RESUMO
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.
Assuntos
Subunidade 1 do Complexo Mediador/metabolismo , Proteínas Oncogênicas v-erbA/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Sítios de Ligação , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Citosol/metabolismo , Fibroblastos/metabolismo , Expressão Gênica , Genes erbA , Células HeLa , Humanos , Carioferinas/metabolismo , Complexo Mediador/metabolismo , Camundongos , Fosforilação , Transporte Proteico , Receptores beta dos Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/metabolismo , TransfecçãoRESUMO
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.
Assuntos
Fatores de Transcrição ARNTL/biossíntese , Fator de Ligação a CCAAT/metabolismo , Relógios Circadianos/fisiologia , Regulação da Expressão Gênica/fisiologia , Elementos de Resposta/fisiologia , Transcrição Gênica/fisiologia , Fatores de Transcrição ARNTL/genética , Animais , Fator de Ligação a CCAAT/genética , Camundongos , Células NIH 3T3 , Proteínas Oncogênicas v-erbA/genética , Proteínas Oncogênicas v-erbA/metabolismo , RNA Interferente Pequeno/genética , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismoRESUMO
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.
Assuntos
Sinais de Localização Nuclear/metabolismo , Receptores alfa dos Hormônios Tireóideos/metabolismo , Receptores beta dos Hormônios Tireóideos/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Motivos de Aminoácidos , Animais , Células HeLa , Humanos , Mutação , Sinais de Localização Nuclear/genética , Proteínas Oncogênicas v-erbA/genética , Proteínas Oncogênicas v-erbA/metabolismo , Estrutura Terciária de Proteína , Ratos , Receptores alfa dos Hormônios Tireóideos/genética , Receptores beta dos Hormônios Tireóideos/genéticaRESUMO
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.
Assuntos
Carcinoma Hepatocelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes erbA , Neoplasias Hepáticas/metabolismo , Proteínas Oncogênicas v-erbA/genética , Receptores dos Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/metabolismo , Animais , Linhagem Celular , Genes Dominantes , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Oncogênicas , Proteínas Oncogênicas v-erbA/metabolismo , Fatores de TempoRESUMO
Aggresome 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 T(3)-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.
Assuntos
Corpos de Inclusão/metabolismo , Proteínas Oncogênicas v-erbA/metabolismo , Alpharetrovirus/genética , Alpharetrovirus/metabolismo , Transporte Biológico , Biomarcadores/metabolismo , Dineínas/metabolismo , Eritroblastos/citologia , Eritroblastos/metabolismo , Eritroblastos/virologia , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Células HeLa , Desacetilase 6 de Histona , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Filamentos Intermediários/metabolismo , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Proteínas Oncogênicas v-erbA/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vimentina/metabolismoRESUMO
Disruption of the transforming growth factor-beta (TGF-beta) pathway is observed in the majority of cancers. To further understand TGF-beta pathway inactivation in cancer, we stably expressed the v-ErbA oncoprotein in TGF-beta responsive cells. v-ErbA participates in erythroleukemic transformation of cells induced by the avian erythroblastosis virus (AEV). Here we demonstrate that expression of v-ErbA was sufficient to antagonize TGF-beta-induced cell growth inhibition and that dysregulation of TGF-beta signaling required that v-ErbA associate with the Smad4 which sequesters Smad4 in the cytoplasm. We also show that AEV-transformed erythroleukemia cells were resistant to TGF-beta-induced growth inhibition and that TGF-beta sensitivity could be recovered by reducing v-ErbA expression. Our results reveal a novel mechanism for oncogenic disruption of TGF-beta signaling and provide a mechanistic explanation of v-ErbA activity in AEV-induced erythroleukemia.
Assuntos
Proteínas Oncogênicas v-erbA/metabolismo , Transdução de Sinais/fisiologia , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , Proliferação de Células , Eritroblastos/metabolismo , Humanos , Mutação , Proteínas Oncogênicas v-erbA/química , Proteínas Oncogênicas v-erbA/genética , Proteína Smad4/análise , Fator de Crescimento Transformador beta/farmacologiaRESUMO
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.
