RESUMO
The GRB2-associated binder 1 (GAB1) docking/scaffold protein is a key mediator of the MET-tyrosine kinase receptor activated by hepatocyte growth factor/scatter factor (HGF/SF). Activated MET promotes recruitment and tyrosine phosphorylation of GAB1, which in turn recruits multiple proteins and mediates MET signaling leading to cell survival, motility, and morphogenesis. We previously reported that, without its ligand, MET is a functional caspase target during apoptosis, allowing the generation of a p40-MET fragment that amplifies apoptosis. In this study we established that GAB1 is also a functional caspase target by evidencing a caspase-cleaved p35-GAB1 fragment that contains the MET binding domain. GAB1 is cleaved by caspases before MET, and the resulting p35-GAB1 fragment is phosphorylated by MET upon HGF/SF binding and can interact with a subset of GAB1 partners, PI3K, and GRB2 but not with SHP2. This p35-GAB1 fragment favors cell survival by maintaining HGF/SF-induced MET activation of AKT and by hindering p40-MET pro-apoptotic function. These data demonstrate an anti-apoptotic role of caspase-cleaved GAB1 in HGF/SF-MET signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/fisiologia , Caspases/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Caspases/genética , Cães , Ativação Enzimática/fisiologia , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/metabolismo , Células HeLa , Células Hep G2 , Fator de Crescimento de Hepatócito/genética , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/genéticaRESUMO
PcG (Polycomb group) proteins are conserved transcriptional repressors essential to regulate cell fate and to maintain epigenetic cellular memory. They work in concert through two main families of chromatin-modifying complexes, PRC1 (Polycomb repressive complex 1) and PRC2-4. In Drosophila, PRC2 contains the H3K27 histone methyltransferase E(Z) whose trimethylation activity towards PcG target genes is stimulated by PCL (Polycomb-like). In the present study, we have examined hPCL3, one of its three human paralogues. Through alternative splicing, hPCL3 encodes a long isoform, hPCL3L, containing an N-terminal TUDOR domain and two PHDs (plant homeodomains) and a smaller isoform, hPCL3S, lacking the second PHD finger (PHD2). By quantitative reverse transcription-PCR analyses, we showed that both isoforms are widely co-expressed at high levels in medulloblastoma. By co-immunoprecipitation analyses, we demonstrated that both isoforms interact with EZH2 through their common TUDOR domain. However, the hPCL3L-specific PHD2 domain, which is better conserved than PHD1 in the PCL family, is also involved in this interaction and implicated in the self-association of hPCL3L. Finally, we have demonstrated that both hPCL3 isoforms are physically associated with EZH2, but in different complexes. Our results provide the first evidence that the two hPCL3 isoforms belong to different complexes and raise important questions about their relative functions, particularly in tumorigenesis.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Processamento Alternativo , Animais , Proteínas de Ligação a DNA/genética , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia , Imunoprecipitação , Proteínas Nucleares/genética , Complexo Repressor Polycomb 2 , Pró-Colágeno-Prolina Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Transcrição/genética , TransfecçãoRESUMO
The tumor suppressor gene HIC1 (Hypermethylated in Cancer 1) that is epigenetically silenced in many human tumors and is essential for mammalian development encodes a sequence-specific transcriptional repressor. The few genes that have been reported to be directly regulated by HIC1 include ATOH1, FGFBP1, SIRT1, and E2F1. HIC1 is thus involved in the complex regulatory loops modulating p53-dependent and E2F1-dependent cell survival and stress responses. We performed genome-wide expression profiling analyses to identify new HIC1 target genes, using HIC1-deficient U2OS human osteosarcoma cells infected with adenoviruses expressing either HIC1 or GFP as a negative control. These studies identified several putative direct target genes, including CXCR7, a G-protein-coupled receptor recently identified as a scavenger receptor for the chemokine SDF-1/CXCL12. CXCR7 is highly expressed in human breast, lung, and prostate cancers. Using quantitative reverse transcription-PCR analyses, we demonstrated that CXCR7 was repressed in U2OS cells overexpressing HIC1. Inversely, inactivation of endogenous HIC1 by RNA interference in normal human WI38 fibroblasts results in up-regulation of CXCR7 and SIRT1. In silico analyses followed by deletion studies and luciferase reporter assays identified a functional and phylogenetically conserved HIC1-responsive element in the human CXCR7 promoter. Moreover, chromatin immunoprecipitation (ChIP) and ChIP upon ChIP experiments demonstrated that endogenous HIC1 proteins are bound together with the C-terminal binding protein corepressor to the CXCR7 and SIRT1 promoters in WI38 cells. Taken together, our results implicate the tumor suppressor HIC1 in the transcriptional regulation of the chemokine receptor CXCR7, a key player in the promotion of tumorigenesis in a wide variety of cell types.
