ABSTRACT: 5-methyl-cytosine DNA methylation regulates gene expression and developmental programming in a broad range of eukaryotes. However, its presence and potential roles in ciliates, complex single-celled eukaryotes with germline-somatic genome specialization via nuclear dimorphism, are largely uncharted. While canonical cytosine methyltransferases have not been discovered in published ciliate genomes, recent studies performed in the stichotrichous ciliate Oxytricha trifallax suggest de novo cytosine methylation during macronuclear development. In this study, we applied bisfulfite genome sequencing, DNA mass spectrometry and antibody-based fluorescence detection to investigate the presence of DNA methylation in Paramecium tetraurelia. While the antibody-based methods suggest cytosine methylation, DNA mass spectrometry and bisulfite sequencing reveal that levels are actually below the limit of detection. Our results suggest that Paramecium does not utilize 5-methyl-cytosine DNA methylation as an integral part of its epigenetic arsenal. Overall design: We performed bisulfite conversion and Illumina DNA sequencing on 3 samples: one from vegetative cells, one from starved vegetative cells, and one from cells undergoing macronuclear development.
Project description:We report the existence of both methylcytosine and hydroxymethylcytosine in the genomic DNA of the ciliate Oxytricha trifallax during its genome rearrangement process. These modifications are dynamically added, de novo, to sequences targeted for elimination and are not present after the rearrangement process (in vegetative cells). We performed methyl-DNA immunoprecipitation-sequencing (meDIP-seq) with antibodies against methylcytosine and hydroxymethylcytosine. We performed methyl-DNA immunoprecipitation-sequencing (meDIP-seq) with antibodies against methylcytosine and hydroxymethylcytosine, and used an IgG control for nonspecific immunoprecpitation. Immunoprecipitation was performed on both vegetative (negative control) and 46h cells (with methylation). The data were normalized to RPKM, then the number of vegetative reads was subtracted from the number of 46h reads, giving excess reads at 46h, which we denote as the methylation/hydroxymethylation "signal". Those data are reported in the tab-deliminated data files included with this dataset.
Project description:Transposable elements are entangled in a constant evolutionary arms race with their host genomes, constantly evolving ways to evade host silencing mechanisms. One silencing mechanism used by many distantly related eukaryotes is dependent on cytosine methylation, an epigenetic mark deposited by C5 cytosine methyltransferases (CMTs). Therefore, it is expected transposable elements would acquire mechanisms to escape from being targeted by cytosine methylation. Here we report how two distantly related eukaryotic lineages have incorporated CMTs into the coding regions of distinct retrotransposon classes. Three of these events have occurred in the dinoflagellates of the genus Symbiodinium, where these CMT-encoding retrotransposons show hundreds of insertions. In a case of convergent evolution, the charophyte Klebsormidium nitens shows an independent expansion of CMT encoding retrotransposons. Concomitantly, we find that Symbiodinium genomes show cytosine methylation patterns unlike any other eukaryote with most of the genome hypermethylated in CpGs, while targeted CH methylation accumulates on transposable elements. Similarly, K. nitens shows CHH and CHG targeted methylation on repressed transposable elements, while CpG methylation is concentrated in gene bodies and transposable elements. Furthermore, we demonstrate the ability of retrotransposon CMTs to de novo methylate CpGs, indicating a putative role in mimicking retrotranscribed DNA as host active genomic DNA. Our results show an unprecedented example of how retrotransposons incorporate host-derived genes involved in DNA methylation as a source of adaptation to their host epigenomic environments. Overall design: Profiling of gene expression and cytosine methylation of Symbiodinium kawagutii and Symbiodinium minutum in two temperature treatments. Profiling of cytosine methylation of Klebsormidium nitens in vegetative growth. Profiling of cytosine methylation of transformant budding yeast lines in duplicates.
