RESUMO
Hypothesis generation is a critical step in research and a cornerstone in the rare disease field. Research is most efficient when those hypotheses are based on the entirety of knowledge known to date. Systematic review articles are commonly used in biomedicine to summarize existing knowledge and contextualize experimental data. But the information contained within review articles is typically only expressed as free-text, which is difficult to use computationally. Researchers struggle to navigate, collect and remix prior knowledge as it is scattered in several silos without seamless integration and access. This lack of a structured information framework hinders research by both experimental and computational scientists. To better organize knowledge and data, we built a structured review article that is specifically focused on NGLY1 Deficiency, an ultra-rare genetic disease first reported in 2012. We represented this structured review as a knowledge graph and then stored this knowledge graph in a Neo4j database to simplify dissemination, querying and visualization of the network. Relative to free-text, this structured review better promotes the principles of findability, accessibility, interoperability and reusability (FAIR). In collaboration with domain experts in NGLY1 Deficiency, we demonstrate how this resource can improve the efficiency and comprehensiveness of hypothesis generation. We also developed a read-write interface that allows domain experts to contribute FAIR structured knowledge to this community resource. In contrast to traditional free-text review articles, this structured review exists as a living knowledge graph that is curated by humans and accessible to computational analyses. Finally, we have generalized this workflow into modular and repurposable components that can be applied to other domain areas. This NGLY1 Deficiency-focused network is publicly available at http://ngly1graph.org/. AVAILABILITY AND IMPLEMENTATION: Database URL: http://ngly1graph.org/. Network data files are at: https://github.com/SuLab/ngly1-graph and source code at: https://github.com/SuLab/bioknowledge-reviewer. CONTACT: asu@scripps.edu.
Assuntos
Pesquisa Biomédica/métodos , Biologia Computacional/métodos , Bases de Dados Factuais , Bases de Conhecimento , Animais , Pesquisa Biomédica/estatística & dados numéricos , Biologia Computacional/estatística & dados numéricos , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/metabolismo , Curadoria de Dados/métodos , Mineração de Dados/métodos , Humanos , Internet , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiência , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Revisões Sistemáticas como AssuntoRESUMO
Fanconi anemia (FA) is a chromosome instability syndrome characterized by increased cancer predisposition. Specifically, the FA pathway functions to protect genome stability during DNA replication. The central FA pathway protein, FANCD2, locates to stalled replication forks and recruits homologous recombination (HR) factors such as CtBP interacting protein (CtIP) to promote replication fork restart while suppressing new origin firing. Here, we identify alpha-thalassemia retardation syndrome X-linked (ATRX) as a novel physical and functional interaction partner of FANCD2. ATRX is a chromatin remodeler that forms a complex with Death domain-associated protein 6 (DAXX) to deposit the histone variant H3.3 into specific genomic regions. Intriguingly, ATRX was recently implicated in replication fork recovery; however, the underlying mechanism(s) remained incompletely understood. Our findings demonstrate that ATRX forms a constitutive protein complex with FANCD2 and protects FANCD2 from proteasomal degradation. ATRX and FANCD2 localize to stalled replication forks where they cooperate to recruit CtIP and promote MRE11 exonuclease-dependent fork restart while suppressing the firing of new replication origins. Remarkably, replication restart requires the concerted histone H3 chaperone activities of ATRX/DAXX and FANCD2, demonstrating that coordinated histone H3 variant deposition is a crucial event during the reinitiation of replicative DNA synthesis. Lastly, ATRX also cooperates with FANCD2 to promote the HR-dependent repair of directly induced DNA double-stranded breaks. We propose that ATRX is a novel functional partner of FANCD2 to promote histone deposition-dependent HR mechanisms in S-phase.
