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• W2020772572 abstract "Ectopic expression of transcription factors has been shown to reprogram somatic cells into induced pluripotent stem (iPS) cells. It remains largely unexplored how this process is regulated by post-translational modifications. Several reprogramming factors possess conserved sumoylation sites, so we investigated whether and how this modification regulates reprogramming of fibroblasts into iPS cells. Substitution of the sole sumoylation site of the Krüppel-like factor (KLF4), a well known reprogramming factor, promoted iPS cell formation. In comparison, much smaller effects on reprogramming were observed for sumoylation-deficient mutants of SOX2 and OCT4, two other classical reprogramming factors. We also analyzed KLF2, a KLF4 homolog and a member of the KLF family of transcription factors with a known role in reprogramming. KLF2 was sumoylated at two conserved neighboring motifs, but substitution of the key lysine residues only stimulated reprogramming slightly. KLF5 is another KLF member with an established link to embryonic stem cell pluripotency. Interestingly, although it was much more efficiently sumoylated than either KLF2 or KLF4, KLF5 was inactive in reprogramming, and its sumoylation was not responsible for this deficiency. Furthermore, sumoylation of KLF4 but not KLF2 or KLF5 stimulated adipocyte differentiation. These results thus demonstrate the importance KLF4 sumoylation in regulating pluripotency and adipocyte differentiation. Ectopic expression of transcription factors has been shown to reprogram somatic cells into induced pluripotent stem (iPS) cells. It remains largely unexplored how this process is regulated by post-translational modifications. Several reprogramming factors possess conserved sumoylation sites, so we investigated whether and how this modification regulates reprogramming of fibroblasts into iPS cells. Substitution of the sole sumoylation site of the Krüppel-like factor (KLF4), a well known reprogramming factor, promoted iPS cell formation. In comparison, much smaller effects on reprogramming were observed for sumoylation-deficient mutants of SOX2 and OCT4, two other classical reprogramming factors. We also analyzed KLF2, a KLF4 homolog and a member of the KLF family of transcription factors with a known role in reprogramming. KLF2 was sumoylated at two conserved neighboring motifs, but substitution of the key lysine residues only stimulated reprogramming slightly. KLF5 is another KLF member with an established link to embryonic stem cell pluripotency. Interestingly, although it was much more efficiently sumoylated than either KLF2 or KLF4, KLF5 was inactive in reprogramming, and its sumoylation was not responsible for this deficiency. Furthermore, sumoylation of KLF4 but not KLF2 or KLF5 stimulated adipocyte differentiation. These results thus demonstrate the importance KLF4 sumoylation in regulating pluripotency and adipocyte differentiation. Post-translational modification is essential for regulating protein functions in diverse organisms. Rather than attachment of small chemical moieties such as phospho, acetyl, and methyl groups, sumoylation adds an ∼10-kDa small ubiquitin-like modifier (SUMO) 5The abbreviations used are: SUMOsmall ubiquitin-like modifieriPSinduced pluripotent stemmEFmouse embryonic fibroblastERRestrogen-related receptorIPimmunoprecipitationKLFKrüppel-like factor. polypeptide to the ϵ-amino group of lysine residues (1Gill G. SUMO and ubiquitin in the nucleus: different functions, similar mechanisms?.Genes Dev. 2004; 18: 2046-2059Crossref PubMed Scopus (621) Google Scholar, 2Hochstrasser M. Origin and function of ubiquitin-like proteins.Nature. 