Identification of Multiple Proteins Coupling Transcriptional Regulation to Genome Stability in Arabidopsis thaliana
ABSTRACT: Eukaryotic genomes are heavily regulated by epigenetic marks that often act to modulate the transcriptional control of genetic elements. In Arabidopsis thaliana the ATXR5 and ATXR6 histone methyltransferases, and their cognate H3K27 monomethylation mark, act in transcriptional silencing while also maintaining genome stability by preventing generation of excess DNA corresponding to pericentromeric heterochromatin. In this study we characterize the atxr5 atxr6 transcriptome and its relationship to the DNA damage response which suggests that the atxr5 atxr6 transcriptional defects may be epistatic to the genome instability defects in the mutants. In addition we isolate several factors that modulate both the transcriptional and genomic instability phenotypes of atxr5 atxr6 mutants, which suggest a mechanism for atxr5 atxr6-induced extra DNA involving conflicts between the replicative and transcriptional processes in the cell. PolyA RNA sequencing (RNA-seq), whole-genome resequencing (DNA-seq), and whole-genome bisulfite sequencing (methyl-seq) was performed on Arabidospsis thaliana mutant and wildtype plants. DNA-seq was used to characterize DNA copy number and map EMS-induced mutations, RNA-seq was used to quantify transcript abundance and map EMS-induced mutations, and methyl-seq was used to assess DNA methylation. Details of the relationship between samples in this series and figures in the associated manuscript can be found in Supplemental Table 4 of the associated manuscript. Unless otherwise noted in the description all lines are ecotype Columbia, and all genotypes should be assumed homozygous unless otherwise indicated with a '/'.
Project description:The earliest stages of Huntington’s disease are marked by changes in gene expression that are caused in an indirect and poorly understood manner by polyglutamine expansions in the huntingtin protein (HTT). To explore the hypothesis DNA methylation may be altered in cells expressing mutated HTT, we use reduced-representation bisulfite sequencing (RRBS) to map sites of DNA methylation in cells carrying either wild-type or mutant HTT. We find that a large fraction of the genes that change in expression in the presence of mutant huntingtin demonstrate significant changes in DNA methylation. Regions with low CpG content, which have previously been shown to undergo methylation changes in response to neuronal activity, are disproportionately affected. Based on the sequence of regions that change in methylation, we identify AP-1 and SOX2 as transcriptional regulators associated with DNA methylation changes, and we confirm these hypotheses using genome-wide chromatin immunoprecipitation (ChIP-Seq). Our findings suggest new mechanisms for the effects of polyglutamine-expanded HTT. These results also raise important questions about the potential effects of changes in DNA methylation on neurogenesis and at later stages, cognitive decline in Huntington’s patients. RRBS in STHdhQ7/Q7 and STHdhQ111/Q111 cells
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).
Project description:Purpose: Description of a spike-adjusting-method (SAM) to normalize ChIP-seq data . Methods: We performed ChIP-seq of POLR3D and POLR2B with mouse liver supplemented with 2.5% of human DNA. Human DNA will be used as an internal control for ChIP-seq quantification. Results: We show that using the SAM for ChIP-seq quantification improve similarity of POLR3D and POLR2B ChIP-seq replicates samples and improve difference between samples originate from different conditions. Conclusions: The SAM improves comparison of ChIP-seq samples, either by increasing similarity between replicates or by emphasise differences between conditions. Chromatin Immuno-precipitations were performed with antibodies directed against POLR3D (Pol III) and POLR2B (Pol II) using mouse liver material supplemented with human DNA. Immuno-precipitated DNA was next sequenced using Illumina HiSeq. Three different concentrations of human spiked DNA were tested for the Pol III ChIP (2.5%, 5% and 10%). We also sequenced the corresponding inputs (crosslinked DNA from mouse liver). Two concentrations of human spiked DNA (5% and 10%) were tested for the Pol 2 ChIP. We also sequenced the corresponding inputs (crosslinked DNA from mouse liver).
