RESUMO
Diverse Polycomb repressive complexes 1 (PRC1) play essential roles in gene regulation, differentiation and development. Six major groups of PRC1 complexes that differ in their subunit composition have been identified in mammals. How the different PRC1 complexes are recruited to specific genomic sites is poorly understood. The Polycomb Ring finger protein PCGF6, the transcription factors MGA and E2F6, and the histone-binding protein L3MBTL2 are specific components of the non-canonical PRC1.6 complex. In this study, we have investigated their role in genomic targeting of PRC1.6. ChIP-seq analysis revealed colocalization of MGA, L3MBTL2, E2F6 and PCGF6 genome-wide. Ablation of MGA in a human cell line by CRISPR/Cas resulted in complete loss of PRC1.6 binding. Rescue experiments revealed that MGA recruits PRC1.6 to specific loci both by DNA binding-dependent and by DNA binding-independent mechanisms. Depletion of L3MBTL2 and E2F6 but not of PCGF6 resulted in differential, locus-specific loss of PRC1.6 binding illustrating that different subunits mediate PRC1.6 loading to distinct sets of promoters. Mga, L3mbtl2 and Pcgf6 colocalize also in mouse embryonic stem cells, where PRC1.6 has been linked to repression of germ cell-related genes. Our findings unveil strikingly different genomic recruitment mechanisms of the non-canonical PRC1.6 complex, which specify its cell type- and context-specific regulatory functions.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , DNA/metabolismo , Fator de Transcrição E2F6/fisiologia , Proteínas Nucleares/fisiologia , Complexo Repressor Polycomb 1/metabolismo , Fatores de Transcrição/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Cultivadas , Fator de Transcrição E2F6/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Células-Tronco Embrionárias Humanas/fisiologia , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/fisiologia , Proteínas Nucleares/genética , Ligação Proteica/genética , Fatores de Transcrição/genéticaRESUMO
Different transcription factors operate together at promoters and enhancers to regulate gene expression. Transcription factors either bind directly to their target DNA or are tethered to it by other proteins. The transcription factor Sp2 serves as a paradigm for indirect genomic binding. It does not require its DNA-binding domain for genomic DNA binding and occupies target promoters independently of whether they contain a cognate DNA-binding motif. Hence, Sp2 is strikingly different from its closely related paralogs Sp1 and Sp3, but how Sp2 recognizes its targets is unknown. Here, we sought to gain more detailed insights into the genomic targeting mechanism of Sp2. ChIP-exo sequencing in mouse embryonic fibroblasts revealed genomic binding of Sp2 to a composite motif where a recognition sequence for TALE homeoproteins and a recognition sequence for the trimeric histone-fold domain protein nuclear transcription factor Y (Nf-y) are separated by 11 bp. We identified a complex consisting of the TALE homeobox protein Prep1, its partner PBX homeobox 1 (Pbx1), and Nf-y as the major partners in Sp2-promoter interactions. We found that the Pbx1:Prep1 complex together with Nf-y recruits Sp2 to co-occupied regulatory elements. In turn, Sp2 potentiates binding of Pbx1:Prep1 and Nf-y. We also found that the Sp-box, a short sequence motif close to the Sp2 N terminus, is crucial for Sp2's cofactor function. Our findings reveal a mechanism by which the DNA binding-independent activity of Sp2 potentiates genomic loading of Pbx1:Prep1 and Nf-y to composite motifs present in many promoters of highly expressed genes.
