title: Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells.
abstract: NANOG (from Irish mythology Tír na nÓg) transcription factor plays a central role in maintaining pluripotency, cooperating with OCT4 (also known as POU5F1 or OCT3/4), SOX2, and other pluripotency factors. Although the physiological roles of the NANOG protein have been extensively explored, biochemical and biophysical properties in relation to its structural analysis are poorly understood. Here we determined the crystal structure of the human NANOG homeodomain (hNANOG HD) bound to an OCT4 promoter DNA, which revealed amino acid residues involved in DNA recognition that are likely to be functionally important. We generated a series of hNANOG HD alanine substitution mutants based on the protein-DNA interaction and evolutionary conservation and determined their biological activities. Some mutant proteins were less stable, resulting in loss or decreased affinity for DNA binding. Overexpression of the orthologous mouse NANOG (mNANOG) mutants failed to maintain self-renewal of mouse embryonic stem cells without leukemia inhibitory factor. These results suggest that these residues are critical for NANOG transcriptional activity. Interestingly, one mutant, hNANOG L122A, conversely enhanced protein stability and DNA-binding affinity. The mNANOG L122A, when overexpressed in mouse embryonic stem cells, maintained their expression of self-renewal markers even when retinoic acid was added to forcibly drive differentiation. When overexpressed in epiblast stem cells or human induced pluripotent stem cells, the L122A mutants enhanced reprogramming into ground-state pluripotency. These findings demonstrate that structural and biophysical information on key transcriptional factors provides insights into the manipulation of stem cell behaviors and a framework for rational protein engineering.
authors.affiliation: Gladstone Institutes of Cardiovascular Disease, San Francisco, CA 94158;
authors.forename: Yohei
authors.initials: Y
authors.lastname: Hayashi
authors.affiliation: Department of Biochemistry and Biophysics, and.
authors.forename: Laura
authors.initials: L
authors.lastname: Caboni
authors.affiliation: Joint Center for Structural Genomics, Stanford Synchrotron Radiation Lightsource, Stanford Linear Accelerator Center National Accelerator Laboratory, Menlo Park, CA 94025;
authors.forename: Debanu
authors.initials: D
authors.lastname: Das
authors.affiliation: Department of Biochemistry and Biophysics, and Structural Biology Research Center, KEK High Energy Accelerator Research Organization, Tsukuba, Ibaraki 305-0801, Japan;
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Yumoto
authors.affiliation: Joint Center for Structural Genomics, Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037;
authors.forename: Thomas
authors.initials: T
authors.lastname: Clayton
authors.affiliation: Joint Center for Structural Genomics, Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037;
authors.forename: Marc C
authors.initials: MC
authors.lastname: Deller
authors.affiliation: Department of Biochemistry and Biophysics, and.
authors.forename: Phuong
authors.initials: P
authors.lastname: Nguyen
authors.affiliation: Joint Center for Structural Genomics, Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037;
authors.forename: Carol L
authors.initials: CL
authors.lastname: Farr
authors.affiliation: Joint Center for Structural Genomics, Stanford Synchrotron Radiation Lightsource, Stanford Linear Accelerator Center National Accelerator Laboratory, Menlo Park, CA 94025;
authors.forename: Hsiu-Ju
authors.initials: HJ
authors.lastname: Chiu
authors.affiliation: Joint Center for Structural Genomics, Stanford Synchrotron Radiation Lightsource, Stanford Linear Accelerator Center National Accelerator Laboratory, Menlo Park, CA 94025;
authors.forename: Mitchell D
authors.initials: MD
authors.lastname: Miller
authors.affiliation: Joint Center for Structural Genomics, Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037;
authors.forename: Marc-André
authors.initials: MA
authors.lastname: Elsliger
authors.affiliation: Joint Center for Structural Genomics, Stanford Synchrotron Radiation Lightsource, Stanford Linear Accelerator Center National Accelerator Laboratory, Menlo Park, CA 94025;
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Deacon
authors.affiliation: Joint Center for Structural Genomics, Center for Research in Biological Systems, University of California, San Diego, La Jolla, CA 92037; Program on Bioinformatics and Systems Biology, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037;
authors.forename: Adam
authors.initials: A
authors.lastname: Godzik
authors.affiliation: Joint Center for Structural Genomics, Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037; Protein Sciences Department, Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121; and.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Lesley
authors.affiliation: Gladstone Institutes of Cardiovascular Disease, San Francisco, CA 94158;
authors.forename: Kiichiro
authors.initials: K
authors.lastname: Tomoda
authors.affiliation: Gladstone Institutes of Cardiovascular Disease, San Francisco, CA 94158; Departments of Medicine, Anatomy, Medical Genetics, and Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158;
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Conklin
authors.affiliation: Joint Center for Structural Genomics, Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037;
authors.forename: Ian A
authors.initials: IA
authors.lastname: Wilson
authors.affiliation: Gladstone Institutes of Cardiovascular Disease, San Francisco, CA 94158; Department of Reprogramming Science, Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan yamanaka@cira.kyoto-u.ac.jp robert.fletterick@ucsf.edu.
authors.forename: Shinya
authors.initials: S
authors.lastname: Yamanaka
authors.affiliation: Department of Biochemistry and Biophysics, and yamanaka@cira.kyoto-u.ac.jp robert.fletterick@ucsf.edu.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Fletterick
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D049109:Cell Proliferation / Q000235:genetics*; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000235:genetics*; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D005855:Germ Layers / Q000166:cytology / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D000071317:Nanog Homeobox Protein; D009690:Nucleic Acid Conformation; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014162:Transfection
chemical_list: D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D004247:DNA
keywords: DNA-binding; NANOG; crystal structure; pluripotent stem cells; reprogramming
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