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. 2015 Apr 14;112(15):4666-71.
doi: 10.1073/pnas.1502855112. Epub 2015 Mar 30.

Structure-based Discovery of NANOG Variant With Enhanced Properties to Promote Self-Renewal and Reprogramming of Pluripotent Stem Cells

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Free PMC article

Structure-based Discovery of NANOG Variant With Enhanced Properties to Promote Self-Renewal and Reprogramming of Pluripotent Stem Cells

Yohei Hayashi et al. Proc Natl Acad Sci U S A. .
Free PMC article

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.

Keywords: DNA-binding; NANOG; crystal structure; pluripotent stem cells; reprogramming.

Conflict of interest statement

Conflict of interest statement: S.Y. is a scientific advisor of iPS Academia Japan without salary.

Figures

Fig. 1.
Fig. 1.
Crystal structure of hNANOG–OCT4 promoter DNA complex. (A) Stereo ribbon diagram of the hNANOG HD in complex with OCT4 promoter DNA. Helices H1, H2, and H3 are labeled. (B) Diagram showing the secondary structure elements of the hNANOG HD superimposed on its primary sequence. The α-helices (H1–H3) and β-turn (β) are indicated. The residues used for making alanine mutants are highlighted in red and boldface type. (C) The L122 residue in hNANOG HD that enhance interaction with OCT4 promoter DNA is shown in green. (D) Residues in hNANOG HD (K137, T141, N145, and R147) that are critical for interaction with OCT4 promoter DNA are shown in green.
Fig. 2.
Fig. 2.
BLI analysis of hNANOG HD mutants binding to the OCT4 promoter DNA. (A) Dose–response curves of hNANOG WT, Y136A, and L122A as representatives of WT-like, or mutants that weaken or enhance DNA binding affinity, respectively. These curves show the binding response (expressed as nanometer shift) to the biotinylated 12-bp fragment of the DNA of a range of hNANOG HD concentrations (20 µM to 0.5 µM). (B) Steady-state analysis to determine the equilibrium dissociation constants (KD) of the protein–DNA interaction divided in three categories: WT-like, and mutants that weaken or enhance DNA binding affinity. The error bars represent three different binding cycles expressed as percent of the maximal response after sensor regeneration with 1 M MgCl2. The equilibrium dissociation constants (KD) and kinetic constant rates for all of the mutants are summarized in Fig. S2 and Table S2.
Fig. 3.
Fig. 3.
Protein stability of NANOG HD mutants in purified protein complex or in mESCs. (A) DSF analysis of the hNANOG HD WT and mutants alone or in complex with OCT4 promoter DNA. The addition of the DNA stabilizes hNANOG HD WT by a thermal shift of around 20 °C. (B) Melting curves of WT and hNANOG HD mutants in presence of the DNA, showing a 3 °C increase in stability for the L122A variant, and around 6 °C reduction in stability for the Q144A and K151A variants. Melting curves are presented as mean values of the percentage of unfolded protein in function of the temperature (°C); values in the table represent mean ± SEM for n = 3; n.d., not determined. Melting temperature (Tm) of all of the mutants are summarized in Table S2. (C) Protein expression of exogenous mNANOG WT or mutants fused with N-terminal 3×FLAG tag in mESCs treated with cycloheximide (CHX) detected by Western blotting. Green and red bands in each sample show the amount of GAPDH and exogenous mNANOG mutants, respectively. The half-life of GAPDH is more than 72 h. (D) Protein half-life of each mNANOG mutants in mESCs. n = 4, values are mean + SEM. P values of Dunnett’s post hoc test against WT were shown as follows: +P < 0.1, *P < 0.05, ***P < 0.001.
Fig. 4.
Fig. 4.
The effects of overexpressing mNANOG mutants on mESC self-renewal. (A) Typical colony morphologies of NT mESCs or mESC transfectants overexpressing WT or L122A mNANOG mutant in Normal, −LIF, or +RA culture conditions. The images were taken 5 d after seeding. (B) Rex1 expression detected by RT-qPCR in mESC transfectants overexpressing each mNANOG mutant in these culture conditions. The amount of an undifferentiated mESC sample was set as 1.0. n = 4, values are mean + SEM. (C) Protein expression of OCT4, SOX2, NANOG (total), ESRRB, and GAPDH in NT mESCs in Normal or +RA conditions or in mESC transfectants overexpressing EGFP, WT, or L122A in +RA conditions detected by Western blotting. Green and red bands in each sample show the amounts of GAPDH and OCT4, ESRRB, SOX2, or total NANOG, respectively. (D) OCT4 protein expression detected by immunocytochemistry in NT mESCs in Normal or +RA conditions or in mESC transfectants overexpressing WT or L122A in +RA conditions. Secondary antibodies were labeled with AlexaFluor488 (green). Nuclei were stained with DAPI (blue). (Scale bars, 100 µm.) The percentage of OCT4+ cells was analyzed from five randomly taken images for each sample, which contain at least 100 cells in each image. n = 5, values are mean + SEM. (E) Heatmap image of RNA-seq data illustrating gene expression profiles for the panel of genes that were differentially expressed between Normal and +RA culture conditions in EGFP-overexpressing mESCs.
Fig. 5.
Fig. 5.
The effect of L122A on EpiSC reprogramming into ground-state pluripotency. (A) AP-staining (shown in red) of NT, WT-overexpressing, or L122A-overexpressing EpiSCs cultured in LIF2i medium for 5 d. The images were taken from a whole well of a six-well plate. (B) AP+ colony numbers counted in a well of a six-well plate. n = 4, values are mean + SEM. (C) Gene expression of Rex1, Klf4, or T in these conditions detected by RT-qPCR. The amount of an undifferentiated mESC sample was set as 1.0. n = 4, values are mean + SEM. (D) Flow cytometry of these EpiSCs using anti-CD31 (PECAM) antibody. The secondary antibody used was conjugated with Allophycocyanin (APC). (E) CD31+ cell ratio calculated from flow cytometry. n = 4, values are mean + SEM. (F) Protein expression of OCT4, ESRRB, T, or GAPDH in the EpiSC transfectants detected by Western blotting analysis. Green and red bands in each sample show the amounts of GAPDH and OCT4, ESRRB, or T, respectively. (G) Contribution of GFP-marked reprogrammed EpiSCs transfected with hNANOG L122A to embryonic day 13.5 chimeric mouse embryo. (H) Chimeric mice obtained by injection of reprogrammed EpiSCs (agouti) transfected with hNANOG L122A into C57BL/6 blastocysts (black) show coat-color contribution.

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