Mediator subunit MED1 modulates intranuclear dynamics of the thyroid hormone receptor.

The thyroid hormone receptors (TRs) mediate thyroid hormone (T3 )-dependent gene expression. The nuclear import and export signals that direct TR shuttling are well characterized, but little is known about factors modulating nuclear retention. We used fluorescence-based nucleocytoplasmic scoring and fluorescence recovery after photobleaching in transfected cells to investigate whether Mediator subunits MED1 and MED13 play a role in nuclear retention of TR. When MED1 was overexpressed, there was a striking shift towards a greater nuclear localization of TRß1 and the oncoprotein v-ErbA, subtypes with cytosolic populations at steady-state, and TRß1 intranuclear mobility was reduced. For TRα1, there was no observable change in its predominantly nuclear distribution pattern or mobility. Consistent with a role for MED1 in nuclear retention, the cytosolic TRα1 and TRß1 population were significantly greater in MED1-/- cells, compared with MED1+/+ cells. Exposure to T3 and epidermal growth factor, which induces MED1 phosphorylation, also altered TR intranuclear dynamics. Overexpression of miR-208a, which downregulates MED13, led to a more cytosolic distribution of nuclear-localized TRα1; however, overexpression of MED13 had no effect on TRß1 localization. The known binding site of MED1 overlaps with a transactivation domain and nuclear export signal in helix 12 of TR's ligand-binding domain (LBD). Coimmunoprecipitation assays demonstrated that TR's LBD interacts directly with exportins 5 and 7, suggesting that binding of exportins and MED1 to TR may be mutually exclusive. Collectively, our data provide evidence that MED1 promotes nuclear retention of TR, and highlight the dual functionality of helix 12 in TR transactivation and nuclear export.

Transcription factor NF-Y is a functional regulator of the transcription of core clock gene Bmal1.

The circadian clock enables organisms to adjust to daily environmental changes and synchronize multiple molecular, biochemical, physiological, and behavioral processes accordingly. In mammalian clock work, Bmal1 is the most important core clock gene, which works with another core clock gene Clock to drive the expression of other clock genes and clock-controlled genes. However, the regulation of Bmal1 has not been fully understood. This work was aimed at identifying the positive regulator(s) of Bmal1 transcription. A series of 5' deletion reporter constructs was generated, and binding site mutations of mouse Bmal1 promoter fragments were cloned into pGL3-basic and pGL3(R2.1)-basic plasmids and transfected into NIH 3T3 cells. Luciferase activity was either measured 48 h after transfection or recorded for 4 days after serum shock. DNA affinity precipitation assay was used to detect the transcription factors binding to Bmal1 promoter. Small interfering RNA against nuclear factor Y, subunit A (NF-YA) and dominant negative NF-YA were employed to study the role of NF-Y in Bmal1 transcription regulation. Deletion and mutation analyses identified two clusters of CCAAT/GC-boxes at the proximal region of Bmal1 promoter as the activating cis-elements. Bmal1 promoter activity was up-regulated by NF-Y and/or Sp1 and repressed by dominant negative NF-YA or siRNA against NF-YA. The activation of Bmal1 promoter activity by NF-Y and Sp1 was inhibited by Rev-Erbα. DNA affinity precipitation assay showed that NF-Y and Sp1 bound to the two CCAAT/GC clusters of Bmal1 promoter. These results indicate that NF-Y is a functional activator of Bmal1 transcription and it cooperates with Sp1 and Rev-Erbα to generate the daily cycle of Bmal1 expression.

Multiple novel signals mediate thyroid hormone receptor nuclear import and export.

