The v-erbA oncoprotein of the AEV transforming retrovirus binds to the promoter region of the erythroid-specific band 3 gene.
Explore the source record for details and available documents.
Alpharetrovirus↗
Biomedical subjects
B Vennström
Publications and source records attributed to B Vennström.
Retroviral capture of c-erbB proto-oncogene sequences: rapid evolution of distinct viral genomes carrying mutant v-erbB genes with different transforming capacities.
The evolution of oncogene-transducing retroviruses was followed by studying the genomes of five new, erbB carrying retroviruses. These viruses, isolated from cells of one chicken infected with Rous Associated virus 1 (RAV-1), had captured c-erbB sequences as a consequence of RAV-1 integration into the host genome. Their genome structures were distinct; however, their v-erbB genes had sustained identical 5' and 3' deletions and the v-erbB-env junctions were identical at the nucleotide level. The results therefore strongly suggest that all five viruses originate from the same capture event. Sequence analyses of the v-erbB genes from three of these viruses revealed that one of them had undergone no further mutation and lacked detectable capacity to transform cells, therefore probably representing an 'early' form of transducing virus. The two other v-erbB genes contained distinct mutations and differed in their potential to induce fibroblast- and erythroblast transformation; they therefore probably represent later derivatives of the virus that captured the erbB oncogene. The data suggest that the initial retrovirus rapidly underwent many alterations after capture of c-erbB sequences, already in the RAV-1 infected bird as well as during subsequent in vitro isolation procedures. The changes involve both major rearrangements of the genome as well as point mutations that activated the erbB oncogene.
Amino Acid Sequence↗
Identification and analysis of all components of a gel retardation assay by combination with immunoblotting.
A better method was developed for analysis and identification of protein and DNA components of gel-shift assays. The protein-DNA complexes, separated in polyacrylamide gels, were transferred onto stacked nitrocellulose and anion-exchange membranes. The proteins, bound to nitrocellulose, were identified by immunoblotting, while the DNA, which bound only to the anion-exchange membrane, was detected by autoradiography. The technique readily identified thyroid hormone receptors interacting with response elements representing inverted or direct repeats of the consensus half-site AGGTCA. In addition, specific antisera identified both the thyroid hormone and the retinoic acid receptors in heterodimeric complexes. Adding a third membrane and digoxigenin-labeled DNA probes allowed separate detection of [125I]T3 (labeled 3,5,3'-L-triiodothyronine), DNA, and protein from a single gel-shift reaction. The usefulness of this technique was also demonstrated by detecting the transcription factors P75gag-v-erbA and Jun in shifted complexes. Finally, proteins and DNA transferred to anion-exchange membranes can be eluted and subjected to further study. The combination of the gel shift and the immunoblot approaches (called "Shift-Western blotting") allows identification of the individual components of protein-DNA complexes containing multiple transcription factors, their cognate DNA elements and, when applicable, also the ligand.
Animals↗
Versatile avian retrovirus vectors.
Explore the source record for details and available documents.
Alpharetrovirus↗
Thyroid hormone alters the DNA binding properties of chicken thyroid hormone receptors alpha and beta.
The effects of thyroid hormone agonists on thyroid hormone receptor (TR)/DNA complex formation was investigated to elucidate the mechanism by which TRs transactivate genes in response to ligand. The data, obtained from gel shift experiments, indicate that thyroid hormones alter the conformation of TRs bound to DNA, irrespective of if the element is occupied by monomeric TR, homodimeric TR/TR, or heterodimeric complexes with the retinoid receptors RAR or RXR. Furthermore, triiodo-thyronine (T3) prevents 2 TR molecules from binding to oligonucleotides containing direct repeats or inverted palindromes of the consensus AGGTCA motif, an effect that was not detected with palindromic elements. Heterodimers bound to direct repeats were less affected: RXR/TR were fully and RAR/TR complexes partially resistant to thyroid hormone. The data suggest that a ligand-induced conformational change in TR prevents double TR occupancy of a response element containing 2 direct repeats of the consensus binding motif, possibly by steric hindrance, whereas such an event does not prevent TR/RXR heterodimers from binding to DNA. Finally, our data show that a monomeric, liganded TR bound preferentially to the second half site in a AGGTCActcaAGGTCA element, and therefore indicate that nucleotides adjacent to the consensus half site contribute to binding specificity.
