RESUMO
The Quail Neuroretina clone 71 gene (QNR-71) is expressed during the differentiation of retinal pigmented epithelia and the epidermis. It encodes a type I transmembrane glycoprotein that shares significant sequence homologies with several melanosomal proteins. We have studied its intracellular traffic in both pigmented and non-pigmented cells. We report that a di-leucine-based sorting signal (ExxPLL) present in the cytoplasmic domain of QNR-71 is necessary and sufficient for its proper targeting to the endosomal/premelanosomal compartments of both pigmented and non-pigmented cells. The intracellular transport of QNR-71 to these compartments is mediated by the AP-3 assembly proteins. As previously observed for the lysosomal glycoproteins Lampl and LimpII, overexpression of QNR-71 increases the amount of AP-3 associated with membranes, and inhibition of AP-3 synthesis increases the routing of QNR-71 towards the cell surface. In addition, expression of QNR-71 induces a misrouting of endogenous LampI to the cell surface. Thus, the targeting of QNR-71 might be similar to that of the lysosomal integral membrane glycoproteins LampI and LimpII. This suggests that sorting to melanosomes and lysosomes requires similar sorting signals and transport machineries.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Melanossomas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Monoméricas de Montagem de Clatrina , Epitélio Pigmentado Ocular/fisiologia , Sinais Direcionadores de Proteínas/fisiologia , Proteínas Adaptadoras de Transporte Vesicular , Animais , Proteínas de Transporte/genética , Embrião de Galinha , Células HeLa , Humanos , Leucina/metabolismo , Proteínas de Membrana/genética , Mutagênese/fisiologia , Epitélio Pigmentado Ocular/citologia , Transporte Proteico/fisiologia , Codorniz , TransfecçãoRESUMO
The human CYP11A1 gene is expressed specifically in steroidogenic tissues and encodes cytochrome P450scc, which catalyzes the first step in steroid synthesis. A region of the 5'-flanking DNA of the gene from nucleotides -155 to -131 (-155/-131) is shown to activate transcription in steroidogenic human placental JEG-3 (1) and adrenal NCI-H295 cells. Using this region of the gene as probe, a cDNA clone of 4.4 kilobase pairs was isolated by screening JEG-3 cell and human placental cDNA expression libraries. The open reading frame encodes three zinc fingers of the C(2)H(2) subtype, and separate regions rich in glutamate, proline, and glutamine, which are indicative of a DNA-binding protein involved in gene transcription. Expression of the cDNA in vitro and in HeLa cells yields a protein of 132 kDa, which concurs with the predicted size. Northern blot analysis demonstrate expression of two TReP-132 transcripts of 4.4 and 7.5 kilobase pairs in the thymus, adrenal cortex, and testis; and expression is also found in the steroidogenic JEG-3, NCI-H295, and MCF-7 cell lines. Immunocytochemistry analysis demonstrates localization of the HA-tagged TReP-132 protein in the nucleus. The expression of exogenous TReP-132 in HeLa cells was demonstrated to interact with the -155/-131 region in bandshift analysis. Transfection of the cDNA in placental JEG-3 and adrenal NCI-H295 cells increases expression of a reporter construct controlled by the P450scc gene 5'-flanking region from nucleotides -1676 to +49. Moreover, a chimeric protein generated by fusion of TReP-132 with the Gal4 DNA-binding domain was able to significantly increase promoter activity of a reporter construct via Gal4-binding sites upstream of the E1b minimal promoter. Coexpression of CREB-binding protein (CBP)/p300 with TReP-132 has an additive effect on promoter activity, and the proteins were demonstrated to interact physically. Thus, these results together indicate the isolation of a novel zinc-finger transcriptional regulating protein of 132 kDa (TReP-132) involved in the regulation of P450scc gene expression.
