CyMATE: a new tool for methylation analysis of plant genomic DNA after bisulphite sequencing

Hetzl, Jennifer; Foerster, Andrea M.; Raidl, Günther R.; Scheid, Ortrun Mittelsten

doi:10.1111/j.1365-313x.2007.03152.x

Cited by 192 publications

(164 citation statements)

References 35 publications

(43 reference statements)

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“…PCR products were cloned into pGEM-T-Easy (Promega) and sequenced. Methylation was analyzed using CyMATE (Hetzl et al 2007) and graphed using a custom Perl script and Microsoft Excel.…”

Section: Cytosine Methylation Analysesmentioning

confidence: 99%

Subnuclear partitioning of rRNA genes between the nucleolus and nucleoplasm reflects alternative epiallelic states

et al. 2013

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Eukaryotes can have thousands of 45S ribosomal RNA (rRNA) genes, many of which are silenced during development. Using fluorescence-activated sorting techniques, we show that active rRNA genes in Arabidopsis thaliana are present within sorted nucleoli, whereas silenced rRNA genes are excluded. DNA methyltransferase (met1), histone deacetylase (hda6), or chromatin assembly (caf1) mutants that disrupt silencing abrogate this nucleoplasmic-nucleolar partitioning. Bisulfite sequencing data indicate that active nucleolar rRNA genes are nearly completely demethylated at promoter CGs, whereas silenced genes are nearly fully methylated. Collectively, the data reveal that rRNA genes occupy distinct but changeable nuclear territories according to their epigenetic state.

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“…PCR products were cloned into pGEM-T-Easy (Promega) and sequenced. Methylation was analyzed using CyMATE (Hetzl et al 2007) and graphed using a custom Perl script and Microsoft Excel.…”

Section: Cytosine Methylation Analysesmentioning

confidence: 99%

Subnuclear partitioning of rRNA genes between the nucleolus and nucleoplasm reflects alternative epiallelic states

et al. 2013

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“…The number of clones analysed is listed in Supplementary Table S4. The sequences were aligned and statistically evaluated using the CyMATE software (Hetzl et al, 2007).…”

Section: Bisulfite Sequencingmentioning

confidence: 99%

Silenced rRNA genes are activated and substitute for partially eliminated active homeologs in the recently formed allotetraploid, Tragopogon mirus (Asteraceae)

et al. 2014

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To study the relationship between uniparental rDNA (encoding 18S, 5.8S and 26S ribosomal RNA) silencing (nucleolar dominance) and rRNA gene dosage, we studied a recently emerged (within the last 80 years) allotetraploid Tragopogon mirus (2n = 24), formed from the diploid progenitors T. dubius (2n = 12, D-genome donor) and T. porrifolius (2n = 12, P-genome donor). Here, we used molecular, cytogenetic and genomic approaches to analyse rRNA gene activity in two sibling T. mirus plants (33A and 33B) with widely different rRNA gene dosages. Plant 33B had~400 rRNA genes at the D-genome locus, which is typical for T. mirus, accounting for~25% of total rDNA. We observed characteristic expression dominance of T. dubius-origin genes in all organs. Its sister plant 33A harboured a homozygous macrodeletion that reduced the number of T. dubius-origin genes to about 70 copies (~4% of total rDNA). It showed biparental rDNA expression in root, flower and callus, but not in leaf where D-genome rDNA dominance was maintained. There was upregulation of minor rDNA variants in some tissues. The RNA polymerase I promoters of reactivated T. porrifolius-origin rRNA genes showed reduced DNA methylation, mainly at symmetrical CG and CHG nucleotide motifs. We hypothesise that active, decondensed rDNA units are most likely to be deleted via recombination. The silenced homeologs could be used as a 'first reserve' to ameliorate mutational damage and contribute to evolutionary success of polyploids. Deletion and reactivation cycles may lead to bidirectional homogenisation of rRNA arrays in the long term.

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“…An equivalent amount of DNA from blastocysts mixed with lambda DNA was treated in parallel for comparison. The data obtained by bisulphite sequencing were analysed using the CyMATE program (Hetzl et al, 2007) to determine the positions and levels of non-CpG methylation. The rate of experimental errors caused by PCR and sequencing was estimated using the observed frequency of thymine to cytosine transition (oocytes, 0.22%; sperm, 0.18%; blastocysts, 0.19%; 12.5 dpc embryos, 0.20%), and was used for data normalisation.…”

Section: Non-cpg Methylation Analysismentioning

confidence: 99%

Dynamic stage-specific changes in imprinted differentially methylated regions during early mammalian development and prevalence of non-CpG methylation in oocytes

et al. 2011

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SUMMARYMammalian imprinted genes are associated with differentially methylated regions (DMRs) that are CpG methylated on one of the two parental chromosomes. In mice, at least 21 DMRs acquire differential methylation in the germline and many of them act as imprint centres. We previously reported the physical extents of differential methylation at 15 DMRs in mouse embryos at 12.5 days postcoitum. To reveal the ontogeny of differential methylation, we determined and compared methylation patterns of the corresponding regions in sperm and oocytes. We found that the extent of the gametic DMRs differs significantly from that of the embryonic DMRs, especially in the case of paternal gametic DMRs. These results suggest that the gametic DMR sequences should be used to extract the features specifying methylation imprint establishment in the germline: from this analysis, we noted that the maternal gametic DMRs appear as unmethylated islands in male germ cells, which suggests a novel component in the mechanism of gamete-specific marking. Analysis of selected DMRs in blastocysts revealed dynamic changes in allelic methylation in early development, indicating that DMRs are not fully protected from the major epigenetic reprogramming events occurring during preimplantation development. Furthermore, we observed non-CpG methylation in oocytes, but not in sperm, which disappeared by the blastocyst stage. Non-CpG methylation was frequently found at maternally methylated DMRs as well as non-DMR regions, suggesting its prevalence in the oocyte genome. These results provide evidence for a unique methylation profile in oocytes and reveal the surprisingly dynamic nature of DMRs in the early embryo.

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CyMATE: a new tool for methylation analysis of plant genomic DNA after bisulphite sequencing

Cited by 192 publications

References 35 publications

Subnuclear partitioning of rRNA genes between the nucleolus and nucleoplasm reflects alternative epiallelic states

Subnuclear partitioning of rRNA genes between the nucleolus and nucleoplasm reflects alternative epiallelic states

Silenced rRNA genes are activated and substitute for partially eliminated active homeologs in the recently formed allotetraploid, Tragopogon mirus (Asteraceae)

Dynamic stage-specific changes in imprinted differentially methylated regions during early mammalian development and prevalence of non-CpG methylation in oocytes

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