RESUMEN
Mycobacterium tuberculosis (M.tb.) infection leads to over 1.5 million deaths annually, despite widespread vaccination with BCG at birth. Causes for the ongoing tuberculosis endemic are complex and include the failure of BCG to protect many against progressive pulmonary disease. Host genetics is one of the known factors implicated in susceptibility to primary tuberculosis, but less is known about the role that host genetics plays in controlling host responses to vaccination against M.tb. Here, we addressed this gap by utilizing Diversity Outbred (DO) mice as a small animal model to query genetic drivers of vaccine-induced protection against M.tb. DO mice are a highly genetically and phenotypically diverse outbred population that is well suited for fine genetic mapping. Similar to outcomes in people, our previous studies demonstrated that DO mice have a wide range of disease outcomes following BCG vaccination and M.tb. challenge. In the current study, we used a large population of BCG-vaccinated/M.tb.-challenged mice to perform quantitative trait loci mapping of complex infection traits; these included lung and spleen M.tb. burdens, as well as lung cytokines measured at necropsy. We found sixteen chromosomal loci associated with complex infection traits and cytokine production. QTL associated with bacterial burdens included a region encoding major histocompatibility antigens that are known to affect susceptibility to tuberculosis, supporting validity of the approach. Most of the other QTL represent novel associations with immune responses to M.tb. and novel pathways of cytokine regulation. Most importantly, we discovered that protection induced by BCG is a multigenic trait, in which genetic loci harboring functionally-distinct candidate genes influence different aspects of immune responses that are crucial collectively for successful protection. These data provide exciting new avenues to explore and exploit in developing new vaccines against M.tb.
Asunto(s)
Mycobacterium bovis , Mycobacterium tuberculosis , Vacunas contra la Tuberculosis , Tuberculosis , Humanos , Animales , Ratones , Vacuna BCG/genética , Tuberculosis/genética , Tuberculosis/prevención & control , Tuberculosis/microbiología , Vacunas contra la Tuberculosis/genética , Vacunación , Sitios Genéticos , Citocinas/genética , Antígenos BacterianosRESUMEN
Current chemotherapy against Mycobacterium tuberculosis (Mtb), an important human pathogen, requires a multidrug regimen lasting several months. While efforts have been made to optimize therapy by exploiting drugdrug synergies, testing new drug combinations in relevant host environments remains arduous. In particular, host environments profoundly affect the bacterial metabolic state and drug efficacy, limiting the accuracy of predictions based on in vitro assays alone. In this study, we utilized conditional Mtb knockdown mutants of essential genes as an experimentally tractable surrogate for drug treatment and probe the relationship between Mtb carbon metabolism and chemicalgenetic interactions (CGIs). We examined the antitubercular drugs isoniazid, rifampicin, and moxifloxacin and found that CGIs are differentially responsive to the metabolic state, defining both environment-independent and -dependent interactions. Specifically, growth on the in vivorelevant carbon source, cholesterol, reduced rifampicin efficacy by altering mycobacterial cell surface lipid composition. We report that a variety of perturbations in cell wall synthesis pathways restore rifampicin efficacy during growth on cholesterol, and that both environment-independent and cholesterol-dependent in vitro CGIs could be leveraged to enhance bacterial clearance in the mouse infection model. Our findings present an atlas of chemicalgeneticenvironmental interactions that can be used to optimize drugdrug interactions, as well as provide a framework for understanding in vitro correlates of in vivo efficacy.
Asunto(s)
Antituberculosos , Carbono , Pared Celular , Interacciones Farmacológicas , Interacción Gen-Ambiente , Mycobacterium tuberculosis , Antituberculosos/farmacología , Carbono/metabolismo , Pared Celular/ultraestructura , Humanos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/ultraestructuraRESUMEN
Upf1, Upf2, and Upf3, the central regulators of nonsense-mediated mRNA decay (NMD), appear to exercise their NMD functions while bound to elongating ribosomes, and evidence for this conclusion is particularly compelling for Upf1. Hence, we used selective profiling of yeast Upf1:ribosome association to define that step in greater detail, understand whether the nature of the mRNA being translated influences Upf1:80S interaction, and elucidate the functions of ribosome-associated Upf1. Our approach has allowed us to clarify the timing and specificity of Upf1 association with translating ribosomes, obtain evidence for a Upf1 mRNA surveillance function that precedes the activation of NMD, identify a unique ribosome state that generates 37-43 nt ribosome footprints whose accumulation is dependent on Upf1's ATPase activity, and demonstrate that a mutated form of Upf1 can interfere with normal translation termination and ribosome release. In addition, our results strongly support the existence of at least two distinct functional Upf1 complexes in the NMD pathway.
