The Cohesin Ring Uses Its Hinge to Organize DNA Using Non-topological as well as Topological Mechanisms

Abstract

As predicted by the notion that sister chromatid cohesion is mediated by entrapment of sister DNAs inside cohesin rings, there is perfect correlation between co-entrapment of circular minichromosomes and sister chromatid cohesion. In most cells where cohesin loads without conferring cohesion, it does so by entrapment of individual DNAs. However, cohesin with a hinge domain whose positively charged lumen is neutralized loads and moves along chromatin despite failing to entrap DNAs. Thus, cohesin engages chromatin in non-topological, as well as topological, manners. Since hinge mutations, but not Smc-kleisin fusions, abolish entrapment, DNAs may enter cohesin rings through hinge opening. Mutation of three highly conserved lysine residues inside the Smc1 moiety of Smc1/3 hinges abolishes all loading without affecting cohesin's recruitment to CEN loading sites or its ability to hydrolyze ATP. We suggest that loading and translocation are mediated by conformational changes in cohesin's hinge driven by cycles of ATP hydrolysis.

Keywords: SMC; chromosome condensation; cohesin; condensin; loop extrusion; sister chromatid cohesion.

Graphical abstract

Figure 1

Entrapment of Single and Sister DNA Molecules by Hetero-trimeric Cohesin Rings (A) Procedure for detecting entrapment of DNAs by cohesin. 6C strains with cysteine pairs at all three ring subunit interfaces (2C Smc3: E570C S1043C, 2C Smc1: G22C K639C and 2C Scc1 C56 A547C) and 5C strains lacking just one of these cysteines (Scc1 A547C) and carrying a 2.3 kb circular minichromosome were treated with BMOE. DNAs associated with cohesin immunoprecipitates (Scc1-PK6) were denatured with SDS and separated by agarose gel electrophoresis. Southern blotting reveals supercoiled monomers and nicked and supercoiled concatemers along with two forms of DNA unique to 6C cells, termed CMs and CDs. (B) CMs and CDs in exponentially growing strains K23644 (5C), K23889 (6C), and K23890 (5C, no cohesin tag). Quantification of the bands (percentage of total) from the 6C crosslinked sample from 3 biological replicates is shown (data are represented as mean ± SD). See also Figure S1B. (C) CMs and CDs in WT (K23889) and scc2-45 (K24267) 6C strains arrested in G1 with α factor at 25°C in YPD medium and released into nocodazole at 37°C. See also Figure S1D. (D) CM and CDs in exponentially growing 6C strains containing ectopically expressed versions of 2C Smc3-PK6: K24173 (WT Smc3), K24174 (smc3 E1155Q), and K24175 (smc3 K38I). (E) CMs and CDs in strains K23644 (5C), K23889 (6C), and those arrested in late G1 by expressing galactose-inducible nondegradable Sic1 K23971 (5C) and K23972 (6C). See also Figure S1E. (F) CMs and CDs in WT (K23889) and cdc4-1 (K24087) 6C strains arrested in G1 at 25°C in YPD medium and released into YPD medium containing nocodazole at 37°C. See also Figure S1F. (G) CMs in α factor-arrested cells expressing non-cleavable 2C Scc1 (K24695). See also Figure S1G. See also Figure S1.

Figure S1

Related to Figures 1 and 2 (A) Western blot of fully circularizable 6C wild-type cohesin crosslinked in vivo using BMOE, probed for the HA-epitope on Smc3. The positions of the different crosslinked species are indicated. (B) Genomic DNA isolated from aliquots of the experiment in Figure 1B were electrophoresed, Southern blotted and detected with the TRP1 probe. (C) The 6C strain (K23889) was grown as in Figure 1B and subjected to 2D gel electrophoresis either in the absence or presence of proteinase K in the second dimension. The positions of the supercoiled monomer, CM and CD species are marked. ^(∗) indicates non-specific background signal. (D) Samples from experiment shown in Figure 1C were subjected to minichromosome IP without in vivo crosslinking; the position of the band containing supercoiled monomers is indicated in the Southern blot. (E) FACS profiles of the strains described in Figure 1E. (F) FACS profiles of the strains described in Figure 1F. (G) FACS profiles of the strain described in Figure 1G. (H) Exponentially growing diploid cells containing 2 copies of 6C cohesin with a tag on just one of the 2C Scc1 copies (K24242) and diploid cells containing 1 copy of 5C cohesin and one copy of 6C cohesin with a tag on just the 2C Scc1 (K24194) were subjected to the minichromosome IP assay. The intensities of the CM and CD bands quantified using AIDA Image Analyzer software are plotted as % of the total lane intensities. See also Figure 2E. (I) Quantification of the gel from Figure 2E showing the lane traces. (J) FACS profiles of the strains described in Figure 2G.