Assuntos
Fatores de Transcrição Kruppel-Like/metabolismo , Receptores CXCR/genética , Adenoviridae/genética , Oxirredutases do Álcool/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Imunoprecipitação da Cromatina , Sequência Conservada , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/genética , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Vetores Genéticos/genética , Humanos , Fatores de Transcrição Kruppel-Like/genética , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Osteossarcoma/genética , Osteossarcoma/patologia , Filogenia , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CXCR/metabolismo , Sirtuína 1 , Sirtuínas/genética , Sirtuínas/metabolismoRESUMO
Tumor suppressor HIC1 (hypermethylated in cancer 1) is a gene that is essential for mammalian development, epigenetically silenced in many human tumors, and involved in a complex pathway regulating P53 tumor suppression activity. HIC1 encodes a sequence-specific transcriptional repressor containing five Krüppel-like C(2)H(2) zinc fingers and an N-terminal BTB/POZ repression domain. Here, we show that endogenous HIC1 is SUMOylated in vivo on a phylogenetically conserved lysine, K314, located in the central region which is a second repression domain. K314R mutation does not influence HIC1 subnuclear localization but significantly reduces its transcriptional repression potential, as does the mutation of the other conserved residue in the psiKXE consensus, E316A, or the overexpression of the deSUMOylase SSP3/SENP2. Furthermore, HIC1 is acetylated in vitro by P300/CBP. Strikingly, the K314R mutant is less acetylated than wild-type HIC1, suggesting that this lysine is a target for both SUMOylation and acetylation. We further show that HIC1 transcriptional repression activity is positively controlled by two types of deacetylases, SIRT1 and HDAC4, which increase the deacetylation and SUMOylation, respectively, of K314. Knockdown of endogenous SIRT1 by the transfection of short interfering RNA causes a significant loss of HIC1 SUMOylation. Thus, this dual-deacetylase complex induces either a phosphorylation-dependent acetylation-SUMOylation switch through a psiKXEXXSP motif, as previously shown for MEF2, or a phosphorylation-independent switch through a psiKXEP motif, as shown here for HIC1, since P317A mutation severely impairs HIC1 acetylation. Finally, our results demonstrate that HIC1 is a target of the class III deacetylase SIRT1 and identify a new posttranslational modification step in the P53-HIC1-SIRT1 regulatory loop.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteína SUMO-1/metabolismo , Sirtuínas/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Sequência Conservada , Proteínas de Ligação a DNA/genética , Histona Desacetilases/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like , Lisina/metabolismo , Dados de Sequência Molecular , Mutação , Fosforilação , Filogenia , RNA Interferente Pequeno/genética , Sirtuína 1 , Sirtuínas/genética , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
HIC1 (Hypermethylated in Cancer 1), as it name implied, was originally isolated as a new candidate tumor suppressor gene located at 17p13.3 because it resides in a CpG island that is hypermethylated in many types of human cancers. HIC1 encodes a transcription factor associating an N-terminal BTB/POZ domain to five C-terminal Krüppel-like C(2)H(2) zinc finger motifs. In this review, we will begin by providing an overview of the current knowledge on HIC1 function, mainly gained from in vitro studies, as a sequence-specific transcriptional repressor interacting with a still growing range of HDAC-dependent and HDAC-independent corepressor complexes. We will then summarize the studies that have demonstrated frequent hypermethylation changes or losses of heterozygosity of the HIC1 locus in human cancers. Next, we will review animal models which have firmly established HIC1 as a bona fide tumor suppressor gene epigenetically silenced and functionally cooperating notably with p53 within a complex HIC1-p53-SIRT1 regulatory loop. Finally, we will discuss how this epigenetic inactivation of HIC1 might "addict" cancer cells to altered survival and signaling pathways or to lineage-specific transcription factors during the early stages of tumorigenesis.