Assuntos
Neoplasias/metabolismo , Proteínas Oncogênicas v-erbA/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Citoplasma/metabolismo , Dimerização , Histona Desacetilases/metabolismo , Histonas/metabolismo , Carioferinas/metabolismo , Ligantes , Camundongos , Células NIH 3T3 , Proteínas Oncogênicas v-erbA/genética , Transporte Proteico/fisiologia , Receptor X Retinoide beta/metabolismo , Receptores alfa dos Hormônios Tireóideos/genética , Receptores alfa dos Hormônios Tireóideos/metabolismo , Proteína Exportina 1RESUMO
The oncoprotein v-erbA is a mutated form of TRalpha1 that is unable to bind thyroid hormone (T3). V-erbA homodimerizes or heterodimerizes with retinoid X receptor (RXR) on core motifs arranged as direct, everted, or inverted repeats (DRs, ERs, or IRs). We created a series of v-erbA mutants in order to obtain a better understanding of the role of v-erbA homodimers versus v-erbA-RXR heterodimers in the dominant negative activity of v-erbA on ERs (the most potent v-erbA response elements). We found that one of these mutants, v-erbA mutant E325A, is able to homodimerize but unable to heterodimerize with RXR on ERs. Our data also suggest that v-erbA homodimers interact preferentially with the corepressor NCoR over SMRT and that the interaction with corepressors is stronger with v-erbA homodimers over v-erbA-RXR heterodimers. Furthermore, functional studies showed that v-erbA homodimers rather than v-erbA-RXR heterodimers mediate the dominant negative activity of v-erbA on ERs.
Assuntos
Proteínas Oncogênicas v-erbA/genética , Proteínas Oncogênicas v-erbA/metabolismo , Sequências Repetitivas de Ácido Nucleico , Sequência de Aminoácidos , Animais , Ligação Competitiva , Células COS , Chlorocebus aethiops , Dimerização , Sequências Hélice-Alça-Hélice/genética , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Oncogênicas v-erbA/química , Mutação Puntual/genética , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Elementos de Resposta , Receptor X Retinoide alfa/química , Receptor X Retinoide alfa/metabolismoRESUMO
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.
Assuntos
Transporte Ativo do Núcleo Celular , Carioferinas/fisiologia , Proteínas Oncogênicas v-erbA/fisiologia , Receptores Citoplasmáticos e Nucleares , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Ácidos Graxos Insaturados/química , Deleção de Genes , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Carioferinas/química , Carioferinas/metabolismo , Ligantes , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Células NIH 3T3 , Proteínas Oncogênicas v-erbA/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Transfecção , Proteína Exportina 1RESUMO
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.
Assuntos
Glioblastoma/metabolismo , Glioblastoma/terapia , Fatores de Transcrição/genética , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Dedos de Zinco/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Glioblastoma/irrigação sanguínea , Glioblastoma/genética , Humanos , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Proteínas Oncogênicas v-erbA/genética , Proteínas Oncogênicas v-erbA/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Transfecção , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Thyroid hormone receptor alpha (TRalpha) and the oncoprotein v-erbA (a mutated form of TRalpha incapable of binding T3) bind as heterodimers with retinoid X receptor (RXR) to DNA sequences with different orientations of AGGTCA half sites. v-erbA can also form homodimers, whereas, TRalpha1 homodimerizes poorly. Therefore, in order to obtain a better understanding for the distinct homodimerization properties between TRalpha1 and v-erbA, we created chimeras between these two receptors and tested their abilities to homodimerize on direct and everted repeats (DRs, ERs). We found that the enhanced homodimerization properties of v-erbA compared to TRalpha1 map to isoleucine at position 339 in conjunction with serine at position 351 and alanine at position 358. Our data indicate that the methyl group in isoleucine at position 339 plays an important role in v-erbA homodimerization, particularly on ER 6. Functional studies with I339V+S351P+A358T, a v-erbA mutant unable to homodimerize but still able to heterodimerize with RXR on ERs and DRs, indicate that v-erbA-RXR heterodimers mediate the dominant negative activity of v-erbA on DRs. However, the repressor activity of this mutant is weaker than that of the wild type v-erbA on ERs, suggesting that v-erbA homodimers rather than v-erbA-RXR heterodimers mediate the potent dominant negative activity of v-erbA on ERs.
