SUMO-1 Modification Represses Sp3 Transcriptional Activation and Modulates Its Subnuclear Localization  Sarah Ross, Jennifer L Best, Leonard I Zon, Grace Gill  Molecular Cell  Volume 10, Issue 4, Pages 831-842 (October 2002) DOI: 10.1016/S1097-2765(02)00682-2

Figure 1 Endogenous Sp3 Is Modified by SUMO-1 (A) Modification of Sp3 by HA-SUMO-1. Protein extracts from control P19 cells or cells transfected with a plasmid expressing HA-SUMO-1 were analyzed by Western blotting with anti-Sp3 antibody. The three major isoforms of Sp3, generated by translation initiation at different positions, are indicated by arrows. An asterisk indicates a major additional Sp3 isoform in the HA-SUMO-1-transfected extracts. (B) Sp3 is modified by HA-SUMO-1. Protein extracts from P19 cells either untransfected (Control) or transfected with a plasmid expressing HA-SUMO-1 were immunoprecipitated with anti-Sp3, anti-HA, or control IgG beads as indicated. Immunoprecipitated material was analyzed by Western blotting with anti-Sp3 (top panel) or anti-HA antibodies (bottom panel). The asterisks indicate HA-SUMO-1-modified forms of Sp3. (C) Endogenous Sp3 is modified by SUMO-1. Proteins were immunoprecipitated from untransfected cell extracts with anti-Sp3 antibody or control IgG beads. The immunoprecipitated material was analyzed by Western blotting with anti-Sp3 (left panel) or anti-SUMO-1 antibodies (right panel). The major SUMO-1-modified forms of Sp3 are indicated by asterisks, and a minor form is indicated by a closed circle. Extracts in (B) and (C) but not (A) were prepared in the presence of N-ethylmaleimide (NEM) to inhibit de-sumoylating enzymes. Molecular Cell 2002 10, 831-842DOI: (10.1016/S1097-2765(02)00682-2)

Figure 2 K539 Is the Major SUMO-1 Acceptor Site in Sp3 and Mutation of K539 Relieves Inhibition of Sp3 (A) Schematic representation of Sp3. The transcriptional activation domains (AD), inhibitory domain (ID), Zinc finger DNA binding domain (Zn fingers), and putative SUMO acceptor sites (K108 and K539) are shown. The two smaller forms of Sp3 arise from translation initiation at internal methionines indicated by the arrows. (B) K539 is the major SUMO-1 acceptor site in Sp3. P19 cells were cotransfected with the indicated Gal4-Sp3 fusions or a control plasmid expressing the Gal4 DNA binding domain (G4) and a plasmid expressing HA-SUMO-1. KR dbl refers to the double mutant K108R, K539R. Proteins were immunoprecipitated with anti-Gal4 agarose or control IgG beads. The immunoprecipitated material was analyzed by Western blotting with anti-SUMO-1 (top panel) or anti-Gal4 antibodies (bottom panel). The major SUMO-1-modified form of Gal4-Sp3 is indicated by an asterisk, and a minor form is indicated by a closed circle. The Gal4 DNA binding alone, G4, is much smaller than Gal4-Sp3 and was run off these gels. (C) K539 is critical for inhibition of Sp3 transcriptional activity. P19 cells were cotransfected with a Gal4-luciferase reporter plasmid (G5-luc) and the indicated Gal4-Sp3 fusions. The luciferase activity is shown relative to the WT Gal4-Sp3 fusion which was set at 1. Molecular Cell 2002 10, 831-842DOI: (10.1016/S1097-2765(02)00682-2)

Figure 3 The SUMO-1 Protease SuPr-1 Specifically Stimulates the Transcriptional Activity of Gal4-Sp3 (A) Dose-dependent stimulation of Gal4-Sp3 activity by the SUMO-1 protease SuPr-1. P19 cells were cotransfected with G5-luc, wild-type Gal4-Sp3, and the indicated amounts of either vector or a plasmid expressing SuPr-1. Shown is luciferase activity relative to Gal4-Sp3 with 10 ng of vector, which was set at 1. (B) SuPr-3 and SuPr-4 do not increase G4-Sp3 activity. P19 cells were cotransfected with G5-luc, wild-type Gal4-Sp3, and either vector or plasmids expressing the SUMO-1 proteases SuPr-1, SuPr-3, or SuPr-4. G4-fusion activity represents luciferase activity relative to Gal4-Sp3 with vector, which was set at 1. (C) Gal4-Sp1 activity is not affected by SuPr-1. P19 cells were cotransfected with G5-luc, either G4-Sp3 or G4-Sp1, and either vector or the SuPr-1 expression plasmid as indicated. Shown is the luciferase activity relative to Gal4-Sp3 cotransfected with vector which was set at 1. Molecular Cell 2002 10, 831-842DOI: (10.1016/S1097-2765(02)00682-2)

