Co-occupancy of the centromeric DNA by histone H3 and Cse4.

<p>A) Only the 214 bp BglII CEN4 fragment and no full-length minichromosome is detected in the ChIP/qPCR assay. DNA isolated from untreated and BglII-treated lysates was size-fractionated on 2% agarose gel and analyzed by qPCR. A PCR product after 30 cycles of amplification in a conventional PCR reaction with the same primers that were used for qPCR is shown below. B) Minichromosomal CEN DNA can be co-immunoprecipitated with H3 and Cse4. BglII-treated chromatin of the strains 1021 (wt), 1407 (H3-HA3), and 1498 (Cse4-HA6) carrying the minichromosome was either not cross-linked or cross-linked with formaldehyde and immunoprecipitated with anti-HA antibody. The immunoprecipitated DNA was purified and size fractionated and subjected to qPCR analysis. C) CEN DNA of the native chromosome IV can be co-immunoprecipitatd with H3 and Cse4. BglII-treated chromatin of the strains 2059 (wt), 2042 (H3-HA3), and 2043 (Cse4-HA6) with CEN DNA of the native chromosome IV flanked with BglII was either not cross-linked or cross-linked with formaldehyde followed by immunoprecipitation as in (B). D) Flowchart of the sequential Cse4-H3 ChIP. E) Sequential ChIP of minichromosomal CEN DNA. BglII-treated chromatin of the strains 1923 (Cse4-Myc6) and 2300 (H3-HA3, Cse4-Myc6) carrying the minichromosome was cross-linked with formaldehyde and immunoprecipitated with anti-Myc or anti-HA antibody as indicated in the figure, the DNA was eluted off the beads and re-immunoprecipitated with anti-HA antibody. The immunoprecipitated DNA was purified, size fractionated on a 2% agarose gel and subjected to qPCR analysis. F) The same as in (E) but performed with the native CEN DNA. The strains, 2562 (Cse4-Myc6), and 2561 (H3-HA3, Cse4-Myc6) had CEN DNA of the native chromosome IV flanked with BglII. The bar graphs represent the average values from several independent experiments with SDs.</p

Models of how H3 and Cse4 can co-occupy the centromeric DNA.

<p>A heterotetramer of H3, H2A, H2B and H4 is colored in green and a heterotetramer containing Cse4 instead of H3 is blue.1) A heterotypic octamer containing both Cse4 and H3. 2) A heterotypic octamer with additional Cse4 bound to it. 3) A Cse4 hemisome incorporated in the loop of a conventional nucleosome. A DNA fragment of 207 bp is sufficient to accommodate this arrangement (without spacer DNA). 4) Two conventional nucleosomes flanking a Cse4 hemisome. The scissors indicate the BglII sites flanking the 214 bp fragment excised in our experiment. In case of model 4 this fragment would be tethered to non-centromeric DNA. The tethering was not observed in our experiments (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002739#pgen-1002739-g001" target="_blank">Figure 1C</a>). See text for discussion and additional details.</p

Composition of the centromeric nucleosome.

<p>A) The CEN-containing minichromosomes can be specifically co-immunoprecipitated with Cse4 and H3. Lysates from strains transformed with the minichromosomes 1021 (wt), 1498 (Cse4-HA6) and 1407 (H3-HA3) were incubated with anti-HA antibody and Dynabeads. DNA was eluted off the beads and separated on a 1% agarose gel. Southern blot was analyzed using a ³²P labeled <i>TRP1</i> probe. The map of the minichromosome is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002739#pgen.1002739.s001" target="_blank">Figure S1</a>. B) Experimental setup for the immunoprecipitation of minichromosomes digested with restriction enzyme. Chromatin is digested with BglII and incubated with anti-HA antibody recognizing tagged histones and protein A Dynabeads. Minichromosome digest with BglII produces three different fragments: a linearized full-length minichromosome (1), a CEN-less fragment (2) which can be detected with <i>TRP1</i> probe and a small CEN fragment (3) which can be detected with an LNA oligonucleotide. The red ellipse is depicting the centromeric nucleosome. C) Cse4 binding is restricted to minichromosomal CEN DNA. BglII-treated chromatin of strains carrying the minichromosome with BglII restriction sites 50 bp upstream and downstream of CEN boundaries was immunoprecipitated with anti-HA antibody. The strains were 1498 (Cse4-HA6), 1577 (H4-HA3), 1576 (H2A-HA3), 1587 (H2B-HA3), 1407 (H3-HA3), 1593 (Scm3-HA6), and 1021 (wt). DNA was analyzed as in (A) with ³²P labeled <i>TRP1</i> probe. D) H3 is associated with the CEN DNA. Top: Scheme of the excised CEN fragment. Double-DIG labeled LNA probe for CDEI/II is indicated. Bottom: Immunoprecipitated DNA from experiments shown in (C) was separated on a 6% denaturing TBE polyacrylamide gel. Southern blot was analyzed using a double-DIG labeled LNA probe for CDEI/II. Western blots showing immunoprecipitation of the tagged proteins are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002739#pgen.1002739.s004" target="_blank">Figure S4A</a>.</p

