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A chromosomal loop anchor mediates bacterial genome organization

Nature Genetics volume 54, pages 194–201 (2022)Cite this article

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Abstract

Nucleoprotein complexes play an integral role in genome organization of both eukaryotes and prokaryotes. Apart from their role in locally structuring and compacting DNA, several complexes are known to influence global organization by mediating long-range anchored chromosomal loop formation leading to spatial segregation of large sections of DNA. Such megabase-range interactions are ubiquitous in eukaryotes, but have not been demonstrated in prokaryotes. Here, using a genome-wide sedimentation-based approach, we found that a transcription factor, Rok, forms large nucleoprotein complexes in the bacterium Bacillus subtilis. Using chromosome conformation capture and live-imaging of DNA loci, we show that these complexes robustly interact with each other over large distances. Importantly, these Rok-dependent long-range interactions lead to anchored chromosomal loop formation, thereby spatially isolating large sections of DNA, as previously observed for insulator proteins in eukaryotes.

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Fig. 1: Identification of DNA associated with large complexes in B. subtilis.
Fig. 2: Rok clusters have more high-affinity Rok-binding motifs.
Fig. 3: Rok clusters specifically interact with each other.
Fig. 4: Rok clusters interact within superclusters.
Fig. 5: Verification of interaction between Rok clusters.
Fig. 6: Interaction between Rok clusters impacts global chromosomal architecture.
Fig. 7: Interaction between Rok clusters alters local chromosomal architecture and transcription.

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Data availability

All raw and processed sequencing datasets generated during this study can be accessed at Gene Expression Omnibus repository under accession number GSE144475.

Code availability

All source code used in this study has been published before and is referenced in the Methods section.

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Acknowledgements

We thank R. Dame and F. Z. Rashid for providing critical input on Hi-C protocol, S. van Leeuwen (MAD, University of Amsterdam) for providing excellent sequencing services and N. Vischer for help with FROS image analysis. This work was funded by EMBO, ALTF 936–2016 (G.D.), European Commission MCSA-IF grant no. 749510 (G.D.), Netherlands Organization for Scientific Research (NWO) VENI grant-VI.VENI.192.103 (G.D.) and Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy EXC-2181/1–390900948, the Heidelberg STRUCTURES Cluster of Excellence (D.W.H.).

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  1. Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, the Netherlands

    Gaurav Dugar & Leendert W. Hamoen

  2. Institute for Theoretical Physics, Heidelberg University, Heidelberg, Germany

    Andreas Hofmann & Dieter W. Heermann

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Contributions

G.D. conceived the project, designed and performed all experiments (including Hi-C library preparation, analysis and visualization), analyzed data and wrote the manuscript. A.H. and D.W.H. performed Hi-C data analysis and visualization, and provided input on manuscript preparation. L.W.H. conceived the project, analyzed data and wrote the manuscript.

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Correspondence to Gaurav Dugar or Leendert W. Hamoen.

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Extended data

Extended Data Fig. 1 Sedimentation based DNA coverage maps and identification of Rok associated large DNA complexes.

a) Illustration of the expected sedimentation based DNA coverage maps. b) DNA coverage maps from the top fraction of wild type (black) and Δrok (orange) strains. The ratio of top fraction coverage plots (Δrok/wt) shows the Rok clusters as peaks (grey). DNA coverage of DNA obtained from the dense fraction is shown in cyan. Rok clusters are defined at sites where both local minima and local maxima are observed in DNA coverage of top fraction and dense fraction, respectively (Supplementary Table 3). Rok ChIP data in red is also shown along the coverage files.

Extended Data Fig. 2 Rok multimerization is essential for interaction between Rok clusters.

a) Normalized Hi-C contact maps of wild type, rok deletion (Δrok), rok complementation (Δrok + rok), complementation with rok mutant with 45 aa truncation from the N-terminus (Δrok + rok45) and complementation with rok mutant with 95 aa truncation from the N-terminus (Δrok + rok95). The interaction between Rok clusters 1 and 6 is shown in the inset for each strain. b) Interaction between Rok terminus supercluster in the wild type and the different rok mutant strains. The region shown in b) is marked in the Hi-C map of wild type strain in a).

Extended Data Fig. 3 Inhibition of replication promotes Rok mediated chromosomal loop formation in exponential phase.

Normalized Hi-C contact maps of wild type strain at exponential phase after treatment with the replication inhibitor hydroxyurea (1 mg/ml). The interaction between Rok clusters 1 and 6 is shown in the inset. Hydoxyurea shows some inhibition of the SMC complex (reduced contacts in the secondary vertical diagonal showing juxtaposition of the two chromosome arms), presumably since this complex traverses from the origin to the terminus and encounters arrested replisomes.