Assuntos
Proteínas Correpressoras/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Chaperonas Moleculares/genética , Proteína Nuclear Ligada ao X/genética , Linhagem Celular , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Replicação do DNA/genética , Anemia de Fanconi/patologia , Técnicas de Inativação de Genes/métodos , Histonas/genética , Humanos , Proteína Homóloga a MRE11/genética , Rad51 Recombinase/genética , Reparo de DNA por Recombinação/genética , Transdução de Sinais/genéticaRESUMO
The Fanconi anemia (FA) protein network is necessary for repair of DNA interstrand crosslinks (ICLs), but its control mechanism remains unclear. Here we show that the network is regulated by a ubiquitin signaling cascade initiated by RNF8 and its partner, UBC13, and mediated by FAAP20, a component of the FA core complex. FAAP20 preferentially binds the ubiquitin product of RNF8-UBC13, and this ubiquitin-binding activity and RNF8-UBC13 are both required for recruitment of FAAP20 to ICLs. Both RNF8 and FAAP20 are required for recruitment of FA core complex and FANCD2 to ICLs, whereas RNF168 can modulate efficiency of the recruitment. RNF8 and FAAP20 are needed for efficient FANCD2 monoubiquitination, a key step of the FA network; RNF8 and the FA core complex work in the same pathway to promote cellular resistance to ICLs. Thus, the RNF8-FAAP20 ubiquitin cascade is critical for recruiting FA core complex to ICLs and for normal function of the FA network.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Ubiquitinação , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Lisina/química , Lisina/genética , Lisina/metabolismo , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The Fanconi Anemia (FA) pathway consists of proteins involved in repairing DNA damage, including interstrand cross-links (ICLs). The pathway contains an upstream multiprotein core complex that mediates the monoubiquitylation of the FANCD2 and FANCI heterodimer, and a downstream pathway that converges with a larger network of proteins with roles in homologous recombination and other DNA repair pathways. Selective killing of cancer cells with an intact FA pathway but deficient in certain other DNA repair pathways is an emerging approach to tailored cancer therapy. Inhibiting the FA pathway becomes selectively lethal when certain repair genes are defective, such as the checkpoint kinase ATM. Inhibiting the FA pathway in ATM deficient cells can be achieved with small molecule inhibitors, suggesting that new cancer therapeutics could be developed by identifying FA pathway inhibitors to treat cancers that contain defects that are synthetic lethal with FA.
RESUMO
Fanconi anemia (FA) is a heritable disease characterized by bone marrow failure, congenital abnormalities, and cancer predisposition. The 15 identified FA genes operate in a molecular pathway to preserve genomic integrity. Within this pathway the FA core complex operates as an ubiquitin ligase that activates the complex of FANCD2 and FANCI to coordinate DNA repair. The FA core complex is formed by at least 12 proteins. However, only the FANCL subunit displays ubiquitin ligase activity. FANCA and FANCG are members of the FA core complex for which no other functions have been described than to participate in protein interactions. In this study we generated mice with combined null alleles for Fanca and Fancg to identify extended functions for these genes by characterizing the double mutant mice and cells. Double mutant a(-/-)/g(-/-) mice were born at near Mendelian frequencies without apparent developmental abnormalities. Histological analysis of a(-/-)/g(-/-) mice revealed a Leydig cell hyperplasia and frequent vacuolization of Sertoli cells in testes, while ovaries were depleted from developing follicles and displayed an interstitial cell hyperplasia. These gonadal aberrations were associated with a compromised fertility of a(-/-)/g(-/-) males and females. During the first year of life a(-/-)/g(-/-) did not develop malignancies or bone marrow failure. At the cellular level a(-/-)/g(-/-), Fanca(-/-), and Fancg(-/-) cells proved equally compromised in DNA crosslink and homology-directed repair. Overall the phenotype of a(-/-)/g(-/-) double knockout mice and cells appeared highly similar to the phenotype of Fanca or Fancg single knockouts. The lack of an augmented phenotype suggest that null mutations in Fanca or Fancg are fully epistatic, making additional important functions outside of the FA core complex highly unlikely.
