Modulation of expression of RA-regulated genes by the oncoprotein v-erbA.

Retinoic acid (RA) modulates the expression of genes involved in embryogenesis, development and differentiation processes in vertebrates. The v-erbA oncogene is known to exert a dominant-negative effect on the expression of RA-responsive genes. v-erbA belongs to a superfamily of transcription factors called nuclear receptors, which includes the retinoic acid receptors (RARs) responsible for mediating the effects of retinoic acid. While RA inhibits cell proliferation and promotes cell differentiation and apoptosis in a variety of tissues, v-erbA seems to play a role in oncogenesis, namely in the development of hepatocellular carcinoma (HCC) in a transgenic mouse model. In order to study the effect of v-erbA on RA-responsive genes, we used microarray analysis to identify genes differentially expressed in murine hepatocytes in culture (AML12 cells) stably transfected with v-erbA and exposed to RA for 3 h or 24 h. We have identified RA-responsive genes that are affected by v-erbA, as well as genes that are regulated by v-erbA alone. We have found that v-erbA can affect gene expression in the presence of RA and at the level of basal transcription. We have also identified a number of v-erbA-responsive genes that are known to be involved in carcinogenesis and which may play a role in the development of HCC.

Involvement of the TGF-beta and mTOR/p70S6Kinase pathways in the transformation process induced by v-ErbA.

v-ErbA is the oncogenic form of TRalpha/c-ErbA which transforms chicken erythrocytic progenitors by blocking differentiation. The oncogenic property of v-ErbA has been correlated with its ability to antagonize ligand-dependent gene activation by TRalpha/c-ErbA and retinoic acid receptors. Nevertheless, its cytoplasmic retention suggests that v-ErbA could interfere with intracellular signaling pathways. We demonstrate that only the transforming form of v-ErbA confers to chicken erythroid progenitors a TGF-beta independency and induces an activation of the mTOR/p70S6K pathway. In these cells, TGF-beta and mTOR/p70S6K pathways regulate the expression of a known target gene of v-ErbA, band3. This is the first demonstration that v-ErbA is able to modulate specifically some signaling pathways leading to changes in the expression level of a gene involved in transformation.

Multiple mutations contribute to repression by the v-Erb A oncoprotein.

The v-Erb A oncoprotein of avian erythroblastosis virus is derived from c-Erb A, a hormone-activated transcription factor. Notably, v-Erb A has sustained multiple mutations relative to c-Erb A and functions as a constitutive transcriptional repressor. We report here an analysis of the contributions of these different mutations to v-Erb A function. Our experiments demonstrate that two amino-acid differences between v-Erb A and c-Erb A, located in the 'I-box,' alter the dimerization properties of the viral protein, resulting in more stable homodimer formation, increased corepressor binding, and increased target gene repression. An additional amino-acid difference between v- and c-Erb A, located in helix 3 of the hormone binding domain, renders corepressor binding by the viral protein more resistant to release by thyroid hormone. Finally, we report that a C-terminal truncation in v-Erb A not only inhibits exchange of corepressor and coactivator, as previously noted, but also permits v-Erb A to recruit both SMRT and N-CoR corepressors, whereas c-Erb A is selective for N-CoR. The latter two mutations in v-Erb A also impair its ability to suppress c-Jun function in response to T3 hormone. We propose that the acquisition of oncogenic potential by the v-Erb A protein was a multistep process involving a series of mutations that alter the transcriptional repressive properties of the viral protein through multiple mechanisms.

Nuclear export of the oncoprotein v-ErbA is mediated by acquisition of a viral nuclear export sequence.

v-ErbA, an oncogenic derivative of the thyroid hormone receptor alpha (TRalpha) carried by the avian erythroblastosis virus, contains several alterations including fusion of a portion of avian erythroblastosis virus Gag to its N terminus, N- and C-terminal deletions, and 13 amino acid substitutions. Nuclear export of v-ErbA occurs through a CRM1-mediated pathway. In contrast, nuclear export of TRalpha and another isoform, TRbeta, is CRM1-independent. To determine which amino acid changes in v-ErbA confer CRM1-dependent nuclear export, we expressed a panel of green and yellow fluorescent protein-tagged mutant and chimeric proteins in mammalian cells. The sensitivity of subcellular trafficking of these mutants to leptomycin B (LMB), a specific inhibitor of CRM1, was assessed by fluorescence microscopy. Our data showed that a nuclear export sequence resides within a 70-amino acid domain in the C-terminal portion of the p10 region of Gag, and in vitro binding assays demonstrated that Gag interacts directly with CRM1. However, a panel of ligand-binding domain mutants of v-ErbA lacking the Gag sequence exhibited greater nuclear localization in the presence of LMB, suggesting that the various amino acid substitutions/deletions may cause a conformation shift, unmasking an additional CRM1-dependent nuclear export sequence. In contrast, the altered DNA-binding domain of the oncoprotein did not contribute to CRM1-dependent nuclear export. Heterokaryon experiments revealed that v-ErbA did not undergo nucleocytoplasmic shuttling when the CRM1 export pathway was blocked by LMB treatment, suggesting that the ability to follow the export pathway used by TRalpha has been lost by the oncoprotein during its evolution. Our findings thus point to the intriguing possibility that acquisition of altered nuclear export capabilities contributes to the oncogenic properties of v-ErbA.

