Although Oct4 is an essential molecule for creating induced pluripotent stem cells (iPSCs), what makes it so important to cellular reprogramming has remained unknown. But now, in a paper published in Nature Cell Biology last week, scientists at the European Molecular Biology Laboratory (EMBL)-Hamburg and the Max Planck Institute (MPI) for Molecular Biomedicine in Münster, Germany have resolved the structure of Oct4, providing some details that could help boost the efficiency of reprogramming in the future (1).
Nobel-prize winning biologist Shinya Yamanaka first showed that a combination of four transcription factors—Oct4, Sox2, Klf4, and c-Myc—could turn adult mouse and human cells into iPSCs (2,3). Since then, additional research has revealed that for three of the factors, substitutions by their respective molecular family members could be made without affecting the reprogramming process. But there’s no replacing Oct4.
The first clue into what makes Oct4 irreplaceable was its amino acid sequence. The protein has two subdomains that bind with DNA. Although these subdomains are highly conserved between Oct4 and its close relatives, the small stretch of sequence joining the two subdomains of the protein—called the linker—are very different.
“The question that we couldn’t answer before getting the structure is whether this linker is the difference between Oct4 and all the other homologs that cannot replace Oct4 in the reprogramming process,” said Vivian Pogenberg, senior technical officer in senior author Matthias Wilmanns’ group at EMBL-Hamburg.
Oct4's structure had never been solved before because the protein is insoluble and thus tricky to analyze with high-intensity X-ray beams. It took several years for the researchers to crystallize Oct4 along with the DNA it binds and solve the structure of the complex. The new data reveal that part of Oct4’s linker, unlike its relatives, folds into an alpha helix.
Subsequently, the researchers found that that they could replace human Oct4’s linker with that of a mouse, which has a similar sequence, and Oct4 was still capable of reprogramming cells. In contrast, when the team replaced the human Oct4 linker with that from a more divergent organism like zebrafish, reprogramming did not occur. “This means that there is something in this linker that's important and that makes Oct4 have an impact on cell reprogramming,” said Pogenberg.
Hans Schöler’s team at MPI then created mutations in the linker by turning each residue into an alanine, one by one. One linker mutation in particular had a strong affect on reprogramming, hampering Oct4’s ability to recruit several reprogramming partners, such as Smarca4, a transcriptional activator that Schöler’s group has shown improves reprogramming efficiency (4).
The new findings are a first step for understanding how the linker recruits its partners, but many more studies are needed to characterize the interacting surfaces of Oct4’s partners. “Then we might be able to imagine how to improve the interactions to accelerate reprogramming,” Pogenberg said.
1. Esch, D., J. Vahokoski, M. R. Groves, V. Pogenberg, V. Cojocaru, H. Vom Bruch, D. Han, H. C. Drexler, M. J. Araúzo-Bravo, C. K. Ng, R. Jauch, M. Wilmanns, and H. R. Schöler. 2013. A unique Oct4 interface is crucial for reprogramming to pluripotency.
Nat Cell Biol. Epub ahead of print
2. Takahashi, K., and S. Yamanaka. 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126(4):663-676.
3. Takahashi, K., K. Tanabe, M. Ohnuki, M. Narita, T. Ichisaka, K. Tomoda, and S. Yamanaka. 2007. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861-872.
4. Singhal, N., J. Graumann, G. Wu, M. J. Araúzo-Bravo, D. W. Han, B. Greber, L. Gentile, M. Mann, and H. R. Schöler. Chromatin-remodeling components of the BAF complex facilitate reprogramming. 2010. Cell 141(6):943-955.