1. Volume 55, Issue 4, Pages 537-551 (August 2014)
A Methylation-Phosphorylation Switch Determines Sox2 Stability and Function in ESC Maintenance or Differentiation 
Lan Fang, Ling Zhang, Wei Wei, Xueling Jin, Ping Wang, Yufeng Tong, Jiwen Li, James X. Du, Jiemin Wong 
Molecular Cell 
Volume 55, Issue 4, Pages (August 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 55, 537-551DOI: (10.1016/j.molcel.2014.06.018)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1 Set7 Methylates Sox2 at K119 both In Vitro and In Vivo
(A) In vitro methylation of recombinant ESC core transcription factors by Set7. Asterisks mark the positions of the substrate peptide or proteins in Coomassie staining gel.
(B) Sox2 methylation revealed by metabolic labeling in 293T cells.
(C) The consensus Set7 target sequence (top) in Sox2 and DNMT1. M, mouse; H, human; B, bovine; G, chicken; and D, zebrafish.
(D) In vitro methylation of various purified Sox2 mutants by Set7.
(E) In vitro methylation of various Sox2 peptides.
(F) Characterization of anti-Sox2 K119me-specific antibody. Top panel, western blot analysis; lower panel, dot blot analysis.
(G) Western blot analysis showing the effect of ectopically expressed Set7 and Set7m on K119me of endogenous Sox2 in CGR8 ESCs. α(r)Set7, rabbit polyclonal Set7 antibody that detects only ectopically expressed Set7.
(H) Western blot analysis showing methylation of Flag-Sox2 and mutants by Set7 in transfected 293 cells.
(I) Coimmunoprecipitation assay analyzing the interaction between various Sox2 mutants and Set7. See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
Methylation by Set7 Induces Sox2 Ubiquitination and Proteasomal Degradation
(A) Double immunofluorescent analysis of the effect of ectopically expressed V5-Set7 and V5-Set7m (V5 tag in green) on Sox2 (red) in CGR8 ESCs.
(B) Western blot analysis showing MG132 treatment blocked V5-Set7-induced Sox2 downregulation in CGR8 ESCs.
(C) The effect of MG132 treatment on Sox2 K119me and T118p. MG132, 8 hr treatment. α(m)Set7, mouse monoclonal Set7 antibody that detects both endogenous and ectopically expressed Set7.
(D) Immunofluorescent analysis of shSet7-transfected CGR8 ESCs (GFP marker) using anti-Sox2 antibody (red).
(E) Protein stability assay showing that knockdown of Set7 increased Sox2 protein stability in CGR8 ESCs.
(F) Protein stability assay comparing the stability of Sox2 K119R mutant and wild-type Sox2 in CGR8 ESCs.
(G) Expression of V5-Set7 but not Set7m enhanced endogenous Sox2 ubiquitination in CGR8 ESCs.
(H) Expression of V5-Set7 enhanced Sox2 and Sox2 K117R but not K119R mutant ubiquitination. See also Figures S1 and S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 Methylation by Set7 Inhibits Sox2 Transcriptional Activity
(A) Luciferase reporter assay showing V5-Set7- but not Set7m-inhibited Sox2 transcriptional activity.
(B) Luciferase reporter assay for the transcriptional activity of Sox2, K117R, and K119R mutants (top).
(C) ChIP assay evaluating the binding of Flag-Sox2, Sox2 K117R, or K119R mutant to the transfected FGF4-luc reporter. ∗p ≤ 0.05.
(D) Luciferase reporter assay for inhibition of transcriptional activity of various Sox2 proteins by V5-Set7.
(E and F) Coimmunoprecipitation assay analyzing the effect of V5-Set7 and Set7m on the interaction between Sox2 and p300 and CBP, respectively. For (A)–(D), the luc data are represented as mean ± SEM.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
WWP2 Mediates Set7-Induced Sox2 Degradation and Its HECT Domain Recognizes Sox2 K119me Specifically
(A) Immunofluorescent staining analysis for transfected Flag-WWP2 or Flag-WWP1 (green) and endogenous Sox2 (red) in CGR8 ESCs.
(B) Western blot analysis for the effect of WWP2 knockdown on Sox2 proteins in CGR8 ESCs.
(C) WWP2 enhanced the ubiquitination of endogenous Sox2 in CGR8 cells.
(D) V5-Set7-induced Sox2 degradation in CGR8 ESCs was blocked by cotransfection of a shWwp2.
(E) Knockdown of WWP2 blocked Sox2 degradation induced by V5-Set7.
(F) IP-western blot analysis for the Set7-dependent interaction between Flag-WWP2 and HA-Sox2.
(G) IP-western blot analysis showing that knockdown of Set7 abolished the interaction between endogenous WWP2 and Sox2 in CGR8 ESCs.
(H) The diagram illustrating the structural domains of WWP2.
