Periodic Reporting for period 4 - ChromatinRemodelling (Single-Molecule And Structural Studies Of ATP-Dependent Chromatin Remodelling)
Reporting period: 2021-09-01 to 2022-08-31
The packaging of DNA into chromatin regulates a wide range of vital processes that depend on direct access to the genetic information. One class of enzymes that can regulate chromatin structure is the ATP-dependent chromatin remodelling enzymes (remodellers), and their functional impairment has been linked to various cancers and multisystem developmental disorders. Dissecting the functional roles of remodellers and developing therapeutics to fight disease states linked to aberrant remodelling require an in-depth mechanistic understanding. Many remodellers alter chromatin structure by translocating nucleosomes on DNA, yet understanding the translocation mechanism remains a major challenge in the field. Furthermore, how remodellers are regulated in adaptation to various functions is not well understood. We have addressed these longstanding questions in two specific aims.
Aim I: We have developed powerful single-molecule imaging methodologies to monitor, in real time, DNA movements during nucleosome remodelling.
Aim II: We have used structural approaches to investigate the vital regulation of remodelling by features of the nucleosome (its histone tails or linker DNA) and by accessory domains of the remodeller.
In summary, we have examined the nucleosome remodelling mechanisms and regulation of ATP-dependent chromatin remodelling enzymes (remodellers). We have devised much-needed and previously unavailable single-molecule imaging strategies and combined them with structural and biochemical approaches to gain key mechanistic insights into the vital process of chromatin remodelling that had been impossible to obtain hitherto. A deeper mechanistic understanding of chromatin remodelling is expected to reveal links between remodeller dysfunction and diseases. In particular the new insights into the regulation of disease-related remodellers that we generated during this project may open up new horizons for developing therapeutic intervention strategies.
Aim I: We have developed powerful single-molecule imaging methodologies to monitor, in real time, DNA movements during nucleosome remodelling.
Aim II: We have used structural approaches to investigate the vital regulation of remodelling by features of the nucleosome (its histone tails or linker DNA) and by accessory domains of the remodeller.
In summary, we have examined the nucleosome remodelling mechanisms and regulation of ATP-dependent chromatin remodelling enzymes (remodellers). We have devised much-needed and previously unavailable single-molecule imaging strategies and combined them with structural and biochemical approaches to gain key mechanistic insights into the vital process of chromatin remodelling that had been impossible to obtain hitherto. A deeper mechanistic understanding of chromatin remodelling is expected to reveal links between remodeller dysfunction and diseases. In particular the new insights into the regulation of disease-related remodellers that we generated during this project may open up new horizons for developing therapeutic intervention strategies.
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).
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).
Chromatin remodelling enzymes play essential roles in a variety of fundamentally important biological processes. Our novel approaches to study the remodelling of individual nucleosomes in real time provide previously inaccessible insights into nucleosome remodelling dynamics. These measurements, in combination with structural and biochemical approaches help dissect the mechanisms underlying the intrinsic activities of the chromatin remodelling enzymes and elucidate how remodellers are regulated.
Our single-molecule assays allow the first direct and real-time observation of how DNA movements at different sites of the nucleosome are coordinated. These methods not only will facilitate chromatin remodelling studies, but can also be applied to the investigation of many other important nucleosome-related processes.
Our multidisciplinary approach has helped to bridge the gap between detailed structural information and dynamic functional assays, thereby opening up the possibility to more completely understand the conformational landscapes of nucleosomes and chromatin remodelling enzymes.
Developing novel therapies to combat diseases related to remodeller dysfunction requires a mechanistic understanding of the biophysical and structural principles underlying chromatin remodelling. Our highly interdisciplinary approach has allowed us to derive critical mechanistic insight into the regulation of disease-related remodellers. Importantly, our work immediately suggested that targeting the oncogenic remodeller ALC1 could provide an alternative strategy for treating homologous recombination deficient cancers. Our studies have therefore opened up new horizons for developing therapeutic intervention strategies.
Our single-molecule assays allow the first direct and real-time observation of how DNA movements at different sites of the nucleosome are coordinated. These methods not only will facilitate chromatin remodelling studies, but can also be applied to the investigation of many other important nucleosome-related processes.
Our multidisciplinary approach has helped to bridge the gap between detailed structural information and dynamic functional assays, thereby opening up the possibility to more completely understand the conformational landscapes of nucleosomes and chromatin remodelling enzymes.
Developing novel therapies to combat diseases related to remodeller dysfunction requires a mechanistic understanding of the biophysical and structural principles underlying chromatin remodelling. Our highly interdisciplinary approach has allowed us to derive critical mechanistic insight into the regulation of disease-related remodellers. Importantly, our work immediately suggested that targeting the oncogenic remodeller ALC1 could provide an alternative strategy for treating homologous recombination deficient cancers. Our studies have therefore opened up new horizons for developing therapeutic intervention strategies.