FBXL10 and the Protection of Polycomb-bound Genes from Hypermethylation (RRBS)
ABSTRACT: Nearly all CpG-dense promoters are occupied by the multi-domain chromosomal protein FBXL10. We show here that complete inactivation of the Fbxl10 gene leads to dense de novo methylation only of the promoters that are co-occupied by both FBXL10 and by Polycomb Repressive Complexes; this results in pervasive defects in embryonic development and death of homozygous Fbxl10 mutant embryos at midgestation. Deletion of key components of Polycomb Repressive Complexes 1 and 2 did not lead to ectopic de novo methylation. These results indicate that FBXL10 defends Polycomb-occupied promoters against ectopic de novo methylation. FBXL10 is the first reported factor whose loss leads to a gain in genomic DNA methylation. DNA methylation analysis using RRBS and expression analysis using RNA-seq was performed on WT and Fbxl10T/T ES cells.
Project description:Nearly all CpG-dense promoters are occupied by the multi-domain chromosomal protein FBXL10. We show here that complete inactivation of the Fbxl10 gene leads to dense de novo methylation only of the promoters that are co-occupied by both FBXL10 and by Polycomb Repressive Complexes; this results in pervasive defects in embryonic development and death of homozygous Fbxl10 mutant embryos at midgestation. Deletion of key components of Polycomb Repressive Complexes 1 and 2 did not lead to ectopic de novo methylation. These results indicate that FBXL10 defends Polycomb-occupied promoters against ectopic de novo methylation. FBXL10 is the first reported factor whose loss leads to a gain in genomic DNA methylation. DNA methylation analysis using RRBS and expression analysis using RNA-seq was performed on WT and Fbxl10T/T ES cells.
Project description:We provide quantitative maps of cytosine methylation at single base resolution by RRBS in E13.5 primordial germ cells and adult sperm purified from mice exposed to Vinclozolin. Pregnant F0 female mice were exposed to two doses (a low dose VD1=1 mg/kg bw/d and a high dose VD2=100mg/kg bw/d) of Vinclozolin in the drinking water during pregnancy. We isolated primordial germ cells at E13.5 and mature sperm from adult F1 males obtained from control and exposed mothers. For PGCs, we sequenced RRBS libraries prepared from one pool isolated from F1 embryos exposed to the low dose, one pool isolated from F1 embryos exposed to the high dose, and two control pools isolated from unexposed embryos. For sperm, we sequenced three RRBS libraries prepared from a pool of sperm isolated from F1 males exposed to the high dose, and five RRBS libraries prepared from control pools isolated from unexposed animals.
Project description:We quantified the targets and kinetics of DNA methylation acquisition in mouse embryos, and determined the contribution of the de novo methyltransferases DNMT3A and DNMT3B to this process. We provide single-base maps of cytosine methylation by RRBS from the blastocysts to post-implantation stages and in embryos lacking DNMT3A or DNMT3B activity, and performed RNA-Seq in embryos lacking DNMT3B activity. We sequenced RRBS libraries prepared from genomic DNA isolated from embryos at consecutive stages of development between E3.5 and E11.5,and adult differentiated cells (sperm, liver). We performed RRBS on blastocysts at E3.5/E4.5, dissected epiblasts at E5.5/E6.5/E7/5, whole embryos at E8.5/E10.5 and limbs at E11.5. RRBS experiments in Dnmt3a-/- and Dnmt3b-/- embryos were performed in biological duplicates on individual embryos. We sequenced RNA-Seq libraries prepared from total RNAs of three WT and Dnmt3b-/- littermate embryos collected at E8.5.