Animals↗
Transcriptional repression of band 3 and CAII in v-erbA transformed erythroblasts accounts for an important part of the leukaemic phenotype.
The v-erbA oncogene confers two prominent properties on transformed erythroblasts: a block of spontaneous differentiation and tolerance to wide variations in the pH or ionic strength of culture medium. V-erbA acts as a constitutive repressor of erythrocyte-specific gene transcription, arresting the expression of at least three different erythroid genes: the erythrocyte anion transporter (band 3), carbonic anhydrase II (CAII) and delta-aminolevulinate synthase (ALA-S). To test whether or not the v-erbA induced repression of these genes is causally related to the v-erbA induced leukaemic phenotype, we have reintroduced the genes for band 3 or CAII into transformed erythroblasts via retrovirus vectors. We show here that such erythroblasts, expressing v-erbA, require the same narrow range of medium pH and ion concentration for growth as do transformed erythroblasts lacking v-erbA, i.e. the v-erbA induced tolerance to pH variation was abrogated. The v-erbA induced differentiation block, however, remained unaffected by the re-expression of band 3 and was only slightly affected by the re-expression of CAII. Our experiments show that the two v-erbA-related 'erythroblast transformation parameters' are separable: suppression of band 3 and CAII accounts for one parameter (pH/ion tolerance), while the second parameter (differentiation block) must involve v-erbA regulation of a different set of target genes.
5-Aminolevulinate Synthetase↗
Detection and temporal appearance of multiple copies of c-erb-B2 genes in advanced mammary carcinoma using fine needle biopsies and the polymerase chain reaction.
Aspiration of tumor cells by the fine-needle biopsy method yields only a small number of cells, which hampers conventional molecular analysis for the presence of multiple copies of oncogenes. We have therefore adopted the polymerase chain reaction (PCR) method to study semi-quantitatively the level of the c-erb-B2 gene in human breast tumor samples. Of 39 patients with mammary carcinoma, 7 (19%) contained multiple copies of c-erb-B2 genes, whereas only two samples failed to give informative data. Next the multiple copies of c-erb-B2 genes, whereas only two samples failed to give informative data. Next the temporal appearance of multiple gene copies was examined in 20 patients with clinical stage IV disease. Tumor samples were obtained every second to third month from the same tumor lesion of each patient. None of the initial samples from each patient contained multiple copies of c-erb-B2. Of 16 patients that showed progressive clinical disease, 5 developed multiple gene copies, showing that the event occurs in clinical stage IV disease.
Aged↗
Binding characteristics of the thyroid hormone receptor homo- and heterodimers to consensus AGGTCA repeat motifs.
Previous studies have shown that thyroid hormone receptors can form homo- and heterodimeric complexes when binding to response elements. We report here the binding characteristics of thyroid hormone receptor (TR) homo- and heterodimers binding to synthetic oligonucleotides with directly and palindromically repeated consensus motifs (AGGTCA). Binding assays showed that TR homodimer formation on DNA had a low specificity and cooperativity, and very fast off rates. In contrast, TRs and retinoic acid receptors readily formed heterodimers with higher specificity and affinity on direct repeats of the AGGTCA motif spaced by four or five nucleotides, although these heterodimer/DNA complexes were only moderately stable when compared to DNA-bound TR/retinoid X receptor heterodimers. Also, TR/retinoic acid receptor heteromeric binding to other elements, including the synthetic T3RE-pal element, was of low specificity. These biochemical results suggest that TRs are unlikely to regulate transcription as homodimers in vivo, and that TR heterodimers mediate the effects of thyroid hormone.
