Periodic Reporting for period 1 - EPIDNA (Structural investigation of interplay between epigenetics, transcriptional regulation and DNA damage)
Período documentado: 2020-04-01 hasta 2022-03-31
The project’s main objective was to characterize selected structural aspects of epigenetic regulation and its coupling to DNA modification. In particular, the following questions were addressed:
- possible obstruction of DNA repair due to the presence of nucleosomes;
- direct changes in the regulation of gene expression caused by oxidative stress;
- structural response of nucleosome core particles to the chromatin remodeling machinery;
- possible functional roles of histone lipidation, a newly discovered epigenetic marker.
These questions contribute to our fundamental understanding of how genetic information is expressed, maintained, and regulated. The structural approach employed in this project provides important context for often serendipitous experimental observations, one that allows for a rational design of future experiments and therapeutic interventions.
Moreover, since the timeline of the project coincided with the COVID-19 pandemic, significant efforts and resources were redirected to address this urgent issue using the unique techniques at our disposal. These additional lines of research investigated the following questions:
- a possible evolutionary advantage of the A222V point mutation, dominant in Spain in summer 2020 and then rediscovered in a sub-strain of Delta;
- the mutational pathway followed during the virus’ zoonotic transition from bat to human;
- the differences in the structural properties conferred by the collection of mutations accumulated by the Omicron strain;
- a review of methodological and applicational advances in the computational research on COVID-19.
Here, the research performed within the project aimed to further our understanding of the driving forces behind the pandemic, understand the pharmacological strategies deployed to mitigate it, and calibrate new methods for massive, rapid and accurate investigation of the consequences of protein mutations.
- possible obstruction of DNA repair due to the presence of nucleosomes;
- direct changes in the regulation of gene expression caused by oxidative stress;
- structural response of nucleosome core particles to the chromatin remodeling machinery;
- possible functional roles of histone lipidation, a newly discovered epigenetic marker.
These questions contribute to our fundamental understanding of how genetic information is expressed, maintained, and regulated. The structural approach employed in this project provides important context for often serendipitous experimental observations, one that allows for a rational design of future experiments and therapeutic interventions.
Moreover, since the timeline of the project coincided with the COVID-19 pandemic, significant efforts and resources were redirected to address this urgent issue using the unique techniques at our disposal. These additional lines of research investigated the following questions:
- a possible evolutionary advantage of the A222V point mutation, dominant in Spain in summer 2020 and then rediscovered in a sub-strain of Delta;
- the mutational pathway followed during the virus’ zoonotic transition from bat to human;
- the differences in the structural properties conferred by the collection of mutations accumulated by the Omicron strain;
- a review of methodological and applicational advances in the computational research on COVID-19.
Here, the research performed within the project aimed to further our understanding of the driving forces behind the pandemic, understand the pharmacological strategies deployed to mitigate it, and calibrate new methods for massive, rapid and accurate investigation of the consequences of protein mutations.
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.
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.
The direct scientific results of the project bring the attention of the field to the structural aspects of epigenetic regulation, showing how local and seemingly minor modifications can have a far-reaching impact on the functional aspects of gene expression regulation and DNA repair. They will also help explain the mutational patterns observed in vivo in cancer patients, and in this way better attribute physiological outcomes to their underlying molecular causes, as well as contribute to the understanding of the impact of oxidative stress on development and genome maintenance. Ultimately, mechanistic knowledge about these functional relationships might guide therapeutic interventions or preventive measures.
From the methodological side, the project defined new standards for the massive computational investigation of the effects of nucleic acid modifications through the calculation of alchemical free energies. The workflows are based on open-source tools and easily reproducible, and additional effort will be made to ensure that they are popularized and made broadly accessible to the community. The development of a Gromacs-specific utility library (Gromologist), as well as an efficient and customizable non-equilibrium free energy tool (Crooks), add to the expected impact of the project in the long run, especially given the momentum gained from the recent collaboration with Gromacs developers.
The research on COVID-19 helped understand the mechanistic impact of the popular A222V mutation on the Spike dynamics, provided information on the thermodynamic driving forces for the shift of host during the zoonotic jump, as well as the dynamic changes conferred by the Omicron variant mutations. While such investigations are always performed post-factum, they ultimately improve our understanding of the evolutionary fitness landscape of a virus that recently underwent a zoonotic transition, a valuable contribution to the first-line reaction to future zoonotic outbreaks. By reviewing the current state-of-the-art in that respect, we also contributed to the consolidation of the novel but scattered knowledge.
From the methodological side, the project defined new standards for the massive computational investigation of the effects of nucleic acid modifications through the calculation of alchemical free energies. The workflows are based on open-source tools and easily reproducible, and additional effort will be made to ensure that they are popularized and made broadly accessible to the community. The development of a Gromacs-specific utility library (Gromologist), as well as an efficient and customizable non-equilibrium free energy tool (Crooks), add to the expected impact of the project in the long run, especially given the momentum gained from the recent collaboration with Gromacs developers.
The research on COVID-19 helped understand the mechanistic impact of the popular A222V mutation on the Spike dynamics, provided information on the thermodynamic driving forces for the shift of host during the zoonotic jump, as well as the dynamic changes conferred by the Omicron variant mutations. While such investigations are always performed post-factum, they ultimately improve our understanding of the evolutionary fitness landscape of a virus that recently underwent a zoonotic transition, a valuable contribution to the first-line reaction to future zoonotic outbreaks. By reviewing the current state-of-the-art in that respect, we also contributed to the consolidation of the novel but scattered knowledge.