Volume 56, Issue 2, Pages 261-274 (October 2014) Modification of ASC1 by UFM1 Is Crucial for ERα Transactivation and Breast Cancer Development  Hee Min Yoo, Sung Hwan Kang, Jae Yeon Kim, Joo Eun Lee, Min Woo Seong, Seong Won Lee, Seung Hyeun Ka, Yu-Shin Sou, Masaaki Komatsu, Keiji Tanaka, Soon Tae Lee, Dong Young Noh, Sung Hee Baek, Young Joo Jeon, Chin Ha Chung  Molecular Cell  Volume 56, Issue 2, Pages 261-274 (October 2014) DOI: 10.1016/j.molcel.2014.08.007 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 56, 261-274DOI: (10.1016/j.molcel.2014.08.007) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 Identification of ASC1 as a Target for Ufmylation and Its Binding to UFBP1 (A) Strategy for identification of targets for ufmylation. (B) Identification of ASC1 as a target for ufmylation. Proteins eluted from the resins were subjected to SDS-PAGE followed by immunoblot or were silver stained. (C) List of the candidate target proteins identified by mass spectrometry. (D) UFBP1 interacts with ASC1. UFBP1-Myc was expressed in HEK293T cells with Flag-ASC1. Cell lysates were subjected to immunoprecipitation with anti-Flag or anti-Myc antibody followed by immunoblot analysis. (E) UFBP1 directly binds to ASC1. Purified His-ASC1 was incubated with GST or GST-UFBP1 followed by pull-down (PD) with glutathione-Sepharose (GSH-resin). The samples were then subjected to immunoblot with anti-GST antibody. (F) ASC1 forms a ternary complex with UFL1 and UFBP1. Cell lysates were subject to immunoprecipitation with IgG or anti-ASC1 or anti-UFBP1 antibody followed by immunoblot analysis. Molecular Cell 2014 56, 261-274DOI: (10.1016/j.molcel.2014.08.007) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 Requirement of UFBP1 for ASC1 Ufmylation (A) UFBP1 promotes ASC1 ufmylation. Myc-tagged UBA5, UFC1, UFL1, and UFBP1 were expressed in HEK293T cells with Flag-UFM1 and HisMax-ASC1 as indicated. Cell lysates were subjected to pull-down with NTA resins followed by immunoblot with anti-Flag or anti-Xpress antibody. (B and C) UFBP1 and UBA5 are required for ASC1 ufmylation. HisMax-ASC1 and UFM1-conjugating system were expressed in cells with shUFBP1 (B) or shUBA5 (C). (D) The K267R mutation of UFBP1 prevents ASC1 ufmylation. Myc-tagged UFBP1 (Wt) or its K267R mutant (KR) was expressed with HisMax-ASC1 and UFM1-conjugating system. (E and F) The K267R mutation blocks the binding of UFBP1 to UFL1, but not to ASC1. Flag-tagged UFBP1 or its K267R mutant was expressed with Myc-tagged ASC1 (E) or UFL1 (F). (G) UBA5 knockdown inhibits the binding of UFBP1 to UFL1, but not to ASC1. Cells transfected with shNS or shUBA5 were subjected to immunoprecipitation with anti-UFBP1 (left) or anti-ASC1 antibody (right) followed by immunoblot analysis. (H and I) Poly-UFM1 chains are formed via K69-linked isopeptide bonds. Each of six Lys residues in ASC1 was replaced by Arg (H). All Lys residues, except one each of them, were replaced by Arg (I). The UFM1 variants were Flag-tagged and expressed in cells with HisMax-ASC1 and UFM1-conjugating system. K0 denotes Lys-less UFM1. (J) The C-terminal Gly of UFM1 is required for ASC1 ufmylation. The C-terminal Gly was replaced by Ala in UFM1 having only Lys69. (K) The 3D structure of UFM1 (PBD ID 1WSX). Molecular Cell 2014 56, 261-274DOI: (10.1016/j.molcel.2014.08.007) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 Identification of UFM1 Accepter Sites in ASC1 (A and B) Identification of ufmylation region. Deletions of ASC1 were generated, tagged with HisMax to their N termini, and expressed in HEK293T cells with Flag-UFM1 and UFM1-conjugating