Thyroid hormone receptor (TR) is a member of the nuclear receptor superfamily that shuttles between the cytosol and nucleus. The fine balance between nuclear import and export of TR has emerged as a critical control point for modulating thyroid hormone-responsive gene expression; however, sequence motifs of TR that mediate shuttling are not fully defined. Here, we characterized multiple signals that direct TR shuttling. Along with the known nuclear localization signal in the hinge domain, we identified a novel nuclear localization signal in the A/B domain of thyroid hormone receptor α1 that is absent in thyroid hormone receptor ß1 and inactive in the oncoprotein v-ErbA. Our prior studies showed that thyroid hormone receptor α1 exits the nucleus through two pathways, one dependent on the export factor CRM1 and the other CRM1-independent. Here, we identified three novel CRM1-independent nuclear export signal (NES) motifs in the ligand-binding domain as follows: a highly conserved NES in helix 12 (NES-H12) and two additional NES sequences spanning helix 3 and helix 6, respectively. Mutations predicted to disrupt the α-helical structure resulted in a significant decrease in NES-H12 activity. The high degree of conservation of helix 12 suggests that this region may function as a key NES in other nuclear receptors. Furthermore, our mutagenesis studies on NES-H12 suggest that altered shuttling of thyroid hormone receptor ß1 may be a contributing factor in resistance to thyroid hormone syndrome. Taken together, our findings provide a detailed mechanistic understanding of the multiple signals that work together to regulate TR shuttling and transcriptional activity, and they provide important insights into nuclear receptor function in general.

Palmitate alters the rhythmic expression of molecular clock genes and orexigenic neuropeptide Y mRNA levels within immortalized, hypothalamic neurons.

The control of energy homeostasis within the hypothalamus is under the regulated control of homeostatic hormones, nutrients and the expression of neuropeptides that alter feeding behavior. Elevated levels of palmitate, a predominant saturated fatty acid in diet and fatty acid biosynthesis, alter cellular function. For instance, a key mechanism involved in the development of insulin resistance is lipotoxicity, through increased circulating saturated fatty acids. Although many studies have begun to determine the underlying mechanisms of lipotoxicity in peripheral tissues, little is known about the effects of excess lipids in the brain. To determine these mechanisms we used an immortalized, clonal, hypothalamic cell line, mHypoE-44, to demonstrate that palmitate directly alters the expression of molecular clock components, by increasing Bmal1 and Clock, or by decreasing Per2, and Rev-erbα, their mRNA levels and altering their rhythmic period within individual neurons. We found that these neurons endogenously express the orexigenic neuropeptides NPY and AgRP, thus we determined that palmitate administration alters the mRNA expression of these neuropeptides as well. Palmitate treatment causes a significant increase in NPY mRNA levels and significantly alters the phase of rhythmic expression. We explored the link between AMPK and the expression of neuropeptide Y using the AMPK inhibitor compound C and the AMP analog AICAR. AMPK inhibition decreased NPY mRNA. AICAR also elevated basal NPY, but prevented the palmitate-mediated increase in NPY mRNA levels. We postulate that this palmitate-mediated increase in NPY and AgRP synthesis may initiate a detrimental positive feedback loop leading to increased energy consumption.

The effect of oncoprotein v-erbA on thyroid hormone-regulated genes in hepatocytes and their potential role in hepatocellular carcinoma.

Mutant forms of thyroid hormone receptor (TR) with dominant negative activity are frequently found in human hepatocellular carcinoma (HCC). Interestingly, the v-erbA oncogene, known to exert a dominant-negative effect on the expression of thyroid hormone (T3)-responsive genes, led to the development of HCC in a transgenic mouse model. Thus it is possible that the oncogenic activity of v-erbA in hepatocytes may be mediated by its dominant negative activity on T3-responsive genes. Microarray analysis was used to identify genes differentially expressed in murine hepatocytes in culture (AML12 cells) stably transfected with v-erbA and exposed to T3. The Affymetrix GeneChip Mouse Genome 430 2.0 array consisted of over 39,000 transcripts representing well-known genes. We have identified twenty T3-responsive genes that are negatively regulated by v-erbA at 3 h, and eighteen genes at 24 h, such as follistatin, activin ßC, thrombomodulin, Six1, Rasgrp3 and Ndrg2, as well as genes that are regulated by v-erbA only, such as angiopoietin 1 and Igfr2. We have identified T3 responsive genes that are dysregulated by v-erbA. These genes are known to be involved in carcinogenesis. Our studies may provide insight into the potential role of mutant forms of TR in the pathogenesis of HCC.

