Single Molecule Fluorescence Studies on Nucleosome Dynamics

Nucleosomes, as the basic packing unit of chromatin, regulate DNA accessibility and have significant influence on gene expression. Two copies of each histone protein (H2A, H2B, H3, H4) build up the protein octamer, around which approximately 150 bp of DNA are wrapped.The N-terminal tails of the histones protrude from the nucleosome; they are important for inter- and intranucleosomal interactions. Nucleosome disassembly may be forced in vitro by increasing salt concentration and may be followed by Forster resonance energy transfer (FRET) between fluorophores. We use reconstituted Xenopus laevis nucleosomes, fluorescently labeled on different positions[2] to compare the disassembly of nucleosomes containing mutated histones or modifications to the wild type.Here we focus on the dynamics of the N-tail of H3. Mutated versions of H3 labeled with Alexa488 were used to examine whether there are interactions between the labeled H3 tail and the labeled DNA or other labeled histones. First results show an interaction of the H3 tail and the DNA in vicinity of the dyad axis.The second part is about the functional relevance of a particular region of H2A. Molecular dynamic simulations on ‘tailless’ variants of H3 and H2A have suggested conformational changes affecting two arginines of H2A. Furthermore a conformational change of the protruding part of the DNA was described, which is due to internal conformational changes of H2A[3].From those observations the question arises whether these amino acids are necessary for stable octamers and/or nucleosomes, and how they influence DNA breathing, unwrapping, and stability of entire nucleosomes. To address these questions we generated recombinant H2A proteins incorporating site-specific mutations (R81A, R88A, R81AR88A, R81E, R88E, R81ER88E). Our results show a decreasing stability associated with position (Wt > R88 > R81 > R81R88) and charge (RA > RE) of the amino acids.