KLF4 and PBX1 Directly Regulate NANOG Expression in Human Embryonic Stem Cells

Authors

  • Ken Kwok-Keung Chan,

    Corresponding author
    1. Stem Cell Group, Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore
    • Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, 06-01 Centros, Singapore 138668
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    • Telephone: 65-6-478-8898, Fax: 65-6-478-9561

  • Jingyao Zhang,

    1. Stem Cell Group, Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore
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  • Na-Yu Chia,

    1. Gene Regulation Laboratory, Genome Institute of Singapore, A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore
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  • Yun-Shen Chan,

    1. Gene Regulation Laboratory, Genome Institute of Singapore, A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore
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  • Hui Shan Sim,

    1. Stem Cell Group, Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore
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  • Ker Sin Tan,

    1. Stem Cell Group, Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore
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  • Steve Kah-Weng Oh,

    1. Stem Cell Group, Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore
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  • Huck-Hui Ng,

    1. Gene Regulation Laboratory, Genome Institute of Singapore, A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore
    2. Department of Biological Sciences and National University of Singapore, Singapore
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  • Andre Boon-Hwa Choo

    1. Stem Cell Group, Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore
    2. Division of Bioengineering, National University of Singapore, Singapore
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  • Author contributions: K.K.-K.C.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; J.Z.: collection and/or assembly of data, data analysis and interpretation, manuscript writing; N.-Y.C.: collection and/or assembly of data, data analysis and interpretation; Y.-S.C.: collection and/or assembly of data, data analysis and interpretation; H.S.S.: collection and/or assembly of data; T.K.S.: collection and/or assembly of data; S.K.-W.O.: financial support, administrative support; H.-H.N.: provision of study material, data analysis and interpretation; A.B.-H.C.: financial support, administrative support.

  • First published online in STEM CELLS EXPRESS June 11, 2009.

Abstract

Insight into the regulation of core transcription factors is important for a better understanding of the molecular mechanisms that control self-renewal and pluripotency of human ESCs (hESCs). However, the transcriptional regulation of NANOG itself in hESCs has largely been elusive. We established a NANOG promoter luciferase reporter assay as a fast read-out for indicating the pluripotent status of hESCs. From the functional cDNA screens and NANOG promoter characterization, we successfully identified a zinc finger transcription factor KLF4 and a homeodomain transcription factor PBX1 as two novel transcriptional regulators that maintain the pluripotent and undifferentiated state of hESCs. We showed that both KLF4 and PBX1 mRNA and protein expression were downregulated during hESC differentiation. In addition, overexpression of KLF4 and PBX1 upregulated NANOG promoter activity and also the endogenous NANOG protein expression in hESCs. Direct binding of KLF4 on NANOG proximal promoter and PBX1 on a new upstream enhancer and proximal promoter were confirmed by chromatin immunoprecipitation and electrophoretic mobility shift assay. Knockdown of KLF4/PBX1 or mutation of KLF4/PBX1 binding motifs significantly downregulated NANOG promoter activity. We also showed that specific members of the SP/KLF and PBX family are functionally redundant at the NANOG promoter and that KLF4 and PBX1 cooperated with OCT4 and SOX2, and transactivated synergistically the NANOG promoter activity. Our results show two novel upstream transcription activators of NANOG that are functionally important for the self-renewal of hESC and provide new insights into the expanded regulatory circuitry that maintains hESC pluripotency. STEM CELLS 2009;27:2114–2125

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