Journal: The Journal of Biological Chemistry

Article Title: The AF-2 cofactor binding region is key for the selective SUMOylation of estrogen receptor alpha by antiestrogens

doi: 10.1016/j.jbc.2022.102757

Figure Lengend Snippet: Fulvestrant induces SUMOylation of ERα but not ERβ . A – D , Western blot analysis of ERα and ERβ modifications after transient transfection of HEK293 cells with their respective expression vectors, with or without a SENP1 expression vector, and treatment of cells at different time points with fulvestrant (1 μM, F) or vehicle (0). ∗ indicates modified ERα forms. Blots are representative of three independent experiments. E , BRET titration curves performed by transient transfection of HEK293 cells with a fixed amount (50 ng) of ERα-RLucII or ERβ-RLucII and varying amounts of YFP-SUMO3 (0–800 ng) expression vectors, either in the presence of fulvestrant (1 μM, F ) or vehicle (0) for 2 h. The net BRET 1 ratio measured in live cells after addition of coelenterazine H (5 μM) is plotted as a function of measured YFP/RLuc ratios. The graph is a compilation of three independent experiments each performed in quadruplicates. Statistical analyses were performed using nonlinear regression analysis and comparison of Bmax values using one-way ANOVA and Tukey’s multiple comparison test in GraphPad Prism 6.07 (∗ p < 0.05). F , BRET kinetics performed by transfecting a fixed amount of ERα-RLucII or ERβ-RLucII (50 ng) and YFP-SUMO3 (500 ng) expression vectors in HEK293 cells in the presence of fulvestrant (1 μM, F ) or vehicle (0). Net BRET 1 ratios were read at different time points (10–240 min) in live cells after addition of coelenterazine H (5 μM). Compiled results from three independent experiments (mean values ± SEM) are shown. Statistical analyses were performed using a Holm–Sidak’s multiple t test assuming the same scatter in GraphPad Prism 6.07 (∗ p < 0.05). BRET, bioluminescence resonance energy transfer; ERα, estrogen receptor α; ERβ, estrogen receptor β; RLucII, Renilla luciferase II; SENP1, SENtrin-specific protease 1; SUMO, small ubiquitin-related MOdifier; YFP, yellow fluorescent protein.

Article Snippet: Statistical analyses were performed using nonlinear regression analysis and comparison of Bmax values using one-way ANOVA and Tukey’s multiple comparison test in GraphPad Prism 6.07 (∗ p < 0.05).

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Modification, Titration, Comparison, Bioluminescence Resonance Energy Transfer, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: The AF-2 cofactor binding region is key for the selective SUMOylation of estrogen receptor alpha by antiestrogens

doi: 10.1016/j.jbc.2022.102757

Figure Lengend Snippet: Spectrum of E R α SUMOylation induced by antiestrogens . A and B , Western blot analyses of endogenous ERα modifications after treatment with 17β-estradiol (E2, 25 nM) or with the acidic side chain SERD AZD9496 (AZD), the basic side chain SERMs 4-hydroxytamoxifen (OHT), lasofoxifene (Las), raloxifene (Ral), or bazedoxifene (Baz), the basic side chain SERD GDC-0927 (GDC), or the long side chain SERDs RU58,668 (RU) or fulvestrant (F) (1 μM), or with vehicle (0) for 1 h without ( A ) or with ( B ) pretreatment with the SAE inhibitor ML-792 (1 μM, 6 h). Blots are representative of three independent experiments. ∗ indicates modified ERα forms. C and D , western blot analyses of ERα modifications in transiently transfected HEK293 cells, in the presence or absence of SENP1. Cells were treated for 1 h with E2 (25 nM), antiestrogens (1 μM), or vehicle. Blots are representative of three independent experiments. ∗ indicates modified ERα forms. E , BRET titration curves performed by transient transfection of HEK293 cells with a fixed amount (50 ng) of ERα-RlucII and varying amounts of YFP-SUMO3 (0–800 ng) expression vectors, in the presence of E2 (25 nM), antiestrogens (1 μM), or vehicle for 2 h. The net BRET 1 ratios measured in live cells after addition of coelenterazine H (5 μM) are plotted as a function of the YFP/RLuc calculated ratios. The curves are a compilation of three independent experiments, each point representing the mean values of technical quadruplicates. Bmax values derived from nonlinear regression analysis were calculated for each biological replicate, and statistical analysis was performed using one-way ANOVA and Tukey’s multiple comparison test in GraphPad Prism 6.07 (∗ p < 0.05). F , BRET assays between ERα-RlucII and YFP-SUMO3 after treatment for 2 h with E2 (25 nM) or antiestrogens (1 μM) in HEK293 cells transfected with a constant amount of ERα-RlucII (50 ng) and YFP-SUMO3 (500 ng). The graph shows mean values ± SEM from three independent experiments performed in technical octuplicates. Statistical analyses were performed using a one-way ANOVA followed by Dunnett’s multiple comparison test in GraphPad Prism 6.07 (∗ p < 0.05). G , Western blot analyses of endogenous ERα levels in MCF7 cells after treatment with 17β-estradiol (E2, 25 nM), antiestrogens (1 μM), or vehicle (0) for 6 h. Blots are representative of three independent experiments. ∗ indicates modified ERα forms. H , luciferase assays in HEK293 cells transiently cotransfected with an expression vector for ERα and a GREB 1 -ERE-Luc reporter vector. Cells were treated 24 h after transfection with vehicle, E2 (2.5 nM), or antiestrogens (1 μM) for another 24 h. The graph shows mean values ± SEM from three independent experiments performed in technical triplicates. Statistical analyses were performed using Holm–Sidak’s multiple t test assuming the same scatter in GraphPad Prism 6.07 (∗ p < 0,05). BRET, bioluminescence resonance energy transfer; ERE, estrogen responsive element; ERα, estrogen receptor α; ERβ, estrogen receptor β; RlucII, Renilla luciferase II; SUMO, small ubiquitin-related modifier; SENP1, SENtrin-specific protease 1; SERDs, selective estrogen receptor downregulators; YFP, yellow fluorescent protein.

