Non-genetically modified somatic cells can only be inefficiently and stochastically reprogrammed to pluripotency by exogenous expression of reprogramming factors. Low competence of natural reprogramming factors may prevent the majority of cells to successfully and synchronously reprogram. Here we screened DNA-interacting amino acid residues in the zinc-finger domain of KLF4 for enhanced reprogramming efficiency using alanine-substitution scanning methods. Identified KLF4 L507A mutant accelerated and stabilized reprogramming to pluripotency in both mouse and human somatic cells. By testing all the variants of L507 position, variants with smaller amino acid residues in the KLF4 L507 position showed higher reprogramming efficiency. L507A bound more to promoters or enhancers of pluripotency genes, such as KLF5, and drove gene expression of these genes during reprogramming. Molecular dynamics simulations predicted that L507A formed additional interactions with DNA. Our study demonstrates how modifications in amino acid residues of DNA-binding domain enable next-generation reprogramming technology with engineered reprogramming factors.
Borisova, Evgeniia and Nishimura, Ken and An, Yuri and Takami, Miho and Li, Jingyue and Song, Dan and Matsuo-Takasaki, Mami and Luijkx, Dorian and Aizawa, Shiho and Kuno, Akihiro and Sugihara, Eiji and Sato, Taka-aki and Yumoto, Fumiaki and Terada, Tohru and Hisatake, Koji and Hayashi, Yohei, Structurally-Discovered KLF4 Variants Accelerate and Stabilize Reprogramming to Pluripotency. Available at SSRN: https://ssrn.com/abstract=3741220 or http://dx.doi.org/10.2139/ssrn.3741220
This version of the paper has not been formally peer reviewed.
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