Dataset Information

Large-scale mutagenesis in p19(ARF)- and p53-deficient mice identifies cancer genes and their collaborative networks.

ABSTRACT: p53 and p19(ARF) are tumor suppressors frequently mutated in human tumors. In a high-throughput screen in mice for mutations collaborating with either p53 or p19(ARF) deficiency, we identified 10,806 retroviral insertion sites, implicating over 300 loci in tumorigenesis. This dataset reveals 20 genes that are specifically mutated in either p19(ARF)-deficient, p53-deficient or wild-type mice (including Flt3, mmu-mir-106a-363, Smg6, and Ccnd3), as well as networks of significant collaborative and mutually exclusive interactions between cancer genes. Furthermore, we found candidate tumor suppressor genes, as well as distinct clusters of insertions within genes like Flt3 and Notch1 that induce mutants with different spectra of genetic interactions. Cross species comparative analysis with aCGH data of human cancer cell lines revealed known and candidate oncogenes (Mmp13, Slamf6, and Rreb1) and tumor suppressors (Wwox and Arfrp2). This dataset should prove to be a rich resource for the study of genetic interactions that underlie tumorigenesis.

SUBMITTER: Uren AG

PROVIDER: S-EPMC2405818 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Large-scale mutagenesis in p19(ARF)- and p53-deficient mice identifies cancer genes and their collaborative networks.

Uren Anthony G AG Kool Jaap J Matentzoglu Konstantin K de Ridder Jeroen J Mattison Jenny J van Uitert Miranda M Lagcher Wendy W Sie Daoud D Tanger Ellen E Cox Tony T Reinders Marcel M Hubbard Tim J TJ Rogers Jane J Jonkers Jos J Wessels Lodewyk L Adams David J DJ van Lohuizen Maarten M Berns Anton A

Cell 20080501 4

p53 and p19(ARF) are tumor suppressors frequently mutated in human tumors. In a high-throughput screen in mice for mutations collaborating with either p53 or p19(ARF) deficiency, we identified 10,806 retroviral insertion sites, implicating over 300 loci in tumorigenesis. This dataset reveals 20 genes that are specifically mutated in either p19(ARF)-deficient, p53-deficient or wild-type mice (including Flt3, mmu-mir-106a-363, Smg6, and Ccnd3), as well as networks of significant collaborative and ...[more]

PMID: 18485879

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Project description:Gram-positive Streptomyces bacteria produce thousands of bioactive secondary metabolites, including antibiotics. To systematically investigate genes affecting secondary metabolism, we developed a hyperactive transposase-based Tn5 transposition system and employed it to mutagenize the model species Streptomyces coelicolor, leading to the identification of 51,443 transposition insertions. These insertions were distributed randomly along the chromosome except for some preferred regions associated with relatively low GC content in the chromosomal core. The base composition of the insertion site and its flanking sequences compiled from the 51,443 insertions implied a 19-bp expanded target site surrounding the insertion site, with a slight nucleic acid base preference in some positions, suggesting a relative randomness of Tn5 transposition targeting in the high-GC Streptomyces genome. From the mutagenesis library, 724 mutants involving 365 genes had altered levels of production of the tripyrrole antibiotic undecylprodigiosin (RED), including 17 genes in the RED biosynthetic gene cluster. Genetic complementation revealed that most of the insertions (more than two-thirds) were responsible for the changed antibiotic production. Genes associated with branched-chain amino acid biosynthesis, DNA metabolism, and protein modification affected RED production, and genes involved in signaling, stress, and transcriptional regulation were overrepresented. Some insertions caused dramatic changes in RED production, identifying future targets for strain improvement.IMPORTANCE High-GC Gram-positive streptomycetes and related actinomycetes have provided more than 100 clinical drugs used as antibiotics, immunosuppressants, and antitumor drugs. Their genomes harbor biosynthetic genes for many more unknown compounds with potential as future drugs. Here we developed a useful genome-wide mutagenesis tool based on the transposon Tn5 for the study of secondary metabolism and its regulation. Using Streptomyces coelicolor as a model strain, we found that chromosomal insertion was relatively random, except at some hot spots, though there was evidence of a slightly preferred 19-bp target site. We then used prodiginine production as a model to systematically survey genes affecting antibiotic biosynthesis, providing a global view of antibiotic regulation. The analysis revealed 348 genes that modulate antibiotic production, among which more than half act to reduce production. These might be valuable targets in future investigations of regulatory mechanisms, for strain improvement, and for the activation of silent biosynthetic gene clusters.

Project description:The p53 tumor suppressor is a stress sensor, driving cell cycle arrest or apoptosis in response to DNA damage or oncogenic signals. p53 activation by oncogenic signals relies on the p19(Arf) tumor suppressor, while p53 activation downstream of acute DNA damage is reported to be p19(Arf)-independent. Accordingly, p19(Arf)-deficient mouse embryo fibroblasts (MEFs) arrest in response to acute DNA damage. However, p19(Arf) is required for replicative senescence, a condition associated with an activated DNA damage response, as p19(Arf)-/- MEFs do not senesce after serial passage. A possible explanation for these seemingly disparate roles for p19(Arf) is that acute and chronic DNA damage responses are mechanistically distinct. Replicative senescence may result from chronic, low-dose DNA damage responses in which p19(Arf) has a specific role. We therefore examined the role of p19(Arf) in cellular responses to chronic, low-dose DNA-damaging agent treatment by maintaining MEFs in low oxygen and administering 0.5?G?y ?-irradiation daily or 150??M hydroxyurea, a replication stress inducer. In contrast to their response to acute DNA damage, p19(Arf)-/- MEFs exposed to chronic DNA damage do not senesce, revealing a selective role for p19(Arf) in senescence upon low-level, chronic DNA damage. We show further that p53 pathway activation in p19(Arf)-/- MEFs exposed to chronic DNA damage is attenuated relative to wild-type MEFs, suggesting a role for p19(Arf) in fine-tuning p53 activity. However, combined Nutlin3a and chronic DNA-damaging agent treatment is insufficient to promote senescence in p19(Arf)-/- MEFs, suggesting that the role of p19(Arf) in the chronic DNA damage response may be partially p53-independent. These data suggest the importance of p19(Arf) for the cellular response to the low-level DNA damage incurred in culture or upon oncogene expression, providing new insight into how p19(Arf) serves as a tumor suppressor. Moreover, our study helps reconcile reports suggesting crucial roles for both p19(Arf) and DNA damage-signaling pathways in tumor suppression.

Dataset Information

Large-scale mutagenesis in p19(ARF)- and p53-deficient mice identifies cancer genes and their collaborative networks.

Publications

Large-scale mutagenesis in p19(ARF)- and p53-deficient mice identifies cancer genes and their collaborative networks.

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