Article Snippet: Statistical analyses were performed using nonlinear regression analysis and comparison of Bmax values using one-way ANOVA and Tukey’s multiple comparison test in GraphPad Prism 6.07 (∗ p < 0.05).

Techniques: Western Blot, Modification, Transfection, Titration, Expressing, Derivative Assay, Comparison, Luciferase, Plasmid Preparation, Bioluminescence Resonance Energy Transfer

Journal: The Journal of Biological Chemistry

Article Title: The AF-2 cofactor binding region is key for the selective SUMOylation of estrogen receptor alpha by antiestrogens

doi: 10.1016/j.jbc.2022.102757

Figure Lengend Snippet: The ligand-binding domain of ERα is required for induction of SUMOylation of chimeras by fulvestrant . A and B , Western analyses of modification of ERα/β chimeras by fulvestrant. HEK293 cells were transiently transfected with expression vectors for ERα, ERβ, or chimeras and treated with fulvestrant (1 μM) or vehicle for 1 h. Blots are representative of three independent experiments. ∗ indicates modified ERα forms. C , SUMO3 BRET assay with ERα/β chimeras in HEK293 cells treated or not with fulvestrant (2 h, 1 μM). D , the graph shows mean values ± SEM from three independent experiments, each performed in triplicates. Luciferase assays in HEK293 cells transiently transfected with an expression vector for ERα, ERβ, or with the βADαEF chimera together with a GREB 1 -ERE-Luc reporter vector. Cells were treated 24 h after transfection with E2 (2.5 nM) with or without fulvestrant (1 μM; E2 + F), OHT, fulvestrant (1 μM each) or vehicle and assayed for luciferase activity after 24 h. The graph shows mean values ± SEM from three independent experiments performed in technical triplicates. Statistical analyses ( C and D ) were performed using Holm–Sidak’s multiple t test in GraphPad Prism 6.07 assuming the same scatter (∗ p < 0,05). BRET, bioluminescence resonance energy transfer; ERα, estrogen receptor α; ERβ, estrogen receptor β; ERE, estrogen responsive element; OHT, 4-hydroxytamoxifen.

Article Snippet: Statistical analyses were performed using nonlinear regression analysis and comparison of Bmax values using one-way ANOVA and Tukey’s multiple comparison test in GraphPad Prism 6.07 (∗ p < 0.05).

Techniques: Ligand Binding Assay, Western Blot, Modification, Transfection, Expressing, Bioluminescence Resonance Energy Transfer, Luciferase, Plasmid Preparation, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: The AF-2 cofactor binding region is key for the selective SUMOylation of estrogen receptor alpha by antiestrogens

doi: 10.1016/j.jbc.2022.102757

Figure Lengend Snippet: The ERα identity of helices H3-H4 in the ligand binding domain is necessary and sufficient for induction of SUMOylation of chimeras by fulvestrant . A–C , Western analyses of fulvestrant-induced modification of ERα/β chimeras. HEK293 cells were transiently transfected with expression vectors for ERα, ERβ, or chimeras as indicated and were treated with fulvestrant (1 μM) or vehicle for 1 h. Blots are representative of three independent experiments. ∗ indicates modified ERα forms. D – F , SUMO3 BRET assay for SUMOylation of ERα, ERβ, or chimeras in transfected HEK293 cells after 2 h of treatment with fulvestrant. The experiments were performed three times in technical triplicates. Graphs show mean values ± SEM from biological repeats. Statistical analyses were performed in GraphPad Prism 6.07 with a multiple t test using the Holm–Sidak method assuming the same scatter (∗ p < 0,05). BRET, bioluminescence resonance energy transfer; ERα, estrogen receptor α; ERβ, estrogen receptor β; SUMO, small ubiquitin-related modifier.

