Structurally-discovered KLF4 variants accelerate and stabilize reprogramming to pluripotency
Evgeniia Borisova,
Ken Nishimura,
Yuri An,
Miho Takami,
Jingyue Li,
Dan Song,
Mami Matsuo-Takasaki,
Dorian Luijkx,
Shiho Aizawa,
Akihiro Kuno,
Eiji Sugihara,
Taka-aki Sato,
Fumiaki Yumoto,
Tohru Terada,
Koji Hisatake,
Yohei Hayashi
Affiliations
Evgeniia Borisova
iPS Cell Advanced Characterization and Development Team, BioResource Research Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan; Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
Ken Nishimura
Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
Yuri An
iPS Cell Advanced Characterization and Development Team, BioResource Research Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan
Miho Takami
iPS Cell Advanced Characterization and Development Team, BioResource Research Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan; Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
Jingyue Li
iPS Cell Advanced Characterization and Development Team, BioResource Research Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan; Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
Dan Song
iPS Cell Advanced Characterization and Development Team, BioResource Research Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan
Mami Matsuo-Takasaki
iPS Cell Advanced Characterization and Development Team, BioResource Research Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan
Dorian Luijkx
iPS Cell Advanced Characterization and Development Team, BioResource Research Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan
Shiho Aizawa
Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
Akihiro Kuno
Laboratory of Animal Resource Center, Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan; Ph.D. Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan
Eiji Sugihara
Research and Development Center for Precision Medicine, University of Tsukuba, 1-2 Kasuga, Tsukuba, Ibaraki 305-8550, Japan; The Center for Joint Research Facilities Support, Research Promotion and Support Headquarters, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan
Taka-aki Sato
Research and Development Center for Precision Medicine, University of Tsukuba, 1-2 Kasuga, Tsukuba, Ibaraki 305-8550, Japan
Fumiaki Yumoto
Institute of Materials Structure Science, High Energy Accelerator Research Organization in Tsukuba, 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan
Tohru Terada
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Koji Hisatake
Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
Yohei Hayashi
iPS Cell Advanced Characterization and Development Team, BioResource Research Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan; Corresponding author
Summary: 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 domains enable next-generation reprogramming technology with engineered reprogramming factors.
