RESUMO
In addition to being essential for gene expression, transcription is crucial for the maintenance of genome integrity. Here, we undertook a systematic approach, to monitor the assembly kinetics of the pre-initiating RNA Polymerase (Pol) II at promoters at steady state and different stages during recovery from UV irradiation-stress, when pre-initiation and initiation steps have been suggested to be transiently shut down. Taking advantage of the reversible dissociation of pre-initiating Pol II after high salt treatment, we found that de novo recruitment of the available Pol II molecules at active promoters not only persists upon UV at all times tested but occurs significantly faster in the early phase of recovery (2 h) than in unexposed human fibroblasts at the majority of active genes. Our method unveiled groups of genes with significantly different pre-initiation complex (PIC) assembly dynamics after UV that present distinct rates of UV-related mutational signatures in melanoma tumours, providing functional relevance to the importance of keeping transcription initiation active during UV recovery. Our findings uncover novel mechanistic insights further detailing the multilayered transcriptional response to genotoxic stress and link PIC assembly dynamics after exposure to genotoxins with cancer mutational landscapes.
Assuntos
RNA Polimerase II , Iniciação da Transcrição Genética , Humanos , Dano ao DNA , Mutagênese , Regiões Promotoras Genéticas , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Transcrição Gênica , Raios Ultravioleta , Fibroblastos/metabolismo , Reparo do DNARESUMO
Idiopathic pulmonary fibrosis (IPF) is characterized by exuberant deposition of extracellular matrix components, leading to the deterioration of lung architecture and respiratory functions. Profibrotic mechanisms are controlled by multiple regulatory molecules, including MAPKs, in turn regulated by multiple phosphorylation cascades. MAP3K8 is an MAPK kinase kinase suggested to pleiotropically regulate multiple pathogenic pathways in the context of inflammation and cancer; however, a possible role in the pathogenesis of IPF has not been investigated. In this report, MAP3K8 mRNA levels were found decreased in the lungs of IPF patients and of mice upon bleomycin-induced pulmonary fibrosis. Ubiquitous genetic deletion of Map3k8 in mice exacerbated the modeled disease, whereas bone marrow transfer experiments indicated that although MAP3K8 regulatory functions are active in both hematopoietic and nonhematopoietic cells, Map3k8 in hematopoietic cells has a more dominant role. Macrophage-specific deletion of Map3k8 was further found to be sufficient for disease exacerbation thus confirming a major role for macrophages in pulmonary fibrotic responses and suggesting a main role for Map3k8 in the homeostasis of their effector functions in the lung. Map3k8 deficiency was further shown to be associated with decreased Cox-2 expression, followed by a decrease in PGE2 production in the lung; accordingly, exogenous administration of PGE2 reduced inflammation and reversed the exacerbated fibrotic profile of Map3k8 -/- mice. Therefore, MAP3K8 has a central role in the regulation of inflammatory responses and Cox-2-mediated PGE2 production in the lung, and the attenuation of its expression is integral to pulmonary fibrosis development.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Inflamação/metabolismo , Pulmão/patologia , MAP Quinase Quinase Quinases/genética , Proteínas Proto-Oncogênicas/genética , Fibrose Pulmonar/metabolismo , Animais , Transplante de Medula Óssea , Células Cultivadas , Fibrose , Humanos , Terapia de Imunossupressão , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Specific capture of chromatin fractions with distinct and well-defined features has emerged as both challenging and a key strategy towards a comprehensive understanding of genome biology. In this context, we developed aniFOUND (accelerated native isolation of factors on unscheduled nascent DNA), an antibody-free method, which can label, capture, map and characterise nascent chromatin fragments that are synthesized in response to specific cues outside S-phase. We used the 'unscheduled' DNA synthesis (UDS) that takes place during the repair of UV-induced DNA lesions and coupled the captured chromatin to high-throughput analytical technologies. By mass-spectrometry we identified several factors with no previously known role in UVC-DNA damage response (DDR) as well as known DDR proteins. We experimentally validated the repair-dependent recruitment of the chromatin remodeller RSF1 and the cohesin-loader NIPBL at sites of UVC-induced photolesions. Developing aniFOUND-seq, a protocol for mapping UDS activity with high resolution, allowed us to monitor the landscape of UVC repair-synthesis events genome wide. We further resolved repair efficacy of the rather unexplored repeated genome, in particular rDNA and telomeres. In summary, aniFOUND delineates the proteome composition and genomic landscape of chromatin loci with specific features by integrating state-of-the-art 'omics' technologies to promote a comprehensive view of their function.
