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Molecular mechanism of action of plant DRM de novo DNA methyltransferases

ABSTRACT: DNA methylation is a conserved epigenetic gene regulation mechanism. DOMAINS REARRANGED METHYLTRANSFERASE (DRM) is a key de novo methyltransferase in plants, but how DRM acts mechanistically is poorly understood. Here, we report the crystal structure of the methyltransferase domain of tobacco DRM (NtDRM) and reveal a molecular basis for its rearranged structure. NtDRM forms a functional homo-dimer critical for catalytic activity. We also show that Arabidopsis DRM2 exists in complex with the siRNA effector ARGONAUTE4 (AGO4) and preferentially methylates one DNA strand, likely the strand acting as the template for non-coding Pol V RNA transcripts. This strand-biased DNA methylation is also positively correlated with strand-biased siRNA accumulation. These data suggest a model in which DRM2 is guided to target loci by AGO4-siRNA and involves base-pairing of associated siRNAs with nascent RNA transcripts.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE54944 | GEO | 2014/05/27

SECONDARY ACCESSION(S): PRJNA238084

REPOSITORIES: GEO

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Molecular mechanism of action of plant DRM de novo DNA methyltransferases

Project description:DNA methylation is a conserved epigenetic gene regulation mechanism. DOMAINS REARRANGED METHYLTRANSFERASE (DRM) is a key de novo methyltransferase in plants, but how DRM acts mechanistically is poorly understood. Here, we report the crystal structure of the methyltransferase domain of tobacco DRM (NtDRM) and reveal a molecular basis for its rearranged structure. NtDRM forms a functional homo-dimer critical for catalytic activity. We also show that Arabidopsis DRM2 exists in complex with the siRNA effector ARGONAUTE4 (AGO4) and preferentially methylates one DNA strand, likely the strand acting as the template for non-coding Pol V RNA transcripts. This strand-biased DNA methylation is also positively correlated with strand-biased siRNA accumulation. These data suggest a model in which DRM2 is guided to target loci by AGO4-siRNA and involves base-pairing of associated siRNAs with nascent RNA transcripts. Whole-genome bisulfite sequencing was done for a wildtype line (ecotype Col) as well as various transgenic lines in a drm2 mutant background (ecotype Col). Each transgenic line expressed a version of the DRM2 protein that was either wildtype or carried induced mutations in order to test the function of various domains in the DRM2 protein. Two sets of whole-genome bisulfite were performed (130615 or 131216) and comparisons were mainly done within sets although comparisons can also be done between sets. The drm2 mutant methylome was also analyzed in this study using a previously published whole-genome bisulfite library (GSE39901).

2014-05-27 | E-GEOD-54944 | biostudies-arrayexpress

Chromosome-biased binding and gene regulation by the C. elegans DRM complex

Project description:This SuperSeries is composed of the following subset Series: GSE28494: Germline and embryo gene expression of wild-type vs. mutants in lin-54, a component of the C. elegans DRM complex GSE28852: Chromosome-biased binding and gene reguation by the C. elegans DRM complex [ChIP-chip] Refer to individual Series

2011-04-27 | E-GEOD-28853 | biostudies-arrayexpress

RNA-directed DNA methylation involves co-transcriptional small RNA-guided slicing of Pol V transcripts in Arabidopsis.

Project description:Small RNAs regulate chromatin modifications such as DNA methylation and gene silencing across eukaryotic genomes. In plants, RNA-directed DNA methylation (RdDM) requires 24-nucleotide (nt) small RNAs (siRNAs) that bind ARGONAUTE4 (AGO4) and target genomic regions for silencing. It also requires non-coding RNAs transcribed by RNA POLYMERASE V (Pol V), that likely serve as scaffolds for binding of AGO4/siRNA complexes. Here we utilized a modified global nuclear run-on (GRO) protocol followed by deep sequencing to capture Pol V nascent transcripts genome-wide. We uncovered unique characteristics of Pol V RNAs, including a uracil (U) common at position 10. This uracil was complementary to the 5’ adenine found in many AGO4-bound 24-nt siRNAs and was eliminated in an siRNA-deficient mutant as well as in the ago4/6/9 triple mutant, suggesting that the +10U signature is due to siRNA-mediated co-transcriptional slicing of Pol V transcripts. Expressing wild-type AGO4 in ago4/6/9 was able to restore slicing of Pol V transcripts but a catalytically inactive AGO4 mutant did not correct the slicing defect. We also found that Pol V transcript slicing required the little understood elongation factor SPT5L. These results highlight the importance of Pol V transcript slicing in RNA-mediated transcriptional gene silencing, which is a conserved process in many eukaryotes.

