RESUMO
Adeno-associated virus (AAV) vectors are one of the leading platforms for gene delivery for the treatment of human genetic diseases, but the antiviral cellular mechanisms that interfere with optimal transgene expression are incompletely understood. Here, we performed two genome-scale CRISPR screens to identify cellular factors that restrict transgene expression from recombinant AAV vectors. Our screens revealed several components linked to DNA damage response, chromatin remodeling, and transcriptional regulation. Inactivation of the Fanconi anemia gene FANCA; the human silencing hub (HUSH)-associated methyltransferase SETDB1; and the gyrase, Hsp90, histidine kinase, and MutL (GHKL)-type ATPase MORC3 led to increased transgene expression. Moreover, SETDB1 and MORC3 knockout improved transgene levels of several AAV serotypes as well as other viral vectors, such as lentivirus and adenovirus. Finally, we demonstrated that the inhibition of FANCA, SETDB1, or MORC3 also enhanced transgene expression in human primary cells, suggesting that they could be physiologically relevant pathways that restrict AAV transgene levels in therapeutic settings. IMPORTANCE Recombinant AAV (rAAV) vectors have been successfully developed for the treatment of genetic diseases. The therapeutic strategy often involves the replacement of a defective gene by the expression of a functional copy from the rAAV vector genome. However, cells possess antiviral mechanisms that recognize and silence foreign DNA elements thereby limiting transgene expression and its therapeutic effect. Here, we utilize a functional genomics approach to uncover a comprehensive set of cellular restriction factors that inhibit rAAV-based transgene expression. Genetic inactivation of selected restriction factors increased rAAV transgene expression. Hence, modulation of identified restriction factors has the potential to enhance AAV gene replacement therapies.
Assuntos
Fatores de Restrição Antivirais , Dependovirus , Vetores Genéticos , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Dependovirus/genética , Dependovirus/imunologia , Fatores de Restrição Antivirais/genética , Fatores de Restrição Antivirais/metabolismo , Transgenes/genética , Regulação Viral da Expressão Gênica/genética , Células A549 , Células K562 , Técnicas de Inativação de Genes , Células Cultivadas , Humanos , Anemia de Fanconi/genéticaRESUMO
The Smc5-Smc6 complex contains a heterodimeric core of two SMC proteins and non-Smc elements (Nse1-6), and plays an important role in DNA repair. We investigated the functional roles of Nse4 and Nse1 in Dictyostelium discoideum. Nse4 and Nse3 expressed as Flag-tagged fusion proteins were highly enriched in nuclei, while Nse1 was localized in whole cells. Using yeast two-hybrid assays, only the interaction between Nse3 and Nse1 was detected among the combinations. However, all of the interactions among these three proteins were recognized by co-immunoprecipitation assay using cell lysates prepared from the cells expressing green fluorescent protein (GFP)- or Flag-tagged fusion proteins. GFP-tagged Nse1, which localized in whole cells, was translocated to nuclei when co-expressed with Flag-tagged Nse3 or Nse4. RNAi-mediated Nse1 and Nse4 knockdown cells (Nse1 KD and Nse4 KD cells) were generated and found to be more sensitive to UV-induced cell death than control cells. Upon starvation, Nse1 and Nse4 KD cells had increases in the number of smaller fruiting bodies that formed on non-nutrient agar plates or aggregates that formed under submerged culture. We found a reduction in the mRNA level of pdsA, in vegetative and 8 h-starved Nse4 KD cells, and pdsA knockdown cells displayed effects similar to Nse4 KD cells. Our results suggest that Nse4 and Nse1 are involved in not only the cellular DNA damage response but also cellular development in D. discoideum.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas Cromossômicas não Histona/fisiologia , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/genética , Proteínas de Protozoários/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Meios de Cultura/farmacologia , Dictyostelium/efeitos dos fármacos , Dictyostelium/metabolismo , Técnicas de Silenciamento de Genes , Dados de Sequência Molecular , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Subunidades Proteicas , Proteínas de Protozoários/genética , Homologia de Sequência de Aminoácidos , Inanição/genéticaRESUMO
Transcriptome analysis of head and neck squamous cell carcinoma (HNSCC) has been pivotal to comprehending the convoluted biology of HNSCC tumors. MAPKAPK2 or MK2 is a critical modulator of the mRNA turnover of crucial genes involved in HNSCC progression. However, MK2-centric transcriptome profiles of tumors are not well known. This study delves into HNSCC progression with MK2 at the nexus to delineate the biological relevance and intricate crosstalk of MK2 in the tumor milieu. We performed next-generation sequencing-based transcriptome profiling of HNSCC cells and xenograft tumors to ascertain mRNA expression profiles in MK2-wild type and MK2-knockdown conditions. The findings were validated using gene expression assays, immunohistochemistry, and transcript turnover studies. Here, we identified a pool of crucial MK2-regulated candidate genes by annotation and differential gene expression analyses. Regulatory network and pathway enrichment revealed their significance and involvement in the HNSCC pathogenesis. Additionally, 3'-UTR-based filtering recognized important MK2-regulated downstream target genes and validated them by nCounter gene expression assays. Finally, immunohistochemistry and transcript stability studies revealed the putative role of MK2 in regulating the transcript turnover of IGFBP2, MUC4, and PRKAR2B in HNSCC. Conclusively, MK2-regulated candidate genes were identified in this study, and their plausible involvement in HNSCC pathogenesis was elucidated. These genes possess investigative values as targets for diagnosis and therapeutic interventions for HNSCC.