Animals↗
Thyroid hormone receptors in chick retinal development: differential expression of mRNAs for alpha and N-terminal variant beta receptors.
Thyroid-hormone-dependent development of the neuroretina has principally been described in amphibia. Here, we show by in situ hybridisation that mRNAs coding for three distinct thyroid hormone receptors (TRs), TR alpha and two TR beta variants, are differentially expressed during chick retinal development. We isolated a cDNA for a novel N-terminal variant of chick TR beta (cTR beta 2) that is predominantly expressed in retinal development. Interestingly, in its N-terminal A/B domain cTR beta 2 is 70% homologous to the rat pituitary-specific TR beta 2. Expression of cTR beta 2 mRNA was high at embryonic day 6 (E6) in the retinal outer nuclear layer (ONL) and decreased to low levels at hatching. mRNA for the previously described chick beta receptor, cTR beta 0, was expressed at low levels in both the ONL and the inner nuclear layer (INL) after E10. In contrast, cTR alpha expression occurred in the ONL, INL and ganglion cell layer at intermediate and later stages. Finally, cTR beta 2 confers a stronger trans-activation of reporter gene transcription than cTR beta 0. The distinctive kinetics and localisation of TR alpha and beta gene expression suggest cell- and stage-specific functions for TRs, both individually and in combinations, in chick neuroretinal development.
Amino Acid Sequence↗
Distinct functions for thyroid hormone receptors alpha and beta in brain development indicated by differential expression of receptor genes.
Thyroid hormones are essential for correct brain development, and since vertebrates express two thyroid hormone receptor genes (TR alpha and beta), we investigated TR gene expression during chick brain ontogenesis. In situ hybridization analyses showed that TR alpha mRNA was widely expressed from early embryonic stages, whereas TR beta was sharply induced after embryonic day 19 (E19), coinciding with the known hormone-sensitive period. Differential expression of TR mRNAs was striking in the cerebellum: TR beta mRNA was induced in white matter and granule cells after the migratory phase, suggesting a main TR beta function in late, hormone-dependent glial and neuronal maturation. In contrast, TR alpha mRNA was expressed in the earlier proliferating and migrating granule cells, and in the more mature granular and Purkinje cell layers after hatching, indicating a role for TR alpha in both immature and mature neural cells. Surprisingly, both TR genes were expressed in early cerebellar outgrowth at E9, before known hormone requirements, with TR beta mRNA restricted to the ventricular epithelium of the metencephalon and TR alpha expressed in migrating cells and the early granular layer. The results implicate TRs with distinct functions in the early embryonic brain as well as in the late phase of hormone requirement.
Animals↗
Chromosomal assignment of retinoic acid receptor (RAR) genes in the human, mouse, and rat genomes.
The human genes encoding the alpha and beta forms of the retinoic acid receptor are known to be located on chromosomes 17 (band q21.1:RARA) and 3 (band p24:RARB). By in situ hybridization, we have now localized the gene for retinoic acid receptor gamma, RARG, on chromosome 12, band q13. We also mapped the three retinoic acid receptor genes in the mouse, by in situ hybridization, on chromosomes 11, band D (Rar-a); 14, band A (Rar-b); and 15, band F (Rar-g), respectively, and in the rat, using a panel of somatic cell hybrids that segregate rat chromosomes, on chromosomes 10 (RARA), 15 (RARB), and 7 (RARG), respectively. These assignments reveal a retention of tight linkage between RAR and HOX gene clusters. They also establish or confirm and extend the following homologies: (i) between human chromosome 17, mouse chromosome 11, and rat chromosome 10 (RARA); (ii) between human chromosome 3, mouse chromosome 14, and rat chromosome 15 (RARB); and (iii) between human chromosome 12, mouse chromosome 15, and rat chromosome 7 (RARG).