Asunto(s)
Degradación de ARNm Mediada por Codón sin Sentido , ARN Helicasas , ARN Helicasas/genética , ARN Helicasas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
RNA exosomopathies, a growing family of diseases, are linked to missense mutations in genes encoding structural subunits of the evolutionarily conserved, 10-subunit exoribonuclease complex, the RNA exosome. This complex consists of a three-subunit cap, a six-subunit, barrel-shaped core, and a catalytic base subunit. While a number of mutations in RNA exosome genes cause pontocerebellar hypoplasia, mutations in the cap subunit gene EXOSC2 cause an apparently distinct clinical presentation that has been defined as a novel syndrome SHRF (short stature, hearing loss, retinitis pigmentosa, and distinctive facies). We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by modeling pathogenic EXOSC2 missense mutations (p.Gly30Val and p.Gly198Asp) in the orthologous S. cerevisiae gene RRP4 The resulting rrp4 mutant cells show defects in cell growth and RNA exosome function. Consistent with altered RNA exosome function, we detect significant transcriptomic changes in both coding and noncoding RNAs in rrp4-G226D cells that model EXOSC2 p.Gly198Asp, suggesting defects in nuclear surveillance. Biochemical and genetic analyses suggest that the Rrp4 G226D variant subunit shows impaired interactions with key RNA exosome cofactors that modulate the function of the complex. These results provide the first in vivo evidence that pathogenic missense mutations present in EXOSC2 impair the function of the RNA exosome. This study also sets the stage to compare exosomopathy models to understand how defects in RNA exosome function underlie distinct pathologies.
Asunto(s)
Exorribonucleasas/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Mutación Missense , ARN de Hongos/genética , Proteínas de Unión al ARN/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Enanismo/enzimología , Enanismo/genética , Enanismo/patología , Exorribonucleasas/química , Exorribonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/química , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Facies , Expresión Génica , Glicina/química , Glicina/metabolismo , Pérdida Auditiva/enzimología , Pérdida Auditiva/genética , Pérdida Auditiva/patología , Humanos , Modelos Biológicos , Modelos Moleculares , Conformación Proteica , ARN de Hongos/química , ARN de Hongos/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Retinitis Pigmentosa/enzimología , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/patología , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , SíndromeRESUMEN
Restricting the localization of the histone H3 variant CENP-A (Cse4 in yeast, CID in flies) to centromeres is essential for faithful chromosome segregation. Mislocalization of CENP-A leads to chromosomal instability (CIN) in yeast, fly and human cells. Overexpression and mislocalization of CENP-A has been observed in many cancers and this correlates with increased invasiveness and poor prognosis. Yet genes that regulate CENP-A levels and localization under physiological conditions have not been defined. In this study we used a genome-wide genetic screen to identify essential genes required for Cse4 homeostasis to prevent its mislocalization for chromosomal stability. We show that two Skp, Cullin, F-box (SCF) ubiquitin ligases with the evolutionarily conserved F-box proteins Met30 and Cdc4 interact and cooperatively regulate proteolysis of endogenous Cse4 and prevent its mislocalization for faithful chromosome segregation under physiological conditions. The interaction of Met30 with Cdc4 is independent of the D domain, which is essential for their homodimerization and ubiquitination of other substrates. The requirement for both Cdc4 and Met30 for ubiquitination is specifc for Cse4; and a common substrate for Cdc4 and Met30 has not previously been described. Met30 is necessary for the interaction between Cdc4 and Cse4, and defects in this interaction lead to stabilization and mislocalization of Cse4, which in turn contributes to CIN. We provide the first direct link between Cse4 mislocalization to defects in kinetochore structure and show that SCF-mediated proteolysis of Cse4 is a major mechanism that prevents stable maintenance of Cse4 at non-centromeric regions, thus ensuring faithful chromosome segregation. In summary, we have identified essential pathways that regulate cellular levels of endogenous Cse4 and shown that proteolysis of Cse4 by SCF-Met30/Cdc4 prevents mislocalization and CIN in unperturbed cells.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Inestabilidad Cromosómica , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas F-Box/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Centrómero/metabolismo , Segregación Cromosómica , Dominios Proteicos , Proteolisis , UbiquitinaciónRESUMEN
