1,034 research outputs found
v-erbA overexpression is required to extinguish c-erbA function in erythroid cell differentiation and regulation of the erbA target gene CAII.
No full textUdgivelsesdato: 1991-NovThe v-erbA oncoprotein represents a retrovirus-transduced oncogenic version of the thyroid hormone (T3/T4) receptor c-erbA (type alpha). It contributes to virus-induced erythroleukemia by efficiently arresting differentiation of red cell progenitors and by suppressing transcription of erythrocyte-specific genes. Here, we show that v-erbA and c-erbA bind directly to sequences within the promoter of the erythrocyte-specific carbonic anhydrase II (CAII), a gene whose transcription is efficiently suppressed by v-erbA. This erbA-binding site confers thyroid hormone responsiveness to a heterologous promoter in transient expression experiments and is a target for efficient down-regulation of CAII transcription by the v-erbA oncoprotein. In stably transformed erythroblasts coexpressing the v-erbA oncoprotein and the c-erbA/T3 receptor at an approximately equimolar ratio, c-erbA activity is dominant over v-erbA. T3 efficiently induced erythroid differentiation in these cells, thus overcoming the v-erbA-mediated differentiation arrest. Likewise, T3 activated CAII transcription as well as transient expression of a T3-responsive reporter gene containing the CAII-specific erbA-binding site. The c-erbA-dependent activation of this CAII reporter construct could only be suppressed by very high amounts of v-erbA. Our results suggest that overexpression of v-erbA is required for its function as an oncoprotein
Transcriptional regulation by the thyroid hormone receptor and its viral counterpart : the P75gag-V-erbA oncoprotein
No full textThyroid hormone binds to specific DNA binding receptors (TRs) in the cell nucleus and thus regulates transcription during development and in some pathological disorders. The gene of the viral TR homologue, v-erbA, is one of two oncogenes encoded in the genome of avian erythroblastosis virus (AEV). Its protein product (P75gag-V-erbA) differs from TRa by several point mutations that result in the loss of hormone binding and in dominant negative effects on gene activation. TR and p75gag-v-erbA belong to a superfamily of steroid hormone receptors which also includes the retinoic acid receptors, RAR and RXR. The regulatory functions of TR and p75gag-v-erbA are pivotal in many regulatory pathways and biological processes. I and my coworkers have studied these functions by in vitro and in vivo methods.We have studied the DNA sequences through which TR and p75gag-v-erbA regulate transcription. Different in vitro binding assays and a PCR based selection approach showed that TR monomers bind with low affinity to DNA and that homodimers maintain a low cooperativity and relatively poor affinity for DNA. In contrast, TR/RAR and in particular TR/RXR heterodimers show a strong interaction with DNA response elements containing two hexameric half-sites in a head to head orientation (direct repeats). A direct repeat element spaced by 4 nucleotides confers the strongest TR/RXR binding and the best transcriptional activation when the spacer consists of a cytidine in the first and a pyrimidine in the third position. The data suggest that TR activates transcription as a heterodimer, and that it has a limited preference for the structure of the target genes.To understand the role of P75gag-v-erbA in oncogenesis, we studied a chicken erythroblast cell line which had been transformed by AEV. We showed that in order to repress TR mediated activation, P75gag-v-erbA depends on the structure of the response elements. P75gag-v-erbA represses activation as a heterodimer with RXR or possibly as a homodimer. P75gag-v-erbA only interacts with RXR on DNA, which is in contrast to TR which also form heterodimers in solution. The strongest repression was seen with an everted repeat. An in vitro selection showed that P75gag-V-erbA/RXR heterodimers have a relaxed specificity regarding the spacer between the two half-sites in both direct and inverted orientations. The results suggest that the target genes for P75gag-v-erbA is of the everted type.We have studied the ability of TR and P75gag-v-erbA to crosstalk with an RXR specific pathway. The results obtained by transient transfections show that unliganded TR and p75gag-v-erbA repress RXR mediated transactivation in chorion carcinoma cells. The response element from the promoter of the cellular retinol binding protein (CRBPII) was likewise repressed by TR, but only to 50% by P75gag-v-erbA We propose that these homologues inhibit gene activation by different mechanisms. Possible mechanisms include the formation of inactive RXR heterodimers, the physical exclusion of co-factors and/or interference with the basal transcription machinery.</p
v-erbA oncogene activation entails the loss of hormone-dependent regulator activity of c-erbA.
