An elaborate pathway required for Ras-mediated epigenetic silencing

Abstract

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 genes. In many instances, inactivation of genes critical for cancer development occurs by epigenetic silencing, often involving hypermethylation of CpG-rich promoter regions. 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 oncogene. 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.

Figure 1. A genome-wide shRNA screen identifies factors required for Ras-mediated epigenetic silencing of Fas

a, Schematic summary of the genome-wide shRNA screen for Ras-mediated epigenetic silencing of Fas. b, Immunoblot analysis monitoring Fas expression in the 28 K-ras NIH 3T3 knockdown (KD) cell lines. Expression of Fas in K-ras NIH 3T3 cells in the presence and absence of 5-aza-2’-deoxycytidine (5-aza) is also shown. K-Ras expression is shown as a loading control.

Figure 2. ChIP analysis and methylation status of the Fas promoter

a, Summary of bisulphite sequencing. Open circle, unmethylated CpG; closed circle, methylated CpG. Each row represents a single clone. CpG dinucleotide positions are shown by vertical lines. b, MeDIP analysis following knockdown of each of the 28 RESEs. NS, nonsilencing shRNA. Values are expressed as the fold-difference relative to input, and have been corrected for background. Error bars indicate standard error (n=3). c, ChIP assay monitoring occupancy of selected RESEs at the core promoter/TSS (CP/TSS) or ~1 kb or ~2 kb upstream of the TSS. d, Summary of the ChIP results. e, ChIP analysis monitoring occupancy of DNMT1.

Figure 3. Ras directs epigenetic silencing of multiple, unrelated genes through a largely common pathway

a, qRT-PCR monitoring expression of Fas, Sfrp1, Par4, Plagl1, H2-K1 and Lox in NIH 3T3 cells, and in K-ras NIH 3T3 cells in the presence and absence of 5-aza. Values are expressed as fold re-expression relative to expression of the gene in K-ras NIH 3T3 cells, which is arbitrarily set to 1. Error bars indicate standard error (n=3). b, Summary of qRT-PCR analysis monitoring re-expression of Fas, Sfrp1, Par4, Plagl1, H2-K1 and Lox following knockdown of each of the 28 RESEs.