RESUMEN
Recent human genome-wide association studies have identified common missense variants in MARC1, p.Ala165Thr and p.Met187Lys, associated with lower hepatic fat, reduction in liver enzymes and protection from most causes of cirrhosis. Using an exome-wide association study we recapitulated earlier MARC1 p.Ala165Thr and p.Met187Lys findings in 540,000 individuals from five ancestry groups. We also discovered novel rare putative loss of function variants in MARC1 with a phenotype similar to MARC1 p.Ala165Thr/p.Met187Lys variants. In vitro studies of recombinant human MARC1 protein revealed Ala165Thr substitution causes protein instability and aberrant localization in hepatic cells, suggesting MARC1 inhibition or deletion may lead to hepatoprotection. Following this hypothesis, we generated Marc1 knockout mice and evaluated the effect of Marc1 deletion on liver phenotype. Unexpectedly, our study found that whole-body Marc1 deficiency in mouse is not protective against hepatic triglyceride accumulation, liver inflammation or fibrosis. In attempts to explain the lack of the observed phenotype, we discovered that Marc1 plays only a minor role in mouse liver while its paralogue Marc2 is the main Marc family enzyme in mice. Our findings highlight the major difference in MARC1 physiological function between human and mouse.
Asunto(s)
Estudio de Asociación del Genoma Completo , Oximas , Animales , Humanos , Ratones , Cirrosis HepáticaRESUMEN
Pathogenic variants in BRCA2 are known to significantly increase the lifetime risk of developing breast and ovarian cancers. Sequencing-based genetic testing has resulted in the identification of thousands of BRCA2 variants that are considered to be variants of uncertain significance (VUS) because the disease risk associated with them is unknown. One such variant is p.Arg3052Gln, which has conflicting interpretations of pathogenicity in the ClinVar variant database. Arginine at position 3052 in BRCA2 plays an important role in stabilizing its C-terminal DNA binding domain. We have generated a knock-in mouse model expressing this variant to examine its role on growth and survival in vivo. Homozygous as well as hemizygous mutant mice are viable, fertile and exhibit no overt phenotype. While we did not observe any hematopoietic defects in adults, we did observe a marked reduction in the in vitro proliferative ability of fetal liver cells that were also hypersensitive to PARP inhibitor, olaparib. In vitro studies performed on embryonic and adult fibroblasts derived from the mutant mice showed significant reduction in radiation induced RAD51 foci formation as well as increased genomic instability after mitomycin C treatment. We observed mis-localization of a fraction of R3052Q BRCA2 protein to the cytoplasm which may explain the observed in vitro phenotypes. Our findings suggest that BRCA2 R3052Q should be considered as a hypomorphic variant.
Asunto(s)
Antineoplásicos , Neoplasias de la Mama , Neoplasias Ováricas , Humanos , Femenino , Ratones , Animales , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Pruebas Genéticas , Neoplasias Ováricas/genética , Homocigoto , Neoplasias de la Mama/genética , Proteína BRCA1/genética , Predisposición Genética a la EnfermedadRESUMEN
The interaction between tumor suppressor BRCA2 and DSS1 is essential for RAD51 recruitment and repair of DNA double stand breaks (DSBs) by homologous recombination (HR). We have generated mice with a leucine to proline substitution at position 2431 of BRCA2, which disrupts this interaction. Although a significant number of mutant mice die during embryogenesis, some homozygous and hemizygous mutant mice undergo normal postnatal development. Despite lack of radiation induced RAD51 foci formation and a severe HR defect in somatic cells, mutant mice are fertile and exhibit normal RAD51 recruitment during meiosis. We hypothesize that the presence of homologous chromosomes in close proximity during early prophase I may compensate for the defect in BRCA2-DSS1 interaction. We show the restoration of RAD51 foci in mutant cells when Topoisomerase I inhibitor-induced single strand breaks are converted into DSBs during DNA replication. We also partially rescue the HR defect by tethering the donor DNA to the site of DSBs using streptavidin-fused Cas9. Our findings demonstrate that the BRCA2-DSS1 complex is dispensable for RAD51 loading when the homologous DNA is close to the DSB.
