Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

ECAT15-2/Dppa2 transgenic rescue into ECAT15-2/Dppa2 deficient ESCs

ABSTRACT: Many transcription factors and DNA binding proteins play essential roles in the development of organs in which they are highly and/or specifically expressed. Embryonic stem cell (ESC) associated transcript (ECAT) 15-1 and ECAT15-2, also known as developmental pluripotency-associated (Dppa) 4 and Dppa2, are enriched in mouse ESCs and pre-implantation embryos, and encode homologous proteins with a common DNA binding domain known as the SAP motif. Previously, ECAT15-1 was shown to be important in the lung development, while it is dispensable in early development. In this study, we generated ECAT15-2 single and ECAT15-1/15-2 double knockout mice and found that almost all mutants, like ECAT15-1 mutants, died around birth with respiratory defects. Paradoxically, the expression of neither ECAT15-1 nor ECAT15-2 was detected in lung organogenesis. Several genes, such as Nkx2-5, Gata4, and Pitx2 were down-regulated in the ECAT15-2-null lung. On the other hand, genomic DNA of these genes showed inactive chromatin statuses in ECAT15-2-null ESCs, but not in wild-type ESCs. Chromatin immuno-precipitation (ChIP) assay revealed that ECAT15-2 binds to the regulatory region of Nkx2-5 in ESCs. These data suggest that ECAT15-2 have important roles in lung development where it is no longer expressed, by leaving epigenetic marks from earlier developmental stage. The ECAT15-2 mutant ESCs (15-1CT/CT, 15-2-/-) were established from E3.5 blasotcysts of 15-1+/CT, 15-2+/- intercross (CT allele; conditional targeted allele). Then the Flag-tagged ECAT15-2/Dppa2 transgenic vector based on piggyBac system were introduced. They were harvested on gelation coated dish with ES medium containing 15% FCS and LIF for three days. The total RNA from two wild type, two mock transduced ECAT15-2 deficient ESCs and two Flag-tagged ECAT15-2 transduced ECAT15-2 deficient ESCs were labeled with Cy3. Samples were hybridized with Whole Mouse Genome Microarray 4 x 44K (G4122F, Agilent), with the one color protocol. Data was analyzed by using GeneSpring GX11 software (Agilent).

ORGANISM(S): Mus musculus

SUBMITTER: Shinya Yamanaka

PROVIDER: E-GEOD-31582 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Essential roles of ECAT15-2/Dppa2 in functional lung development.

Nakamura Tomonori T Nakagawa Masato M Ichisaka Tomoko T Shiota Arufumi A Yamanaka Shinya S

Molecular and cellular biology 20110906 21

Many transcription factors and DNA binding proteins play essential roles in the development of organs in which they are highly and/or specifically expressed. Embryonic stem cell (ESC)-associated transcript 15-1 (ECAT15-1) and ECAT15-2, also known as developmental pluripotency-associated 4 (Dppa4) and Dppa2, respectively, are enriched in mouse ESCs and preimplantation embryos, and their genes encode homologous proteins with a common DNA binding domain known as the SAP motif. Previously, ECAT15-1 ...[more]

PMID: 21896782

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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.

Dataset Information

ECAT15-2/Dppa2 transgenic rescue into ECAT15-2/Dppa2 deficient ESCs

Publications

Essential roles of ECAT15-2/Dppa2 in functional lung development.

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