Unknown,Transcriptomics,Genomics,Proteomics

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Investigating the role of Lef1 in mouse embryonic stem cells.

ABSTRACT: Genome-wide analysis of gene expression in piggybac empty vector and piggybac-lef1 transfected mouse embryonic stem cells (46C mouse ESCs) Analysis of genes regulated by lef1, and we found that lef1 acts biphasically in mouse embryonic stem cells. Total RNA obtained from empty vector (PB)-transfected 46 ESCs or different states (passage 2 and passage 5) of lef1-transfected (PB-Lef1 P2, PB-Lef1 P5) 46C mouse ESCs.

ORGANISM(S): Mus musculus

SUBMITTER: shoudong ye

PROVIDER: E-GEOD-77330 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description: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).

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Investigating the role of Lef1 in mouse embryonic stem cells.

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