The C-terminal pentapeptide of Nanog tryptophan repeat domain interacts with Nac1 and regulates stem cell proliferation but not pluripotency.
Overexpression of Nanog in mouse embryonic stem (ES) cells has been shown to abrogate the requirement of leukemia inhibitory factor for self-renewal in culture. Little is known about the molecular mechanism of Nanog function. Here we describe the role of the tryptophan repeat (WR) domain, one of the two transactivators at its C terminus, in regulating stem cell proliferation as well as pluripotency. We first created a supertransactivator, W2W3x10, by duplicating repeats W2W3 10 times and discovered that it can functionally substitute for wild type WR at sustaining pluripotency, albeit with a significantly slower cell cycle, phenocopying Nanog(9W) with the C-terminal pentapeptide (WNAAP) of WR deleted. ES cells carrying both W2W3x10 and Nanog(9W) have a longer G1 phase, a shorter S phase in cell cycle distribution and progression analysis, and a lower level of pAkt(Ser473) compared with wild type Nanog, suggesting that both mutants impact the cell cycle machinery via the phosphatidylinositol 3-kinase/Akt pathway. Both mutants remain competent in dimerizing with Nanog but cannot form a complex with Nac1 efficiently, suggesting that WNAAP may be involved in Nac1 binding. By tagging Gal4DBD with WNAAP, we demonstrated that this pentapeptide is sufficient to confer Nac1 binding. Furthermore, we can rescue W2W3x10 by placing WNAAP at the corresponding locations. Finally, we found that Nanog and Nac1 synergistically up-regulate ERas expression and promote the proliferation of ES cells. These results suggest that Nanog interacts with Nac1 through WNAAP to regulate the cell cycle of ES cells via the ERas/phosphatidylinositol 3-kinase/Akt pathway, but not pluripotency, thus decoupling cell cycle control from pluripotency.
From the Laboratory of Stem Cell Biology, Department of Biological Sciences and Biotechnology, Institutes of Biomedicine, School of Medicine, Tsinghua University, Beijing 100084, China; Stem Cell and Cancer Biology Group, Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510663, China.
Tianhua
T
Ma
From the Laboratory of Stem Cell Biology, Department of Biological Sciences and Biotechnology, Institutes of Biomedicine, School of Medicine, Tsinghua University, Beijing 100084, China; Stem Cell and Cancer Biology Group, Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510663, China.
Zhe
Z
Wang
From the Laboratory of Stem Cell Biology, Department of Biological Sciences and Biotechnology, Institutes of Biomedicine, School of Medicine, Tsinghua University, Beijing 100084, China; Stem Cell and Cancer Biology Group, Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510663, China.
Yunqian
Y
Guo
Stem Cell and Cancer Biology Group, Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510663, China. Electronic address: pei_duanqing@gibh.ac.cn.
Duanqing
D
Pei
D000595:Amino Acid Sequence; D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D000071317:Nanog Homeobox Protein; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D015689:Oncogene Protein p21(ras) / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000502:physiology; D017434:Protein Structure, Tertiary; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012097:Repressor Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016196:S Phase / Q000502:physiology; D013329:Structure-Activity Relationship; D015533:Transcriptional Activation / Q000502:physiology; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
C474773:ERas protein, mouse; D018398:Homeodomain Proteins; C484605:Nacc1 protein, mouse; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D009419:Nerve Tissue Proteins; D012097:Repressor Proteins; D014364:Tryptophan; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D015689:Oncogene Protein p21(ras)