Regulation of Nucleosome Conformational Dynamics by Post-Translational Histone Modifications Studied with Single-Pair FRET

Nucleosomes form the basic unit of DNA compaction in eukaryotes. Not only do they condense the DNA, nucleosomes also play a crucial role in gene regulation: they modulate access to nucleosomal DNA for DNA-processing proteins. DNA within the nucleosome is made accessible via a combination of conformational changes caused by spontaneous fluctuations (DNA breathing), and by ATP-dependent remodeling enzymes. Both mechanisms are regulated by specific post-translational modifications to the nucleosome histones. Histone acetylation at H3K56, for example, has been shown to induce increased gene expression in vivo.To characterize the effects of specific histone modifications on conformational dynamics of individual nucleosomes, we perform single-pair FRET (spFRET) measurements. We reconstitute nucleosomes from DNA labeled with a FRET pair and either modified or unmodified histones. The modified histones are obtained using a novel genetic code expansion technique that allows for genetically defined incorporation of modified amino acids. By placing FRET labels at different positions on the nucleosomal DNA, we show that transient DNA unwrapping occurs progressively from both nucleosome ends for up to at least 40 basepairs.We follow DNA breathing dynamics of individual nucleosomes by combining spFRET and Alternating Laser Excitation (ALEX) with TIRF microscopy on immobilized nucleosomes. Alternatively, we combine spFRET and ALEX with gel electrophoresis and Fluorescence Correlation Spectroscopy (FCS) to diffusing nucleosomes. We show that a single acetylation at H3K56 increases DNA breathing of the first ∼20 basepairs at least 2-fold. Furthermore, the initial state shifts to a more unwrapped conformation. Comparison with a simple model that assumes unwrapping of the DNA in ten-basepair steps indicates that acetylation at H3K56 causes the first DNA-histone contact point to break.Using these techniques we aim to further quantify epigenetic changes in chromatin at the single-molecule level.