MECHANISMS OF THE MOUSE ORPHAN NUCLEAR RECEPTOR TR2-11-MEDIATED GENE SUPPRESSION

The mouse orphan nuclear receptor TR2-11 functions as a repressor for reporter genes containing a direct repeat-5 or direct repeat-4 hormone response element. The functional domains responsible for its suppress ive activity are defined, including the DNA-binding domain and the lig and-binding domain. The C-terminal 30 amino acid residues can be delet ed without compromising its suppressive activity, whereas a deletion f or 40 amino acids completely abolishes the suppressive activity and re ceptor dimerization, and reduces the DNA binding affinity. Point mutat ion at three conserved leucine residues located on the predicted dimer interface abolishes the suppressive activity, receptor dimerization a nd its DNA binding property. However, mutation at two consecutive glut amate residues located within the hinge between the last two helices o f the ligand-binding domain (helix 10 and helix 11 according to the hu man retinoid receptor X alpha structure) drastically reduces its DNA b inding affinity and abrogates the suppressive activity without comprom ising its ability to dimerize, indicating that receptor dimerization p roperty can be functionally uncoupled from its suppressive activity. A transferable, active silencing activity is encoded within the DEF seg ment of the receptor molecule, as evidenced by the suppression of a GA L4 reporter by a chimeric protein containing the DNA-binding domain of GAL4 and the DEF segment of TR2-11, Moreover, the C-terminal 49 amino acid sequence is required for this trans-suppressive activity. It is suggested that TR2-11 functions as a repressor, mediated by mechanisms requiring high affinity DNA binding, receptor dimerization, and activ e silencing.