title: The pluripotency regulator Zic3 is a direct activator of the Nanog promoter in ESCs.
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'-CC(C)/(T) GCTGGG-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.
authors.affiliation: Stem Cell and Developmental Biology Group, Genome Institute of Singapore, Singapore.
authors.forename: Linda Shushan
authors.initials: LS
authors.lastname: Lim
authors.forename: Felicia Huimei
authors.initials: FH
authors.lastname: Hong
authors.forename: Galih
authors.initials: G
authors.lastname: Kunarso
authors.forename: Lawrence W
authors.initials: LW
authors.lastname: Stanton
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D053595:Embryonic Stem Cells / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D000071317:Nanog Homeobox Protein; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D034622:RNA Interference; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D018398:Homeodomain Proteins; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D014157:Transcription Factors; C491594:Zic3 protein, mouse