Project description:Understanding the impact of DNA methylation within different disease contexts often requires accurate assessment of these modifications in a genome-wide fashion. Frequently, patient-derived tissue stored in long-term hospital tissue banks have been preserved using formalin-fixation paraffin-embedding (FFPE). While these samples can comprise valuable resources for studying disease, the fixation process ultimately compromises the DNA’s integrity and leads to degradation. Degraded DNA can complicate CpG methylome profiling using traditional techniques, particularly when performing methylation sensitive restriction enzyme sequencing (MRE-seq), yielding high backgrounds and resulting in lowered library complexity. Here, we provide results using our new MRE-seq protocol (Capture MRE-seq), tailored to preserving unmethylated CpG information when using samples with highly degraded DNA. The results using Capture MRE-seq correlate well (0.92) with traditional MRE-seq calls when profiling non-degraded samples, and can recover unmethylated regions in highly degraded samples when traditional MRE-seq fails, which we validate using bisulfite sequencing-based data (WGBS) as well as methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq).
Project description:Genome-wide maps of cytosine methylation, cytosine hydroxylmethylation and small non coding RNAs in mouse ES cells and upon guided differentiation to mesoendoderm cells. Mouse embryonic stem cells (E14) were guided differentiated into mesoendoderm lineages by activin-A induction. cells in three time points (day0, day4 and day6) were collected. The genome-wide studies on three cell types were summerized as following: cytosine methylation data were generated using methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq) and DNA digestion by methyl-sensitive restriction enzymes followed by sequencing (MRE-seq); DNA product for 5-hmC_ChIP-seq is generated by a selctive chemical labeling method (Nat. Biotechnol. 2011, 29, 68-72). E14 Day0 data for MRE-seq and MeDIP-seq are released first in previous publication and included in prior series GSE36114 ChIP-seq, 5-hmC-seq, MeDIP-Seq, MRE-Seq, ncRNA-Seq, and RNA-seq on activin-induced differentiating ES cells at 3 time points.
Project description:The induction of pluripotency or trans-differentiation of one cell type to another can be accomplished with cell lineage-specific transcription factors. Here we report that repression of a single RNA binding protein PTB, which occurs during normal brain development via the action of miR-124, is sufficient to induce trans-differentiation of fibroblasts into functional neurons. Besides its traditional role in regulated splicing, we show that PTB has a previously undocumented function in the regulation of microRNA functions, suppressing or enhancing microRNA targeting by competitive binding on target mRNA or altering local RNA secondary structure. A key event during neuronal induction is the relief of PTB-mediated blockage of microRNA action on multiple components of the REST complex, thereby de-repressing a large array of neuronal genes, including miR-124 and multiple neuronal-specific transcription factors, in non-neuronal cells. This converts a negative feedback loop to a positive one to elicit cellular reprogramming to the neuronal lineage. Examination of PTB regulated AGO2/microRNA targeting in Hela cells by CLIP-seq (two biological replicates) , paired-end RNA-seq (control and PTB knockdown) and 3’end stability RNA-seq (control and PTB knockdown)
Project description:Genome-wide maps of cytosine methylation, cytosine hydroxylmethylation and small non coding RNAs in mouse ES cells and upon guided differentiation to mesoendoderm cells. Mouse embryonic stem cells (E14) were guided differentiated into mesoendoderm lineages by activin-A induction. cells in three time points (day0, day4 and day6) were collected. The genome-wide studies on three cell types were summerized as following: cytosine methylation data were generated using methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq) and DNA digestion by methyl-sensitive restriction enzymes followed by sequencing (MRE-seq); DNA product for 5-hmC_ChIP-seq is generated by a selctive chemical labeling method (Nat. Biotechnol. 2011, 29, 68-72). E14 Day0 data for MRE-seq and MeDIP-seq are released first in previous publication and included in prior series GSE36114
Project description:Neuronal microRNAs, miR-9/9* and miR-124 (miR-9/9*-124), exert reprogramming activities to direct cell-fate conversion of adult human fibroblasts to post-mitotic neurons and enable the generation of discrete neuronal subtypes with additional transcription factors. Previously, the molecular events underlying the neurogenic switch mediated by microRNAs during neuronal reprogramming were unknown. Here, we systematically dissected the neurogenic state induced by miR-9/9*-124 alone and reveal the surprising capability of miR-9/9*-124 in coordinately stimulating the reconfiguration of chromatin accessibilities, DNA methylation and transcriptome, leading to the generation of functionally excitable neurons, yet unbiased towards a particular subtype-lineage. We show that the microRNA-induced neuronal state enables additional transcription factors, ISL1 and LHX3, to selectively commit conversion to a highly homogenous population of human spinal cord motor neurons. Taken together, our study reveals a modular synergism between microRNAs and transcription factors that allows lineage-specific neuronal reprogramming, providing a platform for generating distinct subtypes of human neurons.