Abstract
Nucleosomes form the basic unit of DNA compaction in eukaryotes. Besides condensing the DNA, nucleosomes play a crucial role in gene regulation by modulating access to the nucleosomal DNA for DNA-processing proteins. Accessibility of DNA within the nucleosome can be realized by spontaneous unwrapping (DNA breathing), and by ATP-dependent remodeling enzymes. Both mechanisms are regulated by specific post-translational modifications to the nucleosome histones. We applied single-pair FRET measurements to characterize the effect of specific histone modifications on DNA breathing dynamics in individual nucleosomes. DNA labeled with a FRET pair was used to reconstitute nucleosomes with three types of histones: native chicken erythrocyte histones, recombinant unmodified histones, and recombinant histones acetylated at H3K56. Using alternating laser excitation to select for the correct label stoichiometry, in combination with both widefield TIRF microscopy on immobilized nucleosomes and FCS on freely diffusing nucleosomes, we were able to quantify DNA breathing dynamics at timescales ranging from milliseconds to minutes. Uncovering the effect of histone modifications on the dynamic behavior of single nucleosomes provides insight in the physical mechanisms underlying gene regulation.View Large Image | View Hi-Res Image | Download PowerPoint Slide
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