RESUMO
Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron overload among Caucasians of northern European descent. Over 85% of all cases with HH are due to mutations in the hemochromatosis protein (HFE) involved in iron metabolism. Although the importance in iron homeostasis is well recognized, the mechanism of sensing and regulating iron absorption by HFE, especially in the absence of iron response element in its gene, is not fully understood. In this report, we have identified an inverted repeat sequence (ATGGTcttACCTA) within 1700bp (-1675/+35) of the HFE promoter capable to form cruciform structure that binds PARP1 and strongly represses HFE promoter. Knockdown of PARP1 increases HFE mRNA and protein. Similarly, hemin or FeCl3 treatments resulted in increase in HFE expression by reducing nuclear PARP1 pool via its apoptosis induced cleavage, leading to upregulation of the iron regulatory hormone hepcidin mRNA. Thus, PARP1 binding to the inverted repeat sequence on the HFE promoter may serve as a novel iron sensing mechanism as increased iron level can trigger PARP1 cleavage and relief of HFE transcriptional repression.
Assuntos
Regulação da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/genética , Sequências Repetidas Invertidas , Proteínas de Membrana/genética , Poli(ADP-Ribose) Polimerases/fisiologia , Regiões Promotoras Genéticas/fisiologia , Transcrição Gênica , Western Blotting , Cloretos/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Compostos Férricos/farmacologia , Células HCT116 , Células HEK293 , Células HeLa , Proteína da Hemocromatose , Células Hep G2 , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Luciferases/metabolismo , Proteínas de Membrana/metabolismo , Noxas/farmacologia , Fragmentos de Peptídeos/metabolismo , Poli(ADP-Ribose) Polimerase-1 , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Viral promoters are widely utilized in commercial and customized vectors to drive expression of genes of interest including reporter, effector and transfection control, because of their high transcription efficiency in a variety of primary and transformed cell lines. However, we observed altered rate of transcription for these promoters under conditions such as presence of an effector protein. These variations in viral promoter driven expressions can potentially lead to incorrect conclusion, especially in comparative and quantitative experiments. We found significantly reduced viral promoter activity in cells overexpressing tumor suppressor protein p53, whereas markedly induced transcription in cells overexpressing MAP/ERK kinase kinase 1 (Mekk 1). Using deletion constructs generated from the CMV promoter, we found the transcription reduction by p53 is possibly mediated through the TATA motif present in proximal CMV promoter. The activation of the CMV promoter by Mekk 1, on the other hand, is attributed to the proximal CRE binding site in the promoter. These findings may be of interest to investigators who use CMV (or other viral) promoter driven vectors for either comparative or quantitative gene expression, or effect on promoter activity.
Assuntos
Citomegalovirus/genética , Sistema de Sinalização das MAP Quinases , TATA Box , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Sequência de Bases , Sítios de Ligação , Sequência Consenso , Citomegalovirus/metabolismo , Ativação Enzimática , Vetores Genéticos , Células HEK293 , Humanos , MAP Quinase Quinase Quinase 1/genética , MAP Quinase Quinase Quinase 1/metabolismo , Dados de Sequência Molecular , Mapeamento de Interação de Proteínas , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
Mitogen-activated protein kinase (MAPK) cascades regulate a wide variety of cellular processes that ultimately depend on changes in gene expression. We have found a novel mechanism whereby one of the key MAP3 kinases, Mekk1, regulates transcriptional activity through an interaction with p53. The tumor suppressor protein p53 down-regulates a number of genes, including the gene most frequently mutated in autosomal dominant polycystic kidney disease (PKD1). We have discovered that Mekk1 translocates to the nucleus and acts as a co-repressor with p53 to down-regulate PKD1 transcriptional activity. This repression does not require Mekk1 kinase activity, excluding the need for an Mekk1 phosphorylation cascade. However, this PKD1 repression can also be induced by the stress-pathway stimuli, including TNFα, suggesting that Mekk1 activation induces both JNK-dependent and JNK-independent pathways that target the PKD1 gene. An Mekk1-p53 interaction at the PKD1 promoter suggests a new mechanism by which abnormally elevated stress-pathway stimuli might directly down-regulate the PKD1 gene, possibly causing haploinsufficiency and cyst formation.
