DNMT3B targets in RKO cells by ChIP-on-chip

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 majority of the target genes including P16, DCC, DISC1, SLIT1, CAVEOLIN1, 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 and bisulfite genomic sequencing showed that the CGIs encompassing the transcription start sites (TSS) of DCC, TBX5, TBX18, SLIT1 were also methylated in primary colorectal tumors compared to matching normal tissues whereas GNA11 was methylated in both. MassARRAY analysis demonstrated that the CGI located ~4.5 kb upstream of HOXB13 +1 site was tumor-specifically hypermethylated in primary colorectal cancers and cancer cell lines. 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. Chromatin immunoprecipitation with DNMT3B specific antibody in RKO cells was compared to control antibody.