No full textUdgivelsesdato: 1990-Jun-15The 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
Recruitment of the Oncoprotein v-ErbA to Aggresomes
No full textAggresome formation, a cellular response to misfolded protein aggregates, is linked to cancer and neurodegenerative disorders. Previously we showed that Gag-v-ErbA (v-ErbA), a retroviral variant of the thyroid hormone receptor (TRα1), accumulates in and sequesters TRα1 into cytoplasmic foci. Here, we show that foci represent v-ErbA targeting to aggresomes. v-ErbA colocalizes with aggresomal markers, proteasomes, hsp70, HDAC6, and mitochondria. Foci have hallmark characteristics of aggresomes: formation is microtubule-dependent, accelerated by proteasome inhibitors, and they disrupt intermediate filaments. Proteasome-mediated degradation is critical for clearance of v-ErbA and T3-dependent TRα1 clearance. Our studies highlight v-ErbA's complex mode of action: the oncoprotein is highly mobile and trafficks between the nucleus, cytoplasm, and aggresome, carrying out distinct activities within each compartment. Dynamic trafficking to aggresomes contributes to the dominant negative activity of v-ErbA and may be enhanced by the viral Gag sequence. These studies provide insight into novel modes of oncogenesis across multiple cellular compartments.Biolog
Unliganded T3R, but not its oncogenic variant, v-erbA, suppresses RAR-dependent transactivation by titrating out RXR
No full textV-erbA is thought to be an antagonist of thyroid hormone receptor (T3R) function. Here we show that unliganded T3R, but not v-erbA, suppresses retinoic acid (RA)-dependent induction of the RAR-beta 2 promoter by competing for the common dimerization partner, the retinoid X receptor (RXR). Firstly, T3R suppression can be alleviated by co-transfection of RXR. Secondly, T3R, but not v-erbA, competes with RAR for RXR and causes the dissociation of a preformed RAR/RXR-RARE ternary complex in vitro. A single point mutation located in the dimerization interface of v-erbA (Pro349 to Ser) abolishes the transdominant phenotype when introduced at the respective position in T3R. The hypertransforming v-erbA variant r12, in which this mutation is reversed (Ser349 to Pro) suppresses RA-induced differentiation in chicken erythroid progenitors, while v-erbA does not. Our data thus suggest that unliganded T3R and v-erbA act as dominant suppressors through mechanistically distinct pathways
Activation of protein kinase C or cAMP-dependent protein kinase increases phosphorylation of the c-erbA-encoded thyroid hormone receptor and of the v-erbA-encoded protein.
No full textUdgivelsesdato: 1988-AugThe c-erbA proto-oncogene encodes a nuclear receptor for thyroid hormone (T3), which is believed to stimulate transcription from specific target promoters upon binding to cis-acting DNA sequence elements. The v-erbA oncogene of avian erythroblastosis virus (AEV) encodes a ligand-independent version of this nuclear receptor. The v-erbA product inhibits terminal differentiation of avian erythroblasts, presumably by affecting the transcription of specific genes. We show here that the c-erbA-encoded nuclear receptor (p46c-erbA) is phosphorylated on serine residues on two distinct sites. One of these sites, defined by the limit tryptic phosphopeptide 28SSQCLVK, is retained on the v-erbA-encoded P75gag-v-erbA protein. This site is located in the amino-terminal domain of these molecules, 21 amino acids upstream of the DNA-binding region. Phosphorylation of this site in both p46c-erbA and P75gag-v-erbA is enhanced 10-fold following treatment of cells with activators of either protein kinase C or cAMP-dependent protein kinase. Since cAMP-dependent protein kinase phosphorylates both p46c-erbA and P75gag-v-erbA in vitro at the same site as that observed in vivo, at least part of the cAMP-dependent phosphorylation of erbA molecules in cells could result from direct phosphorylation by this enzyme. The possible role phosphorylation may play in the function of the erbA-encoded transcriptional factors is discussed
Mechanism of transformation by v-ErbA: substitution for steroid hormone receptor function in self renewal induction
