The retinoic acid receptor-beta 2 contains two separate cell-specific transactivation domains, at the N-terminus and in the ligand-binding domain.

Expression of retinoic acid receptor ␤ (RAR␤) is spatially and temporally restricted during embryonal development. Also during retinoic acid (RA)-dependent embryonal carcinoma (EC) cell differentiation, RAR␤ expression is initially up-regulated, while in later phases of differentiation expression is down-regulated, by an unknown mechanism. To gain insight into the regulation of RAR␤, we studied the activity of the RAR␤2 promoter and mutants thereof in various cell lines. While the RAR␤2 promoter is activated by RA in a limited number of cell lines, synthetic RA-responsive reporters are activated in most cell types. We show that the expression levels of proteins that bind to the ␤-retinoic acid response element (RAR/retinoid X receptors and orphan receptors) and also the differential expression of a number of coactivators modulate the RA response on both natural and synthetic reporters. We further show that cell type-specific activation of the RAR␤2 promoter is dependent on the promoter architecture including the spacing between retinoic acid response element and TATA-box and initiator sequence (␤INR). Mutation within these regions caused a decrease in the activity of this promoter in responsive EC cells, while an increase in activity in non-EC cell lines was observed. Cell-specific complexes were formed on the ␤INR, suggesting that the ␤INR contributes to cell-specific activation of the promoter. On this basis we propose that promoter context-dependent and more general RA response-determining mechanisms contribute to cellspecific RA-dependent activation of transcription.

Section: Methodsmentioning

confidence: 99%

Promoter Architecture, Cofactors, and Orphan Receptors Contribute to Cell-specific Activation of the Retinoic Acid Receptor β2 Promoter

Folkers

Burg

Saag

1998

“…1, C-F). Cell specificity has been found to play an important role in the activity of the AF-1 and AF-2 domains in the estrogen, glucocorticoid, progesterone receptors, and RARs (66,67). The ability of different activation functions 1 and 2 to operate has been found to vary in relation to the cell line used and the spatio-temporal expression pattern of the specific receptor, suggesting that cell-specific activation mechanisms are involved in the functioning of the different AFs.…”

Section: Nor-1 Trans-activates Gene Expression In a Cell-and Responsementioning

confidence: 99%

The AF-1 Domain of the Orphan Nuclear Receptor NOR-1 Mediates Trans-activation, Coactivator Recruitment, and Activation by the Purine Anti-metabolite 6-Mercaptopurine

Wansa

Harris

Yan

et al. 2003

140

125

NOR-1/NR4A3 is an "orphan member" of the nuclear hormone receptor superfamily. NOR-1 and its close relatives Nurr1 and Nur77 are members of the NR4A subgroup of nuclear receptors. Members of the NR4A subgroup are induced through multiple signal transduction pathways. They have been implicated in cell proliferation, differentiation, T-cell apoptosis, chondrosarcomas, neurological disorders, inflammation, and atherogenesis. However, the mechanism of transcriptional activation, coactivator recruitment, and agonist-mediated activation remain obscure. Hence, we examined the molecular basis of NOR-1-mediated activation. We observed that NOR-1 trans-activates gene expression in a cell-and target-specific manner; moreover, it operates in an activation function (AF)-1-dependent manner. The N-terminal AF-1 domain delimited to between amino acids 1 and 112, preferentially recruits the steroid receptor coactivator (SRC). Furthermore, SRC-2 modulates the activity of the AF-1 domain but not the Cterminal ligand binding domain (LBD). Homology modeling indicated that the NOR-1 LBD was substantially different from that of hROR␤, a closely related AF-2-dependent receptor. In particular, the hydrophobic cleft characteristic of nuclear receptors was replaced with a very hydrophilic surface with a distinct topology. This observation may account for the inability of this nuclear receptor LBD to efficiently mediate cofactor recruitment and transcriptional activation. In contrast, the N-terminal AF-1 is necessary for cofactor recruitment and can independently conscript coactivators. Finally, we demonstrate that the purine anti-metabolite 6-mercaptopurine, a widely used antineoplastic and anti-inflammatory drug, activates NOR-1 in an AF-1-dependent manner. Additional 6-mercaptopurine analogs all efficiently activated NOR-1, suggesting that the signaling pathways that modulate proliferation via inhibition of de novo purine and/or nucleic acid biosynthesis are involved in the regulation NR4A activity. We hypothesize that the NR4A subgroup mediates the genotoxic stress response and suggest that this subgroup may function as sensors that respond to genotoxicity. Nuclear hormone receptors (NRs)1 function as ligand-activated transcription factors that regulate gene expression involved in reproduction, development, and general metabolism (1). NRs function as the pipeline between physiology and gene expression. The importance of NRs in human physiology is underscored by the extensive range of therapeutics that has been created to combat disorders associated with dysfunctional hormone signaling. These diseases affect every discipline of medicine (2). All members of the NR superfamily display a highly conserved structural organization (1) with an aminoterminal region AB (that encodes activation function 1 (AF-1)), followed by the C-region that encodes the DNA binding domain (DBD), a linker region D, and the C-terminal E region. The DE region encodes the ligand binding domain (LBD) and a transcriptional domain, denoted as activation function 2 (AF...

