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The C-terminal Pentapeptide of Nanog Tryptophan Repeat Domain Interacts with Nac1 and Regulates Stem Cell Proliferation but Not Pluripotency

The C-terminal Pentapeptide of Nanog Tryptophan Repeat Domain Interacts with Nac1 and Regulates... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 24, pp. 16071–16081, June 12, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. The C-terminal Pentapeptide of Nanog Tryptophan Repeat Domain Interacts with Nac1 and Regulates Stem Cell Proliferation but Not Pluripotency Received for publication, January 16, 2009, and in revised form, April 7, 2009 Published, JBC Papers in Press, April 14, 2009, DOI 10.1074/jbc.M109.005041 ‡§ ‡§ ‡§ §1 Tianhua Ma , Zhe Wang , Yunqian Guo , and Duanqing Pei From the Laboratory of Stem Cell Biology, Department of Biological Sciences and Biotechnology, Institutes of Biomedicine, School of Medicine, Tsinghua University, Beijing 100084, China and the 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 Overexpression of Nanog in mouse embryonic stem (ES) cells lized to reprogram somatic cells back to the pluripotent state has been shown to abrogate the requirement of leukemia inhib- through the iPS or induced pluripotent stem cell protocol (2–6). itory factor for self-renewal in culture. Little is known about the Nanog is a relatively new arrival into the pluripotent factor molecular mechanism of Nanog function. Here we describe the family (7, 8). Discovered by its in vitro ability to sustain ES cell role of the tryptophan repeat (WR) domain, one of the two self-renewal in the absence of LIF, Nanog was recently shown to transactivators at its C terminus, in regulating stem cell prolif- possess reprogramming potential during the generation of eration as well as pluripotency. We first created a supertransac- human iPS cells, suggesting that it possesses power similar to tivator, W2W310, by duplicating repeats W2W3 10 times and that of other core regulators, such as Oct4 and Sox2. Paradox- discovered that it can functionally substitute for wild type WR at ically, recent work from Chambers et al. (9) has demonstrated sustaining pluripotency, albeit with a significantly slower cell that Nanog works to safeguard, but is not required for, pluripo- cycle, phenocopying Nanog(9W) with the C-terminal penta- tency and appears to play a more direct role in germ line main- peptide (WNAAP) of WR deleted. ES cells carrying both tenance. Through high throughput technologies, several W2W310 and Nanog(9W) have a longer G phase, a shorter S groups have identified the downstream targets of Nanog in the phase in cell cycle distribution and progression analysis, and a genome as well as proteins with which Nanog interacts (10, 11). lower level of pAkt(Ser ) compared with wild type Nanog, sug- Although these prominent studies illustrate the potential com- plexity of the function networks Nanog regulates, they describe gesting that both mutants impact the cell cycle machinery via the phosphatidylinositol 3-kinase/Akt pathway. Both mutants very little how Nanog achieves these activities. The structural remain competent in dimerizing with Nanog but cannot form a basis of Nanog function remains largely undefined. complex with Nac1 efficiently, suggesting that WNAAP may be Embyonic stem cells can undergo unlimited self-renewal, so that the cell cycle appears to be less controlled than the somatic involved in Nac1 binding. By tagging Gal4DBD with WNAAP, ones. For example, although RB plays a key role in the progres- we demonstrated that this pentapeptide is sufficient to confer sion of somatic cell cycle through its phosphorylation by cyclin Nac1 binding. Furthermore, we can rescue W2W310 by plac- ing WNAAP at the corresponding locations. Finally, we found D/CDK4 or cyclin D/CDK6 and subsequent release of E2F to allow the expression of downstream genes critical for the pro- that Nanog and Nac1 synergistically up-regulate ERas expres- gression through the G /S checkpoint, embryonic stem cells sion and promote the proliferation of ES cells. These results execute cell cycles independent of RB phosphorylation and suggest that Nanog interacts with Nac1 through WNAAP to reg- contain only a low level of cyclin D. In addition, although the ulate the cell cycle of ES cells via the ERas/phosphatidylinositol Ras/extracellular signal-regulated kinase pathway promotes 3-kinase/Akt pathway, but not pluripotency, thus decoupling cell cycle progression in somatic cells, extracellular signal-reg- cell cycle control from pluripotency. ulated kinase signaling is dispensable for cell cycle progression in embryonic stem cells. Last, p53 is an important check point to induce cell apoptosis in somatic cells, whereas ES cells lack Recent advances have identified Oct4, Sox2, and Nanog as core such a checkpoint (12). Until now, the only known regulator factors for the mammalian pluripotency program (1). Remarkably, controlling the cell cycle of embryonic stem cells is the phos- some of these pluripotent factors have also been successfully uti- phorylation status of Akt at Ser , which is activated by PI3K and is not regulated by mitogen stimulation (13, 14). We investigated the structure-function relationship of * This work was supported by National Natural Science Foundation of Nanog in a series of studies. Based on these results, Nanog is China Grants 30725012 and 30630039; Chinese Academy of Sciences Grant KSCX2-YW-R-48; Guangzhou Science and Technology Grant divided into the N-terminal domain, DNA binding homeodo- 2006A50104002; Ministry of Science and Technology 973 Grants 2006CB701504, 2006CB943600, 2007CB948002, 2007CB947804, and 2009CB941000; and National High Technology Project 863 Grant The abbreviations used are: ES, embryonic stem; WR, tryptophan repeat; 2005AA210930. CD2, C-terminal domain 2; LIF, leukemia-inhibitory factor; PI3K, phospha- To whom correspondence should be addressed. Tel.: 86-20-3229-0706; tidylinositol 3-kinase; PBS, phosphate-buffered saline; BrdUrd, bromode- E-mail: [email protected]. oxyuridine; RT, reverse transcription; EB, embryonic body. JUNE 12, 2009• VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16071 This is an Open Access article under the CC BY license. Nanog WR Domain and ES Cell Growth main, C-terminal domain 1, tryptophan repeat (WR) domain, Co-immunoprecipitation—3 g of pCBA-hrGFP-c-Myc- and C-terminal domain 2 (CD2) (Fig. 1A). We first demon- Nanog, pCBA-hrGFP-c-Myc-Zfp281, or pCBA-hrGFP-c-Myc- strated that Nanog is a transcription activator possessing two Nac1 together with/without 1 g of pPyCAGIP-Nanog-FLAG, strong transactivators, WR and CD2 (15, 16). Reporter assays were co-transfected with 1 g of pPyCAGIP, pPyCAGIP- demonstrated that deletion of WR had little effect on transcrip- Nanog-FLAG, pPyCAGIP-Nanog(WR)-FLAG, pPyCAGIP- tion activity of Nanog, whereas removal of CD2 reduced its Nanog(W2W310)-FLAG, pPyCAGIP-Nanog(9W)-FLAG, activity severely, suggesting that WR is dispensable for Nanog pPyCAGIP-Nanog(W2W310mu)-FLAG, pCR3.1, pCR3.1- function (15, 16). Consistently, we demonstrated that CD2, FLAG-Gal4DBD, pCR3.1-FLAG-Gal4DBD-WNAAP, or apparently endowed by a few critical aromatic residues with its pCR3.1-FLAG-Gal4DBD-2xWNAAP to HEK293T cells cul- strong transactivation activity, is required for Nanog to mediate tured in a well of a 6-well plate by using the calcium phosphate LIF-independent ES cell self-renewal (17). Unexpectedly, we method. 48 h after transfection, cells were washed two times recently proved that WR plays an important role in Nanog- with PBS buffer and lysed by 200 l of TNE buffer (100 mM Tris, mediated LIF-independent ES cell self-renewal despite its pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM EDTA) plus 5 apparent lack of contribution to Nanog transcription activity. l of protease inhibitor mixture (Sigma). Whole cell lysates The detailed function of other domains, especially the N-termi- were cleared by centrifugation at 15,000 g for 5 min at 4 °C. A nal domain and C-terminal domain 1, are largely unknown. volume of 40 l of cleared whole cell lysates was reserved for Nac1 is a BTB domain-containing protein related to Dro- Western blot analysis, and the remaining supernatants were sophila bric-a-brac/tramtrack, which prevents inappropriate transferred to a new 1.5-ml tube containing 15 l of anti- neural gene expression (18, 19). Recent studies revealed that FLAG-conjugated agarose beads (Sigma) equilibrated by TNE Nac1 is a protein-interacting partner of Nanog and may partic- buffer before use. After rocking for 2 h, at 4 °C, the anti-FLAG ipate in a regulatory network for sustaining pluripotency beads were then washed five times with TNE buffer. After all of (20, 21). the TNE buffer was removed, the beads were boiled for 5 min in In this report, we describe our findings that Nanog interacts 40 lof1 SDS loading buffer (with 5% -mercaptoethanol) to with Nac1 through a pentapeptide WNAAP unit to regulate the elute the bound antibodies and antigens. After centrifuging at proliferation of mouse embryonic stem cells via the ERas/PI3K/ 12,000  g for 5 min, supernatants (8 l) were detected by Akt pathway but not pluripotency. immunoblotting. Western Blot and Antibodies—For Western blot analysis of MATERIALS AND METHODS mouse ES cells, the cells were lysed in radioimmune precipita- Plasmids—All of the WR mutants, W2W310, W3W210, tion lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% WR2, 9W, and W2W310mu, were substituted for sodium deoxycholate, 1% Triton X-100, 0.1% SDS). The pre- the Nanog WR domain to construct Nanog(W2W310), pared samples were loaded on 10% SDS-PAGE and then blotted Nanog(W3W210), Nanog(WR2), Nanog(9W), and onto polyvinylidene difluoride membrane for detection by Nanog(W2W310mu), respectively. These five Nanog WR using specific first antibody and second antibody. The antibod- mutants, wild type Nanog, and WR-truncated mutant ies we used included the following: Akt antibody (catalog num- Nanog(WR), with or without the C-terminal FLAG tag, were ber 9272; Cell Signaling), pAkt(Ser ) antibody (catalog num- subcloned into pPyCAGIP (generously provided by Dr. Cham- ber ab27773; Abcam), Nanog antibody (catalog number bers) by XhoI and NotI sites. N-terminal Myc-tagged Nanog, ab21603; Abcam), -actin antibody (catalog number A2066; Nac1, and Zfp281 were subcloned into pCBA-hrGFP (kindly Sigma), FLAG antibody (catalog number 2368; Cell Signaling), provided by Dr. Kim) or pCAG-IRES-Neo (generously pro- c-Myc antibody (catalog number K03145R; Biodesign), alkaline vided by Dr. Niwa) by XhoI and NotI sites. WNAAP and phosphatase-labeled anti-rabbit IgG antibody (catalog number 2WNAAP were ligated into EcoRV site at the C-terminal end A3687; Sigma), and peroxidase-labeled anti-rabbit IgG anti- of FLAG-Gal4DBD in pCR3.1 (Invitrogen) to construct FLAG- body (catalog number 074-1506; KPL). Gal4DBD-WNAAP and FLAG-Gal4DBD-2WNAAP. Measurement of Expansion in Cell Numbers—We plated ES Cell Culture—HEK293T cells were cultured in Dulbecco’s cells at 2 10 cells/well in 24-well plates and cultured them for modified Eagle’s medium (Hyclone) supplemented with 10% 4 days in ES medium. The cells were trypsinized and counted fetal bovine serum (Invitrogen) and antibiotics (penicillin and with a blood count plate (sample  6) every day. streptomycin; 100 g/ml). CGR8 ES cells were cultured on 0.1% Early Apoptotic Marker Detection—ES cells cultured in ES gelatin-coated substrates in ES medium consisting of Glasgow medium were trypsinized, washed in PBS, and treated with the minimum essential medium (Sigma) supplemented with 20% ApopNexin Annexin V fluorescein isothiocyanate apoptosis kit fetal bovine serum (Invitrogen), 100 mM nonessential amino (catalog number APT750; Chemicon) according to the manu- acids (Invitrogen), 0.55 mM -mercaptoethanol (Invitrogen), 2 facturer’s instructions and analyzed by a FACScalibur flow mML-glutamine (Invitrogen), and 1,000 units/ml human recombinant LIF (Chemicon). The final concentration of cytometer (BD Biosciences). DMSO (Sigma) or LY294002 (Sigma) used in inhibition of Akt Cell Cycle Distribution Analysis—ES cells cultured in ES phosphorylation was 10 M. medium were harvested, fixed in cold 70% ethanol, washed in PBS, treated with 50 g/ml propidium iodide (Sigma) and 5 g/ml RNase A (Sigma) at 37 °C for 30 min, and analyzed by a D. Pei, Y. Guo, T. Ma, J. Zhang, W. Zhou, M. Chen, G. Pan, Y. Rao, Z. Fu, and X. Chi, unpublished results. FACScalibur flow cytometer (BD Biosciences). 