Excision of 5-hydroxymethylcytosine by DEMETER family DNA glycosylases.
ABSTRACT: In plants and animals, 5-methylcytosine (5mC) serves as an epigenetic mark to repress gene expression, playing critical roles for cellular differentiation and transposon silencing. Mammals also have 5-hydroxymethylcytosine (5hmC), resulting from hydroxylation of 5mC by TET family-enzymes. 5hmC is abundant in mouse Purkinje neurons and embryonic stem cells, and regarded as an important intermediate for active DNA demethylation in mammals. However, the presence of 5hmC in plants has not been clearly demonstrated. In Arabidopsis, the DEMETER (DME) family DNA glycosylases efficiently remove 5mC, which results in DNA demethylation and transcriptional activation of target genes. Here we show that DME and ROS1 have a significant 5hmC excision activity in vitro, although we detected no 5hmC in Arabidopsis, suggesting that it is very unlikely for plants to utilize 5hmC as a DNA demethylation intermediate. Our results indicate that both plants and animals have 5mC in common but DNA demethylation systems have independently evolved with distinct mechanisms.
Project description:DNA methylation is a prominent epigenetic modification in plants and animals regulated by similar mechanisms but the process of DNA demethylation is profoundly different. Unlike vertebrates that require a series of enzymatic conversions of 5-methylcytosine (5mC) into other bases for DNA demethylation, plants utilize the DEMETER (DME) family of 5mC DNA glycosylases to catalyze a direct removal of 5mC from DNA. Here we introduced Arabidopsis DME into human HEK-293T cells to allow direct 5mC excision, and observed that direct DNA demethylation activity was successfully implemented by DME expression. In addition, DME induced diverse cellular responses such as cell proliferation inhibition, cell cycle dysregulation and S phase arrest. Microarray and methylome analyses revealed that DME upregulated a number of genes including cell cycle components, heat shock proteins, and notably, various interferon-stimulated genes. Moreover, DME-mediated DNA demethylation activated endogenous repeat elements, which are likely to form dsRNAs as viral mimics and eventually trigger interferon cascades to establish the antiviral state. This work demonstrates that plant DNA demethylase catalyzes DNA demethylation with a bypass of initial base conversion steps, and the interferon signaling plays a pivotal role to alleviate genotoxic stresses associated with DME-induced DNA demethylation in mammalian cells.
Project description:Methylation of cytosine to 5-methylcytosine (5mC) is important for gene expression, gene imprinting, X-chromosome inactivation, and transposon silencing. Active demethylation in animals is believed to proceed by DNA glycosylase removal of deaminated or oxidized 5mC. In plants, 5mC is removed from the genome directly by the DEMETER (DME) family of DNA glycosylases. Arabidopsis thaliana DME excises 5mC to activate expression of maternally imprinted genes. Although the related Repressor of Silencing 1 (ROS1) enzyme has been characterized, the molecular basis for 5mC recognition by DME has not been investigated. Here, we present a structure-function analysis of DME and the related DME-like 3 (DML3) glycosylases for 5mC and its oxidized derivatives. Relative to 5mC, DME and DML3 exhibited robust activity toward 5-hydroxymethylcytosine, limited activity for 5-carboxylcytosine, and no activity for 5-formylcytosine. We used homology modeling and mutational analysis of base excision and DNA binding to identify residues important for recognition of 5mC within the context of DNA and inside the enzyme active site. Our results indicate that the 5mC binding pocket is composed of residues from discrete domains and is responsible for discrimination against 5mC derivatives, and suggest that DME, ROS1, and DML3 utilize subtly different mechanisms to probe the DNA duplex for cytosine modifications.
Project description: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.