Project description:DNA methylation is an epigenetic modification associated with transcriptional repression of promoters and is essential for mammalian development. Establishment of DNA methylation is mediated by the de novo DNA methyltransferases DNMT3A and DNMT3B, whereas DNMT1 ensures maintenance of methylation through replication. Absence of these enzymes is lethal, and somatic mutations in these genes have been associated with several human diseases. How genomic DNA methylation patterns are regulated remains poorly understood, as the mechanisms that guide recruitment and activity of DNMTs in vivo are largely unknown. To gain insights into this matter we determined chromosomal binding and site-specific activity of the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B. We show that both enzymes localize to methylated, CpG dense regions in mouse stem cells, yet are excluded from active promoters and enhancers. By specifically measuring sites of de novo methylation, we observe that enzymatic activity reflects chromosomal binding. De novo methylation increases with CpG density, yet is excluded from nucleosomes. Notably, we observed selective binding of DNMT3B to the bodies of transcribed genes, which leads to their preferential methylation. This targeting to transcribed sequences requires SETD2-mediated methylation of lysine 36 on histone H3 and a functional PWWP domain of DNMT3B. Together these findings reveal how sequence and chromatin cues guide de novo methyltransferase activity to ensure methylome integrity. Whole-genome bisulfite sequencing for Dnmt1,3a,3b-triple-KO ES cells expressing DNMT3A2 or DNMT3B1 and for Dnmt1,3a,3b,Setd2-KO ES cells expressing DNMT3B1
Project description:The heterotic hybrid offspring of Arabidopsis accessions C24 and Landsberg erecta have altered methylomes. Changes occur most frequently at loci where parental methylation levels are different. There are context-specific biases in the non-additive methylation patterns with mCG generally increased and mCHH decreased relative to the parents. These changes are a result of two main mechanisms, Trans Chromosomal Methylation (TCM) and Trans Chromosomal deMethylation (TCdM), where the methylation level of one parental allele alters to resemble that of the other parent. Regions of altered methylation are enriched around genic regions and are often correlated with changes in siRNA levels. We identified examples of genes with altered expression likely to be due to methylation changes and suggest that in crosses between the C24 and Ler accessions, epigenetic controls can be important in the generation of altered transcription levels which may contribute to the increased biomass of the hybrids. C24, Ler, and C24 x Ler
Project description:Seasonal photoperiodic changes have strong impact on development in Nasonia vitripennis. Here, Using high-throughput Reduced Representation Bisulfite Sequencing (RRBS) and single-molecule-based sequencing, we generated DNA methylation maps of female wasps maintained in long vs short day. We have identified differential methylated loci that encode the photoperiodic change. analysis of DNA methylation in female wasps maintained in long vs short day, using RRBS followed by Illumina sequencing
Project description:We measured the effect of methylation potential decrease, characteristic to the betaine homocysteine methyl-transferase (Bhmt) null mice, on liver DNA methylation patterns at 4 weeks. We used reduced representation bisulfite sequencing (RRBS) to measure DNA methylation differences in the livers of Bhmt-null and wild type mice (n= 8/group). We filtered sequencing data for CpGs with at least 10x coverage.
Project description:Obesity is a metabolic disease caused by environmental, genetic, and epigenetic factors. However, the epigenetic mechanisms of obesity are incompletely understood. The aim of our study was to identify skeletal muscle DNA methylation patterns in obesity. Muscle biopsies were obtained basally from lean (n=11) and obese (n=9) participants in combination with euglycemic hyperinsulinemic clamps to assess insulin sensitivity. We performed reduced representation bisulfite sequencing next generation methylation analysis on DNA isolated from vastus lateralis muscle biopsies.
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:More than 2x10E9 sequences made on Illumina platform derived from the genome of E14 embryonic stem cells cultured in our laboratory were used to build a database of about 2.7x10E6 single nucleotide variant. The database was validated using other two sequencing datasets from other laboratory and high overlap was observed. The identified variant are enriched on intergenic regions, but several thousands reside on gene exons and regulatory regions, such as promoters, enhancers, splicing site and untranslated regions of RNA, thus indicating high probability of an important functional impact on the molecular biology of this cells. We created a new E14 genome assembly including the new identified variants and used it to map reads from next generation sequencing data generated in our laboratory or in others on E14 cell line. We observed an increase in the number of mapped reads of about 5%. CpG dinucleotide showed the higher variation frequency, probably because of it could be target of DNA methylation. We performed a reduced representation bisulfite sequencing on E14 cell line to test our new genome assembly with respect to the mm9 genome reference. After mapping and methylation status calling, we obtained an increase of about 120,000 called CpG and we avoided about 20,000 wrong CpG calling. genotyping of E14 embryonic stem cells (ESCs) and Reduced representation Bisulfite Sequencing (RRBS) of E14 ESCs.