Volume 52, Issue 1, Pages 9-24 (October 2013) Parallel SCF Adaptor Capture Proteomics Reveals a Role for SCFFBXL17 in NRF2 Activation via BACH1 Repressor Turnover  Meng-Kwang Marcus Tan, Hui-Jun Lim, Eric J. Bennett, Yang Shi, J. Wade Harper  Molecular Cell  Volume 52, Issue 1, Pages 9-24 (October 2013) DOI: 10.1016/j.molcel.2013.08.018 Copyright © 2013 Elsevier Inc. Terms and Conditions

Molecular Cell 2013 52, 9-24DOI: (10.1016/j.molcel.2013.08.018) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Parallel Adaptor Capture Proteomics (A) Schematic representation of the parallel adaptor capture (PAC) proteomics approach. (B and C) APSMs for known interaction partners for FBXW11/β-TRCP2 (B) and FBXW7α (C) identified as HCIPs by PAC proteomics. Molecular Cell 2013 52, 9-24DOI: (10.1016/j.molcel.2013.08.018) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Application of PAC Proteomics to the FBXL Family of SCF Adaptors (A) Expression of FBXL proteins in HEK293 cells, as determined by analysis of RNA sequencing data from Sultan et al. (2008). (B) Heatmap representation of APSMs for HCIPs from each FLAG-HA-FBXL in untreated (U), MLN4924-treated (M), and Btz-treated (B) cells in biological replicates 1 and 2. (C) Venn diagram comparing the overlap in HCIPs identified in biological replicate experiments, independent of the treatment in which the HCIP was identified. (D) Venn diagram of the 97 HCIPs found in only one PAC proteomics replicate experiments for HCIPs that were identified under only one treatment condition and those found with two or three treatment conditions. See also Tables S1 and S2. Molecular Cell 2013 52, 9-24DOI: (10.1016/j.molcel.2013.08.018) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 Interaction Landscape of the FBXL Adaptor Family (A) Interaction maps for FBXLs examined by PAC proteomics. The interactions shown represent the union between biological replicates 1 and 2. (B) Legend depicting symbols used in (A). See also Table S2. Molecular Cell 2013 52, 9-24DOI: (10.1016/j.molcel.2013.08.018) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Experimental Validation of Candidate FBXL-Interacting Protein Pairs (A) FBXL interacting proteins selected for validation and a summary of validation results for reverse immunoprecipitation western blotting and stabilization with MLN4924. Supporting data are provided in Figures S1 and S2. (B) Selected examples of reciprocal interaction validation by immunoprecipitation western blot analysis. N-terminally MYC- or FLAG-HA-tagged interacting proteins were transiently coexpressed in HEK293T cells, using GFP as a control. After 48 hr, the indicated immune complexes or whole cell extracts (WCE) were immunoblotted with the indicated antibody. (C) Venn diagram showing the results of validation experiments examining the reciprocal association of FBXLs and their interacting proteins tested. Supporting data are provided in Figure S1. (E and F) Validation of substrates as CRL targets via stabilization by MLN4924 treatment. (E) HEK293 cells stably expressing N-terminally FLAG-HA-tagged FBXL interacting proteins were incubated with or without MLN4924 (1 μM, 4 hr), and extracts subjected to immunoblotting with the indicated antibodies. Histone H3 was used as a loading control and p27 was used as a positive control. (F) Venn diagram showing the extent of validation as a CRL target based on increased steady-state abundance in the presence of MLN4924. Supporting data are provided in Figure S2. (G) FBXL2, FBXL5, FBXL7, FBXL12, FBXL13, FBXL14, FBXL16, FBXL17, FBXL18, and FBXL19 were N-terminally tagged with FLAG-HA and stably expressed in HCT116 cells. PAC proteomics was performed on untreated cells and on cells treated with MLN4924 (1 μM, 4 hr). The Venn diagram displays overlap between HCIPS found for these FBXLs in HCT116 and HEK293 cells under these two conditions. Supporting data are provided in Figure S2 and Table S3. See also Figure S3. Molecular Cell 2013 52, 9-24DOI: (10.1016/j.molcel.2013.08.018) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 The SCFFBXL17-BACH1 Interaction Network (A) Proteomic analysis of FBXL17 and BACH1 complexes. APSMs and WDN-scores are provided for HA-BACH1 and HA-FBXL17 complexes isolated from HEK293 cells and for HA-FBXL17 complexes isolated from HCT116 cells. (B) Interaction map for FBXL17 and BACH1 complexes. Blue edges, this study; red edges, Genemania; green edges, STRING (threshold = 900). (C) Association of FBXL17 with SKP1 requires the F-box motif. N-FLAG-HA-tagged GFP, FBXL17, and FBXL17ΔF were coexpressed in HEK293T cells. Forty-eight hours after transfection, the cells were harvested and immunoprecipitated with anti-HA resin and immunoblotted with indicated antibodies. (D) N-FLAG-HA-tagged GFP or FBXL17 was transiently cotransfected with N-MYC-BACH1 into HEK293T cells. Forty-eight hours after transfection, the cells were harvested and immunoprecipitated with α-HA resin and immunoblotted with indicated antibodies. (E and F) FBXL17 associates with BACH1 preferentially in the nucleus at steady state. (E) Nuclear and cytoplasmic fractions from HeLa cells stably expressing N-FLAG-HA-FBXL17 were immunoblotted with the indicated antibodies. (F) Nuclear and cytoplasmic fractions from cells in (E) treated with MLN4924 (1 μM, 4 hr) were subjected to immunoprecipitation with anti-HA resin and immunoblotted with the indicated antibodies. Molecular Cell 2013 52, 