The conversion of a normal cell to a cancer cell occurs in several steps and typically involves the activation of oncogenes and the inactivation of tumour suppressor and pro-apoptotic genes1. In many instances, inactivation of genes critical for cancer development occurs by epigenetic silencing, often involving hypermethylation of CpG-rich promoter regions2,3. It remains to be determined whether silencing occurs by random acquisition of epigenetic marks that confer a selective growth advantage or through a specific pathway initiated by an oncogene4,5,6. Here we perform a genome-wide RNA interference (RNAi) screen in K-ras-transformed NIH 3T3 cells and identify 28 genes required for Ras-mediated epigenetic silencing of the pro-apoptotic Fas gene. At least nine of these RESEs (Ras epigenetic silencing effectors), including the DNA methyltransferase DNMT1, are directly associated with specific regions of the Fas promoter in K-ras-transformed NIH 3T3 cells but not in untransformed NIH 3T3 cells. RNAi-mediated knockdown of any of the 28 RESEs results in failure to recruit DNMT1 to the Fas promoter, loss of Fas promoter hypermethylation, and derepression of Fas expression. Analysis of five other epigenetically repressed genes indicates that Ras directs the silencing of multiple unrelated genes through a largely common pathway. Last, we show that nine RESEs are required for anchorage-independent growth and tumorigenicity of K-ras-transformed NIH 3T3 cells; these nine genes have not previously been implicated in transformation by Ras. Our results show that Ras-mediated epigenetic silencing occurs through a specific, complex, pathway involving components that are required for maintenance of a fully transformed phenotype.
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We thank S. Evans for editorial assistance. C.G. is on leave from the CNRS, Paris, France. This work was funded in part by a grant from the NIH to M.R.G. M.R.G. is an investigator of the Howard Hughes Medical Institute.
Author Contributions S.G. and C.-M.V. prepared the retroviral library. C.G. and N.W. performed all experiments and data analyses. C.G., N.W. and M.R.G. designed the experiments, discussed the interpretation of the results and co-wrote the paper. All authors commented on the manuscript.
The file contains Supplementary Figures 1-12 and Supplementary Tables 1-2 with Legends; Supplementary Methods and additional references. The Supplementary Figures show several control experiments; confirm and extend the generality of Fas silencing and the involvement of RESEs to other mouse and human ras-transformed cell lines; show transcriptional or post-transcriptional upregulation of several RESEs by Ras; confirm the hypermethylated status of additional promoters; and show the requirement for several RESEs for a fully transformed phenotype. The Supplementary Tables provide shRNA and primer sequence information, as well as supplementary methods and references.