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.

DNA methylation is important for the silencing of transposons and other repetitive elements in many higher eukaryotes. However, plant and mammalian genomes have evolved to contain repetitive elements near or inside their genes. How these genes are kept from being silenced by DNA methylation is not well understood. A forward genetics screen led to the identification of the putative chromatin regulator Enhanced Downy Mildew 2 (EDM2) as a cellular antisilencing factor and regulator of genome DNA methylation patterns. EDM2 contains a composite Plant Homeo Domain that recognizes both active and repressive histone methylation marks at the intronic repeat elements in genes such as the Histone 3 lysine 9 demethylase gene Increase in BONSAI Methylation 1 (IBM1) and is necessary for maintaining the expression of these genes by promoting mRNA distal polyadenylation. Because of its role in maintaining IBM1 expression, EDM2 is required for preventing CHG methylation in the bodies of thousands of genes. Our results thus increase the understanding of antisilencing, genome methylation patterns, and regulation of alternative RNA processing by intronic heterochromatin.

DNA methylation is a critical, dynamically regulated epigenetic mark. Small chemicals can be valuable tools in probing cellular processes, but the set of chemicals with broad effects on epigenetic regulation is very limited. Using the Arabidopsis thaliana repressor of silencing1 mutant, in which transgenes are transcriptionally silenced, we performed chemical genetic screens and found sulfamethazine (SMZ) as a chemical suppressor of epigenetic silencing. SMZ treatment released the silencing of transgenes as well as endogenous transposons and other repetitive elements. Plants treated with SMZ exhibit substantially reduced levels of DNA methylation and histone H3 Lys-9 dimethylation, but heterochromatic siRNA levels were not affected. SMZ is a structural analog and competitive antagonist to p-aminobenzoic acid (PABA), which is a precursor of folates. SMZ decreased the plant folate pool size and caused methyl deficiency, as demonstrated by reductions in S-adenosylmethionine levels and in global DNA methylation. Exogenous application of PABA or compounds downstream in the folate biosynthesis pathway restored transcriptional silencing in SMZ-treated plants. Together, our results revealed a novel type of chemical suppressor of epigenetic silencing, which may serve as a valuable tool for studying the roles and mechanisms of epigenetic regulation and underscores an important linkage between primary metabolism and epigenetic gene regulation.

DNA 5-methylcytosine (5-meC) is an important epigenetic mark for transcriptional gene silencing in many eukaryotes. In Arabidopsis, 5-meC DNA glycosylase/lyases actively remove 5-meC to counteract transcriptional gene silencing in a locus-specific manner, and have been suggested to maintain the expression of transposons. However, it is unclear whether plant DNA demethylases can promote the transposition of transposons. Here we report the functional characterization of the DNA glycosylase/lyase DNG701 in rice. DNG701 encodes a large (1,812 amino acid residues) DNA glycosylase domain protein. Recombinant DNG701 protein showed 5-meC DNA glycosylase and lyase activities in vitro. Knockout or knockdown of DNG701 in rice plants led to DNA hypermethylation and reduced expression of the retrotransposon Tos17. Tos17 showed less transposition in calli derived from dng701 knockout mutant seeds compared with that in wild-type calli. Overexpression of DNG701 in both rice calli and transgenic plants substantially reduced DNA methylation levels of Tos17 and enhanced its expression. The overexpression also led to more frequent transposition of Tos17 in calli. Our results demonstrate that rice DNG701 is a 5-meC DNA glycosylase/lyase responsible for the demethylation of Tos17 and this DNA demethylase plays a critical role in promoting Tos17 transposition in rice calli.Oryza sativa | incision activity | Tos17 copy number I n eukaryotes, DNA cytosine methylation at carbon 5 of the pyrimidine ring [5-methylcytosine (5-meC)] is an important epigenetic mark that contributes to gene silencing and plays critical roles in development and genome defense against viruses, transposons, and transgenes (1-3). Heavy cytosine methylation usually occurs at heterochromatin and at regions rich in transposons and repetitive DNA (2, 4). Cytosine methylation of DNA, however, is reversible through demethylation (5, 6). DNA demethylation can be passive or active, and active DNA demethylation is catalyzed by one or more enzymes to remove methylated cytosines and can occur independently of DNA replication (7,8). So far, several models have been proposed to explain mechanisms of DNA demethylation in animals (7,8). One of the models suggests that active DNA demethylation is mediated at least in part by a base excision repair (BER) pathway where the AID/Apobec family of deaminases convert 5-meC to T followed by G/T mismatch repair through the DNA glycosylase MBD4 or TDG with the involvement of Gadd45a (7-9). Recently, a new study suggested that 5-meC hydroxylase TET1 promotes DNA demethylation in mammalian cells through a process that requires the BER pathway (10). Nevertheless, many aspects of these models have not been confirmed, and how DNA demethylation is carried out in animals remains controversial (7,8).In contrast to the uncertainty about the mechanism of DNA demethylation in animals, mechanisms of DNA demethylation in plants are much clearer and widely accepted. In the model plant Arabidopsis, research showed that the 5-meC DNA glycosylase/ lyase-me...