Animals↗
Chromosomal assignment of five cancer-associated rat genes: two thyroid hormone receptor (ERBA) genes, two ERBB genes and the retinoblastoma gene.
Using a panel of somatic cell hybrids that segregate rat chromosomes, the localization of five cancer-related rat genes was determined: (i) two thyroid receptor genes, THRA1/ERBA1 and THRB/ERBA2 on chromosomes 10 and 15 respectively, (ii) two ERBB genes, namely the epidermal growth factor gene (EGFR, also called ERBB1) and the ERBB2 gene (also designated neu) on chromosomes 14 and 10 respectively, and (iii) the retinoblastoma gene, RB1, on chromosome 15. The THRA1/ERBA1 and ERBB2/neu genes are thus included in a synteny group, conserved on rat chromosomes 10 and human chromosome arm 17q.
Alleles↗
v-erbA oncogene activation entails the loss of hormone-dependent regulator activity of c-erbA.
The v-erbA oncogene, one of the two oncogenes of the avian erythroblastosis virus, efficiently blocks erythroid differentiation and suppresses erythrocyte-specific gene transcription. Here we show that the overexpressed thyroid hormone receptor c-erbA effectively modulates erythroid differentiation and erythrocyte-specific gene expression in a T3-dependent fashion, when introduced into erythroid cells via a retrovirus. In contrast, the endogenous thyroid hormone receptor does not detectably affect erythroid differentiation. The analysis of a series of chimeric v-/c-erbA proteins suggests that the v-erbA oncoprotein has lost one type of thyroid hormone receptor function (regulating erythrocyte gene transcription in response to T3), but constitutively displays another function: it represses transcription in the absence of T3. The region responsible for the loss of hormone-dependent regulator activity of v-erbA has been mapped to the very C-terminus of c-erbA, encompassing a cluster of highly conserved amino acid residues with the potential to form an amphipathic alpha-helix.
Alpharetrovirus↗
A major thyroid hormone response element in the third intron of the rat growth hormone gene.
The rat growth hormone (RGH) gene constitutes a well-documented model system for the direct regulation of transcription by thyroid hormones. In order to analyse its interaction with sequences in the RGH gene, we have overproduced the thyroid hormone receptor-alpha (c-erbA) protein using a vaccinia virus expression system. The expressed protein bound T3 and DNA-cellulose with expected affinities, and the major binding site for the receptor protein was found to be located in the third intron of the RGH gene. This site displayed significantly higher affinity for the receptor protein than a previously described thyroid hormone response element (TRE) in the promoter of this gene, and also conferred stronger hormone responsiveness in vivo to a heterologous promoter. The data suggest that this novel TRE plays a major role in the regulation of rat growth hormone gene expression by thyroid hormones.
Animals↗
Contrasting developmental and tissue-specific expression of alpha and beta thyroid hormone receptor genes.
Thyroid hormones and their receptors (TRs) have critical functions in development. Here we show that a chicken TR beta cDNA clone encodes a receptor with a novel, short N-terminal domain. In vitro-expressed TR beta protein bound thyroid hormone with similar affinity as the chicken TR alpha. Comparison of expression of TR alpha and TR beta mRNAs throughout chicken development until 3 weeks post-hatching revealed ubiquitous expression of TR alpha mRNAs (in 14 different tissues) with some variations in levels, from early embryonic stages. In contast, expression of TR beta mRNA was restricted, occurring notably in brain, eye, lung, yolk sac and kidney, and was subject to striking developmental control, especially in brain where levels increased 30-fold upon hatching. Levels also sharply increased in late embryonic lung, but were relatively high earlier in embryonic eye and yolk sac. RNase protection analyses detected no obvious mRNAs for alpha and beta TRs with variant C-termini as demonstrated previously for the rat TR alpha gene. The data suggest a general role for TR alpha and specific developmental functions for TR beta, and that thyroid-dependent development involves temporal and tissue-specific expression of the TR beta gene.