Assuntos
Cromatina/metabolismo , Reparo do DNA , Genômica/métodos , Proteômica/métodos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , DNA/biossíntese , DNA/efeitos da radiação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Espectrometria de Massas , Proteínas Nucleares/metabolismo , Proteoma , Sequências Repetitivas de Ácido Nucleico , Transativadores/metabolismo , Raios UltravioletaRESUMO
During central nervous system (CNS) development, proper and timely induction of neurite elongation is critical for generating functional, mature neurons, and neuronal networks. Despite the wealth of information on the action of extracellular cues, little is known about the intrinsic gene regulatory factors that control this developmental decision. Here, we report the identification of Prox1, a homeobox transcription factor, as a key player in inhibiting neurite elongation. Although Prox1 promotes acquisition of early neuronal identity and is expressed in nascent post-mitotic neurons, it is heavily down-regulated in the majority of terminally differentiated neurons, indicating a regulatory role in delaying neurite outgrowth in newly formed neurons. Consistently, we show that Prox1 is sufficient to inhibit neurite extension in mouse and human neuroblastoma cell lines. More importantly, Prox1 overexpression suppresses neurite elongation in primary neuronal cultures as well as in the developing mouse brain, while Prox1 knock-down promotes neurite outgrowth. Mechanistically, RNA-Seq analysis reveals that Prox1 affects critical pathways for neuronal maturation and neurite extension. Interestingly, Prox1 strongly inhibits many components of Ca2+ signaling pathway, an important mediator of neurite extension and neuronal maturation. In accordance, Prox1 represses Ca2+ entry upon KCl-mediated depolarization and reduces CREB phosphorylation. These observations suggest that Prox1 acts as a potent suppressor of neurite outgrowth by inhibiting Ca2+ signaling pathway. This action may provide the appropriate time window for nascent neurons to find the correct position in the CNS prior to initiation of neurites and axon elongation.
Assuntos
Sinalização do Cálcio , Sistema Nervoso Central/patologia , Proteínas de Homeodomínio/metabolismo , Neuroblastoma/patologia , Crescimento Neuronal , Neurônios/patologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Células Cultivadas , Sistema Nervoso Central/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Neuroblastoma/metabolismo , Neurônios/metabolismo , Fosforilação , Transdução de Sinais , Proteínas Supressoras de Tumor/genéticaRESUMO
Chromatin remodeling is tightly linked to all DNA-transacting activities. To study chromatin remodeling during DNA repair, we established quantitative fluorescence imaging methods to measure the exchange of histones in chromatin in living cells. We show that particularly H2A and H2B are evicted and replaced at an accelerated pace at sites of UV-induced DNA damage. This accelerated exchange of H2A/H2B is facilitated by SPT16, one of the two subunits of the histone chaperone FACT (facilitates chromatin transcription) but largely independent of its partner SSRP1. Interestingly, SPT16 is targeted to sites of UV light-induced DNA damage-arrested transcription and is required for efficient restart of RNA synthesis upon damage removal. Together, our data uncover an important role for chromatin dynamics at the crossroads of transcription and the UV-induced DNA damage response.
Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Dano ao DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Histonas/metabolismo , Transcrição Gênica , Fatores de Elongação da Transcrição/metabolismo , Raios Ultravioleta , Western Blotting , Proteínas de Ciclo Celular , Imunoprecipitação da Cromatina , Reagentes de Ligações Cruzadas/farmacologia , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Células HeLa , Proteínas de Grupo de Alta Mobilidade/genética , Histonas/genética , Humanos , Nucleossomos/genética , RNA/genética , RNA/metabolismo , Fatores de Transcrição , Fatores de Elongação da Transcrição/genéticaRESUMO
Transcription-coupled nucleotide excision repair (TC-NER) allows RNA polymerase II (RNAPII)-blocking lesions to be rapidly removed from the transcribed strand of active genes. Defective TCR in humans is associated with Cockayne syndrome (CS), typically caused by defects in either CSA or CSB. Here, we show that CSB contains a ubiquitin-binding domain (UBD). Cells expressing UBD-less CSB (CSB(del)) have phenotypes similar to those of cells lacking CSB, but these can be suppressed by appending a heterologous UBD, so ubiquitin binding is essential for CSB function. Surprisingly, CSB(del) remains capable of assembling nucleotide excision repair factors and repair synthesis proteins around damage-stalled RNAPII, but such repair complexes fail to excise the lesion. Together, our results indicate an essential role for protein ubiquitylation and CSB's UBD in triggering damage incision during TC-NER and allow us to integrate the function of CSA and CSB in a model for the process.
Assuntos
DNA Helicases , Enzimas Reparadoras do DNA , Reparo do DNA , Ubiquitina/metabolismo , Sequência de Aminoácidos , Linhagem Celular/efeitos da radiação , Núcleo Celular/metabolismo , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Dano ao DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Proteínas de Ligação a Poli-ADP-Ribose , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Tetra-Hidrofolato Desidrogenase/genética , Ubiquitina/genética , Raios UltravioletaRESUMO
Nucleotide excision repair (NER) is the most versatile DNA repair system that deals with the major UV photoproducts in DNA, as well as many other DNA adducts. The early steps of NER are well understood, whereas the later steps of repair synthesis and ligation are not. In particular, which polymerases are definitely involved in repair synthesis and how they are recruited to the damaged sites has not yet been established. We report that, in human fibroblasts, approximately half of the repair synthesis requires both pol kappa and pol delta, and both polymerases can be recovered in the same repair complexes. Pol kappa is recruited to repair sites by ubiquitinated PCNA and XRCC1 and pol delta by the classical replication factor complex RFC1-RFC, together with a polymerase accessory factor, p66, and unmodified PCNA. The remaining repair synthesis is dependent on pol epsilon, recruitment of which is dependent on the alternative clamp loader CTF18-RFC.
Assuntos
Dano ao DNA , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Fibroblastos/enzimologia , ATPases Associadas a Diversas Atividades Celulares , Proteínas de Transporte/metabolismo , Linhagem Celular , Senescência Celular , DNA Polimerase II/metabolismo , DNA Polimerase III/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Fibroblastos/efeitos da radiação , Humanos , Proteínas Nucleares/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Antígeno Nuclear de Célula em Proliferação/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico , Interferência de RNA , Proteínas Recombinantes de Fusão/metabolismo , Proteína de Replicação C/metabolismo , Fatores de Tempo , Transfecção , Ubiquitina-Proteína Ligases , Ubiquitinação , Raios Ultravioleta , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
DNA damage poses a constant threat to genome integrity taking a variety of shapes and arising by normal cellular metabolism or environmental insults. Human syndromes, characterized by increased cancer pre-disposition or early onset of age-related pathology and developmental abnormalities, often result from defective DNA damage responses and compromised genome integrity. Over the last decades intensive research worldwide has made important contributions to our understanding of the molecular mechanisms underlying genomic instability and has substantiated the importance of DNA repair in cancer prevention in the general population. In this chapter, we discuss Nucleotide Excision Repair pathway, the causative role of its components in disease-related pathology and recent technological achievements that decipher mutational landscapes and may facilitate pathological classification and personalized therapy.