16072 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 24 •JUNE 12, 2009 Nanog WR Domain and ES Cell Growth FIGURE 1. Nanog mutants lacking the C-terminal pentapeptide of the WR domain slow down cell proliferation, whereas they maintain pluripotency. A, schematic illustration of full-length Nanog and its WR-truncated mutant Nanog(WR). B, sequences of wild type WR and its mutants, W2W310, W3W210, WR2, and 9W, in which repeated units are highlighted. C, morphology of ES cells constitutively expressing none, Nanog, Nanog(W2W310), Nanog(W3W210), Nanog(WR2), Nanog(9W), and Nanog(WR), after being cultured with or without LIF for 5 days. D, Western blot analysis of mock, Nanog, Nanog(W2W310), Nanog(W3W210), Nanog(WR2), Nanog(9W), and Nanog(WR) ES cells in the presence of LIF. Nanog and its mutants were detected with anti-Nanog antibody and normalized by endogenous -actin with its specific antibody. The molecular weight of endogenous Nanog is indicated on the right. E, growth curves of mock, Nanog, Nanog(W2W310), Nanog(W3W210), Nanog(WR2), Nanog(9W), and Nanog(WR) ES cells in the presence of LIF for 4 days. Cell numbers were counted every day and shown as mean S.E. (n 6, p 0.01). F, quantitative PCR analysis of pluripotent genes (nanog, pou5f1, and zfp42) of ES cells in C. The relative expression levels of nanog (containing both endogenous and exogenous expression), pou5f1, and zfp42 were measured as mean S.E. (n 6, p 0.01), and all of the relative mean values of mock ES cells were arbitrarily designated as 1. The transcript level of -actin was used as an internal reference. G–K, quantitative PCR analysis of differentiated and pluripotent genes in mock, Nanog, Nanog(9W), and Nanog(W2W310) ES cells for 0 days (EB0) or 5 days (EB5) after EB-induced differentiation. The relative expression levels of lamininB1 (endoderm marker; G), islet-1 (ectoderm marker; H), t (mesoderm marker; I), pou5f1 (pluripotent marker; J), and nanog (containing both endogenous and exogenous expression; K) were measured as mean  S.E. (n 3, p 0.05), and all of the relative mean values of mock ES cells (EB0) were arbitrarily designated as 1. The transcript level of -actin was used as an internal reference. IB, immunoblot. BrdUrd Pulse Analysis—ES cells with 70% confluence were Cells were again washed twice in PBS-TB and then resus- pulse-labeled with 10 M BrdUrd (Sigma) for 30 min. After pended in PBS containing 5 g/ml propidium iodide (Sigma) they were washed twice, labeled ES cells were cultured for 0, and 1 g/ml RNase A and analyzed on a FACScalibur flow 7, and 14 h in ES medium. Cells were trypsinized, washed in cytometer (BD Biosciences). PBS, and fixed in cold 70% ethanol for 30 min. Cell pellets Constitutive Expression of Transgenes in Mouse ES Cells— were then incubated in 4 N HCl for 30 min and washed in 0.1 CGR8 ES cells cultured in ES medium were plated in 3.5-cm M sodium borate (pH 8.5) followed by a PBS wash. Cells were dishes and transfected with 2 g of pPyCAGIP, pPyCAGIP- then resuspended in 50 l of PBS-TB (PBS containing 0.5% Nanog, pPyCAGIP-Nanog(W2W310), pPyCAGIP-Nanog- Tween 20 and 0.5% bovine serum albumin) and a 1:250 dilu- (W3W210), pPyCAGIP-Nanog(WR2), pPyCAGIP- tion of anti-BrdUrd antibody (BU-33; Sigma) for 30 min at Nanog(9W), pPyCAGIP-Nanog(WR), or pPyCAGIP- room temperature. After two washes in PBS-TB, cells were Nanog(W2W310mu) by Lipofectamine 2000 (Invitrogen). 24 incubated with fluorescein isothiocyanate-conjugated goat h after transfection, the cells were passaged by 1:50 and plated in anti-mouse IgG secondary antibody (catalog number new 6-cm dishes for puromycin (2g/ml; Invitrogen) selection. 10 sc-2010; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) days later, a single clone was picked up and expanded in a 12-well with a 1:100 dilution in PBS-TB, 30 mM at room temperature. plate and then further identified by Western blot analysis and real JUNE 12, 2009• VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16073 Nanog WR Domain and ES Cell Growth Mock Nanog Nanog(9W) Nanog(W2W3x10) 4 4 4 4 10 10 10 10 3.62% 2.55% 3.74% 3.67% 2.93% 1.76% 2.12% 1.32% 3 3 3 3 10 10 10 10 2 2 2 2 10 10 10 10 1 1 1 1 10 10 10 10 0 1.51% 0 1.71% 0 2.38% 0 1.99% 10 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 +DMSO +LY294002 Propidium Iodide 140 120 120 B 600 Mock Nanog 80 80 G1: 35.1±0.9% G1: 33.6±0.9% 450 450 S: 43.2±2.2% S: 43.6±2.1% 300 300 G2-M: 24.3±0.6% 40 40 G2-M: 23.4±0.7% 150 150 1.00 1.27 0.50 0.26 IB: anti-pAkt(S473) 0 0 1d 2d 3d 4d 1d 2d 3d 4d 0 0 1.00 0.97 0.96 0.99 G1 G2 G1 G2 IB: anti-Akt Mock Nanog Nanog(9W) Nanog(W2W3x10) 600 600 Nanog(9W) Nanog(W2W3x10) 1.00 1.02 0.99 0.98 F IB: anti-β-actin +LY294002/+DMSO G1: 40.1±0.9% 450 G1: 47.1±0.9% 1.2 S: 38.6±1.9% S: 32.1±2.0% 300 300 1.0 G2-M: 22.6±0.6% G2-M: 21.7±0.8% 0.8 0.6 0 0 G1 G2 G1 G2 0.4 IB: anti-pAkt(S473) 1.00 1.25 0.51 0.24 0.32 0.41 0.13 0.06 0.2 Mock Nanog Nanog(9W) Nanog(W2W3x10) IB: anti-Akt 3 3 3 C 3 10 10 10 10 0 1.00 0.98 0.96 0.97 0.98 0.99 0.98 0.95 0d 1d 2d 3d 4d 2 2 2 2 10 10 10 10 +DMSO +LY294002 Mock Nanog(9W) 0h Nanog Nanog(W2W3x10) 1 1 1 1 10 10 10 10 0 0 0 0 10 10 10 10 0 1024 0 1024 0 1024 0 1024 3 3 3 3 10 10 10 10 2 2 2 2 10 10 10 10 7h 1 1 1 1 10 10 10 10 0 0 0 0 10 10 10 10 0 1024 0 1024 0 1024 0 1024 3 3 3 3 10 10 10 10 2 2 2 2 10 10 10 10 14h 1 1 1 10 10 10 10 0 0 0 10 10 10 01024 0 1024 0 1024 0 1024 Propidium Iodide FIGURE 2. Both mutants lacking WNAAP shift cell cycle to longer G and short S phases with lower pAkt(Ser ). A, mock, Nanog, Nanog(9W), and Nanog(W2W310) ES cells were flow cytometrically analyzed for membrane integrity and phosphatidylserine accessibility by staining with propidium iodide and fluorescein isothiocyanate-conjugated annexin V (Annexin V-FITC). The percentage of each population (annexin V PI , annexin V PI ,or annexin V PI cells) is indicated. The data were analyzed and represented by FCS Express software. B, flow cytometric analysis of cell cycle distribution of mock, Nanog, Nanog(9W), and Nanog(W2W310) ES cells by PI staining. The data were analyzed and represented by FCS Express software. The percentages of G ,S,andG -M phases in each cell line were calculated as mean  S.E. (n  4, p  0.05). C, expression level of Akt and phosphorylated 1 2 473 473 Akt at Ser of mock, Nanog, Nanog(9W), and Nanog(W2W310) ES cells cultured with LIF. Akt and pAkt(Ser ) were detected by anti-Akt and anti-pAkt(Ser ) antibodies, respectively, and normalized by endogenous -actin with its specific antibody. All of the bands were quantified by QuantityOne (Bio-Rad). D, mock, Nanog, Nanog(9W), and Nanog(W2W310) ES cells were pulse-labeled with BrdUrd for 30 min and harvested at the time intervals indicated on the left. The cells were then stained with anti-BrdUrd antibody and propidium iodide and cytometrically analyzed to measure DNA synthesis (BrdUrd; y axis) and DNA content (propidium iodide; x axis). The data were analyzed and represented by FCS Express software. E, growth curves of mock, Nanog, Nanog(9W), and Nanog(W2W310) ES cells cultured with LIF plus 10 M DMSO (left)or10 M LY294002 (right) for 4 days. Cell numbers were counted every day and shown as mean  S.E. (n  6, p  0.01). F, the ratio of cell number in the presence of LY294002 versus those of control at each day in E was calculated and plotted. G, expression level of Akt and phosphorylated Akt at Ser of mock, Nanog, Nanog(9W), and Nanog(W2W310) ES cells cultured with LIF plus 10 M DMSO (left)or10 M LY294002 (right). Akt and pAkt(Ser ) were detected by anti-Akt and anti-pAkt(Ser ) antibodies, respectively, and normalized by endogenous -actin with its specific antibody. All of the bands were quantified by QuantityOne (Bio-Rad). IB, immunoblot. time reverse transcription (RT)-PCR analysis. Through detection Real Time RT-PCR Analysis—2 g of total RNA was reverse by anti-Nanog antibody and normalization by endogenous -ac- transcribed in a final volume of 20 l, as previously described tin, different molecular weight and band signal could be used to (22). Real time RT-PCRs were undertaken using the real time discriminate expression levels of the wild type versus mutant PCR master mix (SYBR GREEN) reagent kit (TOYOBO), Nanog in stable cell lines. The total Nanog mRNA level (contain- according to the manufacturer’s protocol. PCR was performed ing both endogenous and exogenous expression) revealed by real in a 15-l total volume for 40 cycles. The primers were as fol- time RT-PCR would also help for this discrimination. The positive lows: -actin, forward (5-agtgtgacgttgacatccgt-3) and reverse clone was named after its transgene. (5-tgctaggagccagagcagta-3); nanog, forward (5-ctcaagtcctga- For establishment of ES cell lines carrying both transgenic ggctgaca-3) and reverse (5-tgaaacctgtccttgagtgc-3); pou5f1, Nanog and Myc-Nac1, 2 g of pCAG-IRES-Neo or pCAG- forward (5-ggaagccgacaacaatgagaa-3) and reverse (5-tcgggc- Myc-Nac1-IRES-Neo was transfected into mock or Nanog ES acttcagaaacatg-3); zfp42, forward (5-cagccagaccaccatctgtc- cells, which were then selected by both puromycin (1 mg/ml) 3) and reverse (5-gtctccgatttgcatatctcctg-3); lamininB1, and G418 (500 g/ml) and identified by Western blot with anti- forward (5-ttggatagcatcaccaagtatttcc-3) and reverse (5-ctcgcgt- Nanog and anti-Myc antibody. gagggcagact-3); islet-1, forward (5-tgtggacattactccctcttaca- 16074 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 24 •JUNE 12, 2009 Mock Nanog Nanog(9W) Nanog(W2W3X10) Mock Nanog Nanog(9W) Nanog(W2W3X10) Mock Nanog Nanog(9W) Nanog(W2W3X10) Count Count Annexin V-FITC BrdU Count Count Relative fraction Cell Number (X10 ) Cell Number (X10 ) Nanog WR Domain and ES Cell Growth Statistical Analysis—All of the data from quantitative RT-PCR, cell number count, and cell cycle distri- bution were shown as mean  S.E. and analyzed by t test. p  0.05 or p  0.01 was considered to indicate statistical significance. RESULTS Nanog with Super-WR Decouples Proliferation from Pluripotency—We reported previously that Nanog possesses two potent transactiva- tion domains, WR and CD2 (15, 16) (Fig. 1A). The WR domain, a 10-tandem pentapeptide repeat starting with a tryptophan residue, has been demonstrated to be indispensable for Nanog to medi- ate pluripotency (23, 24). Further- more, we have uncovered the mechanism of transactivity of the WR domain, based on which we designed a superactive WR mutant, W2W310 (Fig. 1B). To test the effect of this superac- tive mutant on ES cells, we replaced the Nanog WR domain with this mutant to construct Nanog- (W2W310) (Fig. 1B) and trans- fected mouse ES cells with Nanog and Nanog(W2W310), respec- tively. After stable expression clones were selected, we verified their expression level by immunoblotting (Fig. 1D). When culturing mock, Nanog, and Nanog(W2W310) ES cells in the presence of LIF for 5 days, Nanog(W2W310) ES cells formed smaller colonies than both mock and FIGURE 3. WNAAP mediates Nanog-Nac1 interaction. A, Myc-Nanog (labeled at the top) was co-transfected Nanog ES cells (Fig. 1C). This phe- with vector, Nanog-FLAG, Nanog(WR)-FLAG, or Nanog(W2W310)-FLAG (indicated at the bottom) into nomenon suggests that Nanog- HEK293T cells. B, Myc-Nac1 (labeled at the top) was co-transfected with vector, Nanog-FLAG, Nanog(W2W310)-FLAG, or Nanog(WR)-FLAG (indicated at the bottom) into HEK293T cells. C, Myc-Zfp281 (W2W310) would attenuate the (labeled at the top) was co-transfected with vector or Nanog-FLAG, Nanog(W2W310)-FLAG, or Nanog(WR)- proliferation of ES cells. To confirm it, FLAG (indicated at the bottom) into HEK293T cells. D, Myc-Nanog (labeled at the top) was co-transfected with we counted the cell number for a con- vector, Nanog-FLAG, Nanog(WR)-FLAG, or Nanog(9W)-FLAG (indicated at the bottom) into HEK293T cells. E, Myc-Nac1 (labeled at the top) was co-transfected with vector, Nanog-FLAG, Nanog(9W)-FLAG, or tinuous 4 days in the presence of LIF. Nanog(WR)-FLAG (indicated at the bottom) into HEK293T cells. Whole-cell lysates were analyzed with anti- The slower proliferation rate of Myc antibody (top). After immunoprecipitation by anti-FLAG-conjugated agarose beads, the samples were detected by anti-FLAG or anti-Myc antibody (two lower panels). All of the bands were quantified by Quantity- Nanog(W2W310) cells was con- One (Bio-Rad). F, schematic illustration of FLAG-tagged Gal4DBD, Gal4DBD-W10, and Gal4DBD-2W10. sistent with its smaller colony size G, Myc-Nac1 (labeled at the top) was co-transfected with vector, FLAG-Gal4DBD, FLAG-Gal4DBD-W10, or FLAG- (Fig. 1E). Furthermore, in order to Gal4DBD-2xW10 (indicated at the bottom) into HEK293T cells. Whole cell lysates were analyzed with anti-Myc antibody (top). After immunoprecipitation by anti-FLAG-conjugated agarose beads, the samples were investigate whether this proliferation detected by anti-FLAG or anti-Myc antibody (two lower panels). All of the bands were quantified by Quantity- decrease is caused by alteration of One (Bio-Rad). IB, immunoblot; IP, immunoprecipitation. Nanog-mediated pluripotency, we cultured these three cell lines