Histone H3 localizes to the centromeric DNA.

<p>A) H3 is associated with CEN DNA throughout the cell cycle. Strains carrying the minichromosomes with BglII restriction sites 50 bp upstream and downstream of CEN boundaries, 1498 (Cse4-HA6), 1407 (H3-HA3), and 1587 (H2B-HA3) were arrested in G1 with alpha factor and in G2 with nocodazole/benomyl. Chromatin was treated with BglII and immunoprecipitated with anti-HA antibody. DNA was eluted off the beads and resolved on a 6% denaturing TBE polyacrylamide gel. Southern blot was analyzed with a double-DIG labeled LNA probe for CDEI/II. The FACS profiles are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002739#pgen.1002739.s004" target="_blank">Figure S4B</a>. B) H3 is associated with the CEN DNA on a native chromosome IV. BglII-treated chromatin of strains with BglII sites 50 bp upstream and downstream of CEN boundaries on chromosome IV 2059 (wt), 2043 (Cse4-HA3), and 2042 (H3-HA3) was immunoprecipitated with anti-HA antibody. DNA was eluted off the beads, separated on a 6% denaturing TBE polyacrylamide gel and analyzed with a double-DIG labeled LNA probe for CDEI/II. C) Minichromosome-bound histone H3 does not turn over during the immunoprecipitation procedure. Lysates of strains 1021 (wt, carrying the minichromosome), 1407 (H3-HA, carrying the minichromosome), 1407 (H3-HA3, without the minichromosome), and mixed lysate of 1021 (wt with minichromosome) and 1407 (H3-HA3, without the minichromosome) were incubated with anti-HA antibody and Dynabeads. DNA was eluted off the beads, separated on a 1% agarose gel and analyzed using a ³²P labeled <i>TRP1</i> probe.</p

Cse4 association with CDEI/II and CDEIII.

<p>A) Cse4 nucleosome straddles the boundary between CDEII and CDEIII. Left: Map of the minichromosome utilized in the experiment. The construct contains 850 bp of pericentromeric sequence of chromosome IV, <i>TRP1</i> marker, <i>ARS1</i> and pUC19 sequence and has a size of 4.5 kb. There are two BglII sites: between CDEII and CDEIII in the CEN and in the <i>ARS1</i>. Right: BglII-treated chromatin of a strain 1498 (Cse4-HA6) carrying the minichromosome was immunoprecipitated with anti-HA antibody. DNA was eluted off the beads and separated on a 1% agarose gel. Southern blot was analyzed with a ³²P labeled probe for the pericentric CEN4 sequence (to detect the CDEI/II containing fragment) and a ³²P labeled probe for the <i>TRP1</i> gene (to detect the CDEIII containing fragment). B) Both Cse4 and H3 are associated with the CDEI/II fragment. Left: Scheme of CDEI/II fragment excised from the minichromosome. Double-DIG labeled LNA probe for CDEI/II is indicated. Right: BglII-treated chromatin of strain 1498 (Cse4-HA6) and 1407 (H3-HA3) carrying the minichromosome with BglII sites between CDEII and CDEIII and 50 bp upstream of CDEI was cross-linked with formaldehyde and immunoprecipitated with anti-HA antibody. DNA was eluted off the beads and resolved on a 6% denaturing TBE polyacrylamide gel. Southern blot was analyzed with a double-DIG labeled LNA probe for CDEI/II. C) Both the CDEI/II and the CDEIII fragments can be co-immunoprecipitated with Cse4 and H3. Strains 1021 (wt), 1407 (H3-HA3), and 1498 (Cse4-HA6) carried the minichromosome where either the CDEI/II (left) or the CDEIII fragment (right) was flanked with BglII sites. BglII-treated chromatin was either not cross-linked or cross-linked with formaldehyde and immunoprecipitated with anti-HA antibody. The immunoprecipitated DNA was purified, size fractionated, and subjected to qPCR analysis. Bar graphs represent the average values from several independent experiments with SDs.</p