system (A). Cell lysates were subjected to pull-down with NTA resins followed by immunoblot with anti-Flag antibody (B). (C and D) Identification of ufmylation sites. The Lys residues in the amino acid sequence of 300–370 were replaced by Arg as indicated (C). The ASC1 mutants were tagged with HisMax and expressed in cells with Flag-UFM1 and UFM1-conjugating system (D). (E) All of Lys324, Lys325, Lys334, and Lys367 are ufmylation sites. The four Lys residues, except one each of them, were replaced by Arg. (F) The 4KR mutant is capable of interacting with UFBP1. HisMax-tagged ASC1 and its 4KR mutant were expressed in cells with Myc-UFBP1. (G) The C-terminal domain of ASC1 inhibits its ufmylation. HisMax-Δ4 was expressed in cells with increasing amounts of HisMax-Δ3, Flag-UFM1, and UFM1-conjugating system. Molecular Cell 2014 56, 261-274DOI: (10.1016/j.molcel.2014.08.007) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 Reversal of ASC1 Ufmylation by UfSP2 (A and B) UfSP2 interacts with ASC1. HisMax-ASC1 was expressed in HEK293T cells with Flag-tagged UfSP2 or its inactive C302S mutant (CS). Cell lysates were subjected to immunoprecipitation by anti-Flag antibody followed by immunoblot with anti-Xpress or anti-Flag antibody (A). Endogenous ASC1 in the same cells was subjected to immunoprecipitation with anti-ASC1 antibody followed by immunoblot with the same or anti-UfSP2 antibody. (C) UfSP2 deufmylates ASC1. HisMax-ASC1 was expressed with Flag-UFM1 and UFM1-conjugating system in the presence and absence of Flag-tagged UfSP2 or its C302S mutant. Cell lysates were subjected to pull-down with NTA resins followed by immunoblot with anti-UFM1 antibody. (D) UfSP2 knockdown promotes ASC1 ufmylation. HisMax-ASC1 and UFM1-conjugating system were expressed with shNS or shUfSP2 and Flag-UFM1 in the presence or absence of Myc-mUfSP2 (the mouse form of UfSP2). Cell lysates were treated as in (C) except for the use of anti-Flag antibody for immunoblot. Molecular Cell 2014 56, 261-274DOI: (10.1016/j.molcel.2014.08.007) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 Requirement of E2 for the Binding of ASC1 to ERα and ASC1 Ufmylation (A) E2 induces the binding of ERα to ASC1. MCF7 cells were incubated with and without 10 nM E2 for 3 hr. They were then subjected to immunoprecipitation with anti-ASC1 (left) or anti-ERα antibody (right). Henceforth E2 was treated at 10 nM, unless otherwise indicated. (B) The 4KR mutation prevents E2-mediated binding of ASC1 to ERα. ASC1 or its 4KR mutant was expressed in MCF7 cells with and without E2. Cell lysates were subjected to pull-down with NTA resins (left) or immunoprecipitation with anti-ERα antibody (right). SE and LE denote short and long exposure of the gels, respectively. (C) Generation of MCF7 cells that stably express shASC1. iASC1 and i4KR, which are shASC1-insensitive ASC1 and its 4KR mutant, respectively, were expressed in the stable cells. eASC1 denotes endogenous ASC1. (D) E2 induces ASC1 ufmylation. MCF7 cells were incubated for 3 hr without and with 10 nM E2 and/or 100 nM 4-hydroxy-tamoxifen (4-HT). (E and F) UfSP2 competes with ERα for binding to ASC1. Increasing amounts of Flag-tagged UfSP2 (E) or ERα (F) were expressed in HEK293T cells with the fixed amounts of Myc-tagged ERα or UfSP2, respectively, in the presence and absence of E2. (G) UfSP2 binds to the ZF domain of ASC1. MCF7 cells expressing HisMax-tagged ASC1 or its mutant lacking the ZF domain (ΔZF) were incubated with and without E2. (H) UfSP2 knockdown leads to E2-independent ASC1 ufmylation. Cells expressing shNS or shUfSP2 were incubated