Assuntos
Epistasia Genética/genética , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Mutação/genética , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Células da Medula Óssea/citologia , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Quebra Cromossômica/efeitos dos fármacos , Embrião de Mamíferos , Feminino , Fertilidade/genética , Fibroblastos/citologia , Fluorbenzenos/farmacologia , Testes Hematológicos , Humanos , Masculino , Camundongos , Ovário/metabolismo , Ftalazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Testículo/metabolismoAssuntos
Exodesoxirribonucleases/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Alquilantes/farmacologia , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA , Endodesoxirribonucleases , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Humanos , Mitomicina/farmacologia , Enzimas Multifuncionais , UbiquitinaçãoRESUMO
FANCM remodels branched DNA structures and plays essential roles in the cellular response to DNA replication stress. Here, we show that FANCM forms a conserved DNA-remodeling complex with a histone-fold heterodimer, MHF. We find that MHF stimulates DNA binding and replication fork remodeling by FANCM. In the cell, FANCM and MHF are rapidly recruited to forks stalled by DNA interstrand crosslinks, and both are required for cellular resistance to such lesions. In vertebrates, FANCM-MHF associates with the Fanconi anemia (FA) core complex, promotes FANCD2 monoubiquitination in response to DNA damage, and suppresses sister-chromatid exchanges. Yeast orthologs of these proteins function together to resist MMS-induced DNA damage and promote gene conversion at blocked replication forks. Thus, FANCM-MHF is an essential DNA-remodeling complex that protects replication forks from yeast to human.
Assuntos
DNA Helicases/metabolismo , DNA/metabolismo , Instabilidade Genômica , Histonas/metabolismo , Dobramento de Proteína , Multimerização Proteica , Sequência de Aminoácidos , Animais , Linhagem Celular , Galinhas , DNA/genética , Dano ao DNA , DNA Helicases/química , DNA Helicases/genética , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Proteínas de Grupos de Complementação da Anemia de Fanconi , Humanos , Dados de Sequência Molecular , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Alinhamento de Sequência , Troca de Cromátide IrmãRESUMO
Members of the Mix/Bix family of paired class homeobox genes play important roles in the development of vertebrate mesoderm and endoderm. The single Mix/Bix family member identified in the mouse, Mix-like 1 (Mixl1), is required for mesendoderm patterning during gastrulation and promotes mesoderm formation and hematopoiesis in embryonic stem cell (ESC)-derived embryoid bodies. Despite its crucial functions the transcriptional activity and targets of Mixl1 have not been well described. To investigate the molecular mechanisms of Mixl1-mediated transcriptional regulation, we have characterized the DNA-binding specificity and transcriptional properties of this homeodomain protein in differentiating ESCs. Mixl1 binds preferentially as a dimer to an 11-base pair (bp) Mixl1 binding sequence (MBS) that contains two inverted repeats separated by a 3-bp spacer. The MBS mediates transcriptional activation by Mixl1 in both NIH 3T3 cells and in a new application of an inducible ESC differentiation system. Consistent with our previous observation that early induction of Mixl1 expression in ESCs results in premature activation of Goosecoid (Gsc), we have found that Mixl1 occupies two variant MBSs within and activates transcription from the Gsc promoter in vitro and in vivo. These results strongly suggest that Gsc is a direct target gene of Mixl1 during embryogenesis. STEM CELLS 2009;27:2884-2895.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas de Homeodomínio/metabolismo , Ativação Transcricional , Animais , Sequência de Bases , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Proteína Goosecoid/genética , Proteínas de Homeodomínio/genética , Mesoderma/metabolismo , Camundongos , Células NIH 3T3 , Ligação Proteica , Alinhamento de SequênciaRESUMO
Genomic stability requires a functional Fanconi anemia (FA) pathway composed of an upstream "core complex" (FA proteins A/B/C/E/F/G/L/M) that mediates monoubiquitination of the downstream targets FANCD2 and FANCI. Unique among FA core complex members, FANCM has processing activities toward replication-associated DNA structures, suggesting a vital role for FANCM during replication. Using Xenopus egg extracts, we analyzed the functions of FANCM in replication and the DNA damage response. xFANCM binds chromatin in a replication-dependent manner and is phosphorylated in response to DNA damage structures. Chromatin binding and DNA damage-induced phosphorylation of xFANCM are mediated in part by the downstream FA pathway protein FANCD2. Moreover, phosphorylation and chromatin recruitment of FANCM is regulated by two mayor players in the DNA damage response: the cell cycle checkpoint kinases ATR and ATM. Our results indicate that functions of FANCM are controlled by FA- and non-FA pathways in the DNA damage response.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA/fisiologia , DNA Helicases/metabolismo , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Oócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Cromatina , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Oócitos/citologia , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/genética , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
