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Structurally-Discovered KLF4 Variants Accelerate and Stabilize Reprogramming to Pluripotency

89 Pages Posted: 2 Dec 2020 Publication Status: Review Complete

See all articles by Evgeniia Borisova

Evgeniia Borisova

RIKEN BioResource Research Centre - iPS Cell Advanced Characterization and Development Team

Ken Nishimura

University of Tsukuba - Laboratory of Gene Regulation

Yuri An

RIKEN BioResource Research Centre - iPS Cell Advanced Characterization and Development Team

Miho Takami

RIKEN BioResource Research Centre - iPS Cell Advanced Characterization and Development Team

Jingyue Li

RIKEN BioResource Research Centre - iPS Cell Advanced Characterization and Development Team

Dan Song

RIKEN BioResource Research Centre - iPS Cell Advanced Characterization and Development Team

Mami Matsuo-Takasaki

RIKEN BioResource Research Centre - iPS Cell Advanced Characterization and Development Team

Dorian Luijkx

RIKEN BioResource Research Centre - iPS Cell Advanced Characterization and Development Team

Shiho Aizawa

University of Tsukuba - Laboratory of Gene Regulation

Akihiro Kuno

University of Tsukuba - Laboratory of Animal Resource Center

Eiji Sugihara

University of Tsukuba - Research and Development Center for Precision Medicine

Taka-aki Sato

University of Tsukuba - Research and Development Center for Precision Medicine

Fumiaki Yumoto

The Institute of Materials Structure Science, High Energy Accelerator Research Organization in Tsukuba

Tohru Terada

The University of Tokyo - Department of Biotechnology

Koji Hisatake

University of Tsukuba - Laboratory of Gene Regulation

Yohei Hayashi

RIKEN BioResource Research Centre - iPS Cell Advanced Characterization and Development Team

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Abstract

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.

Keywords: Reprogramming, induced pluripotent stem cells (iPSCs), Krüppel-like factor 4 (KLF4), amino acid residue, pluripotency, retrovirus vector, SeVdp (Sendai virus vector), ChIPSeq, RNA-seq, MD (molecular dynamics) simulation

Suggested Citation

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.

Evgeniia Borisova

RIKEN BioResource Research Centre - iPS Cell Advanced Characterization and Development Team ( email )

Tsukuba, Ibaraki
Japan

Ken Nishimura

University of Tsukuba - Laboratory of Gene Regulation ( email )

1-1-1 Tennodai
Tsukuba, Ibaraki 305-8575
Japan

Yuri An

RIKEN BioResource Research Centre - iPS Cell Advanced Characterization and Development Team

Tsukuba, Ibaraki
Japan

Miho Takami

RIKEN BioResource Research Centre - iPS Cell Advanced Characterization and Development Team ( email )

Tsukuba, Ibaraki
Japan

Jingyue Li

RIKEN BioResource Research Centre - iPS Cell Advanced Characterization and Development Team ( email )

Tsukuba, Ibaraki
Japan

Dan Song

RIKEN BioResource Research Centre - iPS Cell Advanced Characterization and Development Team

Tsukuba, Ibaraki
Japan

Mami Matsuo-Takasaki

RIKEN BioResource Research Centre - iPS Cell Advanced Characterization and Development Team ( email )

Tsukuba, Ibaraki
Japan

Dorian Luijkx

RIKEN BioResource Research Centre - iPS Cell Advanced Characterization and Development Team ( email )

Tsukuba, Ibaraki
Japan

Shiho Aizawa

University of Tsukuba - Laboratory of Gene Regulation ( email )

1-1-1 Tennodai
Tsukuba, Ibaraki 305-8575
Japan

Akihiro Kuno

University of Tsukuba - Laboratory of Animal Resource Center ( email )

1-1-1 Tennodai
Tsukuba, Ibaraki 305-8575
Japan

Eiji Sugihara

University of Tsukuba - Research and Development Center for Precision Medicine ( email )

Tsukuba University , Ibaraki Ken
Tsukuba, Ibaraki 305-8573, Ibaraki 3050006
Japan

Taka-aki Sato

University of Tsukuba - Research and Development Center for Precision Medicine ( email )

Tsukuba University , Ibaraki Ken
Tsukuba, Ibaraki 305-8573, Ibaraki 3050006
Japan

Fumiaki Yumoto

The Institute of Materials Structure Science, High Energy Accelerator Research Organization in Tsukuba ( email )

Tohru Terada

The University of Tokyo - Department of Biotechnology ( email )

Japan

Koji Hisatake

University of Tsukuba - Laboratory of Gene Regulation ( email )

1-1-1 Tennodai
Tsukuba, Ibaraki 305-8575
Japan

Yohei Hayashi (Contact Author)

RIKEN BioResource Research Centre - iPS Cell Advanced Characterization and Development Team ( email )

Tsukuba, Ibaraki
Japan

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