A Small Conserved Surface in SUMO Is the Critical Structural Determinant of Its Transcriptional Inhibitory Properties

Abstract

Small ubiquitin-like modifier (SUMO) modification of sequence-specific transcription factors has profound regulatory consequences. By providing an intrinsic inhibitory function, SUMO isoforms can suppress transcriptional activation, particularly at promoters harboring multiple response elements. Through a comprehensive structure-function analysis, we have identified a single critical sector along the second beta sheet and the following alpha helix of SUMO2. This distinct surface is defined by four basic residues (K33, K35, K42, R50) that surround a shallow pocket lined by aliphatic (V30, I34) and polar (T38) residues. Substitutions within this area specifically and dramatically affected the ability of both SUMO2 and SUMO1 to inhibit transcription and revealed that the positively charged nature of the key basic residues is the main feature responsible for their functional role. This highly conserved surface accounts for the inhibitory properties of SUMO on multiple transcription factors and promoter contexts and likely defines the interaction surface for the corepressors that mediate the inhibitory properties of SUMO.Eukaryotic transcriptional control relies strongly on the power of combinatorial regulation (53). In a given cell, genes can draw unique sets of regulatory factors from a relatively small pool based on their specific arrangement of cis-acting elements. The often synergistic functional interactions between these factors give rise to the complex patterns of gene expression required for the appropriate physiological performance of cells (6, 9). The regulatory repertoire of each factor can be further expanded by structural alterations induced by binding of ligands or posttranslational modifications, as in the case of the glucocorticoid receptor (GR) and other steroid hormone receptors (53). Subtle mutations that disturb ligand binding or covalent attachment can lead to profound alterations in the outcome of functional interactions among factors. For example, we have identified a short regulatory motif embedded in GR and many other sequence-specific regulators that is both necessary and sufficient to limit their transcriptional synergy (23). Disruption of these conserved synergy control (SC) motifs selectively enhances synergistic activation at compound response elements without altering the activity driven from a single site. In order to exert their effects, SC motifs need to be recruited to multiple independent sites on the DNA, and this feature accounts for their selective effects at compound response elements (20). The function of SC motifs is not circumscribed to inhibit synergistic activation driven by the factor in which they are embedded (in-cis effects) but are also capable of affecting separate activators bound nearby (intrans effects). Based on the convergence between SC motifs and the consensus sequence for posttranslational modification by members of the small ubiquitin-like (Ubl) modifier (SUMO) family, we (20, 47) and others (51) have shown that SC motifs function as SUMO acceptor sites and...

NXP-2 association with SUMO-2 depends on lysines required for transcriptional repression

“…In contrast, the double charge-reversal mutants K33E and K35E (EE1), K33E and K42E (EE2), or K35E and K42E (EE3) were severely impaired and repressed at only 7%, 4%, and 33%, respectively, of WT SUMO-2 levels, in the context of Gal4-SUMO-2 (Table 1). In the context of transcription activated by a SUMOylation-deficient mutant glucocorticoid receptor, SUMO-2 K33A, K35A, or K42A mutants repress at 20-60% of WT SUMO-2 levels, SUMO-2 K33E, K35E, or K42E lack more of WT SUMO-2 repressive activity, and the SUMO-2 K33E,K42E or homologous SUMO-1 K37E,K46E double mutants lack virtually all repressive activity (9). Thus, in different contexts, dual charge-reversal mutations of SUMO-2 K33, K35, and K42 most consistently abrogate SUMO repressive effects.…”

“…Such proteins would be candidate SUMO corepressors. We have used two different linear fusions of SUMO-2 or -1 to the Gal4 DNA-binding domain to extend the recent comprehensive evaluation of SUMO-2 residues that are critical for repression (9). Paired charge-reversal mutations of SUMO-2 K33, K35, and K42 substantially decreased repression and were, therefore, compared with WT SUMO-2 in differential protein-affinity chromatography.…”

Histone sumoylation is a negative regulator in Saccharomyces cerevisiae and shows dynamic interplay with positive-acting histone modifications

“…5C). As a second control, we created a SUMO-H2B fusion containing substitution mutations in SUMO that decrease the repressive effects of SUMO (Chupreta et al 2005). The fusion containing these mutations completely reversed the repression seen with SUMO-H2B (Fig.…”

“…Some consensus sites occur in negative regulatory domains of transcription factors (Holmstrom et al 2003). Recent studies demonstrate that a few charged and conserved residues in the SUMO ubiquitin-like fold are essential for its transcriptional repressive properties (Chupreta et al 2005).…”

SUMO modification regulates inactivation of the voltage-gated potassium channel Kv1.5

“…Many of these effects appear to require a distinct surface in SUMO (28) capable of recognizing SUMO-interacting motifs in partner proteins (29). To date, SUMOylation has been most extensively studied in the context of nuclear proteins, and, in the case of sequence-specific transcription factors, SUMOylation exerts a direct and context-dependent inhibitory role (27,28). SUMOylation, however, is not restricted to this compartment because the SUMO conjugation machinery is found throughout the cell and multiple proteins not associated with the nuclear compartment, such as the plasma membrane K ϩ channel K2P1 (16), are targets for SUMO modification (30)(31)(32).…”