1. Volume 43, Issue 2, Pages 203-216 (July 2011)
Reversible SUMOylation of TBL1-TBLR1 Regulates β-Catenin-Mediated Wnt Signaling 
Hyo-Kyoung Choi, Kyung-Chul Choi, Jung-Yoon Yoo, Meiying Song, Suk Jin Ko, Chul Hoon Kim, Jin-Hyun Ahn, Kyung-Hee Chun, Jong In Yook, Ho-Geun Yoon 
Molecular Cell 
Volume 43, Issue 2, Pages (July 2011)
DOI: /j.molcel
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Figure 1
Both TBL1 and TBLR1 Are In Vitro and In Vivo SUMOylated on Lysines 560 and 497, Respectively
(A) Identification of SUMOylation events in both TBL1 and TBLR1 using an E. coli-based in vitro SUMOylation assay system. Both TBL1- and TBLT1-fused pT-E1E2S1 plasmids were generated, and cells were cotransformed with PT-E1E2S1 encoding Aos1/Uba2, Ubc9, and SUMO1. After IPTG induction, the purified proteins were analyzed by western blotting with the indicated antibodies. Arrow, GST protein; ∗, GST-TBL1 or GST-TBLR1.
(B) In vivo interaction between SUMO1 and TBL1-TBLR1. 293T cells were transfected with Myc-TBL1, Myc-TBLR1, and Flag-SUMO1 (aa 1–97) as indicated.
(C) Confirmation of in vivo SUMOylation on TBL1-TBLR1. For the Duolink in situ PLA analysis, 293T cells were transfected with the indicated plasmids. The permeabilized 293T cells were incubated with antibodies against Myc and Flag, and PLA probes (PLUS and MINUS) were added. The positive signal was analyzed using confocal microscopy.
(D) Lys560 of TBL1 is SUMOylated. After IPTG induction, the purified proteins were analyzed by western blotting with the indicated antibodies.
(E) Mutation of TBL1 at Lys560 and TBLR1 at Lys497 abolished the interaction of these proteins with SUMO1 in vivo. The cell lysates were analyzed by western blotting with the indicated antibodies.
(F) Lys560 of TBL1 is target sites for the in vivo SUMOylation of TBL1. Permeabilized 293T cells were incubated with anti-Myc antibody, anti-Flag antibody, and PLA probes (PLUS and MINUS).
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3. Figure 2
SUMOylation of TBL1-TBLR1 Is Required for the Enhancement of β-Catenin-Mediated Transcription
(A) Overexpression of TBL1 enhances both LiCl2-induced and β-catenin-mediated transcription. 293T cells were cotransfected with a TOPFLASH reporter or FOPFLASH reporter, pCMV-β-galactosidase, pcDNA3-β-CatS33A, and increasing amounts of pSG5-Myc-TBL1 as indicated. Data were reported as the mean values ± SD of three independent experiments and were expressed as fold increase relative to the basal activity.
(B) SUMOylation of TBL1-TBLR1 enhances the LiCl2-induced Wnt activation. The 293T cells were transfected with the indicated plasmids. Two days after cotransfection, 293T cells were harvested, and luciferase activity was determined. Error bars, SD (n = 3).
(C) TBL1-TBLR1 interacts with Ubc9 in a LiCl2-dependent manner. The 293T cells were cotransfected with the indicated plasmids. Cell lysates were immunoprecipitated and subsequently immunoblotted with their respective antibodies as indicated.
(D) The SUMOylation modification is crucial for the enhancement of LiCl2-induced activation of TOPFLASH activities. The 293T cells were cotransfected with the wild-type or mutant TBL1-TBLR1 plasmids. Two days after cotransfection, 293T cells were harvested, and the luciferase activity was determined. Error bars, SD (n = 3).
