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Cited by 143Genomics and Chromatin DynamicsBacterial Genetics and BiotechnologyDNA Repair Mechanisms

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RNA polymerases as moving barriers to condensin loop extrusion

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Brandão, Hugo B., Paul, Payel, van den Berg, Aafke A., Rudner, David Z., Wang, Xindan, Mirny, Leonid A.·2019·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.1907009116PMC PMC6789630

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Abstract

To separate replicated sister chromatids during mitosis, eukaryotes and prokaryotes have structural maintenance of chromosome (SMC) condensin complexes that were recently shown to organize chromosomes by a process known as DNA loop extrusion. In rapidly dividing bacterial cells, the process of separating sister chromatids occurs concomitantly with ongoing transcription. How transcription interferes with the condensin loop-extrusion process is largely unexplored, but recent experiments have shown that sites of high transcription may directionally affect condensin loop extrusion. We quantitatively investigate different mechanisms of interaction between condensin and elongating RNA polymerases (RNAPs) and find that RNAPs are likely steric barriers that can push and interact with condensins. Supported by chromosome conformation capture and chromatin immunoprecipitation for cells after transcription inhibition and RNAP degradation, we argue that translocating condensins must bypass transcribing RNAPs within ∼1 to 2 s of an encounter at rRNA genes and within ∼10 s at protein-coding genes. Thus, while individual RNAPs have little effect on the progress of loop extrusion, long, highly transcribed operons can significantly impede the extrusion process. Our data and quantitative models further suggest that bacterial condensin loop extrusion occurs by 2 independent, uncoupled motor activities; the motors translocate on DNA in opposing directions and function together to enlarge chromosomal loops, each independently bypassing steric barriers in their path. Our study provides a quantitative link between transcription and 3D genome organization and proposes a mechanism of interactions between SMC complexes and elongating transcription machinery relevant from bacteria to higher eukaryotes.