RESUMO
Silencing of nuclear DNA is an essential feature of innate immune responses to invading pathogens. Early in infection, unintegrated lentiviral cDNA accumulates in the nucleus yet remains poorly expressed. In HIV-1-like lentiviruses, the Vpr accessory protein enhances unintegrated viral DNA expression, suggesting Vpr antagonizes cellular restriction. We previously showed how Vpr remodels the host proteome, identifying multiple cellular targets. We now screen these using a targeted CRISPR-Cas9 library and identify SMC5-SMC6 complex localization factor 2 (SLF2) as the Vpr target responsible for silencing unintegrated HIV-1. SLF2 recruits the SMC5/6 complex to unintegrated lentiviruses, and depletion of SLF2, or the SMC5/6 complex, increases viral expression. ATAC-seq demonstrates that Vpr-mediated SLF2 depletion increases chromatin accessibility of unintegrated virus, suggesting that the SMC5/6 complex compacts viral chromatin to silence gene expression. This work implicates the SMC5/6 complex in nuclear immunosurveillance of extrachromosomal DNA and defines its targeting by Vpr as an evolutionarily conserved antagonism.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Infecções por HIV/metabolismo , HIV-1/fisiologia , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/genética , Interações Hospedeiro-Patógeno , Humanos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Integração Viral , Replicação Viral , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genéticaRESUMO
Expanded CAG repeats form stem-loop secondary structures that lead to fork stalling and collapse. Previous work has shown that these collapsed forks relocalize to nuclear pore complexes (NPCs) in late S phase in a manner dependent on replication, the nucleoporin Nup84, and the Slx5 protein, which prevents repeat fragility and instability. Here, we show that binding of the Smc5/6 complex to the collapsed fork triggers Mms21-dependent sumoylation of fork-associated DNA repair proteins, and that RPA, Rad52, and Rad59 are the key sumoylation targets that mediate relocation. The SUMO interacting motifs of Slx5 target collapsed forks to the NPC. Notably, Rad51 foci only co-localize with the repeat after it is anchored to the nuclear periphery and Rad51 exclusion from the early collapsed fork is dependent on RPA sumoylation. This pathway may provide a mechanism to constrain recombination at stalled or collapsed forks until it is required for fork restart.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Reparo do DNA , Replicação do DNA , Poro Nuclear/metabolismo , Rad51 Recombinase/metabolismo , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Poro Nuclear/genética , Rad51 Recombinase/genética , Recombinação Genética , Proteína SUMO-1/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , SumoilaçãoRESUMO
The Sgs1 helicase and Top3-Rmi1 decatenase form a complex that affects homologous recombination outcomes during the mitotic cell cycle and during meiosis. Previous studies have reported that Sgs1-Top3-Rmi1 function is regulated by SUMOylation that is catalyzed by the Smc5-Smc6-Mms21 complex. These studies used strains in which SGS1 was C-terminally tagged with three or six copies of a human influenza hemagglutinin-derived epitope tag (3HA and 6HA). They identified SGS1 mutants that affect its SUMOylation, which we will refer to as SGS1 SUMO-site mutants. In previous work, these mutants showed phenotypes consistent with substantial loss of Sgs1-Top3-Rmi1 function during the mitotic cell cycle. We find that the reported phenotypes are largely due to the presence of the HA epitope tags. Untagged SGS1 SUMO-site mutants show either wild-type or weak hypomorphic phenotypes, depending on the assay. These phenotypes are exacerbated by both 6HA and 3HA epitope tags in two different S. cerevisiae strain backgrounds. Importantly, a C-terminal 6HA tag confers strong hypomorphic or null phenotypes on an otherwise wild-type Sgs1 protein. Taken together, these results suggest that the HA epitope tags used in previous studies seriously compromise Sgs1 function. Furthermore, they raise the possibilities either that sufficient SUMOylation of the Sgs1-Top3-Rmi1 complex might still occur in the SUMO-site mutants isolated, or that Smc5-Smc6-Mms21-mediated SUMOylation plays a minor role in the regulation of Sgs1-Top3-Rmi1 during recombination.