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

ABSTRACT: 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).

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Prof. Dr. Sebastian Arnold

PROVIDER: MSV000090784 | MassIVE | Fri Nov 25 01:00:00 GMT 2022

SECONDARY ACCESSION(S): PXD038355

REPOSITORIES: MassIVE

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Project description:Epigenetic priming factors establish a permissive epigenetic landscape which is not required until a later developmental or physiological time point, temporally uncoupling the presence of these factors with their phenotypic effects. One classic example of epigenetic priming is in the context of bivalent chromatin, found in pluripotent stem cells and early embryos at key developmental gene promoters marked by both activating-associated H3K4me3 and repressive-associated H3K27me3 histone modifications. It is currently unknown how these bivalent domains are targeted, or precisely how they impact on lineage commitment. Here we show that the small heterodimerising non-enzymatic DNA binding proteins Developmental Pluripotency Associated 2 (Dppa2) and 4 (Dppa4) act as epigenetic priming factors to establish bivalency at a subset of developmental genes. Dppa2/4 localise to the +1 nucleosome position of bivalent genes and while they are not required for pluripotency in embryonic stem cells (ESCs), double knockout cells fail to exit pluripotency and to differentiate efficiently, with delays in upregulating bivalently marked lineage genes. Proteomics reveal that Dppa2/4 interact on chromatin with members of the COMPASS and Polycomb complexes important for H3K4me3 and H3K27me3 deposition, respectively. Epigenetic profiling reveals a striking loss of H3K4me3, H3K27me3, and their associated enzymatic machinery at a significant subset of bivalent promoters in Dppa2/4 mutants, in addition to loss of H2A.Z and chromatin accessibility. In wild-type ESCs, these “Dppa2/4-dependent” bivalent promoters are characterised by low H3K4me3 enrichment and breadth, near-absent expression levels and initiating but not elongating RNA polymerase. Notably, Dppa2/4-dependent promoters are less evolutionarily conserved suggesting that they lack additional safeguard measures to maintain bivalency at these genes in the absence of Dppa2/4. Concomitantly with the loss of bivalency, Dppa2/4-dependent bivalent promoters gain DNA methylation and consequently are no longer able to be effectively activated upon ESC differentiation, leading to defects in cell fate acquisition. Our findings reveal a targeting principle for bivalency to developmental gene promoters poising them for future lineage specific gene activation.

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

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