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Figures

Fig. 1. The posited role of transcription in shaping asymmetric SMC translocation rates. (A) Hi-C map of a wild-type B. subtilis PY79. A, Inset depicts the juxtaposition of chromosome arms (dashed lines) centered on the ori to ter axis. (B) Hi-C map of a B. subtilis strain (see SI Appendix for strain tables) with a single parS site at −94° which has a secondary diagonal that points biasedly away from the ori. (C) Loop-extrusion model schematic depicting the active juxtaposition of chromosome arms performed by the B. subtilis condensin loop-extruding complex. (D) Gene directions point biasedly toward the ter (green arrows); for SMC motors translocating toward the ori (blue), there will be increased frequencies of head-on collisions with transcripts as compared to SMCs translocating toward the ter (red). (E) Interpretation of Hi-C: Condensin translocates bidirectionally from the parS site juxtaposing flanking DNA; motion toward the ter is faster than toward the ori, resulting in the asymmetric secondary diagonal.
Fig. 1. The posited role of transcription in shaping asymmetric SMC translocation rates. (A) Hi-C map of a wild-type B. subtilis PY79. A, Inset depicts the juxtaposition of chromosome arms (dashed lines) centered on the ori to ter axis. (B) Hi-C map of a B. subtilis strain (see SI Appendix for strain tables) with a single parS site at −94° which has a secondary diagonal that points biasedly away from the ori. (C) Loop-extrusion model schematic depicting the active juxtaposition of chromosome arms performed by the B. subtilis condensin loop-extruding complex. (D) Gene directions point biasedly toward the ter (green arrows); for SMC motors translocating toward the ori (blue), there will be increased frequencies of head-on collisions with transcripts as compared to SMCs translocating toward the ter (red). (E) Interpretation of Hi-C: Condensin translocates bidirectionally from the parS site juxtaposing flanking DNA; motion toward the ter is faster than toward the ori, resulting in the asymmetric secondary diagonal.CC BY-NC-ND · PubMed Central
Fig. 2. Model of SMC translocation based on gene position and orientation. (A) Rules for the model of condensin-translocation rates based on gene orientation. (B) A parameter sweep of the model shows the agreement with Hi-C data as a function of γ and ρ parameters (Left); illustrative example trajectories (i–iv) are superimposed on the Hi-C map to show how juxtaposition traces change as a function of these parameters (Right). (C) Examples of using the parS+26° strain-specific optimum parameters (γ = 2 and ρ = 20) (see SI Appendix, Fig. S4 for globally optimum trajectories) to predict the juxtaposition trajectories of other engineered B. subtilis strains. (D) The model of condensin translocation using only gene positions and orientations captures the spatiotemporal behavior of Hi-C secondary diagonal formation during a time-course Hi-C experiment where condensin loading was induced at t = 0 min.
Fig. 2. Model of SMC translocation based on gene position and orientation. (A) Rules for the model of condensin-translocation rates based on gene orientation. (B) A parameter sweep of the model shows the agreement with Hi-C data as a function of γ and ρ parameters (Left); illustrative example trajectories (i–iv) are superimposed on the Hi-C map to show how juxtaposition traces change as a function of these parameters (Right). (C) Examples of using the parS+26° strain-specific optimum parameters (γ = 2 and ρ = 20) (see SI Appendix, Fig. S4 for globally optimum trajectories) to predict the juxtaposition trajectories of other engineered B. subtilis strains. (D) The model of condensin translocation using only gene positions and orientations captures the spatiotemporal behavior of Hi-C secondary diagonal formation during a time-course Hi-C experiment where condensin loading was induced at t = 0 min.CC BY-NC-ND · PubMed Central
Fig. 3. Transcription-dependent and -independent features of SMC-mediated chromosome juxtaposition. (A) Hi-C data from previous experiments [Wang et al. (8)] showing the effect of transcription inhibition by rifampicin for 30 min on chromosome arm juxtaposition; superimposed SMC translocation model trajectories (solid and dashed lines) suggest a transcription-elongation independent effect on asymmetric SMC trajectories (i.e., the factor γ is unchanged before and after treatment; SI Appendix, Fig. S6A); schematic representations of the changes to chromosome juxtaposition are shown on the right. (B) Hi-C before and after rifampicin treatment experiments for a strain (parS+94°) with condensin loading far from the highly transcribed rRNA clusters of the other strain (parS+26°); the unchanging secondary diagonal angle confirms a transcription-independent effect.
Fig. 3. Transcription-dependent and -independent features of SMC-mediated chromosome juxtaposition. (A) Hi-C data from previous experiments [Wang et al. (8)] showing the effect of transcription inhibition by rifampicin for 30 min on chromosome arm juxtaposition; superimposed SMC translocation model trajectories (solid and dashed lines) suggest a transcription-elongation independent effect on asymmetric SMC trajectories (i.e., the factor γ is unchanged before and after treatment; SI Appendix, Fig. S6A); schematic representations of the changes to chromosome juxtaposition are shown on the right. (B) Hi-C before and after rifampicin treatment experiments for a strain (parS+94°) with condensin loading far from the highly transcribed rRNA clusters of the other strain (parS+26°); the unchanging secondary diagonal angle confirms a transcription-independent effect.CC BY-NC-ND · PubMed Central
Fig. 4. Mechanistic models of extrusion-transcription interference. (A) The “moving-barriers” model of condensin transcription interactions: Transcription complexes are posited as impermeable barriers to condensin movement, which results in different translocation dynamics when crossing operons in the co-oriented or convergent fashion. Condensin translocates (i) at its native speed (vSMC) until (ii) it reaches a slowly moving RNAP, then (iii) it proceeds in the direction and at the speed of RNAP (vRNAP) until (iv) RNAP reaches the end of the operon, whereby condensin continues translocation at its original speed and direction. (B) Analytically computed average times to cross an operon for each of the head-to-tail (Left) and head-to-head (Right) cases as a function of operon length and RNAP density. Despite similar “rules,” in the head-to-tail case (B, Left), the SMC always reaches the end of the operon, whereas in the head-to-head case (B, Right), a successful traversal by SMC may occur only if no RNAP is encountered within a cell-division time (dashed line). (C) Extension of the “moving-barriers” model allowing for condensin to bypass transcribing RNAP (Left, schematic); simulations of the locus-crossing times with varying permeability (“bypass”) rates and RNAP densities (Right); blue region indicates the experimentally estimated time for condensin to cross a 3-kb rRNA gene locus (with density ∼10 RNAP/kb).
