The Pluripotency Regulator Zic3 Is a Direct Activator of the Nanog Promoter in ESCs§

Authors

  • Linda Shushan Lim,

    1. Stem Cell and Developmental Biology Group, Genome Institute of Singapore, Singapore
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  • Felicia Huimei Hong,

    1. Stem Cell and Developmental Biology Group, Genome Institute of Singapore, Singapore
    2. Department of Biological Sciences, National University of Singapore, Singapore
    3. NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore
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  • Galih Kunarso,

    1. Stem Cell and Developmental Biology Group, Genome Institute of Singapore, Singapore
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  • Lawrence W. Stanton

    Corresponding author
    1. Stem Cell and Developmental Biology Group, Genome Institute of Singapore, Singapore
    2. Department of Biological Sciences, National University of Singapore, Singapore
    • Stem Cell and Developmental Biology, Genome Institute of Singapore, 60 Biopolis Street, #02-01, Genome, Singapore 138672
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    • Telephone: 65-6478-8006; Fax: 65-6478-9051


  • Author contributions: L.S.L: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; F.H.H: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; G.K.: data analysis and interpretation; L.W.S: conception and design, financial support, data analysis and interpretation, final approval of manuscript. L.S.L. and F.H.H. contributed equally to this article.

  • First published online in STEM CELLS EXPRESS August September 24, 2010.

  • §

    Disclosure of potential conflicts of interest is found at the end of this article.

Abstract

The transcription factor Zic3 is required for maintenance of ESC pluripotency. By genome-wide chromatin immunoprecipitation (ChIP-chip) in ESCs, we have identified 379 direct Zic3 targets, many of which are functionally associated with pluripotency, cell cycle, proliferation, oncogenesis, and early embryogenesis. Through a computational analysis of Zic3 target sequences, we have identified a novel Zic3 consensus binding motif (5′-CCC/TGCTGGG-3′). ChIP results and in vitro DNA binding assays revealed that Zic3 binds with high affinity and specificity on the Nanog promoter. Here, we demonstrate that Zic3 functions as a transcriptional activator of the Nanog promoter in three ways: (a) Nanog transcript levels are sustained with Zic3 overexpression in differentiating ESCs, (b) Zic3 depletion in ESCs downregulates Nanog promoter activity, and (c) Zic3 overexpression leads to increased Nanog promoter activity. Furthermore, the activity of a mutant Nanog promoter with ablated Oct4/Sox2 binding is rescued by Zic3 overexpression to nearly wild-type levels. This indicates that Nanog is a positive transcriptional target of Zic3 in a mechanism that is independent of Oct4/Sox2 binding. Hence, we demonstrate an important pathway for regulation of Nanog expression in pluripotent ESCs through direct activation by Zic3. STEM CELLS 2010;28:1961–1969

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