Structural analysis of nuclear receptor subfamily V orphan nuclear receptors suggests that ligand-independent mechanisms must regulate this subclass of receptors. Here, we report that steroidogenic factor 1 (SF-1) and liver receptor homolog 1 are repressed via posttranslational SUMO modification at conserved lysines within the hinge domain. Indeed, mutating these lysines or adding the SUMO isopeptidase SENP1 dramatically increased both native and Gal4-chimera receptor activities. The mechanism by which SUMO conjugation attenuates SF-1 activity was found to be largely histone deacetylase independent and was unaffected by the AF2 corepressor Dax1. Instead, our data suggest that SUMO-mediated repression involves direct interaction of the DEAD-box protein DP103 with sumoylated SF-1. Of potential E3-SUMO ligase candidates, PIASy and PIASx␣ strongly promoted SF-1 sumoylation, and addition of DP103 enhanced both PIAS-dependent receptor sumoylation and SF-1 relocalization to discrete nuclear bodies. Taken together, we propose that DEAD-box RNA helicases are directly coupled to transcriptional repression by protein sumoylation.Steroidogenic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1) are two closely related transcription factors belonging to the nuclear receptor subfamily V (NR5A) that contain a highly conserved DNA binding domain (DBD), a large hinge domain and a ligand binding domain (LBD) (Fig. 1A). Drosophila melanogaster Ftz-F1 is the founding member of this subfamily and interacts directly with the pair-rule gene product of Ftz to control parasegmentation at early embryonic stages (25). The mammalian orthologs SF-1 and LRH-1 are also critical in tissue development and organogenesis (19,27,33). During development, SF-1 is essential for male differentiation, adrenogonadal morphogenesis, and terminal differentiation of the ventromedial hypothalamus, and in the adult, this receptor regulates genes involved in steroid biosynthesis and endocrine signaling (34,44). Although SF-1 null mice die at birth from adrenal failure, SF-1 heterozygous mice live. However, further analyses of these heterozygous mice show that despite seemingly adequate levels of SF-1, the amount of active SF-1 protein is insufficient to overcome defects in adrenal morphogenesis (2, 3). In humans, SF-1 haploinsufficiency is associated with severe adrenal disease and gonadal dysgenesis (1, 28). LRH-1 acts far earlier in development than SF-1, as evidenced by the embryonic lethality observed in LRH-1 null embryos (33). In vitro and in vivo analyses have implicated LRH-1 in bile acid homeostasis (13,26), where a heterozygous phenotype has also emerged in the intestine (4). In addition, LRH-1 controls tissue conversion of androgens to estrogen by regulating aromatase gene expression (7,17) Despite the fact that the high-resolution crystal structure of LRH-1 revealed a large hydrophobic pocket within the LBD (38), natural ligands have yet to emerge for this subclass of receptors. As such, the question of how subfamily V receptors are regulated is u...

We have previously shown that activation of glucocorticoid receptor (GR) signaling in stressed cells will cause inhibition of the heat shock response as mediated by heat shock transcription factor 1 (HSF1). In that work, a full-length human heat shock protein 70 (Hsp70) promoter was used to measure HSF1 transactivity, and the data suggested inhibition of HSF1 through the transactivation or transrepressive properties of GR. Here, we show that the inhibitory effect of glucocorticoid agonist (dexamethasone) upon Hsp70 promoter activity is rapid, occurring within 1 h of hormone addition. Moreover, addition of hormone during the first hour of recovery from stress was sufficient to inhibit HSF1. Thus, dexamethasone is able to rapidly reverse HSF1 transactivity, suggesting a transrepressive mode of action for GR. Yet, GR transrepression of HSF1 by analysis of putative negative glucocorticoid response elements in the Hsp70 promoter was not found. To further investigate the in vivo nature of this fast-acting mechanism, we used the chromatin immunoprecipitation assay with primers specific to the human Hsp70 promoter. Dexamethasone inhibited HSF1 binding at the Hsp70 promoter in response to heat or chemical shock (sodium arsenite). Moreover, dexamethasone also blocked promoter binding by a constitutively active mutant of HSF1 (hHSF1-E189) expressed under nonstress conditions. In all cases, inhibition of HSF1 recruitment to the promoter by dexamethasone was blocked by the GR antagonist RU486, a result that was consistent with promoter activity based on chloramphenicol acetyl transferase gene expression. The ability of dexamethasone to prevent HSF1 recruitment to the promoter was fast acting (occurring in as little as 15 min), and the hormone also caused release of HSF1 already bound to the promoter. Although these results suggest GR can effectively prevent HSF1 binding to Hsp promoters, fractionation and Western blot experiments showed that stress-activated HSF1 was not released from the nucleus in response to hormone. Thus, this effect of dexamethasone is either specific to the Hsp70 promoter or causes shunting of HSF1 to other high-affinity nuclear sites. These observations provide evidence of a novel mechanism for attenuation of the heat shock response by glucocorticoids: prevention or reversal of HSF1 recruitment to Hsp promoters through the rapid actions of GR.

