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Investigating the functional architecture of microbial genomes with synthetic approaches

Periodic Reporting for period 2 - SynarchiC (Investigating the functional architecture of microbial genomes with synthetic approaches)

Reporting period: 2019-10-01 to 2021-03-31

The interplay between the (3D) organization of chromosomes, bearer of heredity and genetic information, and the regulation of DNA metabolic processes such as gene expression, replication or DNA repair, are actively investigated. Past and recent work have revealed multiple levels of chromosomal hierarchical organization in prokaryotes (i.e. bacteria) and eukaryptes. The overall folding of the genome is, in most species studied so far, 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.) up to epigenetic modifications and binding of proteins to specific regions. The SynarchiC project aims at tackling open questions related to the functional organization of genomes, to draw generic principles from experiments and observations made on distant prokaryotic and eukaryotic microorganisms.
Identifying the shared components regarding genome folding in very different species is key to our understanding of how living organisms adapted to this key question, how to organize the information carried by the DNA molecule so it can be accessed in a timely manned upon change in the extracellular environment (e.g. differentiation, infection, physical changes, etc.) We therefore want to bring new insights on the mechanisms and the evolution of this process, as many proteins involved, such as cohesins, are evolutionary conserved from bacteria to mammals. We also extended this investigation of genomes organization to those of a bacteria and a eukaryotic cell during an infection process, and investigate the repercussion of this dramatic and stressful event on the functional organization of both genomes.
The aim of the SynarchiC project is therefore to unveil the interplay between replication, transcription, segregation and chromosome folding in different species, and how these processes are affected during drastic environmental changes (e.g. infection events). We intended to do so by implementing techniques such as the synthetic genomic assembly of large chromosomal regions to investigate in a controlled manner the nature and regulation of the prevalent 3D structures (loops, domains, etc.) found in bacterial and yeast genomes and their interplay with transcription. By reaching at a better characterization of these features, we hope to be able to perform an in-depth analysis of the influence of the passing replication fork on their maintenance, and investigate the topological constraints exerted on the replicating chromosome. We will in parallel exploit budding yeast both as a tool and as a model organism to investigate the fine organization of its genome during replication and anaphase, and its impact on individualization and segregation. Results from these studies will be integrated to study chromosomal dynamic changes occurring during a bacterial infection of a eukaryotic host.
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. To do so, we developped new tools and methods and combined them with established systems to investigate chromosome folding and its consequences on compaction and DNA repair in budding yeast Saccharomyces cerevisiae. We notably recently showed that in budding yeast the structural of maintenance of chromosome complex called cohesin compacts chromosome arms during replication and metaphase stages of the cell cycle. We further show that this compaction occurs through DNA looping, in a replication independent manner, and characterized the influence of several regulatory proteins on the regulation of loop sizes. Interestingly, the similarities between yeast and human loop regulatory pathways show that yeast is an amenable model to further understand the regulation of this highly conserved machinery. It also shows that the same molecular mechanisms, i.e. chromatin loop formation and regulation, has been maintained over evolution but used to insure different functions. In mammals, the loops appear to play roles in the regulation of gene expression in interphase nuclei, while in budding yeast, the loops are only present along replicating/mitotic chromosomes. We further tackled the influence of these chromatin loops on the regulation of DNA repair, showing that the compaction during mitosis restrain the ability for a DNA double-strand break to be repaired with a distant region. This provides new perspective regarding the maintenance of genome stability, by showing that the repair process is influenced by not only the compaction of the chromosome, but also by the sizes of the loops mediated by the cohesin complex.
In parallel to these studies, we continued to develop artificial and synthetic approaches to investigate the folding of these eukaryotic yeast chromosomes. We also developped a powerful tool, chromosight, to identify and quantify the strenght of any structural features in the contact maps of genomes generated using the genomic approach called "capture of chromosome conformation".

In addition to this work on eukaryote, we pursued our exploration of the regulation of chromosomes of the bacteria Escherechia coli. We identified new structures that accumulate along the circular chromosome of E. coli in the absence of the Topoisomerase IV. This topoisomerase, which roles is to promote the passing of a DNA molecule through another, is responsible for the untangling of intermingled DNA molecules that are formed during the replication of the chromosome and are essential in chromosome segregation. We show that the structures that appear in the absence of TopoIV are dependent on other well-known proteins that regulates the architecture of this bacterial genomes. Altogether, these results suggest that unresolved precatenanes are “pulled” toward a hub where they physically accumulate, to be decatenated prior to segregation. We also investigated the role of transcription on the local folding of the DNA fiber, thanks to the implementation of artificial, inducible systems. These works, which are described into articles submitted for publication, bring new insights on the maintenance of chromosome compaction, and how the genome copes with intermingling DNA molecules generated during the cell cycles. These results are likely to resonnate in what is observed in other species as well, given the ubiquitous nature of the mechanisms investigated.

Finally, we are investigating the genomes of Legionella pneumophilia and its natural host the amoeba Acanthamoeba castellanii. Certain pathogenic bacteria have evolved the ability to invade eukaryotic cells and to replicate intracellularly to escape, to some extent, immune defences of the host organisms, although at the expense of surviving in a hostile, nutrient poor, and toxic environment. This process is accompanied by dramatic modifications of the metabolism of both the bacteria and the host: recent work shows that some bacteria improve their growth conditions by redirecting the host’s metabolism, or by inducing directly or indirectly chromatin epigenetic modifications. L. pneumophila is the causative agent of a severe pneumonia (Legionnaires’ disease), which is a considerable burden for public health due to sporadic, epidemic and nosocomial infections (mortality rate 10-30%). L. pneumophila is a paradigm to study host-pathogen interactions and a model organism to analyse intracellular bacterial infections. Once L. pneumophila is reaching the lungs, it is engulfed by macrophages that entrap the bacterium in a vacuole called phagosome. Within this compartment, the bacterium replicates to high numbers. Intracellular multiplication results in the host cell death and the release of bacteria into the environment. Infection of the ubiquitous A. castellanii amoeba proceeds similarly. We tackled wether genome organization can bring new insights on these metabolic changes in both hosts and pathogens during the time course of an infection. First, we had to assemble chromosome-lenght scaffolds of the amoaba A. castellanii, an ubiquitous and model species that suprisingly is not fully sequenced. We generated new reference genomes for two strains of A. castellanni and proceed with an infection experiment. We generated gene expression and 3D genome maps of these species during infection, that were investigated using the program chromosight described above. We identified new chromatin loops and domains in the genome of the amoaba, that are now described in a publication to be submitted.
While investigating chromosome folding in prokaryotic and eukaryotic microorganisms, we wondered whether the observations we made in these domain of life were also valid in the archaea kingdom. We therefore recently applied a protocol that captured the average 3D organization of the genome of different archaea species, unveiling their folding into DNA loops, and self-interacting domains. These structures appear regulated by transcription and a complex called cohesin, similarly to the mechanisms at play in bacteria and yeast (as well as in most if not all larger eukaryotic genomes investigated so far). These results show the ubiquitous nature of the regulation of chromosome folding in all three domains of life, and have just been published in Molecular Cell (Cockram et al., 2020).