both in gat-3) and reverse (5-tgggagacatgggcgatccacc-3); t, forward the absence and presence of LIF for 5 days. As shown in Fig. 1C, (5-atgcggacaattcatctgctt-3) and reverse (5-caggcactccgaggc- Nanog and Nanog(W2W310) ES cells displayed pluripotent tagac-3); ERas, forward (5-ccaagacgcggcaaggt-3) and reverse morphology with different colony sizes, whereas mock ES cells (5-cctcctgggccctctga-3). exhibited flat morphology, dispersed growth, and other differenti- JUNE 12, 2009• VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16075 Nanog WR Domain and ES Cell Growth ated features. The pluripotent state of these ES cells was also con- maintained a high level in Nanog(W2W310) ES cells, medium firmed by quantitative RT-PCR analysis of two pluripotent mark- level in Nanog ES cells, and low level in mock cells when cultured ers, pou5f1 and zfp42. As shown in Fig. 1F, both pou5f1 and zfp42 without LIF. These results indicated that duplicating WTNPT- 16076 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 24 •JUNE 12, 2009 Nanog WR Domain and ES Cell Growth WSSQT 10 times in Nanog could decouple ES cell proliferation stable expression cell lines from ES cells (Fig. 1, A and B). After from pluripotency. we confirmed their expression levels (Fig. 1D), we found that The obvious difference between W2W310 and wild type Nanog(9W) ES cells grew slowly and formed small colonies, WR is the length of total WR domain (50 amino acids for wild whereas the cells expressing Nanog lacking the entire WR type WR versus 100 amino acids for W2W310). To investi- appeared to proliferate normally (Fig. 1, C and E). Interestingly, gate whether the longer WR domain is the key to mediate slow the proliferation rate of Nanog(9W) ES cell lines ranked proliferation, we established mouse ES cell lines constitutively between the ES cells expressing wild type Nanog and Nanog expressing Nanog mutant containing two tandem WRs (Fig. (W2W310) based on data on colony sizes (Fig. 1C) and cell 1B) and then confirmed the protein expressed by these cells number (Fig. 1E). These findings implicated that WNAAP but (Fig. 1D). Both colony sizes and cell numbers (Fig. 1, C and E) not the other nine pentapeptides of WR plays a specific role in clearly showed that ES cells expressing Nanog with double WR retarded proliferation in ES cells. domains proliferated at a rate similar to that of mock- or wild Furthermore, when we withdrew LIF, the Nanog(9W) ES cell type Nanog-expressed cell lines. When cultured in the absence line exhibited pluripotent morphology (Fig. 1C) and marker of LIF, Nanog(WR2) cells exhibited pluripotent morphology gene expressions (Fig. 1E) comparable with those of the wild and high expressions of pluripotent markers, such as pou5f1 type Nanog cell line, whereas ES cells expressing Nanog lacking and zfp42, that are comparable with the cells expressing wild the entire WR showed significant differentiation (Fig. 1, C and type Nanog (Fig. 1, C and E). These results suggest that the E). These data suggest that the last WNAAP has little effect on doubling of wild type WR length in Nanog has no effects to pluripotency and just regulates proliferation. Indeed, upon reduce proliferation and pluripotency. embryonic body (EB) formation, all ES cell lines that express mock, Besides the length, the major difference distinguishing Nanog, Nanog(9W), and Nanog(W2W310) were able to express W2W310 from wild type WR is positional alteration between differentiation markers, such as lamininB1, islet-1, and t (repre- WTNPT and WSSQT. To exclude the possibility that relative senting three germ layers) and down-regulated pou5f1 (indicating sequence alteration slows down ES proliferation, we made pluripotency) (Fig. 1, G–K), which indicated that ES cells another artificial repeat W3W210 (Fig. 1B) to substitute expressing Nanog and its mutants kept the potential to differ- wild type WR and stably transfected it into mouse ES cells. entiate, as mock ES cells did. In fact, compared with mock ES The protein level of this mutant is shown in Fig. 1D. When cells, the Nanog, Nanog(9W), and Nanog(W2W310) cell cultured in the presence of LIF, Nanog(W2W310) and lines actually suppressed differentiation to certain levels as evi- Nanog(W3W210) cell lines proliferate indistinguishably, denced by expressing higher pou5f1 and lower differentiated as indicated by both colony size (Fig. 1C) and cell number markers at 5 days EB (Fig. 1, G–K, EB5), illustrating indistin- (Fig. 1E). Even in absence of LIF, these two cell lines show guishable functions among Nanog and its two mutants in pre- similar morphology (Fig. 1C) and expression level of pluri- venting EB-induced differentiation. Taking all of the data potent marker (Fig. 1F). These findings suggested that either together, we could conclude that the C-terminal pentapeptide Nanog(W2W310) or Nanog(W3W210) can slow down of the WR domain plays a critical role in regulating ES cell proliferation of ES cells without losing pluripotency. proliferation, not sustaining pluripotency. Deleting the C-terminal Pentapeptide of Nanog WR Domain Both Mutants Lacking WNAAP Shift Cell Cycle to Longer G Slows Down Cell Proliferation without Losing Pluripotency—By and Shorter S Phases with Lower pAkt(Ser )—The smaller col- comparing sequences among W2W310, W3W210, and ony sizes and lower cell numbers we observed in Nanog(9W) WR2, we found their major difference lay on the two pen- and Nanog(W2W310) could be due to cell apoptosis. To tapeptides (WNGQP and WNAAP) at the C-terminal end of certainly exclude this possibility, the early stage apoptotic WR. A previous report comparing the Nanog protein sequence marker, phosphatidylserine, was measured. As shown in Fig. in four species showed that the latter one (WNAAP) was more 2A, we did not see significant differences in apoptotic pop- conserved in these two pentapeptides (25), suggesting that ulations (Annexin PI ) among the cell lines we had tested WNAAP may play a more significant role than other repeating thus far (mock, 2.55%; Nanog, 3.67%; Nanog(9W), 1.76%; units. To test this possibility, we made another two Nanog Nanog(W2W310), 1.32%). Thus, the attenuated prolifera- mutant constructs lacking the last pentapeptides (Nanog 9W) tions of Nanog(9W) and Nanog(W2W310) ES cells were or the entire WR domain (Nanog(WR)) and selected their not contributed to by apoptosis. To further understand FIGURE 4. The pentapeptide WNAAP can restore normal proliferative potential to the Nanog(W2W310) mutant. A, sequences of wild type WR and its mutants, W2W310 and W2W310mu, in which repeated unit and substitutive WNAAP were highlighted. B, Myc-Nac1 (labeled at the top) was co-transfected with vector or Nanog-FLAG, Nanog(W2W310mu)-FLAG, or Nanog(WR)-FLAG (indicated at the bottom) into HEK293T cells. Whole cell lysates were analyzed with anti-Myc antibody (top). After immunoprecipitation by anti-FLAG-conjugated agarose beads, the samples were detected by anti-FLAG or anti-Myc antibody (two lower panels). All of the bands were quantified by QuantityOne (Bio-Rad). C, morphology of ES cells constitutively expressing none, Nanog, Nanog(W2W310), and Nanog(W2W310mu), after they were cultured with or without LIF for 5 days. D, Western blot analysis of mock, Nanog, Nanog(W2W310), and Nanog(W2W310mu) ES cells in the presence of LIF. Nanog and its mutants, Akt, and pAkt(Ser ) were detected with anti- Nanog, anti-Akt, and anti-pAkt(Ser ) antibodies, respectively, and normalized by endogenous -actin with its specific antibody. The molecular weight of endogenous Nanog is indicated on the right. All of the quantitative bands were calculated by QuantityOne (Bio-Rad). E, growth curves of mock, Nanog, Nanog(W2W310), and Nanog(W2W310mu) ES cells in the presence of LIF for 4 days. Cell numbers were counted every day and are shown as mean  S.E. (n 6, p 0.01). F, quantitative PCR analysis of pluripotent genes (nanog, pou5f1, and zfp42) of ES cells in C. The relative expression levels of nanog (containing both endogenous and exogenous expression), pou5f1, and zfp42 were measured as mean  S.E. (n  6, p  0.01), and all of the relative mean values of mock ES cells were arbitrarily designated as 1. The transcript level of -actin was used as an internal reference. IB, immunoblot; IP, immunoprecipitation. JUNE 12, 2009• VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16077 Nanog WR Domain and ES Cell Growth the apparent slowdown of growth in Nanog(9W) and WNAAP Mediates Nanog-Nac1 Interaction—The experi- Nanog(W2W310) cells, we analyzed the cell cycle phase dis- mental evidence presented so far strongly suggests that the last tribution of the above four cell lines (Fig. 2B). Statistical analy- pentapeptide, WNAAP, mediates ES cell proliferation. ses indicated that Nanog(W2W310) and Nanog(9W) ES cells Recently, the WR domain has been shown to be involved in have significant longer G phase (12 and 6%, respectively) transactivation (15, 16), dimerization (23, 24), and interac- and shorter S phase (12 and 6%, respectively) compared tion for Sal4, Nac1, Zfp281, and Dax1 (24). Since we have with Nanog or mock ES cells (Fig. 2B). Furthermore, the longer found that the WR mutant devoid of the last two pentapep- G and shorter S phases for Nanog(W2W310) ES cells com- tides has transcriptional activity similar to that of wild type pared with those of Nanog(9W) (Fig. 2B) are quite consistent WR, we focus on dimerization and interaction with other with the lower growth rate observed for Nanog(W2W310) ES proteins. First, we tested whether Nanog(W2W310) or cells. In addition to counting cell number, we also pulse-labeled Nanog(9W) could dimerize with Nanog. The results four cell lines with BrdUrd and examined cell cycle progression revealed that Nanog(W2W310) or Nanog(9W) could form through flow cytometry (Fig. 2C). As shown, 7 h after BrdUrd a dimer with Nanog, as did wild type Nanog (Fig. 3, A and D). pulse labeling, cells from all four cell lines mainly accumulated As negative control, Nanog(WR) dimerized with Nanog at 2 N (40–45%) and 4 N (35–40%) based on DNA content, obviously weakly (see Fig. 5, A and D). These observations indicating that mitosis was proceeding (Fig. 2C). However, 14 h excluded the possibility that WNAAP participates in Nanog after BrdUrd pulse labeling, significant different distributions dimerization. Then we tested if WNAAP mediates interac- were found in Nanog(9W) and Nanog(W2W310) cell lines (2 tion with other pluripotency sustaining proteins, in which N, 25 and 40%; 2–4 N, 25 and 20%; 4 N, 50 and 40%, Nac1 and Zfp281 were the most likely candidates, because respectively) compared with that from both mock and Nanog knockdown of either would decrease mouse ES cell prolifer- cell lines (2 N, 5%; 2–4 N, 30%; 4 N, 65%). These data ation (20). Due to the profound reduction of growth rate for suggest that cells in Nanog(9W) and Nanog(W2W310) Nanog(W2W310) ES cells, we tested first whether undergo arrest at G /S transition (Fig. 2C). Nanog(W2W310) could interact with Nac1 or Zfp281. To identify a potential mechanism associated with this cell After immunoprecipitation and immunoblot, the results cycle delay, we examined the phosphorylation of Akt at Ser , revealed indistinguishable interactions of Nanog, an indicator of PI3K/Akt activity, known as an important reg- Nanog(W2W310), and Nanog(WR) with Zfp281 (Fig. ulatory pathway for the G /S transition during ES cell prolifer- 3C), whereas Nanog(W2W310) interacted weakly with Nac1 ation (13,14). As shown in Fig. 2D, overexpression of Nanog in (Fig. 3B). Indeed, Nanog(9W) shows the same ability as ES cells appears to have elevated Akt phosphorylation at Ser Nanog(WR) to bind Nac1(Fig. 3E), which is consistent with (1.3-fold), whereas overexpression of Nanog (W2W310) or its intermediate Akt phosphorylation and proliferation rate Nanog(9W) significantly reduced this phosphorylation by 50 between Nanog and Nanog(W2W310) (Figs. 1E and 2D). or75%, respectively (Fig. 2D). In contrast, the amounts of Akt Thus, Nac1 could be the interaction partner for WNAAP. In expression were nearly indistinguishable in all four ES cell lines our experiments, Nanog(WR) could interact with Nanog (Fig. 2D). Compared with both mock and Nanog ES cells, weakly and form a heterodimer with Zfp281 as does wild type delayed G /S transitions in Nanog(W2W310) or Nanog(9W) Nanog, which conflicts with previous reports (24), and these ES cells, corresponding to reduction of cell proliferation, was result might be caused by the enhanced overexpression of the consistent with their lower phosphorylation of Akt at Ser . CAG promoter we used. These results suggested that the reduction in PI3K/Akt activity To further support the idea of WNAAP mediating interac- may be responsible for the observed slowdown of G /S transi- tion