Assuntos
Fator de Ligação a CCAAT/metabolismo , Genômica , Proteínas de Homeodomínio/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B/metabolismo , Fator de Transcrição Sp2/metabolismo , Animais , Fator de Ligação a CCAAT/química , Linhagem Celular , Histonas/metabolismo , Camundongos , Motivos de Nucleotídeos , Ligação Proteica , Transporte Proteico , Fator de Transcrição Sp2/química , Dedos de ZincoRESUMO
Transcription factors are grouped into families based on sequence similarity within functional domains, particularly DNA-binding domains. The Specificity proteins Sp1, Sp2 and Sp3 are paradigmatic of closely related transcription factors. They share amino-terminal glutamine-rich regions and a conserved carboxy-terminal zinc finger domain that can bind to GC rich motifs in vitro. All three Sp proteins are ubiquitously expressed; yet they carry out unique functions in vivo raising the question of how specificity is achieved. Crucially, it is unknown whether they bind to distinct genomic sites and, if so, how binding site selection is accomplished. In this study, we have examined the genomic binding patterns of Sp1, Sp2 and Sp3 in mouse embryonic fibroblasts by ChIP-seq. Sp1 and Sp3 essentially occupy the same promoters and localize to GC boxes. The genomic binding pattern of Sp2 is different; Sp2 primarily localizes at CCAAT motifs. Consistently, re-expression of Sp2 and Sp3 mutants in corresponding knockout MEFs revealed strikingly different modes of genomic binding site selection. Most significantly, while the zinc fingers dictate genomic binding of Sp3, they are completely dispensable for binding of Sp2. Instead, the glutamine-rich amino-terminal region is sufficient for recruitment of Sp2 to its target promoters in vivo. We have identified the trimeric histone-fold CCAAT box binding transcription factor Nf-y as the major partner for Sp2-chromatin interaction. Nf-y is critical for recruitment of Sp2 to co-occupied regulatory elements. Equally, Sp2 potentiates binding of Nf-y to shared sites indicating the existence of an extensive Sp2-Nf-y interaction network. Our results unveil strikingly different recruitment mechanisms of Sp1/Sp2/Sp3 transcription factor members uncovering an unexpected layer of complexity in their binding to chromatin in vivo.
Assuntos
Mapas de Interação de Proteínas/genética , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp2/genética , Fator de Transcrição Sp3/genética , Dedos de Zinco/genética , Animais , Sítios de Ligação , Fator de Ligação a CCAAT/genética , Fator de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Genoma , Histonas/genética , Camundongos , Motivos de Nucleotídeos/genética , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp2/metabolismo , Fator de Transcrição Sp3/metabolismoRESUMO
Oligodendrocytes and Schwann cells are the myelinating glia of the vertebrate nervous system and by generation of myelin sheaths allow rapid saltatory conduction. Previous in vitro work had pointed to a role of the zinc finger containing specificity proteins Sp1 and Sp3 as major regulators of glial differentiation and myelination. Here, we asked whether such a role is also evident in vivo using mice with specific deletions of Sp1 or Sp3 in myelinating glia. We also studied glia-specific conditional Sp2- and constitutive Sp4-deficient mice to include all related glutamine-rich Sp factors into our analysis. Surprisingly, we did not detect developmental Schwann cell abnormalities in any of the mutant mice. Oligodendrocyte development and differentiation was also not fundamentally affected as oligodendrocytes were present in all mouse mutants and retained their ability to differentiate and initiate myelin gene expression. The most severe defect we observed was a 50% reduction in Mbp- and proteolipid protein 1 (Plp1)-positive differentiating oligodendrocytes in Sp2 mutants at birth. Unexpectedly, glial development appeared undisturbed even in the joint absence of Sp1 and Sp3. We conclude that Sp2 has a minor effect on the differentiation of myelinating glia, and that glutamine-rich Sp proteins are not essential regulators of the process.
