Feedback regulation between orphan nuclear receptor TR2 and human papilloma virus type 16

The human TR2 orphan receptor (TR2), initially isolated from testis and prostate cDNA libraries, is a member of the steroid receptor superfamily. TR2 can regulate several target genes via binding to a consensus response element (AGGTCA) in direct repeat orientation (AGGTCAX((n))AGGTCA, n = 0-6). Here we show that TR2 is able to induce the expression of human papilloma virus type 16 (HPV-16) genes via binding to a DR4 response element in the long control region of HPV-16. Additionally, one of the HPV-16 gene products, the E6 oncogene, regulates TR2 gene expression. A likely mechanism for this regulation involves E6-mediated degradation of the tumor suppressor p53, a protein known to suppress TR2 expression. Together our data provide evidence for feedback regulation between TR2 and HPV-16, which represents a novel regulatory pathway involving a member of the steroid receptor superfamily and the HPV-16 DNA tumor virus.     

Regulation of CYP3A4 by pregnane X receptor: The role of nuclear receptors competing for response element binding

Induction of the major drug metabolizing enzyme CYP3A4 by xenobiotics contributes to the pronounced interindividual variability of its expression and often results in clinically relevant drug-drug interactions. It is mainly mediated by PXR, which regulates CYP3A4 expression by binding to several specific elements in the 5′ upstream regulatory region of the gene. Induction itself shows a marked interindividual variability, whose underlying determinants are only partly understood. In this study, we investigated the role of nuclear receptor binding to PXR response elements in CYP3A4, as a potential non-genetic mechanism contributing to interindividual variability of induction. By in vitro DNA binding experiments, we showed that several nuclear receptors bind efficiently to the proximal promoter ER6 and distal xenobiotic-responsive enhancer module DR3 motifs. TRα1, TRβ1, COUP-TFI, and COUP-TFII further demonstrated dose-dependent repression of PXR-mediated CYP3A4 enhancer/promoter reporter activity in transient transfection in the presence and absence of the PXR inducer rifampin, while VDR showed this effect only in the absence of treatment. By combining functional in vitro characterization with hepatic expression analysis, we predict that TRα1, TRβ1, COUP-TFI, and COUP-TFII show a strong potential for the repression of PXR-mediated activation of CYP3A4 in vivo. In summary, our results demonstrate that nuclear receptor binding to PXR response elements interferes with PXR-mediated expression and induction of CYP3A4 and thereby contributes to the interindividual variability of induction.     

孤儿受体TR3与人CNTF受体基因中顺式元件作用机制的研究

应用两对人工合成的寡核苷酸引物,分别通过ＰＣＲ扩增,得到了ＣＮＴＦＲα－Ｉ５ＮＢＲＥ序列两侧的两个扩增片段,将其和在ＥｃｏＲⅤ位点切开的ｐＴ７ｂｌｕｅ一起定向连接,得到了插入在ｐＴ７ｂｌｕｅ的ＥｃｏＲⅤ位点的缺失了ＮＢＲＥ序列的ＣＮＴＦＲα－Ｉ５,然后再将其切下,插入到具有ＳＶ４０起动子的ＣＡＴ基因表达载体的ＢｇｌⅡ位点,构建了ＣＡＴ报道基因．细胞转染和ＣＡＴ实验表明,缺失ＮＢＲＥ后,ＣＮＴＦＲα－Ｉ５仍具有增强子功能,ＴＲ３通过该增强子对ＣＮＴＦＲα的表达具有诱导作用,说明这种诱导作用并不是单一通过ＮＢＲＥ序列进行的．     

Germ Cell Nuclear Factor: An Orphan Receptor in Search of a Function

Germ Cell Nuclear Factor (GCNF) is an orphan member of the nuclearreceptor gene superfamily. Much has been understood about thefunctioning of GCNF which represents a candidate receptor fora novel hormonal signalling pathway. GCNF is not closely relatedto other members of the nuclear receptor superfamily and formsits own branch within the superfamily tree. It has a uniqueexpression pattern that spans both embryonic and adult stagesof development. In the adult, it is expressed in the germ cells:oocytes and spermatogenic cells as well as specific neuronalcells within the brain. In the embryo, GCNF expression is turnedon after gastrulation in all germ layers the ectoderm, mesodermand endoderm. An antero-posterior gradient of GCNF is establishedin the neuroectoderm of the embryo, suggesting a role in regulationof neuronal and germ cell development. Regulation of physiologicalprocesses by a nuclear receptor is achieved through regulationof gene expression. GCNF is the only nuclear receptor to specifcallybind to DR0 hormone response elements to regulate gene expression.In the absense of a ligand, GCNF represses gene expression.GCNF is capable of regulating the expression of the protaminegenes in a response element-dependent manner. At present theligand for GCNF is unknown, but it is hypothesized that GCNFis a receptor for a novel hormonal signalling pathway that effectsits biological response by regulating the expression of a subsetof genes containing DR0 response elements.     

Hepatocyte nuclear factor 4α regulates the expression of the murine pyruvate carboxylase gene through the HNF4-specific binding motif in its proximal promoter

Pyruvate carboxylase (PC) is the first regulatory enzyme of gluconeogenesis. Here we report that the proximal promoter of the murine PC gene contains three binding sites for hepatocyte nuclear factor 4α (HNF4α). These sites include the classical direct repeat 1 (DR1) (− 386/− 374), non-perfect DR1 (− 118/− 106) and HNF4α-specific binding motif (H4-SBM) (− 26/− 14). Under basal conditions, mutation of the non-perfect DR1 decreased promoter activity by 50%, whereas mutation of neither the DR1 nor the H4-SBM had any effect. In marked contrast, only mutation of the H4-SBM decreased HNF4α-transactivation of the promoter activity by 65%. EMSA revealed that HNF4α binds to the DR1site and H4-SBM with similar affinity while it binds poorly to the non-perfect DR1. Interestingly, this non-perfect DR1 also coincides with two E-boxes. Mutation of the non-perfect DR1 together with the nearby E-box reduced USF1- but not USF2-transactivation of promoter activity, suggesting that USF1 partly contributes to the basal activity of the promoter. Substitution of the H4-SBM with the DR1 marginally reduced the basal promoter activity but did not eliminate HNF4α-transactivation, suggesting that HNF4α can exert its effect via DR1 within this promoter context. ChIP-assay confirmed that HNF4α is associated with the H4-SBM. Suppression of HNF4α expression in AML12 cells down-regulated PC mRNA and PC protein by 60% and 50%, respectively, confirming that PC is a target of HNF4α. We also propose a model for differential regulation of P1 promoter of PC gene in adipose tissue and liver.