between Nanog and Nac1, we fused WNAAP or two tan- tion for ES cells expressing Nanog mutants lacking WNAAP. dem WNAAP to the C terminus of the Gal4 DNA binding To further test this idea, we cultured these four ES cell lines in domain (Fig. 3F) and tested whether these two fusion proteins the presence of LY294002, a specific inhibitor of PI3K, which interact with Nac1. Pull-down assays showed that Gal4DBD- could reduce Akt phosphorylation at Ser . As indicated in Fig. WNAAP or -2WNAAP endows Nac1 binding with increas- 2E, all of the ES cell lines treated with LY294002 grew more ing affinity, whereas Gal4DBD alone does not (Fig. 3G). Thus, slowly than control groups treated with DMSO as expected; these results strongly suggest that WNAAP, the C-terminal Nanog ES cells grow faster in all four cell lines cultured with pentapeptide in WR, is sufficient to mediate the interaction of LY294002 and exhibited some resistance to this inhibitor, Nanog with Nac1. which was consistent with its highest level of phosphorylation Pentapeptide WNAAP Can Restore Normal Proliferative of Akt at Ser (Fig. 2D); and the calculated ratio of cell number Potential to Nanog(W2W310) Mutant—To further confirm in the presence of LY294002 versus those of control at each day the function of WNAAP, we then replaced the 10th and 20th in Fig. 2F suggested that Nanog confers partial resistance to pentapeptides (WSSQT) in Nanog(W2W310) with WNAAP LY294002 inhibition, whereas Nanog(W2W310) and and tested whether this replacement can restore normal func- Nanog(9W) ES cells with lower phosphorylation of Akt at tion to Nanog(W2W310mu) (Fig. 4A). First, we performed Ser are in fact more sensitive to this drug. Western blot anal- co-immunoprecipitation and immunoblot to verify that this ysis of Akt phosphorylation essentially confirmed this observa- WNAAP replacement rescued its interaction with Nac1, nearly tion (Fig. 2G). These results further demonstrated that Nanog comparably as wild type Nanog did (Fig. 4B). To evaluate the mutants lacking WNAAP reduce activity of PI3K/Akt, which in role of this replacement mutant on ES cells, we introduced turn attenuates G /S transition and cell growth. Nanog(W2W310mu) into ES cells, and enhanced expression 16078 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 24 •JUNE 12, 2009 Nanog WR Domain and ES Cell Growth was detected in Fig. 4D. Relative to low Akt phosphorylation at Ser in Nanog(W2W310) ES cells, Nanog(W2W310mu) elevated Akt phosphorylation at Ser up to the phosphorylation level in mock ES cell line (Fig. 4D). Consistent with the enhancement of Akt phosphorylation, analysis of colony size (Fig. 4C) and cell number (Fig. 4E) also demonstrated that Nanog(W2W310mu) ES cells grew in a fashion similar to the mock or Nanog ES cell line, in con- trast to the Nanog(W2W310) ES cell at a very low rate. To further evaluate whether this replacement mutation affects the role of Nanog in sustaining ES cell pluripotency independently of LIF, we cultured the Nanog- (W2W310mu) cell line in the absence of LIF. After 5 days, this cell line displayed pluripotent morphology as Nanog ES cells (Fig. 4C) and maintained a rela- tively high level pluripotent marker like the Nanog(W2W310) ES cell line (Fig. 4F). Based on this observation, we confirmed that the last pentapeptide, WNAAP, partic- ipates only in mediating cell prolif- eration, not pluripotency in ES cells. Nanog and Nac1 Synergistically Up-regulate ERas Expression in ES Cells, and Both Nanog Mutants Lacking WNAAP Might Down-regu- late ES Cell Proliferation in a Dom- inant Negative Way—ERas is specif- FIGURE 5. Nanog and Nac1 synergistically up-regulate ERas expression in ES cells, and both Nanog ically expressed by ES cells and has mutants lacking WNAAP might down-regulate ES cell proliferation in a dominant negative way. A, quan- been shown to be crucial to mediate titative PCR analysis of ERas mRNA level in mock, Myc-Nac1, Nanog, and Nanog  Myc-Nac1 ES cells. The relative expression levels of ERas were measured as mean S.E. (n 6, p 0.01), and the relative mean value PI3K/Akt activity (14). Microarray of mock ES cells was arbitrarily designated as 1. The transcript level of-actin was used as an internal reference. analysis of transcription factor tar- B, growth curves of mock, Myc-Nac1, Nanog, and Nanog Myc-Nac1 ES cells in the presence of LIF for 4 days. gets in whole genome scale, fol- Cell numbers were counted every day and are shown as mean S.E. (n 6, p 0.01). C, Western blot analysis of mock, Myc-Nac1, Nanog, and Nanog Myc-Nac1 ES cells in the presence of LIF. Nanog, Myc-Nac1, Akt, and lowed by chromatin immunopre- 473 473 pAkt(Ser ) were detected with anti-Nanog, anti-Myc, anti-Akt, and anti-pAkt(Ser ) antibodies, respectively, cipitation, indicated that ERas could and normalized by endogenous -actin with its specific antibody. The molecular weight of Myc-Nac1 is indi- be regulated by Nanog (21). cated on the right. All of the quantitative bands were calculated by QuantityOne (Bio-Rad). D, both Myc-Nac1 and Nanog-FLAG (labeled at the top) were co-transfected with vector, Nanog(WR), Nanog(9W), Together with our results shown Nanog(W2W310), and Nanog(W2W310mu) (indicated at the bottom) into HEK293T cells. Whole cell lysates here, we reasoned that Nanog and were analyzed with anti-Nanog or anti-Myc antibody (two upper panels). After immunoprecipitation by anti- FLAG-conjugated agarose beads, the samples were detected by anti-FLAG or anti-Myc antibody (two lower Nac1 might regulate transcription panels). Molecular weights of Nanog-FLAG, Nanog(WR), Nanog(9W), Nanog(W2W310), and of ERas in ES cells. To test this Nanog(W2W310mu) are indicated. All of the quantitative bands were calculated by QuantityOne (Bio-Rad). E, hypothesis, we introduced Nanog quantitative PCR analysis of ERas mRNA level in mock, Nanog(WR), Nanog(9W), Nanog(W2W310), and Nanog(W2W310mu) ES cells. The relative expression levels of ERas were measured as mean S.E. (n 6, p and Nac1 into ES cells, either indi- 0.01), and the relative mean value of mock ES cells was arbitrarily designated as 1. The transcript level of-actin vidually or in combination. The was used as an internal reference. F, schematic illustration of the proposed mechanism by which WNAAP, the expressions of these transgenes C-terminal pentapeptide of the Nanog WR domain, regulates mouse ES cell proliferation but not pluripotency. WNAAP mediates tight interaction between Nanog and Nac1, which controls ERas expression, which in turn were verified by Western blot (Fig. regulates ES cell proliferation through PI3K/Akt activity. Furthermore, Nanog mutant lacking WNAAP might 5B). Quantitative RT-PCR analysis down-regulate ES cell proliferation through interfering with the interaction between Nanog and Nac1. IB, immunoblot; IP, immunoprecipitation. showed that ERas mRNA levels JUNE 12, 2009• VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16079 Nanog WR Domain and ES Cell Growth were slightly elevated (1.2–1.4-fold) in ES cells carrying either pentapeptide of the Nanog WR domain, and Nac1 contributes Nanog or Nac1 alone compared with mock cells (Fig. 5A), to the cell cycle control of ES cells through mediation of the whereas they were more dramatically up-regulated in ES cells ERas/PI3K/Akt pathway, whereas the Nanog mutant lacking carrying both Nanog and Nac1 (up to 2.5-fold; Fig. 5A). Con- WNAAP might slow down ES cell proliferation in a dominant sistent with the up-regulated ERas, phosphorylated Akt at S473 negative way (Fig. 5F). also increased accordingly in either Nanog or Nac1 alone or DISCUSSION co-expressed cell lines (Fig. 5D), and again, ES cells expressing both Nanog and Nac1 showed higher levels of phosphorylated Nanog is the first in the class of transcription factors that can Akt than those expressing Nanog or Nac1 alone (Fig. 5D). We confer a LIF-independent self-renewal property to mouse ES then examined the proliferation rate of these cell lines. As cells in culture (7, 8). In vivo evidence suggests that Nanog plays shown in Fig. 5C, ES cells expressing both Nanog and Nac1 a critical role in the inner cell mass formation of early embryo grew much faster than other cell lines, presumably due to and the maintenance of germ line pluripotency (9). These prop- higher levels of ERas and phosphorylated Akt. These findings erties have generated tremendous interest in the mechanisms indicated that ERas might be an immediate downstream effec- through which Nanog can execute these functions both in vitro tor for Nanog and Nac1 to mediate ES cell proliferation. In and in vivo. We have taken a structure-function approach to addition, it is implicated that an unknown mechanism makes dissect the various components of Nanog protein and deter- ES cell proliferation tolerant of small changes in the ERas/PI3K/ mine the related functional correlates. So far, we have been able Akt cascade, which would explain the similar growth rate to divide Nanog into five distinct domains, the N-terminal between ES cells carrying either Nanog or Nac1 alone and mock domain, the DNA binding homeodomain, C-terminal domain ES cells. 1, WR, and CD2. Functionally, we were able to assign WR and Since the mutant Nanog(9W) and Nanog(W2W310) kept CD2 as transactivators embedded in its C terminus (15, 16). the capability to dimerize with wild type Nanog, we reasoned Our further analysis has demonstrated that CD2 is the domi- that these mutants might interfere with wild type Nanog to bind nant transactivator and that its activity is absolutely required its partner Nac1. We then co-transfected wild type Nanog and for Nanog-mediated LIF-independent ES cell self-renewal (17). Nac1 to HEK293T cells and along with various Nanog WR We report here that WNAAP from the WR domain appears to mutants, as indicated in Fig. 5D. The interactions between wild bind to Nac1 and impact the cell cycle machinery rather than type Nanog and Nac1 from various transfection combinations the pluripotency maintenance or self-renewal function of ES were examined by immunoprecipitation. As shown in Fig. 5D, cells. Moreover, the interaction between endogenous Nanog Nanog-Nac1 interaction was reduced to 60 and 30%, and Nac1 appears to be critical to regulate ERas/PI3K/Akt respectively, when co-expressed with Nanog(9W) and pathway responsible for sustaining proliferation in mouse ES Nanog(W2W310) compared with control. As expected, cells. Nanog lacking the entire WR that presumably cannot bind wild Our findings have several implications for stem cell biology. type Nanog showed no effects of interference with the interac- First, since the cell cycle of embryonic stem cells is not well tion (Fig. 5D). Surprisingly, Nanog(W2W310mu) with characterized, our finding that cell cycle and self-renewal can restored capability to bind Nac1 (Fig. 4D) showed little ef- be investigated separately may become an important tool to fect to alter the interaction (Fig. 5D). Nanog(9W) and delineate the molecular circuits regulating ES cell cycle. Sec- Nanog(W2W310) can interfere with wild type Nanog to bind ond, WR is uniquely conserved in Nanog throughout evolution. Nac1, so they might serve as dominant negative mutants upon The fact that the last pentapeptide, WNAAP, is more conserved expression in ES cells. We then expressed Nanog with various in Nanog signifies that it may participate in a critical function. WR mutations in ES cells and examined the expression levels of Our finding that it interacts with Nac1 and impacts the cell ERas by quantitative RT-PCR. As shown in Fig. 5E, expressions cycle may reveal more regulatory circuits beyond ERas/PI3K/ of Nanog(9W) and Nanog(W2W310) in ES cells reduced Akt pathways. Last, if WNAAP plays such an important role, ERas expression to 60 and 30%, respectively, compared one may argue that the other nine pentapeptides may have sim- with mock cells, although ES cells expressing Nanog(WR)or ilar or different roles in ES cell self-renewal or other physiolog- Nanog(W2W310mu) showed little change upon ERas ical property. Further work is needed to further delineate their expression. The decreased levels of ERas transcript in contribution to Nanog function. Nanog(9W) and Nanog(W2W310) ES cells could explain the lower pAkt(S473) and reduced proliferation that we showed Acknowledgments—We thank all of the members of the Pei laboratory earlier, and also in Fig. 1E, ES cells expressing Nanog(WR) for kind assistance, Dr. Guangjin Pan (University of Wisconsin-Mad- ison for manuscript revision) and Liying Du (Peking University) for growing at a normal rate could be explained by the normal ERas fluorescence-activated cell sorting analysis. levels showed in Fig. 5E. 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The C-terminal Pentapeptide of Nanog Tryptophan Repeat Domain Interacts with Nac1 and Regulates Stem Cell Proliferation but Not Pluripotency