Amino Acid Sequence↗
The chicken c-erbA alpha-product induces expression of thyroid hormone-responsive genes in 3,5,3'-triiodothyronine receptor-deficient rat hepatoma cells.
To determine the capacity of the chicken c-erbA (cTR-alpha) gene product in regulating expression of known thyroid hormone-responsive genes, both the cTR-alpha and the viral v-erbA genes were expressed in FAO cells, a rat hepatoma cell line defective for functional thyroid hormone receptors. Upon nuclear expression of the cTR-alpha protein the cells become responsive to thyroid hormone, as detected by expression of a number of genes (malic enzyme, phosphoenolpyruvate carboxykinase, and Na+/K(+)-ATPase) reported to be indirectly induced by the hormone in vivo. In addition, our data show that the c-erbA product directly activates the Moloney murine leukemia virus promoter in a ligand-dependent manner. The data show that the chicken c-erbA-alpha protein can modulate the expression of rat genes under either direct or indirect control by thyroid hormone.
Animals↗
Requirement for the C-terminal domain of the v-erbA oncogene protein for biological function and transcriptional repression.
In contrast to the normal thyroid hormone receptor, the v-erbA product fails to bind hormone due to mutations in the C-terminal ligand binding domain and thus appears to represent a hormone-independent, oncogenic transcription factor. Therefore, we asked whether or not the C-terminal domain of v-erbA is required for its biological activity and putative transcriptional control functions by analysing mutants with altered C-termini. A v-erbA protein truncated in the C-terminal domain lacked detectable biological activity in transformed erythroblasts and its transcriptional repression function with respect to the band 3 gene was abolished. The protein displayed a nuclear location and could still bind to DNA, indicating that the N-terminal region retained DNA-binding activity but was insufficient to produce characteristic v-erbA changes in erythroblasts. Another biologically defective v-erbA variant with a small frameshift towards the extreme C-terminus also failed to repress band 3, indicating a requirement for a specific C-terminal structure in repression. However, this mutant retained partial biological activity, stimulating erythroblasts to grow at a higher rate than cells containing a completely inactive, deleted v-erbA gene. The results demonstrate that the mutated hormone-binding domain, in addition to the DNA-binding region, is critical for v-erbA biological and transcriptional control functions.
Amino Acid Sequence↗
The v-erb A oncogene causes repression of erythrocyte-specific genes and an immature, aberrant differentiation phenotype in normal erythroid progenitors.
We have compared the effects of the v-erb A oncogene on proliferation and differentiation of normal erythroid progenitors with those of tyrosine kinase oncogenes, e.g. v-sea. For this, a v-erb A retrovirus containing the neomycin resistance gene as a selectable marker or, alternatively, a v-erb A-ts v-sea retrovirus were used to infect normal bone marrow cells. V-erb A induced the outgrowth of immature, erythropoietin(EPO)-dependent erythroid cells from infected bone marrow which ceased to proliferate and disintegrated after 9 to 18 divisions. In contrast, ts-v-sea erythroblasts grew for the expected 25 to 40 population doublings in the absence of EPO. Transcription of the erythrocyte genes carbonic anhydrase II and erythrocyte anion transporter was significantly inhibited in v-erb A infected erythroblasts, indicating that v-erb A alone was sufficient for the repression of the erythrocyte-specific genes observed in AEV-transformed leukemic cells. A detailed analysis of the differentiation phenotype induced by v-erb A in erythroblasts (in the presence or absence of a temperature-inactivated ts sea oncogene) indicates that v-erb A-erythroblasts express a partially mature, aberrant phenotype characterized by the coexpression of mature and immature differentiation antigens. This phenotype clearly differs from that induced by tyrosine kinase oncogenes in erythroid cells.
Animals↗