2009; 458: 422-429Crossref PubMed Scopus (596) Google Scholar, 3Geiss-Friedlander R. Melchior F. Concepts in sumoylation: a decade on.Nat. Rev. Mol. Cell Biol. 2007; 8: 947-956Crossref PubMed Scopus (1359) Google Scholar). Sumoylation exists in all eukaryotes and is essential for viability (4Nacerddine K. Lehembre F. Bhaumik M. Artus J. Cohen-Tannoudji M. Babinet C. Pandolfi P.P. Dejean A. The SUMO pathway is essential for nuclear integrity and chromosome segregation in mice.Dev. Cell. 2005; 9: 769-779Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar). In humans, there are four different SUMO proteins, SUMO1, -2, -3, and -4. SUMO2 and SUMO3 are highly homologous (95% identical) and allow both mono- and polysumoylation (5Hay R.T. SUMO: a history of modification.Mol. Cell. 2005; 18: 1-12Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar). SUMO1 is 47% identical to SUMO2 and -3, but it lacks the key lysine residue for polysumoylation, thereby conferring only monosumoylation (2Hochstrasser M. Origin and function of ubiquitin-like proteins.Nature. 2009; 458: 422-429Crossref PubMed Scopus (596) Google Scholar, 3Geiss-Friedlander R. Melchior F. Concepts in sumoylation: a decade on.Nat. Rev. Mol. Cell Biol. 2007; 8: 947-956Crossref PubMed Scopus (1359) Google Scholar). The functional relevance of SUMO4 remains unclear. SUMOs are ∼18% identical to ubiquitin at the sequence level and have three-dimensional structural folds similar to that of ubiquitin (2Hochstrasser M. Origin and function of ubiquitin-like proteins.Nature. 2009; 458: 422-429Crossref PubMed Scopus (596) Google Scholar, 3Geiss-Friedlander R. Melchior F. Concepts in sumoylation: a decade on.Nat. Rev. Mol. Cell Biol. 2007; 8: 947-956Crossref PubMed Scopus (1359) Google Scholar). Like ubiquitination, a conserved enzymatic cascade catalyzes sumoylation, including a heterodimeric E1 activating enzyme (SAE1/SAE2), an E2 conjugating enzyme (UBC9), and multiple E3 ligases (1Gill G. SUMO and ubiquitin in the nucleus: different functions, similar mechanisms?.Genes Dev. 2004; 18: 2046-2059Crossref PubMed Scopus (621) Google Scholar, 3Geiss-Friedlander R. Melchior F. Concepts in sumoylation: a decade on.Nat. Rev. Mol. Cell Biol. 2007; 8: 947-956Crossref PubMed Scopus (1359) Google Scholar). Sentrin-specific proteases remove SUMO polypeptides from target proteins, rendering sumoylation dynamic and reversible (5Hay R.T. SUMO: a history of modification.Mol. Cell. 2005; 18: 1-12Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar). small ubiquitin-like modifier induced pluripotent stem mouse embryonic fibroblast estrogen-related receptor immunoprecipitation Krüppel-like factor. Numerous proteins have been found to be sumoylated in diverse species from yeast to human, and ∼50% of known sumoylation sites conform to the classical consensus sequence ψKXE, where ψ is a bulky hydrophobic amino acid (such as Ile, Leu, and Val), and X is any residue (1Gill G. SUMO and ubiquitin in the nucleus: different functions, similar mechanisms?.Genes Dev. 2004; 18: 2046-2059Crossref PubMed Scopus (621) Google Scholar, 5Hay R.T. SUMO: a history of modification.Mol. Cell. 2005; 18: 1-12Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar, 6Blomster H.A. Hietakangas V. Wu J. Kouvonen P. Hautaniemi S. Sistonen L. Novel proteomics strategy brings insight into the prevalence of SUMO-2 target sites.Mol. Cell. Proteomics. 