RESUMO
The cancer-free photosensitive trichothiodystrophy (PS-TTD) and the cancer-prone xeroderma pigmentosum (XP) are rare monogenic disorders that can arise from mutations in the same genes, namely ERCC2/XPD or ERCC3/XPB Both XPD and XPB proteins belong to the 10-subunit complex transcription factor IIH (TFIIH) that plays a key role in transcription and nucleotide excision repair, the DNA repair pathway devoted to the removal of ultraviolet-induced DNA lesions. Compelling evidence suggests that mutations affecting the DNA repair activity of TFIIH are responsible for the pathological features of XP, whereas those also impairing transcription give rise to TTD. By adopting a relatives-based whole transcriptome sequencing approach followed by specific gene expression profiling in primary fibroblasts from a large cohort of TTD or XP cases with mutations in ERCC2/XPD gene, we identify the expression alterations specific for TTD primary dermal fibroblasts. While most of these transcription deregulations do not impact on the protein level, very low amounts of prostaglandin I2 synthase (PTGIS) are found in TTD cells. PTGIS catalyzes the last step of prostaglandin I2 synthesis, a potent vasodilator and inhibitor of platelet aggregation. Its reduction characterizes all TTD cases so far investigated, both the PS-TTD with mutations in TFIIH coding genes as well as the nonphotosensitive (NPS)-TTD. A severe impairment of TFIIH and RNA polymerase II recruitment on the PTGIS promoter is found in TTD but not in XP cells. Thus, PTGIS represents a biomarker that combines all PS- and NPS-TTD cases and distinguishes them from XP.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Neoplasias/patologia , Síndromes de Tricotiodistrofia/enzimologia , Animais , Células Cultivadas , Sistema Enzimático do Citocromo P-450/genética , Epoprostenol , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/efeitos da radiação , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos da radiação , Camundongos , Pele/patologia , Transcrição Gênica , Síndromes de Tricotiodistrofia/genética , Raios Ultravioleta , Xeroderma Pigmentoso/genéticaRESUMO
CSA and CSB proteins are key players in transcription-coupled nucleotide excision repair (TC-NER) pathway that removes UV-induced DNA lesions from the transcribed strands of expressed genes. Additionally, CS proteins play relevant but still elusive roles in other cellular pathways whose alteration may explain neurodegeneration and progeroid features in Cockayne syndrome (CS). Here we identify a CS-containing chromatin-associated protein complex that modulates rRNA transcription. Besides RNA polymerase I (RNAP1) and specific ribosomal proteins (RPs), the complex includes ferrochelatase (FECH), a well-known mitochondrial enzyme whose deficiency causes erythropoietic protoporphyria (EPP). Impairment of either CSA or FECH functionality leads to reduced RNAP1 occupancy on rDNA promoter that is associated to reduced 47S pre-rRNA transcription. In addition, reduced FECH expression leads to an abnormal accumulation of 18S rRNA that in primary dermal fibroblasts from CS and EPP patients results in opposed rRNA amounts. After cell irradiation with UV light, CSA triggers the dissociation of the CSA-FECH-CSB-RNAP1-RPs complex from the chromatin while it stabilizes its binding to FECH. Besides disclosing a function for FECH within nucleoli, this study sheds light on the still unknown mechanisms through which CSA modulates rRNA transcription.
