Fundamentally distinct roles of thyroid hormone receptor isoforms in a thyrotroph cell line are due to differential DNA binding

Thyroid hormones have a profound influence on human development and disease. The hypothalamic-pituitary-thyroid axis involves finely tuned feedback mechanisms to maintain thyroid hormone (TH) levels. Despite the important role of TH-negative feedback in regulating this axis, the mechanism by which this occurs is not clearly defined. Previous in vivo studies suggest separate roles for the two thyroid hormone receptor isoforms, THRA and THRB, in this axis. We performed studies using a unique pituitary thyrotroph cell line (TαT1.1) to determine the relative roles of THRA and THRB in the regulation of Tshb. Using chromatin immunoprecipitation assays, we found that THRB, not THRA, bound to the Tshb promoter. By selectively depleting THRB, THRA, or both THRA and THRB in TαT1.1 cells, we found that simultaneous knockdown of both THRB and THRA abolished T(3)-mediated down-regulation of Tshb at concentrations as high as 100 nm T(3). In contrast, THRA knockdown alone had no effect on T(3)-negative regulation, whereas THRB knockdown alone abolished T(3)-mediated down-regulation of Tshb mRNA levels at 10 nm but not 100 nm T(3) concentrations. Interestingly, chromatin immunoprecipitation assays showed that THRA becomes enriched on the Tshb promoter after knockdown of THRB. Thus, a likely mechanism for the differential effects of THR isoforms on Tshb may be based on their differential DNA-binding affinity to the promoter.     

Genes encoding the alpha, gamma, delta, and four F0 subunits of ATP synthase constitute an operon in the cyanobacterium Anabaena sp. strain PCC 7120.

A cluster of genes encoding subunits of ATP synthase of Anabaena sp. strain PCC 7120 was cloned, and the nucleotide sequences of the genes were determined. This cluster, denoted atp1, consists of four F0 genes and three F1 genes encoding the subunits a (atpI), c (atpH), b' (atpG), b (atpF), delta (atpD), alpha (aptA), and gamma (atpC) in that order. Closely linked upstream of the ATP synthase subunit genes is an open reading frame denoted gene 1, which is equivalent to the uncI gene of Escherichia coli. The atp1 gene cluster is at least 10 kilobase pairs distant in the genome from apt2, a cluster of genes encoding the beta (atpB) and epsilon (atpE) subunits of the ATP synthase. This two-clustered ATP synthase gene arrangement is intermediate between those found in chloroplasts and E. coli. A unique feature of the Anabaena atp1 cluster is overlap between the coding regions for atpF and atpD. The atp1 cluster is transcribed as a single 7-kilobase polycistronic mRNA that initiates 140 base pairs upstream of gene 1. The deduced translation products for the Anabaena sp. strain PCC 7120 subunit genes are more similar to chloroplast ATP synthase subunits than to those of E. coli.     

Identification and analysis of additional copies of the platelet-derived growth factor receptor and colony stimulating factor 1 receptor genes in fugu

The receptors for the platelet-derived growth factor (PDGFRalpha and PDGFRbeta) belong to a subfamily of protein tyrosine kinase receptors that also includes kit and the colony stimulating factor-1 receptor (CSF1R). In mammals, the genes encoding PDGFRalpha and PDGFRbeta are tandemly linked to the kit and CSF1R genes, respectively. Based on the structural similarity and genomic organization of these four genes, it has been suggested that they arose from an ancestral protein tyrosine kinase receptor gene by two rounds of duplication. We have previously cloned the PDGFRbeta and CSF1R genes from the pufferfish, Fugu rubripes, and shown that they are tandemly linked like the mammalian genes [Genome Res. 6 (1996) 1185]. We have now cloned two additional members of this gene family, fPDGFRbeta2 and fCSF1R2 from the fugu and shown that these two genes are also tandemly linked. This indicates that the PDGFRbeta-CSF1R locus has been duplicated in the lineage leading to fugu. The fugu fPDGFRbeta2 and fCSF1R2 genes contain three and one extra introns, respectively, compared with other members of this family. Polymerase chain reaction cloning of a conserved region of PDGFRbeta gene from other ray-finned fishes identified two copies in the zebrafish (order Cypriniformes) and sunfish (order Tetraodontiformes). These results are discussed in the context of the proposed teleost lineage-specific whole genome duplication hypothesis.     