2009; 8: 1382-1390Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). A subgroup of known sumoylation sites contains one or a few acidic residues located two residues C-terminal from the core motif ψKXE and forms a negatively charged amino acid-dependent sumoylation motif (7Yang S.H. Galanis A. Witty J. Sharrocks A.D. An extended consensus motif enhances the specificity of substrate modification by SUMO.EMBO J. 2006; 25: 5083-5093Crossref PubMed Scopus (167) Google Scholar). The negative charge enhances the affinity for Ubc9 through binding to its positively charged surface close to the sumoylation pocket (7Yang S.H. Galanis A. Witty J. Sharrocks A.D. An extended consensus motif enhances the specificity of substrate modification by SUMO.EMBO J. 2006; 25: 5083-5093Crossref PubMed Scopus (167) Google Scholar, 8Mohideen F. Capili A.D. Bilimoria P.M. Yamada T. Bonni A. Lima C.D. A molecular basis for phosphorylation-dependent SUMO conjugation by the E2 UBC9.Nat. Struct. Mol. Biol. 2009; 16: 945-952Crossref PubMed Scopus (69) Google Scholar). Similar to the negatively charged motif is the phosphorylation-dependent sumoylation motif ψKXEXX(S/T), shared by HSF1, PPARγ, MEF2, estrogen-related receptors (ERRs), and others (9Hietakangas V. Anckar J. Blomster H.A. Fujimoto M. Palvimo J.J. Nakai A. Sistonen L. PDSM, a motif for phosphorylation-dependent SUMO modification.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 45-50Crossref PubMed Scopus (381) Google Scholar, 10Shalizi A. Gaudillière B. Yuan Z. Stegmüller J. Shirogane T. Ge Q. Tan Y. Schulman B. Harper J.W. Bonni A. A calcium-regulated MEF2 sumoylation switch controls postsynaptic differentiation.Science. 2006; 311: 1012-1017Crossref PubMed Scopus (378) Google Scholar, 11Grégoire S. Tremblay A.M. Xiao L. Yang Q. Ma K. Nie J. Mao Z. Wu Z. Giguère V. Yang X.J. Control of MEF2 transcriptional activity by coordinated phosphorylation and sumoylation.J. Biol. Chem. 2006; 281: 4423-4433Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 12Tremblay A.M. Wilson B.J. Yang X.J. Giguère V. Phosphorylation-dependent sumoylation regulates estrogen-related receptor-α and -γ transcriptional activity through a synergy control motif.Mol. Endocrinol. 2008; 22: 570-584Crossref PubMed Scopus (77) Google Scholar). Signal-dependent phosphorylation of the Ser/Thr residue promotes sumoylation and provides a unique mechanism for phosphorylation-dependent sumoylation in response to different signaling pathways (13Yang X.J. Grégoire S. A recurrent phospho-sumoyl switch in transcriptional repression and beyond.Mol. Cell. 2006; 23: 779-786Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The negative charge resulting from phosphorylation enhances the affinity for a positively charged surface on Ubc9 and promotes sumoylation (8Mohideen F. Capili A.D. Bilimoria P.M. Yamada T. Bonni A. Lima C.D. A molecular basis for phosphorylation-dependent SUMO conjugation by the E2 UBC9.Nat. Struct. Mol. Biol. 2009; 16: 945-952Crossref PubMed Scopus (69) Google Scholar). We searched sequence databases with the extended motif ψKXEXX(S/T) and identified additional targets that are potentially subject to phosphorylation-dependent sumoylation (13Yang X.J. Grégoire S. A recurrent phospho-sumoyl switch in transcriptional repression and beyond.Mol. Cell. 2006; 23: 779-786Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Two of them are KLF4 and SOX2, both of which are sumoylated (14Kawai-Kowase K. Ohshima T. Matsui H. Tanaka T. Shimizu T. Iso T. Arai M. Owens G.K. Kurabayashi M. PIAS1 mediates TGFβ-induced SM α-actin gene expression through inhibition of KLF4 function-expression by protein sumoylation.