Assuntos
Proteínas Oncogênicas v-erbA/genética , Receptores alfa dos Hormônios Tireóideos/genética , Sequência de Aminoácidos/fisiologia , Animais , Dimerização , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Oncogênicas v-erbA/química , Proteínas Oncogênicas v-erbA/metabolismo , Mutação Puntual , Estrutura Secundária de Proteína , Receptores do Ácido Retinoico/metabolismo , Proteínas Recombinantes de Fusão/genética , Receptores X de Retinoides , Alinhamento de Sequência , Receptores alfa dos Hormônios Tireóideos/química , Receptores alfa dos Hormônios Tireóideos/metabolismo , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
We have recently observed that many of our sarcoma patients presented also with thyroid disorders. Literature data are almost unavailable on this topic. The relationship between the sarcoma and thyroid disorders is examined. Retrospective analysis of files of patients with sarcoma and clinically overt thyroid disorders was carried out. Of the 375 patients with soft tissue sarcomas (STS) and 235 with bone sarcoma (BS) including small blue round cell tumors (SBRC), 28 patients (4.6%) had an associated significant thyroid disorder. The types of sarcoma were mainly liposarcoma followed by malignant fibrous histiocytoma, leiomyosarcoma and bone sarcoma. The primary sites were mainly limb and trunk. The interval between the diagnosis of the thyroid disorder and the sarcoma varied between -14 years (thyroid first) and +16.5 years (thyroid later) with a median of -0.2 years. Thyroid disorders included goiter, thyroiditis and carcinoma. There are both basic-science and clinical evidence to a possible common pathway that leads to the association between overt thyroid disorders and sarcomas of bone or soft tissues. Oncogene erbA activity is related to thyroid receptors to T3 and to development of sarcoma. Cross talk of the sarcoma oncogene and the erbA might contribute to the development of sarcoma. The thyroid hormone receptor and the highly related viral oncoprotein v-erbA are found exclusively in the nucleus as stable constituents of chromatin. It has been shown that v-erbA can block the spontaneous differentiation in erythroid cells transformed by various retroviral oncogenes. V-erbA can alter the spectrum of neoplasia induced by the v-src oncogene, which causes predominantly sarcomas and erythroblastosis in chicks. The erbA can cooperate with other oncogenes such as v-erbB or with v-fms, v-ras, and c-kit. Cooperation with v-myc may play a role in the development of rhabdomyosarcoma especially in thyroid hormone deficiency state. The possible clinical implications are the need to screen patients with sarcoma to thyroid disorders, and patients with thyroid disorders for malignant diseases.
Assuntos
Neoplasias Ósseas/complicações , Sarcoma/complicações , Sarcoma/etiologia , Doenças da Glândula Tireoide/complicações , Adulto , Idoso , Neoplasias Ósseas/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Oncogênicas v-erbA/metabolismo , Receptor Cross-Talk/fisiologia , Receptores dos Hormônios Tireóideos/metabolismo , Sarcoma/metabolismo , Transdução de Sinais , Doenças da Glândula Tireoide/metabolismoRESUMO
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.
Assuntos
Regulação para Baixo , Integrinas/genética , Proteínas Oncogênicas v-erbA/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Cátions Bivalentes , Adesão Celular/fisiologia , Divisão Celular , Linhagem Celular , Galinhas , Colágeno/metabolismo , Colágeno Tipo I/metabolismo , Eritrócitos/citologia , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Células-Tronco Hematopoéticas/citologia , Integrinas/biossíntese , Proteínas Oncogênicas v-erbA/genética , Receptores de ColágenoRESUMO
To assess the mechanisms of repression of the erythroid-specific carbonic anhydrase II (CAII) locus we used chromatin immunoprecipitation and show that an NCoR-histone deacetylase (HDAC)3 complex is recruited by the nuclear receptor v-ErbA to the intronic HS2 enhancer turning it into a potent silencer. Furthermore we demonstrate that efficient CAII silencing requires binding of a MeCP2-targeted HDAC-containing corepressor complex to the hypermethylated CpG-island at the promoter. Activation of transcription by either AZAdC or thyroid hormone results in loss of one of the two corepressor complexes. Thyroid hormone further replaces the enhancer-bound NCoR-corepressor complex by the TRAP220 coactivator. Treatment with the HDAC inhibitor trichostatin A (TSA) causes activation of CAII transcription and histone H3 and H4 hyperacetylation at the enhancer, apparently without affecting binding of the two corepressor complexes. Unexpectedly, histone H3 and H4 at the fully repressed promoter are already hyperacetylated despite the close apposition of the MeCP2-targeted HDAC complex. Acetylation of histone H4, but not H3, at the promoter is moderately increased following TSA treatment. Our data suggest that the hyperacetylated but repressed CAII promoter is (partially) remodeled and primed for activation in v-ErbA-transformed cells.
