RESUMEN
The presence of ribonucleotides in genomic DNA is undesirable given their increased susceptibility to hydrolysis. Ribonuclease (RNase) H enzymes that recognize and process such embedded ribonucleotides are present in all domains of life. However, in unicellular organisms such as budding yeast, they are not required for viability or even efficient cellular proliferation, while in humans, RNase H2 hypomorphic mutations cause the neuroinflammatory disorder Aicardi-Goutières syndrome. Here, we report that RNase H2 is an essential enzyme in mice, required for embryonic growth from gastrulation onward. RNase H2 null embryos accumulate large numbers of single (or di-) ribonucleotides embedded in their genomic DNA (>1,000,000 per cell), resulting in genome instability and a p53-dependent DNA-damage response. Our findings establish RNase H2 as a key mammalian genome surveillance enzyme required for ribonucleotide removal and demonstrate that ribonucleotides are the most commonly occurring endogenous nucleotide base lesion in replicating cells.
Asunto(s)
Replicación del ADN , Embrión de Mamíferos/metabolismo , Ribonucleasa H/genética , Ribonucleasa H/metabolismo , Ribonucleótidos/metabolismo , Animales , Inestabilidad Cromosómica , ADN Polimerasa Dirigida por ADN/metabolismo , Células Madre Embrionarias/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Topologically associating domains (TADs) have been proposed to both guide and constrain enhancer activity. Shh is located within a TAD known to contain all its enhancers. To investigate the importance of chromatin conformation and TAD integrity on developmental gene regulation, we have manipulated the Shh TAD - creating internal deletions, deleting CTCF sites, and deleting and inverting sequences at TAD boundaries. Chromosome conformation capture and fluorescence in situ hybridisation assays were used to investigate the changes in chromatin conformation that result from these manipulations. Our data suggest that these substantial alterations in TAD structure have no readily detectable effect on Shh expression patterns or levels of Shh expression during development - except where enhancers are deleted - and result in no detectable phenotypes. Only in the case of a larger deletion at one TAD boundary could ectopic influence of the Shh limb enhancer be detected on a gene (Mnx1) in the neighbouring TAD. Our data suggests that, contrary to expectations, the developmental regulation of Shh expression is remarkably robust to TAD perturbations.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Animales , Emparejamiento Base/genética , Factor de Unión a CCCTC , Cromatina/metabolismo , Embrión de Mamíferos/metabolismo , Extremidades/embriología , Genoma , Proteínas Hedgehog/metabolismo , Ratones , Especificidad de Órganos/genética , Fenotipo , Eliminación de Secuencia/genéticaRESUMEN
Aicardi-Goutières syndrome (AGS) provides a monogenic model of nucleic acid-mediated inflammation relevant to the pathogenesis of systemic autoimmunity. Mutations that impair ribonuclease (RNase) H2 enzyme function are the most frequent cause of this autoinflammatory disorder of childhood and are also associated with systemic lupus erythematosus. Reduced processing of eitherRNA:DNAhybrid or genome-embedded ribonucleotide substrates is thought to lead to activation of a yet undefined nucleic acid-sensing pathway. Here, we establishRnaseh2b(A174T/A174T)knock-in mice as a subclinical model of disease, identifying significant interferon-stimulated gene (ISG) transcript upregulation that recapitulates theISGsignature seen inAGSpatients. The inflammatory response is dependent on the nucleic acid sensor cyclicGMP-AMPsynthase (cGAS) and its adaptorSTINGand is associated with reduced cellular ribonucleotide excision repair activity and increasedDNAdamage. This suggests thatcGAS/STINGis a key nucleic acid-sensing pathway relevant toAGS, providing additional insight into disease pathogenesis relevant to the development of therapeutics for this childhood-onset interferonopathy and adult systemic autoimmune disorders.