9-24DOI: (10.1016/j.molcel.2013.08.018) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 Regulation of the NRF2-HMOX1 Pathway via SCFFBXL17-Dependent Control of BACH1 Abundance (A) MLN4924 (1 μM, 4 hr) increases the abundance of endogenous BACH1 in HEK293T cells as determined by immunoblotting with the indicated antibodies. p27 was used as a positive control. (B) Expression of CUL1DN but not GFP as a control increases the abundance of BACH1 in HEK293T cells, as revealed by immunoblotting of cell extracts. p27 was used as a positive control. (C) Overexpression of FBXL17 reduces steady-state BACH1 levels, while overexpression of FBXL17ΔF increases BACH1 abundance in HEK293T cells. (D) Validation of FBXL17 siRNAs. HCT116 cells stably expressing N-FLAG-HA-FBXL17 were transiently transfected with either control siRNA or independent FBXL17 siRNAs. Seventy-two hours later, cells were harvested, lysed, and immunoblotted with the indicated antibodies. (E) HeLa cells were transfected with control or independent FBXL17 siRNAs. Seventy-two hours later, cells were lysed and extracts immunoblotted with the indicated antibodies and mRNA was isolated and used for quantitative PCR (qPCR) to assess the levels of FBXL17, BACH1, and HMOX1 transcripts. GAPDH was used in the normalization of all qPCR analyses. Error bars represent SE from three independent analyses. (F) Increased HMOX1 transcription correlates with decreased BACH1 upon ectopic FBXL17 expression. HeLa cells, transfected with the indicated plasmids, were harvested and processed either for immunoblotting with the indicated antibodies or for qPCR to assess HMOX1 levels. Error bars represent SD from technical triplicates. (G) Schematic diagram showing the “MARE” enhancer region of the HMOX1 gene, where both BACH1 and NRF2 have been shown to bind. (H) FBXL17 regulates the balance between NRF2 and BACH occupancy on the HMOX1 promoter. HeLa cells were transiently transfected with control siRNA, FBXL17_2 siRNA, BACH1 siRNA, or both BACH1 and FBXL17_2 siRNAs. Seventy-two hours posttransfection, cells were either lysed for immunoblotting (right panel) or crosslinked and subjected to ChIP-qPCR analysis using NRF2 or BACH1 antibodies. Error bars represent SE from three independent analyses. (I) FBXL17-dependent ubiquitylation of BACH1. HEK293T cells were transfected with the indicated constructs. Forty hours after transfection, the cells were treated with MG132 (6 hr) prior to either lysis for protein analysis or qPCR to determine FBXL17 mRNA levels. Protein samples were lysed in 2% SDS and used for immunoprecipitation with α-HA antibodies followed by immunoblotting with α-Ub or α-FLAG. Error bars represent SD from technical triplicates. See also Figure S4. Molecular Cell 2013 52, 9-24DOI: (10.1016/j.molcel.2013.08.018) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 7 BACH1 Turnover via SCFFBXL17 Regulates the Response of the HMOX1 Promoter to Cellular Stress (A) Proteasome and CRL inhibition blocks BACH1 loss in response to hemin (4 hr), as determined by immunoblotting of HeLa cell extracts with the indicated antibodies. Error bars represent SD from technical triplicates. (B) FBXL17 depletion reduces hemin-dependent ubiquitylation of BACH1 in response to hemin. HEK293T cells were transfected with the indicated constructs. Forty hours after transfection, the cells were treated with MG132 (6 hr) prior to either lysis for protein analysis or qPCR to determine FBXL17 mRNA levels. Protein samples were lysed in 2% SDS and used for immunoprecipitation with α-HA antibodies followed by immunoblotting with α-Ub or α-FLAG. Error bars represent SD from technical triplicates. (C) FBXL17 depletion stabilizes BACH1 turnover in response to hemin. Sixty hours posttransfection with the indicated siRNAs, HEK293T cells were treated with either hemin and CHX or hemin and MLN4924 as indicated prior to immunoblotting of cell extracts. Parallel cultures were used for qPCR analysis to determine efficiency of FBXL17 depletion. Error bars represent SD from technical triplicates. (D) FBXL17 depletion blocks HMOX1 transcriptional activation in response to hemin. Sixty hours posttransfection of HeLa cells with control of FBXL17 siRNAs, cells were treated with or without 10 μM hemin (4 hr). A portion of cells were used for immunoblotting for BACH1 and the remainder used for FBXL17 and HMOX1 mRNA analysis by qPCR. Error bars represent SD from technical triplicates. (E and F) FBXL17 depletion reduces HMOX1 induction in response to H2O2. HeLa cells were transfected with either control or two different FBXL17 siRNAs. Sixty hours posttransfection of HeLa cells with the indicated siRNAs, cells were labeled with CM-H2DCCFDA for subsequent analysis of oxidative stress and then treated with H202 for 30 min. (E) Cells extracts were subjected to analysis of BACH1 levels by immunoblotting or mRNA was subjected to qPCR for FBXL17, BACH1, and HMOX1. (F) An aliquot of cells was analyzed using flow cytometry to monitor oxidative stress via CM-H2DCCFDA. Error bars represent SE from duplicate analyses (experiments 1 and 2). (G) Schematic representation of the relationship between BACH1 and NRF2 in the control of HMOX1 expression and the role of SCFFBXL17 in controlling the balance between activating and repressive transcriptional control. Molecular Cell 2013 52, 9-24DOI: (10.1016/j.molcel.2013.08.018) Copyright © 2013 Elsevier Inc. Terms and Conditions