Assuntos
Inativação Gênica , Fatores de Transcrição Kruppel-Like/genética , Neoplasias/genética , Animais , Heterozigoto , Histona Desacetilases/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Modelos Biológicos , Neoplasias/metabolismo , Sirtuínas/genética , Sirtuínas/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Dedos de ZincoRESUMO
HIC1 (hypermethylated in cancer) and its close relative HRG22 (HIC1-related gene on chromosome 22) encode transcriptional repressors with five C(2)H(2) zinc fingers and an N-terminal BTB/POZ autonomous transcriptional repression domain that is unable to recruit histone deacetylases (HDACs). Alignment of the HIC1 and HRG22 proteins from various species highlighted a perfectly conserved GLDLSKK/R motif highly related to the consensus CtBP interaction motif (PXDLSXK/R), except for the replacement of the virtually invariant proline by a glycine. HIC1 strongly interacts with mCtBP1 both in vivo and in vitro through this conserved GLDLSKK motif, thus extending the CtBP consensus binding site. The BTB/POZ domain does not interact with mCtBP1, but the dimerization of HIC1 through this domain is required for the interaction with mCtBP1. When tethered to DNA by fusion with the Gal4 DNA-binding domain, the HIC1 central region represses transcription through interactions with CtBP in a trichostatin A-sensitive manner. In conclusion, our results demonstrate that HIC1 mediates transcriptional repression by both HDAC-independent and HDAC-dependent mechanisms and show that CtBP is a HIC1 corepressor that is recruited via a variant binding site.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Oxirredutases do Álcool , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS/metabolismo , Estruturas do Núcleo Celular/metabolismo , Células Cultivadas , Sequência Conservada , Cricetinae , Proteínas de Ligação a DNA/genética , Dimerização , Evolução Molecular , Genes Supressores de Tumor , Histona Desacetilases/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like , Fosfoproteínas/genética , Coelhos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
The BTB/POZ transcriptional repressor HIC1 (Hypermethylated in Cancer 1) is a tumor suppressor gene located at chromosome 17p13.3, a region frequently hypermethylated or deleted in human tumors and in a contiguous-gene syndrome, the Miller-Dieker syndrome. The human and murine HIC1 genes are composed of two alternative 5' exons, 1a and 1b fused to a large second coding exon 2. Exon 1a is a noncoding exon associated with a major G-C-rich promoter whereas exon 1b is a downstream coding exon associated with a minor TATA box promoter. By human-mouse genome comparison, we have identified a short upstream conserved sequence containing G-C boxes which were shown to be functional. Transcripts initiating from this new promoter were detected in various human and mouse tissues and contained a long 5'-UTR sequence, called 1c which encompass the G-C-rich promoter associated with exon 1a and uses the same splice donor site. RT-PCR analyses of two primary breast epithelial cell lines identified two other 5'-UTRs generated by alternative splicing within exon 1c. Our results thus highlight the existence of an unexpected complex transcriptional regulation of HIC1.