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Fenótipo , RecQ Helicases/genética , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , SumoilaçãoRESUMO
A family of Structural Maintenance of Chromosome (SMC) complexes is essential for key cellular processes ensuring proper cohesion, condensation and replication. They share a common SMC-kleisin architecture allowing them to embrace DNA. In SMC5/6, the NSE1 and NSE3 KITE and NSE4 kleisin subunits form a stable subcomplex that binds DNA and regulates essential processes. In addition, NSE5 and NSE6 subunits associate with the core SMC5/6 complex and recruit it to DNA repair sites. The architecture of the SMC5/6 complex is crucial for its proper functioning, and mutations within the human SMC5/6 subunits result in severe syndromes. Therefore, we aimed to analyze interactions within the human SMC5/6 complex and determine its detailed architecture. Firstly, we analyzed different parts of SMC5/6 by crosslinking and MS/MS analysis. Our data suggested domain arrangements of hNSE1-hNSE3 and orientation of hNSE4 within the hNSE1-hNSE3-hNSE4 subcomplex. The crosslinking and electron microscopic analysis of the SMC5/6 core complex showed its rod-like architecture with juxtaposed hSMC5-hSMC6 arms. Additionally, we observed fully or partially opened hSMC5-hSMC6 shapes with the hNSE1-hNSE3-hNSE4 trimer localized in the SMC head domains. To complete mapping of the human SMC5/6 complex architecture, we analyzed positions of hNSE5-hNSE6 at the hSMC5-hSMC6 arms. We showed that hNSE6 binding to hNSE5 and the coiled-coil arm of hSMC6 is mediated by a conserved FAM178 domain, which we therefore renamed CANIN (Coiled-coil SMC6 And NSE5 INteracting) domain. Interestingly, hNSE6 bound both hSMC5 and hSMC6 arms, suggesting that hNSE6 may lock the arms and regulate the dynamics of the human SMC5/6 complex.
Assuntos
Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Reparo do DNA/genética , Humanos , Complexos Multiproteicos/genética , Mutação , Ligação Proteica/genética , Domínios Proteicos/genética , Multimerização Proteica/genéticaRESUMO
As genetic instability drives disease or loss of cell fitness, cellular safeguards have evolved to protect the genome, especially during sensitive cell cycle phases, such as DNA replication. Fission yeast Brc1 has emerged as a key factor in promoting cell survival when replication forks are stalled or collapsed. Brc1 is a multi-BRCT protein that is structurally related to the budding yeast Rtt107 and human PTIP DNA damage response factors, but functional similarities appear limited. Brc1 is a dosage suppressor of a mutation in the essential Smc5-Smc6 genome stability complex and is thought to act in a bypass pathway. In this study, we reveal an unexpectedly intimate connection between Brc1 and Smc5-Smc6 function. Brc1 is required for the accumulation of the Smc5-Smc6 genome stability complex in foci during replication stress and for activation of the intrinsic SUMO ligase activity of the complex by collapsed replication forks. Moreover, we show that the chromatin association and SUMO ligase activity of Smc5-Smc6 require the Nse5-Nse6 heterodimer, explaining how this nonessential cofactor critically supports the DNA repair roles of Smc5-Smc6. We also found that Brc1 interacts with Nse5-Nse6, as well as gamma-H2A, so it can tether Smc5-Smc6 at replicative DNA lesions to promote survival.