Periodic Reporting for period 4 - SynarchiC (Investigating the functional architecture of microbial genomes with synthetic approaches)
Período documentado: 2022-10-01 hasta 2024-03-31
The interaction and potential causal relationships between the (3D) organisation of chromosomes, the carriers of heredity and genetic information, and the regulation of DNA metabolic processes such as gene expression, replication or DNA repair, is being actively studied. Past and recent studies have revealed multiple levels of hierarchical chromosome organisation in prokaryotes (i.e. bacteria) and eukaryotes. In most species studied to date, the overall folding of the genome is a mosaic of intertwined structural features. These structures result from a combination of factors, ranging from the basic physical properties of the DNA molecule (bending, etc.) to epigenetic modifications and the binding of proteins to specific regions. Identifying shared components of genome folding in very different species is essential for understanding how living organisms have adapted to this key question, how to organise the information carried by the DNA molecule so that it can be accessed in a timely manner when there is a change in the extracellular environment (e.g. differentiation, infection, cell cycle...). We therefore wanted to provide new insights into the mechanisms and evolution of this process in species belonging to the three domains of life. We also extended this study of genome organisation to those of a bacterium and a eukaryotic cell during an infection process, and studied the repercussions of this dramatic and stressful event on the functional organisation of the two genomes.
The aim of the SynarchiC project first consisted in exploring interplay between chromosomes 3D folding and as diverse as as replication, transcription, segregation and folding in various species, mostly budding yeast Saccharomyces cerevisiae and the bacteria Escherichia coli. To this end, we implemented techniques such as synthetic genomic assembly of large chromosome regions in order to study, in a controlled manner, the nature and molecular regulation of the predominant 3D structures (loops, domains, etc.) found in these genomes, as well as their interaction with transcription. We also explored and took advantage of the results of these studies to explore the dynamic chromosomal changes that occur during bacterial infection of a eukaryotic host.
The aim of the SynarchiC project first consisted in exploring interplay between chromosomes 3D folding and as diverse as as replication, transcription, segregation and folding in various species, mostly budding yeast Saccharomyces cerevisiae and the bacteria Escherichia coli. To this end, we implemented techniques such as synthetic genomic assembly of large chromosome regions in order to study, in a controlled manner, the nature and molecular regulation of the predominant 3D structures (loops, domains, etc.) found in these genomes, as well as their interaction with transcription. We also explored and took advantage of the results of these studies to explore the dynamic chromosomal changes that occur during bacterial infection of a eukaryotic host.
The SynarchiC project aimed at tackling questions related to the functional organization of bacterial and eukaryotic genome during the cell cycle. We exploited a variety of species (Escherichia coli, Saccharomyces cerevisiae) to investigate various aspects of chromosome 3D folding regulation, and interplay with a number of DNA metabolic processes. The work involved designing new experimental approaches involving synthetic and artificial chromosomes to address these questions. Overall, the different tasks have been completed, and for some of them led to unexpected directions, including exploration of similar questions in the underexplored domain of life archaea, or the exploitation of deep learning for genomics to explore the interplay between sequence composition and chromatin activity. The Synarchic project was organized into three main work packages, that each have led to new discoveries on our understanding of the regulation of genome folding in a variety of organisms.
Our study of how transcription affects the folding and dynamics of the Escherichia coli chromosome, published in Nature Structural & Molecular Biology (Bignaud et al., 2024), demonstrated that the activation of a single transcription pathway in a silent genome creates significant topological constraints, influencing the organisation of the entire genome. This work is following another study on the regulation of sister chromatids intermingling following replication in this species (Conin et al., 2021). In addition, our research into the role of chromosome structural maintenance complexes (SMCs) and transcription in bacterial and archaeal chromosome folding, published in Molecular Cell (Cockram et al., 2020; Yáñez-Cuna et al., 2023), has highlighted the universal nature of these regulatory mechanisms in all areas of life. We have also studied the way in which sequence composition dictates chromatin structure and function (Meneu et al. 2025), showing that DNA introduced into a new nuclear environment is processed on the basis of host sequence rules. In addition, our research into the regulation of eukaryotic chromosome 3D folding by the cohesin complex in S. cerevisiae during replication has been documented in several publications (Dauban et al., Molecular Cell, 2020 ; Bastié et al., Nature SMB, 2022 ; Garcia-Luiz et al., Nature SMB, 2019 ; Elife, 2022 ; Bastié et al., Molecular Cell, 2024). In bacterial cell cycles, we found that inactivation of Topo IV leads to significant chromosome reorganisation, with distinct roles played by Topo III, MatP and MukB in sister chromatid segregation. Finally, our exploration of genome folding in the eukaryotic amoeba Acanthamoeba castellanii during infection with Legionella pneumophila involved the development of new computational methods and the assembly of complete genomes of reference strains, revealing infection-dependent chromatin reorganisation (Matthey-Doret et al., Genome Research, 2022). Our efforts have also led to the creation of several open access programmes associated with these publications.