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 99-106Crossref PubMed Scopus (43) Google Scholar, 15Du J.X. McConnell B.B. Yang V.W. A small ubiquitin-related modifier-interacting motif functions as the transcriptional activation domain of Kruppel-like factor 4.J. Biol. Chem. 2010; 285: 28298-28308Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 16Tsuruzoe S. Ishihara K. Uchimura Y. Watanabe S. Sekita Y. Aoto T. Saitoh H. Yuasa Y. Niwa H. Kawasuji M. Baba H. Nakao M. Inhibition of DNA binding of Sox2 by the SUMO conjugation.Biochem. Biophys. Res. Commun. 2006; 351: 920-926Crossref PubMed Scopus (95) Google Scholar), raising the question whether neighboring phosphorylation regulates sumoylation. Strikingly, both are among the four transcription factors initially found to reprogram mouse fibroblasts to iPS cells (17Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (18948) Google Scholar). The other two reprogramming factors are OCT4 (also known as Pou5f1) and c-MYC. Interestingly, OCT4 also contains a sumoylation site (18Wei F. Schöler H.R. Atchison M.L. Sumoylation of Oct4 enhances its stability, DNA binding, and transactivation.J. Biol. Chem. 2007; 282: 21551-21560Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 19Tolkunova E. Malashicheva A. Parfenov V.N. Sustmann C. Grosschedl R. Tomilin A. PIAS proteins as repressors of Oct4 function.J. Mol. Biol. 2007; 374: 1200-1212Crossref PubMed Scopus (32) Google Scholar), so three of the four reprogramming factors have been shown to be sumoylated. In addition, two KLF4 homologs, KLF2 and KLF5, play a role in reprogramming (20Nakagawa M. Koyanagi M. Tanabe K. Takahashi K. Ichisaka T. Aoi T. Okita K. Mochiduki Y. Takizawa N. Yamanaka S. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts.Nat. Biotechnol. 2008; 26: 101-106Crossref PubMed Scopus (2206) Google Scholar). Although KLF5 is known to be sumoylated (21Du J.X. Bialkowska A.B. McConnell B.B. Yang V.W. SUMOylation regulates nuclear localization of Kruppel-like factor 5.J. Biol. Chem. 2008; 283: 31991-32002Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 22Oishi Y. Manabe I. Tobe K. Ohsugi M. Kubota T. Fujiu K. Maemura K. Kubota N. Kadowaki T. Nagai R. SUMOylation of Kruppel-like transcription factor 5 acts as a molecular switch in transcriptional programs of lipid metabolism involving PPAR-δ.Nat. Med. 2008; 14: 656-666Crossref PubMed Scopus (125) Google Scholar), KLF2 possesses two putative sumoylation sites awaiting characterization. Furthermore, ERRs are subject to phosphorylation-dependent sumoylation and have a role in reprogramming (12Tremblay A.M. Wilson B.J. Yang X.J. Giguère V. Phosphorylation-dependent sumoylation regulates estrogen-related receptor-α and -γ transcriptional activity through a synergy control motif.Mol. Endocrinol. 2008; 22: 570-584Crossref PubMed Scopus (77) Google Scholar, 23Feng B. Jiang J. Kraus P. Ng J.H. Heng J.C. Chan Y.S. Yaw L.P. Zhang W. Loh Y.H. Han J. Vega V.B. Cacheux-Rataboul V. Lim B. Lufkin T. Ng H.H. Reprogramming of fibroblasts into induced pluripotent stem cells with orphan nuclear receptor Esrrb.Nat. Cell Biol. 2009; 11: 197-203Crossref PubMed Scopus (379) Google Scholar). These observations suggest the intriguing possibility that sumoylation regulates iPS cell generation and pluripotency. Since the initial description of iPS cell formation by ectopic expression of only four transcription factors (17Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (18948) Google Scholar), there have been intensive research efforts to apply this technology to disease modeling and autologous cell therapy (24Jaenisch R. Young R. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming.Cell. 2008; 132: 567-582Abstract Full Text Full Text PDF PubMed Scopus (1117) Google Scholar, 25Yamanaka S. Blau H.M. Nuclear reprogramming to a pluripotent state by three approaches.Nature. 