Non-CG methylation has engendered increased interest recently with the recognition of enrichment in stem cells and the development of genome-wide analyses. Our study is one of the first to compare non-CG methylation at CpG islands in cancer and normal epithelial cells. The CpG islands we evaluated have been shown previously to be densely methylated at CG dinucleotides in cancer ]. In contrast, we find that non-CG methylation does not differ between cancer and normal. In addition, we find that in cancer cells, inhibition of DNA methyltransferases using a global inhibitor results in a preferential loss of CG and CC methylation.
Previous studies have reported the more frequent occurrence of non-CG methylation in stem cells compared to more differentiated cells [4
], although others have found wide variation between pluripotent cell lines [6
]. Many cancers contain a stem cell phenotype leading us speculate differences exist between cancer and normal. We utilized several known loci that demonstrate dense CG methylation in cancer cells and are functionally important [14
]. We did not find a significant difference in non-CG methylation between cancer and non-cancer cells at these loci. Others have reported a close association between CG methylation and non-CG methylation on a genome-wide scale in stem cells [4
]. Analysis of FILIP1L
did not demonstrate the unusually high occurrence of non-CG methylation observed at the EVX1
locus, suggesting that non-CG methylation is locus specific and not always correlated with CG methylation in cancer. Our data also suggests that non-CG methylation and CG methylation are not mutually exclusive consistent with earlier reports [11
Previously, Ziller et al.
demonstrated that knockdown of DNMT3a
resulted in a global reduction in non-CG methylation [6
]. Our study is the first study to demonstrate a decrease in non-CG methylation using a well-known DNA methyltransferase inhibitor. Treatment of PC3 cells with 5-azacytidine resulted in a significant 2-fold reduction in CG methylation and a 3-fold reduction in non-CG methylation (P
< 0.0001, ). We further find that 5-azacytidine preferentially reverses only CG and CC methylation, while leaving CA and CT methylation intact. The implication of this finding is not clear but suggests CA and CT methylation may not be dependent on DNA methyltransferases that are inhibited by 5-azacytidine.
Non-CG methylation has been an area of controversy due to the possibility of incomplete bisulfite conversion [13
]. However, there is increasing evidence for the existence of non-CG methylation in mammals with the development of genome-wide techniques for analysis [6
]. In our study, analysis of a second locus, FILIP1L,
revealed much lower frequencies of non-CG methylation compared to EVX1
, a pattern consistent across both normal and cancerous cell lines. If the high frequency of non-CG methylation at EVX1
were due to incomplete conversion, then similar rates of non-conversion would have been observed across different genomic regions. One limitation of our study is that non-CpG methylation was evaluated across only two loci. However, these are biologically significant regions in cancer and demonstrate clear differences in non-CG methylation. Previous reports have found elevated non-CG methylation at functionally important promoters [21
The role of non-CG methylation in mammalian cells remains unclear. It would be expected that if non-CG methylation were maintained at a high fidelity a depletion of these sites were occur through deamination; a process that does not occur. In plants, non-CG methylation plays a role in gene silencing and genomic imprinting [3
]. Whether this occurs in cancer is unknown. Our finding of localized high levels of non-CG methylation at EVX1, and decreased non-CG methylation with inhibition, might suggest a role in expression inhibition. EVX1
is an important gene during development, a time when CG methylation levels are reduced [23
]. Alternately, low levels of non-CG methylation are seen at a locus, FILIP1L, that in a similar fashion to EVX1 undergoes reexpression with AzaC exposure. Further studies are required to fully elucidate the functional significance of non-CG methylation within promoter regions in cancer.