Modulation of nuclear receptor activity by a unique class of corepressors

Nuclear receptors comprise a unique family of transcription factors that are crucial regulators of gene expression involved in diverse physiological and developmental processes. The nuclear receptor field has undergone a very rapid development during the recent two decades due to the recognition of the importance of nuclear receptors as intracellular signal transmitters and pharmaceutical targets. Today we know that nuclear receptors are not only regulated by smallmolecule ligands, but also by posttranslational modifications and coregulatory proteins. Coregulators, which participate in nuclear receptor dependent transcriptional regulation either as coactivators or corepressors, represent a particularly exciting area of current research. First, they may mediate both ligand-selective and ligand-independent signaling pathways, and second, they may determine developmental and cell-type specific activities of nuclear receptors. Central to the classical view of nuclear receptor activation is the role of ligand-dependent conformational changes to induce the release of corepressors and to induce the binding of coactivators in a stepby- step model. While corepressors are suggested to bind to nuclear receptors only in the absence of ligand or in the presence of antagonists, coactivators are thought to bind receptors in their active conformation in the presence of agonists. However, this classical view could be incomplete, as various potential corepressors have been identified that apparently bind nuclear receptors in an agonist-dependent manner. Furthermore, not all nuclear receptors appear to be regulated by ligands, and there are a number of orphan receptors that appear to be constitutively active. Finally, possible mechanisms of transcription attenuation or repression subsequent to activation have not been considered. In this thesis we have studied three members of a unique class of corepressors that associate with nuclear receptors in their “active” conformation. We refer to these corepressors as corepressors class II (CoRs II) to distinguish them from classical corepressors (corepressors class I (CoRs I)). One aim has been to identify mechanisms of CoRs II action leading to repression of nuclear receptor activity. This resulted not only in discovery of novel regulatory pathways but also in identification of the homeobox protein Prox-1 as a CoR II for LRH-1, a ligand-independent orphan member of the nuclear receptor family. We also studied RIP140, which represents the first identified CoR II, and elucidated a unique mechanism of regulating the intracellular localization of this corepressor. A substantial part of this thesis was endeavored to understand the intracellular regulation of DAX-1. This atypical orphan receptor is known to have important functions in steroidogenesis, and mutations in DAX-1 are associated with a human disorder called adrenal hypoplasia congenita. Although these mutations cause altered DAX-1 localization, currently little is known about the regulation of DAX-1 shuttling. We describe here for the first time a novel protein, ZIBRA, that influences both the intracellular localization and the stability of DAX-1 and that creates a link between CoRs II and the ubiquitin system. In addition we identify the androgen receptor as a novel target receptor for DAX-1 and suggest that repression of the activity of this receptor can be achieved through both non-transcriptional and transcriptional pathways. Although RIP140, Prox-1 and DAX-1 have a very different structure, several common features are found in their mode of action. Most importantly, they bind to nuclear receptors via LXXLL-motifs, which originally were characterized as a binding motif typical for coactivators. We suggest that this mechanism may be particularly important in modulating the activity of constitutively “active” orphan receptors, independent of ligands. In conclusion, our results reveal novel aspects of modulating nuclear receptor activity, many of them pertinent to the intracellular distribution of both receptors and coregulators, allowing us to revise the classical view and to propose an extended model for nuclear receptor repression by integrating the action of class II corepressors

Vers l'instant (Portrait d'un inconnu de Nathalie Sarraute)

CAEN-BU Droit Lettres (141182101) / SudocSudocFranceF

Functional conservation of interactions between a homeodomain cofactor and a mammalian FTZ-F1 homologue

Nuclear receptors are master regulators of metazoan gene expression with crucial roles during development and in adult physiology. Fushi tarazu factor 1 (FTZ-F1) subfamily members are ancient orphan receptors with homologues from Drosophila to human that regulate diverse gene expression programs important for developmental processes, reproduction and cholesterol homeostasis in an apparently ligand-independent manner. Thus, developmental and tissue-specific cofactors may be particularly important in modulating the transcriptional activities of FTZ-F1 receptors. In Drosophila, the homeodomain protein Fushi tarazu acts as a cofactor for FTZ-F1 (NR5A3), leading to the hypothesis that a similar type of homeodomain cofactor–nuclear receptor relationship might exist in vertebrates. In this study, we have identified and characterized the homeodomain protein Prox1 as a co-repressor for liver receptor homologue 1 (LRH1/NR5A2), a master regulator of cholesterol homeostasis in mammals. Our study suggests that interactions between LRH1 and Prox1 may fulfil roles both during development of the enterohepatic system and in adult physiology of the liver

E3 Ubiquitin Ligase RNF31 Cooperates with DAX-1 in Transcriptional Repression of Steroidogenesis▿ †

Genetic and experimental evidence points to a critical involvement of the atypical mammalian orphan receptor DAX-1 in reproductive development and steroidogenesis. Unlike conventional nuclear receptors, DAX-1 appears not to function as a DNA-bound transcription factor. Instead, it has acquired the capability to act as a transcriptional corepressor of steroidogenic factor 1 (SF-1). The interplay of DAX-1 and SF-1 is considered a central, presumably ligand-independent element of adrenogonadal development and function that requires tight regulation. This raises a substantial interest in identifying its modulators and the regulatory signals involved. Here, we uncover molecular mechanisms that link DAX-1 to the ubiquitin modification system via functional interaction with the E3 ubiquitin ligase RNF31. We demonstrate that RNF31 is coexpressed with DAX-1 in steroidogenic tissues and participates in repressing steroidogenic gene expression. We provide evidence for the in vivo existence of a corepressor complex containing RNF31 and DAX-1 at the promoters of the StAR and CYP19 genes. Our data suggest that RNF31 functions to stabilize DAX-1, which might be linked to DAX-1 monoubiquitination. In conclusion, RNF31 appears to be required for DAX-1 to repress transcription, provides means to regulate DAX-1 in ligand-independent ways, and emerges as a relevant coregulator of steroidogenic pathways governing physiology and disease

Nuclear Receptor Liver X Receptor Is O-GlcNAc-modified in Response to Glucose*

Post-translational modification of nucleocytoplasmic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has for the last 25 years emerged as an essential glucose-sensing mechanism. The liver X receptors (LXRs) function as nutritional sensors for cholesterol-regulating lipid metabolism, glucose homeostasis, and inflammation. LXRs are shown to be post-translationally modified by phosphorylation, acetylation, and sumoylation, affecting their target gene specificity, stability, and transactivating and transrepressional activity, respectively. In the present study, we show for the first time that LXRα and LXRβ are targets for glucose-hexosamine-derived O-GlcNAc modification in human Huh7 cells. Furthermore, we observed increased hepatic LXRα O-GlcNAcylation in vivo in refed mice and in streptozotocin-induced refed diabetic mice. Importantly, induction of LXRα O-GlcNAcylation in both mouse models was concomitant with increased expression of the lipogenic gene SREBP-1c (sterol regulatory element-binding protein 1c). Furthermore, glucose increased LXR/retinoic acid receptor-dependent activation of luciferase reporter activity driven by the mouse SREBP-1c promoter via the hexosamine biosynthetic pathway in Huh7 cells. Altogether, our results suggest that O-GlcNAcylation of LXR is a novel mechanism by which LXR acts as a glucose sensor affecting LXR-dependent gene expression, substantiating the crucial role of LXR as a nutritional sensor in lipid and glucose metabolism