RESUMO
The human POK family members are transcription factors with a POZ domain and zinc-fingers that act primarily as transcriptional repressors. Several members of this family are involved in oncogenesis and this prompted us to assess whether expression levels of individual POK family members are associated with clinical outcomes in cancer. We have observed that ZBTB4 (zinc-finger and BTB domain containing 4) is downregulated in breast cancer patients, and that its expression is significantly correlated with relapse-free survival. Further integrative analysis of mRNA and microRNA (miR) expression data from the NCI-60 cell lines revealed an inverse correlation between ZBTB4 and oncogenic miRs derived from the miR-17-92 cluster and its paralogs. The experimental results using MDA-MB-231 and MCF-7 human breast cancer cells confirm that miRNAs derived from these clusters, containing miR-17-5p, miR-20a, miR-106a, miR-106b and miR-93, negatively regulate ZBTB4 expression. Overexpression of ZBTB4 or restoration of ZBTB4 by using an antagomir inhibit growth and invasion of breast cancer cells, and this effect is due, in part, to ZBTB4-dependent repression of the specificity protein 1 (Sp1), Sp3 and Sp4 genes, and subsequent downregulation of several Sp-dependent oncogenes, in part, through competition between ZBTB4 and Sp transcription factors for GC-rich promoter sequences. These results confirm that ZBTB4 functions as a novel tumor-suppressor gene with prognostic significance for breast cancer survival, and the oncogenic miR-17-92/ZBTB4/Sp axis may be a potential therapeutic target.
Assuntos
Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Oncogenes , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ligação Competitiva , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Ciclina D1/genética , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Intervalo Livre de Doença , Regulação para Baixo , Feminino , Genes Reporter , Humanos , Estimativa de Kaplan-Meier , Luciferases/biossíntese , Luciferases/genética , MicroRNAs/metabolismo , Prognóstico , Regiões Promotoras Genéticas , Ligação Proteica , Interferência de RNA , RNA Longo não Codificante , Proteínas Repressoras/genética , Fatores de Transcrição Sp/genética , Fatores de Transcrição Sp/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
HIPK2 is a eukaryotic Serine-Threonine kinase that controls cellular proliferation and survival in response to exogenous signals. Here, we show that the human transcription factor ZBTB4 is a new target of HIPK2. The two proteins interact in vitro, colocalize and associate in vivo, and HIPK2 phosphorylates several conserved residues of ZBTB4. Overexpressing HIPK2 causes the degradation of ZBTB4, whereas overexpressing a kinase-deficient mutant of HIPK2 has no effect. The chemical activation of HIPK2 also decreases the amount of ZBTB4 in cells. Conversely, the inhibition of HIPK2 by drugs or by RNA interference causes a large increase in ZBTB4 levels. This negative regulation of ZBTB4 by HIPK2 occurs under normal conditions of cell growth. In addition, the degradation is increased by DNA damage. These findings have two consequences. First, we have recently shown that ZBTB4 inhibits the transcription of p21. Therefore, the activation of p21 by HIPK2 is two-pronged: stimulation of the activator p53, and simultaneous repression of the inhibitor ZBTB4. Second, ZBTB4 is also known to bind methylated DNA and repress methylated sequences. Consequently, our findings raise the possibility that HIPK2 might influence the epigenetic regulation of gene expression at loci that remain to be identified.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Dano ao DNA , Regulação para Baixo , Células HCT116 , Humanos , Imunoprecipitação , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação , Células NIH 3T3 , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Treonina/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
The model organism Saccharomyces cerevisiae is providing new insights into the molecular and cellular changes that are related to aging. The yeast protein Sir2p (Silent Information Regulator 2) is a histone deacetylase involved in transcriptional silencing and the control of genomic stability. Recent results have led to the identification of Sir2p as a crucial determinant of yeast life span. Dosage, intracellular localization, and activity of Sir2p all have important effects on yeast longevity. For instance, calorie restriction apparently increases yeast life span by increasing Sir2p activity. Since Sir2p-related proteins have been identified in many prokaryotic and eukaryotic organisms, the fundamental principles derived from the studies in yeast may prove valuable in directing our future research toward an understanding of the mechanisms of aging in higher eukaryotes. BioEssays 23:327-332, 2001.