with and without E2. (I) UBA5 knockdown reduces the interaction of ASC1 with ERα. Cells expressing shNS or shUBA5 were incubated with and without E2. Molecular Cell 2014 56, 261-274DOI: (10.1016/j.molcel.2014.08.007) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 Requirement of ASC1 Ufmylation for E2-Induced ERα Transactivation (A and B) Ufmylation of ASC1 promotes its binding to p300 and SRC1. ASC1 or 4KR (A) and shNS or shUBA5 (B) was expressed in MCF7 cells in the presence or absence of E2. Cell lysates were subjected to pull-down with NTA resins followed by immunoblot analysis. SE and LE in (A) denote short and long exposure of the gels, respectively. (C) UfSP2 knockdown promotes the binding of ASC1 to p300 and SRC1. Cells expressing shNS or shUfSP2 were incubated with and without E2. (D) Ufmylation of ASC1 is required for the recruitment of p300, SRC1, and itself to the pS2 promoter. HisMax-tagged ASC1 or 4KR was expressed in cells with ERα, SRC1, and p300. Cells were then subjected to ChIP analysis. See also Figure S5A. (E and F) UBA5 and UfSP2 inversely affect the recruitment of ASC1, p300, and SRC1 to the pS2 promoter. shUBA5 (E) or shUfSP2 (F) was expressed in cells with and without shASC1. Cells were then subjected to ChIP analysis. See also Figures S5B and S5C. (G–I) ASC1 ufmylation is required for ERα transactivation. ASC1 and its 4KR mutant were expressed in MCF7 cells with and without UFM1-conjugating system (UFM-S) (G). shUBA5 (H) or shUfSP2 (I) was expressed in cells with and without shASC1. Cells were also transfected with ERE-Luc. Cell lysates were then assayed for the luciferase activity. The activities seen with cells transfected with empty vectors were expressed as 1.0, and the others were as its relative values. (J–L) ASC1 ufmylation is required for the expression of ERα target genes. ASC1 or its 4KR mutant was expressed in MCF7 cells (J). shUBA5 (K) or shUfSP2 (L) was expressed in cells with and without shASC1. The transcript levels of pS2, cyclin D1, and c-Myc were then determined by qPCR. Data in (D)–(L) are the mean ± SD (n = 3). See also Figures S5E and S5F. Molecular Cell 2014 56, 261-274DOI: (10.1016/j.molcel.2014.08.007) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 7 Promotion of Cell Growth and Tumor Formation by ASC1 Ufmylation (A and B) ASC1 ufmylation is required for colony formation. ASC1 with and without shUBA5 or the 4KR mutant was expressed in MCF7 cells (A). shUBA5 or shUfSP2 was also expressed in MCF7 cells with and without shASC1 (B). Cells were then grown on soft agar in the presence or absence of E2 and/or 4-hydroxy-tamoxifen (4-HT). After incubation for 4 weeks, colonies were stained with crystal violet. (C) UBA5 and UfSP2 inversely affect cell proliferation. Cells prepared as in (A) and (B) were incubated with increasing concentrations of E2, and then subjected to MTT assay. Data are the mean ± SD (n = 3). (D–G) ASC1 ufmylation is required for tumor growth. BALB/c nude mice were injected with MCF7 cells stably expressing ASC1 with and without shUBA5 or the 4KR mutant (D and F). They were also injected with cells expressing shASC1, shUBA5, shUfSP2 or indicated combinations (E and G). After injection, orthotopic tumor growth assays were performed every week. After 8 weeks, mice were sacrificed, tumors were dissected out, and their weights were measured. Control indicates the cells transfected with an empty vector. Data in (D)–(G) are the mean ± SD (n = 5). See also Figure S6. Molecular Cell 2014 56, 261-274DOI: (10.1016/j.molcel.2014.08.007) Copyright © 2014 Elsevier Inc. Terms and Conditions