Assuntos
Anidrase Carbônica II/genética , Proteínas Cromossômicas não Histona , Ilhas de CpG , Proteínas de Ligação a DNA/metabolismo , Regulação Enzimológica da Expressão Gênica , Histona Desacetilases/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Ativação Transcricional , Acetilação , Linhagem Celular Transformada , Humanos , Proteína 2 de Ligação a Metil-CpG , Correpressor 1 de Receptor Nuclear , Proteínas Oncogênicas v-erbA/genética , Proteínas Oncogênicas v-erbA/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Tri-Iodotironina/farmacologiaRESUMO
Hematopoietic cytokines are critically required for survival and cell proliferation of myeloid and erythroid progenitors. It is poorly understood how the apoptotic machinery of progenitor cells senses the absence of specific cytokines. Here we show that G1-Cdk activity is essential for cytokine-mediated viability of myeloid and erythroid progenitors. Cytokine deprivation is associated with rapid downregulation of G1-Cdk activity, cell cycle arrest, and apoptosis. Specific inhibition of G1-Cdk activity results in apoptotic cell death in the presence of saturating cytokine levels. In contrast, specific cell cycle arrest in G2/M does not affect viability. When cell proliferation is arrested by cytokine withdrawal, primary erythroid progenitors expressing v-ErbA maintain G1-Cdk activity and undergo delayed apoptosis. Cdk-inhibitors strongly enhance apoptosis in starved v-ErbA cells, indicating that sustained Cdk activity is required for protection from apoptosis by v-ErbA.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Divisão Celular , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Citocinas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Apoptose , Ciclo Celular , Linhagem Celular , Sobrevivência Celular , Ciclina G , Quinase 2 Dependente de Ciclina , Células-Tronco Hematopoéticas/citologia , Proteínas Oncogênicas v-erbA/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
The thyroid hormone receptor alpha (TR alpha) exhibits a dual role as an activator or repressor of gene transcription in response to thyroid hormone (T(3)). Our studies show that TR alpha, formerly thought to reside solely in the nucleus tightly bound to DNA, actually shuttles rapidly between the nucleus and cytoplasm. The finding that TR alpha shuttles reveals an additional checkpoint in receptor control of gene expression. Using Xenopus oocyte microinjection assays, we show that there are two coexisting mechanisms for nuclear entry of TR alpha. First, nuclear import of TR alpha (molecular mass 46 kDa) was not sensitive to general inhibitors of signal-mediated transport, indicating that TR alpha can enter the oocyte nucleus by passive diffusion. Second, when TR alpha was tagged with glutathione-S:-transferase, import of the fusion protein (molecular mass 73 kDa) was completely blocked by these inhibitors, demonstrating that an alternative, signal-mediated import pathway exists for TR alpha. Nuclear retention of TR alpha in oocytes is enhanced in the presence of T(3), suggesting that more intranuclear binding sites are available for the ligand-bound receptor. Using mammalian cells, we show that shuttling of green fluorescent protein (GFP)-tagged and untagged TR alpha is inhibited in both chilled and energy-depleted cells, suggesting that there is an energy-requiring step in the nuclear retention/export process. Nuclear export of TR alpha is not blocked by leptomycin B, a specific inhibitor of the export receptor CRM1, indicating that TR alpha does not require the CRM1 pathway to exit the nucleus. Dominant negative mutants of TR with defects in DNA binding and transactivation accumulate in the cytoplasm at steady state, illustrating that even single amino acid changes in functional domains may alter the subcellular distribution of TR. In contrast to TR alpha, nuclear export of its oncogenic homolog v-ErbA is sensitive to leptomycin B, suggesting that the oncoprotein follows a CRM1-mediated export pathway. Acquisition of altered nuclear export capabilities may contribute to the oncogenic properties of v-ErbA.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Carioferinas , Receptores Citoplasmáticos e Nucleares , Receptores dos Hormônios Tireóideos/metabolismo , Animais , Apirase/farmacologia , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Células Cultivadas , Ácidos Graxos Insaturados/farmacologia , Feminino , Genes Dominantes , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mamíferos , Camundongos , Mutação , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas v-erbA/metabolismo , Oócitos/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Receptores dos Hormônios Tireóideos/genética , Proteínas Ribossômicas/metabolismo , Temperatura , Xenopus , Proteína Exportina 1RESUMO
Transcriptional repression by nuclear hormone receptors is thought to result from a unison of targeting chromatin modification and disabling the basal transcriptional machinery. We used Xenopus oocytes to compare silencing effected by the thyroid hormone receptor (TR) and its mutated version, the oncoprotein v-ErbA, on partly and fully chromatinized TR-responsive templates in vivo. Repression by v-ErbA was not as efficient as that mediated by TR, was significantly more sensitive to histone deacetylase (HDAC) inhibitor treatment and, unlike TR, v-ErbA required mature chromatin to effect repression. We find that both v-ErbA and TR can recruit the corepressor N-CoR, but, in contrast to existing models, show a concomitant enrichment for HDAC3 that occurs without an association with Sin3, HDAC1/RPD3, Mi-2 or HDAC5. We propose a requirement for chromatin infrastructure in N-CoR/HDAC3-effected repression and suggest that the inability of v-ErbA to silence on partly chromatinized templates may stem from its impaired capacity to interfere with basal transcriptional machinery function. In support of this notion, we find v-ErbA to be less competent than TR for binding to TFIIB in vitro and in vivo.