2018-01-29 | GSE108078 | GEO

Chromatin and siRNA pathways cooperate to maintain DNA methylation

Project description:DNA methylation occurs at preferred sites in eukaryotes, although the basis for preference is not known. We use a microarray-based profiling method to explore the involvement of Arabidopsis CMT3 and DRM DNA methyltransferases, a histone H3 lysine-9 methyltransferase (KYP) and an Argonaute-related RNA silencing component (AGO4) in methylating target loci. We find that KYP targets are also CMT3 targets, suggesting that histone methylation maintains CNG methylation genome-wide. CMT3 and KYP targets show similar proximal distributions that corresponds to the overall distribution of transposable elements of all types, whereas DRM targets are distributed more distally along the chromosome. We find an inverse relationship between element size and loss of methylation in ago4 and drm mutants. Our results suggest that RNA-directed DNA methylation is required to silence isolated elements that may be too small to be maintained in a silent state by a chromatin-based mechanism. Thus, parallel pathways would be needed to maintain silencing of transposable elements. Keywords: Methylation profiling using Msp I enzyme

2005-10-25 | GSE3109 | GEO

Chromosome-biased binding and gene reguation by the C. elegans DRM complex [ChIP-chip]

Project description:DRM is a conserved transcription factor complex that includes E2F/DP and pRB family proteins and plays important roles in development and cancer. Here we analyze genome-wide binding and function of the C. elegans DRM subunit LIN-54. We demonstrate that LIN-54 DNA-binding activity is required for the DRM complex to efficiently bind and regulate target genes containing adjacent putative E2F/DP and LIN-54 binding sites. We show that LIN-54 binds to the promoters of genes involved in cell division, development, and reproduction, and acts differently in the germline versus the soma. The E2F/DP-LIN-54 binding motif, individual target genes, and overall DRM function are conserved among worms, flies, and humans. Despite this conservation, we discovered one striking feature of C. elegans DRM not shared in flies or humans: it is depleted from X chromosomes. We show that DRM binding, the E2F-LIN-54 hybrid motif, and LIN-54-regulated genes are all autosome-enriched.

2011-04-27 | GSE28852 | GEO

DRM complex mutant lin-54 vs. H3K36 methyltransferase mutant mes-4 vs. lin-54; mes-4 double mutant vs. wild type C.elegans germline

Project description:Here we uncover antagonistic regulation of transcript levels in the germline of Caenorhabditis elegans hermaphrodites. The histone methyltransferase MES-4 marks genes expressed in the germline with methylated Lys36 on histone H3 (H3K36me) and promotes their transcription; MES-4 also represses genes normally expressed in somatic cells and genes on the X chromosomes. The DRM complex, which includes E2F/DP and Retinoblastoma homologs, affects germline gene expression and prevents excessive repression of X-chromosome genes. Using genome-scale analyses of germline tissue, we show that common germline-expressed genes are activated by MES-4 and repressed by DRM, and that MES-4 and DRM co-bind many germline-expressed genes. Reciprocally, MES-4 represses and DRM activates a set of autosomal soma-expressed genes and overall X-chromosome gene expression. Mutations in mes-4 or the DRM subunit lin-54 oppositely skew target transcript levels and cause sterility; a double mutant restores near wild-type transcript levels and germ cell development. Together, “yin-yang” regulation by MES-4 and DRM ensures transcript levels appropriate for germ cell function, elicits robust but not excessive dampening of X-chromosome-wide transcription, and may poise genes for future expression changes. Our study reveals that conserved transcriptional regulators implicated in development and cancer counteract each other to fine-tune transcript dosage.