Assuntos
Sequência Conservada , Genes Supressores de Tumor , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Animais , Sequência de Bases , Feminino , Humanos , Fatores de Transcrição Kruppel-Like , Camundongos , Dados de Sequência Molecular , Ovário/metabolismo , Coelhos , RatosRESUMO
Hypermethylated in Cancer 1 (HIC1) is a human tumor suppressor gene located at chromosome 17p13.3 which is frequently hypermethylated and transcriptionally silent in many types of tumors. In addition, its location in the Miller-Dieker syndrome's (MDS) deletion region, its embryonic expression pattern in mice and the phenotype of the HIC1-deficient mice have provided strong evidence for its implication in this contiguous-gene syndrome. HIC1 encodes a five C2H2-type zinc finger transcriptional repressor belonging to the BTB/POZ family. We have isolated the true zebrafish orthologue of human HIC1 since it has a comparable intron-exon structure and since its predicted gene product, ZfHIC1 displays much higher sequence similarities in its overall sequence (737 residues) with human HIC1 (714 residues) than the 454 residues encoded by the only zebrafish HIC1 sequence (AF111712) described so far, which has been renamed ZfHIC1alpha. Notably, the C-terminal end and one zinc finger in the DNA-binding domain are missing in ZfHIC1alpha. As a consequence, ZfHIC1 proteins bind the human HIC1 consensus DNA-binding sequence in vitro, whereas ZfHIC1alpha cannot. Analyses of the expression pattern of ZfHIC1 and of its paralogue ZfHRG22 (HIC1 related gene on chromosome 22) show that they share expression domains with their respective orthologous vertebrate genes. ZfHRG22 is prominently expressed in the brain and in neural tissues. Interestingly, the predominant expression of ZfHIC1 in the mesenchyme of the head, around the nose and the eye and in the branchial arches is possibly consistent with some of the abnormalities seen in the HIC1-deficient mice and provides another clue for the implication of HIC1 in MDS.
Assuntos
Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Proteínas de Ligação a DNA , Genes Supressores de Tumor , Fatores de Transcrição Kruppel-Like , Mesoderma/metabolismo , Dados de Sequência Molecular , Alinhamento de Sequência , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genéticaRESUMO
The tumor suppressor gene HIC1 encodes a transcriptional repressor involved in regulatory loops modulating P53-dependent and E2F1-dependent cell survival, growth control, and stress responses. Despite its importance, few HIC1 corepressors and target genes have been characterized thus far. Using a yeast two-hybrid approach, we identify MTA1, a subunit of the NuRD complex, as a new HIC1 corepressor. This interaction is regulated by two competitive posttranslational modifications of HIC1 at lysine 314, promotion by SUMOylation, and inhibition by acetylation. Consistent with the role of HIC1 in growth control, we demonstrate that HIC1/MTA1 complexes bind on two new target genes, Cyclin D1 and p57KIP2 in quiescent but not in growing WI38 cells. In addition, HIC1/MTA1 and HIC1/CtBP complexes differentially bind on two mutually exclusive HIC1 binding sites (HiRE) on the SIRT1 promoter. SIRT1 transcriptional activation induced by short-term serum starvation coincides with loss of occupancy of the distal sites by HIC1/MTA1 and HIC1/CtBP. Upon longer starvation, both complexes are found but on a newly identified proximal HiRE that is evolutionarily conserved and specifically enriched with repressive histone marks. Our results decipher a mechanistic link between two competitive posttranslational modifications of HIC1 and corepressor recruitment to specific genes, leading to growth control.
Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Acetilação , Oxirredutases do Álcool/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p57/genética , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Genes bcl-1 , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Técnicas In Vitro , Interfase , Fatores de Transcrição Kruppel-Like/química , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Camundongos , Modelos Biológicos , Células NIH 3T3 , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Sirtuína 1/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Transativadores , Ativação Transcricional , Técnicas do Sistema de Duplo-HíbridoRESUMO
HIC1 (hypermethylated in cancer 1) is a tumor suppressor gene located at chromosome 17p13.3, a region frequently hypermethylated or deleted in human tumors and in a contiguous-gene syndrome, the Miller-Dieker syndrome. HIC1 is a transcriptional repressor containing five Krüppel-like C(2)H(2) zinc fingers and an N-terminal dimerization and autonomous repression domain called BTB/POZ. Although some of the HIC1 transcriptional repression mechanisms have been recently deciphered, target genes are still to be discovered. In this study, we determined the consensus binding sequence for HIC1 and investigated its DNA binding properties. Using a selection and amplification of binding sites technique, we identified the sequence 5'-(C)/(G)NG(C)/(G)GGGCA(C)/(A) CC-3' as an optimal binding site. In silico and functional analyses fully validated this consensus and highlighted a GGCA core motif bound by zinc fingers 3 and 4. The BTB/POZ domain inhibits the binding of HIC1 to a single site but mediates cooperative binding to a probe containing five concatemerized binding sites, a property shared by other BTB/POZ proteins. Finally, full-length HIC1 proteins transiently expressed in RK13 cells and more importantly, endogenous HIC1 proteins from the DAOY medulloblastoma cell line, repress the transcription of a reporter gene through their direct binding to these sites, as confirmed by chromatin immunoprecipitation experiments. The definition of the HIC1-specific DNA binding sequence as well as the requirement for multiple sites for optimal binding of the full-length protein are mandatory prerequisites for the identification and analyses of bona fide HIC1 target genes.
Assuntos
DNA/metabolismo , Fatores de Transcrição/fisiologia , Transcrição Gênica , Motivos de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Northern Blotting , Western Blotting , Células COS , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cromatina/metabolismo , DNA/química , Análise Mutacional de DNA , Dimerização , Genes Reporter , Glutationa Transferase/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like , Dados de Sequência Molecular , Mutação , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Fatores de Transcrição/biossíntese , Transfecção , Dedos de ZincoRESUMO
HIC1 (hypermethylated in cancer 1) is a transcriptional repressor containing five Krüppel-like C(2)H(2) zinc fingers and an N-terminal dimerization and autonomous repression domain called BTB/POZ. Here, we demonstrate that full-length HIC1 proteins are modified both in vivo and in vitro with O-linked N-acetylglucosamine (O-GlcNAc). This is a highly dynamic glycosylation found within the cytosolic and the nuclear compartments of eukaryotes. Analysis of [(3)H]Gal-labeled tryptic peptides indicates that HIC1 has three major sites for O-GlcNAc glycosylation. Using C-terminal deletion mutants, we have shown that O-GlcNAc modification of HIC1 proteins occurred preferentially in the DNA-binding domain. Nonglycosylated and glycosylated forms of full-length HIC1 proteins separated by wheat germ agglutinin affinity purification, displayed the same specific DNA-binding activity in electrophoretic mobility shift assays proving that the O-GlcNAc modification is not directly implicated in the specific DNA recognition of HIC1. Intriguingly, N-terminal truncated forms corresponding to BTB-POZ-deleted proteins exhibited a strikingly differential activity, as the glycosylated truncated forms are unable to bind DNA whereas the unglycosylated ones do. Electrophoretic mobility shift assays performed with separated pools of glycosylated and unglycosylated forms of a construct exhibiting only the DNA-binding domain and the C-terminal tail of HIC1 (residues 399-714) and supershift experiments with wheat germ agglutinin or RL-2, an antibody raised against O-GlcNAc residues, fully corroborated these results. Interestingly, these truncated proteins are O-GlcNAc modified in their C-terminal tail (residues 670-711) and not in the DNA-binding domain, as for the full-length proteins. Thus, the O-GlcNAc modification of HIC1 does not affect its specific DNA-binding activity and is highly sensitive to conformational effects, notably its dimerization through the BTB/POZ domain.