18-P011 High complexity, random-primed domain libraries for yeast two-hybrid analysis of human and model organism interactomes

The POU-V transcription factor Oct4 is a master regulator of self-renewal and pluripotency in embryonic stem (ES) cells as well as an important regulator of lineage commitment in embryonic development. We have shown that Oct4’s ability to regulate selfrenewal in ES cells is related to a conserved function in regulating embryonic differentiation in certain vertebrates. During Xenopus laevis development three Oct4 homologues (Xlpou25, Xlpou60 and Xlpou91) are expressed and are thought to maintain a pool of undifferentiated multi-potent cells. One of these proteins, Xlpou91, is also capable of functionally substituting for Oct4 in ES cells. Here we explore the regulatory network downstream of the amphibian Oct4 homologues by using loss of function morpholino combinations and microarray analysis. A number of studies have analysed the downstream targets of mammalian Oct4 using ES cells as a model. By using the amphibian embryos our study aims to identify the targets essential for correct germ layer specification in addition to maintenance of the undifferentiated state. Based on in silico analysis of microarray data we selected a subset of 307 differentially regulated genes responsive to the amphibian Oct4 homologues. Known mammalian homologues exist for 202 of these targets and 66 of them have been identified as Oct4 targets in mammalian ES cells. We have also found that over expression of murine Oct4 activator and repressor fusion proteins regulate these targets in a manner consistent with the view that the activator function of PouV proteins plays the major role in the suppression of lineage specification.