High resolution characterization of bacterial chromosome structuring – HiResBaCS

The HiResBaCS project proposes a dedicated strategy, based on recent advances in 1) the so-called “synthetic genome” field 49,50, 2) in SRM and 3) in statistics and modeling to enhance the resolution at which a chromosome structure can be measured and described. These tools will support a genetic deconvolution approach that should allow us to address specific structuring questions independently of environmental noise. First, we will design and assemble large DNA regions dedicated to high resolution Hi-C. The basic pattern of these redesigned chromosomal regions will be a 3C-optimized design. The pertinence of this approach has been validated in budding yeast Saccharomyces cerevisiae as described in the preliminary result section of the proposal (RK, unpublished). The redesigned region will constitute a convenient “chassis” for “plug-and-play” experiments. For instance, we will introduce gene desert(s) where structuring can be analyzed independently from gene expression. Second, we will introduce in bacteria a new labeling method, called oligopaint, which will be combined with super-resolution microscopy. This method will allow painting large (replichores or macrodomains) or small (selected plectonemes) entire regions, and characterizing their localization with respects to specific landmarks. Third, HiResBaCS will rely on genetics and molecular biology experiments to analyze chromosome folding and its consequences in various genetic backgrounds, growth conditions or cell cycle steps. Finally, HiResBaCS will rely on powerful biostatistics and mathematical modeling to interpret in quantitative framework our experimental data.

HiresbacS aims at identifying the factors involved in the formation of bacterial TADs, unveiling the molecular mechanisms of formation and regulation of TAD structure, and investigate the interplay between TAD structures and other DNA management processes such as DNA segregation, replication and gene regulation. We developed a unique, integrated strategy that will rely on genetic, synthetic biology, genomic, advanced microscopy and physical modeling. First, we characterized biophysical properties of natural plasmids and small circular DNA molecules excised from the chromosome. These experiments revealed the interplay between partition systems and nucleoid activity i.e. structuring and expression (Planchenault et al, 2020; Guilhas et al 2020). Second we investigated the interplay between gene expression and chromosome folding (a manuscript involving the 4 groups involved in Hiresbacs is in preparation). Third, we investigated the relationship between DNA topology and chromosome folding (a manuscript involving the RK and OE groups is in preparation). Fourth we developed synthetic biology, imaging and analytical tools to enhance the resolution of the definition of bacterial chromosome structuring (Muller et al 2018, Cardozo-Gizzi, Mol Cell 2019; Cardozo Gizzi, Nature Protocols, 2020, Le Gall, et al, 2017, Matthey-Doret et al. 2020, Carron et al. 2019). The findings of HiResBaCs are relevant to the study chromosome organization in bacteria and mammals, as well as to understanding the roles of DNA structure in bacterial infection and differentiation. The methods developed in HiResBaCS will, in addition, be of interest to many other fields of biology.

At short term, our objectives are to complete synthesis and integration of the synthetic chromosome region. We would like to quantitatively understand the structuring that emanates from transcription bubbles. An ANR HiresbaCS funded post doc involved in these aspects and the characterization of new chromosomal structuring features revealed by super resolution HiC will be hired during the fall 2017 in the Mozziconacci group (UPMC). We will start to investigate chromosme dynamics related to replication and segregation in B. subtilis.

Lioy VS, Cournac A, Marbouty M, Duigou S, Mozziconacci J, Espéli O, Boccard F, Koszul R.Multiscale Structuring of the E. coli Chromosome by Nucleoid-Associated and Condensin Proteins. Cell. 2018 Feb 8;172(4):771-783.e18. doi: 10.1016/j.cell.2017.12.027. Epub 2018 Jan 18.

Muller H, Scolari VF, Agier N, Piazza A, Thierry A, Mercy G, Descorps-Declere S, Lazar-Stefanita L, Espeli O, Llorente B, Fischer G, Mozziconacci J, Koszul R.
Characterizing meiotic chromosomes' structure and pairing using a designer sequence optimized for Hi-C. Mol Syst Biol. 2018 Jul 16;14(7):e8293. d

Planchenault C, Pons MC, Schiavon C, Siguier P, Rech J, Guynet C, Dauverd-Girault J, Cury J, Rocha EPC, Junier I, Cornet F, Espéli O. Intracellular Positioning Systems Limit the Entropic Eviction of Secondary Replicons Toward the Nucleoid Edges in Bacterial Cells. J Mol Biol. 2020 Feb 7;432(3):745-761.

El Sayyed H, Espéli O. Mapping E. coli Topoisomerase IV Binding and Activity Sites. Methods Mol Biol. 2018;1703:87-94.

Cardozo Gizzi AM, Espinola SM, Gurgo J, Houbron C, Fiche JB, Cattoni DI, Nollmann M.
Direct and simultaneous observation of transcription and chromosome architecture in single cells with Hi-M. Nat Protoc. 2020 Mar;15(3):840-876.

Cardozo Gizzi AM, Cattoni DI, Fiche JB, Espinola SM, Gurgo J, Messina O, Houbron C, Ogiyama Y, Papadopoulos GL, Cavalli G, Lagha M, Nollmann M.Microscopy-Based Chromosome Conformation Capture Enables Simultaneous Visualization of Genome Organization and Transcription in Intact Organisms. Mol Cell. 2019 Apr 4;74(1):212-222.e5. doi: 10.1016/j.molcel.2019.01.011.

Le Gall A, Cattoni DI, Nollmann M. Imaging of Bacterial Chromosome Organization by 3D Super-Resolution Microscopy. Methods Mol Biol. 2017;1624:253-268. doi: 10.1007/978-1-4939-7098-8_19.


Carron, L., Morlot, J. B., Matthys, V., Lesne, A., & Mozziconacci, J. (2019). Boost-HiC: computational enhancement of long-range contacts in chromosomal contact maps. Bioinformatics, 35(16), 2724-2729