Embryonic stem cells (ESCs) are unique in that they have the capacity to differentiate into all of the cell types in the body. We know a lot about the complex transcriptional control circuits that maintain the naive pluripotent state under self-renewing conditions but comparatively less about how cells exit from this state in response to differentiation stimuli. Here, we examined the role of Otx2 in this process in mouse ESCs and demonstrate that it plays a leading role in remodeling the gene re ...[more]
Project description:Gene expression was studied in the presence or absence of Otx2 (RNAi) in the Rex1GFPd2 mouse embryonic stem cell line (parental line; E14Tg2a) in undifferentiated and differentiated states.
Project description:Mouse embryonic stem cells were maintained in minimal and chemically-defined culture conditions supporting naive pluripotency. Inhibitors of the Gsk3 (CHIR99021) and Mek/Erk (PD0325901) pathways were withdrawn, cultures maintained for 24 hours, and subsequently sorted by flow cytometry based on fluorescence of a short-half-life Rex1(Zfp42)::GFP reporter into two populations: Rex1-high cells, functionally capable of reversion to naive pluripotency, and Rex1-low cells that have exited the naive state.
Project description:The bHLH transcription factor Tfe3 is a powerful regulator of pluripotency and we report a genome-wide analysis of Tfe3 occupancy in mouse ES cells. Nuclear localization of Tfe3 is inhibited by a protein complex containing the tumor-suppressor Folliculin (Flcn) and we also determine Tfe3 binding sites in ES cells expressing an shRNA targeting Flcn. Specificity is controlled for by using unspecific IgGs and ES cells expressing an shRNA targeting Tfe3. ChIP-Seq profiling of Tfe3 in ES cells
Project description:Gene regulatory elements such as enhancers have profound effects on cellular function, health, and disease. Our understanding of mammalian enhancer function is limited by the lack of a technology that would allow for a rapid and thorough test of their cell type-specific function. Here, we describe a novel Cas9-effector system that enables rapid testing and functional annotation of native enhancers in embryonic stem cells. Total RNA obtained from R26 dCas9-effector mESC lines after viral delivery of sgRNAs directed against proximal promoter or enhancer regions