BACKGROUND: The Fanconi anemia (FA) pathway is a multigene DNA damage response network implicated in the repair of DNA lesions that arise during replication or after exogenous DNA damage. The FA pathway displays synthetic lethal relationship with certain DNA repair genes such as ATM (Ataxia Telangectasia Mutated) that are frequently mutated in tumors. Thus, inhibition of FANCD2 monoubiquitylation (FANCD2-Ub), a key step in the FA pathway, might target tumor cells defective in ATM through synthetic lethal interaction. Curcumin was previously identified as a weak inhibitor of FANCD2-Ub. The aim of this study is to identify derivatives of curcumin with better activity and specificity. RESULTS: Using a replication-free assay in Xenopus extracts, we screened monoketone analogs of curcumin for inhibition of FANCD2-Ub and identified analog EF24 as a strong inhibitor. Mechanistic studies suggest that EF24 targets the FA pathway through inhibition of the NF-kB pathway kinase IKK. In HeLa cells, nanomolar concentrations of EF24 inhibited hydroxyurea (HU)-induced FANCD2-Ub and foci in a cell-cycle independent manner. Survival assays revealed that EF24 specifically sensitizes FA-competent cells to the DNA crosslinking agent mitomycin C (MMC). In addition, in contrast with curcumin, ATM-deficient cells are twofold more sensitive to EF24 than matched wild-type cells, consistent with a synthetic lethal effect between FA pathway inhibition and ATM deficiency. An independent screen identified 4H-TTD, a compound structurally related to EF24 that displays similar activity in egg extracts and in cells. CONCLUSIONS: These results suggest that monoketone analogs of curcumin are potent inhibitors of the FA pathway and constitute a promising new class of targeted anticancer compounds.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Curcumina/análogos & derivados , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/antagonistas & inibidores , Anemia de Fanconi/metabolismo , Cetonas/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Supressoras de Tumor/antagonistas & inibidores , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Curcumina/farmacologia , Proteínas de Ligação a DNA/metabolismo , Sinergismo Farmacológico , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Células HeLa , Humanos , Mitomicina/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , XenopusRESUMO
The Fanconi Anemia (FA) DNA damage response pathway is involved in the processing of DNA interstrand crosslinks (ICLs). As such, inhibition of the FA pathway could chemosensitize FA-competent tumor cells to commonly used ICL agents like cisplatin. Moreover, suppression of the FA pathway is synthetic lethal with deficiencies in several other DNA repair pathways, suggesting that FA pathway inhibitors could be used in targeted therapies against specific tumors. To identify such inhibitors, we designed a novel in vitro screening assay utilizing Xenopus egg extracts. Using the DNA-stimulated monoubiquitylation of Xenopus FANCD2 (xFANCD2-L) as readout, a chemical library screen identified DDN (2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone) as a novel and potent FA pathway inhibitor. DDN inhibited xFANCD2-L formation in a dose-dependent manner in both extracts and human cells without disruption of the upstream FA core complex. DDN also inhibited the characteristic subnuclear FANCD2 foci formation following DNA damage. Moreover, DDN displayed a greater synergistic effect with cisplatin in a FA-proficient cancer cell line compared to its FA-deficient isogenic counterpart, suggesting that DDN might be a good lead candidate as cisplatin chemosensitizer in both FA-deficient and FA-competent tumors. This system constitutes the first cell-free screening assay for identifying compounds that inhibit the FA pathway and provides a new biochemical platform for mapping the functions of its various components with specific chemical inhibitors.
Assuntos
Avaliação Pré-Clínica de Medicamentos/instrumentação , Avaliação Pré-Clínica de Medicamentos/métodos , Anemia de Fanconi/tratamento farmacológico , Anemia de Fanconi/genética , Animais , Sobrevivência Celular , Sistema Livre de Células , Cisplatino/farmacologia , Reagentes de Ligações Cruzadas/farmacologia , DNA/química , Dano ao DNA , Reparo do DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Células HeLa , Humanos , Modelos Biológicos , Xenopus laevisRESUMO
BLM, the helicase mutated in Bloom syndrome, associates with topoisomerase 3alpha, RMI1 (RecQ-mediated genome instability), and RPA, to form a complex essential for the maintenance of genome stability. Here we report a novel component of the BLM complex, RMI2, which interacts with RMI1 through two oligonucleotide-binding (OB)-fold domains similar to those in RPA. The resulting complex, named RMI, differs from RPA in that it lacks obvious DNA-binding activity. Nevertheless, RMI stimulates the dissolution of a homologous recombination intermediate in vitro and is essential for the stability, localization, and function of the BLM complex in vivo. Notably, inactivation of RMI2 in chicken DT40 cells results in an increased level of sister chromatid exchange (SCE)--the hallmark feature of Bloom syndrome cells. Epistasis analysis revealed that RMI2 and BLM suppress SCE within the same pathway. A point mutation in the OB domain of RMI2 disrupts the association between BLM and the rest of the complex, and abrogates the ability of RMI2 to suppress elevated SCE. Our data suggest that multi-OB-fold complexes mediate two modes of BLM action: via RPA-mediated protein-DNA interaction, and via RMI-mediated protein-protein interactions.
