Abstract
Approximately 70% of KRAS-positive colorectal cancers (CRCs) have a CpG island methylator phenotype (CIMP) characterized by aberrant DNA hypermethylation and transcriptional silencing of many genes. The factors involved in, and the mechanistic basis of, CIMP is not understood. Among the CIMP genes are the tumor suppressors p14(ARF), p15(INK4B), and p16(INK4A), encoded by the INK4-ARF locus. In this study, we perform an RNA interference screen and identify ZNF304, a zinc-finger DNA-binding protein, as the pivotal factor required for INK4-ARF silencing and CIMP in CRCs containing activated KRAS. In KRAS-positive human CRC cell lines and tumors, ZNF304 is bound at the promoters of INK4-ARF and other CIMP genes. Promoter-bound ZNF304 recruits a corepressor complex that includes the DNA methyltransferase DNMT1, resulting in DNA hypermethylation and transcriptional silencing. KRAS promotes silencing through upregulation of ZNF304, which drives DNA binding. Finally, we show that ZNF304 also directs transcriptional silencing of INK4-ARF in human embryonic stem cells. DOI: http://dx.doi.org/10.7554/eLife.02313.001.
Highlights
Epigenetic dysregulation of gene expression plays a major role in the initiation and progression of cancer
Our results reveal a KRAS-directed pathway that mediates silencing of the entire INK4-ARF locus, is responsible for CpG island methylator phenotype (CIMP) in Colorectal cancers (CRCs), and is related to the pathway that silences INK4-ARF in human embryonic stem cells
Positive candidates identified in the primary screen were validated by stably transducing DLD-1 cells with an shRNA directed against each candidate gene, followed by the analysis of endogenous p14ARF expression by quantitative RT-PCR
Summary
Epigenetic dysregulation of gene expression plays a major role in the initiation and progression of cancer (reviewed in Baylin and Jones, 2011; Esteller, 2008; Hassler and Egger, 2012). DNA hypermethylation can alter genetic stability and genomic structure, and is associated with transcriptional silencing of gene expression (commonly referred to as epigenetic silencing; reviewed in Baylin and Jones, 2011; Esteller, 2008; Hassler and Egger, 2012). A subset of CRCs have a so-called CpG island methylator phenotype (CIMP) characterized by aberrant DNA hypermethylation of many genes. Specific panels of CIMP marker genes have been developed to classify CRCs into these three subclasses.
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