The GR is a hormone-activated transcription factor that acts to regulate specific gene expression. In the absence of hormone, the GR and other steroid receptors have been shown to form complexes with several mammalian heat shock proteins. As heat shock proteins are produced by cells as an adaptive response to stress, speculation has existed that communication between the heat shock and glucocorticoid hormone signal pathways must exist. Only recently has evidence to support this hypothesis been reported. In almost all cases, the evidence has been of an ability of heat shock to cause a potentiation of the glucocorticoid hormone response. In this proposal, evidence is now presented that heat shock signaling can, in turn, be regulated by glucocorticoids. In mouse L929 cells stably expressing a chloramphenicol acetyltransferase reporter controlled by the human heat shock protein70 promoter and containing known binding sites for heat shock transcription factor 1 treatment with glucocorticoid agonist (dexamethasone) results in a dose-dependent decrease of stress-induced chloramphenicol acetyltransferase gene expression. In these cells, inhibition of heat shock protein70 promoter activity by dexamethasone was completely blocked by GR antagonist (RU486). Similar treatment of L929 cells stably expressing a chloramphenicol acetyltransferase reporter under the control of the constitutively active SV40 promoter showed no such inhibition by dexamethasone. More importantly, dexamethasone was also found to inhibit heat shock-induced expression of the major heat shock proteins-heat shock proteins70, 90, and 110. Thus, the inhibitory effect of dexamethasone appears to apply to most, if not all, heat shock transcription factor 1-regulated genes. Although dexamethasone did not prevent the DNA-binding function of heat shock-activated heat shock transcription factor 1, it did inhibit a constitutively active mutant of human heat shock transcription factor 1 under nonstress conditions, suggesting that dexamethasone repression of heat shock transcription factor 1 was primarily through an inhibition of heat shock transcription factor 1 transcription enhancement activity. To more accurately characterize the stage of GR signaling responsible for inhibition of heat shock transcription factor 1, a series of Chinese hamster ovary cells containing either no GR, wild-type mouse GR, or single-point mutations of GR were employed. Dexamethasone inhibition of heat shock-induced heat shock transcription factor 1 activity was observed in the presence of wild-type GR, but not in Chinese hamster ovary cells lacking GR, suggesting that signaling cascades other than GR were not involved in this effect of dexamethasone. Consistent with this conclusion was the observation that dexamethasone had no effect on activity of the MAPKs (ERK1, ERK2, or c-jun N-terminal kinase), which are known to negatively regulate heat shock transcription factor 1. Dexamethasone inhibition of heat shock transcription factor 1 was not seen in Chinese hamster ovary cells expressing...

To further define the role of heat shock factor 1 (HSF1) in the stress potentiation of glucocorticoid receptor (GR) activity, we placed a constitutively active mutant of human HSF1 (hHSF1-E189) under the control of a doxycycline (DOX)-inducible vector. In mouse L929 cells, DOX-induced expression of hHSF1-E189 correlated with in vivo occupancy of the human heat shock protein 70 (hHsp70) promoter (chromatin-immunoprecipitation assay) and with increased activity under nonstress conditions at the hHsp70 promoter controlling expression of chloramphenicol acetyl transferase (CAT) (p2500-CAT). Comparison of hHSF1-E189 against stress-activated, endogenous HSF1 for DNA-binding, p2500-CAT, and Hsp70 protein expression activities showed the mutant factor to have lower, but clearly detectable, activities as compared with wild-type factor. Thus, the hHSF1-E189 mutant is capable of replicating these key functions of endogenous HSF1, albeit at reduced levels. To assess the involvement of hHSF1-E189 in GR activity, DOX-induced expression of hHSF1-E189 was performed in L929 cells expressing the minimal pGRE(2)E1B-CAT reporter. hHSF1-E189 protein expression in these cells was maximal at 24 h of DOX and remained constant up to 72 h. hHSF1-E189 expressed under these conditions was found both in the cytosolic and nuclear compartments, in a state capable of binding DNA. More importantly, GR activity at the pGRE(2)E1B-CAT promoter was found to increase after DOX-induced expression of hHSF1-E189. The potentiation of GR by hHSF1-E189 occurred at saturating concentrations of hormone and was dependent on at least 48 h of hHSF1-E189 up-regulation, suggesting that time was needed for an HSF1-induced factor to accumulate to a threshold level. Initial efforts to characterize how hHSF1-E189 controls GR signaling showed that it does not occur through alterations of GR protein levels or changes in GR hormone binding capacity. In summary, our observations provide the first molecular evidence for the existence of HSF1-regulated genes that serve to elevate the response of steroid receptors under stress conditions.

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