BACKGROUND: Deep sequencing of transposon mutant libraries (or TnSeq) is a powerful method for probing essentiality of genomic loci under different environmental conditions. Various analytical methods have been described for identifying conditionally essential genes whose tolerance for insertions varies between two conditions. However, for large-scale experiments involving many conditions, a method is needed for identifying genes that exhibit significant variability in insertions across multiple conditions. RESULTS: In this paper, we introduce a novel statistical method for identifying genes with significant variability of insertion counts across multiple conditions based on Zero-Inflated Negative Binomial (ZINB) regression. Using likelihood ratio tests, we show that the ZINB distribution fits TnSeq data better than either ANOVA or a Negative Binomial (in a generalized linear model). We use ZINB regression to identify genes required for infection of M. tuberculosis H37Rv in C57BL/6 mice. We also use ZINB to perform a analysis of genes conditionally essential in H37Rv cultures exposed to multiple antibiotics. CONCLUSIONS: Our results show that, not only does ZINB generally identify most of the genes found by pairwise resampling (and vastly out-performs ANOVA), but it also identifies additional genes where variability is detectable only when the magnitudes of insertion counts are treated separately from local differences in saturation, as in the ZINB model.
Asunto(s)
Elementos Transponibles de ADN/genética , Bases de Datos Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento , Modelos Estadísticos , Animales , Antibacterianos/farmacología , Distribución Binomial , Genes Esenciales , Funciones de Verosimilitud , Modelos Lineales , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genéticaRESUMEN
After immunization or infection, activation-induced cytidine deaminase (AID) initiates diversification of immunoglobulin (Ig) genes in B cells, introducing mutations within the antigen-binding V regions (somatic hypermutation, SHM) and double-strand DNA breaks (DSBs) into switch (S) regions, leading to antibody class switch recombination (CSR). We asked if, during B cell activation, AID also induces DNA breaks at genes other than IgH genes. Using a nonbiased genome-wide approach, we have identified hundreds of reproducible, AID-dependent DSBs in mouse splenic B cells shortly after induction of CSR in culture. Most interestingly, AID induces DSBs at sites syntenic with sites of translocations, deletions, and amplifications found in human B cell lymphomas, including within the oncogene B cell lymphoma11a (bcl11a)/evi9. Unlike AID-induced DSBs in Ig genes, genome-wide AID-dependent DSBs are not restricted to transcribed regions and frequently occur within repeated sequence elements, including CA repeats, non-CA tandem repeats, and SINEs.
Asunto(s)
Linfocitos B/enzimología , Citidina Desaminasa/fisiología , Roturas del ADN de Doble Cadena , Secuencias de Aminoácidos , Animales , Sitios de Unión , Proteínas Portadoras/química , Citidina Desaminasa/metabolismo , Proteínas de Unión al ADN , Genes myc , Cambio de Clase de Inmunoglobulina , Activación de Linfocitos , Ratones , Proteínas Nucleares/química , Secuencias Repetitivas de Ácidos Nucleicos , Proteínas RepresorasRESUMEN
Tn-Seq is an experimental method for probing the functions of genes through construction of complex random transposon insertion libraries and quantification of each mutant's abundance using next-generation sequencing. An important emerging application of Tn-Seq is for identifying genetic interactions, which involves comparing Tn mutant libraries generated in different genetic backgrounds (e.g. wild-type strain versus knockout strain). Several analytical methods have been proposed for analyzing Tn-Seq data to identify genetic interactions, including estimating relative fitness ratios and fitting a generalized linear model. However, these have limitations which necessitate an improved approach. We present a hierarchical Bayesian method for identifying genetic interactions through quantifying the statistical significance of changes in enrichment. The analysis involves a four-way comparison of insertion counts across datasets to identify transposon mutants that differentially affect bacterial fitness depending on genetic background. Our approach was applied to Tn-Seq libraries made in isogenic strains of Mycobacterium tuberculosis lacking three different genes of unknown function previously shown to be necessary for optimal fitness during infection. By analyzing the libraries subjected to selection in mice, we were able to distinguish several distinct classes of genetic interactions for each target gene that shed light on their functions and roles during infection.