Assuntos
Enzima de Clivagem da Cadeia Lateral do Colesterol/biossíntese , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Regulação Enzimológica da Expressão Gênica , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Transativadores/química , Transativadores/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/fisiologia , Dedos de Zinco , Córtex Suprarrenal/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Northern Blotting , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , DNA Complementar/metabolismo , Biblioteca Gênica , Células HeLa , Humanos , Imuno-Histoquímica , Masculino , Microscopia de Fluorescência , Dados de Sequência Molecular , Placenta/metabolismo , Plasmídeos/metabolismo , Testes de Precipitina , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Ribonucleases/metabolismo , Esteroides/metabolismo , Testículo/metabolismo , Timo/metabolismo , Distribuição Tecidual , Transfecção , Células Tumorais CultivadasRESUMO
Rab5 regulates endocytic membrane traffic by specifically recruiting cytosolic effector proteins to their site of action on early endosomal membranes. We have characterized a new Rab5 effector complex involved in endosomal fusion events. This complex includes a novel protein, Rabenosyn-5, which, like the previously characterized Rab5 effector early endosome antigen 1 (EEA1), contains an FYVE finger domain and is recruited in a phosphatidylinositol-3-kinase-dependent fashion to early endosomes. Rabenosyn-5 is complexed to the Sec1-like protein hVPS45. hVPS45 does not interact directly with Rab5, therefore Rabenosyn-5 serves as a molecular link between hVPS45 and the Rab5 GTPase. This property suggests that Rabenosyn-5 is a closer mammalian functional homologue of yeast Vac1p than EEA1. Furthermore, although both EEA1 and Rabenosyn-5 are required for early endosomal fusion, only overexpression of Rabenosyn-5 inhibits cathepsin D processing, suggesting that the two proteins play distinct roles in endosomal trafficking. We propose that Rab5-dependent formation of membrane domains enriched in phosphatidylinositol-3-phosphate has evolved as a mechanism for the recruitment of multiple effector proteins to mammalian early endosomes, and that these domains are multifunctional, depending on the differing activities of the effector proteins recruited.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transporte Vesicular , Proteínas rab5 de Ligação ao GTP/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Transporte/genética , Catepsina D/metabolismo , Linhagem Celular , Clonagem Molecular , Endossomos/química , Imunofluorescência , Células HeLa , Humanos , Lisossomos/química , Lisossomos/metabolismo , Fusão de Membrana , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas Munc18 , Proteínas do Tecido Nervoso/química , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Qa-SNARE , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
The Erg gene belongs to the Ets family encoding a class of transcription factors. To gain new insight on the in vivo functional specificity of the Erg gene within the wide Ets family, we used in situ hybridization to determine its expression pattern during murine embryogenesis. We found that the Erg gene expression predominates in mesodermal tissues, including the endothelial, precartilaginous and urogenital areas. A specific Erg gene expression was also identified in migrating neural crest cells. A comparison with Fli-1, the most closely Erg-related gene, revealed that both gene expressions partially overlap, suggesting that they may contribute to related functions in these tissues. Like other Ets family genes, Erg seems involved in several fundamental developmental steps in murine embryogenesis, including epithelio-mesenchymal transition, cell migration, settlement and differentiation.