Assuntos
Diferenciação Celular/fisiologia , Glutamina/metabolismo , Bainha de Mielina/metabolismo , Neuroglia/metabolismo , Oligodendroglia/metabolismo , Fator de Transcrição Sp2/metabolismo , Animais , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteína Básica da Mielina/metabolismo , Ratos , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismoRESUMO
Sp1 and Sp3 belong to the specificity proteins (Sp)/Krüppel-like transcription factor family. They are closely related, ubiquitously expressed, and recognize G-rich DNA motifs. They are thought to regulate generic processes such as cell-cycle and growth control, metabolic pathways, and apoptosis. Ablation of Sp1 or Sp3 in mice is lethal, and combined haploinsufficiency results in hematopoietic defects during the fetal stages. Here, we show that in adult mice, conditional pan-hematopoietic (Mx1-Cre) ablation of either Sp1 or Sp3 has minimal impact on hematopoiesis, whereas the simultaneous loss of Sp1 and Sp3 results in severe macrothrombocytopenia. This occurs in a cell-autonomous manner as shown by megakaryocyte-specific (Pf4-Cre) double-knockout mice. We employed flow cytometry, cell culture, and electron microscopy and show that although megakaryocyte numbers are normal in bone marrow and spleen, they display a less compact demarcation membrane system and a striking inability to form proplatelets. Through megakaryocyte transcriptomics and platelet proteomics, we identified several cytoskeleton-related proteins and downstream effector kinases, including Mylk, that were downregulated upon Sp1/Sp3 depletion, providing an explanation for the observed defects in megakaryopoiesis. Supporting this notion, selective Mylk inhibition by ML7 affected proplatelet formation and stabilization and resulted in defective ITAM receptor-mediated platelet aggregation.
Assuntos
Plaquetas/citologia , Megacariócitos/citologia , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3/genética , Animais , Azepinas/química , Plaquetas/metabolismo , Medula Óssea/metabolismo , Citometria de Fluxo , Lectinas Tipo C/metabolismo , Camundongos , Camundongos Knockout , Naftalenos/química , Agregação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteoma , Transdução de Sinais , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/metabolismo , Baço/metabolismo , Trombocitopenia/metabolismo , Fatores de Transcrição/metabolismoRESUMO
To elucidate the function of the transcriptional coregulator PRMT1 (protein arginine methyltranferase 1) in interferon (IFN) signaling, we investigated the expression of STAT1 (signal transducer and activator of transcription) target genes in PRMT1-depleted cells. We show here that PRMT1 represses a subset of IFNgamma-inducible STAT1 target genes in a methyltransferase-dependent manner. These genes are also regulated by the STAT1 inhibitor PIAS1 (protein inhibitor of activated STAT1). PIAS1 is arginine methylated by PRMT1 in vitro as well as in vivo upon IFN treatment. Mutational and mass spectrometric analysis of PIAS1 identifies Arg 303 as the single methylation site. Using both methylation-deficient and methylation-mimicking mutants, we find that arginine methylation of PIAS1 is essential for the repressive function of PRMT1 in IFN-dependent transcription and for the recruitment of PIAS1 to STAT1 target gene promoters in the late phase of the IFN response. Methylation-dependent promoter recruitment of PIAS1 results in the release of STAT1 and coincides with the decline of STAT1-activated transcription. Accordingly, knockdown of PRMT1 or PIAS1 enhances the anti-proliferative effect of IFNgamma. Our findings identify PRMT1 as a novel and crucial negative regulator of STAT1 activation that controls PIAS1-mediated repression by arginine methylation.
Assuntos
Arginina/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Linhagem Celular , Proliferação de Células , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Interferon gama/metabolismo , Interferons/metabolismo , Metilação , Regiões Promotoras GenéticasRESUMO
SUMO modification of many transcription factors is linked to transcriptional repression. The molecular mechanisms by which SUMO attachment represses transcription are largely unknown. Here we report a genome-wide RNA interference screen in Drosophila melanogaster cells for components regulating and mediating SUMO-dependent transcriptional repression. Analysis of >21,000 double-stranded RNAs (dsRNAs) identified 120 genes whose dsRNA-mediated knockdowns impaired SUMO-dependent transcriptional repression. Several of these genes encode chromatin-associated proteins, including the ATP-dependent chromatin remodeler Mi-2, the D. melanogaster ortholog of the C. elegans protein MEP-1, and the polycomb protein Sfmbt. Knockdown of these proteins did not impair SUMO conjugation, demonstrating that they act downstream of SUMO attachment. Biochemical analyses revealed that MEP-1, Mi-2, and Sfmbt interact with each other, bind to SUMO, and are recruited to promoters in a SUMOylation-dependent manner. Our results suggest that MEP-1, Mi-2, and Sfmbt are part of a common repression complex established by DNA-bound SUMO-modified transcription factors.