Project description:DNA glycosylases initiate the base excision repair (BER) pathway by excising damaged, mismatched, or otherwise modified bases. Animals and plants independently evolved active BER-dependent DNA demethylation mechanisms important for epigenetic reprogramming. One such DNA demethylation mechanism is uniquely initiated in plants by DEMETER (DME)-class DNA glycosylases. Arabidopsis DME family glycosylases contain a conserved helix-hairpin-helix domain present in both prokaryotic and eukaryotic DNA glycosylases as well as two domains A and B of unknown function that are unique to this family. Here, we employed a mutagenesis approach to screen for DME residues critical for DNA glycosylase activity. This analysis revealed that amino acids clustered in all three domains, but not in the intervening variable regions, are required for in vitro 5-methylcytosine excision activity. Amino acids in domain A were found to be required for nonspecific DNA binding, a prerequisite for 5-methylcytosine excision. In addition, mutational analysis confirmed the importance of the iron-sulfur cluster motif to base excision activity. Thus, the DME DNA glycosylase has a unique structure composed of three essential domains that all function in 5-methylcytosine excision.
Project description:The Arabidopsis DEMETER (DME) DNA glycosylase is required for the maternal allele expression of imprinted Polycomb group (MEDEA and FIS2) and transcription factor (FWA) genes in the endosperm. Expression of DME in the central cell, not in pollen or stamen, establishes gene imprinting by hypomethylating maternal alleles. However, little is known about other genes regulated by DME. To identify putative DME target genes, we generated CaMV:DME plants which ectopically express DME in pollen and stamens. Comparison of mRNA profiles revealed 94 genes induced by ectopic DME expression in both stamen and pollen. Gene ontology analysis identified three molecular functions enriched in the DME-inducible RNA list: DNA or RNA binding, kinase activity, and transcription factor activity. Semi-quantitative RT-PCR verified the candidate genes identified by GeneChip analysis. The putative target genes identified in this study will provide insights into the regulatory mechanism of DME DNA glycosylase and the functions of DNA demethylation.
Project description:Cytosine methylation is an epigenetic mark that promotes gene silencing and plays important roles in development and genome defense against transposons. Methylation patterns are established and maintained by DNA methyltransferases that catalyze transfer of a methyl group from S-adenosyl-L-methionine to cytosine bases in DNA. Erasure of cytosine methylation occurs during development, but the enzymatic basis of active demethylation remains controversial. In Arabidopsis thaliana, DEMETER (DME) activates the maternal expression of two imprinted genes silenced by methylation, and REPRESSOR OF SILENCING 1 (ROS1) is required for release of transcriptional silencing of a hypermethylated transgene. DME and ROS1 encode two closely related DNA glycosylase domain proteins, but it is unknown whether they participate directly in a DNA demethylation process or counteract silencing through an indirect effect on chromatin structure. Here we show that DME and ROS1 catalyze the release of 5-methylcytosine (5-meC) from DNA by a glycosylase/lyase mechanism. Both enzymes also remove thymine, but not uracil, mismatched to guanine. DME and ROS1 show a preference for 5-meC over thymine in the symmetric dinucleotide CpG context, where most plant DNA methylation occurs. Nevertheless, they also have significant activity on both substrates at CpApG and asymmetric sequences, which are additional methylation targets in plant genomes. These findings suggest that a function of ROS1 and DME is to initiate erasure of 5-meC through a base excision repair process and provide strong biochemical evidence for the existence of an active DNA demethylation pathway in plants.