Assuntos
Dano ao DNA , Reparo do DNA , Neoplasias/genética , Doenças Neurodegenerativas/genética , Instabilidade Genômica , HumanosRESUMO
The timing and location of writing and erasing of histone modifications determine gene expression programs and are tightly controlled processes. One such modification is the monoubiquitination of histone H2B (H2Bub), whose precise level during transcription elongation is dynamically regulated by the synergistic action of RNF20/40 ubiquitin-ligase and the de-ubiquitinase (DUB) of the ATXN7L3-containing DUB modules. Here, we characterize the dynamics of H2Bub in transcription and explore its role in perspective with the recently updated model of UV damage-induced transcription reorganization. Employing integrative analysis of genome-wide high-throughput approaches, transcription inhibitors and ATXN7L3-DUB knockdown cells, we find that H2Bub levels and patterns depend on intron-exon architecture both in steady state and upon UV. Importantly, our analysis reveals a widespread redistribution of this histone mark, rather than a uniform loss as previously suggested, which closely mirrors the post-UV dynamics of elongating RNA Polymerase II (RNAPII) at transcribed loci. The observed effects are due to a direct inter-dependence on RNAPII local concentration and speed, and we show that deficient ATXN7L3-mediated DUB activity leads to increased elongation rates in both non-irradiated and irradiated conditions. Our data and the implementation of a high-resolution computational framework reveal that the H2Bub pattern follows that of RNAPII, both in the ATXNL3 knockdown and in response to UV guaranteeing faithful elongation speed, especially in the context of the transcription-driven DNA damage response.
RESUMO
Cellular senescence constitutes a generally irreversible proliferation barrier, accompanied by macromolecular damage and metabolic rewiring. Several senescence types have been identified based on the initiating stimulus, such as replicative (RS), stress-induced (SIS) and oncogene-induced senescence (OIS). These senescence subtypes are heterogeneous and often develop subset-specific phenotypes. Reduced protein synthesis is considered a senescence hallmark, but whether this trait pertains to various senescence subtypes and if distinct molecular mechanisms are involved remain largely unknown. Here, we analyze large published or experimentally produced RNA-seq and Ribo-seq datasets to determine whether major translation-regulating entities such as ribosome stalling, the presence of uORFs/dORFs and IRES elements may differentially contribute to translation deficiency in senescence subsets. We show that translation-regulating mechanisms may not be directly relevant to RS, however uORFs are significantly enriched in SIS. Interestingly, ribosome stalling, uORF/dORF patterns and IRES elements comprise predominant mechanisms upon OIS, strongly correlating with Notch pathway activation. Our study provides for the first time evidence that major translation dysregulation mechanisms/patterns occur during cellular senescence, but at different rates depending on the stimulus type. The degree at which those mechanisms accumulate directly correlates with translation deficiency levels. Our thorough analysis contributes to elucidating crucial and so far unknown differences in the translation machinery between senescence subsets.
