Project description:ChIP-seq for H3K27me3 and Ring1B was performed in WT mESCs and mESCs containing catalytically inactive Ring1B (I53A mutant). Cells expressing catalytically inactive Ring1B maintain the spatial distribution of Ring1B and H3K27me3 but at reduced levels. These findings support the notion that PRC2 recruitment is, in part, dependent on H2A ubiquitination (H2AK119ub).
Project description:ChIP-seq for H3K27me3 and Ring1B was performed in WT mESCs and mESCs containing catalytically inactive Ring1B (I53A mutant). Cells expressing catalytically inactive Ring1B maintain the spatial distribution of Ring1B and H3K27me3 but at reduced levels. These findings support the notion that PRC2 recruitment is, in part, dependent on H2A ubiquitination (H2AK119ub). Two biological replicates were performed for Ring1B and H3K27me3 ChIPs in WT and Ring1B I53A/I53A mouse ESCs. Input chromatin was sequenced for each replicate as a control for ChIP enrichment.
Project description:Epigenetic memory in the form of cytosine methylation is essential for vertebrate development and the formation of cellular identity. Active removal of DNA methylation by the action of the TET hydroxylase family helps enable developmental potency, both in vivo and during creation of induced pluripotent stem cells. Despite this, little is known about how TET proteins are targeted to DNA. We report that mammalian TET enzymes show strong preference (>200 fold) for oxidising certain CG-containing hexamers in vitro, and during global methylation reprogramming in cultured cells and during embryogenesis. These preferred sequences, and also the most poorly targeted motifs, constitute recognition sites for developmental transcription factors whose binding activity is sensitive to DNA methylation. X-ray structural analysis and molecular dynamics simulations suggest that TET proteins use indirect readout to sense the sequence context flanking CG sites. These results are significant for understanding epigenetic reprogramming during development and the biological role TET enzymes play in modulating epigenetic memory.
Project description:TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic domain of mammalian TET enzymes favor CGs embedded within bHLH and bZIP transcription factor binding sites, with up to 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intrasubstrate interactions and CG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germline. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and OCT4, respectively, illuminating TET function in transcriptional responses and pluripotency support.