Assuntos
Cromatina/genética , Drosophila melanogaster/genética , Interferência de RNA , Proteína SUMO-1/metabolismo , Transcrição Gênica , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica , Genoma , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Mamíferos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Regiões Promotoras Genéticas , RNA de Cadeia Dupla/genética , Especificidade da EspécieRESUMO
Lethal(3) malignant brain tumour like 2 (L3MBTL2) is an integral component of the polycomb repressive complex 1.6 (PRC1.6) and has been implicated in transcriptional repression and chromatin compaction. Here, we show that L3MBTL2 is modified by SUMO2/3 at lysine residues 675 and 700 close to the C-terminus. SUMOylation of L3MBTL2 neither affected its repressive activity in reporter gene assays nor it's binding to histone tails in vitro. In order to analyse whether SUMOylation affects binding of L3MBTL2 to chromatin, we performed ChIP-Seq analysis with chromatin of wild-type HEK293 cells and with chromatin of HEK293 cells stably expressing either FLAG-tagged SUMOylation-competent or SUMOylation-defective L3MBTL2. Wild-type FLAG-L3MBTL2 and the SUMOylation-defective FLAG-L3MBTL2 K675/700R mutant essentially occupied the same sites as endogenous L3MBTL2 suggesting that SUMOylation of L3MBTL2 does not affect chromatin binding. However, a subset of L3MBTL2-target genes, particularly those with low L3MBTL2 occupancy including pro-inflammatory genes, was de-repressed in cells expressing the FLAG-L3MBTL2 K675/700R mutant. Finally, we provide evidence that SUMOylation of L3MBTL2 facilitates repression of these PRC1.6-target genes by balancing the local H2Aub1 levels established by the ubiquitinating enzyme RING2 and the de-ubiquitinating PR-DUB complex.
Assuntos
Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Sumoilação , Fatores de Transcrição/metabolismo , Transcrição Gênica , Sítios de Ligação , Fator de Transcrição E2F6/metabolismo , Genoma Humano , Células HEK293 , Histonas/metabolismo , Humanos , Lisina/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Repressoras/química , Proteínas Repressoras/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The transcription factor Sp2 is essential for early mouse development and for proliferation of mouse embryonic fibroblasts in culture. Yet its mechanisms of action and its target genes are largely unknown. In this study, we have combined RNA interference, in vitro DNA binding, chromatin immunoprecipitation sequencing and global gene-expression profiling to investigate the role of Sp2 for cellular functions, to define target sites and to identify genes regulated by Sp2. We show that Sp2 is important for cellular proliferation that it binds to GC-boxes and occupies proximal promoters of genes essential for vital cellular processes including gene expression, replication, metabolism and signalling. Moreover, we identified important key target genes and cellular pathways that are directly regulated by Sp2. Most significantly, Sp2 binds and activates numerous sequence-specific transcription factor and co-activator genes, and represses the whole battery of cholesterol synthesis genes. Our results establish Sp2 as a sequence-specific regulator of vitally important genes.