2010; 465: 704-712Crossref PubMed Scopus (587) Google Scholar, 26Stadtfeld M. Hochedlinger K. Induced pluripotency: history, mechanisms, and applications.Genes Dev. 2010; 24: 2239-2263Crossref PubMed Scopus (596) Google Scholar). A better understanding of the underlying molecular and cellular mechanisms is important for further improvement and eventual optimization of this powerful technology. Although post-translational modifications such as sumoylation are crucial for various transcription factors to function (27Holmberg C.I. Tran S.E. Eriksson J.E. Sistonen L. Multisite phosphorylation provides sophisticated regulation of transcription factors.Trends Biochem. Sci. 2002; 27: 619-627Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 28Latham J.A. Dent S.Y. Cross-regulation of histone modifications.Nat. Struct. Mol. Biol. 2007; 14: 1017-1024Crossref PubMed Scopus (327) Google Scholar, 29Yang X.J. Seto E. Lysine acetylation: codified crosstalk with other posttranslational modifications.Mol. Cell. 2008; 31: 449-461Abstract Full Text Full Text PDF PubMed Scopus (783) Google Scholar), it remains not so clear how such modifications may affect iPS cell generation. We thus analyzed how sumoylation of KLF4, SOX2, and OCT4 may regulate iPS cell formation. Here, we report that sumoylation of these factors inhibits iPS cell induction. Interestingly, KLF4 sumoylation appeared to stimulate adipocyte differentiation. In addition, we analyzed KLF2 and KLF5. Like KLF5, KLF2 was sumoylated at two sumoylation sites. However, substitution of the sumoylation sites on KLF2 or KLF5 had minimal effects on reprogramming or adipocyte differentiation. These findings support the importance of KLF4 sumoylation in pluripotency induction and adipocyte differentiation. HEK293 and 3T3-L1 were cultured and expanded in DMEM, 10% fetal bovine serum (FBS), and penicillin and streptomycin (50 μg/ml each). Mouse embryonic fibroblasts (MEFs) were derived from 13.5-day post-coitus mouse embryos as described previously (30Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (15033) Google Scholar). Briefly, a 13.5-day post-coitus pregnant mouse (FVB strain, Jax) was sacrificed by cervical dislocation under sterile conditions, and the embryos were surgically removed from the uterus. After the head, forelimb, tail, and abdomen were surgically removed, the remaining embryos were nipped into pieces and incubated in 0.25% trypsin for 30 min. Trypsinization was repeated twice, and after each time, trypsinized cells were separated from the tissue pieces and plated onto a 10-cm culture dish. This was considered as passage 0, and MEFs were expanded for 3 to 4 passages. MEFs at passages 2–3 were used for iPS cell generation. For preparation of feeder layers, MEFs were irradiated at 6000 rads or treated with 10 μg/ml mitomycin C (Sigma) for 3 h. FVB mice were maintained, bred, and sacrificed according to an animal use protocol approved by McGill University Animal Care Committee. For MEF preparation, we encountered mysterious contamination by “black swimming dots,” which appeared to be very similar to what was reported to be Achromobacter (31Gray J.S. Birmingham J.M. Fenton J.I. Got black swimming dots in your cell culture? Identification of Achromobacter as a novel cell culture contaminant.Biologicals. 2010; 38: 273-277Crossref PubMed Scopus (18) Google Scholar). In light of the similarity, ciprofloxacin and piperacillin (10 μg/ml each; Sigma, Cat. nos. 17850 and P8396, respectively) were included in the MEF medium. The former is an acid and was prepared in 30 mm NaOH for a 10 mg/ml stock, prior to sterilization by filtration, whereas the latter is a salt and was prepared in water or PBS, with all stocks stored as aliquots at −20 °C). This remedy was effective in eradiating and preventing the contamination. Such contamination has occurred in many other laboratories (31Gray J.S. Birmingham J.M. Fenton J.I. Got black swimming dots in your cell culture? Identification of Achromobacter as a novel cell culture contaminant.Biologicals. 