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Rapid modelling of cooperating genetic events in cancer through somatic genome editing.

ABSTRACT: Cancer is a multistep process that involves mutations and other alterations in oncogenes and tumour suppressor genes. Genome sequencing studies have identified a large collection of genetic alterations that occur in human cancers. However, the determination of which mutations are causally related to tumorigenesis remains a major challenge. Here we describe a novel CRISPR/Cas9-based approach for rapid functional investigation of candidate genes in well-established autochthonous mouse models of cancer. Using a Kras(G12D)-driven lung cancer model, we performed functional characterization of a panel of tumour suppressor genes with known loss-of-function alterations in human lung cancer. Cre-dependent somatic activation of oncogenic Kras(G12D) combined with CRISPR/Cas9-mediated genome editing of tumour suppressor genes resulted in lung adenocarcinomas with distinct histopathological and molecular features. This rapid somatic genome engineering approach enables functional characterization of putative cancer genes in the lung and other tissues using autochthonous mouse models. We anticipate that this approach can be used to systematically dissect the complex catalogue of mutations identified in cancer genome sequencing studies.

SUBMITTER: Sanchez-Rivera FJ

PROVIDER: S-EPMC4292871 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Rapid modelling of cooperating genetic events in cancer through somatic genome editing.

Sánchez-Rivera Francisco J FJ Papagiannakopoulos Thales T Romero Rodrigo R Romero Rodrigo R Tammela Tuomas T Bauer Matthew R MR Bhutkar Arjun A Joshi Nikhil S NS Subbaraj Lakshmipriya L Bronson Roderick T RT Xue Wen W Jacks Tyler T

Nature 20141022 7531

Cancer is a multistep process that involves mutations and other alterations in oncogenes and tumour suppressor genes. Genome sequencing studies have identified a large collection of genetic alterations that occur in human cancers. However, the determination of which mutations are causally related to tumorigenesis remains a major challenge. Here we describe a novel CRISPR/Cas9-based approach for rapid functional investigation of candidate genes in well-established autochthonous mouse models of ca ...[more]

PMID: 25337879

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Project description:BackgroundIdentifying novel regulatory factors and uncovered mechanisms of somatic cell reprogramming will be helpful for basic research and clinical application of induced pluripotent stem cells (iPSCs). Sin3a, a multifunctional transcription regulator, has been proven to be involved in the maintenance of pluripotency in embryonic stem cells (ESCs), but the role of Sin3a in somatic cell reprogramming remains unclear.MethodsRNA interference of Sin3a during somatic cell reprogramming was realized by short hairpin RNAs. Reprogramming efficiency was evaluated by the number of alkaline phosphatase (AP)-positive colonies and Oct4-GFP-positive colonies. RNA sequencing was performed to identify the influenced biological processes after Sin3a knockdown and further confirmed by quantitative RT-PCR (qRT-PCR), western blotting and flow cytometry. The interaction between Sin3a and Tet1 was detected by coimmunoprecipitation. The enrichment of Sin3a and Tet1 at the epithelial gene promoters was measured by chromatin immunoprecipitation. Furthermore, DNA methylation patterns at the gene loci were investigated by hydroxymethylated DNA immunoprecipitation. Finally, Sin3a mutants that disrupt the interaction of Sin3a and Tet1 were also introduced to assess the importance of the Sin3a-Tet1 interaction during the mesenchymal-to-epithelial transition (MET) process.ResultsWe found that Sin3a was gradually increased during OSKM-induced reprogramming and that knockdown of Sin3a significantly impaired MET at the early stage of reprogramming and iPSC generation. Mechanistic studies showed that Sin3a recruited Tet1 to facilitate the hydroxymethylation of epithelial gene promoters. Moreover, disrupting the interaction of Sin3a and Tet1 significantly blocked MET and iPSC generation.ConclusionsOur studies revealed that Sin3a was a novel mediator of MET during early reprogramming, where Sin3a functioned as an epigenetic coactivator, cooperating with Tet1 to activate the epithelial program and promote the initiation of somatic cell reprogramming. These findings highlight the importance of Sin3a in the MET process and deepen our understanding of the epigenetic regulatory network of early reprogramming.

Dataset Information

Rapid modelling of cooperating genetic events in cancer through somatic genome editing.

Publications

Rapid modelling of cooperating genetic events in cancer through somatic genome editing.

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