Assuntos
Síndrome de Cockayne/genética , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Ferroquelatase/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Polimerase I/genética , RNA Ribossômico/genética , Fatores de Transcrição/genética , Linhagem Celular Transformada , Sobrevivência Celular , Imunoprecipitação da Cromatina , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/patologia , Dano ao DNA , DNA Helicases/metabolismo , Reparo do DNA/efeitos da radiação , Enzimas Reparadoras do DNA/metabolismo , Ferroquelatase/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Regulação da Expressão Gênica , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Polimerase I/metabolismo , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Raios UltravioletaRESUMO
Amyotrophic Lateral Sclerosis (ALS) is a neural disorder gradually leading to paralysis of the whole body. Alterations in superoxide dismutase SOD1 gene have been linked with several variants of familial ALS. Here, we investigated a transgenic (Tg) cloned swine model expressing the human pathological hSOD1G93A allele. As in patients, these Tg pigs transmitted the disease to the progeny with an autosomal dominant trait and showed ALS onset from about 27â¯months of age. Post mortem analysis revealed motor neuron (MN) degeneration, gliosis and hSOD1 protein aggregates in brainstem and spinal cord. Severe skeletal muscle pathology including necrosis and inflammation was observed at the end stage, as well. Remarkably, as in human patients, these Tg pigs showed a quite long presymptomatic phase in which gradually increasing amounts of TDP-43 were detected in peripheral blood mononuclear cells. Thus, this transgenic swine model opens the unique opportunity to investigate ALS biomarkers even before disease onset other than testing novel drugs and possible medical devices.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Neurônios Motores/patologia , Doenças Musculares/genética , Degeneração Neural/genética , Superóxido Dismutase-1/genética , Proteinopatias TDP-43/genética , Esclerose Lateral Amiotrófica/genética , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Humanos , Doenças Musculares/patologia , Degeneração Neural/patologia , Suínos , Proteinopatias TDP-43/patologiaRESUMO
The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEß). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEα and TFIIEß) as well as decreased phosphorylation of TFIIEα in cells from both children. Interestingly, decreased phosphorylation of TFIIEα was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP.
Assuntos
Quinases Ciclina-Dependentes/genética , Reparo do DNA , Fatores de Transcrição TFII/genética , Síndromes de Tricotiodistrofia/genética , Sequência de Aminoácidos , Quinases Ciclina-Dependentes/metabolismo , Dano ao DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Inativação Gênica , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Linhagem , Fosforilação , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo , Fatores de Transcrição TFII/metabolismo , Proteína Grupo D do Xeroderma Pigmentoso/genética , Proteína Grupo D do Xeroderma Pigmentoso/metabolismo , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Mutations in the XPD subunit of the DNA repair/transcription factor TFIIH result in distinct clinical entities, including the cancer-prone xeroderma pigmentosum (XP) and the multisystem disorder trichothiodystrophy (TTD), which share only cutaneous photosensitivity. Gene-expression profiles of primary dermal fibroblasts revealed overexpression of matrix metalloproteinase 1 (MMP-1), the gene encoding the metalloproteinase that degrades the interstitial collagens of the extracellular matrix (ECM), in TTD patients mutated in XPD compared with their healthy parents. The defect is observed in TTD and not in XP and is specific for fibroblasts, which are the main producers of dermal ECM. MMP-1 transcriptional up-regulation in TTD is caused by an erroneous signaling mediated by retinoic acid receptors on the MMP-1 promoter and leads to hypersecretion of active MMP-1 enzyme and degradation of collagen type I in the ECM of cell/tissue systems and TTD patient skin. In agreement with the well-known role of ECM in eliciting signaling events controlling cell behavior and tissue homeostasis, ECM alterations in TTD were shown to impact on the migration and wound-healing properties of patient dermal fibroblasts. The presence of a specific inhibitor of MMP activity was sufficient to restore normal cell migration, thus providing a potential approach for therapeutic strategies. This study highlights the relevance of ECM anomalies in TTD pathogenesis and in the phenotypic differences between TTD and XP.
