The structural basis of pioneer factors in reporgramming to pluripotency

Pluripotent reprogramming is still a very time-consuming, unreliable, and inefficient process with underlying mechanism that remains elusive, constituting the major obstacle to its highly anticipated clinical application. To overcome these limitations, we need to gain an understanding of reprogramming process. OSKM impart their reprogramming activity by targeting silent pluripotency genes located within closed chromatin, which is highly packed with nucleosome. Thus, revealing the structural features of OSKM-nucleosome interactions will illuminate our understanding of reprogramming.
This PhD project adopts a multidisciplinary approach, combining expertise from Stem Cells, Epigenetics, Structural Biology and Biophysics, to understand the structural basis underpinning the interaction between TFs and nucleosomes. This project takes advantage of well-established techniques such as nucleosome-reconstitution, recombinant protein purification, and DNA-binding assays and develop new biochemical and biophysical methods based on Transmission Electron Microscopy (TEM), Cryo-Electron Microscopy (Cryo-EM), Size Exclusion Chromatography-Multi-Angle Light Scattering (SEC-MALS) to uncover the three-dimensional arrangement and the biophysical characterization of the reprogramming factor-nucleosome complexes. Particularly, focusing on pioneer TF Oct4 since it targets the most highly nucleosomes enriched sites and it has higher affinity for nucleosomal DNA compared to free DNA (Soufi et al. 2015). Moreover, unlike other reprogramming factors Oct4 cannot be substituted by other POU family member to facilitate reprogramming to pluripotency (Esch et al. 2013). Studying structural features of the interactions between POU domains of Oct4 and the octamer DNA motifs exposed on the nucleosomes (Soufi et al. 2015) and how these interactions may change in nucleosome arrays.