Asunto(s)
Enfermedades Autoinmunes del Sistema Nervioso/genética , Inmunidad Innata/genética , Proteínas de la Membrana/inmunología , Mutación Missense , Malformaciones del Sistema Nervioso/genética , Nucleotidiltransferasas/inmunología , Ribonucleasa H/genética , Ribonucleasas/genética , Animales , Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Enfermedades Autoinmunes del Sistema Nervioso/metabolismo , Autoinmunidad/genética , Daño del ADN , Regulación de la Expresión Génica , Humanos , Interferones/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Mutantes , Malformaciones del Sistema Nervioso/inmunología , Malformaciones del Sistema Nervioso/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Ribonucleasa H/metabolismoRESUMEN
Coordinated gene expression controlled by long-distance enhancers is orchestrated by DNA regulatory sequences involving transcription factors and layers of control mechanisms. The Shh gene and well-established regulators are an example of genomic composition in which enhancers reside in a large desert extending into neighbouring genes to control the spatiotemporal pattern of expression. Exploiting the local hopping activity of the Sleeping Beauty transposon, the lacZ reporter gene was dispersed throughout the Shh region to systematically map the genomic features responsible for expression activity. We found that enhancer activities are retained inside a genomic region that corresponds to the topological associated domain (TAD) defined by Hi-C. This domain of approximately 900â kb is in an open conformation over its length and is generally susceptible to all Shh enhancers. Similar to the distal enhancers, an enhancer residing within the Shh second intron activates the reporter gene located at distances of hundreds of kilobases away, suggesting that both proximal and distal enhancers have the capacity to survey the Shh topological domain to recognise potential promoters. The widely expressed Rnf32 gene lying within the Shh domain evades enhancer activities by a process that may be common among other housekeeping genes that reside in large regulatory domains. Finally, the boundaries of the Shh TAD do not represent the absolute expression limits of enhancer activity, as expression activity is lost stepwise at a number of genomic positions at the verges of these domains.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/fisiología , Animales , Blastocisto/citología , Elementos Transponibles de ADN , Elementos de Facilitación Genéticos , Perfilación de la Expresión Génica , Genes Reporteros , Prueba de Complementación Genética , Proteínas Hedgehog/genética , Heterocigoto , Intrones , Ratones , Ratones Transgénicos , Modelos Genéticos , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , TransgenesRESUMEN
Conservation within intergenic DNA often highlights regulatory elements that control gene expression from a long range. How conservation within a single element relates to regulatory information and how internal composition relates to function is unknown. Here, we examine the structural features of the highly conserved ZRS (also called MFCS1) cis-regulator responsible for the spatiotemporal control of Shh in the limb bud. By systematically dissecting the ZRS, both in transgenic assays and within in the endogenous locus, we show that the ZRS is, in effect, composed of two distinct domains of activity: one domain directs spatiotemporal activity but functions predominantly from a short range, whereas a second domain is required to promote long-range activity. We show further that these two domains encode activities that are highly integrated and that the second domain is crucial in promoting the chromosomal conformational changes correlated with gene activity. During limb bud development, these activities encoded by the ZRS are interpreted differently by the fore limbs and the hind limbs; in the absence of the second domain there is no Shh activity in the fore limb, and in the hind limb low levels of Shh lead to a variant digit pattern ranging from two to four digits. Hence, in the embryo, the second domain stabilises the developmental programme providing a buffer for SHH morphogen activity and this ensures that five digits form in both sets of limbs.
Asunto(s)
Esbozos de los Miembros/embriología , Esbozos de los Miembros/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Animales , Secuencia de Bases , Cromosomas de los Mamíferos/química , Análisis Mutacional de ADN , Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Miembro Posterior/embriología , Miembro Posterior/metabolismo , Hibridación Fluorescente in Situ , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Conformación de Ácido Nucleico , Fenotipo , Mutación Puntual/genética , Eliminación de Secuencia/genéticaRESUMEN
In meiosis, a supramolecular protein structure, the synaptonemal complex (SC), assembles between homologous chromosomes to facilitate their recombination. Mammalian SC formation is thought to involve hierarchical zipper-like assembly of an SYCP1 protein lattice that recruits stabilizing central element (CE) proteins as it extends. Here we combine biochemical approaches with separation-of-function mutagenesis in mice to show that, rather than stabilizing the SYCP1 lattice, the CE protein SYCE3 actively remodels this structure during synapsis. We find that SYCP1 tetramers undergo conformational change into 2:1 heterotrimers on SYCE3 binding, removing their assembly interfaces and disrupting the SYCP1 lattice. SYCE3 then establishes a new lattice by its self-assembly mimicking the role of the disrupted interface in tethering together SYCP1 dimers. SYCE3 also interacts with CE complexes SYCE1-SIX6OS1 and SYCE2-TEX12, providing a mechanism for their recruitment. Thus, SYCE3 remodels the SYCP1 lattice into a CE-binding integrated SYCP1-SYCE3 lattice to achieve long-range synapsis by a mature SC.