Assuntos
Proteínas Fúngicas/fisiologia , Regulação Fúngica da Expressão Gênica , Inativação Gênica , Histona Desacetilases/fisiologia , Saccharomyces cerevisiae/fisiologia , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae , Transativadores/fisiologia , Animais , DNA , Células Eucarióticas , Proteínas Fúngicas/metabolismo , Histona Desacetilases/metabolismo , Humanos , Recombinação Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Sirtuína 1 , Sirtuína 2 , Sirtuínas , Transativadores/metabolismo , Transcrição GênicaRESUMO
Calorie restriction extends life-span in a wide variety of organisms. Although it has been suggested that calorie restriction may work by reducing the levels of reactive oxygen species produced during respiration, the mechanism by which this regimen slows aging is uncertain. Here, we mimicked calorie restriction in yeast by physiological or genetic means and showed a substantial extension in life-span. This extension was not observed in strains mutant for SIR2 (which encodes the silencing protein Sir2p) or NPT1 (a gene in a pathway in the synthesis of NAD, the oxidized form of nicotinamide adenine dinucleotide). These findings suggest that the increased longevity induced by calorie restriction requires the activation of Sir2p by NAD.
Assuntos
Ingestão de Energia , Inativação Gênica , Glucose/metabolismo , Histona Desacetilases/metabolismo , Longevidade , NAD/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae , Transativadores/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , DNA Circular/genética , DNA Circular/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Ativação Enzimática , Histona Desacetilases/genética , Mutação , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Recombinação Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sirtuína 2 , Sirtuínas , Transativadores/genéticaRESUMO
A cause of aging in yeast is the accumulation of circular species of ribosomal DNA (rDNA) arising from the 100-200 tandemly repeated copies in the genome. We show here that mutation of the FOB1 gene slows the generation of these circles and thus extends life span. Fob1p is known to create a unidirectional block to replication forks in the rDNA. We show that Fob1p is a nucleolar protein, suggesting a direct involvement in the replication fork block. We propose that this block can trigger aging by causing chromosomal breaks, the repair of which results in the generation of rDNA circles. These findings may provide a novel link between metabolic rate and aging in yeast and, perhaps, higher organisms.
Assuntos
Replicação do DNA/genética , Proteínas de Ligação a DNA , Proteínas Fúngicas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Reparo do DNA/genética , DNA Circular/genética , DNA Ribossômico/genética , Imunofluorescência , Deleção de Genes , Proteínas de Fluorescência Verde , Proteínas Luminescentes , Proteínas Nucleares/genética , Sequências Repetitivas de Ácido NucleicoRESUMO
A cause of aging in Saccharomyces cerevisiae is the accumulation of extrachromosomal ribosomal DNA circles (ERCs). Introduction of an ERC into young mother cells shortens life span and accelerates the onset of age-associated sterility. It is important to understand the process by which ERCs are generated. Here, we demonstrate that homologous recombination is necessary for ERC formation. rad52 mutant cells, defective in DNA repair through homologous recombination, do not accumulate ERCs with age, and mutations in other genes of the RAD52 class have varying effects on ERC formation. rad52 mutation leads to a progressive delocalization of Sir3p from telomeres to other nuclear sites with age and, surprisingly, shortens life span. We speculate that spontaneous DNA damage, perhaps double-strand breaks, causes lethality in mutants of the RAD52 class and may be an initial step of aging in wild-type cells.