2010; 38: 273-277Crossref PubMed Scopus (18) Google Scholar). The following expression plasmids or cDNA constructs were purchased from Open Biosystems: KLF2 (BC071983); KLF4 (MHS1011-7509690); KLF5 (MHS1011-61504); OCT4 (MHS1768-98081221); SOX2 (MHS1011-169828), and c-Myc (MHS1010-9205764). The following lentivirus plasmids were obtained from Addgene: pLOVE (15948); pLOVE-Klf4 (15950); pLOVE-N-MYC (15951); pSin-EF2-SOX2-Pur (16577), and pSin-EF2-OCT4-Pur (16579). The plasmids for FLAG-tagged wild-type and mutant proteins were constructed by use of standard subcloning and mutagenesis protocols. Lentiviral shuttle vectors were prepared on pENTR11 for homologous recombination with pLOVE via the Gateway system (Invitrogen). The following antibodies were purchased as specified: anti-Gal4 (Santa Cruz Biotechnology, RK5CI); anti-FLAG (Sigma, F3165); anti-HA (Babco/Covance); anti-mouse HRP IgG (Amersham Biosciences, NA93IV); goat anti-rabbit IgG (Fisher, AP307FMI); anti-Nanog (Bethyl Laboratories, BL1662); anti-KLF4 (Santa Cruz Biotechnology, H-180), and anti-Ssea-1 (Cell Signaling Technology, MC-480). The procedure has been described previously (11Grégoire S. Tremblay A.M. Xiao L. Yang Q. Ma K. Nie J. Mao Z. Wu Z. Giguère V. Yang X.J. Control of MEF2 transcriptional activity by coordinated phosphorylation and sumoylation.J. Biol. Chem. 2006; 281: 4423-4433Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Briefly, an HA-tagged SUMO construct and a FLAG-tagged transcription factor construct were co-transfected into HEK293 cells by using the Superfect transfection reagent (Qiagen, 301307). After 48 h, the cells were lysed in buffer S (SDS sample buffer (0.15 m Tris-HCl, pH 6.7, 5% SDS, and 30% glycerol) diluted 1:10 in PBS containing 0.5% Nonidet P-40, and protease inhibitors), followed by 15 s of sonication three times at the power setting of 3.5 (Model Virsonic 100 sonicator) to break up chromatin and decrease the viscosity. Anti-FLAG M2 beads (Sigma, A2220) were used to immunoprecipitate FLAG-tagged proteins according to the manufacturer's instructions. Briefly, prewashed anti-FLAG M2-agarose was mixed with soluble extracts and rotated for 2 h at 4 °C. The agarose was collected by centrifugation at 400 × g for 1 min and washed three times with buffer R (PBS, 0.5% Nonidet P-40, 1 mm PMSF, 12.8 mm β-mercaptoethanol, and protease inhibitors). For elution, the agarose was incubated with buffer R containing 0.2 mg/ml FLAG peptide for 30 min on a rotator at 4 °C. After a brief spin, the supernatant was collected for SDS-PAGE and Western blotting. On the day before transfection, 4 × 104 HEK293 cells or 2 × 104 MEFs were seeded per well onto a 12-well plate. 200 ng of the luciferase reporter Gal4-tk-luc or Nanog-luc (pGL3-Nanog(−2342 to +50), obtained from Takashi Tada, Kyoto University (32Kuroda T. Tada M. Kubota H. Kimura H. Hatano S.Y. Suemori H. Nakatsuji N. Tada T. Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression.Mol. Cell. Biol. 2005; 25: 2475-2485Crossref PubMed Scopus (404) Google Scholar)) were transfected along with 200 ng of expression plasmids. The β-galactosidase expression plasmid CMV-β-Gal (50 ng) was co-transfected as an internal control. The transfection reagents Superfect (Qiagen, catalog no. 301307) and Lipofectamine 2000 (Invitrogen, catalog no. 11668-019) were employed for transfection of HEK293 cells and MEFs, respectively. 