The expression of CYP2B6, CYP2C9 and CYP3A4 genes: a tangle of networks of nuclear and steroid receptors

Numerous chemicals increase the metabolic capability of organisms by their ability to activate genes encoding various xenochemical-metabolizing enzymes, such as cytochromes P450 (CYPs), transferases and transporters. For example, natural and synthetic glucocorticoids (agonists and antagonists) as well as other clinically important drugs induce the hepatic CYP2B, CYP2C and CYP3A subfamilies in man, and these inductions might lead to clinically important drug-drug interactions. Only recently, the key cellular receptors that mediate such inductions have been identified. They include nuclear receptors, such as the constitutive androstane receptor (CAR, NR1I3), the retinoid X receptor (RXR, NR2B1), the pregnane X receptor (PXR, NR1I2), and the vitamin D receptor (VDR, NR1I1) and steroid receptors such as the glucocorticoid receptor (GR, NR3C1). There is a wide promiscuity of these receptors in the induction of CYPs in response to xenobiotics. Indeed, this adaptive system appears now as a tangle of networks, where receptors share partners, ligands, DNA response elements and target genes. Moreover, they influence mutually their relative expression. This review is focused on these different pathways controlling human CYP2B6, CYP2C9 and CYP3A4 gene expression, and the cross-talk between these pathways.     

Nucleotide sequence and transcript analysis of three photosystem II genes from the cyanobacterium Synechococcus sp. PCC7942

The genome of the cyanobacterium Synechococcus sp. PCC7942 contains two genes encoding the D2 polypeptide of photosystem II (PSII), which are designated here as psbDI and psbDII. The psbDI gene, like the psbD gene of plant chloroplasts, is cotranscribed with and overlaps the open reading frame of the psbC gene, encoding the PSII protein CP43. The psbDII gene is not linked to psbC, and appears to be transcribed as a monocistronic message. The two psbD genes encode identical polypeptides of 352 amino acids, which are 86% conserved with the D2 polypeptide of spinach. In plants, the translational start codon of the psbC gene has been reported to be an ATG codon 50 bp upstream from the end of the psbD gene. This triplet is not present in the psbDI sequence of Synechococcus sp., but is replaced by ACG, a codon which is very unlikely to initiate translation. Translation of the psbC gene may begin at a GTG codon which overlap the psbDI open reading frame by 14 bp and is preceded by a block of homology to the 3' end of the 16S ribosomal RNA, a potential ribosome-binding site. There are only two bp differences between the sequences of the two psbD genes; one of these results in substitution in psbDII of GCG for the presumed GTG start codon in psbDI.     

Control of gene expression by the retinoic acid-related orphan receptor alpha in HepG2 human hepatoma cells

Retinoic acid-related Orphan Receptor alpha (RORα; NR1F1) is a widely distributed nuclear receptor involved in several (patho)physiological functions including lipid metabolism, inflammation, angiogenesis, and circadian rhythm. To better understand the role of this nuclear receptor in liver, we aimed at displaying genes controlled by RORα in liver cells by generating HepG2 human hepatoma cells stably over-expressing RORα. Genes whose expression was altered in these cells versus control cells were displayed using micro-arrays followed by qRT-PCR analysis. Expression of these genes was also altered in cells in which RORα was transiently over-expressed after adenoviral infection. A number of the genes found were involved in known pathways controlled by RORα, for instance LPA, NR1D2 and ADIPOQ in lipid metabolism, ADIPOQ and PLG in inflammation, PLG in fibrinolysis and NR1D2 and NR1D1 in circadian rhythm. This study also revealed that genes such as G6PC, involved in glucose homeostasis, and AGRP, involved in the control of body weight, are also controlled by RORα. Lastly, SPARC, involved in cell growth and adhesion, and associated with liver carcinogenesis, was up-regulated by RORα. SPARC was found to be a new putative RORα target gene since it possesses, in its promoter, a functional RORE as evidenced by EMSAs and transfection experiments. Most of the other genes that we found regulated by RORα also contained putative ROREs in their regulatory regions. Chromatin immunoprecipitation (ChIP) confirmed that the ROREs present in the SPARC, PLG, G6PC, NR1D2 and AGRP genes were occupied by RORα in HepG2 cells. Therefore these genes must now be considered as direct RORα targets. Our results open new routes on the roles of RORα in glucose metabolism and carcinogenesis within cells of hepatic origin.     

THRA1 and D17S183 flank an interval of < 4 cM for the breast-ovarian cancer gene (BRCA1) on chromosome 17q21.