Assuntos
Matriz Extracelular/patologia , Metaloproteinase 1 da Matriz/metabolismo , Fator de Transcrição TFIIH/fisiologia , Síndromes de Tricotiodistrofia/enzimologia , Humanos , Metaloproteinase 1 da Matriz/genética , Regiões Promotoras Genéticas , Receptores do Ácido Retinoico/metabolismo , Síndromes de Tricotiodistrofia/patologia , CicatrizaçãoRESUMO
BACKGROUND: It is still unclear whether oxidative stress (OS) is a disease consequence or is directly involved in the etiology of neurodegenerative disorders (NDs) onset and/or progression; however, many of these conditions are associated with increased levels of oxidation markers and damaged cell components. Previously we demonstrated the accumulation of reactive oxygen species (ROS) and increased SOD1 gene expression in H2O2-treated SH-SY5Y cells, recapitulating pathological features of Amyotrophic Lateral Sclerosis (ALS). Since we observed a post-transcriptional regulation of SOD1 gene in this cellular model, we investigated the transcriptional regulation of SOD1 mRNA under oxidative stress (OS). RESULTS: In response to H2O2 treatment, PolII increased its association to SOD1 promoter. Electrophoretic mobility shift assays and mass spectrometry analyses on SOD1 promoter highlighted the formation of a transcriptional complex bound to the ARE sequences. Western Blotting experiments showed that in our in vitro model, H2O2 exposure increases Nrf2 expression in the nuclear fraction while immunoprecipitation confirmed its phosphorylation and release from Keap1 inhibition. However, H2O2 treatment did not modify Nrf2 binding on SOD1 promoter, which seems to be regulated by different transcription factors (TFs). CONCLUSIONS: Although our data suggest that SOD1 is transcriptionally regulated in response to OS, Nrf2 does not appear to associate with SOD1 promoter in this cellular model of neurodegeneration. Our results open new perspectives in the comprehension of two key antioxidant pathways involved in neurodegenerative disorders.
Assuntos
Esclerose Lateral Amiotrófica/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fator 2 Relacionado a NF-E2/biossíntese , Degeneração Neural/genética , Superóxido Dismutase/biossíntese , Transcrição Gênica , Esclerose Lateral Amiotrófica/patologia , Linhagem Celular , Regulação da Expressão Gênica/genética , Humanos , Peróxido de Hidrogênio/toxicidade , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Proteína 1 Associada a ECH Semelhante a Kelch , Fator 2 Relacionado a NF-E2/genética , Degeneração Neural/induzido quimicamente , Degeneração Neural/patologia , Estresse Oxidativo/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
Mutations in the XPD subunit of the transcription/DNA repair factor (TFIIH) give rise to trichothiodystrophy (TTD), a rare hereditary multisystem disorder with skin abnormalities. Here, we show that TTD primary dermal fibroblasts contain low amounts of collagen type VI alpha1 subunit (COL6A1), a fundamental component of soft connective tissues. We demonstrate that COL6A1 expression is downregulated by the sterol regulatory element-binding protein-1 (SREBP-1) whose removal from the promoter is a key step in COL6A1 transcription upregulation in response to cell confluence. We provide evidence for TFIIH being involved in transcription derepression, thus highlighting a new function of TFIIH in gene expression regulation. The lack of COL6A1 upregulation in TTD is caused by the inability of the mutated TFIIH complexes to remove SREBP-1 from COL6A1 promoter and to sustain the subsequent high rate of COL6A1 transcription. This defect might account for the pathologic features that TTD shares with hereditary disorders because of mutations in COL6A genes.
Assuntos
Colágeno Tipo VI/genética , Regulação para Baixo , Fator de Transcrição TFIIH/metabolismo , Transcrição Gênica , Síndromes de Tricotiodistrofia/genética , Proteína Grupo D do Xeroderma Pigmentoso/genética , Colágeno Tipo VI/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Fator de Transcrição TFIIH/genética , Síndromes de Tricotiodistrofia/metabolismo , Proteína Grupo D do Xeroderma Pigmentoso/metabolismoRESUMO
Recent evidence points to homeotic proteins as actors in the crosstalk between development and DNA replication. The present work demonstrates that HOXC13, previously identified as a new member of human DNA replicative complexes, is a stable component of early replicating chromatin in living cells: it displays a slow nuclear dynamics due to its anchoring to the DNA minor groove via the arginine-5 residue of the homeodomain. HOXC13 binds in vivo to the lamin B2 origin in a cell-cycle-dependent manner consistent with origin function; the interaction maps with nucleotide precision within the replicative complex. HOXC13 displays in vitro affinity for other replicative complex proteins; it interacts also in vivo with the same proteins in a cell-cycle-dependent fashion. Chromatin-structure modifying treatments, disturbing origin function, reduce also HOXC13-origin interaction. The described interactions are not restricted to a single origin nor to a single homeotic protein (also HOXC10 binds the lamin B2 origin in vivo). Thus, HOX complexes probably contribute in a general, structure-dependent manner, to origin identification and assembly of replicative complexes thereon, in presence of specific chromatin configurations.