No full textV-ErbA, a mutated thyroid hormone receptor (TR) alpha cooperates with tyrosine kinase oncoproteins to induce fatal erythroleukemia in chicks. In vitro, v-ErbA employs a similar cooperation to induce sustained proliferation and arrest differentiation of committed erythroid progenitors. V-ErbA has been proposed to function as a dominant-negative c-ErbA/TR alpha, since it lacks an AF-2 transactivation domain and cannot be activated by hormone but retains the capacity to bind corepressors. However, v-ErbA fails to heterodimerize with the coreceptor RXR, exhibits an altered DNA binding specificity and fails to suppress the action of coexpressed TR alpha/c-ErbA in erythroblasts. In this paper, we identify a novel mechanism by which v-ErbA contributes to leukemogenesis. Recently, the glucocorticoid receptor (GR) was identified as a key regulator of proliferation and differentiation in normal erythroid progenitors. For this, the GR required to cooperate with endogenous receptor tyrosine kinases (c-Kit) and with the estrogen receptor (ER). Here, we demonstrate that v-ErbA can substitute for the ligand-activated GR and ER, inducing proliferation and arresting differentiation in the presence of specific GR and ER antagonists. Like the GR, v-ErbA required to cooperate with c-Kit for both proliferation induction and differentiation arrest, being devoid of biological activity in the absence of an active c-Kit. In self-renewing erythroblasts, v-ErbA not only repressed known v-ErbA target genes but also maintained high expression of c-myb. These biological activities of v-ErbA depended on distinct mutations in the DNA-binding domain. Additionally, v-ErbA acted as a partial, weak repressor of c-ErbA/TR alpha function in normal erythroblasts. It could be converted into a truly dominant-negative receptor by restoring its ability to heterodimerize with RX
New mechanisms of v-ErbA oncogene action revealed by SAGE analysis
No full textNational audienceThe v-erbA oncogene, carried by the Avian Erythroblastosis Virus, derives from the c-erbAalpha proto-oncogene which encodes the nuclear receptor for the thyroid hormone triiodothyronine (T3). In vitro, v-ErbA transforms erythroid progenitors by blocking their differentiation. It has been proposed that v-ErbA acts as a transcriptional repressor for genes normally activated by T3 and retinoic acid (RA), upregulated during the differentiation process. However, v-ErbA target genes responsible for transformation have yet to be identified. We used Serial Analysis of Gene Expression (SAGE) to analyze the transcriptome of avian erythroid progenitors (T2ECs), the natural target cells of v-erbA, expressing either an oncogenic form or a non-transforming form of verbA. The comparison of these two libraries revealed 83 genes differentially expressed between these two conditions. So far, the differential expression for 16 of them has been confirmed by real-time PCR on multiple independent repetitions. We observed that, among these v-ErbA target genes, some are activated by T3 and RA. This confirms that activation by T3 and RA receptors is indeed inhibited by v-ErbA. However, the expression of a vast majority of v-ErbA target genes did not vary in response to T3 and RA. These results suggest that v-ErbA must also act by T3- and RA-independent mechanisms in the transformation process. Furthermore, most v-erbA target genes do not vary during the differentiation process, in contrast to the expected role of v-erbA. In order to determine which major functions are deregulated by v-ErbA, we clustered the target genes identified according to the cellular function encoded by their corresponding proteins. We found that many of them are involved in the protein translation process. In order to understand the molecular mechanisms responsible for the coordinated variation of the v-ErbA target gene, we analyzed their promoter sequences and found the presence of c-myb binding sites as a signature motif of v-erbA target genes. This suggests a role for c-myb in the v-erbA-induced transformation process. Altogether, these studies demonstrate the involvement of new mechanisms pointing toward an unanticipated complexity of v-erbA oncogene action
C-erbA and v-erbA modulate growth and gene expression of a mouse glial precursor cell line
No full text8 pages, 7 figures.-- et al.The c-erbAa protooncogene coding for the thyroid
hormone (T3) receptor (TRa1 ) and the viral, mutated v-erbA oncogene were expressed in an immortal mouse glial cell line (B3.1 ) using retroviral vedors. c-erbAa
expression led to a decrease in cell proliferation in high
and low serum conditions, both in the presence and in
the absence of T3. In serum-free medium, c-erbAexpressing
cells (B3.1 + TRa1 ) were completely
arrested, whereas cells expressing v-erbA (B3.1 +
v-erbA) showed a higher DNA synthesis rate than
normal B3.1 cells. Although proliferation of all three cell
types was stimulated by platelet-derived growth factor
and basic fibroblast growth factor, differences were also
observed in the response to these agents. B3.1 + TRa1
cells were more sensitive to platelet-derived growth
factor than B3.1 and B3.1 + v-erbA cells. In contrast,
B3.1 cells responded to basic fibroblast growth factor
better than B3.1 + TRa1 or B3.1 + v-erbA cells. Insulinlike
growth factor I potentiated the action of plateletderived
growth factor and basic fibroblast growth factor.