Assuntos
Regulação Enzimológica da Expressão Gênica , MAP Quinase Quinase Quinase 1/metabolismo , Regiões Promotoras Genéticas , Canais de Cátion TRPP/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Humanos , Camundongos , Microscopia de Fluorescência/métodos , Dados de Sequência Molecular , Mutagênese , Estresse Oxidativo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Osteoarthritis (OA) is the most common form of joint disease in middle-aged and older individuals. Previous studies have shown that over-expression of matrix-degrading proteinases and proinflammatory cytokines is associated with osteoarthritic cartilage degradation. However, it remains unclear which transcription factors regulate the expression of these cartilage-degrading molecules in articular chondrocytes. This study demonstrated that mice lacking Nfat1, a member of the nuclear factor of activated T cells (NFAT) transcription factors, exhibited normal skeletal development but displayed loss of type II collagen (collagen-2) and aggrecan with over-expression of specific matrix-degrading proteinases and proinflammatory cytokines in young adult articular cartilage of load-bearing joints. These initial changes are followed by articular chondrocyte proliferation/clustering, progressive articular surface destruction, periarticular chondro-osteophyte formation and exposure of thickened subchondral bone, all of which resemble human OA. Forced expression of Nfat1 delivered with lentiviral vectors in cultured 3 month-old primary Nfat1 knockout (Nfat1(-/-)) articular chondrocytes partially or completely rescued the abnormal catabolic and anabolic activities of Nfat1(-/-) articular chondrocytes. These new findings revealed a previously unrecognized critical role of Nfat1 in maintaining the physiological function of differentiated adult articular chondrocytes through regulating the expression of specific matrix-degrading proteinases and proinflammatory cytokines. Nfat1 deficiency causes OA due to an imbalance between the catabolic and anabolic activities of adult articular chondrocytes, leading to articular cartilage degradation and failed repair activities in and around articular cartilage. These results may provide new insights into the aetiology, pathogenesis and potential therapeutic strategies for osteoarthritis.
Assuntos
Artrite Experimental/patologia , Cartilagem Articular/patologia , Condrócitos/fisiologia , Fatores de Transcrição NFATC/deficiência , Osteoartrite/patologia , Animais , Apoptose , Artrite Experimental/metabolismo , Cartilagem Articular/metabolismo , Cartilagem Articular/fisiopatologia , Células Cultivadas , Condrócitos/metabolismo , Vetores Genéticos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lentivirus/genética , Camundongos , Camundongos Knockout , Fatores de Transcrição NFATC/metabolismo , Osteoartrite/metabolismo , Fenótipo , Suporte de Carga/fisiologiaRESUMO
Rapsyn is a synapse-specific protein that is required for clustering acetylcholine receptors at the neuromuscular junction. Analysis of the rapsyn promoter revealed a consensus site for the transcription factor Kaiso within a region that is mutated in a subset of patients with congenital myasthenic syndrome. Kaiso is a POZ-zinc finger family transcription factor which recognizes the specific core consensus sequence CTGCNA (where N is any nucleotide). Previously, the only known binding partner for Kaiso was the cell adhesion cofactor, p120 catenin. Here we show that delta-catenin, a brain-specific member of the p120 catenin subfamily, forms a complex with Kaiso. Antibodies against Kaiso and delta-catenin recognize proteins in the nuclei of C2C12 myocytes and at the postsynaptic domain of the mouse neuromuscular junction. Endogenous Kaiso in C2C12 cells coprecipitates with the rapsyn promoter in vivo as shown by chromatin immunoprecipitation assay. Minimal promoter assays demonstrated that the rapsyn promoter can be activated by Kaiso and delta-catenin; this activation is apparently muscle specific. These results provide the first experimental evidence that rapsyn is a direct sequence-specific target of Kaiso and delta-catenin. We propose a new model of synapse-specific transcription that involves the interaction of Kaiso, delta-catenin, and myogenic transcription factors at the neuromuscular junction.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Regulação da Expressão Gênica , Proteínas Musculares/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Antibióticos Antineoplásicos/farmacologia , Proteínas do Domínio Armadillo , Sequência de Bases , Cateninas , Moléculas de Adesão Celular , Linhagem Celular , Galinhas , Ácidos Graxos Insaturados/farmacologia , Genes Reporter , Humanos , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/química , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Síndromes Miastênicas Congênitas/genética , Síndromes Miastênicas Congênitas/metabolismo , Junção Neuromuscular/fisiologia , Fosfoproteínas , Alinhamento de Sequência , delta CateninaRESUMO
Duchenne muscular dystrophy is a fatal childhood disease caused by mutations that abolish the expression of dystrophin in muscle. Utrophin is a paralogue of dystrophin and can functionally replace it in skeletal muscle. A potential therapeutic approach is to increase utrophin levels in muscle. One way to achieve this aim is to increase the expression of the utrophin gene at a transcriptional level via promoter activation. In this study, we have shown that utrophin A mRNA levels can be induced by okadaic acid in murine myogenic C2C12 cells. We have found that a utrophin A promoter reporter can be induced by Sp1 in C2C12 myoblasts, but not in myotubes. This activation can be enhanced by okadaic acid treatment. Our data suggest that this induction is due to Sp1 phosphorylation during myogenesis and thus, utrophin expression in muscle could be regulated by treatment with phosphatase inhibitors. Control of utrophin promoter activation could then be used to increase the expression of utrophin, and thus ameliorate the symptoms of Duchenne muscular dystrophy.