Fig. 4. Mechanistic models of extrusion-transcription interference. (A) The “moving-barriers” model of condensin transcription interactions: Transcription complexes are posited as impermeable barriers to condensin movement, which results in different translocation dynamics when crossing operons in the co-oriented or convergent fashion. Condensin translocates (i) at its native speed (vSMC) until (ii) it reaches a slowly moving RNAP, then (iii) it proceeds in the direction and at the speed of RNAP (vRNAP) until (iv) RNAP reaches the end of the operon, whereby condensin continues translocation at its original speed and direction. (B) Analytically computed average times to cross an operon for each of the head-to-tail (Left) and head-to-head (Right) cases as a function of operon length and RNAP density. Despite similar “rules,” in the head-to-tail case (B, Left), the SMC always reaches the end of the operon, whereas in the head-to-head case (B, Right), a successful traversal by SMC may occur only if no RNAP is encountered within a cell-division time (dashed line). (C) Extension of the “moving-barriers” model allowing for condensin to bypass transcribing RNAP (Left, schematic); simulations of the locus-crossing times with varying permeability (“bypass”) rates and RNAP densities (Right); blue region indicates the experimentally estimated time for condensin to cross a 3-kb rRNA gene locus (with density ∼10 RNAP/kb).CC BY-NC-ND · PubMed Central
Fig. 5. Evidence for the “permeable moving barriers” model of condensin–transcription interactions. (A) Comparison of SMC ChIP-seq tracks (anti-SMC) with RNAP tracks (anti-GFP and RpoC-GFP) showing that RNAP colocalizes with SMC, but SMC peaks may occur without RNAP; operon locations are shown. (B) Fold difference of ChIP-seq signal for SMC tracks normalized by RNAP tracks; average signal is shown for all genes of length up to length 1 kb, separated by the direction of SMC translocation relative to transcription direction (SI Appendix, section 1.3). (C) Simulation SMC ChIP-seq for 1-kb gene, demonstrating the differential accumulation of SMC within the gene body for the “moving barriers with permeable boundaries” model; a permeability rate of 0.5 s−1 well describes the ∼2-fold change in experimentally observed head-to-tail versus head-to-head SMC accumulation. (D) Summary model: Condensins (possibly oligomers) translocate away from the parS loading site by 2 independent (blue and red) motor activities; condensin motors can bypass steric barriers (like transcription machinery or other DNA-bound proteins) which are of similar size or larger than the condensin lumen; while condensin attempts to bypass a steric barrier, it may be “pushed” by other translocating factors like RNAP, leading to transcription-dependent SMC translocation rates.
Fig. 5. Evidence for the “permeable moving barriers” model of condensin–transcription interactions. (A) Comparison of SMC ChIP-seq tracks (anti-SMC) with RNAP tracks (anti-GFP and RpoC-GFP) showing that RNAP colocalizes with SMC, but SMC peaks may occur without RNAP; operon locations are shown. (B) Fold difference of ChIP-seq signal for SMC tracks normalized by RNAP tracks; average signal is shown for all genes of length up to length 1 kb, separated by the direction of SMC translocation relative to transcription direction (SI Appendix, section 1.3). (C) Simulation SMC ChIP-seq for 1-kb gene, demonstrating the differential accumulation of SMC within the gene body for the “moving barriers with permeable boundaries” model; a permeability rate of 0.5 s−1 well describes the ∼2-fold change in experimentally observed head-to-tail versus head-to-head SMC accumulation. (D) Summary model: Condensins (possibly oligomers) translocate away from the parS loading site by 2 independent (blue and red) motor activities; condensin motors can bypass steric barriers (like transcription machinery or other DNA-bound proteins) which are of similar size or larger than the condensin lumen; while condensin attempts to bypass a steric barrier, it may be “pushed” by other translocating factors like RNAP, leading to transcription-dependent SMC translocation rates.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Brandão, H. B., Paul, P., van den Berg, A. A., Rudner, D. Z., Wang, X., & Mirny, L. A.
Affiliations
  • Harvard University (US)
  • Indiana University Bloomington (US)
  • Massachusetts Institute of Technology (US)
Corresponding author
  • Wang, Xindan
  • Mirny, Leonid A.
ORCID
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
2019-09-23
Volume
116
Issue
41
Pages
20489-20499
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
DOI
10.1073/pnas.1907009116
PMC
PMC6789630
OpenAlex
W2975167630
Cited by
143
References
92
Citations by year
2026: 15 · 2025: 20 · 2024: 14 · 2023: 28 · 2022: 23 · 2021: 21 · 2020: 14 · 2019: 7
FWCI
5.38
Type
journal article
Language
en
Open access
Licence
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
Funders
  • HHS | National Institutes of Health (GM086466, GM073831, GM114190)
  • IU | Indiana University Bloomington (Start-up funds)
  • Gouvernement du Canada | Natural Sciences and Engineering Research Council of Canada (PGS-D)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
Keywords
condensin, DNA loop extrusion, RNA polymerase, sister chromatid separation, SMC complex, steric barrier, transcription, 3D genome organization, ribosomal DNA, operon, chromosome conformation capture, chromatin immunoprecipitation
MeSH terms
Adenosine Triphosphatases, Bacillus subtilis, Bacterial Proteins, Chromosomes, Bacterial, DNA-Binding Proteins, Protein Binding, DNA-Directed RNA Polymerases, RNA, Ribosomal, Transcription, Genetic, Genome, Bacterial, Multiprotein Complexes

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