title: KLF4 and PBX1 directly regulate NANOG expression in human embryonic stem cells.
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.
authors.affiliation: Stem Cell Group, Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore. ken_chan@bti.a-star.edu.sg
authors.forename: Ken Kwok-Keung
authors.initials: KK
authors.lastname: Chan
authors.forename: Jingyao
authors.initials: J
authors.lastname: Zhang
authors.forename: Na-Yu
authors.initials: NY
authors.lastname: Chia
authors.forename: Yun-Shen
authors.initials: YS
authors.lastname: Chan
authors.forename: Hui Shan
authors.initials: HS
authors.lastname: Sim
authors.forename: Ker Sin
authors.initials: KS
authors.lastname: Tan
authors.forename: Steve Kah-Weng
authors.initials: SK
authors.lastname: Oh
authors.forename: Huck-Hui
authors.initials: HH
authors.lastname: Ng
authors.forename: Andre Boon-Hwa
authors.initials: AB
authors.lastname: Choo
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D053595:Embryonic Stem Cells; D018507:Gene Expression Regulation, Developmental; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism*; D000071317:Nanog Homeobox Protein; D000075062:Pre-B-Cell Leukemia Transcription Factor 1; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D000075062:Pre-B-Cell Leukemia Transcription Factor 1; D011518:Proto-Oncogene Proteins; C083400:PBX1 protein, human