Article Snippet: Statistical analyses were performed using nonlinear regression analysis and comparison of Bmax values using one-way ANOVA and Tukey’s multiple comparison test in GraphPad Prism 6.07 (∗ p < 0.05).

Techniques: Ligand Binding Assay, Western Blot, Modification, Transfection, Expressing, Bioluminescence Resonance Energy Transfer

Journal: The Journal of Biological Chemistry

Article Title: The AF-2 cofactor binding region is key for the selective SUMOylation of estrogen receptor alpha by antiestrogens

doi: 10.1016/j.jbc.2022.102757

Figure Lengend Snippet: Divergent amino acids in H3-H4 located on the outside rim of AF-2 confer induction of SUMOylation by PIAS1. A , luciferase assays in HEK293 cells transiently transfected with an expression vector for ERα, ERβ, αAFβH3H4, or αAFβH3H6 together with a GREB 1 -ERE-Luc reporter vector. Cells were treated 24 h after transfection with vehicle, E2 (2,5 nM) with or without fulvestrant (1 μM; E2 + F), OHT or fulvestrant (1 μM each) and assayed for luciferase activity after 24 h. The graph shows mean values ± SEM from three independent experiments performed in technical triplicates. Statistical analyses were performed using Holm–Sidak’s multiple t test in GraphPad Prism 6.07 assuming the same scatter (∗ p < 0,05). B , residues in human ERα H3-H4 region diverging with rat ERβ are highlighted in magenta in the human ERα sequence and in green in the rat ERβ sequence. K362 in human ERα, conserved between the two receptors, is highlighted in red . C , superposition of the human ERα (from the 3ERT structure, cyan ) and of the rat ERβ complexed to ICI164,384 (1HJ1 structure, green ), whose side chain is inserted in the coactivator binding groove. Residues in the human ERα H3-H4 region diverging with rat ERβ are highlighted in magenta and those unique to rat ERβ are shown in stick representation ( green ). D , SUMO1 BRET was performed with ERα, ERβ, or chimeras exchanging the H3-H6 or H3-H4 regions in the presence ( striped bars ) or absence ( solid colors ) of PIAS1. The graph shows the mean values ± SEM from two independent experiments; each point represents the average from technical triplicates. Statistical analyses were performed using Holm-Sidak’s multiple t test assuming the same scatter in GraphPad Prism 6.07 (∗ p < 0,05). BRET, bioluminescence resonance energy transfer; ERE, estrogen responsive element; ERα, estrogen receptor α; ERβ, estrogen receptor β; PIAS1, protein inhibitor of activated STAT 1; SUMO, small ubiquitin-related modifier.

Article Snippet: Statistical analyses were performed using nonlinear regression analysis and comparison of Bmax values using one-way ANOVA and Tukey’s multiple comparison test in GraphPad Prism 6.07 (∗ p < 0.05).

Techniques: Luciferase, Transfection, Expressing, Plasmid Preparation, Activity Assay, Sequencing, Binding Assay, Bioluminescence Resonance Energy Transfer

Journal: The Journal of Biological Chemistry

Article Title: The AF-2 cofactor binding region is key for the selective SUMOylation of estrogen receptor alpha by antiestrogens

doi: 10.1016/j.jbc.2022.102757

Figure Lengend Snippet: PIAS proteins have a differential impact on SUMOylation of ERα in the presence of fulvestrant. SUMO3 BRET assay for SUMOylation of ERα in the absence or presence of wt or catalytically dead mutant PIAS proteins in transfected HEK293 cells treated or not with fulvestrant for 2 h. HEK293 cells were transfected with a fixed amount of ERα (50 ng) and SUMO3 (500 ng) with or without overexpression (400 ng) of PIAS proteins (PIAS1, PIAS2α, PIAS2β, PIAS3, or PIAS4), catalytically dead mutants of PIAS proteins (PIAS1 C351S, PIAS2α or PIAS2β C362A, PIAS3 C308S or PIAS4 C342A) or empty vector (pCMV-Flag). The experiments were performed three times in technical triplicates. The graph shows mean values ± SEM from biological repeats. Statistical analyses were performed in GraphPad Prism 6.07 using a 2-way ANOVA with a Tukey’s multiple comparison test (∗ p < 0,05). BRET, bioluminescence resonance energy transfer; ERα, estrogen receptor α; ERβ, estrogen receptor β; PIAS1, protein inhibitor of activated STAT 1; RlucII, Renilla luciferase II; SUMO, small ubiquitin-related modifier; YFP, yellow fluorescent protein.

Article Snippet: Statistical analyses were performed using nonlinear regression analysis and comparison of Bmax values using one-way ANOVA and Tukey’s multiple comparison test in GraphPad Prism 6.07 (∗ p < 0.05).

Techniques: Bioluminescence Resonance Energy Transfer, Mutagenesis, Transfection, Over Expression, Plasmid Preparation, Comparison, Luciferase