Assuntos
Regulação da Expressão Gênica , Fator de Transcrição Sp2/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Proliferação de Células , DNA/química , DNA/metabolismo , Mineração de Dados , Deleção de Genes , Perfilação da Expressão Gênica , Genoma , Células HEK293 , Células HeLa , Humanos , Camundongos , Matrizes de Pontuação de Posição Específica , Regiões Promotoras Genéticas , Interferência de RNA , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp2/antagonistas & inibidores , Fator de Transcrição Sp2/genéticaRESUMO
SUMO modification of transcription factors is linked to repression of transcription. The physiological significance of SUMO attachment to a particular transcriptional regulator, however, is largely unknown. We have employed the ubiquitously expressed murine transcription factor Sp3 to analyze the role of SUMOylation in vivo. We generated mice and mouse embryonic fibroblasts (MEFs) carrying a subtle point mutation in the SUMO attachment sequence of Sp3 (IKEE(553)D mutation). The E(553)D mutation impedes SUMOylation of Sp3 at K(551)in vivo, without affecting Sp3 protein levels. Expression profiling revealed that spermatocyte-specific genes, such as Dmc1 and Dnahc8, and neuronal genes, including Paqr6, Rims3, and Robo3, are de-repressed in non-testicular and extra-neuronal mouse tissues and in mouse embryonic fibroblasts expressing the SUMOylation-deficient Sp3E(553)D mutant protein. Chromatin immunoprecipitation experiments show that transcriptional de-repression of these genes is accompanied by the loss of repressive heterochromatic marks such as H3K9 and H4K20 tri-methylation and impaired recruitment of repressive chromatin-modifying enzymes. Finally, analysis of the DNA methylation state of the Dmc1, Paqr6, and Rims3 promoters by bisulfite sequencing revealed that these genes are highly methylated in Sp3wt MEFs but are unmethylated in Sp3E(553)D MEFs linking SUMOylation of Sp3 to tissue-specific CpG methylation. Our results establish SUMO conjugation to Sp3 as a molecular beacon for the assembly of repression machineries to maintain tissue-specific transcriptional gene silencing.
Assuntos
Inativação Gênica , Neurônios/metabolismo , Fator de Transcrição Sp3/genética , Espermatócitos/metabolismo , Sumoilação/genética , Animais , Cromatina/metabolismo , Metilação de DNA/genética , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Marcação de Genes , Masculino , Meiose/genética , Camundongos , Camundongos Mutantes , Neurônios/citologia , Especificidade de Órgãos/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fator de Transcrição Sp3/metabolismoRESUMO
Modification of many transcription factors including Sp3 and steroidogenic factor 1 with the small ubiquitin-like modifier (SUMO) is associated with transcriptional repression. Here, we show that SUMOylation of transcription factors bound to DNA provokes the establishment of compacted repressive chromatin with characteristics of heterochromatin. Chromatin immunoprecipitation experiments revealed SUMO-dependent recruitment of the chromatin remodeller Mi-2, MBT-domain proteins, heterochromatic protein 1, and the histone methyltransferases SETDB1 and SUV4-20H, concomitant with the establishment of histone modifications associated with repressed genes, including H3K9 and H4K20 trimethylation. These results indicate that SUMOylation has a crucial role in regulating gene expression by initiating chromatin structure changes that render DNA inaccessible to the transcription machinery.
Assuntos
Inativação Gênica , Heterocromatina/genética , Proteína SUMO-1/metabolismo , Fator de Transcrição Sp3/metabolismo , Transcrição Gênica/genética , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Histona-Lisina N-Metiltransferase , Humanos , Camundongos , Modelos Biológicos , Reação em Cadeia da Polimerase/métodos , Proteínas Metiltransferases/genética , Proteínas Metiltransferases/metabolismo , Proteína SUMO-1/genética , Fator de Transcrição Sp3/genética , TransfecçãoRESUMO
The cyclin-dependent kinase inhibitor p21/WAF1/CIP1 is an important regulator of cell cycle progression, senescence, and differentiation. Genotoxic stress leads to activation of the tumor suppressor p53 and subsequently to induction of p21 expression. Here we show that the tumor suppressor p53 cooperates with the transcription factor Sp1 in the activation of the p21 promoter, whereas histone deacetylase 1 (HDAC1) counteracts p53-induced transcription from the p21 gene. The p53 protein binds directly to the C terminus of Sp1, a domain which was previously shown to be required for the interaction with HDAC1. Induction of p53 in response to DNA-damaging agents resulted in the formation of p53-Sp1 complexes and simultaneous dissociation of HDAC1 from the C terminus of Sp1. Chromatin immunoprecipitation experiments demonstrated the association of HDAC1 with the p21 gene in proliferating cells. Genotoxic stress led to recruitment of p53, reduced binding of HDAC1, and hyperacetylation of core histones at the p21 promoter. Our findings show that the deacetylase HDAC1 acts as an antagonist of the tumor suppressor p53 in the regulation of the cyclin-dependent kinase inhibitor p21 and provide a basis for understanding the function of histone deacetylase inhibitors as antitumor drugs.
