Abstract Aberrant epigenetic changes which include alterations in DNA methylation, nucleosome positioning and histone modifications have been studied extensively in the context of tumorigenesis. The role of DNA methylation in the organization of genome-wide chromatin structure and its interaction with other epigenetic modifiers in cancer, however, remains unclear. Here, we use the Nucleosome Occupancy and Methylation whole-genome sequencing (NOMe-seq) assay to compare a colorectal cancer cell line, HCT116, with its derivatives that carry homozygous deletions of DNMT3B and are hypomorphic for DNMT1, DKO1, to examine the role of DNA methylation in the functional organization of the cancer epigenome. Upon the global loss of DNA methylation, we observe a widespread chromatin remodeling where specifically, DKO1 cells gain nucleosome depleted regions (NDRs) and/or well-phased nucleosome that are not present in the parent HCT116 cells. Interestingly, this genome-wide nucleosome repositioning in DKO1 cells is accompanied by significant histone modification changes and low level expression increase only in CpG islands (CGI), where promoters may gain permissive histone marks H2A.Z, H3K4me3 and H3K4me1 as well as the polycomb repressive mark, H3K27me3. Non-CGI promoters on the other hand, do not gain permissive or repressive histone modifications despite dramatic nucleosome reorganization, which suggests that distinct mechanisms regulate the equilibrium between DNA methylation, nucleosome and gene expression in CGI and non-CGI promoters. Taken together, our study demonstrates the cross-talk between epigenetic mechanisms wherein DNA methylation may directly control the organization of chromatin architecture. Citation Format: Fides D. Lay, Yaping Liu, Theresa K. Kelly, Heather Witt, Adam Blattler, Peggy J. Farnham, Ben P. Berman, Peter A. Jones. The effects of the global loss of DNA methylation on the functional organization of the epigenome. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4780. doi:10.1158/1538-7445.AM2014-4780