Project description: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, and how it is recruited to these differing chromatin landscapes is unknown. The C-terminal DME catalytic core consists of three conserved regions required for catalysis in vitro. We show that the catalytic core of DME guides active demethylation at endogenous targets, rescuing the developmental and genomic hypermethylation phenotypes of DME mutants. However, without the N-terminus, heterochromatin demethylation is significantly impeded, and abundant CG-methylated genic sequences are ectopically demethylated. We used comparative analysis to reveal that the conserved DME N-terminal domains are only present in the flowering plants, whereas the domain architecture of DME-like proteins in non-vascular plants mainly resembles the catalytic core, suggesting that it might represent the ancestral form of the 5mC DNA glycosylase found in all 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. Overall design: DNA methylation was measured in C-terminal DME-complemented (dme-2/dme-2; nDMECTD/nDMECTD) or full length DME-complemented (dme-2/dme-2; DMEFL/DMEFL) dme-2 Arabidopsis endosperm
Project description:The DEMETER (DME) DNA glycosylase initiates active DNA demethylation via the base-excision repair pathway and is vital for reproduction in Arabidopsis thaliana DME-mediated DNA demethylation is preferentially targeted to small, AT-rich, and nucleosome-depleted euchromatic transposable elements, influencing expression of adjacent genes and leading to imprinting in the endosperm. In the female gametophyte, DME expression and subsequent genome-wide DNA demethylation are confined to the companion cell of the egg, the central cell. Here, we show that, in the male gametophyte, DME expression is limited to the companion cell of sperm, the vegetative cell, and to a narrow window of time: immediately after separation of the companion cell lineage from the germline. We define transcriptional regulatory elements of DME using reporter genes, showing that a small region, which surprisingly lies within the DME gene, controls its expression in male and female companion cells. DME expression from this minimal promoter is sufficient to rescue seed abortion and the aberrant DNA methylome associated with the null dme-2 mutation. Within this minimal promoter, we found short, conserved enhancer sequences necessary for the transcriptional activities of DME and combined predicted binding motifs with published transcription factor binding coordinates to produce a list of candidate upstream pathway members in the genetic circuitry controlling DNA demethylation in gamete companion cells. These data show how DNA demethylation is regulated to facilitate endosperm gene imprinting and potential transgenerational epigenetic regulation, without subjecting the germline to potentially deleterious transposable element demethylation.
Project description:Patterns of DNA methylation, an important epigenetic modification involved in gene silencing and development, are disrupted in cancer cells. Understanding the functional significance of aberrant methylation in tumors remains challenging, due in part to the lack of suitable tools to actively modify methylation patterns. DNA demethylation caused by mammalian DNA methyltransferase inhibitors is transient and replication-dependent, whereas that induced by TET enzymes involves oxidized 5mC derivatives that perform poorly understood regulatory functions. Unlike animals, plants possess enzymes that directly excise unoxidized 5mC from DNA, allowing restoration of unmethylated C through base excision repair. Here, we show that expression of Arabidopsis 5mC DNA glycosylase DEMETER (DME) in colon cancer cells demethylates and reactivates hypermethylated silenced loci. Interestingly, DME expression causes genome-wide changes that include both DNA methylation losses and gains, and partially restores the methylation pattern observed in normal tissue. Furthermore, such methylome reprogramming is accompanied by altered cell cycle responses and increased sensibility to anti-tumor drugs, decreased ability to form colonospheres, and tumor growth impairment in vivo. Our study shows that it is possible to reprogram a human cancer DNA methylome by expression of a plant DNA demethylase.
Project description:DNA demethylases function in conjunction with DNA methyltransferases to modulate genomic DNA methylation levels in plants. The Arabidopsis genome contains four DNA demethylase genes, DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1) also known as DEMETER-LIKE 1 (DML1), DML2, and DML3. While ROS1, DML2, and DML3 were shown to function in disease response in somatic tissues, DME has been thought to function only in reproductive tissues to maintain the maternal-specific expression pattern of a subset of imprinted genes. Here we used promoter:?-glucuronidase (GUS) fusion constructs to show that DME is constitutively expressed throughout the plant, and that ROS1, DML2, and DML3 have tissue-specific expression patterns. Loss-of-function mutations in DME cause seed abortion and therefore viable DME mutants are not available for gene function analysis. We knocked down DME expression in a triple ros1 dml2 dml3 (rdd) mutant background using green tissue-specific expression of a hairpin RNA transgene (RNAi), generating a viable 'quadruple' demethylase mutant line. We show that this rdd DME RNAi line has enhanced disease susceptibility to Fusarium oxysporum infection compared to the rdd triple mutant. Furthermore, several defence-related genes, previously shown to be repressed in rdd, were further repressed in the rdd DME RNAi plants. DNA methylation analysis of two of these genes revealed increased differential promoter DNA methylation in rdd DME RNAi plants compared to WT, beyond the difference observed in the parental rdd plants. These results indicate that DME contributes to DNA demethylase activity and disease response in somatic tissues.