Asunto(s)
Epistasis Genética , Genes Bacterianos , Análisis de Secuencia de ADN/métodos , Algoritmos , Proteínas Bacterianas/genética , Teorema de Bayes , Elementos Transponibles de ADN , Interpretación Estadística de Datos , Frecuencia de los Genes , Técnicas de Inactivación de Genes , Biblioteca de Genes , Modelos Genéticos , Método de Montecarlo , Mutagénesis Insercional , Mycobacterium tuberculosis/genéticaRESUMEN
Activation-induced cytidine deaminase (AID) is required for initiation of Ig class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes during immune responses. AID has also been shown to induce chromosomal translocations, mutations, and DNA double-strand breaks (DSBs) involving non-Ig genes in activated B cells. To determine what makes a DNA site a target for AID-induced DSBs, we identify off-target DSBs induced by AID by performing chromatin immunoprecipitation (ChIP) for Nbs1, a protein that binds DSBs, followed by deep sequencing (ChIP-Seq). We detect and characterize hundreds of off-target AID-dependent DSBs. Two types of tandem repeats are highly enriched within the Nbs1-binding sites: long CA repeats, which can form Z-DNA, and tandem pentamers containing the AID target hotspot WGCW. These tandem repeats are not nearly as enriched at AID-independent DSBs, which we also identified. Msh2, a component of the mismatch repair pathway and important for genome stability, increases off-target DSBs, similar to its effect on Ig switch region DSBs, which are required intermediates during CSR. Most of the off-target DSBs are two-ended, consistent with generation during G1 phase, similar to DSBs in Ig switch regions. However, a minority are one-ended, presumably due to conversion of single-strand breaks to DSBs during replication. One-ended DSBs are repaired by processes involving homologous recombination, including break-induced replication repair, which can lead to genome instability. Off-target DSBs, especially those present during S phase, can lead to chromosomal translocations, deletions and gene amplifications, resulting in the high frequency of B cell lymphomas derived from cells that express or have expressed AID.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Citidina Desaminasa/fisiología , Roturas del ADN de Doble Cadena , Proteínas Nucleares/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Inmunoprecipitación de Cromatina , ADN Intergénico/genética , Proteínas de Unión al ADN , Ratones Endogámicos C57BL , Ratones Transgénicos , Unión Proteica , Bazo/citología , Bazo/enzimología , Secuencias Repetidas en TándemRESUMEN
In a recent issue of Molecular Cell, Pidoux et al. (2009) and Williams et al. (2009) identify S. pombe Scm3 as the proximate factor in the Cnp1/CENP-A deposition pathway, providing a direct connection to centromere-localized Mis16-Mis18.
Asunto(s)
Autoantígenos/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Centrómero/metabolismo , Proteína A Centromérica , Modelos Biológicos , Transporte de Proteínas , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
The kinetochore (centromeric DNA and associated proteins) is a key determinant for high fidelity chromosome transmission. Evolutionarily conserved Scm3p is an essential component of centromeric chromatin and is required for assembly and function of kinetochores in humans, fission yeast, and budding yeast. Overexpression of HJURP, the mammalian homolog of budding yeast Scm3p, has been observed in lung and breast cancers and is associated with poor prognosis; however, the physiological relevance of these observations is not well understood. We overexpressed SCM3 and HJURP in Saccharomyces cerevisiae and HJURP in human cells and defined domains within Scm3p that mediate its chromosome loss phenotype. Our results showed that the overexpression of SCM3 (GALSCM3) or HJURP (GALHJURP) caused chromosome loss in a wild-type yeast strain, and overexpression of HJURP led to mitotic defects in human cells. GALSCM3 resulted in reduced viability in kinetochore mutants, premature separation of sister chromatids, and reduction in Cse4p and histone H4 at centromeres. Overexpression of CSE4 or histone H4 suppressed chromosome loss and restored levels of Cse4p at centromeres in GALSCM3 strains. Using mutant alleles of scm3, we identified a domain in the N-terminus of Scm3p that mediates its interaction with CEN DNA and determined that the chromosome loss phenotype of GALSCM3 is due to centromeric association of Scm3p devoid of Cse4p/H4. Furthermore, we determined that similar to other systems the centromeric association of Scm3p is cell cycle regulated. Our results show that altered stoichiometry of Scm3p/HJURP, Cse4p, and histone H4 lead to defects in chromosome segregation. We conclude that stringent regulation of HJURP and SCM3 expression are critical for genome stability.