Extended Data Fig. 4 Virtual 4 C analysis of Rok clusters at the origin supercluster.

Interactions of Rok clusters 6 and 7 with the whole genome during stationary phase in wild type (wt) and Δrok strains. Rok clusters are marked using grey bars. DNA coverage of top fraction obtained from the wild type strain is shown as dotted line.

Extended Data Fig. 5 Virtual 4 C analysis of Rok clusters at the terminus supercluster.

a) Interactions of Rok cluster 2 with the whole genome during stationary phase in wild type and Δrok strains. Rok clusters are marked using grey bars. b) Interactions of a Rok binding site (located between Rok cluster 2 and 3, near yonX gene) with the whole genome during stationary phase in wild type and Δrok strains. This site was found to interact with both Rok cluster 2 and 3 (see Fig. 4b) and is recruited to the Rok terminus supercluster during stationary phase. Rok ChIP data1 (orange) at the terminus supercluster is shown below. The Rok binding site is marked using green bar. DNA coverage of top fraction obtained from the wild type strain is shown as dotted line.

Extended Data Fig. 6 Three other Rok ChIP sites interact with the Rok origin supercluster.

a) Normalized Hi-C contact maps of wild type and Δrok strains near Rok cluster 1 at stationary phase. Rok ChIP data (red) is also shown along the genome below highlighting the other Rok binding sites as green dots. b) Difference plot shows Rok dependent interaction of three other Rok binding sites (green dots) with Rok clusters (1, 6,7 and 8) from the origin supercluster. c) Illustration shows association of Rok clusters 1,6,7 and 8 to form the origin supercluster and their interaction with the three nearby Rok-binding sites recruited to the origin supercluster at stationary phase.

Extended Data Fig. 7 Relocated Rok cluster 8 (RC8) interacts with other Rok clusters.

a) Location of Rok clusters in the wild type and modified genomes. RC8 was deleted and complemented at an ectopic locus (amyE) within the origin supercluster in both sense (+RC8S) and reverse (+RC8R) orientation. b) Virtual 4 C analysis to determine the interactions of the amyE locus (containing the complemented RC8) with the whole genome during stationary phase in ΔRC8 and the complemented strains. Rok clusters are marked using grey bars and RC8 complementation at the amyE locus is marked using a green bar. c) Screenshot showing relative cDNA reads (RNA-seq data) mapped to RC8 in the wild type, RC8 deletion and the RC8 complementation strains (without yybN promoter). The transcription start site and the terminator around the yybN gene obtained from SubtiWiki is marked in the annotation below. d) Normalized Hi-C contact maps and difference plots of wild type and the RC8 mutant strains near the amyE complementation locus at stationary phase. The genomic regions shown is boxed in a).

Extended Data Fig. 8 SMC complex mediates Rok cluster interactions.

a) Circular representation of DNA coverage from top fraction of differential sedimentation assay (wild type coverage from Fig. 1) after smoothing. The other three Rok binding sites which were found to interact with the origin supercluster (see Extended Data Fig. 6) are marked using an asterisk. b) Normalized Hi-C contact maps of wild type (wt) and ΔscpB strains along with the difference plot at stationary growth phase in minimal media (SMM). The SMC protein forms a homodimer, and together with the kleisen protein ScpA and the kite protein ScpB it forms the SMC complex. Deletion of scpB inactivates the SMC-complex. The interaction between Rok clusters 1 and 6 is shown in the inset for both strain. The ΔscpB strain is only viable when grown in minimal medium. However, Rok cluster are also formed in wild type strain during stationary growth phase in minimal medium (c).

Extended Data Fig. 9 Role of DnaA in Rok mediated chromosomal loop formation.

a) Normalized Hi-C contact maps of wild type and dnaA deletion strains at exponential phase. b) Normalized Hi-C contact maps of wild type, dnaA deletion and rok deletion strains at stationary phase. The interaction between Rok clusters 1 and 7/8 is shown in the inset.

Extended Data Fig. 10 Topological domains boundaries are formed by interaction between Rok clusters.

a) Scalogram and DI analysis (200 Kb scale) of wild type and Δrok strains near Rok cluster 1 and 3 other Rok binding sites (green dots) recruited to the origin supercluster at stationary growth phase (see Extended Data Fig. 6). b) DI analysis (400 Kb scale) of RC8 mutant strains (see Extended Data Fig. 7) at stationary growth phase. The RC8 complementation locus is marked using a dotted line.

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Dugar, G., Hofmann, A., Heermann, D.W. et al. A chromosomal loop anchor mediates bacterial genome organization. Nat Genet 54, 194–201 (2022). https://doi.org/10.1038/s41588-021-00988-8

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