Probing the mechanisms of synergy control motifs in the glucocorticoid receptor: A role for posttranslational modification by the small ubiquitin -like modifier (SUMO)
The recruitment of a given factor to multiple response elements within the promoter of a gene often results in a more than additive, or synergistic, transcriptional response compared to that seen from a single element. Our group has identified a novel class of peptide sequences, termed synergy control, or SC motifs, that function in multiple regulators by selectively limiting the activity emanting from multiple, but not single, response elements. This dissertation examines the mechanisms of action of SC motifs using the Glucocorticoid Receptor (GR) as a paradigm. Through in vivo and in vitro approaches, we demonstrate that SC motifs serve as sites for post-translational modification by members of the small ubiquitin-like modifier (SUMO) protein family. This modification is directly responsible for the inhibitory effects of SC motifs. The basis for selective action of SC motifs is not due to targeting of synergy per se, but, rather, is dependent on binding of SUMO-modified factors to multiple sites on DNA. A comprehensive mutational analysis to probe the structural determinants of SUMO required for its inhibitory properties, identified a cluster of basic and hydrophobic residues that define a critical surface essential for transcriptional inhibition. An analysis of receptor-specific requirements for SC motif function revealed that the inhibitory actions of SUMO are dependent on stable binding of GR to DNA. The systematic alteration of the number and arrangement of response elements revealed that the repressive effects of SUMO extend over a relatively short distance (∼80bps). We propose that the stable binding of SC motif-bearing regulators to multiple sites on DNA allows for the recruitment of specific co-repressors through their interaction with a critical functional surface of SUMO.
0307: Molecular biology