Effects of CpG Methylation on the Structure and Assembly of a Nucleosome

Abstract

We studied the mechanism of nucleosome assembly with a histone chaperon Nap1 and the effects of CpG methylation on the structure and the assembly of nucleosomes with fluorescence techniques. Based on single molecule and ensemble fluorescence measurements, we found that CpG methylation induces overwrapping of nucleosomal DNA accompanied by a topology change. Real-time monitoring of nucleosome assembly with the Selex 601 sequence and Nap1 revealed that nucleosome assembly takes place largely in four steps. Reduced kinetic rates for DNA wrapping during the assembly upon CpG methylation strongly suggest that DNA becomes rigid with methylated CpG dinucleotides. Free energy profiles of the nucleosome assembly process were also constructed based on the results. According to the profiles, all the intermediate states are kinetically stabilized but thermodynamically destabilized upon CpG methylation. The decreased kinetic rates upon CpG methylation may be due to the elevated transition state energy caused by the restricted conformational space of the rigidified DNA in the transition states. Based on these results, we propose a hypothesis where rigidified DNA upon CpG methylation inhibits nucleosome disassembly by destabilizing the transition states.