Figure 4 The SUMO-1 Protease Activity of SuPr-1 Is Required to Stimulate Transcription by Gal4-Sp3 (A) SuPr-1 hydrolyzes SUMO-1 from Gal4-Sp3. Protein extracts from cells cotransfected with wild-type Gal4-Sp3 and either vector or HA-SuPr-1 were immunoprecipitated with anti-Gal4 agarose or IgG beads alone. Immunoprecipitates were analyzed by Western blot analysis with anti-SUMO-1 (top), anti-Gal4 (middle), or anti-HA antibodies (bottom). The major sumoylated form of Gal4-Sp3 is indicated by an asterisk. (B) Stimulation of Gal4-Sp3 by SuPr-1 requires the Sp3 SUMO-1 acceptor sites. P19 cells were cotransfected with G5-luc, either the wild-type (WT) or a SUMO-1 acceptor mutant (KR dbl) of Gal4-Sp3, and the indicated amounts of the SuPr-1 expression plasmid. Shown is the luciferase activity relative to WT Gal4-Sp3 with 0 ng of SuPr-1, which was set at 1. (C) The catalytic activity of SuPr-1 is required to activate Gal4-Sp3. P19 cells were cotransfected with G5-luc, wild-type Gal4-Sp3, and the indicated amounts of plasmids expressing either wild-type (HA-SuPr-1) or a catalytic mutant (HA-C466S) of SuPr-1. Shown is the luciferase activity relative to Gal4-Sp3 with 0 ng of SuPr-1, which was set at 1. Molecular Cell 2002 10, 831-842DOI: (10.1016/S1097-2765(02)00682-2)

Figure 5 N-Terminal Fusion of SUMO-1 Represses Sp3 Transcriptional Activity SUMO-1 (1–96), lacking the C-terminal Gly-Gly characteristic of mature SUMO-1, was fused in frame to the Gal4 DNA binding domain alone, G4, Gal4-Sp3, or Gal4-Sp3 K539R as indicated in the schematic drawing. Cells were cotransfected with the indicated Gal4 fusions and a Gal4-dependent luciferase reporter gene. The luciferase activity relative to Gal4-Sp3 wild-type, which was set at 1, is shown. The activity of Gal4, Gal4-Sp3, and Gal4-Sp3 K539R is indicated by open bars, and the activity of the corresponding SUMO-1 fusions is indicated by shaded bars. Molecular Cell 2002 10, 831-842DOI: (10.1016/S1097-2765(02)00682-2)

Figure 6 Endogenous Sp3 Localizes to the Nuclear Periphery and to Discrete Nuclear Dots (A) Sp3 localizes to the nuclear periphery. Immunofluorescence of P19 cells with anti-Sp3 antibodies (red). Nuclei were visualized by Hoechst staining for DNA (blue). (B) A fraction of Sp3 in nuclear dots colocalizes with PML. Immunofluorescence of P19 cells with anti-Sp3 (red) and anti-PML (green) antibodies. Colocalization of Sp3 and PML is indicated in the merge (yellow). (C) Nuclear localization of Sp3 and Sp1 is different. Immunofluorescence of P19 cells with anti-Sp1 antibodies (red) is shown. Molecular Cell 2002 10, 831-842DOI: (10.1016/S1097-2765(02)00682-2)

Figure 7 Localization of Gal4-Sp3 Is Regulated by SUMO-1 and Correlates with Activity (A) SUMO-1-modified, repressed forms of Gal4-Sp3 localize to the nuclear periphery and nuclear dots. P19 cells were transfected with constructs expressing wild-type Gal4-Sp3 (left) and Gal4-SUMO-1-K539R (right) and the subcellular localization of the fusions determined by indirect immunofluorescence using anti-Gal4 antibody (red). Nuclei were visualized by Hoechst staining for DNA (blue). (B) Transcriptionally active forms of Gal4-Sp3, lacking SUMO-1, are diffusely localized in the nucleus. P19 cells were transfected with Gal4-Sp3 K539R (left) or wild-type Gal4-Sp3 together with a plasmid expressing HA-SuPr-1 (right). Cells expressing Gal4 fusions (red) and HA-SuPr-1 (green) were visualized by indirect immunofluorescence using anti-Gal4 and anti-HA antibodies. Nuclei were visualized by Hoechst staining for DNA (blue). Although SuPr-1 has been reported to localize to PML nuclear bodies (Best et al., 2002), in these studies SuPr-1 was not exclusively found in nuclear dots, perhaps due to higher levels of expression. Four representative transfected cells are shown for each Gal4-fusion. Molecular Cell 2002 10, 831-842DOI: (10.1016/S1097-2765(02)00682-2)