The computational, applicational work within the planned epigenetic part of the project covered 4 main topics: (a) the impact of nucleobase modifications in terms of their preferred rotational and translational positioning of nucleosomes; (b) the effect of base oxidation on the sequence-specific binding of transcription factors; (c) the mechanism of bubble diffusion in complexes of nucleosomes with chromatin remodelers; (d) the potential functional role of nucleosome lipidation, a novel epigenetic modification identified recently by our collaborators. Here, novel state-of-the-art workflows were used to perform massive free energy calculations, revealing the opposite positioning effects exerted on nucleosomes by photoinduced thymine dimers (both cyclobutane dimers and 6-4 photoproducts) and selected other lesions (abasic sites, thymine glycol). Results showed among others the susceptibility of CTCF – a known chromatin topology modulator – to DNA oxidation, as well as an exceptional ability of nucleosomes to stably accommodate bulges as small as 4-5 base pairs long.
In parallel, the COVID-19 pandemic, whose start coincided with the beginning of the project, drastically affected the scientific priorities in our group. We launched or joined several research projects related to the progression of the pandemic, and our experience in alchemical free energy calculations proved critical to many of these. In brief, we identified the structural impact of the A222V “Spanish” mutation that reappeared in one Delta subvariant, investigated the zoonotic transition between the bat and human hosts, and recently uncovered the differences in conformational dynamics between the wild-type and Omicron Spike protein in the structural transition crucial to infection. Finally, we reviewed the pandemic-related efforts of the computational community in a recently published review article.
Accordingly, the experimental side of the project suffered from the pandemic-caused disruption, first due to the lockdown and involvement of the experimental lab in testing activities, and then due to delays in hiring the PhD student working on the project. Nevertheless, experimental works are underway and will soon deliver much-needed validations for the computational predictions made in the epigenetic part of the project.
On the theoretical and software side, much effort was devoted to developing better strategies for alchemical free energy calculations and automated editing of system topologies. Within the first topic, I worked on combining the idea of Weighted Ensemble sampling with non-equilibrium free energy calculations based on Crooks’ theorem; after several surprising findings, we have narrowed down the problem and have now been working on further developments in collaboration with researchers who inspired the idea. In parallel, the sustained addition of new features and capabilities contributed to the development of Gromologist, a fully functional utility library for Gromacs, and we are now working directly with Gromacs developers on better addressing the needs of the community.