Assuntos
Ciclinas/genética , Histona Desacetilases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Antineoplásicos/farmacologia , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21 , Dano ao DNA , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Histona Desacetilase 1 , Inibidores de Histona Desacetilases , Humanos , Proteínas da Gravidez/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fator de Transcrição Sp1/metabolismo , TransfecçãoRESUMO
For long it has been recognized that CCAAT boxes and GC-rich elements co-occur in many human and murine promoters within 100bp upstream of the transcription start site. The trimeric transcription factor NF-Y is the major CCAAT box-binding factor, and members of the SP family of transcription factors are the major GC box-binding proteins. Recent chromatin immunoprecipitations coupled with high throughput sequencing (ChIP-seq) have examined binding of NF-Y and the ubiquitous SP factors SP1, SP2 and SP3 genome-wide, allowing for comprehensive comparison of NF-Y and SP factor actions in the context of chromatin. Here, I attempt a synthesis of the earlier single-promoter type of analysis with the more recent genome-wide studies. In particular, I also discuss different modes of genomic interactions between SP factors and NF-Y that have emerged recently, and identify a key technical issue, which needs to be taken into account in a critical evaluation of genome-wide studies. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.
Assuntos
Fator de Ligação a CCAAT/metabolismo , Elementos de Resposta/fisiologia , Fatores de Transcrição Sp/metabolismo , Animais , Fator de Ligação a CCAAT/genética , Imunoprecipitação da Cromatina , Estudo de Associação Genômica Ampla , Humanos , Camundongos , Fatores de Transcrição Sp/genéticaRESUMO
The molecular mechanisms involved in modulation of the antioxidant cell defence by survival signals remain largely unexplored. Here, we report a mechanistic connection between the survival signal elicited by nerve growth factor (NGF) and the antioxidant cell defence represented by heme oxygenase-1 (HO-1) at the level of a newly identified Sp1 site in the human ho1 proximal promoter. By using luciferase reporter constructs we identified a PI3K-responsive region containing a GC-box that resembled the response element for Sp1. Indeed, transfection of Sp1-deficient SL2 cells, electrophoretic mobility shift assays, the use of the GC-box binding drug mithramycin, and mutation of the GC-box provided evidence for a Sp1-like site in the PI3K-sensitive region. Then, we observed with the use of a Sp1-Gal4 chimera that PI3K regulates the transactivating capacity of Sp1. Cotransfection of active PI3K and PKC-zeta expression vectors resulted in substantial increase of Sp1 phosphorylation and in synergistic activation of both Sp1-Gal4 and endogenous Sp1. Moreover, these effects were mimicked by cotransfection of active MEK and ERK expression vectors and were blocked by the MEK inhibitor PD98059. Inhibition of HO-1 with Sn protoporphyrin IX and blockage of Sp-1-mediatied upregulation of HO-1 with mithramycin attenuated antioxidant and cytoprotective functions of NGF against hydrogen peroxide. This study elucidates how NGF contributes to protection of target cells against oxidative stress.
Assuntos
Regulação Enzimológica da Expressão Gênica , Heme Oxigenase-1/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C/metabolismo , Fator de Transcrição Sp1/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Fator de Crescimento Neural/farmacologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA Interferente Pequeno , Ratos , Ativação Transcricional , Regulação para CimaRESUMO
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors, originally implicated in the regulation of lipid and glucose homeostasis. In addition, natural and synthetic PPAR activators may control inflammatory processes by inhibition of distinct proinflammatory genes. As signaling via the vascular endothelial growth factor receptor-2 (VEGFR2) pathway is critical for angiogenic responses during chronic inflammation, we explored whether known antiinflammatory effects of PPAR ligands are mediated in part through diminished VEGFR2 expression. In this study, PPARalpha agonists are found to inhibit endothelial VEGFR2 expression, whereas predominant PPARgamma ligands remained without discernible effects. Time- and concentration-dependent inhibition is demonstrated both at the level of protein and mRNA VEGFR2 expression. Inhibitory effects of PPARalpha agonists on transcriptional activity of the VEGFR2 promoter are conveyed by an element located between base pairs -60 and -37 that contains two adjacent consensus Sp1 transcription factor binding sites. Constitutive Sp1-containing complex formation to this sequence is decreased by PPARalpha treatment, indicating that VEGFR2 gene expression is inhibited by repressing Sp1 site-dependent DNA binding and transactivation. Our coimmunoprecipitation experiments revealed enhanced protein interactions between PPARalpha and Sp1 on PPARalpha activation, thus constituting a probable mechanism by which PPARalpha activators decrease Sp-dependent binding activity to the VEGFR2 promoter. Hence, molecular mechanisms by which PPARs modulate the rate of gene transcription may include direct interactions between specific transcription factors and PPARs that ultimately result in reduced DNA binding to their respective response elements.