Assuntos
Desenvolvimento Embrionário e Fetal/genética , Mesoderma/metabolismo , Crista Neural/metabolismo , Proteínas Oncogênicas/genética , Proteínas Proto-Oncogênicas , Fatores de Transcrição/genética , Animais , Movimento Celular , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário e Fetal/fisiologia , Expressão Gênica , Camundongos , Crista Neural/citologia , Proteínas Oncogênicas/biossíntese , Proteína Proto-Oncogênica c-fli-1 , Transativadores/genética , Regulador Transcricional ERG , Sistema UrogenitalRESUMO
The Ets transcription factors of the PEA3 group--E1AF/PEA3, ETV1/ER81 and ERM--are almost identical in the ETS DNA-binding and the transcriptional acidic domains. To accelerate our understanding of the molecular basis of putative diseases linked to ETV1 such as Ewing's sarcoma we characterized the human ETV1 and the mouse ER81 genes. We showed that these genes are both encoded by 13 exons in more than 90 kbp genomic DNA, and that the classical acceptor and donor splicing sites are present in each junction except for the 5' donor site of intron 9 where GT is replaced by TT. The genomic organization of the ETS and acidic domains in the human ETV1 and mouse ER81 (localized to chromosome 12) genes is similar to that observed in human ERM and human E1AF/PEA3 genes. Moreover, as in human ERM and human E1AF/PEA3 genes, a first untranslated exon is upstream from the first methionine, and the mouse ER81 gene transcription is regulated by a 1.8 kbp of genomic DNA upstream from this exon. In human, the alternative splicing of the ETV1 gene leads to the presence (ETV1 alpha) or the absence (ETV1 beta) of exon 5 encoding the C-terminal part of the transcriptional acidic domain, but without affecting the alpha helix previously described as crucial for transactivation. We demonstrated here that the truncated isoform (human ETV1 beta) and the full-length isoform (human ETV1 alpha) bind similarly specific DNA Ets binding sites. Moreover, they both activate transcription similarly through the PKA-transduction pathway, so suggesting that this alternative splicing is not crucial for the function of this protein as a transcription factor. The comparison of human ETV1 alpha and human ETV1 beta expression in the same tissues, such as the adrenal gland or the bladder, showed no clear-cut differences. Altogether, these data open a new avenue of investigation leading to a better understanding of the functional role of this transcription factor.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Genes , Isoformas de Proteínas/genética , Splicing de RNA , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/patologia , Mapeamento Cromossômico , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/química , Éxons/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Ligação Proteica , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/química , Estrutura Terciária de Proteína , Coelhos , Especificidade da Espécie , Fatores de Transcrição/biossíntese , Fatores de Transcrição/química , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Regulation of the human CYP11A gene encoding cytochrome P450scc, which catalyzes the first step of steroid synthesis, is regulated by many trans-acting transcription factors including steroidogenic factor 1 (SF-1). Transfection experiments in human adrenal NCI-H295 cells demonstrate regulation of the P450scc gene promoter region that contains several putative SF-1 binding sites. Cotransfection of SF-1 with a luciferase reporter construct containing the P450scc gene 5'-flanking region from nucleotides -1676 to +49 increased promoter activity, and deletion of the nucleotide sequence from position -1676 to -1620, which removes a putative cAMP response element (CRE), did not affect the stimulatory response to SF-1. As well, further deletion of the promoter region to nucleotide -110, which contains only one SF-1 binding site, still retained the ability to respond to exogenous SF-1. However, mutation of the remaining site which abolished SF-1 protein/DNA interaction also abrogated any functional response to the factor. All the P450scc reporter constructs which responded to SF-1 were further stimulated by exogenous p300 and CREB-binding protein (CBP), suggesting interaction between SF-1 and p300/CBP. As well, mutation of the binding site that abrogated the response to SF-1 also abolished the response to p300 and CBP. Cotransfection of the adenovirus E1A oncoprotein, which has been shown to interact with p300/CBP and interfere with its function, decreased the stimulatory effect of SF-1 and p300/CBP. Cotransfection of a mutated E1A protein, RG2, which does not interact with p300/CBP, did not alter the stimulatory effect of SF-1 and p300/CBP on the P450scc promoter. Deletion of the region from amino acid residues 2-67 in E1A, which has been postulated to interact with p300/CBP, also abolished the inhibitory effect of E1A, whereas deletion of the region from residues 120 to 140 had no effect. Two regions of CBP from amino acids 1 to 451 and from 1460 to 1891 were demonstrated to interact with SF-1 in vitro. Coexpression of fragments of the p300 protein fused to the VP16 protein in the presence of SF-1 and the -110 P450scc reporter construct indicated in vivo the interaction of two regions of p300 with SF-1, thus confirming the in vitro results. Taken together these results indicate that regulation of the human P450scc gene by SF-1 is mediated by p300/CBP. Due to the many putative roles of SF-1 to regulate many genes, its interaction with p300/CBP is potentially a key component effecting important physiological processes.