48 h post-transfection, the cells were lysed in situ, and soluble extracts were prepared for measurement of luciferase and β-galactosidase activities with a 96-well plate luminometer (Dynex). d-(−)-Luciferin (Roche Applied Science) and Galacto-Light Plus (Tropix) were used as the substrates for luciferase and β-galactosidase, respectively. The procedure has been described previously (33Dufour C.R. Wilson B.J. Huss J.M. Kelly D.P. Alaynick W.A. Downes M. Evans R.M. Blanchette M. Giguère V. Genome-wide orchestration of cardiac functions by the orphan nuclear receptors ERRα and -γ.Cell Metab. 2007; 5: 345-356Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). On irradiated MEF feeders precultured on three 10-cm gelatinized culture dishes, mouse R1 ES cells were grown to ∼90% confluency, resulting in ∼1.5 × 107 cells for ChIP. 0.27 ml of 37% formaldehyde was added directly to each dish to achieve a final concentration of 1%. The three dishes were transferred to an orbital shaker and shaken for 10 min at room temperature. This cross-linking reaction was stopped by addition of glycine to a final concentration of 0.125 m, which was followed by a 5-min incubation at room temperature. The dishes were washed twice with ice-cold PBS and scraped on ice to harvest the cells in some residual PBS. The cell suspension was combined into one Falcon tube and centrifuged at 220 × g for 10 min at 4 °C. The cell pellet was lysed in 150 μl of the lysis buffer (50 mm Tris-HCl, pH 8.0, 1% SDS, 10 mm EDTA, and protease inhibitors) and sonicated three times on ice for 20 s each at setting 6 (Virsonic 100 sonicator). The cell lysate was centrifuged at 16,000 × g for 10 min at 4 °C. The resulting supernatant was diluted in 1.2 ml of ChIP dilution buffer (20 mm Tris-HCl, pH 8.0, 1% Triton X-100, 2 mm EDTA, and 150 mm NaCl). Sheared salmon sperm DNA was mixed with protein A-agarose (Upstate) and incubated with the diluted lysate for pre-clearance. The suspension was rotated for 1 h at 4 °C. After brief centrifugation, the supernatant was incubated with the primary antibody overnight at 4 °C. The next day, 40 μl of salmon sperm DNA/protein A-agarose bead suspension was added into the lysate/antibody mixture and incubated for 2 h at 4 °C. The beads were then separated from the lysate by centrifugation at 750 × g for 1 min, resuspended in 1 ml of wash buffer 1 (low salt buffer: 20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.1% SDS, 1% Triton, and 2 mm EDTA), and rotated for 10 min. The beads were separated from buffer 1 by brief centrifugation and resuspended in 1 ml of buffer 2 (high salt buffer: 20 mm Tris-HCl, pH 8.0, 500 mm NaCl, 0.1% SDS, 1% Triton, and 2 mm EDTA). The same washing step was repeated in freshly prepared buffer 3 (LiCl buffer: 10 mm Tris-HCl, pH 8.0, 0.25 m LiCl, 1% Nonidet P-40, 1 mm EDTA, and 1% sodium deoxycholate) and cold buffer TE (10 mm Tris-HCl, pH 7.5, and 1 mm EDTA). 150 μl of de-crosslink buffer (0.1 m NaHCO3 and 1% SDS) was added onto the beads and incubated at 65 °C for 6–18 h. The supernatant was mixed with 5 μl of proteinase K (1 mg/ml) and incubated for 1 h at 55 °C. Afterward, DNA was purified with the QIAQuick PCR purification column kit (Qiagen) for PCR. For the Nanog promoter, the PCR primers were 5′-GTGAAATGAGGTAAAGCCTCTTTT-3′ and 5′-AAGGCCAACGGCTCAAGGCGATAG-3′. For the Lefty promoter, the primers 5′-AAGCTGCAGACTTCATTCCA-3′ and 5′-CGGGGGATAGATGAAGAAAC-3′ were used (34Nakatake Y. Fukui N. Iwamatsu Y. Masui S. Takahashi K. Yagi R. Yagi K. Miyazaki J. Matoba R. Ko M.S. Niwa H. Klf4 cooperates with Oct3/4 and Sox2 to activate the Lefty1 core promoter in embryonic stem cells.Mol. Cell. Biol. 2006; 26: 7772-7782Crossref