Assuntos
Proliferadores de Peroxissomos/farmacologia , Fator de Transcrição Sp1/metabolismo , Ativação Transcricional/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Sítios de Ligação/genética , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fenofibrato/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Luciferases/genética , Luciferases/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Pirimidinas/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Rosiglitazona , Tiazolidinedionas/farmacologia , Fatores de Transcrição/agonistas , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Most small cell lung cancer (SCLC) patients relapse within 12 months of starting combination chemotherapy plus radio-therapy, due to the development of acquired chemo- and radio-resistance. This phenomenon relates to the induction of tumour differentiation, resulting in apoptosis-resistant, morphologically variant (v-SCLC) cells, which lack the neuroendocrine expression of classic (c-) SCLC cells. In this study spontaneously adherent SCLC sublines were shown by differential gene expression analysis to provide an in vitro model of variant differentiation in SCLC, with down-regulation of neuroendocrine markers and up-regulation of epithelial differentiation markers cyclin D1, endothelin, the cell adhesion molecules CD 44 and integrin subunits alpha2, beta3 and beta4. The sensitivity of adherent SCLC sublines to etoposide, cyclophosphamide and gamma radiation was significantly diminished relative to parent suspension cell lines. Western blot analysis using phosphorylation-specific antibodies to Akt and MAP kinase revealed markedly elevated activation in adherent SCLC sublines, paralleled by increased levels of phosphorylated Bad protein and activated NF-kappaB. Subcultivation of the adherent sublines on uncoated surfaces reversed their adherent phenotype immediately and under these conditions Akt activity reverted to low levels. These results suggest that c-SCLC cells can differentiate spontaneously to v-SCLC and that the associated cellular adhesion may trigger Akt-dependent inhibition of apoptosis in SCLC cells, thus leading to acquired chemo- and radio-resistance.
Assuntos
Carcinoma de Células Pequenas/patologia , Adesão Celular , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Antineoplásicos/farmacologia , Sequência de Bases , Carcinoma de Células Pequenas/tratamento farmacológico , Carcinoma de Células Pequenas/enzimologia , Carcinoma de Células Pequenas/radioterapia , Primers do DNA , Raios gama , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/radioterapia , Proteínas Proto-Oncogênicas c-akt , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated nuclear receptor regulating adipogenesis, glucose homeostasis and inflammatory responses. The activity of PPARγ is controlled by post-translational modifications including SUMOylation and phosphorylation that affects its biological and molecular functions. Several important aspects of PPARγ SUMOylation including SUMO isoform-specificity and the impact of ligand binding on SUMOylation remain unresolved or contradictory. Here, we present a comprehensive study of PPARγ1 SUMOylation. We show that PPARγ1 can be modified by SUMO1 and SUMO2. Mutational analyses revealed that SUMOylation occurs exclusively within the N-terminal activation function 1 (AF1) domain predominantly at lysines 33 and 77. Ligand binding to the C-terminal ligand-binding domain (LBD) of PPARγ1 reduces SUMOylation of lysine 33 but not of lysine 77. SUMOylation of lysine 33 and lysine 77 represses basal and ligand-induced activation by PPARγ1. We further show that lysine 365 within the LBD is not a target for SUMOylation as suggested in a previous report, but it is essential for full LBD activity. Our results suggest that PPARγ ligands negatively affect SUMOylation by interdomain communication between the C-terminal LBD and the N-terminal AF1 domain. The ability of the LBD to regulate the AF1 domain may have important implications for the evaluation and mechanism of action of therapeutic ligands that bind PPARγ.