Asunto(s)
Roturas del ADN de Doble Cadena , Recombinasa Rad51 , Animales , ADN , Reparación del ADN/genética , Recombinación Homóloga , Ratones , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismoRESUMEN
Increased blood levels of low-density lipoprotein cholesterol (LDL-C) and fibrinogen are independent risk factors for cardiovascular disease. We identified associations between an Amish-enriched missense variant (p.Asn352Ser) in a functional domain of beta-1,4-galactosyltransferase 1 (B4GALT1) and 13.9 milligrams per deciliter lower LDL-C (P = 4.1 × 1019) and 29 milligrams per deciliter lower plasma fibrinogen (P = 1.3 × 105). B4GALT1 genebased analysis in 544,955 subjects showed an association with decreased coronary artery disease (odds ratio = 0.64, P = 0.006). The mutant protein had 50% lower galactosyltransferase activity compared with the wild-type protein. N-linked glycan profiling of human serum found serine 352 allele to be associated with decreased galactosylation and sialylation of apolipoprotein B100, fibrinogen, immunoglobulin G, and transferrin. B4galt1 353Ser knock-in mice showed decreases in LDL-C and fibrinogen. Our findings suggest that targeted modulation of protein galactosylation may represent a therapeutic approach to decreasing cardiovascular disease.
Asunto(s)
LDL-Colesterol/sangre , Fibrinógeno/análisis , Galactosiltransferasas/genética , Mutación Missense , Animales , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/prevención & control , Femenino , Galactosa/metabolismo , Galactosiltransferasas/metabolismo , Técnicas de Sustitución del Gen , Técnicas de Silenciamiento del Gen , Glicoproteínas/sangre , Glicosilación , Humanos , Hígado/enzimología , Masculino , Ratones , Ácido N-Acetilneuramínico/metabolismo , Polisacáridos/sangre , Secuenciación Completa del GenomaRESUMEN
CRISPR-based transcriptional activation is a powerful tool for functional gene interrogation; however, delivery difficulties have limited its applications in vivo. Here, we created a mouse model expressing all components of the CRISPR-Cas9 guide RNA-directed Synergistic Activation Mediator (SAM) from a single transcript that is capable of activating target genes in a tissue-specific manner. We optimized Lipid Nanoparticles and Adeno-Associated Virus guide RNA delivery approaches to achieve expression modulation of one or more genes in vivo. We utilized the SAM mouse model to generate a hypercholesteremia disease state that we could bidirectionally modulate with various guide RNAs. Additionally, we applied SAM to optimize gene expression in a humanized Transthyretin mouse model to recapitulate human expression levels. These results demonstrate that the SAM gene activation platform can facilitate in vivo research and drug discovery.
Asunto(s)
Sistemas CRISPR-Cas/genética , Hipercolesterolemia/genética , Liposomas/farmacología , Prealbúmina/metabolismo , Activación Transcripcional/genética , Animales , Línea Celular , Expresión Génica/genética , Regulación de la Expresión Génica/genética , Ingeniería Genética/métodos , Células HEK293 , Humanos , Hipercolesterolemia/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nanopartículas , Prealbúmina/genética , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismoRESUMEN
Human breast cancer susceptibility gene, BRCA2, encodes a 3418-amino acid protein that is essential for maintaining genomic integrity. Among the proteins that physically interact with BRCA2, Partner and Localizer of BRCA2 (PALB2), which binds to the N-terminal region of BRCA2, is vital for its function by facilitating its subnuclear localization. A functional redundancy has been reported between this N-terminal PALB2-binding domain and the C-terminal DNA-binding domain of BRCA2, which undermines the relevance of the interaction between these two proteins. Here, we describe a genetic approach to examine the functional significance of the interaction between BRCA2 and PALB2 by generating a knock-in mouse model of Brca2 carrying a single amino acid change (Gly25Arg, Brca2G25R) that disrupts this interaction. In addition, we have combined Brca2G25R homozygosity as well as hemizygosity with Palb2 and Trp53 heterozygosity to generate an array of genotypically and phenotypically distinct mouse models. Our findings reveal defects in body size, fertility, meiotic progression, and genome stability, as well as increased tumor susceptibility in these mice. The severity of the phenotype increased with a decrease in the interaction between BRCA2 and PALB2, highlighting the significance of this interaction. In addition, our findings also demonstrate that hypomorphic mutations such as Brca2G25R have the potential to be more detrimental than the functionally null alleles by increasing genomic instability to a level that induces tumorigenesis, rather than apoptosis.
