Evolutionary consequences of DNA methylation on the GC content in vertebrate genomes
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ABSTRACT: The genomes of many vertebrates show a characteristic variation in GC content. To explain its origin and evolution mainly three mechanisms have been proposed, selection for GC content, mutation bias and GC-biased gene conversion. At present the mechanism of GC-biased gene conversion, i.e. short-scale, unidirectional exchanges between homologous chromosomes in the neighborhood of recombination-initiating double-strand breaks in favor for GC nucleotides, is the most widely accepted hypothesis. We here suggest that DNA methylation also plays an important role in the evolution of GC content in vertebrate genomes. To test this hypothesis we investigated one mammalian (human; GSE30340) and one avian (chicken) genome. We used bisulfite sequencing to generate a whole-genome methylation map of chicken sperm. Human processed data files (spermdonor1, #reads>=1) were downloaded from the NGSmethDB database (http://bioinfo2.ugr.es/NGSmethDB/database.php). Inclusion of these methylation maps into a model of GC content evolution provided significant support for the impact of DNA methylation on the local equilibrium GC content. Moreover, two different estimates of equilibrium GC content, one which neglects and one which incorporates the impact of DNA methylation and the concomitant CpG hypermutability, give estimates that differ about 15% in both genomes, arguing for a strong impact of DNA methylation on the evolution of GC content. Thus, our results put forward that previous estimates of equilibrium GC content, which neglect the hypermutability of CpG dinucleotides, need to be reevaluated. Genomic DNA from chicken mature sperm was isolated, bisulfite converted and sequenced on a Illumina HiSeq instrument
Project description:The genomes of many vertebrates show a characteristic variation in GC content. To explain its origin and evolution mainly three mechanisms have been proposed, selection for GC content, mutation bias and GC-biased gene conversion. At present the mechanism of GC-biased gene conversion, i.e. short-scale, unidirectional exchanges between homologous chromosomes in the neighborhood of recombination-initiating double-strand breaks in favor for GC nucleotides, is the most widely accepted hypothesis. We here suggest that DNA methylation also plays an important role in the evolution of GC content in vertebrate genomes. To test this hypothesis we investigated one mammalian (human; GSE30340) and one avian (chicken) genome. We used bisulfite sequencing to generate a whole-genome methylation map of chicken sperm. Human processed data files (spermdonor1, #reads>=1) were downloaded from the NGSmethDB database (http://bioinfo2.ugr.es/NGSmethDB/database.php). Inclusion of these methylation maps into a model of GC content evolution provided significant support for the impact of DNA methylation on the local equilibrium GC content. Moreover, two different estimates of equilibrium GC content, one which neglects and one which incorporates the impact of DNA methylation and the concomitant CpG hypermutability, give estimates that differ about 15% in both genomes, arguing for a strong impact of DNA methylation on the evolution of GC content. Thus, our results put forward that previous estimates of equilibrium GC content, which neglect the hypermutability of CpG dinucleotides, need to be reevaluated.
Project description:DNA methylation and the Polycomb Repression System are epigenetic mechanisms that play important roles in maintaining transcriptional repression. Recent evidence suggests that DNA methylation can attenuate the binding of Polycomb protein components to chromatin and thus plays a role in determining their genomic targeting. However, whether this role of DNA methylation is important in the context of transcriptional regulation is unclear. By genome-wide mapping of the Polycomb Repressive Complex 2 (PRC2)-signature histone mark, H3K27me3, in severely DNA hypomethylated mouse somatic cells, we show that hypomethylation leads to widespread H3K27me3 redistribution, in a manner that reflects the local DNA methylation status in wild-type cells. Unexpectedly, we observe striking loss of H3K27me3 and PRC2 from Polycomb-target gene promoters in DNA hypomethylated cells, including Hox gene clusters. Importantly, we show that many of these genes become ectopically expressed in DNA hypomethylated cells, consistent with loss of Polycomb-mediated repression. An intact DNA methylome is required for appropriate Polycomb-mediated gene repression by constraining PRC2 targeting. These observations identify a previously unappreciated role for DNA methylation in gene regulation and therefore influence our understanding of how this epigenetic mechanism contributes to normal development and disease. comparison of Dnmt1+/+ vs Dnmt1-/- mouse embryonic fibroblasts by Reduced Representation Bisulfite Sequencing
Project description:Stochastic changes in cytosine methylation are a source of heritable epigenetic and phenotypic diversity in plants. Using the model plant Arabidopsis thaliana, we derive robust estimates of the rate at which methylation is spontaneously gained (forward epimutation) or lost (backward epimutation) at individual cytosines and construct a comprehensive picture of the epimutation landscape in this species. We demonstrate that the dynamic interplay between forward and backward epimutations is modulated by genomic context and show that subtle contextual differences have profoundly shaped patterns of methylation diversity in A. thaliana natural populations over evolutionary timescales. Theoretical arguments indicate that the epimutation rates reported here are high enough to rapidly uncouple genetic from epigenetic variation, but low enough for new epialleles to sustain long-term selection responses. Our results provide new insights into methylome evolution and its population-level consequences. MethylC-seq of Arabidopsis thaliana
Project description:High-throughput sequencing of small RNAs in chicken somite embryos Dissected tissue white leghorn chicken embryos was disaggregated and RNA extracted using the miRVana kit (Ambion). Small RNA fraction between 19 and 24 nt was isolated from 15% denaturing polyacrylamide gel and 15 micro gram was ligated to Solexa adaptors (Illumina) without de-phosphorylating and re-phosphorylating. The short RNAs were converted to DNA by RT-PCR following the Illumina protocol.
