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

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...

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Published inStem cells (Dayton, Ohio) Vol. 28; no. 11; pp. 1961 - 1969
Main Authors Lim, Linda Shushan, Hong, Felicia Huimei, Kunarso, Galih, Stanton, Lawrence W.
Format Journal Article
LanguageEnglish
Published Hoboken Wiley Subscription Services, Inc., A Wiley Company 01.11.2010
Subjects
Animals
Binding Sites
Cell Line
Chromatin Immunoprecipitation
Electrophoretic Mobility Shift Assay
Embryonic Stem Cells - metabolism
ESCs
Homeodomain Proteins - genetics
Homeodomain Proteins - metabolism
Mice
Nanog
Nanog Homeobox Protein
Polymerase Chain Reaction
Promoter Regions, Genetic - genetics
Protein Binding
RNA Interference
Self‐renewal
Transcription factors
Transcription Factors - genetics
Transcription Factors - metabolism
Zic3
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Summary: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
Bibliography:First published online in STEM CELLS
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.
Disclosure of potential conflicts of interest is found at the end of this article.
Telephone: 65‐6478‐8006; Fax: 65‐6478‐9051
August September 24, 2010.
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ISSN:1066-5099
1549-4918
DOI:10.1002/stem.527