Asunto(s)
Inestabilidad Cromosómica/genética , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Histonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Centrómero/genética , Centrómero/metabolismo , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/genética , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Histonas/metabolismo , Humanos , Cinetocoros/metabolismo , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The outcome of an encounter with Mycobacterium tuberculosis (Mtb) depends on the pathogen's ability to adapt to the variable immune pressures exerted by the host. Understanding this interplay has proven difficult, largely because experimentally tractable animal models do not recapitulate the heterogeneity of tuberculosis disease. We leveraged the genetically diverse Collaborative Cross (CC) mouse panel in conjunction with a library of Mtb mutants to create a resource for associating bacterial genetic requirements with host genetics and immunity. We report that CC strains vary dramatically in their susceptibility to infection and produce qualitatively distinct immune states. Global analysis of Mtb transposon mutant fitness (TnSeq) across the CC panel revealed that many virulence pathways are only required in specific host microenvironments, identifying a large fraction of the pathogen's genome that has been maintained to ensure fitness in a diverse population. Both immunological and bacterial traits can be associated with genetic variants distributed across the mouse genome, making the CC a unique population for identifying specific host-pathogen genetic interactions that influence pathogenesis.
Asunto(s)
Ratones de Colaboración Cruzada/genética , Predisposición Genética a la Enfermedad , Variación Genética , Interacciones Huésped-Patógeno/genética , Mycobacterium tuberculosis/genética , Tuberculosis/microbiología , Animales , Modelos Animales de Enfermedad , Genotipo , Masculino , Ratones , Mycobacterium tuberculosis/patogenicidad , FenotipoRESUMEN
Effective tuberculosis treatment requires at least 6 months of combination therapy. Alterations in the physiological state of the bacterium during infection are thought to reduce drug efficacy and prolong the necessary treatment period, but the nature of these adaptations remain incompletely defined. To identify specific bacterial functions that limit drug effects during infection, we employed a comprehensive genetic screening approach to identify mutants with altered susceptibility to the first-line antibiotics in the mouse model. We identified many mutations that increase the rate of bacterial clearance, suggesting new strategies for accelerating therapy. In addition, the drug-specific effects of these mutations suggested that different antibiotics are limited by distinct factors. Rifampin efficacy is inferred to be limited by cellular permeability, whereas isoniazid is preferentially affected by replication rate. Many mutations that altered bacterial clearance in the mouse model did not have an obvious effect on drug susceptibility using in vitro assays, indicating that these chemical-genetic interactions tend to be specific to the in vivo environment. This observation suggested that a wide variety of natural genetic variants could influence drug efficacy in vivo without altering behavior in standard drug-susceptibility tests. Indeed, mutations in a number of the genes identified in our study are enriched in drug-resistant clinical isolates, identifying genetic variants that may influence treatment outcome. Together, these observations suggest new avenues for improving therapy, as well as the mechanisms of genetic adaptations that limit it.IMPORTANCE Understanding how Mycobacterium tuberculosis survives during antibiotic treatment is necessary to rationally devise more effective tuberculosis (TB) chemotherapy regimens. Using genome-wide mutant fitness profiling and the mouse model of TB, we identified genes that alter antibiotic efficacy specifically in the infection environment and associated several of these genes with natural genetic variants found in drug-resistant clinical isolates. These data suggest strategies for synergistic therapies that accelerate bacterial clearance, and they identify mechanisms of adaptation to drug exposure that could influence treatment outcome.