Ma, Tianhua; Wang, Zhe; Guo, Yunqian; Pei, Duanqing
Journal of Biological Chemistry – Jun 1, 2009
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10.1074/jbc.m109.005041
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 24, pp. 16071–16081, June 12, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. The C-terminal Pentapeptide of Nanog Tryptophan Repeat Domain Interacts with Nac1 and Regulates Stem Cell Proliferation but Not Pluripotency Received for publication, January 16, 2009, and in revised form, April 7, 2009 Published, JBC Papers in Press, April 14, 2009, DOI 10.1074/jbc.M109.005041 ‡§ ‡§ ‡§ §1 Tianhua Ma , Zhe Wang , Yunqian Guo , and Duanqing Pei From the Laboratory of Stem Cell Biology, Department of Biological Sciences and Biotechnology, Institutes of Biomedicine, School of Medicine, Tsinghua University, Beijing 100084, China and the 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 Overexpression of Nanog in mouse embryonic stem (ES) cells lized to reprogram somatic cells back to the pluripotent state has been shown to abrogate the requirement of leukemia inhib- through the iPS or induced pluripotent stem cell protocol (2–6). itory factor for self-renewal in culture. Little is known about the Nanog is a relatively new arrival into the pluripotent factor molecular mechanism of Nanog function. Here we describe the family (7, 8). Discovered by its in vitro ability to sustain ES cell role of the tryptophan repeat (WR) domain, one of the two self-renewal in the absence of LIF, Nanog was recently shown to transactivators at its C terminus, in regulating stem cell prolif- possess reprogramming potential during the generation of eration as well as pluripotency. We first created a supertransac- human iPS cells, suggesting that it possesses power similar to tivator, W2W310, by duplicating repeats W2W3 10 times and that of other core regulators, such as Oct4 and Sox2. Paradox- discovered that it can functionally substitute for wild type WR at ically, recent work from Chambers et al. (9) has demonstrated sustaining pluripotency, albeit with a significantly slower cell that Nanog works to safeguard, but is not required for, pluripo- cycle, phenocopying Nanog(9W) with the C-terminal penta- tency and appears to play a more direct role in germ line main- peptide (WNAAP) of WR deleted. ES cells carrying both tenance. Through high throughput technologies, several W2W310 and Nanog(9W) have a longer G phase, a shorter S groups have identified the downstream targets of Nanog in the phase in cell cycle distribution and progression analysis, and a genome as well as proteins with which Nanog interacts (10, 11). lower level of pAkt(Ser ) compared with wild type Nanog, sug- Although these prominent studies illustrate the potential com- plexity of the function networks Nanog regulates, they describe gesting that both mutants impact the cell cycle machinery via the phosphatidylinositol 3-kinase/Akt pathway. Both mutants very little how Nanog achieves these activities. The structural remain competent in dimerizing with Nanog but cannot form a basis of Nanog function remains largely undefined. complex with Nac1 efficiently, suggesting that WNAAP may be Embyonic stem cells can undergo unlimited self-renewal, so that the cell cycle appears to be less controlled than the somatic involved in Nac1 binding. By tagging Gal4DBD with WNAAP, ones. For example, although RB plays a key role in the progres- we demonstrated that this pentapeptide is sufficient to confer sion of somatic cell cycle through its phosphorylation by cyclin Nac1 binding. Furthermore, we can rescue W2W310 by plac- ing WNAAP at the corresponding locations. Finally, we found D/CDK4 or cyclin D/CDK6 and subsequent release of E2F to allow the expression of downstream genes critical for the pro- that Nanog and Nac1 synergistically up-regulate ERas expres- gression through the G /S checkpoint, embryonic stem cells sion and promote the proliferation of ES cells. These results execute cell cycles independent of RB phosphorylation and suggest that Nanog interacts with Nac1 through WNAAP to reg- contain only a low level of cyclin D. In addition, although the ulate the cell cycle of ES cells via the ERas/phosphatidylinositol Ras/extracellular signal-regulated kinase pathway promotes 3-kinase/Akt pathway, but not pluripotency, thus decoupling cell cycle progression in somatic cells, extracellular signal-reg- cell cycle control from pluripotency. ulated kinase signaling is dispensable for cell cycle progression in embryonic stem cells. Last, p53 is an important check point to induce cell apoptosis in somatic cells, whereas ES cells lack Recent advances have identified Oct4, Sox2, and Nanog as core such a checkpoint (12). Until now, the only known regulator factors for the mammalian pluripotency program (1). Remarkably, controlling the cell cycle of embryonic stem cells is the phos- some of these pluripotent factors have also been successfully uti- phorylation status of Akt at Ser , which is activated by PI3K and is not regulated by mitogen stimulation (13, 14). We investigated the structure-function relationship of * This work was supported by National Natural Science Foundation of Nanog in a series of studies. Based on these results, Nanog is China Grants 30725012 and 30630039; Chinese Academy of Sciences Grant KSCX2-YW-R-48; Guangzhou Science and Technology Grant divided into the N-terminal domain, DNA binding homeodo- 2006A50104002; Ministry of Science and Technology 973 Grants 2006CB701504, 2006CB943600, 2007CB948002, 2007CB947804, and 2009CB941000; and National High Technology Project 863 Grant The abbreviations used are: ES, embryonic stem; WR, tryptophan repeat; 2005AA210930. CD2, C-terminal domain 2; LIF, leukemia-inhibitory factor; PI3K, phospha- To whom correspondence should be addressed. Tel.: 86-20-3229-0706; tidylinositol 3-kinase; PBS, phosphate-buffered saline; BrdUrd, bromode- E-mail: [email protected]. oxyuridine; RT, reverse transcription; EB, embryonic body. JUNE 12, 2009• VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16071 This is an Open Access article under the CC BY license. Nanog WR Domain and ES Cell Growth main, C-terminal domain 1, tryptophan repeat (WR) domain, Co-immunoprecipitation—3 g of pCBA-hrGFP-c-Myc- and C-terminal domain 2 (CD2) (Fig. 1A). We first demon- Nanog, pCBA-hrGFP-c-Myc-Zfp281, or pCBA-hrGFP-c-Myc- strated that Nanog is a transcription activator possessing two Nac1 together with/without 1 g of pPyCAGIP-Nanog-FLAG, strong transactivators, WR and CD2 (15, 16). Reporter assays were co-transfected with 1 g of pPyCAGIP, pPyCAGIP- demonstrated that deletion of WR had little effect on transcrip- Nanog-FLAG, pPyCAGIP-Nanog(WR)-FLAG, pPyCAGIP- tion activity of Nanog, whereas removal of CD2 reduced its Nanog(W2W310)-FLAG, pPyCAGIP-Nanog(9W)-FLAG, activity severely, suggesting that WR is dispensable for Nanog pPyCAGIP-Nanog(W2W310mu)-FLAG, pCR3.1, pCR3.1- function (15, 16). Consistently, we demonstrated that CD2, FLAG-Gal4DBD, pCR3.1-FLAG-Gal4DBD-WNAAP, or apparently endowed by a few critical aromatic residues with its pCR3.1-FLAG-Gal4DBD-2xWNAAP to HEK293T cells cul- strong transactivation activity, is required for Nanog to mediate tured in a well of a 6-well plate by using the calcium phosphate LIF-independent ES cell self-renewal (17). Unexpectedly, we method. 48 h after transfection, cells were washed two times recently proved that WR plays an important role in Nanog- with PBS buffer and lysed by 200 l of TNE buffer (100 mM Tris, mediated LIF-independent ES cell self-renewal despite its pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM EDTA) plus 5 apparent lack of contribution to Nanog transcription activity. l of protease inhibitor mixture (Sigma). Whole cell lysates The detailed function of other domains, especially the N-termi- were cleared by centrifugation at 15,000 g for 5 min at 4 °C. A nal domain and C-terminal domain 1, are largely unknown. volume of 40 l of cleared whole cell lysates was reserved for Nac1 is a BTB domain-containing protein related to Dro- Western blot analysis, and the remaining supernatants were sophila bric-a-brac/tramtrack, which prevents inappropriate transferred to a new 1.5-ml tube containing 15 l of anti- neural gene expression (18, 19). Recent studies revealed that FLAG-conjugated agarose beads (Sigma) equilibrated by TNE Nac1 is a protein-interacting partner of Nanog and may partic- buffer before use. After rocking for 2 h, at 4 °C, the anti-FLAG ipate in a regulatory network for sustaining pluripotency beads were then washed five times with TNE buffer. After all of (20, 21). the TNE buffer was removed, the beads were boiled for 5 min in In this report, we describe our findings that Nanog interacts 40 lof1 SDS loading buffer (with 5% -mercaptoethanol) to with Nac1 through a pentapeptide WNAAP unit to regulate the elute the bound antibodies and antigens. After centrifuging at proliferation of mouse embryonic stem cells via the ERas/PI3K/ 12,000  g for 5 min, supernatants (8 l) were detected by Akt pathway but not pluripotency. immunoblotting. Western Blot and Antibodies—For Western blot analysis of MATERIALS AND METHODS mouse ES cells, the cells were lysed in radioimmune precipita- Plasmids—All of the WR mutants, W2W310, W3W210, tion lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% WR2, 9W, and W2W310mu, were substituted for sodium deoxycholate, 1% Triton X-100, 0.1% SDS). The pre- the Nanog WR domain to construct Nanog(W2W310), pared samples were loaded on 10% SDS-PAGE and then blotted Nanog(W3W210), Nanog(WR2), Nanog(9W), and onto polyvinylidene difluoride membrane for detection by Nanog(W2W310mu), respectively. These five Nanog WR using specific first antibody and second antibody. The antibod- mutants, wild type Nanog, and WR-truncated mutant ies we used included the following: Akt antibody (catalog num- Nanog(WR), with or without the C-terminal FLAG tag, were ber 9272; Cell Signaling), pAkt(Ser ) antibody (catalog num- subcloned into pPyCAGIP (generously provided by Dr. Cham- ber ab27773; Abcam), Nanog antibody (catalog number bers) by XhoI and NotI sites. N-terminal Myc-tagged Nanog, ab21603; Abcam), -actin antibody (catalog number A2066; Nac1, and Zfp281 were subcloned into pCBA-hrGFP (kindly Sigma), FLAG antibody (catalog number 2368; Cell Signaling), provided by Dr. Kim) or pCAG-IRES-Neo (generously pro- c-Myc antibody (catalog number K03145R; Biodesign), alkaline vided by Dr. Niwa) by XhoI and NotI sites. WNAAP and phosphatase-labeled anti-rabbit IgG antibody (catalog number 2WNAAP were ligated into EcoRV site at the C-terminal end A3687; Sigma), and peroxidase-labeled anti-rabbit IgG anti- of FLAG-Gal4DBD in pCR3.1 (Invitrogen) to construct FLAG- body (catalog number 074-1506; KPL). Gal4DBD-WNAAP and FLAG-Gal4DBD-2WNAAP. Measurement of Expansion in Cell Numbers—We plated ES Cell Culture—HEK293T cells were cultured in Dulbecco’s cells at 2 10 cells/well in 24-well plates and cultured them for modified Eagle’s medium (Hyclone) supplemented with 10% 4 days in ES medium. The cells were trypsinized and counted fetal bovine serum (Invitrogen) and antibiotics (penicillin and with a blood count plate (sample  6) every day. streptomycin; 100 g/ml). CGR8 ES cells were cultured on 0.1% Early Apoptotic Marker Detection—ES cells cultured in ES gelatin-coated substrates in ES medium consisting of Glasgow medium were trypsinized, washed in PBS, and treated with the minimum essential medium (Sigma) supplemented with 20% ApopNexin Annexin V fluorescein isothiocyanate apoptosis kit fetal bovine serum (Invitrogen), 100 mM nonessential amino (catalog number APT750; Chemicon) according to the manu- acids (Invitrogen), 0.55 mM -mercaptoethanol (Invitrogen), 2 facturer’s instructions and analyzed by a FACScalibur flow mML-glutamine (Invitrogen), and 1,000 units/ml human recombinant LIF (Chemicon). The final concentration of cytometer (BD Biosciences). DMSO (Sigma) or LY294002 (Sigma) used in inhibition of Akt Cell Cycle Distribution Analysis—ES cells cultured in ES phosphorylation was 10 M. medium were harvested, fixed in cold 70% ethanol, washed in PBS, treated with 50 g/ml propidium iodide (Sigma) and 5 g/ml RNase A (Sigma) at 37 °C for 30 min, and analyzed by a D. Pei, Y. Guo, T. Ma, J. Zhang, W. Zhou, M. Chen, G. Pan, Y. Rao, Z. Fu, and X. Chi, unpublished results. FACScalibur flow cytometer (BD Biosciences). 