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

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. Whenmore » 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.« less
Authors:
 [1] ;  [2] ;  [3] ;  [4] ;  [5] ;  [5] ;  [2] ;  [5] ;  [6] ;  [6] ;  [5] ;  [6] ;  [7] ;  [8] ;  [1] ;  [9] ;  [5] ;  [10] ;  [2]
  1. Gladstone Institutes of Cardiovascular Disease, San Francisco, CA (United States)
  2. Univ. of California, San Francisco, CA (United States)
  3. The Scripps Research Institute, La Jolla, CA (United States); SLAC National Accelerator Lab., Menlo Park, CA (United States)
  4. Univ. of California, San Francisco, CA (United States); Structural Biology Research Center, Tsukuba, Ibaraki (Japan)
  5. The Scripps Research Institute, La Jolla, CA (United States)
  6. The Scripps Research Institute, La Jolla, CA (United States); SLAC National Accelerator Lab., Menlo Park, CA (United States)
  7. 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)
  8. The Scripps Research Institute, La Jolla, CA (United States); Genomics Institute of the Novartis Research Foundation, San Diego, CA (United States)
  9. Gladstone Institutes of Cardiovascular Disease, San Francisco, CA (United States);
  10. Gladstone Institutes of Cardiovascular Disease, San Francisco, CA (United States); Kyoto Univ., Kyoto (Japan)
Publication Date:
OSTI Identifier:
1224059
Grant/Contract Number:
GM094614; GM094586; AC03-76SF00515
Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 112; Journal Issue: 15; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Research Org:
SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES NANOG; crystal structure; pluripotent stem cells; DNA-binding; reprogramming