Project description:Mouse embryonic stem cells (ESCs) primed for differentiation display dynamic heterogeneity characterised by stochastic switching between transcriptional states, and recent advances have highlighted the heterogeneous and dynamic nature of DNA methylation in these. Using single cell sequencing we report global oscillations in ESC DNA methylation that affect particularly CpG poor regions including distal enhancers. These oscillations are dependent on DNA methylation turnover by Dnmt3 and Tet enzymes and influence the probability of transcriptional state switching. Our observations suggest that regulated DNA methylation heterogeneity may contribute to lineage priming in cells poised for differentiation.
Project description:Mouse embryonic stem cells (ESCs) primed for differentiation display dynamic heterogeneity characterised by stochastic switching between transcriptional states, and recent advances have highlighted the heterogeneous and dynamic nature of DNA methylation in these. Using single cell sequencing we report global oscillations in ESC DNA methylation that affect particularly CpG poor regions including distal enhancers. These oscillations are dependent on DNA methylation turnover by Dnmt3 and Tet enzymes and influence the probability of transcriptional state switching. Our observations suggest that regulated DNA methylation heterogeneity may contribute to lineage priming in cells poised for differentiation.
Project description:Mouse embryonic stem cells (ESCs) primed for differentiation display dynamic heterogeneity characterised by stochastic switching between transcriptional states, and recent advances have highlighted the heterogeneous and dynamic nature of DNA methylation in these. Using single cell sequencing we report global oscillations in ESC DNA methylation that affect particularly CpG poor regions including distal enhancers. These oscillations are dependent on DNA methylation turnover by Dnmt3 and Tet enzymes and influence the probability of transcriptional state switching. Our observations suggest that regulated DNA methylation heterogeneity may contribute to lineage priming in cells poised for differentiation.
Project description:Mouse embryonic stem cells (ESCs) primed for differentiation display dynamic heterogeneity characterised by stochastic switching between transcriptional states, and recent advances have highlighted the heterogeneous and dynamic nature of DNA methylation in these. Using single cell sequencing we report global oscillations in ESC DNA methylation that affect particularly CpG poor regions including distal enhancers. These oscillations are dependent on DNA methylation turnover by Dnmt3 and Tet enzymes and influence the probability of transcriptional state switching. Our observations suggest that regulated DNA methylation heterogeneity may contribute to lineage priming in cells poised for differentiation.
Project description:We report a method for specific capture of an arbitrary subset of genomic targets for single molecule bisulfite sequencing, and for digital quantitation of DNA methylation at a single nucleotide resolution. We used targeted bisulfite sequencing to characterize the changes of DNA methylation during the de-differentiation of human fibroblasts into hybrid stem cells, and into induced pluripotent stem cells. We compared the methylation level of approximately 66,000 CpG sites within 2020 CpG islands on chromosome 12, chromosome 20, and 34 selected regions. A total of 288 differentially methylated regions were identified between fibroblasts and pluripotent cells. Methylation cluster analysis revealed distinct methylation patterns between fibroblasts and pluripotent cells. Furthermore iPS cells are globally more methylated than human embryonic stem cells, which could be due to the reprogramming process. This targeted bisulfite sequencing method is particularly useful for efficient and large-scale analysis of DNA methylation in organisms with large genomes. Experiment Overall Design: Comparison of DNA methylation on 2020 CpG islands and 34 other selected regions among eleven human ES, iPS and fibroblast lines.
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.
Project description:We report a method for specific capture of an arbitrary subset of genomic targets for single molecule bisulfite sequencing, and for digital quantitation of DNA methylation at a single nucleotide resolution. We used targeted bisulfite sequencing to characterize the changes of DNA methylation during the de-differentiation of human fibroblasts into hybrid stem cells, and into induced pluripotent stem cells. We compared the methylation level of approximately 66,000 CpG sites within 2020 CpG islands on chromosome 12, chromosome 20, and 34 selected regions. A total of 288 differentially methylated regions were identified between fibroblasts and pluripotent cells. Methylation cluster analysis revealed distinct methylation patterns between fibroblasts and pluripotent cells. Furthermore iPS cells are globally more methylated than human embryonic stem cells, which could be due to the reprogramming process. This targeted bisulfite sequencing method is particularly useful for efficient and large-scale analysis of DNA methylation in organisms with large genomes.