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.

A conformational switch in nuclear hormone receptors is involved in coupling hormone binding to corepressor release.

Nuclear hormone receptors are ligand-regulated transcription factors that modulate gene expression in response to small, hydrophobic hormones, such as retinoic acid and thyroid hormone. The thyroid hormone and retinoic acid receptors typically repress transcription in the absence of hormone and activate it in the presence of hormone. Transcriptional repression is mediated, in part, through the ability of these receptors to physically associate with ancillary polypeptides called corepressors. We wished to understand the mechanism by which corepressors are recruited to unliganded nuclear hormone receptors and are released on the binding of hormone. We report here that an alpha-helical domain located at the thyroid hormone receptor C terminus appears to undergo a hormone-induced conformational change required for release of corepressor and that amino acid substitutions that abrogate this conformational change can impair or prevent corepressor release. In contrast, retinoid X receptors appear neither to undergo an equivalent conformational alteration in their C termini nor to release corepressor in response to cognate hormone, consistent with the distinct transcriptional regulatory properties displayed by this class of receptors.

The erbA oncogene represses the actions of both retinoid X and retinoid A receptors but does so by distinct mechanisms.

Genetic lesions that function as dominant negative mutations in microbial systems have long been recognized. It is only relatively recently, however, that similar dominant negative mutations have been implicated as a basis for genetic and neoplastic disorders in vertebrates. We describe here a dissection of the actions of the erbA oncogene protein, an aberrant form of thyroid hormone receptor that acts as a dominant negative inhibitor of other nuclear hormone receptors. We demonstrate that the ErbA oncoprotein interferes with thyroid hormone and trans-retinoic acid receptors by competing for binding to the corresponding response elements. Heterodimerization of the ErbA oncoprotein with these receptors does not play an observable role in repression. In contrast, however, the ErbA oncoprotein does efficiently form a heterodimer with the retinoid X receptor (RXR) class of nuclear hormone receptors; complex formation enhances the DNA-binding properties of the ErbA protein but dramatically interferes with the ability of the RXR component to activate gene expression. Our results indicate that the erbA oncogene may play a previously unanticipated role in neoplasia by interfering with RXR function.

Nuclear hormone receptors involved in neoplasia: erb A exhibits a novel DNA sequence specificity determined by amino acids outside of the zinc-finger domain.

The erb A oncogene is a dominant negative allele of a thyroid hormone receptor gene and acts in the cancer cell by encoding a transcriptional repressor. We demonstrate here that the DNA sequence recognition properties of the oncogenic form of the erb A protein are significantly altered from those of the normal thyroid hormone receptors and more closely resemble those of the retinoic acid receptors; this alteration appears to play an important role in defining the targets of erb A action in neoplasia. Unexpectedly, the novel DNA recognition properties of erb A are encoded by an N-terminal region not previously implicated as playing this function in current models of receptor-DNA interaction. Two N-terminal erb A amino acids in particular, histidine 12 and cysteine 32, contribute to this phenomenon, acting in conjunction with amino acids in the zinc finger domain. The effects of the N-terminal domain can be observed at the level of both DNA binding and transcriptional modulation. Our results indicate that unanticipated determinants within the nuclear hormone receptors participate in DNA sequence recognition and may contribute to the differential target gene specificity displayed by different receptor forms.

Negative protein 1, which is required for function of the chicken lysozyme gene silencer in conjunction with hormone receptors, is identical to the multivalent zinc finger repressor CTCF.

