Combining molecular dynamics simulations and scoring method to computationally model ubiquitylated linker histones in chromatosomes

The chromatin in eukaryotic cells plays a fundamental role in all processes during a cell’s life cycle. This nucleoprotein is normally tightly packed but needs to be unpacked for expression and division. The linker histones are critical for such packaging processes and while most experimental and simulation works recognize their crucial importance, the focus is nearly always set on the nucleosome as the basic chromatin building block. Linker histones can undergo several modifications, but only few studies on their ubiquitylation have been conducted. Mono-ubiquitylated linker histones (HUb), while poorly understood, are expected to influence DNA compaction. The size of ubiquitin and the globular domain of the linker histone are comparable and one would expect an increased disorder upon ubiquitylation of the linker histone. However, the formation of higher order chromatin is not hindered and ubiquitylation of the linker histone may even promote gene expression. Structural data on chromatosomes is rare and HUb has never been modeled in a chromatosome so far. Descriptions of the chromatin complex with HUb would greatly benefit from computational structural data. In this study we generate molecular dynamics simulation data for six differently linked HUb variants with the help of a sampling scheme tailored to drive the exploration of phase space. We identify conformational sub-states of the six HUb variants using the sketch-map algorithm for dimensionality reduction and iterative HDBSCAN for clustering on the excessively sampled, shallow free energy landscapes. We present a highly efficient geometric scoring method to identify sub-states of HUb that fit into the nucleosome. We predict HUb conformations inside a nucleosome using on-dyad and off-dyad chromatosome structures as reference and show that unbiased simulations of HUb produce significantly more fitting than non-fitting HUb conformations. A tetranucleosome array is used to show that ubiquitylation can even occur in chromatin without too much steric clashes. Author summary In eukaryotic cells the linker histones play a crucial role in the formation of higher order nucleoprotein complex of DNA, especially for the arrangement of the nucleosomes. Histones can undergo several modifications, but modification of a linker histone with a single udiquitin (mono-ubiquitylation) remains one of the least understood epigenetic modifications. One reason is the inaccessibility of homogeneously modified linker histones for experimental methods, which are crucial for distinct studies. We combine molecular dynamics simulations with machine learning-based approaches to study the influence of mono-ubiquitylation in linker histones on DNA interaction and their ability to form higher order chromatin structures. We were able to determine the probable states in six differently linked histone-ubiquitin complexes via accelerating classical molecular dynamics simulations and using advanced state characterization techniques. As it is computationally unfeasible to simulate the whole chromatosome with different modified histones we developed efficient geometric scoring technique to select biologically relevant structures of all six mono-ubiquitylated linker histone that can bound to nucleosome.