RESUMEN
Centromeric localization of CENP-A (Cse4 in Saccharomyces cerevisiae, CID in flies, CENP-A in humans) is essential for faithful chromosome segregation. Mislocalization of overexpressed CENP-A contributes to aneuploidy in yeast, flies, and humans, and is proposed to promote tumorigenesis in human cancers. Hence, defining molecular mechanisms that promote or prevent mislocalization of CENP-A is an area of active investigation. In budding yeast, evolutionarily conserved histone chaperones Scm3 and chromatin assembly factor-1 (CAF-1) promote localization of Cse4 to centromeric and noncentromeric regions, respectively. Ubiquitin ligases, such as Psh1 and Slx5, and histone chaperones (HIR complex) regulate proteolysis of overexpressed Cse4 and prevent its mislocalization to noncentromeric regions. In this study, we have identified sumoylation sites lysine (K) 215/216 in the C terminus of Cse4, and shown that sumoylation of Cse4 K215/216 facilitates its genome-wide deposition into chromatin when overexpressed. Our results showed reduced levels of sumoylation of mutant Cse4 K215/216R/A [K changed to arginine (R) or alanine (A)] and reduced interaction of mutant Cse4 K215/216R/A with Scm3 and CAF-1 when compared to wild-type Cse4 Consistent with these results, levels of Cse4 K215/216R/A in the chromatin fraction and localization to centromeric and noncentromeric regions were reduced. Furthermore, in contrast to GAL-CSE4, which exhibits Synthetic Dosage Lethality (SDL) in psh1∆, slx5∆, and hir2∆ strains, GAL-cse4K215/216R does not exhibit SDL in these strains. Taken together, our results show that deposition of Cse4 into chromatin is facilitated by its C-terminal sumoylation.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sumoilación , Factor 1 de Ensamblaje de la Cromatina/genética , Factor 1 de Ensamblaje de la Cromatina/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Dominios Proteicos , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Mutaciones Letales Sintéticas , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Evolutionarily conserved polo-like kinase, Cdc5 (Plk1 in humans), associates with kinetochores during mitosis; however, the role of cell cycle-dependent centromeric ( CEN) association of Cdc5 and its substrates that exclusively localize to the kinetochore have not been characterized. Here we report that evolutionarily conserved CEN histone H3 variant, Cse4 (CENP-A in humans), is a substrate of Cdc5, and that the cell cycle-regulated association of Cse4 with Cdc5 is required for cell growth. Cdc5 contributes to Cse4 phosphorylation in vivo and interacts with Cse4 in mitotic cells. Mass spectrometry analysis of in vitro kinase assays showed that Cdc5 phosphorylates nine serine residues clustered within the N-terminus of Cse4. Strains with cse4-9SA exhibit increased errors in chromosome segregation, reduced levels of CEN-associated Mif2 and Mcd1/Scc1 when combined with a deletion of MCM21. Moreover, the loss of Cdc5 from the CEN chromatin contributes to defects in kinetochore integrity and reduction in CEN-associated Cse4. The cell cycle-regulated association of Cdc5 with Cse4 is essential for cell viability as constitutive association of Cdc5 with Cse4 at the kinetochore leads to growth defects. In summary, our results have defined a role for Cdc5-mediated Cse4 phosphorylation in faithful chromosome segregation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces/metabolismo , Proteínas de Ciclo Celular/fisiología , Centrómero/metabolismo , Proteína A Centromérica/fisiología , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Cinetocoros/metabolismo , Mitosis , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/fisiología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomycetales/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
Despite the administration of multiple drugs that are highly effective in vitro, tuberculosis (TB) treatment requires prolonged drug administration and is confounded by the emergence of drug-resistant strains. To understand the mechanisms that limit antibiotic efficacy, we performed a comprehensive genetic study to identify Mycobacterium tuberculosis genes that alter the rate of bacterial clearance in drug-treated mice. Several functionally distinct bacterial genes were found to alter bacterial clearance, and prominent among these was the glpK gene that encodes the glycerol-3-kinase enzyme that is necessary for glycerol catabolism. Growth on glycerol generally increased the sensitivity of M. tuberculosis to antibiotics in vitro, and glpK-deficient bacteria persisted during antibiotic treatment in vivo, particularly during exposure to pyrazinamide-containing regimens. Frameshift mutations in a hypervariable homopolymeric region of the glpK gene were found to be a specific marker of multidrug resistance in clinical M. tuberculosis isolates, and these loss-of-function alleles were also enriched in extensively drug-resistant clones. These data indicate that frequently observed variation in the glpK coding sequence produces a drug-tolerant phenotype that can reduce antibiotic efficacy and may contribute to the evolution of resistance.IMPORTANCE TB control is limited in part by the length of antibiotic treatment needed to prevent recurrent disease. To probe mechanisms underlying survival under antibiotic pressure, we performed a genetic screen for M. tuberculosis mutants with altered susceptibility to treatment using the mouse model of TB. We identified multiple genes involved in a range of functions which alter sensitivity to antibiotics. In particular, we found glycerol catabolism mutants were less susceptible to treatment and that common variation in a homopolymeric region in the glpK gene was associated with drug resistance in clinical isolates. These studies indicate that reversible high-frequency variation in carbon metabolic pathways can produce phenotypically drug-tolerant clones and have a role in the development of resistance.
