Embryonic stem cells (ESCs) are established by blastocyst in vitro culture and have an ability to proliferate infinitely with maintaining differentiation ability into three germ layers, named pluripotency. Previously, we identified ECATs (ES Cells Associated Transcripts) as genes which are expressed in undifferentiated ESCs and germ cells but not in somatic cells. Functional analysis revealed that many ECATs, for example Nanog(Ecat4), Eras(ECAT5) and Sall4(Ecat24), are important for undifferentiated ESC properties. However, there are still many other genes remains their functions unclear.
Here, we focused on ECAT15-1/Dppa4 (Developmental pluripotency-associated 4) and ECAT15-2/Dppa2. ECAT15-1 and ECAT15-2 located on chromosome 16th tandemly, and encode homologous proteins with a common DNA binding domain known as the SAP motif. To reveal the functions and relationships of those genes in vivo, we generated ECAT15-1 single, ECAT15-2 single and ECAT15-1/15-2 double knockout mice and found that almost all mutants died around birth with respiratory defects. Interestingly, ECAT15-2 single mutant mice showed the severest phenotypes among three types of mutant mice. Paradoxically, the expression of neither ECAT15-1 nor ECAT15-2 was detected in lung organogenesis.
Why do three mutants have respiratory defects without detectable expression? Due to the DNA binding domain, the SAP motif, we hypothesized that ECAT15-1 and ECAT15-2 affect lung development by modifying the epigenetic status of critical genes. To address this, we established ECAT15-2-null ESCs, and found that genomic DNA of genes which are differentially expressed in the ECAT15-2 mutant lungs 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 those genes in ESCs. Furthermore, ECAT15-1 and ECAT15-2 have protein-protein interaction in ESCs. These data suggest that molecular complex of ECAT15-1 and ECAT15-2 have important roles in lung development where it is no longer expressed, by leaving epigenetic marks from earlier developmental stages.
Nakamura T, Nakagawa M, Ichisaka T, Shiota A, & Yamanaka S, Essential roles of ECAT15-2/Dppa2 in functional lung development. Mol Cell Biol. 2011 [Epub ahead of print]