In order to pinpoint the locale of the gene for early-onset familial breast and ovarian cancer (BRCA1), polymorphisms were developed within the locus for thyroid hormone receptor alpha (THRA1) and for several anonymous sequences at chromosome 17q12-q21. The THRA1 polymorphism is a dinucleotide repeat with 10 alleles and heterozygosity.79. Gene mapping in extended families with inherited, early-onset breast and ovarian cancer indicates that BRCA1 is distal to THRA1 and proximal to D17S183 (SCG43), an interval of < 4 cM. This locale excludes HER2, THRA1, WNT3, HOX2, NGFR, PHB, COLIA1, NME1, and NME2 as candidates for BRCA1 but does not exclude RARA or EDH17B. Resolving the remaining recombination events in these families by new polymorphisms in the THRA1-D17S183 interval will facilitate positional cloning of the breast-ovarian cancer gene on chromosome 17q12-q21.     

Rearrangement and expression of T-cell antigen receptor genes in human T-lymphocyte tumor lines and normal human T-cell clones: evidence for allelic exclusion of Ti beta gene expression and preferential use of a J beta 2 gene segment.

The gene encoding the beta chain of the human T-cell receptor for antigen is composed of variable (V), diversity (D), joining (J), and constant (C) gene segments which undergo specific rearrangements during T-lymphocyte ontogeny. Southern blot analyses of seven human T-cell tumor lines and normal human T-lymphocyte clones revealed that most of these T-cell lines rearrange their Ti beta genes differently. The T-cell tumor line HPB-MLT rearranges and transcribes both of its Ti beta genes. Cloning and sequencing of the Ti beta cDNAs corresponding to these rearrangements revealed that one of the rearranged Ti beta genes is defective, while the other is functional and corresponds to the Ti beta protein expressed on the surface of these cells. Thus, this cell line displays a pattern of allelic exclusion of Ti beta gene expression. A comparison of four C beta 2-containing Ti beta cDNAs from three different cell lines revealed that three of the four utilize the same J beta 2.5 gene segment joined to different D beta and V beta genes, suggesting that there may be preferential use of this J gene during J beta 2 rearrangements. Hybridization analyses with probes for the alpha and beta genes of the T-cell receptor and the T-cell-specific T gamma gene revealed that HPB-MLT cells appear to express approximately equivalent amounts of RNA corresponding to each of the rearranged Ti alpha and Ti beta genes. However, they express a much lower level of T gamma RNA.     

Human T lymphocytes express N-methyl-D-aspartate receptors functionally active in controlling T cell activation

The aim of this study was to investigate the expression and the functional role of N-methyl-D-aspartate (NMDA) receptors in human T cells. RT-PCR analysis showed that human resting peripheral blood lymphocytes (PBL) and Jurkat T cells express genes encoding for both NR1 and NR2B subunits: phytohemagglutinin (PHA)-activated PBL also expresses both these genes and the NR2A and NR2D genes. Cytofluorimetric analysis showed that NR1 expression increases as a consequence of PHA (10 microg/ml) treatment. D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5), and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine [(+)-MK 801], competitive and non-competitive NMDA receptor antagonists, respectively, inhibited PHA-induced T cell proliferation, whereas they did not affect IL-2 (10 U/ml)-induced proliferation of PHA blasts. These effects were due to the prevention of T cell activation (inhibition of cell aggregate formation and CD25 expression), but not to cell cycle arrest or death. These results demonstrate that human T lymphocytes express NMDA receptors, which are functionally active in controlling cell activation.     

Structure and expression of two nuclear receptor genes in marsupials: insights into the evolution of the antisense overlap between the α-thyroid hormone receptor and Rev-erbα

Background  

Alternative processing of α-thyroid hormone receptor (TRα, NR1A1) mRNAs gives rise to two functionally antagonistic nuclear receptors: TRα1, the α-type receptor, and TRα2, a non-hormone binding variant that is found only in mammals. TRα2 shares an unusual antisense coding overlap with mRNA for Rev-erbα (NR1D1), another nuclear receptor protein. In this study we examine the structure and expression of these genes in the gray short-tailed opossum, Monodelphis domestica, in comparison with that of eutherian mammals and three other marsupial species, Didelphis virginiana, Potorous tridactylus and Macropus eugenii, in order to understand the evolution and regulatory role of this antisense overlap.     

A chlorate-resistant mutant defective in the regulation of nitrate reductase gene expression in Arabidopsis defines a new HY locus.