Assuntos
Proteínas do Citoesqueleto/genética , Proteínas de Membrana/genética , Mioblastos Esqueléticos/efeitos dos fármacos , Ácido Okadáico/farmacologia , Regiões Promotoras Genéticas/genética , Regulação para Cima/efeitos dos fármacos , Animais , Sequência de Bases/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos , Dados de Sequência Molecular , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia , Mioblastos Esqueléticos/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp1/farmacologia , Regulação para Cima/genética , UtrofinaRESUMO
BACKGROUND: MicroRNA-34a (miR-34a) is a transcriptional target of p53 and is down-regulated in pancreatic cancer. This study aimed to investigate the functional significance of miR-34a in pancreatic cancer progression through its epigenetic restoration with chromatin modulators, demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-dC) and HDAC inhibitor Vorinostat (SAHA). METHODOLOGY/PRINCIPAL FINDINGS: Re-expression of miR-34a in human pancreatic cancer stem cells (CSCs) and in human pancreatic cancer cell lines upon treatment with 5-Aza-dC and SAHA strongly inhibited the cell proliferation, cell cycle progression, self-renewal, epithelial to mesenchymal transition (EMT) and invasion. In pancreatic CSCs, modulation of miR-34a induced apoptosis by activating caspase-3/7. Treatment of pancreatic CSCs with the chromatin-modulating agents resulted in the inhibition of Bcl-2, CDK6 and SIRT1, which are the putative targets of miR-34a. MiR-34a upregulation by these agents also induced acetylated p53, p21(WAF1), p27(KIP1) and PUMA in pancreatic CSCs. Inhibition of miR-34a by antagomiR abrogates the effects of 5-Aza-dC and SAHA, suggesting that 5-Aza-dC and SAHA regulate stem cell characteristics through miR-34a. In CSCs, SAHA inhibited Notch pathway, suggesting its suppression may contribute to inhibition of the self-renewal capacity and induction of apoptosis. Interestingly, treatment of pancreatic CSCs with SAHA resulted in the inhibition of EMT with the transcriptional up-regulation of E-Cadherin and down-regulation of N-Cadherin. Expression of EMT inducers (Zeb-1, Snail and Slug) was inhibited in CSCs upon treatment with SAHA. 5-Aza-dC and SAHA also retard in vitro migration and invasion of CSCs. CONCLUSIONS: The present study thus demonstrates the role of miR-34a as a critical regulator of pancreatic cancer progression by the regulating CSC characteristics. The restoration of its expression by 5-Aza-dC and SAHA in CSCs will not only provide mechanistic insight and therapeutic targets for pancreatic cancer but also promising reagents to boost patient response to existing chemotherapies or as a standalone cancer drug by eliminating the CSC characteristics.