Again, different responses to treatment with insulin-like
growth factor I alone were observed; B3.1 + TRa1 cells
did not respond to it, whereas B3.1 + v-erbA cells
showed a dramatic stimulation by this agent.
Interestingly, in the presence of 13, the blockade in
B3.1 + TRa1 cell proliferation was accompanied by the
down-regulation of the typical astrocytic genes, glial
fibrillary acidic protein and vimentin. These hormone
effects were not found in v-erbA-expressing cells. In
addition, v-erbA inhibited the basal expression of the
cyclic nucleotide phosphodiesterase gene, an
oligodendrocytic marker. In summary, our results show
that c-erbAa inhibits cell proliferation, whereas v-erbA
has the opposite effect, causing an increased cell
growth. Both genes also affect distinctly gene expression
in B3.1 cells. Together, these data suggest a role for
erbA genes in glial cell proliferation and differentiation.This work was supported by grants from the Plan Nacional de l+D (SAF92-
0396) and Fundación Ramón Areces. T. I. was supported by a postdoctoral
fellowship from the Gobierno Vasco and S. L. from the Programa de Formación
del Personal Investigador del Ministerio de Educación y ciencia.Peer reviewe
New mechanisms of v-ErbA oncogene action revealed by SAGE analysis
No full textNational audienceThe v-erbA oncogene, carried by the Avian Erythroblastosis Virus, derives from the c-erbAalpha proto-oncogene which encodes the nuclear receptor for the thyroid hormone triiodothyronine (T3). In vitro, v-ErbA transforms erythroid progenitors by blocking their differentiation. It has been proposed that v-ErbA acts as a transcriptional repressor for genes normally activated by T3 and retinoic acid (RA), upregulated during the differentiation process. However, v-ErbA target genes responsible for transformation have yet to be identified. We used Serial Analysis of Gene Expression (SAGE) to analyze the transcriptome of avian erythroid progenitors (T2ECs), the natural target cells of v-erbA, expressing either an oncogenic form or a non-transforming form of verbA. The comparison of these two libraries revealed 83 genes differentially expressed between these two conditions. So far, the differential expression for 16 of them has been confirmed by real-time PCR on multiple independent repetitions. We observed that, among these v-ErbA target genes, some are activated by T3 and RA. This confirms that activation by T3 and RA receptors is indeed inhibited by v-ErbA. However, the expression of a vast majority of v-ErbA target genes did not vary in response to T3 and RA. These results suggest that v-ErbA must also act by T3- and RA-independent mechanisms in the transformation process. Furthermore, most v-erbA target genes do not vary during the differentiation process, in contrast to the expected role of v-erbA. In order to determine which major functions are deregulated by v-ErbA, we clustered the target genes identified according to the cellular function encoded by their corresponding proteins. We found that many of them are involved in the protein translation process. In order to understand the molecular mechanisms responsible for the coordinated variation of the v-ErbA target gene, we analyzed their promoter sequences and found the presence of c-myb binding sites as a signature motif of v-erbA target genes. This suggests a role for c-myb in the v-erbA-induced transformation process. Altogether, these studies demonstrate the involvement of new mechanisms pointing toward an unanticipated complexity of v-erbA oncogene action
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