The thyroid-hormone receptors are hormone-dependent transcription factors that control expression of many target genes. This regulation is presumably a consequence of hormone-dependent contacts between the receptors and the basal transcription machinery. We used the yeast two-hybrid system to identify a candidate human transcriptional mediator that interacts with both the thyroid-hormone receptor and the retinoid-X receptor in a ligand-dependent fashion. This protein, Trip1 (for thyroid-hormone-receptor interacting protein), shares striking sequence conservation with the yeast transcriptional mediator Sug1 (refs 6, 7). Here we show that Trip1 can functionally substitute for Sug1 in yeast, and that both proteins interact in vitro with the thyroid-hormone receptor, and with the transcriptional activation domains of yeast GAL4 and of herpes virus VP16.

BackgroundS1PL is an aldehyde-lyase that irreversibly cleaves sphingosine 1-phosphate (S1P) in the terminal step of sphingolipid catabolism. Because S1P modulates a wide range of physiological processes, its concentration must be tightly regulated within both intracellular and extracellular environments.MethodologyIn order to better understand the function of S1PL in this regulatory pathway, we assessed the in vivo effects of different levels of S1PL activity using knockout (KO) and humanized mouse models.Principal FindingsOur analysis showed that all S1PL-deficient genetic models in this study displayed lymphopenia, with sequestration of mature T cells in the thymus and lymph nodes. In addition to the lymphoid phenotypes, S1PL KO mice (S1PL−/−) also developed myeloid cell hyperplasia and significant lesions in the lung, heart, urinary tract, and bone, and had a markedly reduced life span. The humanized knock-in mice harboring one allele (S1PLH/−) or two alleles (S1PLH/H) of human S1PL expressed less than 10 and 20% of normal S1PL activity, respectively. This partial restoration of S1PL activity was sufficient to fully protect both humanized mouse lines from the lethal non-lymphoid lesions that developed in S1PL−/− mice, but failed to restore normal T-cell development and trafficking. Detailed analysis of T-cell compartments indicated that complete absence of S1PL affected both maturation/development and egress of mature T cells from the thymus, whereas low level S1PL activity affected T-cell egress more than differentiation.SignificanceThese findings demonstrate that lymphocyte trafficking is particularly sensitive to variations in S1PL activity and suggest that there is a window in which partial inhibition of S1PL could produce therapeutic levels of immunosuppression without causing clinically significant S1P-related lesions in non-lymphoid target organs.

The acidic transcriptional activation motif functions in all eukaryotes, which suggests that it makes contact with some universal component of the transcriptional apparatus. Transcriptional activation by the yeast regulatory protein GAL4 requires an acidic region at its carboxyl terminus. Here we implement a selection scheme to determine whether GAL4 can still function when this C-terminal domain has been deleted. It can, when accompanied by a mutation in the SUG1 gene which is an essential gene in yeast. Analysis of mutant SUG1 in combination with various alleles of GAL4 indicates that SUG1 acts through a transcriptional pathway that depends on GAL4, but requires a region of GAL4 other than the C-terminal acidic activation domain. The predicted amino-acid sequence of SUG1 closely resembles that of two human proteins, TBP1 and MSS1, which modulate expression mediated by the human immunodeficiency virus tat gene.

Biochemical and genetic studies suggest the existence of mediators that work between the activation domains (ADs) of regulatory proteins and the basic transcriptional machinery. We have previously shown genetically that Sug1 interacts with the AD of the yeast activator Ga14. Here we provide evidence that the Sug1 protein of yeast binds directly to the ADs of Ga14 and the viral activator, VP16. Sug1 protein is associated with the TATA-binding protein in vivo and binds to it in vitro, consistent with a mediator function. We also demonstrate that Sug1 is not a component of the 26S proteasome, contrary to previous reports. Sug1 is a member of a large, highly conserved family of ATPases, implying a role for ATP hydrolysis in the activation of transcription.

During nearly a decade of research dedicated to the study of sphingosine signaling pathways, we identified sphingosine-1-phosphate lyase (S1PL) as a drug target for the treatment of autoimmune disorders. S1PL catalyzes the irreversible decomposition of sphingosine-1-phosphate (S1P) by a retro-aldol fragmentation that yields hexadecanaldehyde and phosphoethanolamine. Genetic models demonstrated that mice expressing reduced S1PL activity had decreased numbers of circulating lymphocytes due to altered lymphocyte trafficking, which prevented disease development in multiple models of autoimmune disease. Mechanistic studies of lymphoid tissue following oral administration of 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI) 3 showed a clear relationship between reduced lyase activity, elevated S1P levels, and lower levels of circulating lymphocytes. Our internal medicinal chemistry efforts discovered potent analogues of 3 bearing heterocycles as chemical equivalents of the pendant carbonyl present in the parent structure. Reduction of S1PL activity by oral administration of these analogues recapitulated the phenotype of mice with genetically reduced S1PL expression.