Assuntos
Epigênese Genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cromatina/metabolismo , Epigênese Genética/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , MicroRNAs/metabolismo , Invasividade Neoplásica , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/patologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Ensaio Tumoral de Célula-Tronco , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , VorinostatRESUMO
The development of disease-modifying pharmacologic therapy for osteoarthritis (OA) currently faces major obstacles largely because the regulatory mechanisms for the function of adult articular chondrocytes remain unclear. We previously demonstrated that lack of Nfat1, one of the nuclear factor of activated T cells (NFAT) transcription factors, causes OA-like changes in adult mice. This study aimed to identify whether Nfat1 specifically regulates adult articular chondrocyte function and its age-dependent regulatory mechanism using both Nfat1-deficient and wild-type mice. Deletion of Nfat1 did not induce OA-like articular chondrocyte dysfunction (e.g., overexpression of proinflammatory cytokines and matrix-degrading proteinases) until the adult stage. RNAi-mediated Nfat1 knockdown caused dysfunction of wild-type adult articular chondrocytes. Nfat1 expression in wild-type articular chondrocytes was low in the embryonic but high in the adult stage. Chromatin immunoprecipitation assays demonstrated that an increase in Nfat1 expression in articular chondrocytes was associated with increased H3K4me2 (a histone modification linked to transcriptional activation), whereas a decrease in Nfat1 expression in articular chondrocytes was correlated with increased H3K9me2 (a histone modification linked to transcriptional repression). Knockdown of lysine-specific demethylase-1 (Lsd1) in embryonic articular chondrocytes upregulated Nfat1 expression concomitant with increased H3K4me2 at the Nfat1 promoter. Knockdown of Jmjc-containing histone demethylase-2a (Jhdm2a) in 6-month articular chondrocytes downregulated Nfat1 expression concomitant with increased H3K9me2 at the Nfat1 promoter. These results suggest that Nfat1 is an essential transcriptional regulator of chondrocyte homeostasis in adult articular cartilage. Age-dependent Nfat1 expression in articular chondrocytes is regulated by dynamic histone methylation, one of the epigenetic mechanisms that regulate gene transcription.
Assuntos
Envelhecimento/genética , Cartilagem Articular/patologia , Condrócitos/metabolismo , Epigênese Genética , Histonas/metabolismo , Fatores de Transcrição NFATC/metabolismo , Animais , Sequência de Bases , Condrócitos/patologia , Imunoprecipitação da Cromatina , Técnicas de Silenciamento de Genes , Histona Desmetilases , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lisina/metabolismo , Metilação , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição NFATC/deficiência , Osteoartrite/metabolismo , Osteoartrite/patologia , Oxirredutases N-Desmetilantes/metabolismo , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Regulação para Cima/genéticaRESUMO
The retinoic acids all-trans retinoic acid (AT-RA) and 9-cis retinoic acid (9C-RA) and the retinoic acid receptors RAR and RXR significantly induce transcriptional activity from a 200-bp PKD1 proximal promoter in transfected mammalian cells. This PKD1 promoter region contains Ets, p53, and GC box motifs, but lacks a canonical RAR/RXR motif. Mutagenesis of the Ets sites did not affect RA induction. In contrast, GC box mutations completely blocked stimulation by AT-RA and by RXRbeta or RARbeta. Mithramycin A, which prevents Sp1 binding, significantly reduced basal promoter activity and suppressed upregulation by AT-RA and RXR. The 200-bp proximal promoter could not be induced by AT-RA in Drosophila SL2 cells, which lack Sp1, but could be activated in these cells transfected with exogenous Sp1. Small interfering RNA knockdown of Sp1 in mammalian cells completely blocked RXRbeta upregulation of the promoter. These data indicate that induction of the PKD1 promoter by retinoic acid is mediated through Sp1 elements. RT-PCR showed that AT-RA treatment of HEK293T cells increased the levels of endogenous PKD1 RNA, and chromatin immunoprecipitation showed the presence of both RXR and Sp1 at the PKD1 proximal promoter. These results suggest that retinoids and their receptors may play a role in PKD1 gene regulation.
Assuntos
Regiões Promotoras Genéticas , Canais de Cátion TRPP/genética , Ativação Transcricional/efeitos dos fármacos , Tretinoína/farmacologia , Tretinoína/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Genes Reporter , Humanos , Rim/embriologia , Luciferases/genética , Dados de Sequência Molecular , Plasmídeos , Receptores do Ácido Retinoico/fisiologia , Receptores X de Retinoides/fisiologiaRESUMO
The Na,K-ATPase is an essential enzyme of the plasma membrane that plays a key role in numerous cell processes that depend on the transcellular gradients of Na(+) and K(+). Among the various isoforms of the catalytic subunit of the Na,K-ATPase, alpha4 exhibits the most limited pattern of expression, being restricted to male germ cells. Activity of alpha4 is essential for sperm function, and alpha4 is upregulated during spermatogenesis. The present study addressed the transcriptional control of the human Na,K-ATPase alpha4 gene, ATP1A4. We describe that a 5' untranslated region of the ATP1A4 gene (designated -339/+480 based on the ATP1A4 transcription initiation site) has promoter activity in luciferase reporter assays. Computer analysis of this promoter region revealed consensus sites (CRE) for the cyclic AMP (cAMP) response element modulator (CREM). Accordingly, dibutyryl cAMP (db-cAMP) and ectopic expression of CREMtau, a testis specific splice variant of CREM were able to activate the ATP1A4 promoter driven expression of luciferase in HEK 293 T, JEG-3 and GC-1 cells. Further characterization of the effect of db-cAMP and CREMtau on deleted constructs of the ATP1A4 promoter (-339/+80, and +25/+480), and on the -339/+480 region carrying mutations in the CRE sites showed that db-cAMP and CREMtau effect required the CRE motif located 263 bp upstream the transcription initiation site. EMSA experiments confirmed the CRE sequence as a bonafide CREMtau binding site. These results constitute the first demonstration of the transcriptional control of ATP1A4 gene expression by cAMP and by CREMtau, a transcription factor essential for male germ cell gene expression.
