KLF4 and PBX1 Directly Regulate NANOG Expression in Human Embryonic Stem Cells
Autor: | Na-Yu Chia, Andre Choo, Yun-Shen Chan, Hui Shan Sim, Jingyao Zhang, Ker Sin Tan, Steve Oh, Ken Kwok-Keung Chan, Huck-Hui Ng |
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Rok vydání: | 2009 |
Předmět: |
Homeobox protein NANOG
Chromatin Immunoprecipitation Rex1 Blotting Western Kruppel-Like Transcription Factors Electrophoretic Mobility Shift Assay Biology Cell Line Kruppel-Like Factor 4 SOX2 Proto-Oncogene Proteins Humans Promoter Regions Genetic Transcription factor Embryonic Stem Cells reproductive and urinary physiology Homeodomain Proteins Zinc finger transcription factor Reverse Transcriptase Polymerase Chain Reaction Pre-B-Cell Leukemia Transcription Factor 1 fungi Gene Expression Regulation Developmental Nanog Homeobox Protein Cell Biology Molecular biology DNA-Binding Proteins KLF4 embryonic structures Molecular Medicine RNA Interference biological phenomena cell phenomena and immunity Chromatin immunoprecipitation Protein Binding Developmental Biology |
Zdroj: | Stem Cells. 27:2114-2125 |
ISSN: | 1549-4918 1066-5099 |
Popis: | 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. Disclosure of potential conflicts of interest is found at the end of this article. |
Databáze: | OpenAIRE |
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