Project description:There is increasing evidence that interindividual epigenetic variation is an etiological factor in common human diseases. Such epigenetic variation could be genetic or non-genetic in origin, and epigenome-wide association studies (EWASs) are underway for a wide variety of diseases/phenotypes. However, performing an EWAS is associated with a range of issues not typically encountered in genome-wide association studies (GWASs), such as the tissue to be analyzed. In many EWASs, it is not possible to analyze the target tissue in large numbers of live humans, and consequently surrogate tissues are employed, most commonly blood. But there is as yet no evidence demonstrating that blood is more informative than buccal cells, the other easily accessible tissue. To assess the potential of buccal cells for use in EWASs, we performed a comprehensive analysis of a buccal cell methylome using whole-genome bisulfite sequencing. Strikingly, a buccal vs. blood comparison reveals >6X as many hypomethylated regions in buccal. These tissue-specific differentially methylated regions (tDMRs) are strongly enriched for DNaseI hotspots. Almost 75% of these tDMRs are not captured by commonly used DNA methylome profiling platforms such as Reduced Representational Bisulfite Sequencing and the Illumina Infinium HumanMethylation450 BeadChip, and they also display distinct genomic properties. Buccal hypo-tDMRs show a statistically significant enrichment near SNPs associated to disease identified through GWASs. Finally, we find that, compared with blood, buccal hypo-tDMRs show significantly greater overlap with hypomethylated regions in other tissues. We propose that for non-blood based diseases/phenotypes, buccal will be a more informative tissue for EWASs. Buccal Profile generated from 14 Buccal Individuals
Project description:Bisulfite-seq data sets were generated for peripheral blood lymphocyte (PBL) and hair follicle (HF) DNA from each of two healthy males. Examination of genome-wide CpG methylation two tissues (hair follicle and peripheral blood lymphocyte) from 2 healthy male individuals.
Project description:Whole genome bisulfite sequencing was performed on Chromomethylase-2 and Dicer-like-3 knockout mutants in order to confirm the results from genome wide association mapping and to identify the respective genomic regions that they target. Bisulfite sequencing of knockout mutants and WT controls
Project description:Dnmt2 is a widely conserved protein, which is closely related to eukaryotic DNA methyltransferases. However, Dnmt2 shows a robust tRNA methyltransferase activity and only limited activity towards DNA. Interestingly, a recent study has provided evidence for a biologically important function of Dnmt2-dependent DNA methylation in the blood fluke Schistosoma mansoni, which seemed to contradict the weak activity of the enzyme in other organisms. We now used whole-genome bisulfite sequencing to comprehensively analyze the methylome of adult worms and could not detect any evidence for biologically relevant DNA methylation patterns. We also characterized the methylome of Drosophila melanogaster embryos and did not find any evidence for DNA methylation. Unconverted cytosine residues were detectable only at very low levels and shared many attributes with bisulfite deamination artifacts. Our results thus strongly argue against a DNA methyltransferase activity of Dnmt2 and suggest that Dnmt2-dependent phenotypes are caused by reduced tRNA methylation. Whole genome methylation analysis of S. mansoni. One sample was analyzed, containing DNA from adult male worms.
Project description:Dnmt2 is a widely conserved protein, which is closely related to eukaryotic DNA methyltransferases. However, Dnmt2 shows a robust tRNA methyltransferase activity and only limited activity towards DNA. Interestingly, a recent study has provided evidence for a biologically important function of Dnmt2-dependent DNA methylation in the blood fluke Schistosoma mansoni, which seemed to contradict the weak activity of the enzyme in other organisms. We now used whole-genome bisulfite sequencing to comprehensively analyze the methylome of adult worms and could not detect any evidence for biologically relevant DNA methylation patterns. We also characterized the methylome of Drosophila melanogaster embryos and did not find any evidence for DNA methylation. Unconverted cytosine residues were detectable only at very low levels and shared many attributes with bisulfite deamination artifacts. Our results thus strongly argue against a DNA methyltransferase activity of Dnmt2 and suggest that Dnmt2-dependent phenotypes are caused by reduced tRNA methylation. Whole genome methylation analysis of D. melanogaster. Two samples were analyzed, one sample containing DNA from WT embryos, one sample containing DNA from Dnmt2-/- embryos.
Project description:The results of the study uncover conserved features of cancer methylomes and provide a mechanistic explanation for the tumor-promoting effects of Dnmt3a mutations. Whole genome methylation analysis of Mus musculus. Five samples were analyzed, one control sample containing normal healthy lung tissue, two samples containing big (WTB) or small (WTS) Dnmt3a WT tumors, two samples containing big (KOB) or small (KOS) Dnmt3a knock-out tumors
Project description:Several organisms belonging to diverse animal groups have retained Dnmt2 as their only bona fide DNA methyltransferase gene. However, recent studies have shown that Dnmt2 functions as a tRNA methyltransferase, which prompted us to analyze the methylomes of Dnmt2-only organisms at single-base resolution. Using whole-genome bisulfite sequencing we show here that the genomes of Schistosoma mansoni and Drosophila melanogaster lack detectable DNA methylation patterns. Residual unconverted cytosine residues shared many attributes with bisulfite deamination artifacts and were observed at comparable levels in a Dnmt2-deficient fly strain. Furthermore, genetically modified mouse embryonic stem cells that had retained Dnmt2 as their only bona fide DNA methyltransferase gene, did not show any detectable DNA methylation patterns. Our results thus uncover fundamental differences among animal methylomes and suggest that Dnmt2-only organisms lack biologically relevant DNA methylation patterns. Whole methylome analysis of Mus musculus. One sample was analyzed containing DNA from Dnmt1-/-, Dnmt3a-/- and Dnmt3b-/- mice.