Dimerization interfaces of v-erbA homodimers and heterodimers with retinoid X receptor alpha.

The oncoprotein v-ErbA, a member of the zinc finger transcription factor superfamily, is a mutated version of thyroid hormone receptor alpha1 that is virtually incapable of binding T3. v-ErbA and other members of this family can bind as homodimers and heterodimers with retinoid X receptors to specific DNA sequences arranged as direct, inverted, or everted repeats. At least two regions in the C-terminal domain, the I box (10 and 11 helices in v-ErbA and thyroid hormone receptors) and the 20-amino acid region are involved in dimerization. However, it has not been entirely understood how these receptors dimerize on differently oriented core motifs and whether the domain(s) responsible for homodimerization and heterodimerization are identical. Therefore, deletions of the entire 20-amino acid region, the 10 helix, the 11 helix, and point mutations within these regions of v-ErbA were made by site-directed mutagenesis. The mutant proteins were tested for their ability to form v-ErbA homodimers and heterodimers with retinoid X receptor alpha on differently oriented core motifs by electrophoretic mobility shift assay. Transient transfections were performed to determine the dominant negative activity of the v-ErbA mutants. The data indicate that different dimerization interfaces are used for v-ErbA homodimerization and heterodimerization with retinoid X receptor alpha, and different dimerization interfaces are used on differently oriented core motifs. The data are of general interest because the information improves our understanding of the role of these dimerization interfaces in the mechanism of action not only of v-ErbA but also of other members of the superfamily.

Thyroid hormone receptor, v-ErbA, and chromatin.

The thyroid hormone receptor and the highly related viral oncoprotein v-erbA are found exclusively in the nucleus as stable constituents of chromatin. Unlike most transcriptional regulators, the thyroid hormone receptor binds with comparable affinity to naked and nucleosomal DNA. In vitro reconstitution experiments and in vivo genomic footprinting have delineated the chromatin structural features that facilitate association with the receptor. Chromatin bound thyroid hormone receptor and v-erbA generate Dnase I hypersensitive sites independent of ligand. The unliganded thyroid hormone receptor and v-erbA associate with a corepressor complex containing NCoR, SIN3, and histone deacetylase. The enzymatic activity of the deacetylase and a chromatin environment are essential for the dominant repression of transcription by both the unliganded thyroid hormone receptor and v-erbA. In the presence of ligand, the thyroid hormone receptor undergoes a conformational change that weakens interactions with the corepressor complex while facilitating the recruitment of transcriptional coactivators such as p300 and PCAF possessing histone acetyltransferase activity. The ligand-bound thyroid hormone receptor directs chromatin disruption events in addition to histone acetylation. Thus, the thyroid hormone receptor and v-erbA make very effective use of their stable association with chromatin and their capacity to alter the chromatin environment as a major component of the transcription regulation process. This system provides an exceptionally useful paradigm for investigating the structural and functional consequences of targeted chromatin modification.

Requirements for repression of retinoid X receptor by the oncoprotein P75gag-v-erbA and the thyroid hormone receptors.