Light acts both directly as a signal and indirectly through photosynthesis to regulate the expression of genes encoding nitrate reductase (NR). Here, we report the isolation and characterization of a novel chlorate-resistant mutant that is defective in the regulation of NR gene expression. The response of NR2, but not NR1 or the gene encoding nitrate reductase (NiR), to light signals was impaired in this Arabidopsis mutant, designated cr88. In addition to NR2, the light regulation of the genes encoding the chlorophyll a/b binding protein (CAB) and the small subunit of ribulose bisphosphate carboxylase (RBCS) was also impaired in this mutant. These results suggest that the pathway through which light regulates the expression of NR2, CAB, and RBCS genes is different from those that regulate the expression of NR1 and NiR. An examination of the deetiolation process under different light spectrum showed that cr88 is defective in red light-mediated deetiolation. Complementation tests with various long hypocotyl (hy) mutants indicated that CR88 identifies a new HY locus. The possible functions of CR88 are discussed.     

A somatic cell hybrid map of the long arm of human chromosome 17, containing the familial breast cancer locus (BRCA1).

We describe a detailed somatic cell hybrid map of human chromosome 17q11.2-q23, containing the familial breast and ovarian cancer locus (BRCA1) and highly informative closely linked markers. An X-irradiation panel of 38 hamster/human and mouse/human hybrids with fragments of chromosome 17 was generated and characterized with 22 STS markers from this chromosome. A detailed map of 61 probes onto chromosome 17q, subdividing the chromosome arm into 25 regions, was done by using a panel of hybrids with well-defined breakpoints and nine chromosome-mediated gene transfectants. Our localization of RARA, TOP2, EDH17B1 and 2, and possibly WNT3, between THRA1 and D17S181, two markers known to flank BRCA1, suggests that any of these is a potential candidate for the BRCA1 locus. The marker D17S579 (Mfd188), which is believed to be very close to BRCA1, maps closest to the EDH17B genes.     

Correlating physiology with gene expression in striatal cholinergic neurones

The expression of 34 transmitter-related genes has been examined in the cholinergic neurones of rat striatal brain slices, with the aim of correlating gene expression with functional activity. The mRNAs encoding types I, II/IIA, and III alpha subunits of the voltage-sensitive sodium channels were detected, suggesting the presence of these three types of sodium channel. Similarly, mRNAs encoding all four alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-type glutamate receptor subunits and the NR1 and NR2A, 2B, and 2D subunits of the NMDA-type glutamate receptors were detected, suggesting that various combinations of these subunits mediate the cellular response to synaptically released glutamate. Other mRNAs detected included the NK1 and NK3 tachykinin receptors, all four known adenosine receptors, and the GABA-synthesising enzyme glutamate decarboxylase. Subpopulations of these cholinergic neurones have been identified on the basis of the expression of the NK3 tachykinin receptor in 5% and the trkC neurotrophin receptor in 12% of the cells investigated.     

Cloning, Functional Coexpression, and Pharmacological Characterisation of Human cDNAs Encoding NMDA Receptor NR1 and NR2A Subunits

Abstract: Using expression cloning, and more recently using polymerase chain reaction cloning approaches, a family of rat N -methyl- d -aspartate (NMDA) receptor subunit cDNAs has been described (NR1, NR2A, NR2B, NR2C, and NR2D). Here we report cloning and sequencing of cDNAs encoding isoforms of the human NR1 subunit (NR1a, NR1d, and NR1e) that differ at their C-terminal end as a result of alternative splicing and also of a cDNA encoding the human NR2A subunit. The deduced amino acid sequences of the human NR1 subunit isoforms differed from the published rat NR1 subunit sequences at only eight positions, all of which were N-terminal to the alternatively spliced domains. The human NR2A subunit deduced amino acid sequence differed from the published rat NR2A subunit sequence at 81 of the 1,464 amino acids, with most of the substitutions being located in the C-terminal half of the subunit. The gene for NR2A has been localised to human chromosome 16. We also report the expression and pharmacological characterisation of recombinant human NR1a/NR2A heteromeric receptors in Xenopus oocytes. These receptors had EC₅₀ values of 2.14 and 2.05 μ M for glutamate and glycine, respectively, and an IC₅₀ of 46.8 μ M for Mg²⁺. Responses were antagonised by d -2-amino-5-phosphonovalerate, L-689,560, pH 6.3, zinc, and MK-801. No modulatory effect was observed on application of ifenprodil, confirming previous observations with rat NR1 + NR2A recombinant receptors.