TRH is the principal positive regulator of TSH synthesis and secretion in man. T3 is able to control TRH synthesis through feedback inhibition at the transcriptional level, presumably by binding to its receptor which interacts with one or more negative thyroid hormone response elements (TREs) present within the human TRH promoter. In the present study we have identified the specific negative TREs within the TRH promoter and characterized their ability to interact with thyroid hormone receptors (TRs), and the retinoid X receptor (RXR). Our analysis demonstrates that ligand-independent and dependent regulation of the human TRH promoter is restricted to the TR beta 1 isoform. Deletional analysis of the TRH promoter identified two discrete regions that are responsible for mediating ligand-dependent negative regulation of the TRH promoter. Mutagenesis of potential TR binding half-sites within these regions identified three separate half-sites (site 4 from -55 to -60 base pairs (bp); site 5, +14 to +19 bp; and site 6, +37 to +42 bp) which act in combination to allow for negative regulation. Mutation and/or deletion of each of these sites leads to a loss of negative regulation of the TRH promoter by T3. Gel-mobility shift assays of site 4 and its surrounding nucleotides revealed that this region of the promoter is capable of binding TR monomers, homodimers, and TR-RXR heterodimers. Mutagenesis of site 4 leads to a loss of all binding to this region. The region encompassing sites 5 and 6 binds only TR monomer, and the addition of RXR to the binding reaction leads to a loss of specific monomeric binding. To assess the functional importance of site 4 and its surrounding nucleotides we cotransfected RXR isoforms along with TR beta with TRH promoter constructs containing either site 4 or its mutant. In the presence of wild type site 4 sequence, cotransfected RXR enhanced negative regulation of the TRH promoter. Mutation and or deletion of site 4 leads to a loss of this enhancement. These data demonstrate that two structurally different negative TREs cooperate to allow for negative regulation of the human TRH promoter and that negative regulation is TR isoform-specific and modulated by the RXR-signaling pathway through a novel negative TRE.

Cryptococcus neoformans produces large amounts of the acyclic hexitol mannitol in culture and infected animals, but the functional and pathogenic significance of mannitol production by this fungus is not known. We exposed C. neoformans H99 (Cn H99) to UV irradiation (1 x LD, , ) and screened survivors for mannitol production. A mutant, Cn MLP (Mannitol Low Producer), synthesized less mannitol from glucose (2.7 vs 8-2 nmol per lo8 cells min-' a t 37 "C) and contained less intracellular mannitol (1 vs 11 pmol per lo6 cells a t 37 "C) than did Cn H99. Cn MLP and Cn H99 were similar with respect to carbon assimilation patterns, rates of glucose consumption, growth rates a t 30 "C, urease and phenoloxidase activities, morphology, capsule formation, mating type, electrophoretic karyotype, rapid amplification of polymorphic DNA (RAPD) patterns and antifungal susceptibility. However, Cn MLP was more susceptible than was Cn H99 to growth inhibition and killing by heat and high NaCl concentrations. Also, the LD, , values in mice injected intravenously were 3.7 x lo6 c.f.u. for Cn MLP compared to 6 9 x lo2 c.f.u. for Cn H99. Moreover, 500 c.f.u. Cn H99 intravenously killed 12 of 12 mice by 60 d, whereas all mice given the same inoculum of Cn MLP survived. Classical genetic studies were undertaken to determine if these differences were due to a single mutation, but the basidiospores were nonviable. These results suggest that the abilities of C. neoformans to produce and accumulate mannitol may influence its tolerance to heat and osmotic stresses and its pathogenicity in mice.

The role of retinoid X receptors (RXRs) on negative thyroid hormone response elements (nTREs) is not well understood. In this report, we demonstrate that an orientation-specific monomeric thyroid hormone receptor (T3R) DNA-binding site mediates thyroid hormone inhibition in the thyrotropin beta subunit gene (TSH-beta) from human and murine species. Unlike positive TREs, addition of the ligand 9-cis retinoic acid (9-cis RA) to cells transfected with a T3R beta 1 expression vector significantly reduces thyroid hormone inhibition of the TSH-beta gene, indicating that endogenous retinoid receptors antagonize T3R function. Cotransfection of an RXR-alpha but not a retinoic acid receptor-alpha expression vector further antagonizes thyroid hormone inhibition, but only in the presence of 9-cis RA. Antagonism by RXR requires both an intact DNA- and ligand-binding domain. Removal of monomeric T3R binding to the TSH-beta nTRE also requires both RXR domains. A model is proposed whereby monomeric T3R is removed from a nTRE by RXR occupied by its ligand 9-cis RA. This is the first report of 9-cis RA-dependent antagonism of thyroid hormone inhibition via negative TREs.

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