Project description:Since the discovery that cytosine deoxynucleotides could be methylated (dC5m), not much is known in higher eukaryotes about modifications affecting other deoxynucleotides. Here, we now report that we detected N-6-methyl-deoxyadenosine (dA6m) not only in frog DNA, but also in other species including mouse and human. Our methylome analysis revealed that dA6m is widely distributed across the eukaryotic genome, is present in different cell types, but commonly depleted from gene exons. Since dA6m is a direct modification of DNA, it has the potential to generate a significant impact on different biological areas. Ultimately, our work shows that deoxycytidine modifications might not be the only ones in higher eukaryotes, suggesting that such direct DNA modifications might be more widespread than previously thought.
Project description:Cytosine methylation is an important epigenetic modification of DNA that is involved in genome defense and transcriptional regulation in eukaryotes. Contribution of DNA methylation to biology of microbial eukaryotes is largely unknown to date. Here we used RNA-seq to examine the impact of DNA methylation on transcriptional output in the genome of a model pathogenic fungus, Magnaporthe oryzae by comparing expression profiles of wild-type and methylation-deficient mutant strains. Overall design: Samples from wild type mycelia and two DNA methyltransferase absent isolates (dmt1 and dmt2)
Project description:Cytosine methylation is an important epigenetic modification of DNA that is involved in genome defense and transcriptional regulation in eukaryotes. Despite extensive efforts to understand genome-wide distribution and function of DNA methylation in mammals and plants, contribution of DNA methylation to biology of microbial eukaryotes is largely unknown to date. Here we used genetic manipulations and high-throughput bisulphite sequencing on the model plant pathogenic fungus, Magnaporthe oryzae to elucidate the dynamics and mechanics of DNA methylation during pathogenic development. Overall design: Samples from three different developmental satges (mycelia, conidia and appressorium) and two DNA methyltransferase absent isolates (dmt1 and dmt2)
Project description:Methylation of histone H3 lysine 9 (H3K9me) and small RNA are associated with constitutively silent chromatin in diverse eukaryotes including plants. In plants, silent transposons are also marked by cytosine methylation, especially at non-CpG sites. The transposon-specific non-CpG methylation in plants is controlled by small RNA and H3K9me. Although it is often assumed that small RNA directs H3K9me, interaction between small RNA and H3K9me has not been directly shown in plants. We have previously shown that a mutation in a chromatin remodeling gene DDM1 (decrease in DNA methylation) induces a global decrease as well as local increase of cytosine methylation and accumulation of small RNA in a locus called BONSAI. Here we show that the de novo BONSAI methylation does not depend on RNAi but depends on H3K9me. Notably, in mutant of H3K9 methylase gene KRYPTONITE or H3K9me-dependent DNA methylase gene CHROMOMETHYALSE3, the ddm1-induced de novo cytosine methylation was abolished for all three contexts, CpG, CpHpG, and CpHpH. Furthermore, RNAi mutants showed strong developmental defects when combined with ddm1 mutation. Our results revealed unexpected interactions of epigenetic modifications, which could be conserved among diverse eukaryotes. comparison of DNA methylation between WT, 2G ddm1 (2 replications), 8G ddm1 (2 replications), and 8G ddm1 kyp
Project description:Cytosine DNA methylation regulates the expression of eukaryotic genes and transposable elements. Methylation is copied by DNA methyltransferases after DNA replication, which results in faithful transmission of methylation patterns during cell division and, at least in flowering plants, across generations. Trans-generational inheritance is mediated by a small group of cells that includes gametes and their progenitors. However, methylation is usually analyzed in somatic tissues that do not contribute to the next generation, and the mechanisms of trans-generational inheritance are inferred from such studies. To gain a better understanding of how DNA methylation is inherited, we analyzed purified Arabidopsis thaliana sperm and vegetative cells – the cell types that comprise pollen – with mutations in the DRM, CMT2 and CMT3 methyltransferases. Overall design: We isolated sperm and vegetative nuclei from pollen of Col-0, and cmt2, cmt3, drm1 drm2 (d1d2), and h1.1 h1.2 (h1) mutant Arabidopsis thaliana plants, respectively. DNA is extracted from the isolated nuclei and used for bisulfite-sequencing.
