Structural basis of pioneer factors in reprogramming to pluripotency

Abstract

Induced pluripotent stem cells have been for the first time derived from human fibroblasts by ectopic expression of four transcription factors - OCT4, SOX2, KLF4, and c-MYC (OSKM). To date, pluripotent reprogramming is a very unreliable and inefficient process with an underlying mechanism that remains elusive. In terminally differentiated cells such as fibroblasts, pluripotency genes are transcriptionally silent and found within closed chromatin regions. The high nucleosome density of these silent chromatin regions occludes the access of most DNA-binding proteins to their gene targets. Previously, OSK have been shown to act as pioneer factors that can access closed chromatin by directly interacting with nucleosomes. To examine how OCT4 interacts with nucleosomes during reprogramming, I used cryogenic-electron microscopy (cryo-EM) and computational modelling to reveal the three-dimensional arrangement of OCT4 in complex with a nucleosome containing native Lin28B or Esrrb enhancer sequence, which are both targeted by OCT4 in early reprogramming. First, I have optimized native nucleosome reconstitution to make it suitable for cryo-EM studies. As a result, I present structural data of two native nucleosomes at low resolution as well as a high-resolution cryo-EM analysis of one native nucleosome. This has revealed regions within nucleosome with enhanced local DNA flexibility. Further computational modelling illustrates OCT4 binding at three different target sites on nucleosome. Together, these results propose that OCT4 adopts a specific conformation to recognise one part of its motif specifically using its POUS DNA-binding domain, while the other POUHD DNA-binding domain interacts with DNA non-specifically. This is consistent with the idea that OCT4 uses one of its two POU DNA-binding domains to bind a partial motif exposed on the nucleosome surface in a sequence-specific manner. Overall, revealing the structural features of OCT4-nucleosome complex could help us understand the molecular mechanism by which OCT4 engages its DNA binding sites in closed chromatin and elucidates changes in local chromatin landscape during cell-fate reprogramming.