Assuntos
Proteínas de Homeodomínio/fisiologia , Origem de Replicação , Animais , Linhagem Celular , Cromatina/química , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Proteínas de Homeodomínio/análise , Proteínas de Homeodomínio/metabolismo , Humanos , Lamina Tipo B/análise , Camundongos , Células NIH 3T3RESUMO
Rac3, a neuronal GTP-binding protein of the Rho family, induces neuritogenesis in primary neurons. Using yeast two-hybrid analysis, we show that Neurabin I, the neuronal F-actin binding protein, is a direct Rac3-interacting molecule. Biochemical and light microscopy studies indicate that Neurabin I copartitions and colocalizes with Rac3 at the growth cones of neurites, inducing Neurabin I association to the cytoskeleton. Moreover, Neurabin I antisense oligonucleotides abolish Rac3-induced neuritogenesis, which in turn is rescued by exogenous Neurabin I but not by Neurabin I mutant lacking the Rac3-binding domain. These results show that Neurabin I mediates Rac3-induced neuritogenesis, possibly by anchoring Rac3 to growth cone F-actin.
Assuntos
Cones de Crescimento/fisiologia , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuritos/fisiologia , Fatores de Transcrição/metabolismo , Actinas/metabolismo , Animais , Células Cultivadas , Citoesqueleto/química , Citoesqueleto/metabolismo , Cones de Crescimento/química , Cones de Crescimento/metabolismo , Imunoprecipitação , Camundongos , Proteínas dos Microfilamentos/análise , Proteínas dos Microfilamentos/genética , Mutação , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/genética , Neuritos/química , Neurônios/citologia , Neurônios/metabolismo , Neurônios/fisiologia , Coativador 3 de Receptor Nuclear , Oligodesoxirribonucleotídeos Antissenso/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Ratos , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
The cis-acting elements necessary for the activity of DNA replication origins in metazoan cells are still poorly understood. Here we report a thorough characterization of the DNA sequence requirements of the origin associated with the human lamin B2 gene. A 1.2-kb DNA segment, comprising the start site of DNA replication and located within a large protein-bound region, as well as a CpG island, displays origin activity when moved to different ectopic positions. Genomic footprinting analysis of both the endogenous and the ectopic origins indicates that the large protein complex is assembled in both cases around the replication start site. Replacement of this footprinted region with an unrelated sequence, maintaining the CpG island intact, abolishes origin activity and the interaction with hORC2, a subunit of the origin recognition complex. Conversely, the replacement of 17 bp within the protected region reduces the extension of the protection without affecting the interaction with hORC2. This substitution does not abolish the origin activity but makes it more sensitive to the integration site. Finally, the nearby CpG island positively affects the efficiency of initiation. This analysis reveals the modular structure of the lamin B2 origin and supports the idea that sequence elements close to the replication start site play an important role in origin activation.
Assuntos
Lamina Tipo B/genética , Origem de Replicação , Replicon , Sequência de Bases , Ilhas de CpG/genética , Pegada de DNA , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutação , Complexo de Reconhecimento de OrigemRESUMO
To date, a complete understanding of the molecular events leading to DNA replication origin activation in mammalian cells still remains elusive. In this work, we report the results of a high resolution chromatin immunoprecipitation study to detect proteins interacting with the human Lamin B2 replication origin. In addition to the pre-RC component ORC4 and to the transcription factors USF and HOXC13, we found that 2 components of the AP-1 transcription factor, c-Fos and c-Jun, are also associated with the origin DNA during the late G1 phase of the cell cycle and that these factors interact with ORC4. Both DNA replication and AP-1 factor binding to the origin region were perturbed by cell treatment with merbarone, a topoisomerase II inhibitor, suggesting that DNA topology is essential for determining origin function.