The transcriptional repressor negative protein 1 (NeP1) binds specifically to the F1 element of the chicken lysozyme gene silencer and mediates synergistic repression by v-ERBA, thyroid hormone receptor, or retinoic acid receptor. Another protein, CCCTC-binding factor (CTCF), specifically binds to 50-bp-long sequences that contain repetitive CCCTC elements in the vicinity of vertebrate c-myc genes. Previously cloned chicken, mouse, and human CTCF cDNAs encode a highly conserved 11-Zn-finger protein. Here, NeP1 was purified and DNA bases critical for NeP1-F1 interaction were determined. NeP1 is found to bind a 50-bp stretch of nucleotides without any obvious sequence similarity to known CTCF binding sequences. Despite this remarkable difference, these two proteins are identical. They have the same molecular weight, and NeP1 contains peptide sequences which are identical to sequences in CTCF. Moreover, NeP1 and CTCF specifically recognize each other's binding DNA sequence and induce identical conformational alterations in the F1 DNA. Therefore, we propose to replace the name NeP1 with CTCF. To analyze the puzzling sequence divergence in CTCF binding sites, we studied the DNA binding of 12 CTCF deletions with serially truncated Zn fingers. While fingers 4 to 11 are indispensable for CTCF binding to the human c-myc P2 promoter site A, a completely different combination of fingers, namely, 1 to 8 or 5 to 11, was sufficient to bind the lysozyme silencer site F1. Thus, CTCF is a true multivalent factor with multiple repressive functions and multiple sequence specificities.

A conserved C-terminal sequence that is deleted in v-ErbA is essential for the biological activities of c-ErbA (the thyroid hormone receptor).

The thyroid hormone (T3) receptor type alpha, the c-ErbA alpha proto-oncoprotein, stimulates transcription of T3-dependent promoters, interferes with AP-1 activity, and induces erythroid differentiation in a ligand-dependent manner. The v-ErbA oncoprotein does not bind hormone and has lost all of these activities. Using c-ErbA/v-ErbA chimeras, we found that a deletion of 9 amino acids, conserved among many members of the nuclear receptor superfamily, which are located at the extreme carboxy terminus of c-ErbA alpha is responsible for loss of both transactivation and transcriptional interference activities. Single, double, and triple amino acid substitutions within this region completely abolished T3-dependent transcriptional activation, interference with AP-1 activity, and decreased T3 binding by c-ErbA alpha. However, the lower T3 binding by these mutants does not fully account for the loss of transactivation and transcriptional interference, since a c-ErbA/v-ErbA chimera which was similarly reduced in T3 binding activity has retained both of these functions. Deletion of homologous residues in the retinoic acid receptor alpha (RAR alpha) resulted in a similar loss of transactivation and transcriptional interference activities. The ability of c-ErbA alpha to induce differentiation of transformed erythroblasts is also impaired by all of the mutations introduced into the conserved carboxy-terminal sequence. We conclude that this 9-amino-acid conserved region is essential for normal biological function of c-ErbA alpha and RAR alpha and possibly other T3 and RA receptors.

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.

Cancer promoted by the oncoprotein v-ErbA may be due to subcellular mislocalization of nuclear receptors.

The retroviral v-ErbA oncoprotein is a highly mutated variant of the thyroid hormone receptor alpha (TRalpha), which is unable to bind T(3) and interferes with the action of TRalpha in mammalian and avian cancer cells. v-ErbA dominant-negative activity is attributed to competition with TRalpha for T(3)-responsive DNA elements and/or auxiliary factors involved in the transcriptional regulation of T(3)-responsive genes. However, competition models do not address the altered subcellular localization of v-ErbA and its possible implications in oncogenesis. Here, we report that v-ErbA dimerizes with TRalpha and the retinoid X receptor and sequesters a significant fraction of the two nuclear receptors in the cytoplasm. Recruitment of TRalpha to the cytoplasm by v-ErbA can be partially reversed in the presence of ligand and when chromatin is disrupted by the histone deacetylase inhibitor trichostatin A. These results define a new mode of action of v-ErbA and illustrate the importance of cellular compartmentalization in transcriptional regulation and oncogenesis.