Assuntos
PPAR gama/química , PPAR gama/metabolismo , Sumoilação , Células HEK293 , Células HeLa , Humanos , Ligantes , Lisina/metabolismo , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Repressoras/metabolismo , Serina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Relação Estrutura-Atividade , Transcrição GênicaRESUMO
OBJECTIVE: The transcription factor PAX5 is essential for the activation of B-cell-specific genes and for the silencing of myeloid-specific genes. We previously determined the molecular mechanism by which PAX5 silences the myeloid-specific colony-stimulating-factor-receptor (Csf1R) gene and showed that PAX5 directly binds to the Csf1r promoter as well as to an intronic enhancer that generates an antisense transcript in B cells. Here we examine the role of PAX5 in the regulation of sense and antisense transcription in B cells. MATERIALS AND METHODS: We performed PAX5-specific chromatin immunoprecipitation analyses across the Csfr1 locus. We investigated the role of PAX5 in regulating Csf1r sense and antisense promoter activity by transient transfections and by employing a Pax5(-/-) pro-B-cell line expressing an inducible PAX5 protein. PAX5 interacting factors were identified by pull-down experiments. The role of the transcription factor Sp3 in driving antisense promoter expression was examined in B cells from Sp3 knockout mice. RESULTS: PAX5 differentially regulates the Csf1r promoter and the promoter of the antisense transcript. PAX5 interferes with PU.1 transactivation at the sense promoter by binding to a PAX5 consensus sequence. At the antisense promoter, PAX5 does not specifically recognize DNA, but interacts with Sp3 to upregulate antisense promoter activity. Antisense promoter activation by PAX5 is dependent on the presence of its partial homeo-domain. CONCLUSIONS: We demonstrate that PAX5 regulates Csf1r in B cells by reducing the frequency of binding of the basal transcription machinery to the promoter and by activating antisense RNA expression.
Assuntos
Linfócitos B/metabolismo , Regulação da Expressão Gênica , Fator de Transcrição PAX5/genética , Regiões Promotoras Genéticas/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Imunoprecipitação da Cromatina , DNA Antissenso/genética , Camundongos , Camundongos Knockout , Mutação , Fator de Transcrição PAX5/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição Sp3/genética , Fator de Transcrição Sp3/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
BACKGROUND: The zinc finger protein Sp2 (specificity protein 2) is a member of the glutamine-rich Sp family of transcription factors. Despite its close similarity to Sp1, Sp3 and Sp4, Sp2 does not bind to DNA or activate transcription when expressed in mammalian cell lines. The expression pattern and the biological relevance of Sp2 in the mouse are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Whole-mount in situ hybridization of mouse embryos between E7.5 and E9.5 revealed abundant expression in most embryonic and extra-embryonic tissues. In order to unravel the biological relevance of Sp2, we have targeted the Sp2 gene by a tri-loxP strategy. Constitutive Sp2null and conditional Sp2cko knockout alleles were obtained by crossings with appropriate Cre recombinase expressing mice. Constitutive disruption of the mouse Sp2 gene (Sp2null) resulted in severe growth retardation and lethality before E9.5. Mouse embryonic fibroblasts (MEFs) derived from Sp2null embryos at E9.5 failed to grow. Cre-mediated ablation of Sp2 in Sp2cko/cko MEFs obtained from E13.5 strongly impaired cell proliferation. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that Sp2 is essential for early mouse development and autonomous proliferation of MEFs in culture. Comparison of the Sp2 knockout phenotype with the phenotypes of Sp1, Sp3 and Sp4 knockout strains shows that, despite their structural similarity and evolutionary relationship, all four glutamine-rich members of the Sp family of transcription factors have distinct non-redundant functions in vivo.