Bacillus subtilis SMC complexes juxtapose chromosome arms as they travel from origin to terminus

Abstract

Structural maintenance of chromosomes (SMC) complexes play critical roles in chromosome dynamics in virtually all organisms, but how they function remains poorly understood. In the bacterium Bacillus subtilis, SMC-condensin complexes are topologically loaded at centromeric sites adjacent to the replication origin. Here we provide evidence that these ring-shaped assemblies tether the left and right chromosome arms together while traveling from the origin to the terminus (>2 megabases) at rates >50 kilobases per minute. Condensin movement scales linearly with time, providing evidence for an active transport mechanism. These data support a model in which SMC complexes function by processively enlarging DNA loops. Loop formation followed by processive enlargement provides a mechanism by which condensin complexes compact and resolve sister chromatids in mitosis and by which cohesin generates topologically associating domains during interphase.

Fig. 1. DNA juxtaposition propagates from condensin's loading site

(A-B) Normalized Hi-C interaction maps displaying contact frequencies for pairs of 10 kb bins. The strain (BWX3352) contains a single parS site at -1° and an IPTG-inducible gfp-parB fusion. Maps show interaction frequencies before (A) and after induction (B) with IPTG. Axes present genome positions in degrees and are oriented with the replication origin at the center. Schematics of the juxtaposed regions are shown. The scale bar depicts Hi-C interaction scores for all contact maps presented in this study. The dotted lines indicate the -1° parS site (black) and the leading edge of the juxtaposed DNA (blue). (C) Immunoblot analysis of the same samples from (A) showing GFP-ParB accumulation, the levels of the condensin complex (SMC, ScpA, ScpB) and SigA to control for loading. (D) Anti-SMC ChIP-seq performed under the same condition as in (A-B). Sequencing reads from ChIP and input samples were normalized to the total number of reads. The ratio of ChIP enrichment (ChIP/Input) at each time point relative to time 0 is shown in 1 kb bins. (E) Schematic interpretation of the Hi-C and ChIP-seq data. Progressive juxtaposition enlarges a DNA loop centered on parS.

Figure 2. Condensin is specifically enriched along juxtaposed DNA

(A) Hi-C contact maps of strains harboring single parS sites at -59° (BWX3377), -94° (BWX3270), and -117° (BWX3381). To visualize interactions in the terminus region, the genome was oriented with the terminus (ter) at the center of the maps. The position of the parS sites and the extent of DNA juxtaposition are indicated by black and blue dotted lines, respectively. (B) Anti-SMC ChIP-seq was performed on the same samples as in (A). ChIP enrichment (ChIP/Input) was plotted in 1 kb bins. Schematics of the juxtaposed regions are shown. SMC enrichment at the highly transcribed genes outside the juxtaposed regions is probably nonspecific (see Figure S3).

Figure 3. Condensin complexes loaded at parS travel down the flanking DNA

Cells (BWX3690) harboring a single parS site at -94° with wild-type SMC and an IPTG-inducible gfp-smc fusion we analyzed before an after induction. (A) Immunoblot analysis of GFP-SMC levels (using anti-SMC antibodies) during the induction time course. (B) ChIP-seq enrichment using anti-GFP antibodies. The ratio of ChIP enrichment at the indicated time point relative to time 0 (before induction) is plotted in 1 kb bins. Dotted lines indicate the position of the parS site (black) and the extent of ChIP enrichment of untagged SMC at time 0 (blue). (C) The accumulation of GFP-SMC from the parS towards the origin (red squares) and terminus (black circles) are plotted. The rates of GFP-SMC accumulation were calculated using the first three time points. See Figure S8 for unprocessed data and additional analysis.

Figure 4. Highly transcribed genes influence DNA juxtaposition

(A) Hi-C contact maps of strains harboring a single parS site at +26° (BWX3403) or -27° (BWX3268). The maps are oriented with the origin at the center of the axes. Dotted lines indicate the positions of the origin (yellow) and the parS sites (black). Schematics show DNA juxtaposition. Red lines highlight the asymmetric juxtaposition adjacent to the highly transcribed genes (red arrows). (B) Hi-C contact maps of the strain harboring the +26° parS site (BWX3403) in the absence or presence of 25 μg/ml rifampicin (rif) for 10 and 30 min. (C) Representative images of cells from (B). DAPI-stained DNA (green) and membranes (red) are shown. Bar, 4 μm. (D) Hi-C contact maps of a strain (BWX3352) harboring a single parS site at -1° and gfp-parB under IPTG control. Cells were induced for 20 min with IPTG, then treated with or without 25 μg/ml rifampicin for 15 min. (E) Schematic model for condensin function. (a) Condensin rings topologically loaded by ParB bound to parS travel down the flanking DNA as handcuffs, (b) resolving newly replicated sister origins by processive loop enlargement. Alternatively, a single ring composed of one or more condensin complexes could encircle the DNA on both sides of parS (not shown). (c) Condensin tethers encounter supercoiled plectonemes, DNA binding proteins, RNA polymerase, and ribosomes translating nascent transcripts. (d) Schematic model of encounters between tethered rings and either a plectoneme (gray) or a transcription complex.