BACKGROUND: A hallmark of cancer cells is hypermethylation of CpG islands 
(CGIs), which probably arises from upregulation of one or more DNA 
methyltransferases. The purpose of this study was to identify the targets of 
DNMT3B, an essential DNA methyltransferase in mammals, in colon cancer.
METHODOLOGY/PRINCIPAL FINDINGS: Chromatin immunoprecipitation with DNMT3B 
specific antibody followed by CGI microarray identified genes with or without 
CGIs, repeat elements and genomic contigs in RKO cells. ChIP-Chop analysis 
showed that the majority of the target genes including P16, DCC, DISC1, SLIT1, 
CAVEOLIN1, GNA11, TBX5, TBX18, HOXB13 and some histone variants, that harbor CGI 
in their promoters, were methylated in multiple colon cancer cell lines but not 
in normal colon epithelial cells. Further, these genes were reactivated in RKO 
cells after treatment with 5-aza-2'-deoxycytidine, a DNA hypomethylating agent. 
COBRA showed that the CGIs encompassing the promoter and/or coding region of 
DCC, TBX5, TBX18, SLIT1 were methylated in primary colorectal tumors but not in 
matching normal colon tissues whereas GNA11 was methylated in both. MassARRAY 
analysis demonstrated that the CGI located approximately 4.5 kb upstream of 
HOXB13 +1 site was tumor-specifically hypermethylated in primary colorectal 
cancers and cancer cell lines. HOXB13 upstream CGI was partially hypomethylated 
in DNMT1(-/-) HCT cells but was almost methylation free in cells lacking both 
DNMT1 and DNMT3B. Analysis of tumor suppressor properties of two aberrantly 
methylated transcription factors, HOXB13 and TBX18, revealed that both inhibited 
growth and clonogenic survival of colon cancer cells in vitro, but only HOXB13 
abolished tumor growth in nude mice.
CONCLUSIONS/SIGNIFICANCE: This is the first report that identifies several 
important tumor suppressors and transcription factors as direct DNMT3B targets 
in colon cancer and as potential biomarkers for this cancer. Further, this study 
shows that methylation at an upstream CGI of HOXB13 is unique to colon cancer.