Assuntos
Adenosina Trifosfatases , Cromatina/metabolismo , DNA Helicases , Histona Desacetilases/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas v-erbA/metabolismo , Receptores dos Hormônios Tireóideos/genética , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae , Animais , Autoantígenos/metabolismo , Galinhas , Regulação da Expressão Gênica , Histona Desacetilase 1 , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Correpressor 1 de Receptor Nuclear , Oócitos , Ligação Proteica , Fatores de Transcrição/metabolismo , Transcrição Gênica , Xenopus laevisRESUMO
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.
Assuntos
Proteínas Oncogênicas v-erbA/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Elementos de Resposta , Hormônios Tireóideos/metabolismo , Ativação Transcricional , Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , DNA/metabolismo , Mutagênese Sítio-Dirigida , Nucleotídeos , Proteínas Oncogênicas v-erbA/genética , Codorniz , Receptores dos Hormônios Tireóideos/genéticaRESUMO
In general, translation efficiency of ferritin mRNAs is modulated by variations in iron supply. In primary avian erythroblasts undergoing short-term proliferation, however, ferritin heavy chain (ferH) mRNA is repressed at all iron levels. Yet, expression of v-ErbA oncoprotein is sufficient to reinduce ferH mRNA utilization at physiological iron concentrations. Since overexpression of the receptor tyrosine kinase c-Kit and erythropoietin receptor (EpoR) stimulates long-term proliferation of primary erythroblasts like v-ErbA, we analyzed the impact of cooperation between c-Kit and EpoR on the regulation of iron storage. Whereas endogenous c-Kit in combination with exogenous EpoR had no significant effect, ectopic overexpression of both receptors abolished translational repression of ferH mRNA upon iron administration. Thus, high-intensity signaling through c-Kit plus EpoR pathways mimics the v-ErbA-mediated regulatory phenotype.
Assuntos
Eritroblastos/metabolismo , Ferritinas/genética , Proteínas Oncogênicas v-erbA/metabolismo , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-kit/farmacologia , RNA Mensageiro/genética , Receptores da Eritropoetina/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Galinhas , Eritroblastos/efeitos dos fármacosRESUMO
Members of the thyroid hormone receptor (TR) family act on vertebrate development and homeostasis by activating or repressing transcription of specific target genes in a ligand-dependent way. Repression by TR in the absence of ligand is mediated by an active silencing mechanism. The oncogene v-ErbA is a variant form of TR unable to bind hormone and thus acts as a constitutive repressor. Functional studies and mutation analysis revealed that the TR/v-ErbA silencing domain is composed of three silencing subdomains (SSD1-3) which, although nonfunctional individually, synergize such that silencing activity is restored when they are combined in a heteromeric complex. Here we demonstrate, using protein interaction assays in vitro and in vivo, that the inactive v-ErbA point mutant L489R within helix 5/6 in SSD2 fails to interact with the two corepressors N-CoR (nuclear receptor corepressor) or SMRT (silencing mediator of retinoic acid and thyroid hormone receptor). Furthermore, mutants in SSD1 and SSD3 exhibit a reduced corepressor recruitment corresponding to their weak residual silencing activity. In mammalian two-hybrid assays, only the combination of all three silencing subdomains, SSD1-3, leads to a cooperative binding to the corepressors N-CoR or SMRT comparable to that of the full-length v-ErbA repression domain. In conclusion, full silencing activity requires corepressor interaction with all three silencing subdomains, SSD1-3. Among these, SSD2 is a new target for N-CoR and SMRT and is essential for corepressor binding and function.