Asunto(s)
Emparejamiento Cromosómico , Complejo Sinaptonémico , Animales , Ratones , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/genética , Mamíferos/genética , Meiosis , Proteínas Nucleares/metabolismo , Complejo Sinaptonémico/metabolismoRESUMEN
Disruption of the long-range cis-regulation of developmental gene expression is increasingly recognized as a cause of human disease. Here, we report a novel type of long-range cis-regulatory mutation, in which ectopic expression of a gene is driven by an enhancer that is not its own. We have termed this gain of regulatory information as "enhancer adoption." We mapped the breakpoints of a de novo 7q inversion in a child with features of a holoprosencephaly spectrum (HPES) disorder and severe upper limb syndactyly with lower limb synpolydactyly. The HPES plausibly results from the 7q36.3 breakpoint dislocating the sonic hedgehog (SHH) gene from enhancers that are known to drive expression in the early forebrain. However, the limb phenotype cannot be explained by loss of known SHH enhancers. The SHH transcription unit is relocated to 7q22.1, â¼190 kb 3' of a highly conserved noncoding element (HCNE2) within an intron of EMID2. We show that HCNE2 functions as a limb bud enhancer in mouse embryos and drives ectopic expression of Shh in vivo recapitulating the limb phenotype in the child. This developmental genetic mechanism may explain a proportion of the novel or unexplained phenotypes associated with balanced chromosome rearrangements.
Asunto(s)
Inversión Cromosómica/genética , Elementos de Facilitación Genéticos/genética , Proteínas Hedgehog/genética , Holoprosencefalia/genética , Sindactilia/genética , Animales , Preescolar , Cromosomas Humanos Par 7/genética , Extremidades/embriología , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/metabolismo , Humanos , Esbozos de los Miembros/embriología , Ratones , Ratones Transgénicos , MutaciónRESUMEN
Enhancers that are conserved deep in evolutionary time regulate characteristics held in common across taxonomic classes. Here, deletion of the highly conserved Shh enhancer SBE2 (Shh brain enhancer 2) in mouse markedly reduced Shh expression within the embryonic brain specifically in the rostral diencephalon; however, no abnormal anatomical phenotype was observed. Secondary enhancer activity was subsequently identified which likely mediates low levels of expression. In contrast, when crossing the SBE2 deletion with the Shh null allele, brain and craniofacial development were disrupted; thus, linking SBE2 regulated Shh expression to multiple defects and further enabling the study of the effects of differing levels of Shh on embryogenesis. Development of the hypothalamus, derived from the rostral diencephalon, was disrupted along both the anterior-posterior (AP) and the dorsal-ventral (DV) axes. Expression of DV patterning genes and subsequent neuronal population induction were particularly sensitive to Shh expression levels, demonstrating a novel morphogenic context for Shh. The role of SBE2, which is highlighted by DV gene expression, is to step-up expression of Shh above the minimal activity of the second enhancer, ensuring the necessary levels of Shh in a regional-specific manner. We also show that low Shh levels in the diencephalon disrupted neighbouring craniofacial development, including mediolateral patterning of the bones along the cranial floor and viscerocranium. Thus, SBE2 contributes to hypothalamic morphogenesis and ensures there is coordination with the formation of the adjacent midline cranial bones that subsequently protect the neural tissue.
RESUMEN
Precise spatial and temporal control of developmental genes is crucial during embryogenesis. Regulatory mutations that cause the misexpression of key developmental genes may underlie a number of developmental abnormalities. The congenital abnormality preaxial polydactyly, extra digits, is an example of this novel class of mutations and is caused by ectopic expression of the signalling molecule Sonic Hedgehog (SHH) in the developing limb bud. Mutations in the long-distant, limb-specific cis-regulator for SHH, called the ZRS, are responsible for the ectopic expression which underlies the abnormality. Here, we show that populations of domestic cats which manifest extra digits, including the celebrated polydactylous Hemingway's cats, also contain mutations within the ZRS. The polydactylous cats add significantly to the number of mutations previously reported in mouse and human and to date, all are single nucleotide substitutions. A mouse transgenic assay shows that these single nucleotide substitutions operate as gain-of-function mutations that activate Shh expression at an ectopic embryonic site; and that the sequence context of the mutation is responsible for a variable regulatory output. The plasticity of the regulatory response correlates with both the phenotypic variability and with species differences. The polydactyly mutations define a new genetic mechanism that results in human congenital abnormalities and identifies a pathogenetic mechanism that may underlie other congenital diseases.