The oncogenic counterpart of thyroid hormone receptor-alpha (TRRalpha), denoted P75gag-v-erbA, has served as a paradigm for the ability of TRs to repress basal levels of transcription. We show here that the retinoid X receptor (RXR), when activated by its specific ligand SR11237, is repressed by both the normal TRalpha and the P75gag-v-erbA. The repression caused by the two proteins is distinct and dependent on both the cell type and the hormone-response element through which RXR acts. In HeLa cells only TR repressed efficiently through the palindromic 2xIR0 element, whereas the proteins were equally efficient in JEG cells. This demonstrates that proteins distinct in the two cell types mediate the repression. RXR-dependent induction via the natural response element of the cellular retinol-binding protein (CRBPII) gene was likewise (> or = 50%) repressed by TR, whereas P75gag-v-erbA did not repress during the same conditions. Furthermore, P75gag-v-erbA and its variants v-erbAtd359 (lacking repressing activity on TR) and v-erbAr12 (a highly active repressor of TR) efficiently repressed induction by a hybrid protein consisting of the DNA- binding domain of Gal4 and the ligand-binding region of RXR. The viral proteins did not, however, associate with RXR unless the two partners were allowed to heterodimerize upon binding to a specific response element, such as the 2xIR0 element or that of the CRBPII gene. In conclusion, we suggest that the efficient repression seen with the the 2xIR0 element is due to heterodimerization of TR or the viral oncoproteins with RXR and a concomitant inhibition of binding of the RXR-specific ligand that results in an inability of RXR to attract a cell type-specific cofactor. In addition, the data suggest that the interaction between RXR and P75gag-v-erbA on the CRBPII element is too weak to inhibit RXR from binding a ligand and therefore also to repress.

Role of the different RAR isoforms in controlling the erythrocytic differentiation sequence. Interference with the v-erbA and p135gag-myb-ets nuclear oncogenes.

Little is known as to how the nuclear oncogenes v-erbA and p135gag-myb-ets do transform cells. The elucidation of their molecular mechanisms of action requires the identification of relevant target genes. We analysed the possibility for the RARbeta gene to represent such a target gene. We first show that the RARbeta gene induction is a specific and direct process, requiring the continuous presence of retinoids and under the control of the RARalpha isoform exclusively. We then show that the expression of either the v-erbA or the p135gag-myb-ets oncogene is not sufficient to block the RARbeta gene induction. We confirmed the loss of RARbeta gene response in certain cell lines but we discarded the possibility that this loss might represent a necessary step for cell lines immortalization. We further show that the RARalpha isoform activation is necessary and sufficient to induce the growth inhibition and the differentiation stimulation characteristic for the commitment-inducing ability of retinoids in chicken erythrocytic progenitor cells. We therefore propose a model showing that RARalpha but not RARbeta is the key mediator for commitment to differentiation and that it should control two different set of genes whose expression is differentially affected by the v-erbA and the p135gag-myb-ets oncogenes.

v-erbA oncogene initiates ultrastructural changes characteristic of early and intermediate events of meiotic maturation in Xenopus oocytes.

The growth-promoting properties of the retroviral v-erbA oncogene, a highly mutated version of the chicken thyroid hormone receptor (TR) alpha, have so far exclusively been linked to dominant repression of the antimitogenic roles of TR and retinoic acid receptors. Here we show that when expressed in Xenopus oocytes v-ErbA induced ultrastructural changes characteristic of early and intermediate events of meiotic maturation by activating gene transcription. v-ErbA-induced maturation events occurred without activation of the cAMP/maturation-promoting factor signal pathway and were arrested prior to meiotic spindle formation. The effects of v-ErbA were not mimicked by a dominant negative in vitro-generated mutant of human TR, suggesting that v-ErbA can contribute to cell cycle reentry by interference with regulatory pathways distinct from those involving TR. Interestingly, a portion of v-ErbA expressed in oocytes was present at the cytoplasmic fibrils of the nuclear pore complexes, suggesting that in addition to its intranuclear function v-ErbA may modulate nucleocytoplasmic transport.

Mechanism of transformation by v-ErbA: substitution for steroid hormone receptor function in self renewal induction.

V-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 RXR.

Fatty acids involved in signal cross-talk between cell membrane and nucleus.

The results presented underline the fact that the nature and the concentration of the non-esterified fatty acids (NEFAs) liberated from membrane lipids, particularly the essential ones issued from lipid nutrition, clearly belong to a large group of factors (hormones, retinoids, growth factors, cytokines...) which control the shift between cell multiplication and differentiation. NEFAs act on this shift, per se or after being metabolized, by influencing, as second messengers or modulators, the intertwined mechanisms of action of growth factors and steroid hormones. These results may explain the molecular links which exist between endocrinology, oncology and nutrition.

The oncoprotein P75gag-v-erbA represses thyroid hormone induced transcription only via response elements containing palindromic half-sites.