Figure 2

Sister Chromatid Cohesion Is Generated by Entrapment of Sister DNAs within Individual Cohesin Rings (A) CMs and CDs in WT (K23889), eco1-1 (K23579), and eco1-1 wplΔ (K23578) strains arrested in G1 at 25°C in YPD medium and released into YPD medium containing nocodazole at 37°C. (B) CMs and CDs in exponentially growing WT (K23889) and eco1Δ wplΔ (Κ25287) 6C strains. (C) CMs and CDs in WT (K23889) and pds5-101 (K24030) 6C strains arrested in G1 at 25°C in YPD medium and released into YPD medium containing nocodazole at 37°C. (D) CMs and CDs in exponentially growing WT (K23889) and pds5-101 (K24030) 6C strains arrested in G2 by addition of nocodazole at 25°C and shifted to 37°C. Data are shown from the same Southern blot, with irrelevant lanes removed. (E) CMs and CDs in exponentially growing tetraploid cells containing 4 copies of 6C cohesin with a tag on just one of the 2C Scc1 copies (K24561) and tetraploid cells containing 3 copies of 5C cohesin and one copy of 6C cohesin with a tag on just the 2C Scc1 (K24560). See also Figures S1H and S1I. (F) CMs and CDs in exponentially growing 6C strains containing ectopically expressed versions of 2C Scc1, K24205 (WT), and K26413 (V137K) arrested in G2/M with nocodazole. (G) CMs and CDs in 6C strains with ts scc1-73 allele at the endogenous locus and either WT (2C) Scc1 (K26600) or (2C) Scc1 V137K mutant (K26591) at an ectopic locus. Cells were arrested in G1 at 25°C in YPD medium and released into YPD medium containing nocodazole at 37°C. See also Figure S1J. See also Figure S1.

Figure 3

DNA Entrapment Is Necessary for Cohesion but Not for Loading or Translocation (A) Structure of the mouse hinge domain, highlighting positively charged residues in its central channel neutralized by smc1K554D K661D smc3R665A K668A R669A mutations (DDAAA). (B) CMs and CDs in exponentially growing K23644 (5C) and two 6C strains (K26210 with an ectopic WT 2C SMC1 and K26215 with endogenous 2C smc3AAA and ectopic 2C smc1DD (DDAAA)). Over three biological replicates, the intensities of CM and CD bands in the DDAAA mutant were reduced to around 20% and 3% of the WT levels, respectively. (C) Exponentially growing strains WT (K15426, Smc3-HA) and DDAAA mutant (K15424, smc3AAA-HA) were analyzed by calibrated ChIP-sequencing. ChIP profiles along chromosomes II and VIII are shown. See also Figure S2C. (D) ATPase activity of purified WT and DDAAA mutant tetramer stimulated by Scc2. The rate of ATP hydrolysis was measured either in the presence or absence of DNA. (E) Strains K26797 (containing endogenous 3× miniAID-tagged SMC3 and ectopic WT SMC3), K26611 (containing endogenous 3× miniAID tagged-SMC3 and endogenous smc1DD and ectopic smc3AAA), and K26767 (3× miniAID-tagged SMC3 and no ectopic SMC3) were arrested in G1 and synthetic auxin (indole-3-acetic acid) added to 1 mM 30 min before release. Cultures were released into YPD containing 1 mM auxin and nocodazole. 60 min after release from the G1 arrest, cultures were harvested and chromosomes spread (STAR Methods). Micrographs of chromosome masses of the two strains were quantified from three independent experiments (n = 100) and categorized as “1 loop” (showing fully condensed rDNA loops), “2 loops” (showing fully condensed rDNA loops that are split because of loss of cohesion), and “puffed” (showing unstructured, puffed rDNA morphology). See also Figure S4. Data are represented as mean ± SD. See also Figures S2, S3, and S4.