Asunto(s)
Tipificación del Cuerpo/genética , Proteínas Hedgehog/genética , Mutación Puntual , Polidactilia/genética , Animales , Secuencia de Bases , Gatos , Miembro Anterior/anomalías , Miembro Anterior/embriología , Regulación del Desarrollo de la Expresión Génica , Genes Reguladores , Proteínas Hedgehog/fisiología , Humanos , Operón Lac , Ratones , Mutagénesis Sitio-Dirigida , Linaje , Polidactilia/embriología , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
BACKGROUND: Alterations in gene splicing occur in human sporadic colorectal cancer (CRC) and may contribute to tumour progression. The K-ras proto-oncogene encodes two splice variants, K-ras 4A and 4B, and K-ras activating mutations which jointly affect both isoforms are prevalent in CRC. Past studies have established that splicing of both the K-ras oncogene and proto-oncogene is altered in CRC in favour of K-ras 4B. The present study addressed whether the K-Ras 4A proto-oncoprotein can suppress tumour development in the absence of its oncogenic allele, utilising the ApcMin/+ (Min) mouse that spontaneously develops intestinal tumours that do not harbour K-ras activating mutations, and the K-rastmDelta4A/tmDelta4A mouse that can express the K-ras 4B splice variant only. By this means tumorigenesis in the small intestine was compared between ApcMin/+, K-ras+/+ and ApcMin/+, K-rastmDelta4A/tmDelta4A mice that can, and cannot, express the K-ras 4A proto-oncoprotein respectively. METHODS: The relative levels of expression of the K-ras splice variants in normal small intestine and small intestinal tumours were quantified by real-time RT-qPCR analysis. Inbred (C57BL/6) ApcMin/+, K-ras+/+ and ApcMin/+, K-rastmDelta4A/tmDelta4A mice were generated and the genotypes confirmed by PCR analysis. Survival of stocks was compared by the Mantel-Haenszel test, and tumour number and area compared by Student's t-test in outwardly healthy mice at approximately 106 and 152 days of age. DNA sequencing of codons 12, 13 and 61 was performed to confirm the intestinal tumours did not harbour a K-ras activating mutation. RESULTS: The K-ras 4A transcript accounted for about 50% of K-ras expressed in the small intestine of both wild-type and Min mice. Tumours in the small intestine of Min mice showed increased levels of K-ras 4B transcript expression, but no appreciable change in K-ras 4A transcript levels. No K-ras activating mutations were detected in 27 intestinal tumours derived from Min and compound mutant Min mice. K-Ras 4A deficiency did not affect mouse survival, or tumour number, size or histopathology. CONCLUSION: The K-Ras 4A proto-oncoprotein does not exhibit tumour suppressor activity in the small intestine, even though the K-ras 4A/4B ratio is reduced in adenomas lacking K-ras activating mutations.
Asunto(s)
Adenoma/genética , Regulación Neoplásica de la Expresión Génica/genética , Genes ras/genética , Neoplasias Intestinales/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Animales Modificados Genéticamente , Genes APC/fisiología , Mutación de Línea Germinal/genética , Intestino Delgado/metabolismo , Ratones , Ratones Endogámicos C57BL , Proto-Oncogenes Mas , Transcripción GenéticaRESUMEN
There is an increasing need for more efficient generation of transgenic constructs. Here we present a universal multi-site Gateway vector for use in recombineering reactions. Using transgenic mouse models, we show its use for the generation of BAC transgenics and targeting vectors. The modular nature of the vector allows for rapid modification of constructs to generate different versions of the same construct. As such it will help streamline the generation of series of related transgenic models.
Asunto(s)
Cromosomas Artificiales Bacterianos/genética , Técnicas de Sustitución del Gen/métodos , Ingeniería Genética/métodos , Vectores Genéticos/genética , Recombinación Genética , Animales , Femenino , RatonesRESUMEN
Sonic hedgehog (Shh) expression during limb development is crucial for specifying the identity and number of digits. The spatial pattern of Shh expression is restricted to a region called the zone of polarizing activity (ZPA), and this expression is controlled from a long distance by the cis-regulator ZRS. Here, members of two groups of ETS transcription factors are shown to act directly at the ZRS mediating a differential effect on Shh, defining its spatial expression pattern. Occupancy at multiple GABPα/ETS1 sites regulates the position of the ZPA boundary, whereas ETV4/ETV5 binding restricts expression outside the ZPA. The ETS gene family is therefore attributed with specifying the boundaries of the classical ZPA. Two point mutations within the ZRS change the profile of ETS binding and activate Shh expression at an ectopic site in the limb bud. These molecular changes define a pathogenetic mechanism that leads to preaxial polydactyly (PPD).