Assuntos
Síndrome de Bloom/metabolismo , Proteínas de Transporte/metabolismo , DNA Helicases/fisiologia , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Proteínas Nucleares/metabolismo , Proteína de Replicação A/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Núcleo Celular/metabolismo , Células Cultivadas , Galinhas , Dano ao DNA , Reparo do DNA , DNA Topoisomerases Tipo I/fisiologia , DNA Cruciforme , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Mitose , Dados de Sequência Molecular , Proteínas Nucleares/genética , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Fosforilação , Dobramento de Proteína , RecQ Helicases , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Recombinação Genética , Proteína de Replicação A/genética , Homologia de Sequência de Aminoácidos , Troca de Cromátide IrmãRESUMO
Fanconi anemia (FA) is associated with variable developmental abnormalities, bone marrow failure and cancer susceptibility. FANCG/XRCC9 is member of the FA core complex, a group of proteins that control the monoubiquitylation of FANCD2, an event that plays a critical role in maintaining genomic stability. Here we report the identification of the Xenopus laevis ortholog of human FANCG (xFANCG), its expression during development, and its molecular interactions with a partner protein, xFANCA. The xFANCG protein sequence is 47% similar to its human ortholog, with highest conservation in the two putative N-terminal leucine zippers and the tetratricopeptide repeat (TPR) motifs. xFANCG is maternally and zygotically transcribed. Prior to the midblastula stage, a single xFANCG transcript is observed but two additional alternatively spliced mRNAs are detected after the midblastula transition. One of the variants is predicted to encode a novel isoform of xFANCG lacking exon 2. The mutual association between FANCG and FANCA required for their nuclear import is conserved in Xenopus egg extracts. Our data demonstrate that interactions between FANCA and FANCG occur at the earliest stage of vertebrate development and raise the possibility that functionally different isoforms of xFANCG may play a role in early development.
Assuntos
Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Regulação da Expressão Gênica no Desenvolvimento , Xenopus laevis/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Conservada , DNA Complementar/isolamento & purificação , Embrião não Mamífero , Éxons , Proteína do Grupo de Complementação A da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação G da Anemia de Fanconi/isolamento & purificação , Proteína do Grupo de Complementação G da Anemia de Fanconi/metabolismo , Humanos , Dados de Sequência Molecular , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , RNA Mensageiro Estocado/isolamento & purificação , RNA Mensageiro Estocado/metabolismo , Homologia de Sequência , Xenopus laevis/embriologiaRESUMO
The Fanconi anemia (FA) core complex plays a central role in the DNA damage response network involving breast cancer susceptibility gene products, BRCA1 and BRCA2. The complex consists of eight FA proteins, including a ubiquitin ligase (FANCL) and a DNA translocase (FANCM), and is essential for monoubiquitination of FANCD2 in response to DNA damage. Here, we report a novel component of this complex, termed FAAP100, which is essential for the stability of the core complex and directly interacts with FANCB and FANCL to form a stable subcomplex. Formation of this subcomplex protects each component from proteolytic degradation and also allows their coregulation by FANCA and FANCM during nuclear localization. Using siRNA depletion and gene knockout techniques, we show that FAAP100-deficient cells display hallmark features of FA cells, including defective FANCD2 monoubiquitination, hypersensitivity to DNA crosslinking agents, and genomic instability. Our study identifies FAAP100 as a new critical component of the FA-BRCA DNA damage response network.
Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Células HeLa , Humanos , Modelos Moleculares , Oligonucleotídeos , Interferência de RNARESUMO
The Fanconi anemia (FA) protein FANCE is an essential component of the nuclear FA core complex, which is required for monoubiquitination of the downstream target FANCD2, an important step in the FA pathway of DNA cross-link repair. FANCE is predominantly localized in the nucleus and acts as a molecular bridge between the FA core complex and FANCD2, through direct binding of both FANCC and FANCD2. At present, it is poorly understood how the nuclear accumulation of FANCE is regulated and therefore we investigated the nuclear localization of this FA protein. We found that FANCE has a strong tendency to localize in the nucleus, since the addition of a nuclear export signal does not interfere with the nuclear localization of FANCE. We also demonstrate that the nuclear accumulation of FANCE does not rely solely on its nuclear localization signal motifs, but also on FANCC. The other FA proteins are not involved in the nuclear accumulation of FANCE, indicating a tight relationship between FANCC and FANCE, as suggested from their direct interaction. Finally, we show that the region of FANCE interacting with FANCC appears to be different from the region involved in binding FANCD2. This strengthens the idea that FANCE recruits FANCD2 to the core complex, without interfering with the binding of FANCC.
Assuntos
Proteína do Grupo de Complementação C da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação E da Anemia de Fanconi/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação C da Anemia de Fanconi/química , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Proteína do Grupo de Complementação E da Anemia de Fanconi/química , Proteína do Grupo de Complementação E da Anemia de Fanconi/genética , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , Sinais de Exportação Nuclear/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Fanconi anemia (FA) is a multigene cancer susceptibility disorder characterized by cellular hypersensitivity to DNA interstrand cross-linking agents such as mitomycin C (MMC). FA proteins are suspected to function at the interface between cell cycle checkpoints, DNA repair, and DNA replication. Using replicating extracts from Xenopus eggs, we developed cell-free assays for FA proteins (xFA). Recruitment of the xFA core complex and xFANCD2 to chromatin is strictly dependent on replication initiation, even in the presence of MMC indicating specific recruitment to DNA lesions encountered by the replication machinery. The increase in xFA chromatin binding following treatment with MMC is part of a caffeine-sensitive S-phase checkpoint that is controlled by xATR. Recruitment of xFANCD2, but not xFANCA, is dependent on the xATR-xATR-interacting protein (xATRIP) complex. Immunodepletion of either xFANCA or xFANCD2 from egg extracts results in accumulation of chromosomal DNA breaks during replicative synthesis. Our results suggest coordinated chromatin recruitment of xFA proteins in response to replication-associated DNA lesions and indicate that xFA proteins function to prevent the accumulation of DNA breaks that arise during unperturbed replication.
Assuntos
Proteínas de Transporte/metabolismo , Dano ao DNA/fisiologia , Replicação do DNA , Proteína do Grupo de Complementação A da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Proteínas de Xenopus/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Cafeína/farmacologia , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Reparo do DNA/fisiologia , Feminino , Técnicas In Vitro , Mitomicina/farmacologia , Dados de Sequência Molecular , Oócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fase S/efeitos dos fármacos , Fase S/fisiologia , Homologia de Sequência de Aminoácidos , Xenopus laevisRESUMO
The Fanconi anemia (FA) protein FANCF is an essential component of a nuclear core complex that protects the genome against chromosomal instability, but the specific function of FANCF is still poorly understood. Based upon the homology between human and Xenopus laevis FANCF, we carried out an extensive mutagenesis study to examine which domains are functionally important and to gain more insight into the function of FANCF. In contrast to previous suggestions, we show that FANCF does not have a ROM-like function. We found that the C terminus of FANCF interacts directly with FANCG and allows the assembly of other FA proteins into a stable complex. The N terminus appears to stabilize the interaction with FANCA and FANCG and is essential for the binding of the FANCC/FANCE subcomplex. We identified several important amino acids in this N-terminal region but, surprisingly, many amino acid changes failed to affect the function of the FANCF protein. Our data demonstrate that FANCF acts as a flexible adaptor protein that plays a key role in the proper assembly of the FA core complex.