2013-11-05 | GSE52064 | GEO

DRM complex mutant lin-54 vs. H3K36 methyltransferase mutant mes-4 vs. lin-54; mes-4 double mutant vs. wild type C.elegans germline

Project description:Here we uncover antagonistic regulation of transcript levels in the germline of Caenorhabditis elegans hermaphrodites. The histone methyltransferase MES-4 marks genes expressed in the germline with methylated Lys36 on histone H3 (H3K36me) and promotes their transcription; MES-4 also represses genes normally expressed in somatic cells and genes on the X chromosomes. The DRM complex, which includes E2F/DP and Retinoblastoma homologs, affects germline gene expression and prevents excessive repression of X-chromosome genes. Using genome-scale analyses of germline tissue, we show that common germline-expressed genes are activated by MES-4 and repressed by DRM, and that MES-4 and DRM co-bind many germline-expressed genes. Reciprocally, MES-4 represses and DRM activates a set of autosomal soma-expressed genes and overall X-chromosome gene expression. Mutations in mes-4 or the DRM subunit lin-54 oppositely skew target transcript levels and cause sterility; a double mutant restores near wild-type transcript levels and germ cell development. Together, 'yin-yang' regulation by MES-4 and DRM ensures transcript levels appropriate for germ cell function, elicits robust but not excessive dampening of X-chromosome-wide transcription, and may poise genes for future expression changes. Our study reveals that conserved transcriptional regulators implicated in development and cancer counteract each other to fine-tune transcript dosage. We compared germline gene expression profile of wild-type N2 C. elegans, lin-54(n3423) M+Z- mutant, mes-4(ok2326) M+Z- mutant, and lin-54(n3423);mes-4(ok2326) M+Z-mutant grown at 20C. 50~70 Germlines were dissected from young adults (24hours after L4 stage), and region from the tip until late pachytene stage of meiosis were collected. Three biological replicates for each strain were performed.

2013-11-05 | E-GEOD-52064 | biostudies-arrayexpress

Chromosome-biased binding and gene reguation by the C. elegans DRM complex [ChIP-chip]

2011-04-27 | E-GEOD-28852 | biostudies-arrayexpress

RNA-directed DNA methylation involves co-transcriptional small RNA-guided slicing of Pol V transcripts in Arabidopsis

Project description:Small RNAs regulate chromatin modifications such as DNA methylation and gene silencing across eukaryotic genomes. In plants, RNA-directed DNA methylation (RdDM) requires 24-nucleotide (nt) small RNAs (siRNAs) that bind ARGONAUTE4 (AGO4) and target genomic regions for silencing. It also requires non-coding RNAs transcribed by RNA POLYMERASE V (Pol V), although their function is largely unknown. We utilized a modified global nuclear run-on (GRO) protocol followed by deep sequencing to capture Pol V nascent transcripts genome-wide. We uncovered unique characteristics of Pol V RNAs, including a uracil (U) common at position 10. This uracil was complementary to the 5’ adenine found in many AGO4-bound 24-nt siRNAs and was eliminated in an siRNA-deficient mutant. This indicates co-transcriptional slicing of Pol V transcripts by AGO4-bound siRNAs. Pol V transcript slicing also required the elongation factor SPT5L. These results highlight a novel step in RNA-mediated transcriptional gene silencing, which is a conserved process in many eukaryotes.

2018-01-29 | GSE100010 | GEO

Germline and embryo gene expression of wild-type vs. mutants in lin-54, a component of the C. elegans DRM complex

Project description:DRM is a conserved transcription factor complex that includes E2F/DP and pRB family proteins and plays important roles in development and cancer. Here we perform microarray expression profiling analysis of lin-54, a DNA-binding member of the DRM complex. To identify genes regulated by LIN-54 in soma and germline, we analyzed wild-type and lin-54 mutant C. elegans embryos and isolated germlines. We chose embryos because they consist primarily of somatic cells, at a developmental stage with both active cell divisions and dynamic developmental gene expression programs. Since lin-54 null animals are sterile, embryos were obtained from a strain carrying the partial loss-of-function allele lin-54(n2990). Germlines were dissected from lin-54(n3423) null adults that lack detectable transcript and protein. The results revealed conserved roles for DRM in regulating genes involved in cell division, development, and reproduction. We find LIN-54 promotes expression of reproduction genes in the germline, but prevents ectopic activation of germline-specific genes in embryonic soma. Strikingly, genomics and cytological analyses show that DRM binding, a DRM binding motif, and LIN-54-regulated genes are all autosome-enriched. One paradoxical exception occurs the germline, where DRM binds autosomes but genes down-regulated in DRM mutants are enriched on X chromosomes.

2011-04-09 | GSE28494 | GEO

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