Assuntos
Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , ATPase Trocadora de Sódio-Potássio/genética , Espermatogênese/genética , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Masculino , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Sítio de Iniciação de TranscriçãoRESUMO
The Ets family of transcription factors consists of a group of highly conserved sequence-specific DNA binding proteins that functionally cooperate with other transcription factors to regulate a number of diverse cellular processes including proliferation, differentiation, and apoptosis. We have analyzed a 3.3kb 5'-upstream region of the human PKD1 promoter, using transient transfection in HEK293T cells and Drosophila SL2 cells, to demonstrate that the PKD1 promoter is a target of Ets family transcription factors. Our studies showed that PKD1 promoter-luciferase reporter gene expression is downregulated by cotransfected Fli-1 and is upregulated by cotransfected Ets-1. Using deletion constructs, we demonstrated that the sequences responding to Fli-1 and Ets-1 lie within the -200 to +33bp proximal promoter. This region was found to contain two putative Ets response elements (EREs): an upstream (Ets-A) sequence 5'-CGGAA-3' (-181 to -185) and a downstream (Ets-B) sequence 5'-CGGAT-3' (-129 to -133). Site-directed mutagenesis indicated that both EREs are functional. A Fli-1 DNA binding domain mutant construct (W321R), which is incapable of binding DNA, was unable to inhibit basal promoter activity. In contrast, a Fli-1 DNA binding domain truncation mutant construct, which only contains the DNA binding domain and lacks the transactivation domain, was able to inhibit. These results suggest that the effect of Fli-1 is through direct binding to these EREs. Direct binding of Fli-1 and Ets-1 to the Ets-A and Ets-B sites was supported by electrophoretic mobility shift assays. Lastly, competition between Fli-1 and Ets-1 for the two EREs was demonstrated by showing that increasing amounts of Ets-1 could overcome Fli-1 repression of promoter activity. Taken together, these experiments define the proximal PKD1 promoter region as a potential target of Ets family transcription factors.
Assuntos
Gelsolina/genética , Regiões Promotoras Genéticas/genética , Proteínas/genética , Proteínas Proto-Oncogênicas c-ets/genética , Receptores Citoplasmáticos e Nucleares/genética , Ativação Transcricional/genética , Animais , Células Cultivadas , Drosophila , Regulação da Expressão Gênica/genética , Proteínas dos Microfilamentos , Canais de Cátion TRPP , TransativadoresRESUMO
The last intron of the PKD1 gene (intron 45) was found to have exceptionally high sequence conservation across four mammalian species: human, mouse, rat, and dog. This conservation did not extend to the comparable intron in pufferfish. Pairwise comparisons for intron 45 showed 91% identity (human vs. dog) to 100% identity (mouse vs. rat) for an average for all four species of 94% identity. In contrast, introns 43 and 44 of the PKD1 gene had average pairwise identities of 57% and 54%, and exons 43, 44, and 45 and the coding region of exon 46 had average pairwise identities of 80%, 84%, 82%, and 80%. Intron 45 is 90 to 95 bp in length, with the major region of sequence divergence being in a central 4-bp to 9-bp variable region. RNA secondary structure analysis of intron 45 predicts a branching stem-loop structure in which the central variable region lies in one loop and the putative branch point sequence lies in another loop, suggesting that the intron adopts a specific stem-loop structure that may be important for its removal. Although intron 45 appears to conform to the class of small, G-triplet-containing introns that are spliced by a mechanism utilizing intron definition, its high sequence conservation may be a reflection of constraints imposed by a unique mechanism that coordinates splicing of this last PKD1 intron with polyadenylation.