Asunto(s)
Proteína BRCA2/genética , Neoplasias de la Mama/genética , Proteínas Nucleares/genética , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Apoptosis/genética , Proteína BRCA1/genética , Proteína BRCA2/metabolismo , Neoplasias de la Mama/patología , Carcinogénesis/genética , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Femenino , Técnicas de Sustitución del Gen , Predisposición Genética a la Enfermedad , Inestabilidad Genómica/genética , Humanos , Ratones , Mutación , Proteínas Nucleares/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Genome maintenance in germ cells is critical for fertility and the stable propagation of species. While mechanisms of meiotic DNA repair and chromosome behavior are well-characterized, the same is not true for primordial germ cells (PGCs), which arise and propagate during very early stages of mammalian development. Fanconi anemia (FA), a genomic instability syndrome that includes hypogonadism and testicular failure phenotypes, is caused by mutations in genes encoding a complex of proteins involved in repair of DNA lesions associated with DNA replication. The signaling mechanisms underlying hypogonadism and testicular failure in FA patients or mouse models are unknown. We conducted genetic studies to show that hypogonadism of Fancm mutant mice is a result of reduced proliferation, but not apoptosis, of PGCs, resulting in reduced germ cells in neonates of both sexes. Progressive loss of germ cells in adult males also occurs, overlaid with an elevated level of meiotic DNA damage. Genetic studies indicated that ATM-p53-p21 signaling is partially responsible for the germ cell deficiency.
Asunto(s)
Anemia de Fanconi/genética , Proteína p53 Supresora de Tumor/biosíntesis , Proteínas de Unión al GTP rho/biosíntesis , Animales , Apoptosis/genética , Proteínas de la Ataxia Telangiectasia Mutada/biosíntesis , Proteínas de la Ataxia Telangiectasia Mutada/genética , Reparación del ADN/genética , Replicación del ADN/genética , Anemia de Fanconi/patología , Inestabilidad Genómica , Células Germinativas/metabolismo , Células Germinativas/patología , Humanos , Hipogonadismo/genética , Hipogonadismo/patología , Ratones , Mutación , Transducción de Señal/genética , Proteína p53 Supresora de Tumor/genética , Proteínas de Unión al GTP rho/genéticaRESUMEN
Mammalian male fertility relies on complex inter- and intracellular signaling during spermatogenesis. Here we describe three alleles of the widely expressed A-kinase anchoring protein 9 (Akap9) gene, all of which cause gametogenic failure and infertility in the absence of marked somatic phenotypes. Akap9 disruption does not affect spindle nucleation or progression of prophase I of meiosis but does inhibit maturation of Sertoli cells, which continue to express the immaturity markers anti-Mullerian hormone and thyroid hormone receptor alpha in adults and fail to express the maturation marker p27(Kip1). Furthermore, gap and tight junctions essential for blood-testis barrier (BTB) organization are disrupted. Connexin43 (Cx43) and zona occludens-1 are improperly localized in Akap9 mutant testes, and Cx43 fails to compartmentalize germ cells near the BTB. These results identify and support a novel reproductive tissue-specific role for Akap9 in the coordinated regulation of Sertoli cells in the testis.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Células de Sertoli/citología , Espermatogénesis/genética , Proteínas de Anclaje a la Quinasa A/genética , Animales , Hormona Antimülleriana/genética , Hormona Antimülleriana/metabolismo , Conexina 43/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Uniones Comunicantes/ultraestructura , Masculino , Meiosis/genética , Ratones , Ratones Mutantes , Proteínas Asociadas a Microtúbulos/genética , Especificidad de Órganos , Transporte de Proteínas , Células de Sertoli/metabolismo , Espermatozoides/citología , Espermatozoides/metabolismo , Huso Acromático/metabolismo , Receptores alfa de Hormona Tiroidea/genética , Receptores alfa de Hormona Tiroidea/metabolismo , Uniones Estrechas/ultraestructura , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
Effective DNA replication is critical to the health and reproductive success of organisms. The six MCM2-7 proteins, which form the replicative helicase, are essential for high-fidelity replication of the genome. Many eukaryotes have a divergent paralog, MCM9, that was reported to be essential for loading MCM2-7 onto replication origins in the Xenopus oocyte extract system. To address the in vivo role of mammalian MCM9, we created and analyzed the phenotypes of mice with various mutations in Mcm9 and an intronic DNA replication-related gene Asf1a. Ablation of Mcm9 was compatible with cell proliferation and mouse viability, showing that it is nonessential for MCM2-7 loading or DNA replication. Mcm9 mutants underwent p53-independent embryonic germ-cell depletion in both sexes, with males also exhibiting defective spermatogonial stem-cell renewal. MCM9-deficient cells had elevated genomic instability and defective cell cycle reentry following replication stress, and mutant animals were prone to sex-specific cancers, most notably hepatocellular carcinoma in males. The phenotypes of mutant mice and cells suggest that MCM9 evolved a specialized but nonessential role in DNA replication or replication-linked quality-control mechanisms that are especially important for germ-line stem cells, and also for tumor suppression and genome maintenance in the soma.