Asunto(s)
Embrión de Mamíferos/metabolismo , Proteínas Hedgehog/metabolismo , Esbozos de los Miembros/embriología , Esbozos de los Miembros/metabolismo , Polidactilia/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Animales , Western Blotting , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Embrión de Mamíferos/citología , Elementos de Facilitación Genéticos/genética , Factor de Transcripción de la Proteína de Unión a GA/genética , Factor de Transcripción de la Proteína de Unión a GA/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Hibridación in Situ , Esbozos de los Miembros/citología , Ratones , Ratones Transgénicos , Mutación Puntual/genética , Proteína Proto-Oncogénica c-ets-1/genética , Proteína Proto-Oncogénica c-ets-1/metabolismo , Proteínas Proto-Oncogénicas c-ets/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Elementos Reguladores de la Transcripción , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The epicardial epithelial-mesenchymal transition (EMT) is hypothesized to generate cardiovascular progenitor cells that differentiate into various cell types, including coronary smooth muscle and endothelial cells, perivascular and cardiac interstitial fibroblasts and cardiomyocytes. Here we show that an epicardial-specific knockout of the gene encoding Wilms' tumor-1 (Wt1) leads to a reduction in mesenchymal progenitor cells and their derivatives. We show that Wt1 is essential for repression of the epithelial phenotype in epicardial cells and during embryonic stem cell differentiation through direct transcriptional regulation of the genes encoding Snail (Snai1) and E-cadherin (Cdh1), two of the major mediators of EMT. Some mesodermal lineages do not form in Wt1-null embryoid bodies, but this effect is rescued by the expression of Snai1, underscoring the importance of EMT in generating these differentiated cells. These new insights into the molecular mechanisms regulating cardiovascular progenitor cells and EMT will shed light on the pathogenesis of heart diseases and may help the development of cell-based therapies.
Asunto(s)
Cadherinas/genética , Células Madre/metabolismo , Factores de Transcripción/genética , Transcripción Genética , Proteínas WT1/genética , Animales , Cadherinas/metabolismo , Sistema Cardiovascular/citología , Diferenciación Celular , Células Cultivadas , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Epitelio/metabolismo , Epitelio/patología , Regulación del Desarrollo de la Expresión Génica , Técnicas de Sustitución del Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Immunoblotting , Mesodermo/metabolismo , Mesodermo/patología , Ratones , Ratones Noqueados , Pericardio/anomalías , Pericardio/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción de la Familia Snail , Células Madre/citología , Factores de Transcripción/metabolismo , Proteínas WT1/metabolismoRESUMEN
Denys-Drash syndrome (DDS) is caused by heterozygous mutations of the Wilms' tumour suppressor gene, WT1, characterised by early-onset diffuse mesangial sclerosis often associated with male pseudohermaphroditism and/or Wilms' tumourigenesis. Previously, we reported that the Wt1tmT396 allele induces DDS kidney disease in mice. In the present study heterozygotes (Wt1tmT396/+) were generated on inbred (129/Ola), crossbred (B6/129) and MF1 second backcross (MF1-N2) backgrounds. Whereas male heterozygotes on each background were fertile, inbred heterozygous females were infertile. Kidney disease (proteinuria and sclerosis) was not congenital and developed significantly earlier in inbred mice, although with variable onset. Disease onset in MF1-N2 stocks occurred later in Wt1tmT396/+ mice than reported previously for Wt1R394W/+ mice, and while no kidney disease has been reported in B6/129 Wt1+/- mice, B6/129 Wt1tmT396/+ mice were affected. Offspring of both male and female B6/129 and MF1-N2 Wt1tmT396/+ mice developed kidney disease, but its incidence was significantly higher in offspring of female heterozygotes. Wt1tmT396/tmT396 embryos exhibited identical developmental abnormalities to those reported for Wt1-/- embryos. The results indicate that the Wt1 (tmT396) allele does not predispose to Wilms' tumourigenesis or male pseudohermaphroditism, its effect on kidney disease and female fertility depends on genetic background, stochastic factors may affect disease onset, and disease transmission is subject to a partial parent-of-origin effect. Since the Wt1tmT396 allele has no detectable intrinsic functional activity in vivo, and kidney disease progression is affected by the type of Wt1 mutation, the data support the view that DDS nephropathy results from a dominant-negative action rather than WT1 haploinsufficiency or gain-of-function.