The v-erbA oncoprotein P75gag-v-erbA, derived from the thyroid hormone receptor alpha (TR alpha), functions as a transdominant transcriptional repressor. The mechanism by which P75gag-v-erbA acts is however poorly characterized. Here, we show that repression of TR alpha mediated transcription by P75gag-v-erbA in transformed erythroblasts is dependent on the structure of the thyroid hormone response element to which it binds. A very efficient repression was seen with hormone response elements having half-sites organized as everted repeats (ER), whereas repression was inefficient with directly repeated half-sites (DR). Promoters containing half-sites organized as an inverted palindrome (IR) gave an intermediate repression. Although P75gag-v-erbA failed to associate with the ligand binding domain of retinoid X (RXR) receptor in a two-hybrid test, the oncoprotein in nuclear extracts from transformed cells heterodimerised quantitatively with RXR upon binding to response elements of the DR type. On the other hand, both RXR/P75gag-v-erb heterodimers and other types of dimers formed on ER elements. P75gag-v-erbA also failed to bind to elements that contained only one half-site in vivo and in vitro. The data demonstrate that P75gag-v-erbA represses gene expression efficiently as a dimer, and suggest that thyroid hormone responsive genes that may be targets for the action of the oncoprotein are repressed most efficiently if they contain elements of the ER type.

An identical effect mediated by thyroid deficiency or oncogene v-erbA in the chick embryo.

We have shown earlier that the association of v-myc and v-erbA (MAHEVA construct) is responsible for the appearance of a specific phenotype in chick embryos inoculated at E3. This phenotype comprises rapidly growing heart rhabdomyomas (induced by v-myc alone) and within these tumors secondarily appearing cartilage nodules (Bachnou et al., Oncogene 6: 1041-1047, 1991). Here we report that v-erbA can be replaced by thyroid deficiency. When decapitated embryos were inoculated with virus MC29 (v-myc alone) or when v-myc inoculated embryos were treated with thiourea, 100% of the embryos reaching E17 to E19 displayed tumoral hearts bearing cartilage nodules. We thus report in vivo evidence that v-erbA acts by antagonizing the effects of thyroid hormones. Remarkably, thyroid deficiency rendered embryos more sensitive to the effect of v-myc, since 100% developed heart rhabdomyomas and cartilage nodules, versus about 70% affected when either v-myc or MAHEVA were inoculated. Thyroid deficiency did not alter the species-specific character of transdifferentiation, since only chick but not quail embryos developed cartilage nodules after thyroidectomy or MAHEVA infection.

Enhancement of nuclear receptor transcriptional signalling.

Glucocorticoids and thyroid hormones induce complex responses in about every mammalian tissue. These effects are mediated by the transcription factor function of the corresponding nuclear receptors, which in most cases achieve the observed regulatory strength in synergy with other factors. Here we describe the functional interaction of the glucocorticoid receptor (GR) with liver-specific transcription factors, the functional synergy of GR with the thyroid hormone receptor (TR), the synergizing sub-domains of the TR, and finally the direct interaction of the GR with other proteins.

Constitutive transactivation by the thyroid hormone receptor and a novel pattern of activity of its oncogenic homolog v-ErbA in Xenopus oocytes.

In Xenopus oocytes, the rat thyroid hormone receptor alpha (rTR alpha), but not its oncogenic homolog v-ErbA, constitutively activated thyroid hormone (T3)-responsive reporter genes at four positive thyroid hormone-responsive elements (TREs). At a subset of the positive TREs tested, the addition of T3 resulted in a further enhancement of reporter gene activation. In contrast, both rTR alpha and v-ErbA functioned as constitutive activators when bound to the clone 122 TREs, which are induced by unliganded TR in mammalian cells. Therefore, the responses of the ligand-independent activation domains of TR and v-ErbA to cell-specific and TRE-mediated induction are not equivalent. Coexpression of the human retinoid X receptor alpha (hRXR alpha) enhanced both ligand-dependent and ligand-independent activation functions of rTR alpha and human TR beta (hTR beta) at a palindromic TRE (TREp). An endogenous TR activity mediated T3 induction of TREp, while being repressed by an in vitro-generated dominant negative mutant of TR. T3-mediated gene activation, by exogenous or endogenous TR, was repressed by v-ErbA at three positive TREs, but not at the TRE from the third intron of the rat GH gene (rGH3 TRE). Interestingly, preinjection of nuclear protein extract from anterior pituitary cells converted v-ErbA into a constitutive activator at rGH3 TRE. The pituitary-specific factor Pit-1/GHF-1 or hRXR alpha did not induce activation by v-ErbA at rGH3 TRE, suggesting that the dominant negative phenotype of v-ErbA can be abolished by direct or indirect interactions with other nuclear factors.