(E) SUMOylation of TBL1-TBLR1 is required for the enhanced transcription of Wnt target gene. The 293T cells were cotransfected with indicated plasmids. One day after cotransfection, 293T cells were treated with 20 mM LiCl2. After the cells were harvested, mRNA levels were analyzed by qRT-PCR. Error bars, SD (n = 3).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Restoration of SUMOylation to TBL1-TBLR1 Restored the Activation of β-Catenin-Mediated Transcription
(A and D) A schematic diagram of the various deletion mutants of TBL1-TBLR1 and SUMO1-fused TBL1-TBLR1 plasmids. (B and E) Restoration of SUMOylation to TBL1-TBLR1 restored the activating function of these proteins to β-catenin-mediated transcription. The 293T cells were cotransfected with the indicated plasmids. Two days after cotransfection, 293T cells were harvested, and the luciferase activity was determined. Arrow indicates the β-catenin. Error bars, SD (n = 3). (C and F) Restoration of SUMOylation to TBL1-TBLR1 restored the activating function of these proteins in the LiCl2-induced activation of Wnt signaling. 293T cells were cotransfected with increasing amounts of the indicated plasmids. One day after cotransfection, 293T cells were treated with LiCl2, and then luciferase activity was determined. Error bars, SD (n = 3). (G and H) Restoration of SUMOylation to TBL1-TBLR1 restored transcriptional activation of Wnt target genes. The 293T cells were treated with LiCl2 and then harvested. The mRNA levels were analyzed by qRT-PCR. Error bars, SD (n = 3).
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5. Figure 4
SENP1 DeSUMOylated TBL1-TBLR1 to Suppress β-Catenin-Mediated Transcriptional Activation
(A) Both Wnt3a and LiCl2 treatment efficiently induces the SUMOylation of TBL1. Both 293T and stable 293T cells express the Flag-tagged TBL1 were treated with Wnt3a or LiCl2 for different times. SUMOylated proteins were visualized by western blot analysis. Arrow, SUMOylated TBL1; ∗, Flag-tagged TBL1.
(B) TBL1 is in vivo SUMOylated in a LiCl2-dependent manner. Permeabilized 293T cells were incubated with anti-TBL1, anti-SUMO antibody, and PLA probes (PLUS and MINUS).
(C) In vivo validation of TBL1 SUMOylation. The 293T cells were transfected with the indicated plasmids. Permeabilized 293T cells were treated with LiCl2 and incubated with anti-Myc antibody, anti-SUMO antibody, and PLA probes (PLUS and MINUS).
(D) SENP1 specifically interacts with TBL1. The 293 cells were transfected with the indicated plasmids. Cell lysates were immunoprecipitated and subsequently immunoblotted with their respective antibodies as indicated.
(E) SENP1 is the enzyme responsible for the deSUMOylation of TBL1. SUMOylated proteins were visualized by western blotting analysis.
(F) In vivo visualization of SENP1-mediated deSUMOylation of TBL1. Permeabilized 293T cells were treated with LiCl2 and incubated with anti-TBL1 antibody, anti-SUMO antibody, and PLA probes (PLUS and MINUS).
(G) SENP1 is required for DKK-mediated deSUMOylation of TBL1-TBLR1. The stable SENP1-knocking down 293T cells were transfected with or without Myc-tagged DKK plasmid. Cell lysates were immunoprecipitated and subsequently immunoblotted with their respective antibodies as indicated.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
SUMOylation of TBL1-TBLR1 Dismisses TBL1-TBLR1 from the NCoR/SMRT/HDAC3 Corepressor Complex to Enhance the Formation of the TBLR1-TBL1-TCF4-β-Catenin Complex
(A) The subcellular localization of NCoR/HDAC3/TBL1 corepressor complex was not affected by LiCl2 treatment. Cell fractionation was performed using a nuclear/cytosolic fractionation kit, and the fractions were subsequently immunoblotted with their respective antibodies as indicated.
(B) LiCl2-induced Wnt activation reduced the association of the NCoR/HDAC3 corepressor complex with TBL1-TBLR1. The 293T cells were treated with LiCl2 and then harvested. Cell lysates were immunoprecipitated and subsequently immunoblotted with their respective antibodies as indicated.
(C) Activation of Wnt signaling dissociates TBL1-TBLR1 from the NCoR corepressor complex and increases the formation of the TBL1-TBLR1-β-catenin complex. The 293T cells were cotransfected with the indicated plasmids.
(D) LiCl2-induced SUMOylation of TBL1 via K560 is required for dissociation of TBL1 from the NCoR/HDAC3 corepressor complex. The 293T cells were treated with LiCl2 and then harvested. Cell lysates were immunoprecipitated and subsequently immunoblotted with their respective antibodies as indicated.
(E) SUMOylation of TBL1 triggers the dismissal of TBL1 from the NCoR/SMRT corepressor complex. The 293T cells were cotransfected with the indicated plasmids. The 293T cells were treated with or without LiCl2 and then harvested. Cell lysates were immunoprecipitated and subsequently immunoblotted with their respective antibodies, as indicated. Arrow, HDAC3.
