Modulation of nuclear receptor activity by a unique class of corepressors

Abstract

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 step-by-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.