DNA methylation inhibitors such as 5-aza-2'-deoxycytidine (5-Aza-CdR) are 
currently used for the treatment of myelodysplastic syndrome. Although global 
DNA demethylation has been observed after treatment, it is unclear to what 
extent demethylation induces changes in nucleosome occupancy, a key determinant 
of gene expression. We use the colorectal cancer cell line HCT116 as a model to 
address this question and determine that <2% of regions demethylated by 
5-Aza-CdR treatment assume an open configuration. Consolidating our findings, we 
detect nucleosome retention at sites of global DNA methylation loss in DKO1, an 
HCT116-derived non-tumorigenic cell-line engineered for DNA methyltransferase 
disruption. Notably, regions that are open in both HCT116 cells after treatment 
and in DKO1 cells include promoters belonging to tumor suppressors and genes 
under-expressed in colorectal cancers. Our results indicate that only a minority 
of demethylated promoters are associated with nucleosome remodeling, and these 
could potentially be the epigenetic drivers causing the loss of tumorigenicity. 
Furthermore, we show that the chromatin opening induced by the histone 
deacetylase inhibitor suberoylanilide hydroxamic acid has strikingly distinct 
targets compared with those of 5-Aza-CdR, providing a mechanistic explanation 
for the importance of combinatorial therapy in eliciting maximal de-repression 
of the cancer epigenome.