Assuntos
Senescência Celular , Ribossomos , Senescência Celular/genética , Ribossomos/genética , Ribossomos/metabolismo , Biossíntese de ProteínasRESUMO
Studies based on cell-free systems and on in vitro-cultured living cells support the concept that many cellular processes, such as transcription initiation, are highly dynamic: individual proteins stochastically bind to their substrates and disassemble after reaction completion. This dynamic nature allows quick adaptation of transcription to changing conditions. However, it is unknown to what extent this dynamic transcription organization holds for postmitotic cells embedded in mammalian tissue. To allow analysis of transcription initiation dynamics directly into living mammalian tissues, we created a knock-in mouse model expressing fluorescently tagged TFIIH. Surprisingly and in contrast to what has been observed in cultured and proliferating cells, postmitotic murine cells embedded in their tissue exhibit a strong and long-lasting transcription-dependent immobilization of TFIIH. This immobilization is both differentiation driven and development dependent. Furthermore, although very statically bound, TFIIH can be remobilized to respond to new transcriptional needs. This divergent spatiotemporal transcriptional organization in different cells of the soma revisits the generally accepted highly dynamic concept of the kinetic framework of transcription and shows how basic processes, such as transcription, can be organized in a fundamentally different fashion in intact organisms as previously deduced from in vitro studies.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Fator de Transcrição TFIIH/metabolismo , Transcrição Gênica , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Cultivadas , Cerebelo/citologia , Cerebelo/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Imunoprecipitação da Cromatina , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Fibroblastos/citologia , Recuperação de Fluorescência Após Fotodegradação , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição TFIIH/genéticaRESUMO
BACKGROUND: Synovial fibroblasts (SFs) are specialized cells of the synovium that provide nutrients and lubricants for the proper function of diarthrodial joints. Recent evidence appreciates the contribution of SF heterogeneity in arthritic pathologies. However, the normal SF profiles and the molecular networks that govern the transition from homeostatic to arthritic SF heterogeneity remain poorly defined. METHODS: We applied a combined analysis of single-cell (sc) transcriptomes and epigenomes (scRNA-seq and scATAC-seq) to SFs derived from naïve and hTNFtg mice (mice that overexpress human TNF, a murine model for rheumatoid arthritis), by employing the Seurat and ArchR packages. To identify the cellular differentiation lineages, we conducted velocity and trajectory analysis by combining state-of-the-art algorithms including scVelo, Slingshot, and PAGA. We integrated the transcriptomic and epigenomic data to infer gene regulatory networks using ArchR and custom-implemented algorithms. We performed a canonical correlation analysis-based integration of murine data with publicly available datasets from SFs of rheumatoid arthritis patients and sought to identify conserved gene regulatory networks by utilizing the SCENIC algorithm in the human arthritic scRNA-seq atlas. RESULTS: By comparing SFs from healthy and hTNFtg mice, we revealed seven homeostatic and two disease-specific subsets of SFs. In healthy synovium, SFs function towards chondro- and osteogenesis, tissue repair, and immune surveillance. The development of arthritis leads to shrinkage of homeostatic SFs and favors the emergence of SF profiles marked by Dkk3 and Lrrc15 expression, functioning towards enhanced inflammatory responses and matrix catabolic processes. Lineage inference analysis indicated that specific Thy1+ SFs at the root of trajectories lead to the intermediate Thy1+/Dkk3+/Lrrc15+ SF states and culminate in a destructive and inflammatory Thy1- SF identity. We further uncovered epigenetically primed gene programs driving the expansion of these arthritic SFs, regulated by NFkB and new candidates, such as Runx1. Cross-species analysis of human/mouse arthritic SF data determined conserved regulatory and transcriptional networks. CONCLUSIONS: We revealed a dynamic SF landscape from health to arthritis providing a functional genomic blueprint to understand the joint pathophysiology and highlight the fibroblast-oriented therapeutic targets for combating chronic inflammatory and destructive arthritic disease.
Assuntos
Artrite Reumatoide , Análise de Célula Única , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Fibroblastos/metabolismo , Humanos , Inflamação/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Membrana Sinovial/metabolismo , Membrana Sinovial/patologiaRESUMO
Cockayne syndrome (CS) is a DNA repair syndrome characterized by a broad spectrum of clinical manifestations such as neurodegeneration, premature aging, developmental impairment, photosensitivity and other symptoms. Mutations in Cockayne syndrome protein B (CSB) are present in the vast majority of CS patients and in other DNA repair-related pathologies. In the literature, the role of CSB in different DNA repair pathways has been highlighted, however, new CSB functions have been identified in DNA transcription, mitochondrial biology, telomere maintenance and p53 regulation. Herein, we present an overview of identified structural elements and processes that impact on CSB activity and its post-translational modifications, known to balance the different roles of the protein not only during normal conditions but most importantly in stress situations. Moreover, since CSB has been found to be overexpressed in a number of different tumors, its role in cancer is presented and possible therapeutic targeting is discussed.