Cohesin Rings Devoid of Scc3 and Pds5 Maintain Their Stable Association with the DNA

<div><p>Cohesin is a protein complex that forms a ring around sister chromatids thus holding them together. The ring is composed of three proteins: Smc1, Smc3 and Scc1. The roles of three additional proteins that associate with the ring, Scc3, Pds5 and Wpl1, are not well understood. It has been proposed that these three factors form a complex that stabilizes the ring and prevents it from opening. This activity promotes sister chromatid cohesion but at the same time poses an obstacle for the initial entrapment of sister DNAs. This hindrance to cohesion establishment is overcome during DNA replication via acetylation of the Smc3 subunit by the Eco1 acetyltransferase. However, the full mechanistic consequences of Smc3 acetylation remain unknown. In the current work, we test the requirement of Scc3 and Pds5 for the stable association of cohesin with DNA. We investigated the consequences of Scc3 and Pds5 depletion <em>in vivo</em> using degron tagging in budding yeast. The previously described DHFR–based N-terminal degron as well as a novel Eco1-derived C-terminal degron were employed in our study. Scc3 and Pds5 associate with cohesin complexes independently of each other and require the Scc1 “core” subunit for their association with chromosomes. Contrary to previous data for Scc1 downregulation, depletion of either Scc3 or Pds5 had a strong effect on sister chromatid cohesion but not on cohesin binding to DNA. Quantity, stability and genome-wide distribution of cohesin complexes remained mostly unchanged after the depletion of Scc3 and Pds5. Our findings are inconsistent with a previously proposed model that Scc3 and Pds5 are cohesin maintenance factors required for cohesin ring stability or for maintaining its association with DNA. We propose that Scc3 and Pds5 specifically function during cohesion establishment in S phase.</p> </div

Interaction of Pds5, Scc3 and Wpl1 with cohesin ring.

<p>Lysates of nocodazole/benomyl arrested yeast cultures were incubated with IgG sepharose to precipitate Scc1-TAP or Smc3-TAP. The presence of different proteins on the IgG beads was analysed by Western blot probed with anti-HA (12CA5), anti-MYC (71D10) and PAP (P1291, Sigma). The strains were in (A): 1771 (<i>SCC3-MYC18, PDS5-HA6</i>), 1829 (<i>SCC3-MYC18, PDS5-HA6</i>-degron, <i>SCC1-TAP</i>), 1958 (<i>SCC3-MYC18, PDS5-HA6, SCC1-TAP</i>); in (B): 1734 (<i>PDS5-MYC18, SCC3-HA6</i>), 1834 (<i>PDS5-MYC18, SCC3-HA6</i>-degron, <i>SCC1-TAP</i>), 1956 (<i>PDS5-MYC18, SCC3-HA6, SCC1-TAP</i>); in (C): 1882 (<i>WPL1-MYC18, PDS5-HA6</i>), 2014 (<i>WPL1-MYC18, PDS5-HA6, SCC1-TAP</i>), 2016 (<i>WPL1-MYC18, PDS5-HA6</i>-degron, <i>SCC1-TAP</i>); in (D): 1880 (<i>WPL1-MYC18, SCC3-HA6</i>), 2012 (<i>WPL1-MYC18, SCC3-HA6, SCC1-TAP</i>), 2018 (<i>WPL1-MYC18, SCC3-HA6</i>-degron, <i>SCC1-TAP</i>); in (E): 1771 (<i>SCC3-MYC18, PDS5-HA6</i>), 2251 (<i>SCC3-MYC18, PDS5-HA6, SMC3-TAP</i>), 2290 (<i>SCC3-MYC18, PDS5-HA6</i>-degron, <i>SMC3-TAP</i>); in (F): 1734 (<i>PDS5-MYC18, SCC3-HA6</i>), 2249 (<i>PDS5-MYC18, SCC3-HA6, SMC3-TAP</i>), 2264 (<i>PDS5-MYC18, SCC3-HA6</i>-degron, <i>SMC3-TAP</i>); in (G): 1882 (<i>WPL1-MYC18, PDS5-HA6</i>), 2253 (<i>WPL1-MYC18, PDS5-HA6, SMC3-TAP</i>), 2265 (<i>WPL1-MYC18, PDS5-HA6</i>-degron, <i>SMC3-TAP</i>); in (H): 1882 (<i>WPL1-MYC18, PDS5-HA6</i>), 2261 (<i>WPL1-MYC18, SCC3-HA6, SMC3-TAP</i>), 2271 (<i>WPL1-MYC18, SCC3-HA6</i>-degron, <i>SMC3-TAP</i>).</p

Depletion of Scc3 and Pds5 with a “conventional” temperature-sensitive degron.