PubMed Scopus (206) Google Scholar). Coverslips were flamed and put onto wells of 12- or 24-well dishes for culturing cells. Coverslips containing cultured cells were washed twice with PBS prior to fixation with 2% paraformaldehyde for 20 min at room temperature. Cells were then washed three times with PBS and permeabilized with 0.2% Triton X-100 solution/PBS for 10 min. Afterward, the coverslips were rinsed three times with 100 mm glycine/PBS and blocked in the blocking solution (2% BSA prepared in the IF buffer (PBS, 0.2% Triton X-100 and 0.05% Tween 20)). After 30 min, the coverslips were incubated for 1 h at room temperature with primary antibodies diluted 1:100 or 1:200 in the blocking solution. Cells were then washed three times with the IF buffer and incubated with fluorophore-conjugated secondary antibodies (diluted 1:500 to 1:1000) for 45 min. Afterward, the coverslips were rinsed three times with the IF buffer, briefly exposed to DAPI or Hoechst 33258 to stain the nuclei, and mounted for examination under a Zeiss Axiovert 135 fluorescence microscope. 293FT cells (Invitrogen) were maintained in DMEM/10% FBS medium containing 400 μg/ml neomycin (geneticin, Invitrogen, catalog no. 11811-098) per the manufacturer's instructions and incubated in the antibiotic-free medium for at least 8 h prior to transfection with Lipofectamine 2000. 10 μg of expression plasmid was mixed with 6.5 μg of psPAX2, 3.5 μg of pMD2.G, 50 μl of Lipofectamine 2000, and 1 ml of Opti-MEM to transfect 8 × 106 293FT cells in a 10-cm dish according to the manufacturer's instructions (Invitrogen). 24 h post-transfection, the medium was collected as the viral supernatant everyday for 3 days and was subjected to centrifugation at 76,000 × g for 1.5 h. The viral pellet was then suspended in DMEM, 10% heat-inactivated FBS and rotated overnight at 4 °C. The resulting virus supernatant was used to infect cells directly or was flash-frozen in aliquots on dry ice for long term storage at −80 °C. The procedure for drug selection-free iPS cell induction has been described previously (35Blelloch R. Venere M. Yen J. Ramalho-Santos M. Generation of induced pluripotent stem cells in the absence of drug selection.Cell Stem Cell. 2007; 1: 245-247Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Briefly, the day before virus infection, MEFs (passage 2 or 3) were plated at 0.8–1 × 105 cells per well for a 12-well plate or 3 × 105 cells per well of a 6-well plate and incubated overnight in DMEM containing 10% FBS and penicillin/streptomycin (50 μg/ml each) inside a 37 °C CO2 incubator. The next day, the cells were washed once with PBS or plain DMEM and refed with DMEM, 10% heat-inactivated FBS, 8 μg/ml Polybrene. After addition of the concentrated virus stock (suspended in DMEM, 10% heat-inactivated FBS, see above) to the medium, the cells were incubated in a 37 °C CO2 incubator for 2 days. The virus-containing medium was then removed, and the cells were then washed once with PBS or plain DMEM for culturing in the mESC medium (DMEM high glucose, 1% nonessential amino acids (100× stock, Invitrogen), 1% sodium pyruvate (100× stock, Invitrogen), 0.1 mm β-mercaptoethanol, 15% FBS, penicillin, and streptomycin (50 μg/ml each), and 1000 units of murine leukemia inhibitory factor/ml (Millipore, ESGRO®)). The medium was changed every 2 days. iPS colonies appeared 5–6 days after infection. For alkaline phosphatase stain" @default.
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• W2020772572 title "Sumoylation of Krüppel-like Factor 4 Inhibits Pluripotency Induction but Promotes Adipocyte Differentiation" @default.
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