Assuntos
Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Proteínas de Ligação a DNA/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas Nucleares/fisiologia , Transativadores , Fatores de Transcrição/fisiologia , Proteínas E1A de Adenovirus/metabolismo , Proteína de Ligação a CREB , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição Fushi Tarazu , Proteínas de Homeodomínio , Humanos , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Receptores Citoplasmáticos e Nucleares , Proteínas Recombinantes/genética , Fator Esteroidogênico 1 , Fatores de Transcrição/metabolismoRESUMO
The ERM protein belongs to the family of Ets transcription factors. We show here that the human ERM gene is organized into 14 exons distributed along 65 kb of genomic DNA on chromosome 3. The two main functional domains of ERM, the acidic domain and the DNA-binding ETS domain, are overlapped by three different exons each. The 3'-untranslated region of ERM is 2.1 kb, whereas the 5'-untranslated region is about 0.3 kb; this allows the transcription of ERM transcripts of approximately 4 kb. The human ERM gene is localized to the q27-q29 region of chromosome 3.
Assuntos
Cromossomos Humanos Par 3/genética , Proteínas de Ligação a DNA/genética , Genes , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Éxons/genética , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Família Multigênica , Fatores de Transcrição/classificaçãoRESUMO
The recently cloned human Ets transcription factor ERM is closely related to the ER81 and PEA3 genes. Here, we report the functional analysis of the DNA-binding and transactivation properties of ERM. Specific DNA-binding by ERM requires the ETS domain, conserved in all members of the Ets family and is inhibited by an 84 residue long central region and the carboxy-terminal tail. Two fragments of ERM are transferrable activation domains: alpha, which sits in the 72 first residues and encompasses the acidic domain conserved between ERM, ER81 and PEA3, and the carboxy-terminal tail which also bears a DNA-binding inhibition function. Deletion of alpha strongly reduces transactivation by ERM. Moreover, alpha and the carboxy-terminal tail exhibit functional synergism, suggesting that they activate transcription through different mechanisms. In support of this idea, we demonstrate that VP16 squelches transactivation by alpha but not by the carboxy-terminal tail. This result also indicates that alpha and VP16 may share common limiting cofactors. alpha and the carboxy-terminal tail do not seem to be conserved within the whole Ets family, indicating that the specificity of ERM may rely on interactions with distinct cofactors.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição/fisiologia , Ativação Transcricional/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Proteína Vmw65 do Vírus do Herpes Simples/genética , Proteína Vmw65 do Vírus do Herpes Simples/metabolismo , Humanos , Dados de Sequência Molecular , Conformação Proteica , Coelhos , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The ets gene superfamily encodes a class of transcription factors that bind to a purine rich sequence through a 85 amino-acid ETS domain. Among them, the human erg gene has been found to be involved in Ewing's sarcoma, primitive neurectodermal tumour of childhood and acute myeloid leukaemia. Nevertheless, little is known about human erg expression. Northern blot analyses have shown a human erg expression restricted to few cell lines and thymus, but the status concerning expression during development remains unknown probably because no homologue of this gene has yet been isolated and studied in other vertebrates. We thus choose to clone the chicken erg gene (ck-erg) and to study its expression during chicken development. We obtained a bona fide clone of ck-erg and defined the transcriptional modulating properties of its product. The ck-Erg protein acts as a transcriptional activator through a conventional consensus ETS binding site. Northern blot studies on various chicken tissues, in situ analyses and comparison with the well-characterised c-ets-1 expression show that ck-erg is expressed in mesoderm- and, to a lesser extent, in ectoderm-derived tissues. During chicken development, two salient features could be observed. From stage E1 to E3.5, ck-erg expression was widely distributed in mesodermal derivatives and neural crest, resembling c-ets-1 expression. However, by E6, the expression of ck-erg exhibited, unlike c-ets-1, a drastically new and strong signal in precartilaginous condensation zones and cartilaginous skeletal primordia. These stages are the first steps of bone formation during skeletal elaboration. Our results show for the first time a possible specific involvement of ck-erg in cartilage morphogenesis.