Figure S2

Related to Figures 3, 4, 5, and 6 (A) Two replicates of the experiment performed in Figure 3B. (B) Left panel: Efficiency of the hinge crosslinking was compared between the wild-type and the DDAAA strains: Wild-type (K26085) and DDAAA (K26086) strains were subjected to western blotting with and without in vivo crosslinking and the blots probed with anti-Myc (Smc1) and anti-HA (Smc3) antibodies to detect the un-crosslinked and crosslinked Smc1/Smc3 species. Right panels: Samples from experiment described in Figures 3B and 4F were subjected to western blotting after in vivo crosslinking and the blots probed with either anti-Myc (left panel) or anti-PK antibody (right panel). The positions of the fully circularized species are indicated. (C) Average ChIP profiles of the experiment described in Figure 3C. The ChIP profiles are showing the number of reads at each base pair away from the CDEIII element averaged over all 16 chromosomes. (D) Exponentially growing diploid strains K17660 (expressing Mtw1-RFPand Smc1-eGFP), K18194 (expressing Mtw1-RFP and Scc1-eGFP), and K26700 (expressing Mtw1-RFP and with smc3AAA expressed from the endogenous locus and expressing smc1DD from an ectopic locus) were grown in YEPD medium at 25°C and were placed on 2.5% agarose pads made of synthetic complete medium containing glucose. Live cell imaging was performed under a spinning disk confocal system at 25°C. (E) Coomassie stained gels showing Smc1/3 hinge exchange. Purified heterodimeric hinge domains that had either wild-type or DDD mutant Smc1 associated with Smc3 hinge containing a cysteine substation (E570C) were mixed with purified MBP-tagged Smc1 containing a cysteine substitution (K639C). We measured the exchange of the wild-type and DDD mutant Smc1 with the MBP-Smc1 by adding homo-bifunctional crosslinker bBBr for the indicated times. (F) Crosslinking was compared between the wild-type, Smc3-Scc1 and Scc1-Smc1 fusion strains: Wild-type (K23889), Smc3-Scc1 (K24838) and Scc1-Smc1 (K25696) strains were subjected to western blotting with and without in vivo crosslinking and the blots probed with anti-PK antibody against Scc1/fusion proteins to detect un-crosslinked and crosslinked species. The position of the fully circularized species is indicated.

Figure S3

Related to Figures 3, 4, and 6 (A) Cells from K26797 (containing endogenous 3×miniAID-tagged SMC3 and ectopic wild-type SMC3), K26611 (containing endogenous 3×miniAID tagged-SMC3 and endogenous smc1DD and ectopic smc3AAA) were arrested in G1 and synthetic auxin (indole-3-acetic acid) added to 1 mM 30 min before release. Cultures were released into YPD containing 1mM auxin and nocodazole to arrest the cultures in G2/M and analyzed by calibrated ChIP-sequencing. ChIP profiles show the number of reads at each base pair away from the CDEIII element averaged over all 16 chromosomes. (B) Calibrated ChIP-seq profiles along chromosome II and Chromosome X from the experiment described in Figure 4E. (C) Calibrated ChIP-seq of exponentially growing wild-type (K23889) and Smc3-Scc1 fusion strain (K24838). ChIP profiles show the number of reads at each base pair away from the CDEIII element averaged over all 16 chromosomes. See Figure 6B for representative individual chromosome traces.

Figure S4

Related to Figure 3E Examples of chromosome spreads of the wild-type, DDAAA mutant and the smc3 depletion (smc3 null) strains from the experiment described in Figure 3E.

Figure 4

Residues within Its Hinge Domain Dictate Cohesin’s Ability to Load onto Chromosomes (A) Structure of the mouse hinge depicting mutated Smc1 residues (smc1 K554D K650D K661D) (DDD). (B) Haploid segregants following tetrad dissection of asci from diploid strains (ura3::smc1DD /ura3::smc1DD smc1Δ/smc1Δ containing two mutations of all possible combinations from K554D, K650D, and K661D). (C) Calibrated ChIP-seq of exponentially growing strains with a deletion of the endogenous SMC1 gene and expressing ectopically either WT SMC1 (K15324), smc1K554D K661D (K15322), or smc1K650D K661D mutant (K15226). (D) Haploid segregants following tetrad dissection of asci from diploid strains (SMC1/smc1Δ ura3::SMC1/ura3::SMC1) and (SMC1/smc1Δ ura3::smc1DDD/ura3::smc1DDD). (E) Calibrated ChIP-seq of exponentially growing strains K24327 (ectopic SMC1), K26756 (ectopic smc1DDD), and K699 (untagged control). (F) Minichromosome IP assay of exponentially growing strains K24327 (expressing ectopic WT 2C SMC1) and K26610 (expressing ectopic 2C smc1DDD). See also Figures S2, S3, S5, S6, and S7.

Figure S5

Related to Figures 4 and 5 Multiple sequence alignment indicating conservation of Smc1 resides K554, K650 and K661 in S. cerevisiae across various other eukaryotes. These residues are highlighted in the hinge structure shown in Figure 4A.

Figure 5

smc1DDD Mutation Does Not Affect Scc2-Stimulated ATP Hydrolysis Cycle (A) Calibrated ChIP-seq of exponentially growing strains each containing Scc2-PK6, K26839 (ectopic SMC1), K26840 (ectopic smc1DDD E1158Q), and K25646 (ectopic smc1 E1158Q). (B) ATPase activity of purified WT and DDD mutant tetramer stimulated by Scc2. ATP hydrolysis was measured with and without DNA. (C) Exponentially growing strains K26756 (expressing the WT Smc3 from the endogenous locus and the smc1DDD mutant from an ectopic locus), K26757 (expressing smc1 DDD mutant from an ectopic locus and smc3AAA mutant from the endogenous locus), and K24327 (expressing WT Smc1 and Smc3) were analyzed by calibrated ChIP sequencing (Smc1-PK IP). The ChIP profiles are showing the number of reads at each base pair away from the CDEIII element averaged over all 16 chromosomes. See also Figures S2, S5, and S7.