These works, and others, were presented at international conferences and research centers. The funding has also been instrumental in developing new technologies and computational tools, which have been essential for our research. Altogether, they represent significant advancements made in understanding chromosome dynamics, with the ERC grant enabling us to expand these approaches to new biological questions, forming the basis for our future research.
Our study of how transcription affects the folding and dynamics of the Escherichia coli chromosome, published in Nature Structural & Molecular Biology (Bignaud et al., 2024), demonstrated that the activation of a single transcription pathway in a silent genome creates significant topological constraints, influencing the organisation of the entire genome. This work is following another study on the regulation of sister chromatids intermingling following replication in this species (Conin et al., 2021). In addition, our research into the role of chromosome structural maintenance complexes (SMCs) and transcription in bacterial and archaeal chromosome folding, published in Molecular Cell (Cockram et al., 2020; Yáñez-Cuna et al., 2023), has highlighted the universal nature of these regulatory mechanisms in all areas of life. We have also studied the way in which sequence composition dictates chromatin structure and function (Meneu et al. 2025), showing that DNA introduced into a new nuclear environment is processed on the basis of host sequence rules. In addition, our research into the regulation of eukaryotic chromosome 3D folding by the cohesin complex in S. cerevisiae during replication has been documented in several publications (Dauban et al., Molecular Cell, 2020 ; Bastié et al., Nature SMB, 2022 ; Garcia-Luiz et al., Nature SMB, 2019 ; Elife, 2022 ; Bastié et al., Molecular Cell, 2024). In bacterial cell cycles, we found that inactivation of Topo IV leads to significant chromosome reorganisation, with distinct roles played by Topo III, MatP and MukB in sister chromatid segregation. Finally, our exploration of genome folding in the eukaryotic amoeba Acanthamoeba castellanii during infection with Legionella pneumophila involved the development of new computational methods and the assembly of complete genomes of reference strains, revealing infection-dependent chromatin reorganisation (Matthey-Doret et al., Genome Research, 2022). Our efforts have also led to the creation of several open access programmes associated with these publications.
These works, and others, were presented at international conferences and research centers. The funding has also been instrumental in developing new technologies and computational tools, which have been essential for our research. Altogether, they represent significant advancements made in understanding chromosome dynamics, with the ERC grant enabling us to expand these approaches to new biological questions, forming the basis for our future research.
Among the unexpected results of the project, was the fact that we explored chromosome organization regulation in the archeae domains of life, underlining shared principles with eukaryotes and prokaryotes that regulate this feature. Notably, the conserved roles of transcription and SMC complexes in regulation chromosome folding. In addition, our tools and questions have also led us to develop new synthetic genomics approaches based on decision making informed by deep learning. In collaboration we have shown that neural networks trained on a native yeast genome were capable of predicting features on DNA sequences on which they had not been trained (Meneu et al., Science, 2025). This led us to exploit these trained networks to design de novo large DNA sequences predicted to behave as desired. For example, as a proof of concept, we have designed in silico, and experimentally tested, 10 kb sequences that exhibit correct nucleosome positioning, regular GC%, and no transcription at all, when introduced into the yeast genome. This opens the way to multiple applications. We believe that this approach can provide unexpected solutions to questions hitherto limited by experimental constraints based on the ‘evolved’ genome of an host.