(I) In vitro peptide pull-down assay analyzing the binding of various WWP2 domains to unmodified, T118, or K119me Sox2 aa 113–126 peptides.
(J) The peptide pull-down assays with purified recombinant GST-HECT and control GST proteins.
(K) Molecular modeling showing two aromatic residue clusters on the surface of WWP2 HECT domain.
(L) Peptide pull-down assay for analyzing the binding of various WWP2 aromatic residue mutants to the Sox2 K119me peptide.
(M) Molecular modeling illustrating the potential Sox2 K119me binding aromatic pocket within the WWP2 HECT domain. See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
T118p by AKT1 Stabilizes Sox2 and Predominates over K119me by Set7
(A) Characterization of a Sox2 T118p-specific antibody by western (upper) and dot blot (lower) analyses.
(B) In vitro phosphorylation assay using various Sox2 peptide substrates followed by western blot analysis.
(C) The effect of knockdown of AKT1 in CGR8 on the levels of Sox2 proteins and T118p and K119me.
(D) The effect of AKT inhibitor IV (1 μM) on the level of Sox2 proteins and T118p and K119me in CGR8 cells.
(E) The effect of AKT1 knockdown in CGR8 on the Sox2 protein stability.
(F and G) AKT1 inhibits K119me by Set7 and its consequent Sox2 ubiquitination in CGR8 and 293T cells, respectively. HA-Ub was cotransfected and MG132 treatment was for 8 hr. Two nonspecific bands in western blot by anti-His antibody are marked by asterisks.
(H) Immunofluorescent analysis for the effect of expressing V5-Set7 and Flag-AKT1alone or together on endogenous Sox2 in CGR8 ESCs. In each image, the transfected cells are marked by arrows.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
AKT1 Maintains Sox2 Stability and Self-Renewal of ESCs by Protecting Sox2 from Set7- Induced Degradation
(A) Quantitative analysis of AP staining-positive colonies. The data are represented as mean ± SEM.
(B) The representative images of AP staining-positive ESC colonies at 96 hr.
(C) Western blot analysis showing reduced level of endogenous Sox2 upon overexpression of V5-Set7 but not V5-Set7m in CGR8. The anti-Set7 antibody detected both endogenous and ectopically expressed V5-Set7.
(D) Knockdown of WWP2 blocked ESC differentiation induced by overexpression of V5-Set7.
(E) Coexpression of HA-AKT1 with V5-Set7 blocked ESC differentiation induced by V5-Set7.
(F) Double knockdown of AKT1 and Set7 partially restored ESC colony formation in CGR8 ESCs.
(G) Western blot analysis showing the effect of AKT1 knockdown and double knockdown of AKT1 and Set7 on the levels of Sox2, Sox2 T118p, and K119me in CGR8 ESCs.
(H) Stable Sox2 K119R-expressing CGR8 ESCs maintained ESC self-renewal upon overexpression of V5-Set7 or knockdown of AKT1. ES, control CGR8 ESCs; +F-Sox2, CGR8 ESCs stably expressing Flag-Sox2; +F-K119R, CGR8 ESCs stably expressing Flag-Sox2 K119R mutant.
(I) Western blot analysis showing the effect of V5-Set7 overexpression or AKT1 knockdown on the endogenous as well as stably expressed Flag-Sox2 or Flag-Sox2 K119R mutant. See also Figures S4 and S5.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
Increased Set7 Expression in Development May Contribute to Appropriate Cell Lineage Specification
(A) Expression profiles of Set7, Sox2, and other proteins during 9 day EB differentiation. IP-western blot analysis was also performed to determine the status of Sox2 T118p and K119me during EB differentiation.
(B) Western blot analysis for the levels of Sox2 in EBs derived from CGR8 ESCs with knockdown of Set7, overexpression of Set7, or Sox2 as indicated.
(C) Double immunofluorescent staining analysis for Set7 and Sox2 or Set7 and AFP- positive cells using the day 6 EBs.
(D) qRT-PCR analysis of day 9 EBs for endodermal, mesodermal, and ectodermal markers. The data are represented as mean ± SEM.
(E) Knockdown of Set7 skewed differentiation of CGR8 ESCs into Tuji-positive neuroectodermal cells and impaired differentiation toward AFP-positive endodermal lineage. ESCs were induced for differentiation by LIF withdrawal plus RA treatment.
(F) The CGR8 cells stably expressing Flag-Sox2 K119R mutant skewed differentiation toward Tuji- and Nestin-positive neuronal ectodermal lineage and against the AFP-positive endodermal lineage.
(G) The working model illustrating that the methyl-phospho switch by AKT1 and Set7 determines the stability of Sox2 and its role in ESC maintenance or differentiation. See also Figures S5 and S6.
Molecular Cell  , DOI: ( /j.molcel )
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