Repression of vascular endothelial growth factor A in glioblastoma cells using engineered zinc finger transcription factors.

Angiogenic factors are necessary for tumor proliferation and thus are attractive therapeutic targets. In this study, we have used engineered zinc finger protein (ZFP) transcription factors (TFs) to repress expression of vascular endothelial growth factor (VEGF)-A in human cancer cell lines. We create potent transcriptional repressors by fusing a designed ZFP targeted to the VEGF-A promoter with either the ligand-binding domain of thyroid hormone receptor alpha or its viral relative, vErbA. Moreover, this ZFP-vErbA repressor binds its intended target site in vivo and mediates the specific deacetylation of histones H3 and H4 at the targeted promoter, a result that emulates the natural repression mechanism of these domains. The potential therapeutic relevance of ZFP-mediated VEGF-A repression was addressed using the highly tumorigenic glioblastoma cell line U87MG. Despite the aberrant overexpression of VEGF-A in this cell line, engineered ZFP TFs were able to repress the expression of VEGF-A by >20-fold. The VEGF-A levels observed after ZFP TF-mediated repression were comparable to those of a nonangiogenic cancer line (U251MG), suggesting that the degree of repression obtained with the ZFP TF would be sufficient to suppress tumor angiogenesis. Thus, engineered ZFP TFs are shown to be potent regulators of gene expression with therapeutic promise in the treatment of disease.

A choice between transcriptional enhancement and repression by the v-erbA oncoprotein governed by one nucleotide in a thyroid hormone responsive half site.

The v-erbA oncoprotein (P75gag-v-erbA) can repress thyroid hormone receptor induced transcriptional activation of target genes. A central question is how hormone responsive elements in a target gene determine the transcriptional regulation mediated by P75gag-v-erbA. We addressed this with receptors chimeric between P75gag-v-erbA and thyroid hormone receptor (TR) by testing their regulatory activities on thyroid hormone response elements (TREs) differing in the sequence of the consensus core recognition motif AGGTCA. We report here that enhances, TR dependent transcriptional activation is conferred by P75gag-v-erbA when the thymidine in the half site recognition motif is exchanged for an adenosine. The enhancement was independent of the DNA binding region of P75gag-v-erbA, whereas increased expression of corepressor abolished the enhancing effect. The data indicate that the enhancement results from an impaired DNA binding by the oncoprotein combined with an effective scavenging of corepressors. Our data thus suggest the P75gag-v-erbA indirectly can contribute to enhancement of thyroid hormone induced gene expression.

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.

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.

The v-erbA oncogene blocks expression of alpha2/beta1 integrin a normal inhibitor of erythroid progenitor proliferation.

T2EC are chicken erythrocytic progenitors that balance between self-renewal and differentiation as a function of response to specific growth factors. Their transformation by the v-erbA oncogene locks them into the self-renewal program. We show here that the expression of the VLA-2 integrin alpha2 subunit mRNA is downregulated by v-erbA and that VLA-2 engagement and clustering, brought about by treatment with an alpha2-specific antibody or by culture on the VLA-2 ligand collagen I, inhibits T2EC proliferation. From competition studies using antibodies, VLA-2 was shown to be involved in the collagen-induced response. While engagement of VLA-2 inhibited proliferation, it was not sufficient to induce differentiation. The transformation of T2EC by v-erbA decreased their interaction with collagen I and the VLA-2 brake on cell proliferation, which may account for the increased proliferation potential of transformed erythrocytic progenitors and for their shedding into the blood of infected chickens. Our data suggest that the interaction between erythroid progenitors and collagen, mediated by VLA-2, play a major role in the control of erythropoiesis in vitro and that this pathway is a target of the v-erbA oncogene.

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.