1 promoter region serves as a DNA methylation monitoring sequence (MEMS) that senses DNA methylation and active DNA demethylation activities. Therefore, the ROS1 promoter functions like a thermostat (i.e., methylstat) to sense DNA methylation levels and regulates DNA methylation by controlling ROS1 expression.
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Project description:DNA methylation is a stable epigenetic mark for transcriptional gene silencing in diverse organisms including plants and many animals. In contrast to the well characterized mechanism of DNA methylation by methyltransferases, the mechanisms and function of active DNA demethylation have been controversial. Genetic evidence suggested that the DNA glycosylase domain-containing protein ROS1 of Arabidopsis is a putative DNA demethylase, because loss-of-function ros1 mutations cause DNA hypermethylation and enhance transcriptional gene silencing. We report here the biochemical characterization of ROS1 and the effect of its overexpression on the DNA methylation of target genes. Our data suggest that the DNA glycosylase activity of ROS1 removes 5-methylcytosine from the DNA backbone and then its lyase activity cleaves the DNA backbone at the site of 5-methylcytosine removal by successive beta- and delta-elimination reactions. Overexpression of ROS1 in transgenic plants led to a reduced level of cytosine methylation and increased expression of a target gene. These results demonstrate that ROS1 is a 5-methylcytosine DNA glycosylase/lyase important for active DNA demethylation in Arabidopsis.
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Project description:DNA demethylation is important for the erasure of DNA methylation. The role of DNA demethylation in plant development remains poorly understood. Here, we found extensive DNA demethylation in the CHH context around pericentromeric regions and DNA demethylation in the CG, CHG, and CHH contexts at discrete genomic regions during ectopic xylem tracheary element (TE) differentiation. While loss of pericentromeric methylation occurs passively, DNA demethylation at a subset of regions relies on active DNA demethylation initiated by DNA glycosylases ROS1, DML2, and DML3. The ros1 and rdd mutations impair ectopic TE differentiation and xylem development in the young roots of Arabidopsis seedlings. Active DNA demethylation targets and regulates many genes for TE differentiation. The defect of xylem development in rdd is proposed to be caused by dysregulation of multiple genes. Our study identifies a role of active DNA demethylation in vascular development and reveals an epigenetic mechanism for TE differentiation.
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Project description:MEDEA (MEA) is an Arabidopsis Polycomb group gene that is imprinted in the endosperm. The maternal allele is expressed and the paternal allele is silent. MEA is controlled by DEMETER (DME), a DNA glycosylase required to activate MEA expression, and METHYLTRANSFERASE I (MET1), which maintains CG methylation at the MEA locus. Here we show that DME is responsible for endosperm maternal-allele-specific hypomethylation at the MEA gene. DME can excise 5-methylcytosine in vitro and when expressed in E. coli. Abasic sites opposite 5-methylcytosine inhibit DME activity and might prevent DME from generating double-stranded DNA breaks. Unexpectedly, paternal-allele silencing is not controlled by DNA methylation. Rather, Polycomb group proteins that are expressed from the maternal genome, including MEA, control paternal MEA silencing. Thus, DME establishes MEA imprinting by removing 5-methylcytosine to activate the maternal allele. MEA imprinting is subsequently maintained in the endosperm by maternal MEA silencing the paternal allele.
| S-EPMC4106368 | biostudies-literature