“…We investigated and compared the ability of full-length Nur77, Nur77 lacking the AB region (Nur77⌬AB construct containing amino acids 269 -601), and Nur77 lacking the DE region (Nur77⌬LBD construct encoding amino acids 1-356) to transactivate NBRE in muscle and non-muscle cells in the absence of agonists. Cell specificity has been found to play an important role in the activation functions of the AB and DE domains in the estrogen, glucocorticoid, progesterone, and retinoic acid receptors (45,46). The ability of different AFs to function has been found to (i) vary in relation to the cell line used and (ii) depend on the spatio-temporal expression pattern of the specific receptor, indicating that cell-specific activation mechanisms are involved in the functioning of the different AFs.…”

Section: The Ab (Af-1) Region Of Nur77 Is Necessary For Agonistindepementioning

confidence: 99%

The Activation Function-1 Domain of Nur77/NR4A1 Mediates Trans-activation, Cell Specificity, and Coactivator Recruitment

Wansa

Harris

Muscat

2002

134

116

Nur77/NR4A1 is an "orphan member" of the nuclear hormone receptor superfamily. Nur77 and its close relatives Nurr1 and NOR-1 bind as monomers to a consensus binding site, the nerve growth factor induced protein I-B (NGFI-B)-binding response element (NBRE). The Nur77/ NURR1/NOR1 nuclear receptors are classified as immediate early response genes which are induced through multiple signal transduction pathways. They have been implicated in cell proliferation, differentiation, and apoptosis. However, the mechanism of coactivation and ligand independent trans-activation remains unclear. Hence we examined the molecular basis of Nur77-mediated cofactor recruitment and activation. We observed that Nur77 trans-activates gene expression in a cell-specific manner, and operates in an activation function-1 (AF-1)-dependent manner. The AB region encodes an uncommonly potent N-terminal AF-1 domain delimited to between amino acids 50 and 160 and is essential for the ligand-independent activation of gene expression. Steroid receptor coactivator-2 (SRC-2) modulates the activity of the N-terminal AF-1 domain. Moreover, SRC-2 dramatically potentiates the retinoid induced RXR-dependent activation of the Nur77 ligand binding domain (LBD). Interestingly, the N-terminal AB region (not the LBD) facilitates coactivator recruitment and directly interacts with SRC, p300, PCAF, and DRIP-205. Consistent with this, homology modeling indicated that the Nur77 LBD coactivator binding cleft was substantially different from that of retinoic acid receptor ␥, a closely related AF-2-dependent receptor. In particular, the hydrophobic cleft characteristic of nuclear receptors was replaced with a much more hydrophilic surface with a distinct topology. This observation accounts for the inability of this nuclear receptor LBD to directly mediate cofactor recruitment. Furthermore, the AF-1 domain physically associates with the Nur77 C-terminal LBD and synergizes with the retinoid X receptor LBD. Thus, the AF-1 domain plays a major role in Nur77-mediated transcriptional activation, cofactor recruitment, and intra-and intermolecular interactions.