Project description:The goal of this study was to compare the host's response (chicken tracheal epithelium) to laboratory attenuated vaccine versus pathogenic strains of M. gallisepticum
Project description:Morphological characters are the result of developmental gene expression. Hence the identity of a character is ultimately grounded in the gene regulatory network directing development and thus whole genome gene expression data can provide evidence about character identity. Here we use transcriptomic data to address one of the most enduring paradoxes in evolutionary biology, the identity of the avian wing digits. Deep Sequencing of mRNA from embryonic chicken digits is performed and the gene expression profiles are analyzed. Analysis of mRNA-seq data from 16 samples of chicken digits covering two embryonic stages.
Project description:All large-scale LC-MS/MS post-translational methylation site discovery experiments require methylpeptide spectrum matches (methyl-PSMs) to be identified at acceptably low false discovery rates (FDRs). To meet estimated methyl-PSM FDRs, methyl-PSM filtering criteria are often determined using the target-decoy approach. The efficacy of this methyl-PSM filtering approach has, however, yet to be thoroughly evaluated. Here we conduct a systematic analysis of methyl-PSM FDRs across a range of sample preparation workflows (each differing in their exposure to the alcohols methanol and isopropanol) and mass spectrometric instrument platforms (each employing a different mode of MS/MS dissociation). Through 13CD3-methionine labeling (heavy-methyl SILAC) of S. cerevisiae cells and in-depth manual data inspection, accurate lists of true positive methyl-PSMs were determined, allowing methyl-PSM FDRs to be compared to target-decoy approach-derived methyl-PSM FDR estimates. These results show that global FDR estimates produce extremely unreliable methyl-PSM filtering criteria; we demonstrate that this is an unavoidable consequence of the high number of amino acid combinations capable of producing peptide sequences that are isobaric to methylated peptides of a different sequence. Separate methyl-PSM FDR estimates were also found to be unreliable due to prevalent sources of false positive methyl-PSMs that produce high peptide identity score distributions. Incorrect methylation site localizations, peptides containing cysteinyl-S-β-propionamide, and methylated glutamic or aspartic acid residues can partially, but not wholly, account for these false positive methyl-PSMs. Together these results indicate that the target-decoy approach is an unreliable means of estimating methyl-PSM FDRs and methyl-PSM filtering criteria. We suggest that orthogonal methylpeptide validation (e.g. heavy-methyl SILAC or its offshoots) should be considered a prerequisite for obtaining high confidence methyl-PSMs in large-scale LC-MS/MS methylation site discovery experiments, and make recommendations on how to reduce methyl-PSM FDRs in samples not amenable to heavy isotope labeling.
Project description:Methylation is a common post-translational modification of lysine and arginine in eukaryotic proteins. To date, these methylomes are best characterised in model organisms and higher eukaryotes. Herein, we integrated bioinformatics, proteomics and high-content drug-screening to comprehensively explore protein methylation in the human gastrointestinal parasite and deep-branching eukaryote, Giardia duodenalis. We demonstrate Giardia and other Diplomonadida species lack arginine-methyltransferases and have remodelled RGG/RG motifs preferred by these enzymes. Further, Giardia had no detectable methylarginine in vitro, demonstrating the first eukaryote with no arginine methylome. In contrast, we performed detailed curation of 11 putative lysine-methyltransferases, including highly-diverged SET-domain proteins, and novel annotations demonstrating conserved eEF1a methyllysine. We identified >200 high-confidence methyllysine sites, highlighting methyllysine within coiled-coil features, and for Giardia cytoskeletal regulation. Lastly, using known methylation-inhibitors, we demonstrate inhibition of methylation plays a key role in replication and cyst formation in this parasite.
Project description:Protein methylation catalyzed by SAM-dependent methyltransferase represents a major PTM involved in important biological processes. Because methylation can occur on nitrogen, oxygen and sulfur centers and multiple methylation states exist on the nitrogen centers, methylproteome remains poorly documented. Here we present the methylation by isotope labeled SAM (MILS) strategy for a highly-confident analysis of the methylproteome of the SAM-auxotrophic Saccharomyces cerevisiae based on the online multidimensional μHPLC/MS/MS technology. We identified 117 methylated proteins, containing 182 methylation events associated with 174 methylation sites. About 90% of these methylation events were previously unknown. Our results indicated, 1) over 6% of the yeast proteome are methylated, 2) the amino acid residue preference of protein methylation follows the order Lys >> Arg > Asp > Glu ≈ Gln ≈ Asp > His > Cys, 3) the methylation state on nitrogen center is largely exclusive, and 4) the methylated proteins are located mainly in nucleus/ribosome associated with translation/transcription and DNA/RNA processing. Our dataset is the most comprehensive methylproteome known-to-date of all living organisms, and should significantly contribute to the field of protein methylation and related research.