(F) In vivo visualization of dissociation of TBL1-TBLR1 from the NCoR corepressor complex and the resultant increases in the formation of the TBL1-TBLR1-β-catenin complex. Permeabilized 293T cells were treated with LiCl2 and incubated with the indicated antibodies and PLA probes (PLUS and MINUS).
(G) Restriction of the corepressive function of TBL1-TBLR1 is crucial for β-catenin-mediated transcriptional activation. The 293T cells were cotransfected with the indicated plasmids. Two days after cotransfection, cells were harvested, and the luciferase activity was determined. Error bars, SD (n = 3).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions

7. Figure 6
SUMOylation of TBL1-TBLR1 Enhanced the Recruitment of the TBLR1-TBL1-TCF4-β-Catenin Complex to Chromatin for Wnt Signaling Activation
(A) LiCl2 treatment increased the recruitment of the TBL1-TBLR1-β-catenin complex to the promoter regions of Wnt target genes. The 293T cells were treated with or without LiCl2, and then ChIP assays were performed with the indicated antibodies. The precipitated samples were analyzed by real-time PCR, and results are given as the percentage of input as means ± SD of three independent experiments.
(B) LiCl2 treatment induced the recruitment of the TBL1SUMO-TBLR1SUMO-β-catenin complex to the promoter region of Wnt target genes. ChIP and reChIP assays were performed with the indicated antibodies. Error bars, SD (n = 3).
(C) SUMOylation of TBL1 and TBLR1 enhanced the binding of β-catenin to chromatin at Wnt target genes. The 293T cells were transfected with the indicated plasmids followed by ChIP assays with the indicated antibodies. Error bars, SD (n = 3).
(D) SENP1-mediated deSUMOylation attenuated the constitutive activation of Wnt signaling via enhanced formation of TBL1-TBLR1-NCoR corepressor complex. The mRNA levels in both HT29 and SW480 cells were analyzed by qRT-PCR without LiCl2 treatment (left and upper), and cell lysates were immunoprecipitated and then analyzed by western blotting (right). SW480 cells were transfected with SENP1 and/or SENP1C603S plasmids. The mRNA levels were analyzed by qRT-PCR (left and lower), and cell lysates were immunoprecipitated and then analyzed by western blotting (right). ∗p < 0.05; #p < Error bars, SD (n = 3).
(E) SW480 cells were transfected with SENP1 and/or SENP1C603S plasmids. The cell lysates were immunoprecipitated and then analyzed by western blotting with indicated antibodies.
(F) SENP1-mediated deSUMOylation induced the dissociation of the TBL1-TBLR1-β-catenin complex from the promoter of the Wnt target genes in SW480 cells. ChIP assays were carried out with the indicated antibodies. Error bars, SD (n = 3).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions

8. Figure 7
SUMOylation of TBL1 and TBLR1 Enhances the Tumorigenicity of SW480 Cells
(A and C) The SUMOylation of TBL1 enhanced the tumorigenic growth of SW480 colon cancer cells. SW480 cells were cotransfected with TBL1 plasmids. Two days after transfection, the number of cells in soft agar was determined under a microscope. To measure the invasiveness of SW480 cells, the cells were transfected with the indicated plasmids and then seeded in the upper chamber. Data represent the means ± SD of at least three independent experiments. Error bars, SD (n = 3). (B) SENP1 suppresses the TBL1-enhanced tumorigenic growth of SW480 cells. SW480 cells were cotransfected with TBL1, SENP1, or SENP1C603S plasmids as indicated. Error bars, SD (n = 3). (D) Overexpression of SENP1 suppressed the tumor growth. Stable SENP1-expressed SW80 or SENP1C603S-expressed SW480 cells were injected subcutaneously into the right flank of nude mice with 2 × 106 cells per site, and tumor growth was measured for 4 weeks. Values are means ± SD for eight mice from a representative experiment. P callus are shown from Student's t test analysis. ∗p < (E) Depletion of SENP1 enhanced the tumorigenecity of SW480 cells. ∗p < (F) SUMOylation of TBL1-TBLR1 enhanced the in vivo tumorigenicity of SW480 cells. ∗p < (G) Model of SUMOylation of TBL1-TBLR1. SUMOylation triggers the chromatin targeting of the TBL1SUMO-TBLR1SUMO-β-catenin complex to Wnt target genes via dismissal of TBL1-TBLR1 from the NCoR/SMRT corepressor complex. Conversely, SENP1-mediated deSUMOyaltion of TBL1-TBLR1 leads to suppression of Wnt signaling.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions