Characterisation of interactions between the pluripotency transcription factors Nanog, Oct4 and Sox2

Abstract

ABSTRACTThe pluripotency transcription factors (TFs) Nanog, Sox2, and Oct4 are at the centre of the gene regulatory network that controls cell identity in embryonic stem (ES) cells. However, the mechanisms by which these factors control cell fate, and their interactions with one another are not fully understood. Here we combine biophysical and novel biochemical assays to assess how these factors interact with each other quantitatively. A new confocal microscopy method to detect binding of a target protein to a fluorescently labelled partner (coimmunoprecipitation bead imaging microscopy [CBIM]) is presented and used to demonstrate homotypic binding of Nanog and heterotypic binding between Nanog and Sox2 and between Nanog and Oct4. Using fluorescence correlation spectroscopy we show that in solution, Nanog but not Oct4 or Sox2 can form homodimers. Fluorescence Cross Correlation Spectroscopy shows that the affinity of Nanog for dimer formation is in the order Sox2 > Nanog > Oct4. Importantly, live cell analysis demonstrate the existence of Nanog homodimers in vivo. While Sox2 and Oct4 bind one another in a DNA-dependent manner, Nanog appears not to bridge Sox2 and Oct4, even though Nanog binds both Sox2 and Oct4 individually. Together these findings extend understanding of the molecular interactions occurring between these central mediators of the pluripotency gene regulatory network at the single molecule level.