TRANSCRIPTIONAL SILENCING BY UNLIGANDED THYROID-HORMONE RECEPTOR-BETAREQUIRES A SOLUBLE COREPRESSOR THAT INTERACTS WITH THE LIGAND-BINDINGDOMAIN OF THE RECEPTOR

Unliganded thyroid hormone receptor (TR) functions as a transcriptiona l repressor of genes bearing thyroid hormone response elements in thei r promoters, Binding of hormonal ligand to the receptor releases the t ranscriptional silencing and leads to gene activation, Previous studie s showed that the silencing activity of TR is located within the C-ter minal ligand-binding domain (LED) of the receptor, To dissect the role of the LED in receptor-mediated silencing, we used a cell-free transc ription system containing HeLa nuclear extracts in which exogenously a dded unliganded TR beta repressed the basal level of RNA polymerase II -driven transcription from a thyroid hormone response element-linked t emplate, We designed competition experiments with a peptide fragment c ontaining the entire LED (positions 145 to 456) of TR beta. This pepti de, which lacks the DNA-binding domain, did not affect basal RNA synth esis from the thyroid hormone response element-linked promoter when ad ded to a cell-free transcription reaction mixture, However, the additi on of the LED peptide to a reaction mixture containing TR beta led to a complete reversal of receptor-mediated transcriptional silencing in the absence of thyroid hormone, An LED peptide harboring point mutatio ns, which severely impair receptor dimerization, also inhibited effici ently the silencing activity of TR, indicating that the relief of repr ession by the LED was not due to the sequestration of TR or its hetero dimeric partner retinoid X receptor into inactive homo- or heterodimer s. We postulate that the LED peptide competed with TR for a regulatory molecule, termed a corepressor, that exists in the HeLa nuclear extra cts and is essential for efficient receptor-mediated gene repression, We have identified the region from positions 145 to 260 (the D domain) of the LED as a potential binding site of the putative corepressor, W e observed further that a peptide containing the LED of retinoic acid receptor (RAR) competed for TR-mediated silencing, suggesting that the RAR LED may bind to the same corepressor activity as the TR LED. Inte restingly, the RAR LED complexed with its cognate ligand, all-trans re tinoic acid, failed to compete for transcriptional silencing by TR bet a, indicating that the association of the LED with the corepressor is ligand dependent. Finally, we provide strong biochemical evidence supp orting the existence of the corepressor activity in the HeLa nuclear e xtracts. Our studies demonstrated that the silencing activity of TR wa s greatly reduced in the nuclear extracts preincubated with immobilize d, hormone-free glutathione S-transferase-LBD fusion proteins, indicat ing that the corepressor activity was depleted from these extracts thr ough protein-protein interactions with the LED. Similar treatment with immobilized, hormone-bound glutathione S-transferase-LBD, on the othe r hand, failed to deplete the corepressor activity from the nuclear ex tracts: indicating that ligand binding to the LED disrupts its interac tion with the corepressor, From these results, we propose that a corep ressor binds to the LED of unliganded TR and critically influences the interaction of the receptor with the basal transcription machinery to promote silencing, Ligand binding to TR results in the release of the corepressor from the LED and triggers the reversal of silencing by al lowing the events leading to gene activation to proceed.