ECAT15-2/Dppa2 deficient ESCs

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. ESCs were established from E3.5 blasotcysts of 15-1+/CT, 15-2+/- intercross (CT allele; conditional targeted allele). 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 and two ECAT15-2 deficient ESCs (15-1CT/CT, 15-2-/-) was 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).