Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells

Hayashi, Yohei; Caboni, Laura; Das, Debanu; Yumoto, Fumiaki; Clayton, Thomas; Deller, Marc C.; Nguyen, Phuong; Farr, Carol L.; Chiu, Hsiu -Ju; Miller, Mitchell D.; Elsliger, Marc -André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Tomoda, Kiichiro; Conklin, Bruce R.; Wilson, Ian A.; Yamanaka, Shinya; Fletterick, Robert J.

doi:10.1073/pnas.1502855112

Title: Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells

Journal Article · Mon Mar 30 00:00:00 EDT 2015 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.1502855112· OSTI ID:1224059

Hayashi, Yohei ^[1]; Caboni, Laura ^[2]; Das, Debanu ^[3]; Yumoto, Fumiaki ^[4]; Clayton, Thomas ^[5]; Deller, Marc C. ^[5]; Nguyen, Phuong ^[2]; Farr, Carol L. ^[5]; Chiu, Hsiu -Ju ^[6]; Miller, Mitchell D. ^[6]; Elsliger, Marc -André ^[5]; Deacon, Ashley M. ^[6]; Godzik, Adam ^[7]; Lesley, Scott A. ^[8]; Tomoda, Kiichiro ^[1]; Conklin, Bruce R. ^[9]; Wilson, Ian A. ^[5]; Yamanaka, Shinya ^[10]; Fletterick, Robert J. ^[2]

Gladstone Institutes of Cardiovascular Disease, San Francisco, CA (United States)
Univ. of California, San Francisco, CA (United States)
The Scripps Research Institute, La Jolla, CA (United States); SLAC National Accelerator Lab., Menlo Park, CA (United States)
Univ. of California, San Francisco, CA (United States); Structural Biology Research Center, Tsukuba, Ibaraki (Japan)
The Scripps Research Institute, La Jolla, CA (United States)
The Scripps Research Institute, La Jolla, CA (United States); SLAC National Accelerator Lab., Menlo Park, CA (United States)
The Scripps Research Institute, La Jolla, CA (United States); Univ. of California, San Diego, CA (United States); Sanford-Burnham Medical Research Institute, La Jolla, CA (United States)
The Scripps Research Institute, La Jolla, CA (United States); Genomics Institute of the Novartis Research Foundation, San Diego, CA (United States)
Gladstone Institutes of Cardiovascular Disease, San Francisco, CA (United States);
Gladstone Institutes of Cardiovascular Disease, San Francisco, CA (United States); Kyoto Univ., Kyoto (Japan)

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 indicate 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.

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Research Organization:: SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC)

Grant/Contract Number:: GM094614; GM094586; AC03-76SF00515

OSTI ID:: 1224059

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Vol. 112, Issue 15; ISSN 0027-8424

Publisher:: National Academy of Sciences, Washington, DC (United States)Copyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 35 works

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Deep learning at base-resolution reveals motif syntax of the cis-regulatory code Avsec, Žiga; Weilert, Melanie; Shrikumar, Avanti Nature Genetics https://doi.org/10.1101/737981	journal	July 2020
Epigenetic Contribution of High-Mobility Group A Proteins to Stem Cell Properties Giancotti, Vincenzo; Bergamin, Natascha; Cataldi, Palmina International Journal of Cell Biology, Vol. 2018 https://doi.org/10.1155/2018/3698078	journal	January 2018
Automated Adherent Cell Elimination by a High-Speed Laser Mediated by a Light-Responsive Polymer Hayashi, Yohei; Matsumoto, Junichi; Kumagai, Shohei Communications Biology https://doi.org/10.1101/280487	journal	March 2018
Autotaxin-mediated lipid signaling intersects with LIF and BMP signaling to promote the naive pluripotency transcription factor program Kime, Cody; Sakaki-Yumoto, Masayo; Goodrich, Leeanne Proceedings of the National Academy of Sciences, Vol. 113, Issue 44 https://doi.org/10.1073/pnas.1608564113	journal	October 2016
Structural mechanism of DNA-mediated Nanog–Sox2 cooperative interaction Yesudhas, Dhanusha; Anwar, Muhammad Ayaz; Choi, Sangdun RSC Advances, Vol. 9, Issue 14 https://doi.org/10.1039/c8ra10085c	journal	January 2019
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Automated adherent cell elimination by a high-speed laser mediated by a light-responsive polymer Hayashi, Yohei; Matsumoto, Junichi; Kumagai, Shohei Communications Biology, Vol. 1, Issue 1 https://doi.org/10.1038/s42003-018-0222-4	journal	December 2018
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Naive-like ESRRB+ iPSCs with the Capacity for Rapid Neural Differentiation Kisa, Fumihiko; Shiozawa, Seiji; Oda, Keisuke Stem Cell Reports, Vol. 9, Issue 6 https://doi.org/10.1016/j.stemcr.2017.10.008	journal	December 2017
Plumbagin suppresses epithelial to mesenchymal transition and stemness via inhibiting Nrf2-mediated signaling pathway in human tongue squamous cell carcinoma cells Zhou, Shu-Feng; Pan, Shu-Ting; Qin, Yiru Drug Design, Development and Therapy https://doi.org/10.2147/dddt.s89621	journal	October 2015
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59 BASIC BIOLOGICAL SCIENCES
NANOG
crystal structure
pluripotent stem cells
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reprogramming

Title: Structure-based discovery of NANOG variant with enhanced properties to promote self-renewal and reprogramming of pluripotent stem cells

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