AbstractThe FES locus encodes a unique nonreceptor protein‐tyrosine kinase (FES) traditionally viewed as a proto‐oncogene but more recently implicated as a tumor suppressor in colorectal cancer (CRC). Recent studies have demonstrated that while FES is expressed in normal colonic epithelium, expression is lost in tumor tissue and colorectal cancer cell lines, a finding common among tumor suppressors. Here we provide compelling evidence that promoter methylation is an important mechanism responsible for downregulation of FES gene expression in colorectal cancer cells. Treatment with the DNA methyltransferase inhibitor 5‐aza‐2′‐deoxycytidine resulted in the expression of functional FES transcripts in all CRC cell lines examined, including Caco‐2, COLO 320, DLD‐1, HCT 116, SNU‐1040, SW‐480, and HT‐29. Bisulfite sequencing of genomic DNA isolated from 5‐aza‐2′‐deoxycytidine‐treated HT‐29 cells identified methylated CpG dinucleotides immediately upstream from the FES transcription initiation sites. In contrast, this region of the FES promoter was hypomethylated in genomic DNA from normal colonic epithelium. In addition, methylation completely blocked the activity of the FES promoter in reporter gene assays. Promoter methylation is a previously unrecognized mechanism by which FES expression is suppressed in CRC cell lines, and is consistent with a tumor suppressor role for FES in this tumor site despite its tyrosine kinase activity. © 2008 Wiley‐Liss, Inc.