Asunto(s)
Replicación del ADN/fisiología , Proteínas de Unión al ADN/genética , Células Germinativas/metabolismo , Células Madre/metabolismo , Animales , Carcinoma Hepatocelular/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/deficiencia , Gametogénesis/genética , Componentes del Gen , Inestabilidad Genómica/genética , Inmunohistoquímica , Neoplasias Hepáticas/genética , Masculino , Ratones , Pruebas de Micronúcleos , Proteínas de Mantenimiento de Minicromosoma , Chaperonas Moleculares , Mutación/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The transcriptional regulation of mammalian meiosis is poorly characterized, owing to few genetic and ex vivo models. From a genetic screen, we identify the transcription factor MYBL1 as a male-specific master regulator of several crucial meiotic processes. Spermatocytes bearing a novel separation-of-function allele (Mybl1(repro9)) had subtle defects in autosome synapsis in pachynema, a high incidence of unsynapsed sex chromosomes, incomplete double-strand break repair on synapsed pachytene chromosomes and a lack of crossing over. MYBL1 protein appears in pachynema, and its mutation caused specific alterations in expression of diverse genes, including some translated postmeiotically. These data, coupled with chromatin immunoprecipitation (ChIP-chip) experiments and bioinformatic analysis of promoters, identified direct targets of MYBL1 regulation. The results reveal that MYBL1 is a master regulator of meiotic genes that are involved in multiple processes in spermatocytes, particularly those required for cell cycle progression through pachynema.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Meiosis/fisiología , Proteínas Proto-Oncogénicas c-myb/metabolismo , Espermatocitos/fisiología , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Roturas del ADN de Doble Cadena , Femenino , Perfilación de la Expresión Génica , Humanos , Infertilidad Masculina/genética , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Análisis por Micromatrices , Datos de Secuencia Molecular , Mutación , Fase Paquiteno/fisiología , Proteínas Proto-Oncogénicas c-myb/genética , Alineación de Secuencia , Espermatocitos/citología , Espermatogénesis/fisiología , Transactivadores/genética , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
A novel mutation, repro5, was isolated in a forward genetic screen for infertility mutations induced by ENU mutagenesis. Homozygous mutant mice were phenotypically normal but were infertile. Oocytes from mutant females appeared normal, but were severely maturation-defective in that they had reduced ability to progress to metaphase II (MII), and those reaching MII were unable to progress beyond the two pronuclei stage following in vitro fertilization (IVF). Mutant males exhibited defective spermiogenesis, resulting in oligoasthenoteratospermia. Genetic mapping, positional cloning, and complementation studies with a disruption allele led to the identification of a mutation in Brwd1 (Bromodomain and WD repeat domain containing 1) as the causative lesion. Bromodomain-containing proteins typically interact with regions of chromatin containing histones hyperacetylated at lysine residues, a characteristic of chromatin in early spermiogenesis before eventual replacement of histones by the protamines. Previous data indicated that Brwd1 is broadly expressed, encoding a putative transcriptional regulator that is believed to act on chromatin through interactions with the Brg1-dependent SWI/SNF chromatin-remodeling pathway. Brwd1 represents one of a small number of genes whose elimination disrupts gametogenesis in both sexes after the major events of meiotic prophase I have been completed.
Asunto(s)
Oogénesis , Espermatogénesis , Animales , Clonación Molecular , Etilnitrosourea , Masculino , Meiosis , Ratones , Ratones Endogámicos C57BL , Mutagénesis , Oocitos/citología , Oocitos/metabolismo , Caracteres SexualesRESUMEN
Mcm4 (minichromosome maintenance-deficient 4 homolog) encodes a subunit of the MCM2-7 complex (also known as MCM2-MCM7), the replication licensing factor and presumptive replicative helicase. Here, we report that the mouse chromosome instability mutation Chaos3 (chromosome aberrations occurring spontaneously 3), isolated in a forward genetic screen, is a viable allele of Mcm4. Mcm4(Chaos3) encodes a change in an evolutionarily invariant amino acid (F345I), producing an apparently destabilized MCM4. Saccharomyces cerevisiae strains that we engineered to contain a corresponding allele (resulting in an F391I change) showed a classical minichromosome loss phenotype. Whereas homozygosity for a disrupted Mcm4 allele (Mcm4(-)) caused preimplantation lethality, Mcm(Chaos3/-) embryos died late in gestation, indicating that Mcm4(Chaos3) is hypomorphic. Mutant embryonic fibroblasts were highly susceptible to chromosome breaks induced by the DNA replication inhibitor aphidicolin. Most notably, >80% of Mcm4(Chaos3/Chaos3) females succumbed to mammary adenocarcinomas with a mean latency of 12 months. These findings suggest that hypomorphic alleles of the genes encoding the subunits of the MCM2-7 complex may increase breast cancer risk.