Assuntos
Senescência Celular/genética , Reparo do DNA/genética , DNA Circular/genética , DNA Ribossômico/genética , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae , Proteínas de Ligação a DNA/genética , Epistasia Genética , Citometria de Fluxo , Imunofluorescência , Proteínas Fúngicas/genética , Genótipo , Infertilidade/genética , Modelos Biológicos , Mutação/genética , Proteína Rad52 de Recombinação e Reparo de DNA , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae , Telômero/genética , Transativadores/genéticaRESUMO
The past year has confirmed the great potential of the yeast Saccharomyces cerevisiae as a model to study aging. Ground breaking papers have revealed similarities between aging in yeast and in mammals, and have identified genetic instability of the ribosomal DNA array as the first known cause of aging in yeast cells.
Assuntos
Senescência Celular , Saccharomyces cerevisiae/fisiologia , Envelhecimento/fisiologia , Animais , Senescência Celular/genética , DNA Circular/genética , DNA Circular/fisiologia , DNA Fúngico/genética , DNA Fúngico/fisiologia , DNA Ribossômico/genética , DNA Ribossômico/fisiologia , Humanos , Saccharomyces cerevisiae/genéticaRESUMO
The PEA3 group of transcription factors belongs to the Ets family and is composed of PEA3, ERM, and ER81, which are more than 95% identical within the DNA-binding domain--the ETS domain--and which demonstrate 50% aa identity overall. We present here a review of the current knowledge of these transcription factors, which possess functional domains responsible for DNA-binding, DNA-binding inhibition, and transactivation. Recent data suggest that these factors are targets for signaling cascades, such as the Ras-dependent ones, and thus may contribute first to the nuclear response to cell stimulation and second to Ras-induced cell transformation. The expression of the PEA3 group members in numerous developing murine organs, and, especially, in epithelial-mesenchymal interaction events, suggests a key role in murine organogenesis. Moreover, their expression in certain breast cancer cells suggests a possible involvement of these genes in the appearance, progression, and invasion of malignant cells.
Assuntos
Proteínas de Ligação a DNA/genética , Família Multigênica , Proteínas Oncogênicas , Estrutura Terciária de Proteína , Fatores de Transcrição/genética , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-ets , Sarcoma de Ewing/genética , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
ERM, ER81 and PEA3 are three highly related transcription factors belonging to the ETS family. Together they form the PEA3 group within this family. Little data is yet available regarding the roles of these three genes during embryonic development. A prerequisite to investigations in this field is to obtain an accurate spatio-temporal expression map for the erm, er81 and pea3 genes. To this end, we have used in situ hybridization to compare their expression patterns during critical stages of murine embryogenesis. We report that all three genes are expressed in numerous developing organs coming from different embryonic tissues. The three genes appeared co-expressed in different organs but presented specific sites of expression, so that the resultant expression pattern could in fact reveal several distinct functions depending upon isolated and/or various combinations of the PEA3 member expression. These results suggest that erm, er81 and pea3 genes are differentially regulated, probably to serve important functions as cell proliferation control, tissue interaction mediator or cell differentiation, all over successive steps of the mouse organogenesis.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Sistema Nervoso Central/embriologia , DNA Complementar/isolamento & purificação , Proteínas de Ligação a DNA/genética , Ectoderma/metabolismo , Desenvolvimento Embrionário e Fetal , Endoderma/metabolismo , Expressão Gênica , Mesoderma/metabolismo , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição/genéticaRESUMO
Transcription factors are modular entities built up of discrete domains, some devoted to DNA binding and others permitting transcriptional modulation. The structure of DNA binding domains has been thoroughly investigated and structural classes clearly defined. In sharp contrast, the structural constraints put on transactivating regions, if any, are mostly unknown. Our investigations focus on ERM, a eukaryotic transcription factor of the ETS family. We have previously shown that ERM harbours two transactivating domains (TADs) with distinct functional features: AD1 lies in the first 72 amino acids of ERM, while AD2 sits in the last 62. Here we show that AD1 is a bona fide acidic TAD, for it activated transcription in yeast cells, while AD2 did not. AD1 contains a 20 amino acid stretch predicted to form an alpha-helix that is found unchanged in the related PEA3 and ER81 transcription factors. Circular dichroism analysis revealed that a 32 amino acid peptide encompassing this region is unstructured in water but folds into a helix when the hydrophobic solvent trifluoroethanol is added. The isolated helix was sufficient to activate transcription and mutations predicted to disrupt it dramatically affected AD1-driven transactivation, whereas mutations decreasing its acidity had more gentle effects. A phenylalanine residue within the helix was particularly sensitive to mutations. Finally, we observed that ERM bound TAFII60 via AD1 and bound TBP and TAFII40, presumably via other activation domains.