Project description:Genome-wide analysis of histone modification (H2AZ, H3K27ac, H3K27me3, H3K36me3, H3K4me1, H3K4me2, H3K4me3 and H3K9me3), protein-DNA binding (TAF1, P300, Pou5f1 and Nanog), cytosine methylation and transcriptome data in mouse and human ES cells and pig iPS cells We generated histone modification data (H2AZ, H3K27ac, H3K27me3, H3K36me3, H3K4me1, H3K4me2, H3K4me3 and H3K9me3) and protein-DNA binding data (TAF1, P300, Pou5f1 and Nanog) using Chromatin Immunoprecipitation followed by short sequencing (ChIP-seq), cytosine methylation data using methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq) and DNA digestion by methyl-sensitive restriction enzymes followed by sequencing (MRE-seq), transcriptome data with RNA short sequencing (RNA-seq) in human embryonic stem cells, mouse embryonic stem cells, pig induced pluripotent stem cells and mouse embryonic stem cells under activin-A-induced-differentiation. Examination of 8 histone modifications, 4 protein-DNA binding, cytosine methylation and transcriptome in human embryonic stem cells, mouse embryonic stem cells, pig induced pluripotent stem cells and mouse embryonic stem cells under activin-A-induced-differentiation.
Project description:Blocking histone deacetylation with trichostatin A (TSA) or blocking cytosine methylation using 5-aza-2'-deoxycytosine (aza-dC) can derepress silenced genes in multicellular eukaryotes, including animals and plants. We questioned whether DNA methylation and histone deacetylation overlap in the regulation of endogenous plant genes by monitoring changes in expression of ~7800 Arabidopsis thaliana genes following treatment with azadC, TSA, or both chemicals together. RNA levels for ~4% of the genes were reproducibly changed 3-fold or more by at least one treatment. Distinct subsets of genes are up-regulated or down-regulated in response to aza-dC, TSA, or simultaneous treatment with both chemicals, with little overlap among subsets. Surprisingly, the microarray data indicate that TSA and aza-dC are often antagonistic rather than synergistic in their effects. Analysis of green fluorescent protein transgenic plants confirmed this finding, showing that TSA can block the up-regulation of silenced green fluorescent protein transgenes in response to aza-dC or a ddm1 (decrease in DNA methylation 1) mutation. Our results indicate that global inhibition of DNA methylation or histone deacetylation has complex, nonredundant effects for the majority of responsive genes and suggest that activation of some genes requires one or more TSA-sensitive deacetylation events in addition to cytosine demethylation.
Project description:Methylation of carbon 5 in cytosine (5-methylcytosine; m5C) is a well-characterized DNA modification, and is also predominantly reported in highly abundant noncoding RNAs, such as rRNA and tRNA, in both prokaryotes and eukaryotes. However, the distribution and biological functions of m5C in plant mRNAs remain largely unknown. Here we develop an m5C RNA immunoprecipitation followed by deep sequencing approach (m5C-RIP-seq) to achieve transcriptome-wide profiling of RNA m5C in Arabidopsis thaliana. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and dot blot analyses reveal a dynamic pattern of m5C mRNA modification in various tissues and at different developmental stages. m5C-RIP-seq analysis identifies 6,045 putative m5C peaks in 4,465 expressed genes in young seedlings. m5C is enriched in coding sequences with two peaks located immediately after start codons and before stop codons, and is associated with mRNAs with low translation activity. We further show that a RNA (cytosine-5)-methyltransferase, tRNA specific methyltransferase 4B (TRM4B), exhibits the m5C mRNA methyltransferase activity. Mutations in TRM4B display defects in root development and decreased m5C levels in root mRNA. Furthermore, TRM4B affects transcript levels of the genes involved in root development, which is positively correlated with their mRNA stability and m5C levels. Our results suggest that m5C in mRNA is a new epitranscriptome marker widely distributed in plant genes, and that regulation of this modification is an integral part of gene regulatory networks underlying plant development. Overall design: RNA-seq in Arabidopsis thaliana (Col-0) wild-type and trm4b-4, two replicates for each sample