Assuntos
Núcleo Celular/fisiologia , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Animais , Proteína do Grupo de Complementação F da Anemia de Fanconi , Humanos , Dados de Sequência Molecular , Mutagênese , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/genética , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , XenopusRESUMO
Fanconi anemia (FA) is an autosomal recessive syndrome featuring diverse symptoms including progressive bone marrow failure and early occurrence of acute myeloid leukemia. Nine genetic subtypes have been described for FA (A, B, C, D1, D2, E, F, G, and L), all of which have been connected to distinct disease genes, except B. Here we report on 8 unrelated FA patients who were excluded from the known subtypes on the basis of phenotypic correction or genetic data. Four of these cell lines failed to complement each other in somatic cell hybrids and therefore represent a new group, termed FA-I. The remaining cell lines complemented group FA-I but did not complement each other, thus representing a second new group, FA-J. Both FA-I and -J cell lines were capable of forming an FA multiprotein core complex. This complex is required for activation of the FANCD2 protein by mono-ubiquitination, a key downstream event in the FA pathway. In FA-I cells FANCD2 was not mono-ubiquitinated, indicating a defect upstream in the FA pathway, whereas in FA-J cells FANCD2 was mono-ubiquitinated, indicating a downstream defect. Our results suggest that the FA pathway of genome stabilization may be controlled by at least 11 different genes, including FANCI and FANCJ.
Assuntos
Anemia de Fanconi/classificação , Anemia de Fanconi/genética , Polimorfismo Genético , Divisão Celular , Fusão Celular , Linhagem Celular , Criança , Pré-Escolar , Anemia de Fanconi/patologia , Genes Recessivos , Teste de Complementação Genética , Humanos , TransfecçãoRESUMO
Fanconi anaemia (FA) is an inherited form of progressive pancytopenia associated with developmental defects, chromosomal instability, and cancer predisposition. At least seven distinct FA proteins function in concert to protect the genome, a key step being the activation of FANCD2 by mono-ubiquitination. This paper reports an immunohistochemical analysis of FANCD2 expression in normal human tissue. The highest expression was observed in maturing spermatocytes and fetal oocytes (consistent with a role for FANCD2 in meiosis) and in germinal centre cells of the spleen, tonsil, and lymph nodes (consistent with a role in proliferation). FANCD2 expression was also seen in tissues predisposed to cancer development in FA patients: haematopoietic cells, especially in the fetus, and squamous cell epithelia, particularly in the head and neck region and uterine cervix. FANCD2 expression was also occasionally seen in the breast and Fallopian tube epithelium, the respiratory epithelium of the trachea, and the exocrine cells of the pancreas, indicating that these tissues may also be cancer-prone in FA. FANCD2 expression is frequently expressed in proliferating cells as demonstrated by Ki-67 immunofluorescence double staining, consistent with a function of FANCD2 in DNA replication.
Assuntos
Biomarcadores Tumorais/análise , Anemia de Fanconi/patologia , Proteínas Nucleares/análise , Adulto , Biomarcadores/análise , Divisão Celular , Linhagem Celular Transformada , Replicação do DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi , Feminino , Feto/química , Células Germinativas/química , Humanos , Imuno-Histoquímica/métodos , Antígeno Ki-67/análise , Masculino , Meiose , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Células-Tronco/química , Distribuição TecidualRESUMO
Fanconi anemia is a recessively inherited disease characterized by congenital defects, bone marrow failure and cancer susceptibility. Cells from individuals with Fanconi anemia are highly sensitive to DNA-crosslinking drugs, such as mitomycin C (MMC). Fanconi anemia proteins function in a DNA damage response pathway involving breast cancer susceptibility gene products, BRCA1 and BRCA2 (refs. 1,2). A key step in this pathway is monoubiquitination of FANCD2, resulting in the redistribution of FANCD2 to nuclear foci containing BRCA1 (ref. 3). The underlying mechanism is unclear because the five Fanconi anemia proteins known to be required for this ubiquitination have no recognizable ubiquitin ligase motifs. Here we report a new component of a Fanconi anemia protein complex, called PHF9, which possesses E3 ubiquitin ligase activity in vitro and is essential for FANCD2 monoubiquitination in vivo. Because PHF9 is defective in a cell line derived from an individual with Fanconi anemia, we conclude that PHF9 (also called FANCL) represents a novel Fanconi anemia complementation group (FA-L). Our data suggest that PHF9 has a crucial role in the Fanconi anemia pathway as the likely catalytic subunit required for monoubiquitination of FANCD2.