Assuntos
Sequência Conservada , Íntrons , Proteínas/genética , Animais , Sequência de Bases , Cães , Humanos , Camundongos , Conformação de Ácido Nucleico , Mutação Puntual , RNA Mensageiro/química , Ratos , Canais de Cátion TRPPRESUMO
Polycystic kidney disease (PKD) results from loss-of-function mutations in the PKD1 gene. There are also reports showing abnormally high levels of PKD1 expression in cystic epithelial cells. At present, nothing is known about the molecular mechanisms regulating the normal expression of the PKD1 gene or whether transcriptional disregulation of the PKD1 gene has a role in cyst formation. We have analyzed a 3.3-kb 5'-proximal portion of the human PKD1 gene. Sequence analysis revealed the presence of consensus sequences for numerous transactivating factors, including four T-cell factor (TCF) binding elements (TBEs). Transcriptional activity of the 3.3-kb fragment and a series of deletion constructs was assayed in HEK293T cells. A 2.0-kb proximal promoter region containing one of the four TBEs (TBE1) was inducible up to 6-fold by cotransfection with beta-catenin. beta-catenin-mediated induction was inhibited by dominant-negative TCF and by deletion of the TBE1 sequence. 15- or 109-bp sequences containing the TBE1 site, when cloned upstream of a minimal promoter, were shown to respond to beta-catenin induction. Gel shift assays confirmed that the TBE1 site is capable of forming complexes with TCF and beta-catenin. To determine whether expression of the endogenous PKD1 gene responds to beta-catenin, HT1080 cells were treated with LiCl, and HeLa cells were stably transfected with beta-catenin. In both cases, endogenous PKD1 mRNA levels were elevated in response to these treatments. Taken together, these studies define an active PKD1 promoter region and suggest that the PKD1 gene is a target of the beta-catenin/TCF pathway.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Regiões Promotoras Genéticas , Proteínas/genética , Transativadores/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismo , Canais de Cátion TRPP , beta CateninaRESUMO
Acetylation controls the activity of numerous proteins involved in regulating gene transcription as well as many other cellular processes. In this report we show that the CREB-binding protein (CBP) acetyltransferase acetylates beta-catenin protein in vivo. beta-Catenin is a central component of the Wnt signaling pathway, which is of key importance in development as well as being heavily implicated in a variety of human cancers. We show that the CBP-mediated acetylation of beta-catenin occurs at a single site, lysine 49. Importantly, this lysine is frequently found mutated in cancer and is in a region of importance to the regulation of beta-catenin. We show that mutation of this site leads specifically to an increase in the ability of beta-catenin to activate the c-myc gene but not other beta-catenin-regulated genes. This suggests that acetylation of beta-catenin is involved in regulating Wnt signaling in a promoter-specific fashion.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Acetilação , Sequência de Aminoácidos , Sequência de Bases , Proteína de Ligação a CREB , Linhagem Celular , Proteínas do Citoesqueleto/química , Primers do DNA , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , beta CateninaRESUMO
Cux-1 is a murine homeobox gene that is highly expressed in the developing kidney with expression restricted to the nephrogenic zone. Cux-1 is highly expressed in cyst epithelium of polycystic kidneys from C57BL/6J-cpk/cpk mice, but not in kidneys isolated from age-matched phenotypically normal littermates. To further elucidate the role of Cux-1 in renal development, we generated transgenic mice expressing Cux-1 under the control of the CMV immediate early gene promoter. Mice constitutively expressing Cux-1 developed multiorgan hyperplasia and organomegaly, but not an overall increase in body size. Transgenic kidneys were enlarged 50% by 6 weeks of age, with the increased growth primarily restricted to the cortex. Proliferating cells were found in proximal and distal tubule epithelium throughout the cortex, and the squamous epithelium that normally lines Bowman's capsule was replaced with proximal tubule epithelium. However, the total number of nephrons was not increased. In the developing kidneys of transgenic mice, Cux-1 was ectopically expressed in more highly differentiated tubules and glomeruli, and this was associated with reduced expression of the cyclin kinase inhibitor, p27. Transient transfection experiments revealed that Cux-1 is an inhibitor of p27 promoter activity. These results suggest that Cux-1 regulates cell proliferation during early nephrogenesis by inhibiting expression of p27.