Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012;7(6):e39606.
doi: 10.1371/journal.pone.0039606. Epub 2012 Jun 22.

SUMOylation represses Nanog expression via modulating transcription factors Oct4 and Sox2

Yongyan Wu  1 , Zekun Guo, Haibo Wu, Xiaohai Wang, Lixia Yang, Xiaoyan Shi, Juan Du, Bo Tang, Wenzhong Li, Liping Yang, Yong Zhang
Affiliations

SUMOylation represses Nanog expression via modulating transcription factors Oct4 and Sox2

Yongyan Wu et al. PLoS One. 2012.

Abstract

Nanog is a pivotal transcription factor in embryonic stem (ES) cells and is essential for maintaining the pluripotency and self-renewal of ES cells. SUMOylation has been proved to regulate several stem cell markers' function, such as Oct4 and Sox2. Nanog is strictly regulated by Oct4/Sox2 heterodimer. However, the direct effects of SUMOylation on Nanog expression remain unclear. In this study, we reported that SUMOylation repressed Nanog expression. Depletion of Sumo1 or its conjugating enzyme Ubc9 increased the expression of Nanog, while high SUMOylation reduced its expression. Interestingly, we found that SUMOylation of Oct4 and Sox2 regulated Nanog in an opposing manner. SUMOylation of Oct4 enhanced Nanog expression, while SUMOylated Sox2 inhibited its expression. Moreover, SUMOylation of Oct4 by Pias2 or Sox2 by Pias3 impaired the interaction between Oct4 and Sox2. Taken together, these results indicate that SUMOylation has a negative effect on Nanog expression and provides new insights into the mechanism of SUMO modification involved in ES cells regulation.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. SUMOylation represses Nanog expression in F9 embryonal carcinoma cells.
(A) Endogenous Sumo1 expression in control and Sumo1-knockdown F9 EC cells. After 48 hours post-transfection with a Sumo1-specific shRNA construct (sh-Sumo1) or negative control (Vector and sh-scramble), Sumo1 expression was determined by qPCR and western blot. (B) Endogenous Ubc9 expression in control and Ubc9-knockdown F9 EC cells. After 48 hours post-transfection with an Ubc9-specific shRNA construct (sh-Ubc9) or negative control (Vector and sh-scramble), Ubc9 expression was determined by qPCR and western blot. (C) Overexpression of Sumo1 and/or Ubc9 in F9 EC cells. F9 EC cells were transfected with pCMV-HA-Sumo1, pCMV-HA-Ubc9 and empty vector as indicated, Sumo1 and Ubc9 mRNA levels were detected by qPCR respectively. (D) qPCR analysis of Nanog expression in F9 EC cells in response to knockdown of Sumo1/Ubc9. (E) qPCR analysis of Nanog expression in F9 EC cells in response to overexpression of Sumo1/Ubc9. (F) Endogenous Nanog protein in F9 EC cells were determined by western blot after transient transfection with the indicated constructs. (G) Transcriptional activity of the Nanog proximal promoter in response to SUMOylation. After 48 hours post-transfection with the indicated plasmids, luciferase activity was determined and normalized against control empty vector transfection. qPCR data were normalized to GAPDH. Data are presented as the mean +/− SD and are derived from three independent experiments. *, p<0.05; **, p<0.01; WB: western blot.
Figure 2
Figure 2. SUMOylation of Oct4 enhances Nanog expression.
(A) Oct4 is modified by Sumo1 at Lysine 118. Wild-type Oct4 or the SUMO receptor site mutant Oct4 K118R was expressed in combination with HA-Sumo1 and HA-Ubc9 in F9 EC cells. (B) qPCR analysis of Nanog mRNA in response to various levels of SUMOylated Oct4. The levels of the transcripts were normalized against control empty vector transfection. (C) Western blot analysis of Nanog in F9 EC cells under a varying SUMOylation status of Oct4. (D) SUMOylation of Oct4 enhances the Nanog proximal promoter transcription. Transcriptional activities of the Nanog promoter (−230 to +50 bp relative to the transcription start site) in response to various levels of SUMOylated Oct4 were determined by dual-luciferase reporter assays. qPCR data were normalized to GAPDH. Data are presented as the mean +/− SD and are derived from three independent experiments. *, p<0.05; **, p<0.01; WB: western blot.
Figure 3
Figure 3. SUMOylation of Sox2 represses Nanog expression.
(A) Covalent modification of Sox2 by Sumo1 at Lysine 247. Wild-type Sox2 and mutant Sox2 K247R were coexpressed with HA-Sumo1 and HA-Ubc9. (B) qPCR analysis of Nanog mRNA in response to various levels of SUMOylated Sox2. (C) Western blot analysis of Nanog in F9 EC cells under a varying status of SUMOylated Sox2. (D) Covalent modification of Sox2 with Sumo1 inhibits the transcriptional activity of the Nanog proximal promoter. Transcriptional activities of the Nanog proximal promoter (−230 to +50 bp relative to the transcription start site) in response to various levels of SUMOylated Sox2 were determined by dual-luciferase reporter assays. qPCR data were normalized to GAPDH. Data are presented as the mean +/− SD and are derived from three independent experiments. *, p<0.05; **, p<0.01; WB: western blot.