Asunto(s)
Antituberculosos/farmacología , Glicerol Quinasa/genética , Mycobacterium tuberculosis/genética , Farmacorresistencia Bacteriana Múltiple/genética , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacosRESUMEN
Host genetics plays an important role in determining the outcome of Mycobacterium tuberculosis infection. We previously found that Collaborative Cross (CC) mouse strains differ in their susceptibility to M. tuberculosis and that the CC042/GeniUnc (CC042) strain suffered from a rapidly progressive disease and failed to produce the protective cytokine gamma interferon (IFN-γ) in the lung. Here, we used parallel genetic and immunological approaches to investigate the basis of CC042 mouse susceptibility. Using a population derived from a CC001/Unc (CC001) × CC042 intercross, we mapped four quantitative trait loci (QTL) underlying tuberculosis immunophenotypes (Tip1 to Tip4). These included QTL that were associated with bacterial burden, IFN-γ production following infection, and an IFN-γ-independent mechanism of bacterial control. Further immunological characterization revealed that CC042 animals recruited relatively few antigen-specific T cells to the lung and that these T cells failed to express the integrin alpha L (αL; i.e., CD11a), which contributes to T cell activation and migration. These defects could be explained by a CC042 private variant in the Itgal gene, which encodes CD11a and is found within the Tip2 interval. This 15-bp deletion leads to aberrant mRNA splicing and is predicted to result in a truncated protein product. The ItgalCC042 genotype was associated with all measured disease traits, indicating that this variant is a major determinant of susceptibility in CC042 mice. The combined effect of functionally distinct Tip variants likely explains the profound susceptibility of CC042 mice and highlights the multigenic nature of tuberculosis control in the Collaborative Cross.IMPORTANCE The variable outcome of Mycobacterium tuberculosis infection observed in natural populations is difficult to model in genetically homogeneous small-animal models. The newly developed Collaborative Cross (CC) represents a reproducible panel of genetically diverse mice that display a broad range of phenotypic responses to infection. We explored the genetic basis of this variation, focusing on a CC line that is highly susceptible to M. tuberculosis infection. This study identified multiple quantitative trait loci associated with bacterial control and cytokine production, including one that is caused by a novel loss-of-function mutation in the Itgal gene, which is necessary for T cell recruitment to the infected lung. These studies verify the multigenic control of mycobacterial disease in the CC panel, identify genetic loci controlling diverse aspects of pathogenesis, and highlight the utility of the CC resource.
