Promoter activity and regulation of the Pou5f1 homolog from a teleost, Nile tilapia

Mammalian POU5F1 (also known as OCT4) is an essential transcription factor that induces and controls stemness in the inner cell mass and embryonic stem (ES) cells. Its expression results from intricate regulatory networks involving its 5' upstream DNA elements and numerous transcription factors. Pou5f3, the ortholog of POU5F1, has been identified in non-mammalians including fish. However, little is known about the molecular mechanisms controlling its expression up to date. Here we report the promoter activity and regulation of Nile tilapia (Oreochromis niloticus) pou5f3 (Onpou5f3) in fish early-stage embryos and ES cells. A 3.1-kb Onpou5f3 promoter region was cloned, analyzed and constructed into pT2AL-GFP vector. Multiple potential regulatory elements including potential octamer sequence for Pou domain and retinoic acid-responsive elements were found in the 5' upstream region. In vivo and in vitro transfection assays reveal that the 3.1-kb DNA sequence was sufficient to drive strong GFP expression in blastula-stage embryos and ES cells, but low or undetectable expression in either late developmental stage embryos or differentiated cells, suggesting the feasibility as a tool to monitor the pluripotency state in fish stem cells. Deletion luciferase assays reveal that the region from -726 to -219 contains positive regulatory elements, whereas both the regions from -3056 to -1306 and -1306 to -729 contain negative regulatory elements. Notably, just like mammalian POU5F1, OnPou5f3 significantly enhanced its own expression in a dose-dependent manner, whereas RA treatment dramatically reduced its expression. Taken together, our study not only provides a tool for monitoring the pluripotency state of fish stem cells in vitro, but also experimentally demonstrates the molecular mechanisms underlying the Pou5f1 homolog expression might be conserved to some content between mammals and fish.