A functional role for Ubc9 in the T3-dependent regulation of thyroid hormone receptor beta1-mediated transactivation.

The dramatic structural change that the thyroid hormone receptor (TR) undergoes in response to the binding of its cognate ligands, triodothyronine (T3) and thyroxine (T4) represents the primary operational switch for TR-mediated transcriptional activity. The result is the presentation of a surface within the C-terminal, ligand binding domain (LBD), referred to as transcriptional activation function-2 (AF-2), which functions to recruit coregulatory proteins that bridge TR to the cellular transcriptional machinery. A secondary activation function (AF-1) found within the N-terminal A/B domain works in tandem with AF-2 to insure the proper regulation of TR target gene expression. Evidence suggests that AF-1 can modulate promoter-specific, ligand-dependent (AF-2) responses in a cell-type-specific context, conferring isoform-specific functions to TRs. Ubc9 was identified in a yeast 2-hybrid screen for proteins that interact specifically with the A/B domain of TRbeta1. The beta1 A/B domain contains a specific consensus motif that is recognized and bound by Ubc9 which is not found in the N-terminus of any other TR isoform. An additional Ubc9 interaction motif, subsequently identified within the TR LBD, is however well conserved among the TR isoforms. Yeast interaction data suggests that the Ubc9 interaction motif that is exclusive to the beta1 A/B domain may confer an isoform-specific function to TRbeta1. Ubc9 functions primarily to covalently attach its sole substrate, the small ubiquitin-like modifier (SUMO), to the lysine that lies within Ubc9 binding motif. Results from this study show that TRbeta1 is SUMO modified in mammalian cells in the absence of T3-binding, and suggests a possible role for SUMO modification in TR stability. It was also shown that Ubc9 overexpression enhances TRbeta1 T3-mediated transcriptional activity and that it requires Ubc9 enzymatic activity; however SUMO modification of TRbeta1 is not a requirement. Ubc9 siRNA knockdown results confirmed the coactivator-like function of Ubc9 in T3-dependent transcriptional activity. Collectively, the results from these studies indicate that Ubc9 contributes to the regulation of TRbeta1 function.