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Proteínas de Saccharomyces cerevisiae , Fatores Associados à Proteína de Ligação a TATA , Fatores de Transcrição/metabolismo , Ativação Transcricional , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Sequência Conservada , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Éxons , Células HeLa , Humanos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Coelhos , Saccharomyces cerevisiae , Relação Estrutura-Atividade , Transativadores/metabolismo , Fator de Transcrição TFIID , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição TFII/metabolismo , Transcrição GênicaRESUMO
Matrix metalloproteinases belong to a family of structurally related enzymes that plays important role in tissue morphogenesis, differentiation, and wound healing. Their expression is negatively regulated by several members of the steroid hormone receptor family. This is thought to occur through interaction of the steroid receptors with the transcription factor AP-1 that is otherwise required for positive regulation. Here, we demonstrate that AP-1 is not always a target for down-regulation of expression of matrix metalloproteinases by steroid receptors. Androgen receptor negatively regulates matrix metalloproteinase-1 expression not through AP-1 but through a family of Ets-related transcription factors that are also required for positive regulation. This negative regulation is specific for the androgen receptor. It does not require the DNA binding activity but needs amino-terminal sequences of the receptor. These results identify a novel regulatory pathway for negative regulation utilized by a member of the steroid hormone receptor family for down-regulating the expression of matrix metalloproteinases.
Assuntos
Androgênios/farmacologia , Colagenases/genética , Proteínas de Ligação a DNA/metabolismo , Metaloproteinase 3 da Matriz/genética , Metaloendopeptidases/genética , Receptores Androgênicos/fisiologia , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/fisiologia , Regulação para Baixo , Humanos , Metaloproteinase 1 da Matriz , Metaloproteinase 7 da Matriz , Regiões Promotoras Genéticas , Relação Estrutura-Atividade , Fator de Transcrição AP-1/fisiologiaRESUMO
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
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 ets-related transcription factors PEA3 and ER81 have recently been isolated and characterized in the mouse. They share 95% identity in a 85 amino acid (AA) domain termed the ETS domain which is responsible for DNA binding, and therefore they form an Ets family group. By screening a human testis cDNA library with a probe containing the mouse PEA3 ETS domain, we isolated a 2.2 kb clone containing a 510 AA open reading frame. Since the ETS domain, which is localized in the carboxy terminal region of the encoded protein, is 95% and 96% identical to that of PEA3 and ER81, respectively, we named this new member 'Ets Related Molecule PEA3-like' (ERM). Although the first 120 AA in the amino-terminal region of ERM share 47% identity with PEA3 and 66% with ER81, ERM contains a central region of approximately 35 AA not found in the two mouse proteins. Gel shift analysis indicates that the full-length ERM protein is able to bind specifically to an oligonucleotide containing the consensus nucleotide core sequence GGAA recognized by the Ets proteins. Moreover, in vitro translation of 83 AA of the ERM ETS domain led to the production of a truncated protein which also binds to DNA. Though differential expression is observed in primary tumors and normal lymphocytes do not express ERM, this gene is almost ubiquitously expressed in human normal tissues. ERM mRNA is highly expressed in brain as well as in placenta and, to a lesser degree, in lung, pancreas, and heart. Moreover, almost all human cell lines tested express it at varying levels. In mouse tissues, we showed that PEA3 and ER81 mRNAs display restricted expression, whereas ERM is almost ubiquitously expressed as observed for human tissues. Altogether these results indicate that ERM is clearly a new ets family member and not the human equivalent of PEA3 or ER81.