Thyroid hormone response element sequence and the recruitment of retinoid X receptors for thyroid hormone responsiveness

Thyroid hormone receptors (TRs) are transcription factors that bind to thyr oid hormone response elements (TREs) in the regulatory regions of target ge nes. TRs are thought to activate transcription primarily as heterodimers wi th retinoid X receptors (RXRs), with RXR binding upstream to the two direct ly repeated half-sites in a typical TRE. However, given that TRs and RXRs p refer to bind to different DNA sequences (T(A/G)AGGTCA and GGGGTCA), we pos tulate that only certain TREs require RXR-TR heterodimerization, depending on the TRE sequence. We have tested this hypothesis by comparing in Sacchar omyces cerevisiae the functional activity of TR +/- RXR on 10 naturally occ urring mammalian TREs. S. cerevisiae was used as a model system because yea st lack endogenous nuclear receptors and thus can be manipulated to express TRs and/or RXRs. We first studied ligand-independent reporter gene activat ion, which reflects the activity of the activator function 1 (AF-1) domain. The 10 TREs formed a continuous spectrum from being fully dependent on RXR for TR AF-1 activity to being essentially independent of RXR, Relative ind ependence of RXR generally was seen when the TRE upstream half-site has a T A or TG 5' to the core hexamer, Gel mobility shift assays revealed that fun ctional independence of RXR correlates with the strong binding of TR alone, whereas more RXR dependence correlates with higher binding of RXR-TR heter odimers. Restoration of ligand-dependent (AF-2 domain) reporter gene activa tion was achieved by expression of the coactivator TIF2. This ligand-induce d stimulation was stronger in the presence of TR alone than with RXR plus T R, suggesting a preference for TIF2 activation of TR homodimers. Overall th e data support the notion that the TRE sequence plays an important role in determining the nuclear hormone receptor and coactivator requirements for T R action.