Direct and distinguishable inhibitory roles for SUMO isoforms in the control of transcriptional synergy

Abstract

Functional interactions between factors bound at multiple sites on DNA often lead to a synergistic or more-than-additive transcriptional response. We previously defined a class of peptide sequences termed synergy control motifs (SC motifs) that function in multiple regulators by selectively inhibiting synergistic activity driven from multiple but not single response elements. By studying the prototypic SC motifs of the glucocorticoid receptor, we show that SC motifs inhibit transcription per se both in cis and in trans, and that a requirement for multiple contacts with DNA renders them selective for compound response elements. Notably, SC motifs are sites for SUMOylation, and the degree of modification correlates strongly with the extent of synergy control. Recruiting SUMO to the promoter either independently or as a fusion to the glucocorticoid receptor is sufficient to recapitulate the in trans and in cis inhibition by SC motifs without apparent changes in subcellular localization. Moreover, we find that the core ubiquitin fold domain of SUMO is sufficient for inhibition and that, independently of their potential for polySUMO chain formation, SUMO-2 and SUMO-3 are more effective inhibitors than SUMO-1.

Fig. 1.

SC motifs inhibit GR activity in trans. CV-1 cells were transfected with vectors for either WT (p6RGR) or SC mutant (p6RGR K297R/K313R) GR (30 ng) and the indicated amounts of vectors for Gal4 DBD fusions to either WT (pG4(SC)₂) or mutant (pG4(SCmut)₂) SC motifs of GR. The reporter plasmids used were pΔ(Gal)₂(TAT)₃-Luc (A), pΔ(Gal)₂(TAT)₁-Luc (B), pΔ(Gal)₁(TAT)₃-Luc (C), and pΔ(Gal)₁(TAT)₁-Luc (D).

Fig. 2.

The SC motifs in GR are sites for SUMO modification. COS-7 cells were transfected with vectors for WT (p6RGR) or SC mutant (p6RGR K297R/K313R) GR- and pCDNA3-based vectors for HA-tagged SUMO-1, -2, and -3. After a 16-h incubation with either vehicle or Dex, GR immunoprecipitates were resolved by SDS/PAGE and processed for immunoblotting by using anti-HA (A) or BuGR (B) antibodies. (C) In vitro SUMOylation reactions were performed as described in Methods by using in vitro-transcribed and -translated [³⁵S]-labeled WT or SC mutant (K297E/K313E) GR as substrates in the presence of the indicated components.

Fig. 3.

SUMO modification correlates with extent of synergy control. (A) CV-1 cells were transfected with pΔTAT₁-luc or pΔTAT₃-luc reporters and 30 ng of expression vectors for either WT (p6RGR) or SC mutant forms of GR containing a K297R mutation in the first SC motif alone or in combination with the indicated mutations in the second motif. The number above the brackets represents the TAT3/TAT1 activity ratio. The sequence above represents GR amino acids 294–317 containing the two SC motifs. (B) COS-7 cells cotransfected with HA-SUMO-1 and the GR vectors used in A were processed as in Fig. 2.

Fig. 4.

SUMO inhibits GR activity in trans. CV-1 cells were transfected with expression vectors for WT (p6R GR) or SC mutant (p6RGR K297R/K313R) GR (30 ng) and the indicated amounts of empty (pCDNA3) or Gal4 DBD fusion expression vectors to either HA-tagged SUMO-1 or SUMO-2 or the HA epitope alone. The reporter plasmids were the same as in Fig. 1.

Fig. 5.

Specific inhibition by SUMO isoforms does not require their N-terminal region. (A) Expression of Gal4 DBD fusions in CV-1 cells was confirmed by immunoblotting for the HA epitope as described in Methods. (B) CV-1 cells were transfected with 30 ng of expression vectors for WT (p6RGR) or SC mutant (p6RGR K297R/K313R) GR and 30 ng of pCDNA3.1(+)-based vectors for the indicated Gal4 DBD fusions. The reporter was pΔ(Gal)₂(TAT)₃-Luc.

Fig. 6.

Colinear fusion of SUMO inhibits GR synergy. CV-1 cells were transfected with the indicated amounts of p6R-based vectors for the expression of WT or SC mutant GR (K297R/K313R) fused to HA alone, HA-SUMO-1, or SUMO-2 and 100 ng of either pΔTAT₁-Luc (A) or pΔTAT₃-Luc (B) reporters. Average activity values for SC mutant GR (25 ng) in the presence of Dex were 4.5 ± 0.2 (A) and 78.1 ± 6.7 (B). (C) Expression of GR variants in CV-1 cells was confirmed by immunoblotting for the HA epitope as described in Methods.

Fig. 7.

Disruption of SC motifs or colinear fusion to SUMO does not alter the subcellular localization of GR. CV-1 cells were transfected with the same expression plasmids as in Fig. 6. After 1 h of Dex (10 nM) or vehicle treatment, cells were fixed in 100% methanol and stained with anti-HA primary and Oregon Green-labeled secondary antibodies. After Hoechst 33258 (Molecular Probes) staining, slides were processed for immunofluorescence digital camera imaging as described in Methods. Representative fields are shown, and transfected cells are marked with an asterisk.