Assuntos
DNA/metabolismo , Lamina Tipo B/metabolismo , Linhagem Celular Tumoral , Replicação do DNA/efeitos dos fármacos , Fase G1 , Células HeLa , Proteínas de Homeodomínio/metabolismo , Humanos , Imunoprecipitação , Lamina Tipo B/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Complexo de Reconhecimento de Origem/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Origem de Replicação/efeitos dos fármacos , Tiobarbitúricos/farmacologia , Inibidores da Topoisomerase II/farmacologia , Fator de Transcrição AP-1/metabolismo , Fatores Estimuladores Upstream/metabolismoRESUMO
DNA replication occupies a central position in the cell cycle and, therefore, in the development and life of multicellular organisms. During the last 10 years, our comprehension of this important process has considerably improved. Although the mechanisms that coordinate DNA replication with the other moments of the cell cycle are not yet fully understood, it is known that they mainly operate through DNA replication origins and the protein complexes bound to them. In eukaryotes, the packaging status of chromatin seems to be part of the mechanism that controls whether or not and when during the S-phase a particular origin will be activated. Intriguingly, the protein complexes bound to DNA replication origins appear to be directly involved in controlling chromatin packaging. In this manner they can also affect gene expression. In this review we focus on DNA replication origins in metazoan cells and on the relationship between these elements and the structural and functional organization of the genome.
Assuntos
Sequência de Bases/genética , Ciclo Celular/fisiologia , Cromatina/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Origem de Replicação/genética , Animais , Proteínas de Ligação a DNA/metabolismo , Complexo de Reconhecimento de Origem , PlantasRESUMO
The enzyme NAD(+) synthetase (NadE) catalyzes the last step of NAD biosynthesis. Given NAD vital role in cell metabolism, the enzyme represents a valid target for the development of new antimycobacterial agents. In the present study we expressed and purified two putative forms of Mycobacterium tuberculosis NAD(+) synthetase, differing in the polypeptide chain length (NadE-738 and NadE-679). Furthermore, we evaluated several systems for the heterologous expression and large scale purification of the enzyme. In particular, we compared the efficiency of production, the yield of purification, and the catalytic activity of recombinant enzyme in different hosts, ranging from Escherichia coli strains to cultured High Five (Trichoplusia ni BTI-TN-5B1-4) insect cells. Among the systems assayed, we found that the expression of a thioredoxin-NadE fusion protein in E. coli Origami(DE3) is the best system in obtaining highly pure, active NAD(+) synthetase. The recombinant enzyme maintained its activity even after proteolytic cleavage of thioredoxin moiety. Biochemical evidence suggests that the shorter form (NadE-679) may be the real M. tuberculosis NAD(+) synthetase. These results enable us to obtain a purified product for structure-function analysis and high throughput assays for rapid screening of compounds which inhibit enzymatic activity.
Assuntos
Amida Sintases/isolamento & purificação , Amida Sintases/metabolismo , Mycobacterium tuberculosis/enzimologia , NAD/metabolismo , Amida Sintases/química , Amida Sintases/genética , Sequência de Aminoácidos , Animais , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Insetos/citologia , Insetos/genética , Cinética , Dados de Sequência Molecular , Mycobacterium tuberculosis/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de SequênciaRESUMO
Hox proteins are transcription factors involved in controlling axial patterning, leukaemias and hereditary malformations. Here, we show that HOXC10 oscillates in abundance during the cell cycle, being targeted for degradation early in mitosis by the ubiquitin-dependent proteasome pathway. Among abdominal-B subfamily members, the mitotic proteolysis of HOXC10 appears unique, since the levels of the paralogous HOXD10 and the related homeoprotein HOXC13 are constant throughout the cell cycle. When two destruction box motifs (D-box) are mutated, HOXC10 is stabilized and cells accumulate in metaphase. HOXC10 appears to be a new prometaphase target of the anaphase-promoting complex (APC), since its degradation coincides with cyclin A destruction and is suppressed by expression of a dominant-negative form of UbcH10, an APC-associated ubiquitin-conjugating enzyme. Moreover, HOXC10 co-immunoprecipitates the APC subunit CDC27, and its in vitro degradation is reduced in APC-depleted extracts or by competition with the APC substrate cyclin A. These data imply that HOXC10 is a homeoprotein with the potential to influence mitotic progression, and might provide a link between developmental regulation and cell cycle control.