Figure 6

Entrapment of DNAs When Smc and Kleisin Subunits Are Fused Together (A) CMs and CDs in an exponentially growing 6C WT (K23889) strain and a strain containing 2C SMC1 and expressing an SMC3-SCC1 fusion containing cysteines in Smc3’s hinge and Scc1’s C terminus (K24838) as the sole source of Smc1 and Scc1. Data are shown from the same Southern blot, with one irrelevant lane removed. The fractions of CD and CM of the total DNA immunoprecipitates from WT and fusion strains across 3 biological replicates are shown (data are represented as mean ± SD). (B) Calibrated ChIP-seq of exponentially growing WT (K23889) and Smc3-Scc1 fusion strain (K24838). Calibrated ChIP-seq profiles of representative chromosomes I and X are shown. See Figure S3B for a representation of the average of all 16 chromosomes. (C) CMs and CDs in exponentially growing 6C WT (K23889) strain and a 4C strain (containing an Smc3-Scc1 fusion protein with cysteine pairs at the hinge and Scc1/Smc1 interfaces but not at the Smc3/Scc1 interface) expressing a PK3-Scc1-Smc1 fusion as the sole source of Scc1 and Smc1 (K25696). See also Figures S2 and S3.

Figure 7

Covalent Closure of Cohesin’s Hinge Interface Fails to Block Loading (A) Coomassie-stained gel showing the Xenopus cohesin tetramer purified from baculovirus-infected Sf-9 cells. (B) Mock- and Scc2-depleted interphase low-speed supernatants (LSS) Xenopus egg extracts were supplemented with the recombinant Xenopus tetramer and sperm nuclei and incubated at 23°C for 90 min. The isolated chromatin fraction and the soluble extracts were analyzed by western blotting using indicated antibodies. (C) Recombinant Xenopus tetramer was treated with TEV protease or buffer for 60 min at 16°C. The reaction was then mixed with LSS interphase Xenopus egg extracts and treated as in (B); the chromatin and soluble fractions were analyzed by western blotting using indicated antibodies. (D) LSS interphase extract was treated with either purified recombinant geminin (60 nM) or buffer for 15 min on ice. The extracts were then supplemented with recombinant Xenopus tetramer and sperm chromatin and treated as in (A). The chromatin and soluble fractions were analyzed by western blotting using indicated antibodies. (E) Recombinant WT cohesin and cohesin complex containing cysteine substitutions in the hinge domain (Hinge Cys) were treated with DMSO (–), 125 μM bBBr (+), or 125 μM bBBr with 10 mM DTT (+/−). Samples were also supplemented with tetramethylrhodamine (TMR) HaloTag ligand and incubated on ice for 10 min and then run on a 3%–10% gradient gel. The crosslinking efficiency was quantified via TMR fluorescence. (F) WT and hinge substituted Xenopus tetramer was treated with DMSO or bBBr on ice for 10 min, and excess crosslinker was then quenched by adding 10 mM DTT. The reactions were then supplemented with interphase extracts, TMR ligand, and sperm chromatin and treated as in (B). The soluble and chromatin fractions were analyzed by TMR fluorescence and indicated antibodies. (G) Crosslinking reactions described in (F) were supplemented with extracts pre-treated with buffer or recombinant geminin and western blots performed as described in (D). (H) Hinge substituted cohesin was crosslinked and supplemented with interphase extracts and 3 ng BAC DNA/μL. After a 90 min incubation, chromatin fractions were isolated, and the chromatin pellets were washed with buffer containing indicated amounts of KCl and analyzed by western blotting. (I) Hinge substituted Xenopus tetramer was crosslinked and loaded onto chromatin as in (F). The isolated chromatin pellet washed with buffer containing 300 mM KCl. The pellet was then re-suspended in Xenopus B (XB) buffer supplemented with anti-V5 antibody and benzonase (1 U/μL) and incubated at 12°C overnight. The immunoprecipitates were analyzed by western blot. See also Figure S6.

Figure S6

Related to Figure 7 Interphase Xenopus egg extract was treated with either DMSO or aphidicholine for 15 min. The extracts were then supplemented with 3 ng BAC DNA/μl. After a 90 min incubation, chromatin fractions were isolated, the chromatin pellets washed with buffer containing indicated amounts of KCl and analyzed by western blotting.

Figure S7

Related to Figures 4 and 5 The conserved positively charged residues that lie inside the lumen of condensin hinge domain are marked in the structure of Smc2-Smc4 hinge domain.