Asunto(s)
Adenocarcinoma/genética , Inestabilidad Cromosómica/genética , ADN Helicasas/genética , Neoplasias Mamarias Animales/genética , Secuencia de Aminoácidos , Animales , Células Cultivadas , Mapeo Cromosómico , Análisis Mutacional de ADN , Femenino , Viabilidad Fetal/genética , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Componente 4 del Complejo de Mantenimiento de Minicromosoma , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
A region-specific ENU mutagenesis screen was conducted to elucidate the functional content of proximal mouse Chr 5. We used the visibly marked, recessive, lethal inversion Rump White (Rw) as a balancer in a three-generation breeding scheme to identify recessive mutations within the approximately 50 megabases spanned by Rw. A total of 1003 pedigrees were produced, representing the largest inversion screen performed in mice. Test-class animals, homozygous for the ENU-mutagenized proximal Chr 5 and visibly distinguishable from nonhomozygous littermates, were screened for fertility, hearing, vestibular function, DNA repair, behavior, and dysmorphology. Lethals were identifiable by failure to derive test-class animals within a pedigree. Embryonic lethal mutations (total of 34) were overwhelmingly the largest class of mutants recovered. We characterized them with respect to the time of embryonic death, revealing that most act at midgestation (8.5-10.5) or sooner. To position the mutations within the Rw region and to guide allelism tests, we performed complementation analyses with a set of new and existing chromosomal deletions, as well as standard recombinational mapping on a subset of the mutations. By pooling the data from this and other region-specific mutagenesis projects, we calculate that the mouse genome contains approximately 3479-4825 embryonic lethal genes, or about 13.7%-19% of all genes.
Asunto(s)
Cromosomas , Desarrollo Embrionario/genética , Genes Letales , Mutación , Animales , Conducta Animal/fisiología , Cromosomas/efectos de los fármacos , Etilnitrosourea/toxicidad , Femenino , Fertilidad/genética , Prueba de Complementación Genética , Edad Gestacional , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Mutagénesis , Pruebas de Función VestibularRESUMEN
The genetic control of mammalian gametogenesis is inadequately characterized because of a lack of mutations causing infertility. To further the discovery of genes required for mammalian gametogenesis, phenotype-driven screens were performed in mice using random chemical mutagenesis of whole animals and embryonic stem cells. Eleven initial mutations are reported here that affect proliferation of germ cells, meiosis, spermiogenesis, and spermiation. Nine of the mutations have been mapped genetically. These preliminary studies provide baselines for estimating the number of genes required for gametogenesis and offer guidance in conducting new genetic screens that will accelerate and optimize mutant discovery. This report demonstrates the efficacy and expediency of mutagenesis to identify new genes required for mammalian gamete development.
Asunto(s)
Gametogénesis/genética , Mutación/genética , Reproducción/genética , Animales , Mapeo Cromosómico , Etilnitrosourea/farmacología , Femenino , Genotipo , Infertilidad/genética , Masculino , Meiosis/genética , Ratones , Mutagénesis/efectos de los fármacos , Pruebas de Mutagenicidad , Mutágenos/farmacología , Oocitos/fisiología , Fenotipo , EmbarazoRESUMEN
There is increasing evidence that defects in DNA double-strand-break (DSB) repair can cause chromosome instability, which may result in cancer. To identify novel DSB repair genes in mice, we performed a phenotype-driven mutagenesis screen for chromosome instability mutants using a flow cytometric peripheral blood micronucleus assay. Micronucleus levels were used as a quantitative indicator of chromosome damage in vivo. Among offspring derived from males mutagenized with the germline mutagen N-ethyl-N-nitrosourea (ENU), we identified a recessive mutation conferring elevated levels of spontaneous and radiation- or mitomycin C-induced micronuclei. This mutation, named chaos1 (chromosome aberration occurring spontaneously 1), was genetically mapped to a 1.3-Mb interval on chromosome 16 containing Polq, encoding DNA polymerase theta. We identified a nonconservative mutation in the ENU-derived allele, making it a strong candidate for chaos1. POLQ is homologous to Drosophila MUS308, which is essential for normal DNA interstrand crosslink repair and is unique in that it contains both a helicase and a DNA polymerase domain. While cancer susceptibility of chaos1 mutant mice is still under investigation, these data provide a practical paradigm for using a forward genetic approach to discover new potential cancer susceptibility genes using the surrogate biomarker of chromosome instability as a screen.