The biochemical and biophysical basis for the enhanced reprogramming activity of an artificially enhanced Pit-Oct-Unc transcription factor : ePOU

Abstract

Cellular reprogramming has the potential to revolutionize medicine through personalized disease models, ex vivo drug testing, and regenerative therapies. Our group engineered the scaffold of a Pit-Oct-Unc (POU) factor in the context of iPSC reprogramming and identified an enhanced POU factor (ePOU) that substantially outperforms wild-type Oct4 in terms of speed and efficiency. However, the mechanism behind its enhanced ability is unknown. To address this with biochemical and biophysical assays, proteins were first recombinantly expressed and purified to homogeneity. The proteins were then used to perform two complementary high throughput DNA binding assays, to study DNA-dependent dimerization and to record thermal unfolding profiles. Results show that binding toward primary binding sites such as the Octamer sequence and the heterodimerization with Sox factors are similar. Through biophysical and cell-based assays, ePOU was found to be thermally stabilized leading to a slower turnover rate in reprogramming cells. Using genome-wide analysis and in vitro binding assays, ePOU was found to preferentially bind a palindromic DNA element as a homodimer with positive cooperativity. These results show that a balance between maintaining core-binding mechanisms and accessing additional binding sites can enhance reprogramming. Furthermore, as enhanced thermal stability correlates with improved reprogramming it can be an additional parameter amenable to optimization when engineering transcription factors.