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Coactivators constitute a diverse group of proteins that are essential for optimal transcriptional activity of nuclear receptors. In the past few years many coactivators have been identified but it is still unclear whether these proteins interact indiscriminately with all nuclear receptors and whether there is some redundancy in their functions. We have previously cloned and characterized RAP250 (ASC-2/PRIP/TRBP/NRC), an LXXLLcontaining coactivator for nuclear receptors. In order to study its biological role, Rap250 null mice were generated by gene targeting. Here we show that genetic disruption of Rap250 results in embryonic lethality at embryonic day (E) 13.5. Histological examination of placentas revealed a dramatically reduced spongiotrophoblast layer, a collapse of blood vessels in the region bordering the spongiotrophoblast, and labyrinthine layers in placentas from Rap250 ؊/؊ embryos. These findings suggest that the lethality of Rap250 ؊/؊ embryos is the result of obstructed placental blood circulation. Moreover, the transcriptional activity of PPAR␥ is reduced in fibroblasts derived from Rap250 ؊/؊ embryos, suggesting that RAP250 is an essential coactivator for this nuclear receptor in the placenta. Our results demonstrate that RAP250 is necessary for placental development and thus essential for embryonic development.Nuclear receptors comprise a family of transcription factors that regulate gene expression in a ligand-dependent manner. Upon ligand binding these proteins activate transcription of specific genes involved in the control of diverse physiological processes, such as cellular growth, development, differentiation, and homeostasis (20). Binding of ligands to the ligandbinding domain of nuclear receptors causes conformational changes of the receptor, enabling the recruitment of coactivators harboring LXXLL motifs (see below). A number of coactivators for nuclear receptors have been identified (for a review, see reference 6). Among the most studied is the steroid receptor coactivator 1 (SRC-1) family, which contains three related coactivators, referred to as SRC-1, SRC-2/GRIP1/ TIF2, and SRC-3/p/CIP/RAC3/ACTR/AIB1/TRAM1 (1,4,7,17,23,28,30,33). These factors, together with CBP/p300 and pCAF, bind nuclear receptors and gain access to target promoter regions through histone acetyltransferase-mediated nucleosome remodeling. Another class of coactivators is represented by the TRAP/DRIP/ARC multiprotein complex (5, 22, 26), with one major nuclear receptor interacting subunit, PBP/ TRAP220/DRIP205 (25,31,41,45), that appears to act independently of histone acetyltransferase activity.Nuclear receptor-activating protein 250 (RAP250) (3), also called ASC-2, PRIP, TRBP, and NRC (13,15,19,42), was recently cloned and described as a novel nuclear receptor coactivator which interacts with the ligand-binding domain of ligand-bound nuclear receptors. The interaction is mediated through a short hydrophobic motif called the LXXLL motif (or NR box) which is found in most nuclear receptor coactivators. In contrast to ...

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.

Proteins belonging to the 14--3-3 family interact with various regulatory proteins involved in cellular signaling, cell cycle regulation, or apoptosis. 14--3-3 proteins have been suggested to act by regulating the cytoplasmic/nuclear localization of their target proteins or by acting as molecular scaffolds or chaperones. We have previously shown that overexpression of 14--3-3 enhances the transcriptional activity of the glucocorticoid receptor (GR), which is a member of the nuclear receptor family. In this study, we show that 14--3-3 interacts with the nuclear receptor corepressor RIP140. In transfection assays, RIP140 antagonizes 14--3-3- enhanced GR transactivation. Using colocalization studies we demonstrate that 14--3-3 can export RIP140 out of the nucleus and, interestingly, can also change its intranuclear localization. Moreover, we also observed that 14--3-3 can bind various other nuclear receptors and cofactors. In summary, our findings suggest that 14--3-3-mediated intracellular relocalization of the GR corepressor RIP140 might be a novel mechanism to enhance glucocorticoid responsiveness of target genes. They furthermore indicate a more general role for 14--3-3 protein by influencing the nuclear availability of nuclear receptor-associated cofactors.

DAX-1 (NROB1) is an atypical member of the nuclear receptor family that is predominantly expressed in mammalian reproductive tissues. While a receptor function of DAX-1 remains enigmatic, previous work has indicated that DAX-1 inhibits the activity of the orphan receptor steroidogenic factor 1 and the estrogen receptors (ERs), presumably via direct occupation of the coactivator-binding surface and subsequent recruitment of additional corepressors. In vivo evidence points at a particular role of DAX-1 for the development and maintenance of male reproductive functions. In this study, we have identified the androgen receptor (AR) NR3C4 as a novel target for DAX-1. We show that DAX-1 potently inhibits ligand-dependent transcriptional activation as well as the interaction between the N- and C-terminal activation domains of AR. We provide evidence for direct interactions of the two receptors that involve the N-terminal repeat domain of DAX-1 and the C-terminal ligand-binding and activation domain of AR. Moreover, DAX-1, known to shuttle between the cytoplasm and the nucleus, is capable of relocalizing AR in both cellular compartments, suggesting that intracellular tethering is associated with DAX-1 inhibition. These results implicate novel inhibitory mechanisms of DAX-1 action with particular relevance for the modulation of androgen-dependent gene transcription in the male reproductive system.

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