Dynamics of Histone H3 Tails in Mononucleosomes Studied by Single-Molecule FRET and MD Simulations

Abstract

The nucleosome, consisting of two copies of each of the four histone proteins H2A, H2B, H3, H4, and a 147bp long DNA, is the basic unit of chromatin compaction and key to gene regulation. The N-terminal tails of the four core histone proteins and the C-terminal tail of H2A are protruding from the core particle in a disorder manner. Even though, the nucleosome has been widely studied in the field of epigenetics, surprisingly little is known about the actual conformation of the tails and their dynamics. Here we focus on the conformation and dynamics of the H3 N-terminal tail at various NaCl concentrations and linker DNA length in experiments and MD simulations. Salt dependent conformational changes were measured by FRET with reconstitute Widom 601 mononucleosomes with a DNA length of 170 bp, 190 bp or 210 bp. Nucleosomes were labeled on the H3 tail at position K9 and at various positions on the intranucleosomal DNA or linker DNA. Both, bulk experiments using microplate scanning FRET spectroscopy (µpsFRET), and single molecule experiments using alternating laser excitation (ALEX) or multidimensional confocal fluorescence spectroscopy with pulsed interleaved excitation (MFD-PIE) were performed. Our results confirm the previously described binding preference of the H3 tail towards the linker DNA at low salt concentrations as well as an influence of the linker DNA length on the H3 tail:DNA interaction. At higher salt concentrations (> 600 mM), when nucleosome disassembly occurs, interaction of the H3 tail with the linker DNA is disrupted and interaction with the inner DNA gyre is favorized. The designed constructs enable us for the first time to our knowledge to monitor conformational changes of the H3 tail, which underpins the proposed dynamic nature of the H3 tail.