Asunto(s)
Síndrome de Denys-Drash/genética , Fertilidad/genética , Marcación de Gen/métodos , Crecimiento y Desarrollo/genética , Enfermedades Renales/genética , Alelos , Animales , Clonación Molecular , Cruzamientos Genéticos , Embrión de Mamíferos , Femenino , Dosificación de Gen/fisiología , Genes Dominantes/fisiología , Pérdida de Heterocigocidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Proteínas WT1/genéticaRESUMEN
To examine the roles of endogenous K-ras 4A and K-ras 4B splice variants in tumorigenesis, murine lung carcinogenesis was induced by N-methyl-N-nitrosourea (MNU), which causes a K-ras mutation (G12D) that jointly affects both isoforms. Compared with age-matched K-ras(tmDelta4A/-) mice (where tumours can express mutationally activated K-ras 4B only), tumour number and size were significantly higher in K-ras(+/-) mice (where tumours can also express mutationally activated K-ras 4A), and significantly lower in K-ras(tmDelta4A/tmDelta4A) mice (where tumours can express both wild-type and activated K-ras 4B). MNU induced significantly more, and larger, tumours in wild-type than K-ras(tmDelta4A/tmDelta4A) mice which differ in that only tumours in wild-type mice can express wild-type and activated K-ras 4A. Lung tumours in all genotypes were predominantly papillary adenomas, and tumours from K-ras(+/-) and K-ras(tmDelta4A/-) mice exhibited phospho-Erk1/2 and phospho-Akt staining. Hence (1) mutationally activated K-ras 4B is sufficient to activate the Raf/MEK/ERK(MAPK) and PI3-K/Akt pathways, and initiate lung tumorigenesis, (2) when expressed with activated K-ras 4B, mutationally activated K-ras 4A further promotes lung tumour formation and growth (both in the presence and absence of its wild-type isoform) but does not affect either tumour pathology or progression, and (3) wild-type K-ras 4B, either directly or indirectly, reduces tumour number and size.
Asunto(s)
Neoplasias Pulmonares/etiología , Proteínas Mutantes , Isoformas de Proteínas , Proteínas ras/genética , Animales , Progresión de la Enfermedad , Neoplasias Pulmonares/patología , Metilnitrosourea , Ratones , Ratones Noqueados , Mutagénesis/genética , Transducción de Señal , Carga Tumoral/genética , Proteínas ras/fisiologíaRESUMEN
Ras proteins function as molecular switches in signal transduction pathways, and, here, we examined the effects of the K-ras4A and 4B splice variants on cell function by comparing wild-type embryonic stem (ES) cells with K-ras(tmDelta4A/tmDelta4A) (exon 4A knock-out) ES cells which express K-ras4B only and K-ras(-/-) (exons 1-3 knock-out) ES cells which express neither splice variant, and intestinal epithelium from wild-type and K-ras(tmDelta4A/tmDelta4A) mice. RT-qPCR analysis found that K-ras4B expression was reduced in K-ras(tmDelta4A/tmDelta4A) ES cells but unaffected in small intestine. K-Ras deficiency did not affect ES cell growth, and K-Ras4A deficiency did not affect intestinal epithelial proliferation. K-ras(tmDelta4A/tmDelta4A) and K-ras(-/-) ES cells showed a reduced capacity for differentiation following LIF withdrawal, and K-ras(-/-) cells were least differentiated. K-Ras4A deficiency inhibited etoposide-induced apoptosis in ES cells and intestinal epithelial cells. However, K-ras(tmDelta4A/tmDelta4A) ES cells were more resistant to etoposide-induced apoptosis than K-ras(-/-) cells. The results indicate that (1) K-Ras4A promotes apoptosis while K-Ras4B inhibits it, and (2) K-Ras4B, and possibly K-Ras4A, promotes differentiation. The findings raise the possibility that alteration of the K-Ras4A/4B isoform ratio modulates tumorigenesis by differentially affecting stem cell survival and/or differentiation. However, K-Ras4A deficiency did not affect life expectancy or spontaneous overall tumor incidence in aging mice.