Asunto(s)
Mycobacterium tuberculosis/fisiología , Tuberculosis/genética , Animales , Ratones de Colaboración Cruzada , Modelos Animales de Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Interferón gamma/genética , Interferón gamma/inmunología , Pulmón/inmunología , Pulmón/microbiología , Masculino , Ratones , Mycobacterium tuberculosis/genética , Sitios de Carácter Cuantitativo , Linfocitos T/inmunología , Tuberculosis/inmunología , Tuberculosis/microbiologíaRESUMEN
Stringent regulation of cellular levels of evolutionarily conserved centromeric histone H3 variant (CENP-A in humans, CID in flies, Cse4 in yeast) prevents its mislocalization to non-centromeric chromatin. Overexpression and mislocalization of CENP-A has been observed in cancers and leads to aneuploidy in yeast, flies, and human cells. Ubiquitin-mediated proteolysis of Cse4 by E3 ligases such as Psh1 and Sumo-Targeted Ubiquitin Ligase (STUbL) Slx5 prevent mislocalization of Cse4. Previously, we identified Siz1 and Siz2 as the major E3 ligases for sumoylation of Cse4. In this study, we have identified lysine 65 (K65) in Cse4 as a site that regulates sumoylation and ubiquitin-mediated proteolysis of Cse4 by Slx5. Strains expressing cse4 K65R exhibit reduced levels of sumoylated and ubiquitinated Cse4 in vivo Furthermore, co-immunoprecipitation experiments reveal reduced interaction of cse4 K65R with Slx5, leading to increased stability and mislocalization of cse4 K65R under normal physiological conditions. Based on the increased stability of cse4 K65R in psh1∆ strains but not in slx5∆ strains, we conclude that Slx5 targets sumoylated Cse4 K65 for ubiquitination-mediated proteolysis independent of Psh1. In summary, we have identified and characterized the physiological role of Cse4 K65 in sumoylation, ubiquitin-mediated proteolysis, and localization of Cse4 for genome stability.
Asunto(s)
Centrómero/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Proteolisis , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sumoilación , Segregación Cromosómica , Lisina/metabolismo , Proteínas Mutantes/metabolismo , Mutación/genética , Unión Proteica , Transporte de Proteínas , UbiquitinaciónRESUMEN
Cell viability and gene expression profiles are altered in cellular models of neurodegenerative disorders such as Huntington's Disease (HD). Using the yeast model system, we show that the SUMO-targeted ubiquitin ligase (STUbL) Slx5 reduces the toxicity and abnormal transcriptional activity associated with a mutant, aggregation-prone fragment of huntingtin (Htt), the causative agent of HD. We demonstrate that expression of an aggregation-prone Htt construct with 103 glutamine residues (103Q), but not the non-expanded form (25Q), results in severe growth defects in slx5Δ and slx8Δ cells. Since Slx5 is a nuclear protein and because Htt expression affects gene transcription, we assessed the effect of STUbLs on the transcriptional properties of aggregation-prone Htt. Expression of Htt 25Q and 55Q fused to the Gal4 activation domain (AD) resulted in reporter gene auto-activation. Remarkably, the auto-activation of Htt constructs was abolished by expression of Slx5 fused to the Gal4 DNA-binding domain (BD-Slx5). In support of these observations, RNF4, the human ortholog of Slx5, curbs the aberrant transcriptional activity of aggregation-prone Htt in yeast and a variety of cultured human cell lines. Functionally, we find that an extra copy of SLX5 specifically reduces Htt aggregates in the cytosol as well as chromatin-associated Htt aggregates in the nucleus. Finally, using RNA sequencing, we identified and confirmed specific targets of Htt's transcriptional activity that are modulated by Slx5. In summary, this study of STUbLs uncovers a conserved pathway that counteracts the accumulation of aggregating, transcriptionally active Htt (and possibly other poly-glutamine expanded proteins) on chromatin in both yeast and in mammalian cells.
RESUMEN
Upon inhibition of respiration, which occurs in hypoxic or nitric oxide-containing host microenvironments, Mycobacterium tuberculosis (Mtb) adopts a non-replicating "quiescent" state and becomes relatively unresponsive to antibiotic treatment. We used comprehensive mutant fitness analysis to identify regulatory and metabolic pathways that are essential for the survival of quiescent Mtb. This genetic study identified a protein acetyltransferase (Mt-Pat/Rv0998) that promoted survival and altered the flux of carbon from oxidative to reductive tricarboxylic acid (TCA) reactions. Reductive TCA requires malate dehydrogenase (MDH) and maintains the redox state of the NAD+/NADH pool. Genetic or chemical inhibition of MDH resulted in rapid cell death in both hypoxic cultures and in murine lung. These phenotypic data, in conjunction with significant structural differences between human and mycobacterial MDH enzymes that could be exploited for drug development, suggest a new strategy for eradicating quiescent bacteria.