Figure 4
Figure 4. SUMOylation regulates transactivity of Oct4 and Sox2.
(A) NIH3T3 cells lack the expression of pluripotency genes. Detection of Oct4, Sox2 and Nanog protein expression in wild-type and Flag-Oct4 or Myc-Sox2-transfected NIH3T3 cells, F9 EC cells lysate was used as a positive control. (B) SUMOylation of Oct4 enhances the Nanog proximal promoter transcription in NIH3T3 cells. NIH3T3 cells were transfected with the indicated plasmids, and dual luciferase assays were performed at 48 hours post-transfection. (C) SUMOylation inhibits the transcriptional activity of Sox2 in NIH3T3 cells. NIH3T3 cells were transfected with the indicated plasmids, and then dual luciferase assays were performed at 48 hours post-transfection. (D) Schematic representation of Octamer/Sox single site reporter constructs. The construct consisted of the firefly luciferase gene driven by the SV40 promoter and three tandem copies of the Octamer/Sox element. (E) SUMOylation of Oct4 promotes the transcriptional activity of pGL3-3×Oct. NIH3T3 cells were cotransfected with pGL3-3×Oct and various combinations of plasmids, and then luciferase activity was determined at 48 hours post-transfection. (F) SUMOylation of Sox2 decreases the transcriptional activity of pGL3-3×Sox. NIH3T3 cells were cotransfected with pGL3-3×Sox and various combinations of plasmids, and then luciferase activity was determined at 48 hours post-transfection. Data are presented as the mean +/− SD and are derived from three independent experiments. WB: western blot.
Figure 5
Figure 5. SUMOylation does not alter the subcellular localization of Oct4 and Sox2.
(A) Subcellular localization of Oct4 and Oct4K118R. F9 EC cells were cotransfected with red fluorescent protein tagged Oct4/Oct4 K118R plasmids and HA-Sumo1 or HA-Ubc9. There is no obvious difference in the subcellular localization of Sumo1-modified and unmodified Oct4. (B) The distribution of Sox2 and Sox2 K247R in F9 EC cells. Cotransfection of F9 EC cells with pDsRed-Sox2/pDsRed-Sox2 K247R and HA-Sumo1 or HA-Ubc9. Both SUMOylated Sox2 and unmodified Sox2 K247R localize in the nuclei. Nuclei were stained with DAPI (blue). Cells were observed and photographed under a Nikon confocal microscope at ×400 magnification.
Figure 6
Figure 6. SUMOylation impairs the protein-protein interaction between Oct4 and Sox2.
NIH3T3 cells were cotransfected with various combinations of Oct4/Oct4 K118R, Sox2/Sox2 K247R, HA-Sumo1 and HA-Ubc9 expression plasmids as indicated. Cell extracts were respectively co-immunoprecipitated with anti-Oct4 and anti-Sox2 antibody-coated affinity beads. Whole-cell lysates (input) and immunoprecipitated proteins were separated by 12% SDS-PAGE, followed by western blot with anti-Sox2, anti-Oct4, or anti-GAPDH antibodies. Western blot images were analyzed using Image J. (A) The protein-protein interaction between wild-type Oct4 and Sox2, the relative band intensity values of samples to controls were presented in bar histogram. (B) The protein-protein interaction between wild-type Oct4/Sox2 and mutant Sox2 K247R/Oct4 K118R, the relative band intensity values of samples to controls were presented in bar histogram. CoIP: co-immunoprecipitation; WB: western blot.
Figure 7
Figure 7. SUMO E3 ligases PIAS proteins mediate substrates-specific SUMOylation and regulate Nanog transcription.
(A and B) Pias2 and Pias3 promote SUMOylation of Oct4 and Sox2, respectively. NIH3T3 cells were transfected with various combinations of plasmids as indicated. SUMOylated Oct4 and Sox2 was enriched by CoIP using an anti-Sumo1 antibody, and detected by western blot with anti-Oct4 and anti-Sox2 antibodies, respectively (upper panel). (C and D) Nanog transcription is up-regulated by Pias3, but down-regulated by Pias2. Transfection of F9 EC cells with various combinations of plasmids as indicated. The levels of Nanog transcripts were normalized against GAPDH expression. Data are presented as the mean +/− SD and are derived from three independent experiments. *: p<0.05; **: p<0.01. CoIP: co-immunoprecipitation; WB: western blot.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A. 1981;78:7634–7638. - PMC - PubMed
    1. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature. 1981;292:154–156. - PubMed
    1. Chambers I, Colby D, Robertson M, Nichols J, Lee S, et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell. 2003;113:643–655. - PubMed
    1. Lavial F, Acloque H, Bertocchini F, Macleod DJ, Boast S, et al. The Oct4 homologue PouV and Nanog regulate pluripotency in chicken embryonic stem cells. Development. 2007;134:3549–3563. - PubMed
    1. Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell. 2003;113:631–642. - PubMed

Publication types

MeSH terms

Substances