Bicistronic retroviral vector reveals capacity of v-erbA to induce erythroleukemia and to co-operate with v-myb.

Previous studies have shown that v-erbA and v-myb can induce the proliferation of avian erythroid cells in culture. To study the combined effects of v-erbA and v-myb, the two oncogenes were engineered into a retrovirus bicistronic vector with an internal ribosomal entry site (IRES) or into a vector with a splice acceptor (SPL). This allowed coexpression of the two proteins and a comparison with the same vector containing either v-erbA or v-myb only. Both the erbA IRES and the erbA/myb IRES virus constructs transformed erythroid cells after infection of bone marrow or blastoderm cultures. The erbA/myb IRES virus exhibited a 5-10-fold higher transformed colony forming efficiency than the erbA IRES virus in the blastoderm assay. Surprisingly, when injected into chicken embryos in the presence of helper virus, both viruses induced an erythroleukemia in about half of the animals. In contrast, no leukemia was observed with a myb IRES virus, with spliced vectors containing v-erbA alone or v-erbA in combination with v-myb, nor with erbA IRES and erbA/myb IRES viruses produced in the absence of helper virus. The average latency of leukemia induction was shorter for the erbA/myb IRES virus (3.5 weeks) than for the erbA IRES virus (5 weeks). Nevertheless, for both viruses the leukemic blasts retained full factor dependence for growth. These results show that v-erbA is capable of inducing an erythroleukemia when expressed by a high titer bicistronic retrovirus under conditions of virus spreading and that its in vitro and in vivo transforming potential can be enhanced by v-myb.

Two silencing sub-domains of v-erbA synergize with each other, but not with RXR.

The thyroid hormone receptor (TR) and the retinoic acid receptor (RAR) induce gene expression in the presence of specific ligand and repress transcription in the absence of hormone. This repression is mediated by an active silencing mechanism rather then by interference with DNA binding activators. V-erbA, a variant form of TR which is unable to bind hormone, represents a constitutive repressor. Here we show, using fusion proteins with the GAL4 DNA binding domain, that the minimal silencing domain of v-erbA extends from amino acids 389 to 632 and that internal deletions within this domain retain at least some repression function. Co-transfection experiments of different deletion mutants indicate that the silencing domain is composed of at least two sub-domains which are non-functional when tested individually. When combined in a heterodimeric complex, they synergize such that silencing activity is regained. In contrast to the retinoic acid receptor the retinoid X receptor does not contain a silencing domain. In addition it is unable to cooperate with the repression function of TR or v-erbA in a heterodimer.

Factors influencing nuclear receptors in transcriptional repression.

Members of the steroid receptor superfamily, like other transcription factors, can function as transcriptional inducers as well as repressors of transcription. The mechanisms by which repression is achieved seem to be specific for the factors and regulatory sequences involved. Silencing activity is conferred by the DNA bound v-ERBA, which is able to repress the activity of a complete or of a minimal promoter. Removal of the T3 or RA ligands converts the activated form of TR or RAR into a silencing conformation. Ligand-free TR, RAR or v-ERBA synergize with the DNA-bound negative protein 1 (NeP1) in a specific silencer sequence. In contrast to silencing, competitive repression is seen for specific negative hormone response elements. These elements are characterized by the presence of binding sites for other transcription factors.

Negative transcriptional regulation by nuclear receptors.

Steroid and thyroid hormones, and vitamins A and D bind to nuclear receptors, which act as ligand-modulated transcription factors. In many cases, ligand-activated nuclear receptor binds to positively acting hormone response elements (p-HREs) to induce gene transcription. However, ligand-activated receptors also repress transcription of specific genes and several mechanisms that account for negative regulation have recently emerged. One major form of negative regulation is based on transcriptional interference between nuclear receptors and other transcription factors, such as AP-1. In this case, the liganded receptor prevents AP-1 or other positively acting transcription factors from fruitful interaction with the transcription initiation complex. A second form of negative regulation is based on binding of nuclear receptors to specialized negative HREs (n-HREs). Binding of unliganded receptor to such an element results in constitutive activation, which is terminated by the binding of ligand. While transcriptional interference with AP-1 has been described for many members of the nuclear receptor family, negative regulation through n-HREs so far has been shown only for one of the thyroid hormone receptors. However, this type of negative regulation is likely to be widespread.