16072 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 24 •JUNE 12, 2009 Nanog WR Domain and ES Cell Growth FIGURE 1. Nanog mutants lacking the C-terminal pentapeptide of the WR domain slow down cell proliferation, whereas they maintain pluripotency. A, schematic illustration of full-length Nanog and its WR-truncated mutant Nanog(WR). B, sequences of wild type WR and its mutants, W2W310, W3W210, WR2, and 9W, in which repeated units are highlighted. C, morphology of ES cells constitutively expressing none, Nanog, Nanog(W2W310), Nanog(W3W210), Nanog(WR2), Nanog(9W), and Nanog(WR), after being cultured with or without LIF for 5 days. D, Western blot analysis of mock, Nanog, Nanog(W2W310), Nanog(W3W210), Nanog(WR2), Nanog(9W), and Nanog(WR) ES cells in the presence of LIF. Nanog and its mutants were detected with anti-Nanog antibody and normalized by endogenous -actin with its specific antibody. The molecular weight of endogenous Nanog is indicated on the right. E, growth curves of mock, Nanog, Nanog(W2W310), Nanog(W3W210), Nanog(WR2), Nanog(9W), and Nanog(WR) ES cells in the presence of LIF for 4 days. Cell numbers were counted every day and shown as mean S.E. (n 6, p 0.01). F, quantitative PCR analysis of pluripotent genes (nanog, pou5f1, and zfp42) of ES cells in C. The relative expression levels of nanog (containing both endogenous and exogenous expression), pou5f1, and zfp42 were measured as mean S.E. (n 6, p 0.01), and all of the relative mean values of mock ES cells were arbitrarily designated as 1. The transcript level of -actin was used as an internal reference. G–K, quantitative PCR analysis of differentiated and pluripotent genes in mock, Nanog, Nanog(9W), and Nanog(W2W310) ES cells for 0 days (EB0) or 5 days (EB5) after EB-induced differentiation. The relative expression levels of lamininB1 (endoderm marker; G), islet-1 (ectoderm marker; H), t (mesoderm marker; I), pou5f1 (pluripotent marker; J), and nanog (containing both endogenous and exogenous expression; K) were measured as mean  S.E. (n 3, p 0.05), and all of the relative mean values of mock ES cells (EB0) were arbitrarily designated as 1. The transcript level of -actin was used as an internal reference. IB, immunoblot. BrdUrd Pulse Analysis—ES cells with 70% confluence were Cells were again washed twice in PBS-TB and then resus- pulse-labeled with 10 M BrdUrd (Sigma) for 30 min. After pended in PBS containing 5 g/ml propidium iodide (Sigma) they were washed twice, labeled ES cells were cultured for 0, and 1 g/ml RNase A and analyzed on a FACScalibur flow 7, and 14 h in ES medium. Cells were trypsinized, washed in cytometer (BD Biosciences). PBS, and fixed in cold 70% ethanol for 30 min. Cell pellets Constitutive Expression of Transgenes in Mouse ES Cells— were then incubated in 4 N HCl for 30 min and washed in 0.1 CGR8 ES cells cultured in ES medium were plated in 3.5-cm M sodium borate (pH 8.5) followed by a PBS wash. Cells were dishes and transfected with 2 g of pPyCAGIP, pPyCAGIP- then resuspended in 50 l of PBS-TB (PBS containing 0.5% Nanog, pPyCAGIP-Nanog(W2W310), pPyCAGIP-Nanog- Tween 20 and 0.5% bovine serum albumin) and a 1:250 dilu- (W3W210), pPyCAGIP-Nanog(WR2), pPyCAGIP- tion of anti-BrdUrd antibody (BU-33; Sigma) for 30 min at Nanog(9W), pPyCAGIP-Nanog(WR), or pPyCAGIP- room temperature. After two washes in PBS-TB, cells were Nanog(W2W310mu) by Lipofectamine 2000 (Invitrogen). 24 incubated with fluorescein isothiocyanate-conjugated goat h after transfection, the cells were passaged by 1:50 and plated in anti-mouse IgG secondary antibody (catalog number new 6-cm dishes for puromycin (2g/ml; Invitrogen) selection. 10 sc-2010; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) days later, a single clone was picked up and expanded in a 12-well with a 1:100 dilution in PBS-TB, 30 mM at room temperature. plate and then further identified by Western blot analysis and real JUNE 12, 2009• VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16073 Nanog WR Domain and ES Cell Growth Mock Nanog Nanog(9W) Nanog(W2W3x10) 4 4 4 4 10 10 10 10 3.62% 2.55% 3.74% 3.67% 2.93% 1.76% 2.12% 1.32% 3 3 3 3 10 10 10 10 2 2 2 2 10 10 10 10 1 1 1 1 10 10 10 10 0 1.51% 0 1.71% 0 2.38% 0 1.99% 10 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 +DMSO +LY294002 Propidium Iodide 140 120 120 B 600 Mock Nanog 80 80 G1: 35.1±0.9% G1: 33.6±0.9% 450 450 S: 43.2±2.2% S: 43.6±2.1% 300 300 G2-M: 24.3±0.6% 40 40 G2-M: 23.4±0.7% 150 150 1.00 1.27 0.50 0.26 IB: anti-pAkt(S473) 0 0 1d 2d 3d 4d 1d 2d 3d 4d 0 0 1.00 0.97 0.96 0.99 G1 G2 G1 G2 IB: anti-Akt Mock Nanog Nanog(9W) Nanog(W2W3x10) 600 600 Nanog(9W) Nanog(W2W3x10) 1.00 1.02 0.99 0.98 F IB: anti-β-actin +LY294002/+DMSO G1: 40.1±0.9% 450 G1: 47.1±0.9% 1.2 S: 38.6±1.9% S: 32.1±2.0% 300 300 1.0 G2-M: 22.6±0.6% G2-M: 21.7±0.8% 0.8 0.6 0 0 G1 G2 G1 G2 0.4 IB: anti-pAkt(S473) 1.00 1.25 0.51 0.24 0.32 0.41 0.13 0.06 0.2 Mock Nanog Nanog(9W) Nanog(W2W3x10) IB: anti-Akt 3 3 3 C 3 10 10 10 10 0 1.00 0.98 0.96 0.97 0.98 0.99 0.98 0.95 0d 1d 2d 3d 4d 2 2 2 2 10 10 10 10 +DMSO +LY294002 Mock Nanog(9W) 0h Nanog Nanog(W2W3x10) 1 1 1 1 10 10 10 10 0 0 0 0 10 10 10 10 0 1024 0 1024 0 1024 0 1024 3 3 3 3 10 10 10 10 2 2 2 2 10 10 10 10 7h 1 1 1 1 10 10 10 10 0 0 0 0 10 10 10 10 0 1024 0 1024 0 1024 0 1024 3 3 3 3 10 10 10 10 2 2 2 2 10 10 10 10 14h 1 1 1 10 10 10 10 0 0 0 10 10 10 01024 0 1024 0 1024 0 1024 Propidium Iodide FIGURE 2. Both mutants lacking WNAAP shift cell cycle to longer G and short S phases with lower pAkt(Ser ). A, mock, Nanog, Nanog(9W), and Nanog(W2W310) ES cells were flow cytometrically analyzed for membrane integrity and phosphatidylserine accessibility by staining with propidium iodide and fluorescein isothiocyanate-conjugated annexin V (Annexin V-FITC). The percentage of each population (annexin V PI , annexin V PI ,or annexin V PI cells) is indicated. The data were analyzed and represented by FCS Express software. B, flow cytometric analysis of cell cycle distribution of mock, Nanog, Nanog(9W), and Nanog(W2W310) ES cells by PI staining. The data were analyzed and represented by FCS Express software. The percentages of G ,S,andG -M phases in each cell line were calculated as mean  S.E. (n  4, p  0.05). C, expression level of Akt and phosphorylated 1 2 473 473 Akt at Ser of mock, Nanog, Nanog(9W), and Nanog(W2W310) ES cells cultured with LIF. Akt and pAkt(Ser ) were detected by anti-Akt and anti-pAkt(Ser ) antibodies, respectively, and normalized by endogenous -actin with its specific antibody. All of the bands were quantified by QuantityOne (Bio-Rad). D, mock, Nanog, Nanog(9W), and Nanog(W2W310) ES cells were pulse-labeled with BrdUrd for 30 min and harvested at the time intervals indicated on the left. The cells were then stained with anti-BrdUrd antibody and propidium iodide and cytometrically analyzed to measure DNA synthesis (BrdUrd; y axis) and DNA content (propidium iodide; x axis). The data were analyzed and represented by FCS Express software. E, growth curves of mock, Nanog, Nanog(9W), and Nanog(W2W310) ES cells cultured with LIF plus 10 M DMSO (left)or10 M LY294002 (right) for 4 days. Cell numbers were counted every day and shown as mean  S.E. (n  6, p  0.01). F, the ratio of cell number in the presence of LY294002 versus those of control at each day in E was calculated and plotted. G, expression level of Akt and phosphorylated Akt at Ser of mock, Nanog, Nanog(9W), and Nanog(W2W310) ES cells cultured with LIF plus 10 M DMSO (left)or10 M LY294002 (right). Akt and pAkt(Ser ) were detected by anti-Akt and anti-pAkt(Ser ) antibodies, respectively, and normalized by endogenous -actin with its specific antibody. All of the bands were quantified by QuantityOne (Bio-Rad). IB, immunoblot. time reverse transcription (RT)-PCR analysis. Through detection Real Time RT-PCR Analysis—2 g of total RNA was reverse by anti-Nanog antibody and normalization by endogenous -ac- transcribed in a final volume of 20 l, as previously described tin, different molecular weight and band signal could be used to (22). Real time RT-PCRs were undertaken using the real time discriminate expression levels of the wild type versus mutant PCR master mix (SYBR GREEN) reagent kit (TOYOBO), Nanog in stable cell lines. The total Nanog mRNA level (contain- according to the manufacturer’s protocol. PCR was performed ing both endogenous and exogenous expression) revealed by real in a 15-l total volume for 40 cycles. The primers were as fol- time RT-PCR would also help for this discrimination. The positive lows: -actin, forward (5-agtgtgacgttgacatccgt-3) and reverse clone was named after its transgene. (5-tgctaggagccagagcagta-3); nanog, forward (5-ctcaagtcctga- For establishment of ES cell lines carrying both transgenic ggctgaca-3) and reverse (5-tgaaacctgtccttgagtgc-3); pou5f1, Nanog and Myc-Nac1, 2 g of pCAG-IRES-Neo or pCAG- forward (5-ggaagccgacaacaatgagaa-3) and reverse (5-tcgggc- Myc-Nac1-IRES-Neo was transfected into mock or Nanog ES acttcagaaacatg-3); zfp42, forward (5-cagccagaccaccatctgtc- cells, which were then selected by both puromycin (1 mg/ml) 3) and reverse (5-gtctccgatttgcatatctcctg-3); lamininB1, and G418 (500 g/ml) and identified by Western blot with anti- forward (5-ttggatagcatcaccaagtatttcc-3) and reverse (5-ctcgcgt- Nanog and anti-Myc antibody. gagggcagact-3); islet-1, forward (5-tgtggacattactccctcttaca- 16074 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 24 •JUNE 12, 2009 Mock Nanog Nanog(9W) Nanog(W2W3X10) Mock Nanog Nanog(9W) Nanog(W2W3X10) Mock Nanog Nanog(9W) Nanog(W2W3X10) Count Count Annexin V-FITC BrdU Count Count Relative fraction Cell Number (X10 ) Cell Number (X10 ) Nanog WR Domain and ES Cell Growth Statistical Analysis—All of the data from quantitative RT-PCR, cell number count, and cell cycle distri- bution were shown as mean  S.E. and analyzed by t test. p  0.05 or p  0.01 was considered to indicate statistical significance. RESULTS Nanog with Super-WR Decouples Proliferation from Pluripotency—We reported previously that Nanog possesses two potent transactiva- tion domains, WR and CD2 (15, 16) (Fig. 1A). The WR domain, a 10-tandem pentapeptide repeat starting with a tryptophan residue, has been demonstrated to be indispensable for Nanog to medi- ate pluripotency (23, 24). Further- more, we have uncovered the mechanism of transactivity of the WR domain, based on which we designed a superactive WR mutant, W2W310 (Fig. 1B). To test the effect of this superac- tive mutant on ES cells, we replaced the Nanog WR domain with this mutant to construct Nanog- (W2W310) (Fig. 1B) and trans- fected mouse ES cells with Nanog and Nanog(W2W310), respec- tively. After stable expression clones were selected, we verified their expression level by immunoblotting (Fig. 1D). When culturing mock, Nanog, and Nanog(W2W310) ES cells in the presence of LIF for 5 days, Nanog(W2W310) ES cells formed smaller colonies than both mock and FIGURE 3. WNAAP mediates Nanog-Nac1 interaction. A, Myc-Nanog (labeled at the top) was co-transfected Nanog ES cells (Fig. 1C). This phe- with vector, Nanog-FLAG, Nanog(WR)-FLAG, or Nanog(W2W310)-FLAG (indicated at the bottom) into nomenon suggests that Nanog- HEK293T cells. B, Myc-Nac1 (labeled at the top) was co-transfected with vector, Nanog-FLAG, Nanog(W2W310)-FLAG, or Nanog(WR)-FLAG (indicated at the bottom) into HEK293T cells. C, Myc-Zfp281 (W2W310) would attenuate the (labeled at the top) was co-transfected with vector or Nanog-FLAG, Nanog(W2W310)-FLAG, or Nanog(WR)- proliferation of ES cells. To confirm it, FLAG (indicated at the bottom) into HEK293T cells. D, Myc-Nanog (labeled at the top) was co-transfected with we counted the cell number for a con- vector, Nanog-FLAG, Nanog(WR)-FLAG, or Nanog(9W)-FLAG (indicated at the bottom) into HEK293T cells. E, Myc-Nac1 (labeled at the top) was co-transfected with vector, Nanog-FLAG, Nanog(9W)-FLAG, or tinuous 4 days in the presence of LIF. Nanog(WR)-FLAG (indicated at the bottom) into HEK293T cells. Whole-cell lysates were analyzed with anti- The slower proliferation rate of Myc antibody (top). After immunoprecipitation by anti-FLAG-conjugated agarose beads, the samples were detected by anti-FLAG or anti-Myc antibody (two lower panels). All of the bands were quantified by Quantity- Nanog(W2W310) cells was con- One (Bio-Rad). F, schematic illustration of FLAG-tagged Gal4DBD, Gal4DBD-W10, and Gal4DBD-2W10. sistent with its smaller colony size G, Myc-Nac1 (labeled at the top) was co-transfected with vector, FLAG-Gal4DBD, FLAG-Gal4DBD-W10, or FLAG- (Fig. 1E). Furthermore, in order to Gal4DBD-2xW10 (indicated at the bottom) into HEK293T cells. Whole cell lysates were analyzed with anti-Myc antibody (top). After immunoprecipitation by anti-FLAG-conjugated agarose beads, the samples were investigate whether this proliferation detected by anti-FLAG or anti-Myc antibody (two lower panels). All of the bands were quantified by Quantity- decrease is caused by alteration of One (Bio-Rad). IB, immunoblot; IP, immunoprecipitation. Nanog-mediated pluripotency, we cultured these three cell lines both in gat-3) and reverse (5-tgggagacatgggcgatccacc-3); t, forward the absence and presence of LIF for 5 days. As shown in Fig. 1C, (5-atgcggacaattcatctgctt-3) and reverse (5-caggcactccgaggc- Nanog and Nanog(W2W310) ES cells displayed pluripotent tagac-3); ERas, forward (5-ccaagacgcggcaaggt-3) and reverse morphology with different colony sizes, whereas mock ES cells (5-cctcctgggccctctga-3). exhibited flat morphology, dispersed growth, and other differenti- JUNE 12, 2009• VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16075 Nanog WR Domain and ES Cell Growth ated features. The pluripotent state of these ES cells was also con- maintained a high level in Nanog(W2W310) ES cells, medium firmed by quantitative RT-PCR analysis of two pluripotent mark- level in Nanog ES cells, and low level in mock cells when cultured ers, pou5f1 and zfp42. As shown in Fig. 1F, both pou5f1 and zfp42 without LIF. These results indicated that duplicating WTNPT- 16076 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 24 •JUNE 12, 2009 Nanog WR Domain and ES Cell Growth WSSQT 10 times in Nanog could decouple ES cell proliferation stable expression cell lines from ES cells (Fig. 1, A and B). After from pluripotency. we confirmed their expression levels (Fig. 1D), we found that The obvious difference between W2W310 and wild type Nanog(9W) ES cells grew slowly and formed small colonies, WR is the length of total WR domain (50 amino acids for wild whereas the cells expressing Nanog lacking the entire WR type WR versus 100 amino acids for W2W310). To investi- appeared to proliferate normally (Fig. 1, C and E). Interestingly, gate whether the longer WR domain is the key to mediate slow the proliferation rate of Nanog(9W) ES cell lines ranked proliferation, we established mouse ES cell lines constitutively between the ES cells expressing wild type Nanog and Nanog expressing Nanog mutant containing two tandem WRs (Fig. (W2W310) based on data on colony sizes (Fig. 1C) and cell 1B) and then confirmed the protein expressed by these cells number (Fig. 1E). These findings implicated that WNAAP but (Fig. 1D). Both colony sizes and cell numbers (Fig. 1, C and E) not the other nine pentapeptides of WR plays a specific role in clearly showed that ES cells expressing Nanog with double WR retarded proliferation in ES cells. domains proliferated at a rate similar to that of mock- or wild Furthermore, when we withdrew LIF, the Nanog(9W) ES cell type Nanog-expressed cell lines. When cultured in the absence line exhibited pluripotent morphology (Fig. 1C) and marker of LIF, Nanog(WR2) cells exhibited pluripotent morphology gene expressions (Fig. 1E) comparable with those of the wild and high expressions of pluripotent markers, such as pou5f1 type Nanog cell line, whereas ES cells expressing Nanog lacking and zfp42, that are comparable with the cells expressing wild the entire WR showed significant differentiation (Fig. 1, C and type Nanog (Fig. 1, C and E). These results suggest that the E). These data suggest that the last WNAAP has little effect on doubling of wild type WR length in Nanog has no effects to pluripotency and just regulates proliferation. Indeed, upon reduce proliferation and pluripotency. embryonic body (EB) formation, all ES cell lines that express mock, Besides the length, the major difference distinguishing Nanog, Nanog(9W), and Nanog(W2W310) were able to express W2W310 from wild type WR is positional alteration between differentiation markers, such as lamininB1, islet-1, and t (repre- WTNPT and WSSQT. To exclude the possibility that relative senting three germ layers) and down-regulated pou5f1 (indicating sequence alteration slows down ES proliferation, we made pluripotency) (Fig. 1, G–K), which indicated that ES cells another artificial repeat W3W210 (Fig. 1B) to substitute expressing Nanog and its mutants kept the potential to differ- wild type WR and stably transfected it into mouse ES cells. entiate, as mock ES cells did. In fact, compared with mock ES The protein level of this mutant is shown in Fig. 1D. When cells, the Nanog, Nanog(9W), and Nanog(W2W310) cell cultured in the presence of LIF, Nanog(W2W310) and lines actually suppressed differentiation to certain levels as evi- Nanog(W3W210) cell lines proliferate indistinguishably, denced by expressing higher pou5f1 and lower differentiated as indicated by both colony size (Fig. 1C) and cell number markers at 5 days EB (Fig. 1, G–K, EB5), illustrating indistin- (Fig. 1E). Even in absence of LIF, these two cell lines show guishable functions among Nanog and its two mutants in pre- similar morphology (Fig. 1C) and expression level of pluri- venting EB-induced differentiation. Taking all of the data potent marker (Fig. 1F). These findings suggested that either together, we could conclude that the C-terminal pentapeptide Nanog(W2W310) or Nanog(W3W210) can slow down of the WR domain plays a critical role in regulating ES cell proliferation of ES cells without losing pluripotency. proliferation, not sustaining pluripotency. Deleting the C-terminal Pentapeptide of Nanog WR Domain Both Mutants Lacking WNAAP Shift Cell Cycle to Longer G Slows Down Cell Proliferation without Losing Pluripotency—By and Shorter S Phases with Lower pAkt(Ser )—The smaller col- comparing sequences among W2W310, W3W210, and ony sizes and lower cell numbers we observed in Nanog(9W) WR2, we found their major difference lay on the two pen- and Nanog(W2W310) could be due to cell apoptosis. To tapeptides (WNGQP and WNAAP) at the C-terminal end of certainly exclude this possibility, the early stage apoptotic WR. A previous report comparing the Nanog protein sequence marker, phosphatidylserine, was measured. As shown in Fig. in four species showed that the latter one (WNAAP) was more 2A, we did not see significant differences in apoptotic pop- conserved in these two pentapeptides (25), suggesting that ulations (Annexin PI ) among the cell lines we had tested WNAAP may play a more significant role than other repeating thus far (mock, 2.55%; Nanog, 3.67%; Nanog(9W), 1.76%; units. To test this possibility, we made another two Nanog Nanog(W2W310), 1.32%). Thus, the attenuated prolifera- mutant constructs lacking the last pentapeptides (Nanog 9W) tions of Nanog(9W) and Nanog(W2W310) ES cells were or the entire WR domain (Nanog(WR)) and selected their not contributed to by apoptosis. To further understand FIGURE 4. The pentapeptide WNAAP can restore normal proliferative potential to the Nanog(W2W310) mutant. A, sequences of wild type WR and its mutants, W2W310 and W2W310mu, in which repeated unit and substitutive WNAAP were highlighted. B, Myc-Nac1 (labeled at the top) was co-transfected with vector or Nanog-FLAG, Nanog(W2W310mu)-FLAG, or Nanog(WR)-FLAG (indicated at the bottom) into HEK293T cells. Whole cell lysates were analyzed with anti-Myc antibody (top). After immunoprecipitation by anti-FLAG-conjugated agarose beads, the samples were detected by anti-FLAG or anti-Myc antibody (two lower panels). All of the bands were quantified by QuantityOne (Bio-Rad). C, morphology of ES cells constitutively expressing none, Nanog, Nanog(W2W310), and Nanog(W2W310mu), after they were cultured with or without LIF for 5 days. D, Western blot analysis of mock, Nanog, Nanog(W2W310), and Nanog(W2W310mu) ES cells in the presence of LIF. Nanog and its mutants, Akt, and pAkt(Ser ) were detected with anti- Nanog, anti-Akt, and anti-pAkt(Ser ) antibodies, respectively, and normalized by endogenous -actin with its specific antibody. The molecular weight of endogenous Nanog is indicated on the right. All of the quantitative bands were calculated by QuantityOne (Bio-Rad). E, growth curves of mock, Nanog, Nanog(W2W310), and Nanog(W2W310mu) ES cells in the presence of LIF for 4 days. Cell numbers were counted every day and are shown as mean  S.E. (n 6, p 0.01). F, quantitative PCR analysis of pluripotent genes (nanog, pou5f1, and zfp42) of ES cells in C. The relative expression levels of nanog (containing both endogenous and exogenous expression), pou5f1, and zfp42 were measured as mean  S.E. (n  6, p  0.01), and all of the relative mean values of mock ES cells were arbitrarily designated as 1. The transcript level of -actin was used as an internal reference. IB, immunoblot; IP, immunoprecipitation. JUNE 12, 2009• VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16077 Nanog WR Domain and ES Cell Growth the apparent slowdown of growth in Nanog(9W) and WNAAP Mediates Nanog-Nac1 Interaction—The experi- Nanog(W2W310) cells, we analyzed the cell cycle phase dis- mental evidence presented so far strongly suggests that the last tribution of the above four cell lines (Fig. 2B). Statistical analy- pentapeptide, WNAAP, mediates ES cell proliferation. ses indicated that Nanog(W2W310) and Nanog(9W) ES cells Recently, the WR domain has been shown to be involved in have significant longer G phase (12 and 6%, respectively) transactivation (15, 16), dimerization (23, 24), and interac- and shorter S phase (12 and 6%, respectively) compared tion for Sal4, Nac1, Zfp281, and Dax1 (24). Since we have with Nanog or mock ES cells (Fig. 2B). Furthermore, the longer found that the WR mutant devoid of the last two pentapep- G and shorter S phases for Nanog(W2W310) ES cells com- tides has transcriptional activity similar to that of wild type pared with those of Nanog(9W) (Fig. 2B) are quite consistent WR, we focus on dimerization and interaction with other with the lower growth rate observed for Nanog(W2W310) ES proteins. First, we tested whether Nanog(W2W310) or cells. In addition to counting cell number, we also pulse-labeled Nanog(9W) could dimerize with Nanog. The results four cell lines with BrdUrd and examined cell cycle progression revealed that Nanog(W2W310) or Nanog(9W) could form through flow cytometry (Fig. 2C). As shown, 7 h after BrdUrd a dimer with Nanog, as did wild type Nanog (Fig. 3, A and D). pulse labeling, cells from all four cell lines mainly accumulated As negative control, Nanog(WR) dimerized with Nanog at 2 N (40–45%) and 4 N (35–40%) based on DNA content, obviously weakly (see Fig. 5, A and D). These observations indicating that mitosis was proceeding (Fig. 2C). However, 14 h excluded the possibility that WNAAP participates in Nanog after BrdUrd pulse labeling, significant different distributions dimerization. Then we tested if WNAAP mediates interac- were found in Nanog(9W) and Nanog(W2W310) cell lines (2 tion with other pluripotency sustaining proteins, in which N, 25 and 40%; 2–4 N, 25 and 20%; 4 N, 50 and 40%, Nac1 and Zfp281 were the most likely candidates, because respectively) compared with that from both mock and Nanog knockdown of either would decrease mouse ES cell prolifer- cell lines (2 N, 5%; 2–4 N, 30%; 4 N, 65%). These data ation (20). Due to the profound reduction of growth rate for suggest that cells in Nanog(9W) and Nanog(W2W310) Nanog(W2W310) ES cells, we tested first whether undergo arrest at G /S transition (Fig. 2C). Nanog(W2W310) could interact with Nac1 or Zfp281. To identify a potential mechanism associated with this cell After immunoprecipitation and immunoblot, the results cycle delay, we examined the phosphorylation of Akt at Ser , revealed indistinguishable interactions of Nanog, an indicator of PI3K/Akt activity, known as an important reg- Nanog(W2W310), and Nanog(WR) with Zfp281 (Fig. ulatory pathway for the G /S transition during ES cell prolifer- 3C), whereas Nanog(W2W310) interacted weakly with Nac1 ation (13,14). As shown in Fig. 2D, overexpression of Nanog in (Fig. 3B). Indeed, Nanog(9W) shows the same ability as ES cells appears to have elevated Akt phosphorylation at Ser Nanog(WR) to bind Nac1(Fig. 3E), which is consistent with (1.3-fold), whereas overexpression of Nanog (W2W310) or its intermediate Akt phosphorylation and proliferation rate Nanog(9W) significantly reduced this phosphorylation by 50 between Nanog and Nanog(W2W310) (Figs. 1E and 2D). or75%, respectively (Fig. 2D). In contrast, the amounts of Akt Thus, Nac1 could be the interaction partner for WNAAP. In expression were nearly indistinguishable in all four ES cell lines our experiments, Nanog(WR) could interact with Nanog (Fig. 2D). Compared with both mock and Nanog ES cells, weakly and form a heterodimer with Zfp281 as does wild type delayed G /S transitions in Nanog(W2W310) or Nanog(9W) Nanog, which conflicts with previous reports (24), and these ES cells, corresponding to reduction of cell proliferation, was result might be caused by the enhanced overexpression of the consistent with their lower phosphorylation of Akt at Ser . CAG promoter we used. These results suggested that the reduction in PI3K/Akt activity To further support the idea of WNAAP mediating interac- may be responsible for the observed slowdown of G /S transi- tion between Nanog and Nac1, we fused