Project description:RNA Sequencing of H1 WT hESCs, H1 QSER1 KO hESCs, H1 TET1 KO hESCs, H1 QSER1/TET1 DKO hESCs, WT Day10 embryoid bodies (EBs), QSER1 KO Day10 EBs, TET1 KO Day10 EBs, QSER1/TET1 DKO Day10 EBs, WT pancreatic progenitors (PP1), QSER1 KO PP1, TET1 KO PP1, and QSER1/TET1 DKO PP1. DNA methylation is essential to mammalian development, and dysregulation can cause serious pathological conditions. Key enzymes responsible for deposition and removal of DNA methylation are known, but how they cooperate to tightly regulate the methylation landscape remains a central question. Utilizing a knockin DNA methylation reporter, we performed a genome-wide CRISPR/Cas screen in human embryonic stem cells to discover DNA methylation regulators. The top screen hit was an uncharacterized gene QSER1, which proved to be a key guardian of bivalent promoters and poised enhancers of developmental genes, especially those residing in DNA methylation valleys (or canyons). We further demonstrate cooperation of QSER1 and TET1 through genetic and biochemical interactions to inhibit DNMT3-mediated de novo methylation and safeguard developmental programs.
Project description:DNA methylation is essential to mammalian development, and aberrant regulation can lead to disease. The enzymes responsible for deposition and removal of DNA methylation have been identified, but how tightly regulated DNA methylation patterns are achieved globally remains a central question. To discover regulators of DNA methylation in human Embryonic Stem Cells (hESCs), we engineered a DNA methylation reporter line and performed a genome-wide CRISPR/Cas screen. Through our screen, we identified the functionally uncharacterized gene QSER1, which proved to be essential for protection from DNA hypermethylation at bivalent promoters and poised enhancers of developmental genes, especially those residing in DNA Methylation Valleys (DMVs). Further mechanistic enquiry revealed that QSER1 protein depends on TET1 for efficient recruitment to DNA, and QSER1 occupancy inhibits the binding of DNMT3B. Our discovery of QSER1 is a major advance in understanding TET-dependent protection from DNA hypermethylation and provides mechanistic insight into how epigenetic factors can cooperate to target locus-specific regulation.
Project description:The TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine, which can lead to DNA demethylation. However, direct connections between TET-mediated DNA demethylation and transcriptional output are difficult to establish due to challenges of distinguishing global versus locus-specific effects. Here we show that TET1/2/3 triple knockout (TKO) human embryonic stem cells (hESCs) exhibit preferential hypermethylation at bivalent promoters without corresponding gene expression changes in undifferentiated hESCs. In the absence of the TET proteins, abnormal accumulation of DNMT3B at bivalent promoters results in hypermethylation and impaired gene activation upon differentiation. Broadly, the competitive balance between the TET proteins and de novo methyltransferases at bivalent promoters could facilitate rapid changes of their methylation state to either activate or silence transcription in a cell-lineage and gene dependent manner.
Project description:Mammalian somatic cells can be directly reprogrammed into induced pluripotent stem cells (iPSCs) by introducing defined sets of transcription factors. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem (ES) cells. Human ES cells contain 5-hydroxymethylcytosine (5hmC), which is generated though the oxidation of 5-methylcytosine (5mC) by the TET family of enzymes. Here we show that 5hmC level increases significantly during reprogramming due to the activation of TET1. During this process, dynamic genome-wide 5hmC modification occurs across the genome with more modifications at telomere-proximal regions. Compared with hES cells, we found iPS cells tend to form large-scale (100kb-1.3Mb) aberrant reprogramming hotspots in subtelomeric regions, most of which display incomplete hydroxymethylation. Strikingly, these 5hmC aberrant hotspots largely coincide (>80%) with previously reported aberrant non-CG methylation regions. Our results suggest that 5hmC modification could play important roles during reprogramming to pluripotency, and contribute to the differences between iPSCs and hESCs. we generated comprehensive genome-wide profiles of 5hmC in somatic cells, iPS cell lines derived from a variety of origins, and multiple hES cell lines.
Project description:Mammalian somatic cells can be directly reprogrammed into induced pluripotent stem cells (iPSCs) by introducing defined sets of transcription factors. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem (ES) cells. Human ES cells contain 5-hydroxymethylcytosine (5hmC), which is generated though the oxidation of 5-methylcytosine (5mC) by the TET family of enzymes. Here we show that 5hmC level increases significantly during reprogramming due to the activation of TET1. During this process, dynamic genome-wide 5hmC modification occurs across the genome with more modifications at telomere-proximal regions. Compared with hES cells, we found iPS cells tend to form large-scale (100kb-1.3Mb) aberrant reprogramming hotspots in subtelomeric regions, most of which display incomplete hydroxymethylation. Strikingly, these 5hmC aberrant hotspots largely coincide (>80%) with previously reported aberrant non-CG methylation regions. Our results suggest that 5hmC modification could play important roles during reprogramming to pluripotency, and contribute to the differences between iPSCs and hESCs.
Project description:Ten-eleven translocation (Tet) hydroxylases (Tet1-3) oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). In neurons increased 5hmC levels within gene bodies correlate positively with gene expression. Here, we studied 5hmC profiles (hMeDIP) during retinal maturation between postnatal week 2 and postnatal week 3 using HiSeq 2000 instrument. Study of retinal 5hmC profile dynamics in 2-week old and 3-week old wild type (WT) mouse.
Project description:5-hydroxymethylcytosine (5hmC) is the first oxidative product of the TET-mediated 5-methylcytosine (5mC) demethylation pathway. It is a key intermediate in cytosine demethylation, and have potential regulatory functions with emerging importance in mammalian biology. In this work, we used a chemical capture-based technique that coupled with next-generation sequencing to investigate the global 5hmC methylation in five brain subregions (cerebellum, cortex, hippocampus, hypothalamus and thalamus) and liver tissues from female and male adult mice. We also performed total RNA sequencing to study the association between 5hmC and gene expression. The enriched 5-hmC library was sequenced on a HiSeq2500 by paired-end sequencing with 100 bp read length.