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DNA methylation is an important epigenetic mark that is known to induce chromatin condensation and gene silencing. We used a time-domain fluorescence lifetime measurement to quantify the effects of DNA hypermethylation on the conformation and dynamics of a nucleosome. Nucleosomes reconstituted on an unmethylated and a methylated DNA both exhibit dynamic conformations under physiological conditions. The DNA end breathing motion and the H2A-H2B dimer destabilization dominate the dynamic behavior of nucleosomes at low to medium ionic strength. Extensive DNA CpG methylation, surprisingly, does not help to restrain the DNA breathing motion, but facilitates the formation of a more open nucleosome conformation. The presence of the divalent cation, Mg²⁺, essential for chromatin compaction, and the methyl donor molecule SAM, required for DNA methyltransferase reaction, facilitate the …