Assuntos
Síndrome de Cockayne/genética , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Neoplasias/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Animais , Síndrome de Cockayne/metabolismo , Dano ao DNA , DNA Helicases/química , DNA Helicases/metabolismo , Reparo do DNA , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Modelos Moleculares , Mutação , Neoplasias/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/química , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Conformação Proteica , Processamento de Proteína Pós-TraducionalRESUMO
Inhibition of transcription caused by DNA damage-impaired RNA polymerase II (Pol II) elongation conceals a local increase in de novo transcription, slowly progressing from Transcription Start Sites (TSSs) to gene ends. Although associated with accelerated repair of Pol II-encountered lesions and limited mutagenesis, it is still unclear how this mechanism is maintained during genotoxic stress-recovery. Here we uncover a widespread gain in chromatin accessibility and preservation of the active H3K27ac mark after UV-irradiation. The concomitant increase in Pol II escape from promoter-proximal pause (PPP) sites of most active genes, PROMPTs and enhancer RNAs favors unrestrained initiation, as evidenced by the synthesis of nascent RNAs including start RNAs. Accordingly, drug-inhibition of PPP-release replenishes levels of pre-initiating Pol II at TSSs after UV. Our data show that such continuous engagement of Pol II molecules ensures maximal transcription-driven repair throughout expressed genes and regulatory loci. Importantly, revealing this unanticipated regulatory layer of UV-response provides physiological relevant traction to the emerging concept that Pol II initiation rate is determined by pause-release dynamics.
Assuntos
Reparo do DNA , Sítio de Iniciação de Transcrição , Transcrição Gênica , Cromatina/genética , Cromatina/metabolismo , Dano ao DNA , Regulação da Expressão Gênica , Humanos , Regiões Promotoras Genéticas , RNA/genética , RNA/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Sequências Reguladoras de Ácido NucleicoRESUMO
The rad18 gene of Schizosaccharomyces pombe is an essential gene that is involved in several different DNA repair processes. Rad18 (Smc6) is a member of the structural maintenance of chromosomes (SMC) family and, together with its SMC partner Spr18 (Smc5), forms the core of a high-molecular-weight complex. We show here that both S. pombe and human Smc5 and -6 interact through their hinge domains and that four independent temperature-sensitive mutants of Rad18 (Smc6) are all mutated at the same glycine residue in the hinge region. This mutation abolishes the interactions between the hinge regions of Rad18 (Smc6) and Spr18 (Smc5), as does mutation of a conserved glycine in the hinge region of Spr18 (Smc5). We purified the Smc5-6 complex from S. pombe and identified four non-SMC components, Nse1, Nse2, Nse3, and Rad62. Nse3 is a novel protein which is related to the mammalian MAGE protein family, many members of which are specifically expressed in cancer tissue. In initial steps to understand the architecture of the complex, we identified two subcomplexes containing Rad18-Spr18-Nse2 and Nse1-Nse3-Rad62. The subcomplexes are probably bridged by a weaker interaction between Nse2 and Nse3.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/fisiologia , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/fisiologia , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona , Dano ao DNA , Reparo do DNA , DNA Complementar/metabolismo , Relação Dose-Resposta à Radiação , Eletroforese em Gel de Poliacrilamida , Deleção de Genes , Glutationa Transferase/metabolismo , Glicina/química , Humanos , Imunoprecipitação , Espectrometria de Massas , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/metabolismo , Fases de Leitura Aberta , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/metabolismo , Temperatura , Fatores de Tempo , Transcrição Gênica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Defects in the XPD gene can result in several clinical phenotypes, including xeroderma pigmentosum (XP), trichothiodystrophy, and, less frequently, the combined phenotype of XP and Cockayne syndrome (XP-D/CS). We previously showed that in cells from two XP-D/CS patients, breaks were introduced into cellular DNA on exposure to UV damage, but these breaks were not at the sites of the damage. In the present work, we show that three further XP-D/CS patients show the same peculiar breakage phenomenon. We show that these breaks can be visualized inside the cells by immunofluorescence using antibodies to either gamma-H2AX or poly-ADP-ribose and that they can be generated by the introduction of plasmids harboring methylation or oxidative damage as well as by UV photoproducts. Inhibition of RNA polymerase II transcription by four different inhibitors dramatically reduced the number of UV-induced breaks. Furthermore, the breaks were dependent on the nucleotide excision repair (NER) machinery. These data are consistent with our hypothesis that the NER machinery introduces the breaks at sites of transcription initiation. During transcription in UV-irradiated XP-D/CS cells, phosphorylation of the carboxy-terminal domain of RNA polymerase II occurred normally, but the elongating form of the polymerase remained blocked at lesions and was eventually degraded.