The cyclins together with highly conserved cyclin-dependent kinases regulate cell cycle progression in plants. Although extensive and systematic study on cell cycle mechanisms and cyclin functions in yeasts and animals has been carried out, only a small number of plant cyclins have been characterized and classified functionally and phylogenetically. We identified several types of cyclin genes in the rice genome and characterized them by phylogenetic, tandem and segmental duplications analyses. Our results indicated that there were at least 49 predicted rice cyclin genes in the rice genome, and they were distributed on 12 chromosomes. Of these cyclins, one possessed only cyclin_C domain and no cyclin_N domain, and the remaining 48 cyclins with cyclin_N domains were classified as nine types based on evolutionary relationships. Eight of these nine types were common between rice and Arabidopsis, whereas only one, known as F-type cyclins, was unique to rice. No homologues of the F-type cyclins in plants could be retrieved from the public databases, and reverse transcription-PCR analysis supported an existence of the F-type cyclin genes. Sequence alignment suggested that the cyclin genes in the rice genome experienced a mass of gene tandem and segmental duplications occurred on seven chromosomes related to the origins of new cyclin genes. Our study provided an opportunity to facilitate assessment and classification of new members, serving as a guide for further functional elucidation of rice cyclins.

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Arabidopsis Senescence-Associated Subtilisin Protease (SASP) has previously been reported to participate in leaf senescence and in the development of inflorescences and siliques. Here, we describe a new role of SASP in the regulation of abscisic acid (ABA) signaling. SASP encodes a subtilase and its expression was considerably induced by darkness, ABA, and ethylene treatments. sasp knockout mutants displayed obvious developmental phenotypes such as early flowering and smaller leaves. In particular, the sasp mutants exhibited enhanced ABA sensitivity during seed germination and seedling growth, heightened ABA-mediated leaf senescence, and increased production of reactive oxygen species (ROS). Importantly, the sasp mutants also showed remarkably increased tolerance to drought, with expression of six ABA signaling-related genes being either up- or down-regulated following ABA treatment. Interaction assays demonstrated that SASP physically interacts with OPEN STOMATA 1 (OST1) at the cell periphery. Co-expression of SASP and OST1 led to degradation of OST1, whereas this degradation was impaired in sasp-1 protoplasts. ROS attenuation assays demonstrated that in sasp-1 mutant guard cells the attenuation rate markedly decreased. Taken together, the results demonstrate that SASP plays an important role in regulating ABA signaling and drought tolerance through interaction with OST1.

The availability of diversified germplasm resources is the most important for developing improved rice varieties with higher seed yield or tolerance to various biotic or abiotic stresses. Here we report an efficient tool to create increased variations in rice by maize Ac/Ds transposon (a gene trap system) insertion mutagenesis. We have generated around 20,000 Ds insertion rice lines of which majority are homozygous for Ds element. We subjected these lines to phenotypic and abiotic stress screens and evaluated these lines with respect to their seed yields and other agronomic traits as well as their tolerance to drought, salinity and cold. Based on this evaluation, we observed that random Ds insertions into rice genome have led to diverse variations including a range of morphological and conditional phenotypes. Such differences in phenotype among these lines were accompanied by differential gene expression revealed by GUS histochemical staining of gene trapped lines. Among the various phenotypes identified, some Ds lines showed significantly higher grain yield compared to wild-type plants under normal growth conditions indicating that rice could be improved in grain yield by disrupting certain endogenous genes. In addition, several 1,000s of Ds lines were subjected to abiotic stresses to identify conditional mutants. Subsequent to these screens, over 800 lines responsive to drought, salinity or cold stress were obtained, suggesting that rice has the genetic potential to survive under abiotic stresses when appropriate endogenous genes were suppressed. The mutant lines that have higher seed yielding potential or display higher tolerance to abiotic stresses may be used for rice breeding by conventional backcrossing combining with molecular marker-assisted selection. In addition, by exploiting the behavior of Ds to leave footprints upon remobilization, we have shown an alternative strategy to develop new rice varieties without foreign DNA sequences in their genome.