Assuntos
Cartilagem/embriologia , Embrião de Galinha/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Mesoderma/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Cartilagem/citologia , Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Hibridização In Situ , Dados de Sequência Molecular , Transcrição GênicaRESUMO
Prognosis factors such as mutated or amplified oncogenes are used in the treatment of breast cancer. We have recently shown that the members of the PEA3 group (ER81, ERM and PEA3) from the transcription factor family of the ets genes are overexpressed in breast cancer tumors arising from MMTV-neu transgenic animals. Moreover, we have shown that ER81, and in a lesser extent ERM and PEA3, are not expressed in the estrogen and/or progesterone receptor-positive mammary cancer cell lines, whereas they are expressed in the receptor negative ones. Our research interest in now focused on the role(s) of these oncogenes in the development and the regulation of breast tumors.
Assuntos
Neoplasias da Mama/genética , Neoplasias Mamárias Experimentais/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Feminino , Humanos , Alinhamento de Sequência , Fatores de Transcrição/químicaRESUMO
The chicken c-ets-1 locus encodes two transcription factors, p54c-ets-1 and p68c-ets-1 that differ in their N-termini, encoded respectively by the I54 and alpha beta exons. p68c-ets-1 equivalents are only found in birds and reptiles while p54c-ets-1 is widely conserved in vertebrates, from amphibians to mammals. Thus, the classical view concerning the evolution of the c-ets-1 gene has been to consider that I54 is of ancient origin whereas alpha and beta, which provide an additional activating domain in p68c-ets-1, would have been acquired much more recently. Sequencing the alpha and beta exons in various species pinpointed a highly conserved region of 13 amino acids which is rich in acidic and hydrophobic residues, a feature of some other transactivating domains. Strikingly, this subdomain is also present in the otherwise unrelated N-terminal activating region of p58c-ets-2 and was thus named BEC for Ets-1-beta/Ets-2-Conserved sequence. Moreover, the two N-termini share the BEC sequence at a homologous position in their highly similar genomic organization indicating a common origin. This structural homology underlies a functional similarity since fusion of the heterologous GAL4 DNA-binding domain with either of the two isolated domains demonstrates that BEC is essential in both cases for the transactivating activity. The function of the alpha beta domain in the context of p68c-ets-1 also strictly depends on the presence of the BEC sequence. Finally, the whole N-terminus of p58c-ets-2 can functionally substitute for its counterpart in p68c-ets-1 further demonstrating that p68c-ets-1 and p58c-ets-2 are structurally and functionally more closely related than previously thought. Besides, we also found BEC in the N-terminus of the Drosophila pointed gene which may be considered as closely related to the uncommitted 'ets1/2' common ancestor. These data demonstrate that the alpha and beta exons are not a recent and specific acquisition but stem, like the p58c-ets-2 N-terminus, from the invertebrate unduplicated 'ets 1/2' gene. This work unravels a new model for the ets-1/ets-2 gene's evolution, based for the first time on both structural and functional evidences. Accordingly, p68c-ets-1 and p58c-ets-2 are the direct descendants of the ancestral 'ets1/2' gene whereas I54 may have been acquired as a second promoter in the c-ets-1 gene after the duplication. Indeed, I54 is not found in the Drosophila pointed gene. The high degree of similarity, and hence of functional redundancy, between p68c-ets-1 and p58c-ets-2 may have led to the rapid divergence (and even loss in mammals) of alpha and beta during evolution whereas I54, which provided a novel function unique to c-ets-1, was maintained within the presently widespread p54c-ets-1 version.