We have made substantial progress in terms of implementing both aims of the action. Regarding Aim I, my laboratory has developed, in collaboration with Greg Bowman at Johns Hopkins University, a new and simple method for selectively and asymmetrically fluorophore-labelling nucleosomes (Levendosky et al., eLife 2016). This enabled my group to carry out the first bona fide three-colour single-molecule FRET measurements on the nucleosome. For the first time, we could directly examine the real-time coordination of remodeller-induced DNA movements on both sides of the nucleosome, thereby overcoming a blindspot that has stymied a more detailed mechanistic understanding of chromatin remodelling. Our work (Sabantsev et al., Nature Communications 2019) described how during sliding by Chd1 and SNF2h remodellers, DNA is shifted discontinuously, with movement of entry-side DNA preceding that of exit-side DNA. I have presented these results at several international conferences, including the 2019 workshop on Single Molecule Biophysics in Aspen, Colorado and the 2018 Telluride workshop on Chromatin Structure and Dynamics. Insights gleaned from our single-molecule imaging approaches complement a recent flurry of important structural, biochemical, and biophysical work on chromatin remodelling from many research groups. Together, these data allowed me to summarize a core mechanistic framework explaining how nucleosomes are actively repositioned throughout the genome (Bowman and Deindl, Science 2019).
The measurement throughput in our 3-color experiments was limited, and the quantitative dissection of complex dynamics over multiple sequential turnovers of the remodelling enzyme remained challenging. To address these issues, we developed a new method for controlling NTP-driven reactions in single-molecule experiments via the local generation of NTPs (LAGOON) that markedly increases the measurement throughput and enables single-turnover observations (Sabantsev et al., featured on the cover of Nature Chemical Biology 2022). We have recently disseminated this new method at various national and international conferences, for example at the 2022 GRC “Chromatin structure and function” in Barcelona or the 2022 Dutch Chromatin Meeting in Leiden, where I was an invited keynote speaker. We have also leveraged our 3-colour single-molecule fluorescence imaging approaches to study sequence-specific DNA binding proteins in the regulation of gene expression (Marklund et al., Nature 2020 and Marklund et al., Science 2022). Moreover, fluorescence-based approaches similar to the ones developed within the framework of Aim I have synergistically facilitated additional research output from my group (e.g. Romilly et al., PNAS 2019).
In the framework of Aim II, we have successfully applied an integrative structural biology approach in combination with biochemical and cell-based approaches to investigate how the macro domain of the cancer-associated chromatin remodelling enzyme ALC1 regulates its nucleosome translocation activity (Lehmann et al., Molecular Cell 2017). We further showed, in collaboration with Simon Boulton’s group, that nucleosome remodelling by ALC1 is required to access occluded DNA lesions within nucleosomes (Hewitt et al., Molecular Cell 2021). This work immediately suggested that targeting ALC1 alone or in combination with PARP inhibitors could provide an alternative strategy for treating homologous recombination deficient cancers. Finally, we have continued our mechanistic studies of the ALC1 remodeller to further understand its regulation in a DNA damage context (Bacic et al., Cell Reports 2020; Bacic et al., eLife 2021).
