Structure and Expression of the Orphan Nuclear ReceptorSHP Gene

Abstract

clones were isolated from human and mouse genomic libraries. The SHP gene was composed of two exons interrupted by a single intron spanning approximately 1.8 kilobases in human and 1.2 kilobases in mouse. Genomic Southern blot analysis and fluorescence in situ hybridization of human metaphase chromosomes indicated that the SHP gene is located at the human chromosome 1p36.1 subband. The 5-flanking regions of human and mouse SHP genes were highly conserved, showing 77% homology in the region of approximately 600 nucleotides upstream from the transcription start site. Primer extension analysis was carried out to determine the transcription start site of human SHP to 32 nucleotides downstream of a potential TATA box. The human SHP gene was specifically expressed in fetal liver, fetal adrenal gland, adult spleen, and adult small intestine. As expected from this expression pattern, the activity of the mouse SHP promoter measured by transient transfection was significantly higher in the adrenal-derived Y1 cells than HeLa cells.The nuclear receptor superfamily is a group of transcription factors regulated by small hydrophobic hormones such as retinoic acid, thyroid hormone, and steroids and also includes a large number of related proteins that do not have known ligands, referred to as orphan nuclear receptors (for reviews see Refs. 1, 2). The nuclear receptors directly regulate transcription by binding to specific DNA sequences named hormone response elements, generally located in promoters of target genes. The nuclear hormone receptors share a common domain structure. The central DNA binding domain (DBD) 1 includes two zinc binding modules, which consist of a series of invariant cysteine residues. A conserved helical region termed the P box within the DBD (3) makes base-specific contacts and serves as one of the main criteria used for classification of the nuclear receptor superfamily. The C-terminal ligand binding domain (LBD) binds to the cognate ligands. This domain also contains dimerization and transcriptional activation functions. A less well conserved hinge domain that separates DBD and the ligand binding domain has been thought to serve merely as a flexible linker. However, recent results demonstrate that it is also involved with transcriptional repression, at least for a subset of receptors (4). In addition, it was also shown to contain nuclear localization signals (1, 2). A quite variable N-terminal domain includes a transcriptional activation function with some receptors.Although ligands have not been identified for orphan nuclear receptors, a variety of results indicate that they have important functions. The simplest is that knockout mutations of these orphans in mice frequently have shown much more dramatic defects than similar mutations of the conventional receptor genes (5-7). We have recently reported an unusual orphan member of the nuclear receptors that contains a ligand binding domain but lacks the conserved DBD (8). This orphan receptor interacts, both in vitro and in the yeast two-hybrid system wi...

Differential Regulation of Human and Mouse Orphan Nuclear Receptor Small Heterodimer Partner Promoter by Sterol Regulatory Element Binding Protein-1

“…After viral infection for 0, 3, 6, 12, and 24 h, infected cells were harvested for RNA isolation using the TRIzol reagent (Invitrogen). Northern blot analysis was carried out as described previously (13). The mRNAs of hSHP and hCYP7A1 were analyzed by RT-PCR as described previously (22).…”

“…SHP is expressed in a wide variety of tissues, including heart, brain, liver, spleen, adrenal gland, small intestine, and pancreas (2,12). The human SHP gene is located on chromosome 1p36.1 and consists of two exons separated by an intron (13). SHP gene transcription is regulated by several members of the nuclear receptor superfamily, including the bile acid receptor farnesoid X receptor (14 -16), steroidogenic factor-1 (17), HNF4␣ (18), liver receptor homologue 1 (14), and estrogen-related receptor-␥ (9).…”

Cloning and characterization of a nuclear S6 kinase, S6 kinase-related kinase (SRK); A novel nuclear target of Akt

“…In addition, p70S6K was maximally activated at 30 min after stimulation (Chung et al, 1992), but SRK needed about 2 h to be fully activated. Subcellular localization di erences and/or other activa- (A) + mRNA from the indicated human tissues was hybridized with a SRK cDNA fragment (nucleotides 280 ± 570) as described (Lee et al, 1998). (c) A Southern blot containing 4 mg of EcoRI-digested genomic DNAs from di erent species was hybridized with the human SRK cDNA probe as described (Lee et al, 1998) Figure 2 (a) The S6 phosphotransferase activities of SRK.…”

“…Subcellular localization di erences and/or other activa- (A) + mRNA from the indicated human tissues was hybridized with a SRK cDNA fragment (nucleotides 280 ± 570) as described (Lee et al, 1998). (c) A Southern blot containing 4 mg of EcoRI-digested genomic DNAs from di erent species was hybridized with the human SRK cDNA probe as described (Lee et al, 1998) Figure 2 (a) The S6 phosphotransferase activities of SRK. Cos cells transfected with pJ3H-SRK by the DEAE-dextran method (Chou and Blenis, 1996) were serum starved for 24 h, and then stimulated with 10% fetal bovine serum (SER), 25 ng/ml EGF (EGF), 100 ng/ml PMA (PMA), or 100 mM cycloheximide (CYH) for the time periods (min) as indicated.…”

Identification of polymorphisms in the human SHP1 gene

“…Nuclear receptors are transcription factors regulated by small hydrophobic hormones such as retinoic acid, thyroid hormone, and steroids (Lee et al 1998). This superfamily also includes orphan nuclear receptors, which are related proteins with unspecified ligands.…”