RESUMO
DNA compaction and accessibility in eukaryotes are governed by nucleosomes and orchestrated through interactions between DNA and DNA-binding proteins. Using QuantAFM, a method for automated image analysis of atomic force microscopy (AFM) data, we performed a detailed statistical analysis of structural properties of mono-nucleosomes. QuantAFM allows fast analysis of AFM images, including image preprocessing, object segmentation, and quantification of different structural parameters to assess DNA accessibility of nucleosomes. A comparison of nucleosomes reconstituted with and without linker histone H1 quantified H1's already described ability of compacting the nucleosome. We further employed nucleosomes bearing two charge-modifying mutations at position R81 and R88 in histone H2A (H2A R81E/R88E) to characterize DNA accessibility under destabilizing conditions. Upon H2A mutation, even in presence of H1, the DNA opening angle at the entry/exit site was increased and the DNA wrapping length around the histone core was reduced. Interestingly, a distinct opening of the less bendable DNA side was observed upon H2A mutation, indicating an enhancement of the intrinsic asymmetry of the Widom-601 nucleosomes. This study validates AFM as a technique to investigate structural parameters of nucleosomes and highlights how the DNA sequence, together with nucleosome modifications, can influence the DNA accessibility.