Assuntos
Síndrome de Cockayne/genética , Dano ao DNA , Reparo do DNA , Transcrição Gênica , Xeroderma Pigmentoso/genética , Síndrome de Cockayne/complicações , DNA/efeitos da radiação , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Histonas/análise , Humanos , Fosforilação , Poli Adenosina Difosfato Ribose/análise , RNA Polimerase II/metabolismo , Raios Ultravioleta , Xeroderma Pigmentoso/complicaçõesRESUMO
UV-damaged DNA-binding protein (UV-DDB) is essential for global genome repair (GGR) of UV-induced cyclobutane pyrimidine dimers (CPD). Unlike human cells, rodent epidermal cells are deficient in GGR of CPDs and express a subunit of UV-DDB, DDB2, at a low level. In this study, we generated mice (K14-DDB2) ectopically expressing mouse DDB2 at elevated levels. Enhanced expression of DDB2 both delayed the onset of squamous cell carcinoma and decreased the number of tumors per mouse in chronically UV-B light-exposed hairless mice. Enhanced expression of DDB2 improved repair of both CPDs and pyrimidine(6-4)pyrimidone photoproducts (6-4PP) in dermal fibroblasts. However, GGR of CPDs in K14-DDB2 mice did not reach the level of efficiency of human cells, suggesting that another repair protein may become rate limiting when DDB2 is abundantly present. To complement these studies, we generated mice in which the DDB2 gene was disrupted. DDB2-/- and DDB2+/- mice were found to be hypersensitive to UV-induced skin carcinogenesis. On the cellular level, we detected a delay in the repair of 6-4PPs in DDB2-/- dermal fibroblasts. Neither the absence nor the enhanced expression of DDB2 affected the levels of UV-induced apoptosis in epidermal keratinocytes or cultured dermal fibroblasts. Our results show an important role for DDB2 in the protection against UV-induced cancer and indicate that this protection is most likely mediated by accelerating the repair of photolesions.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Neoplasias Cutâneas/prevenção & controle , Animais , Apoptose/fisiologia , Apoptose/efeitos da radiação , Reparo do DNA , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Masculino , Camundongos , Camundongos Pelados , Camundongos Transgênicos , Oligopeptídeos , Peptídeos/genética , Peptídeos/metabolismo , Dímeros de Pirimidina/metabolismo , Tolerância a Radiação/fisiologia , Pele/citologia , Pele/metabolismo , Pele/efeitos da radiação , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Raios UltravioletaRESUMO
Complex molecular responses preserve gene expression accuracy and genome integrity in the face of environmental perturbations. Here we report that, in response to UV irradiation, RNA polymerase II (RNAPII) molecules are dynamically and synchronously released from promoter-proximal regions into elongation to promote uniform and accelerated surveillance of the whole transcribed genome. The maximised influx of de novo released RNAPII correlates with increased damage-sensing, as confirmed by RNAPII progressive accumulation at dipyrimidine sites and by the average slow-down of elongation rates in gene bodies. In turn, this transcription elongation 'safe' mode guarantees efficient DNA repair regardless of damage location, gene size and transcription level. Accordingly, we detect low and homogenous rates of mutational signatures associated with UV exposure or cigarette smoke across all active genes. Our study reveals a novel advantage for transcription regulation at the promoter-proximal level and provides unanticipated insights into how active transcription shapes the mutagenic landscape of cancer genomes.