MassIVE MSV000090784

Partial Public PXD038355

EOMES establishes mesoderm and endoderm differentiation potential through SWI/SNF-mediated global enhancer remodeling

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Description

In this study we analysed the dynamic changes in the epigenetic landscape during the first embryonic lineage decision following pluripotency exit. At gastrulation, mouse pluripotent epiblast cells segregate towards mesoderm and endoderm (ME) or neuroectoderm (NE). The analysis of chromatin accessibility, histone modifications and accompanied gene expression confirmed a bias of the epigenetic landscape towards NE fate while ME cis regulatory elements (CREs) are becoming accessible and active during lineage specification. This is driven by the binding of two T-box transcription factors (TFs) EOMES and TBXT. We aimed to characterise interaction partners of EOMES and TBXT while bound on chromatin during gastrulation via ChIP-MS approach. We detected the ATP-dependent chromatin remodelling complex SWI/SNF as interaction partner of the TFs to modulate CREs accessibility. For ChIP-MS, mouse embryonic stem cells deficient for both T-box TFs Eomes and Tbxt (dKO), which express GFP-tagged EOMES or TBXT from a dox-inducible locus (dKOEoGFP or dKOTbxtGFP), were differentiated for 4 days as embryoid bodies (EBs) under Activin A treatment. EBs were singularised and cross-linked with formaldehyde for 8 min. The chromatin fraction was isolated and fragmented using sonication. Proteins precipitated with anti-GFP anti-body (ab290, abcam) were digested using trypsin, and desalted in HyperSep spin columns. Uninduced dKOEoGFP or dKOTbxtGFP EBs (nodox) were used as controls. Pulldowns were done for three biological replicates. Desalted peptides were analysed by LC-MS/MS with a Q-Exactive Plus mass spectrometer (Thermo Scientific, San Jose, CA) coupled to an EASY-nLCTM 1000 UHPLC system (Thermo Scientific). [doi:10.25345/C5KH0F404] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Gastrulation ; ChIP-MS ; Differentiation ; T-box transcription factors

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Principal Investigators:
(in alphabetical order) 		Prof. Dr. Sebastian Arnold, University of Freiburg, Germany
Submitting User: ChiaraSchroeder
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