WNAAP or two tan- tion for ES cells expressing Nanog mutants lacking WNAAP. dem WNAAP to the C terminus of the Gal4 DNA binding To further test this idea, we cultured these four ES cell lines in domain (Fig. 3F) and tested whether these two fusion proteins the presence of LY294002, a specific inhibitor of PI3K, which interact with Nac1. Pull-down assays showed that Gal4DBD- could reduce Akt phosphorylation at Ser . As indicated in Fig. WNAAP or -2WNAAP endows Nac1 binding with increas- 2E, all of the ES cell lines treated with LY294002 grew more ing affinity, whereas Gal4DBD alone does not (Fig. 3G). Thus, slowly than control groups treated with DMSO as expected; these results strongly suggest that WNAAP, the C-terminal Nanog ES cells grow faster in all four cell lines cultured with pentapeptide in WR, is sufficient to mediate the interaction of LY294002 and exhibited some resistance to this inhibitor, Nanog with Nac1. which was consistent with its highest level of phosphorylation Pentapeptide WNAAP Can Restore Normal Proliferative of Akt at Ser (Fig. 2D); and the calculated ratio of cell number Potential to Nanog(W2W310) Mutant—To further confirm in the presence of LY294002 versus those of control at each day the function of WNAAP, we then replaced the 10th and 20th in Fig. 2F suggested that Nanog confers partial resistance to pentapeptides (WSSQT) in Nanog(W2W310) with WNAAP LY294002 inhibition, whereas Nanog(W2W310) and and tested whether this replacement can restore normal func- Nanog(9W) ES cells with lower phosphorylation of Akt at tion to Nanog(W2W310mu) (Fig. 4A). First, we performed Ser are in fact more sensitive to this drug. Western blot anal- co-immunoprecipitation and immunoblot to verify that this ysis of Akt phosphorylation essentially confirmed this observa- WNAAP replacement rescued its interaction with Nac1, nearly tion (Fig. 2G). These results further demonstrated that Nanog comparably as wild type Nanog did (Fig. 4B). To evaluate the mutants lacking WNAAP reduce activity of PI3K/Akt, which in role of this replacement mutant on ES cells, we introduced turn attenuates G /S transition and cell growth. Nanog(W2W310mu) into ES cells, and enhanced expression 16078 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 24 •JUNE 12, 2009 Nanog WR Domain and ES Cell Growth was detected in Fig. 4D. Relative to low Akt phosphorylation at Ser in Nanog(W2W310) ES cells, Nanog(W2W310mu) elevated Akt phosphorylation at Ser up to the phosphorylation level in mock ES cell line (Fig. 4D). Consistent with the enhancement of Akt phosphorylation, analysis of colony size (Fig. 4C) and cell number (Fig. 4E) also demonstrated that Nanog(W2W310mu) ES cells grew in a fashion similar to the mock or Nanog ES cell line, in con- trast to the Nanog(W2W310) ES cell at a very low rate. To further evaluate whether this replacement mutation affects the role of Nanog in sustaining ES cell pluripotency independently of LIF, we cultured the Nanog- (W2W310mu) cell line in the absence of LIF. After 5 days, this cell line displayed pluripotent morphology as Nanog ES cells (Fig. 4C) and maintained a rela- tively high level pluripotent marker like the Nanog(W2W310) ES cell line (Fig. 4F). Based on this observation, we confirmed that the last pentapeptide, WNAAP, partic- ipates only in mediating cell prolif- eration, not pluripotency in ES cells. Nanog and Nac1 Synergistically Up-regulate ERas Expression in ES Cells, and Both Nanog Mutants Lacking WNAAP Might Down-regu- late ES Cell Proliferation in a Dom- inant Negative Way—ERas is specif- FIGURE 5. Nanog and Nac1 synergistically up-regulate ERas expression in ES cells, and both Nanog ically expressed by ES cells and has mutants lacking WNAAP might down-regulate ES cell proliferation in a dominant negative way. A, quan- been shown to be crucial to mediate titative PCR analysis of ERas mRNA level in mock, Myc-Nac1, Nanog, and Nanog  Myc-Nac1 ES cells. The relative expression levels of ERas were measured as mean S.E. (n 6, p 0.01), and the relative mean value PI3K/Akt activity (14). Microarray of mock ES cells was arbitrarily designated as 1. The transcript level of-actin was used as an internal reference. analysis of transcription factor tar- B, growth curves of mock, Myc-Nac1, Nanog, and Nanog Myc-Nac1 ES cells in the presence of LIF for 4 days. gets in whole genome scale, fol- Cell numbers were counted every day and are shown as mean S.E. (n 6, p 0.01). C, Western blot analysis of mock, Myc-Nac1, Nanog, and Nanog Myc-Nac1 ES cells in the presence of LIF. Nanog, Myc-Nac1, Akt, and lowed by chromatin immunopre- 473 473 pAkt(Ser ) were detected with anti-Nanog, anti-Myc, anti-Akt, and anti-pAkt(Ser ) antibodies, respectively, cipitation, indicated that ERas could and normalized by endogenous -actin with its specific antibody. The molecular weight of Myc-Nac1 is indi- be regulated by Nanog (21). cated on the right. All of the quantitative bands were calculated by QuantityOne (Bio-Rad). D, both Myc-Nac1 and Nanog-FLAG (labeled at the top) were co-transfected with vector, Nanog(WR), Nanog(9W), Together with our results shown Nanog(W2W310), and Nanog(W2W310mu) (indicated at the bottom) into HEK293T cells. Whole cell lysates here, we reasoned that Nanog and were analyzed with anti-Nanog or anti-Myc antibody (two upper panels). After immunoprecipitation by anti- FLAG-conjugated agarose beads, the samples were detected by anti-FLAG or anti-Myc antibody (two lower Nac1 might regulate transcription panels). Molecular weights of Nanog-FLAG, Nanog(WR), Nanog(9W), Nanog(W2W310), and of ERas in ES cells. To test this Nanog(W2W310mu) are indicated. All of the quantitative bands were calculated by QuantityOne (Bio-Rad). E, hypothesis, we introduced Nanog quantitative PCR analysis of ERas mRNA level in mock, Nanog(WR), Nanog(9W), Nanog(W2W310), and Nanog(W2W310mu) ES cells. The relative expression levels of ERas were measured as mean S.E. (n 6, p and Nac1 into ES cells, either indi- 0.01), and the relative mean value of mock ES cells was arbitrarily designated as 1. The transcript level of-actin vidually or in combination. The was used as an internal reference. F, schematic illustration of the proposed mechanism by which WNAAP, the expressions of these transgenes C-terminal pentapeptide of the Nanog WR domain, regulates mouse ES cell proliferation but not pluripotency. WNAAP mediates tight interaction between Nanog and Nac1, which controls ERas expression, which in turn were verified by Western blot (Fig. regulates ES cell proliferation through PI3K/Akt activity. Furthermore, Nanog mutant lacking WNAAP might 5B). Quantitative RT-PCR analysis down-regulate ES cell proliferation through interfering with the interaction between Nanog and Nac1. IB, immunoblot; IP, immunoprecipitation. showed that ERas mRNA levels JUNE 12, 2009• VOLUME 284 • NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 16079 Nanog WR Domain and ES Cell Growth were slightly elevated (1.2–1.4-fold) in ES cells carrying either pentapeptide of the Nanog WR domain, and Nac1 contributes Nanog or Nac1 alone compared with mock cells (Fig. 5A), to the cell cycle control of ES cells through mediation of the whereas they were more dramatically up-regulated in ES cells ERas/PI3K/Akt pathway, whereas the Nanog mutant lacking carrying both Nanog and Nac1 (up to 2.5-fold; Fig. 5A). Con- WNAAP might slow down ES cell proliferation in a dominant sistent with the up-regulated ERas, phosphorylated Akt at S473 negative way (Fig. 5F). also increased accordingly in either Nanog or Nac1 alone or DISCUSSION co-expressed cell lines (Fig. 5D), and again, ES cells expressing both Nanog and Nac1 showed higher levels of phosphorylated Nanog is the first in the class of transcription factors that can Akt than those expressing Nanog or Nac1 alone (Fig. 5D). We confer a LIF-independent self-renewal property to mouse ES then examined the proliferation rate of these cell lines. As cells in culture (7, 8). In vivo evidence suggests that Nanog plays shown in Fig. 5C, ES cells expressing both Nanog and Nac1 a critical role in the inner cell mass formation of early embryo grew much faster than other cell lines, presumably due to and the maintenance of germ line pluripotency (9). These prop- higher levels of ERas and phosphorylated Akt. These findings erties have generated tremendous interest in the mechanisms indicated that ERas might be an immediate downstream effec- through which Nanog can execute these functions both in vitro tor for Nanog and Nac1 to mediate ES cell proliferation. In and in vivo. We have taken a structure-function approach to addition, it is implicated that an unknown mechanism makes dissect the various components of Nanog protein and deter- ES cell proliferation tolerant of small changes in the ERas/PI3K/ mine the related functional correlates. So far, we have been able Akt cascade, which would explain the similar growth rate to divide Nanog into five distinct domains, the N-terminal between ES cells carrying either Nanog or Nac1 alone and mock domain, the DNA binding homeodomain, C-terminal domain ES cells. 1, WR, and CD2. Functionally, we were able to assign WR and Since the mutant Nanog(9W) and Nanog(W2W310) kept CD2 as transactivators embedded in its C terminus (15, 16). the capability to dimerize with wild type Nanog, we reasoned Our further analysis has demonstrated that CD2 is the domi- that these mutants might interfere with wild type Nanog to bind nant transactivator and that its activity is absolutely required its partner Nac1. We then co-transfected wild type Nanog and for Nanog-mediated LIF-independent ES cell self-renewal (17). Nac1 to HEK293T cells and along with various Nanog WR We report here that WNAAP from the WR domain appears to mutants, as indicated in Fig. 5D. The interactions between wild bind to Nac1 and impact the cell cycle machinery rather than type Nanog and Nac1 from various transfection combinations the pluripotency maintenance or self-renewal function of ES were examined by immunoprecipitation. As shown in Fig. 5D, cells. Moreover, the interaction between endogenous Nanog Nanog-Nac1 interaction was reduced to 60 and 30%, and Nac1 appears to be critical to regulate ERas/PI3K/Akt respectively, when co-expressed with Nanog(9W) and pathway responsible for sustaining proliferation in mouse ES Nanog(W2W310) compared with control. As expected, cells. Nanog lacking the entire WR that presumably cannot bind wild Our findings have several implications for stem cell biology. type Nanog showed no effects of interference with the interac- First, since the cell cycle of embryonic stem cells is not well tion (Fig. 5D). Surprisingly, Nanog(W2W310mu) with characterized, our finding that cell cycle and self-renewal can restored capability to bind Nac1 (Fig. 4D) showed little ef- be investigated separately may become an important tool to fect to alter the interaction (Fig. 5D). Nanog(9W) and delineate the molecular circuits regulating ES cell cycle. Sec- Nanog(W2W310) can interfere with wild type Nanog to bind ond, WR is uniquely conserved in Nanog throughout evolution. Nac1, so they might serve as dominant negative mutants upon The fact that the last pentapeptide, WNAAP, is more conserved expression in ES cells. We then expressed Nanog with various in Nanog signifies that it may participate in a critical function. WR mutations in ES cells and examined the expression levels of Our finding that it interacts with Nac1 and impacts the cell ERas by quantitative RT-PCR. As shown in Fig. 5E, expressions cycle may reveal more regulatory circuits beyond ERas/PI3K/ of Nanog(9W) and Nanog(W2W310) in ES cells reduced Akt pathways. Last, if WNAAP plays such an important role, ERas expression to 60 and 30%, respectively, compared one may argue that the other nine pentapeptides may have sim- with mock cells, although ES cells expressing Nanog(WR)or ilar or different roles in ES cell self-renewal or other physiolog- Nanog(W2W310mu) showed little change upon ERas ical property. Further work is needed to further delineate their expression. The decreased levels of ERas transcript in contribution to Nanog function. Nanog(9W) and Nanog(W2W310) ES cells could explain the lower pAkt(S473) and reduced proliferation that we showed Acknowledgments—We thank all of the members of the Pei laboratory earlier, and also in Fig. 1E, ES cells expressing Nanog(WR) for kind assistance, Dr. Guangjin Pan (University of Wisconsin-Mad- ison for manuscript revision) and Liying Du (Peking University) for growing at a normal rate could be explained by the normal ERas fluorescence-activated cell sorting analysis. levels showed in Fig. 5E. 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Published: Jun 1, 2009

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