
Nucleosomes are the basic packaging unit of chromatin compaction. They have significant influence on gene expression and regulate DNA accessibility. Two copies of each core histone protein (H2A, H2B, H3, H4) build up the protein octamer, around which 147 bp of DNA are wrapped. We study the disassembly process of reconstituted Xenopus laevis nucleosomes containing mutated histones or modifications in comparison to the wild type. In vitro nucleosome disassembly may be forced by increasing salt concentration and followed by Förster resonance energy transfer (FRET) between fluorophores. Therefore, nucleosomes were fluorescently labeled at various positions of the DNA and the histone proteins. This tool set was used to analyze the functional relevance of the alpha-3- domain of H2A, which might take part in a long range interaction with the N-terminal tail of H3. This was first observed in molecular dynamic simulations on 'tailless' variants of H3 and H2A suggesting conformational changes affecting two arginines of H2A (R81 and R88). The altered conformation of those amino acids lead to conformational changes of the DNA exit arms and to an altered electrostatic environment in vicinity of the alpha-3-domain of H2A. To investigate the influence of the electrostatic environment of this region on DNA breathing, unwrapping, and stability of entire nucleosomes we generated recombinant H2A proteins incorporating site-specific mutations (R81A, R88A, R81AR88A, R81E, R88E, R81ER88E). Our results show a decrease in stability associated with position (Wt > R88 > R81 > R81R88) and charge (RA > RE) of the introduced amino acids. Although the sequence of the nucleosomal disassembly steps remains, the distribution of the intermediate states is affected in the same manner. Furthermore, we developed a labelling strategy to analyze the dynamics and intranucleosomal interactions of the N-terminal tail of H3. Combining this with our H2A-mutants or other modified histones will allow the analysis of long range effects on the H3-tail dynamics and intranucleosomal interactions.

Nucleosomes are the basic packaging unit of chromatin compaction. They have significant influence on gene expression and regulate DNA accessibility. Two copies of each core histone protein (H2A, H2B, H3, H4) build up the protein octamer, around which 147 bp of DNA are wrapped.

We study the disassembly process of reconstituted Xenopus laevis nucleosomes containing mutated histones or modifications in comparison to the wild type. In vitro nucleosome disassembly may be forced by increasing salt concentration and followed by Förster resonance energy transfer (FRET) between fluorophores. Therefore, nucleosomes were fluorescently labeled at various positions of the DNA and the histone proteins. This tool set was used to analyze the functional relevance of the alpha-3- domain of H2A, which might take part in a long range interaction with the N-terminal tail of H3. This was first observed in molecular dynamic simulations on 'tailless' variants of H3 and H2A suggesting conformational changes affecting two arginines of H2A (R81 and R88). The altered conformation of those amino acids lead to conformational changes of the DNA exit arms and to an altered electrostatic environment in vicinity of the alpha-3-domain of H2A. To investigate the influence of the electrostatic environment of this region on DNA breathing, unwrapping, and stability of entire nucleosomes we generated recombinant H2A proteins incorporating site-specific mutations (R81A, R88A, R81AR88A, R81E, R88E, R81ER88E). Our results show a decrease in stability associated with position (Wt > R88 > R81 > R81R88) and charge (RA > RE) of the introduced amino acids. Although the sequence of the nucleosomal disassembly steps remains, the distribution of the intermediate states is affected in the same manner.

Furthermore, we developed a labelling strategy to analyze the dynamics and intranucleosomal interactions of the N-terminal tail of H3. Combining this with our H2A-mutants or other modified histones will allow the analysis of long range effects on the H3-tail dynamics and intranucleosomal interactions.