<p>(A–C) Strains 2395 (<i>SCC1-HA6</i>), 2452 (<i>SCC1-HA6</i>, degron-<i>MYC18-PDS5</i>), 2455 (<i>SCC1-HA6</i>, degron-<i>MYC18</i>-<i>SCC3</i>) and 2456 (<i>SCC1-HA6</i>, degron-<i>MYC18- PDS5, degron-MYC18-SCC3</i>) were arrested with nocodazole in YEP raffinose at 30°C for 2 hours, resuspended in YEP galactose containing nocodazole and incubated for 45 minutes at 30°C to induce the expression of Ubr1. Cells were shifted to 37°C in YEP galactose containing nocodazole and doxycycline to deplete Pds5 and/or Scc3. (A) Chromosomal spreads were prepared at the indicated time points and stained with DAPI for DNA, anti-HA (mouse, 16B12) and anti-MYC (rabbit, 71D10) antibodies. The secondary antibodies were Alexa Fluor 488 anti-mouse and Alexa Fluor 568 anti-rabbit. Protein fluorescence was quantified using Metamorph software. At every time point fluorescence of 50 nuclei was determined. Error bars represent standard deviation. (B) Western blot of TCA protein extracts probed with anti-HA (16B12), anti-MYC (71D10) and anti-Cdc28 (sc-28550, Santa Cruz). (C) FACS analysis of cellular DNA content. (D–F) Strains were staged in G1 with <i>α</i>-factor in YEP raffinose at 30°C, resuspended in YEP galactose containing <i>α</i>-factor and incubated for 45 minutes at 30°C to induce the expression of Ubr1. Cells were then shifted to 37°C in YEP galactose containing doxycycline and <i>α</i>-factor, incubated for 90 minutes to deplete Pds5 and/or Scc3 and subsequently released in YEP galactose containing nocodazole and doxycycline at 37°C. Chromosomal spreads (D), Western blot (E), and FACS analysis of cellular DNA content (F) are shown.</p

Depletion of Scc3 and Pds5 does not affect cohesin association with chromatin.

<p>Yeast strains were staged in G1 with <i>α</i>-factor and released into media with nocodazole. Chromosomal spreads were prepared as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002856#pgen-1002856-g002" target="_blank">Figure 2</a>. At every time point fluorescence of 50 nuclei was determined. Error bars represent standard deviation. FACS analysis of cellular DNA content is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002856#pgen.1002856.s013" target="_blank">Figure S13</a>. The strains were in (A): 1813 (<i>SCC1-Myc18</i>, <i>SCC3-HA6</i>), 1625 (<i>SCC1-Myc18</i>, <i>SCC3-HA6</i>-degron), 1815 (<i>SCC1-Myc18</i>, <i>PDS5-HA6</i>), 1818 (<i>SCC1-Myc18</i>, <i>PDS5-HA6</i>-degron), in (B): 1771 (<i>SCC3-Myc18</i>, <i>PDS5-HA6</i>), 1796 (<i>SCC3-Myc18</i>, <i>PDS5-HA6</i>-degron), 1734 (<i>PDS5-MYC18</i>, <i>SCC3-HA6</i>) and 1744 (<i>PDS5-Myc18</i>, <i>SCC3-HA6</i>-degron) in (C): 1479 (<i>SCC3-HA6</i>), 1864 (<i>SCC3-HA6, Δwpl1</i>), 1677 (<i>PDS5-HA6</i>), 1866 (<i>PDS5-HA6, Δwpl1</i>), 10589 (<i>SCC1-Myc18</i>) and 1906 (<i>SCC1-Myc18, Δwpl1</i>).</p

Cohesin rings devoid of Scc3 and Pds5 topologically embrace circular DNA.

<p>Strains 1021 (untagged), 1813 (<i>SCC3-HA6</i>, <i>SCC1-Myc18</i>), 1625 (<i>SCC3-HA6-</i>degron, <i>SCC1-Myc18</i>), 2525 (<i>PDS5-HA6</i>, <i>SCC1-Myc18</i>) and 1818 (<i>PDS5-HA6</i>-degron, <i>SCC1-Myc18</i>) carried the centromeric minichromosomes. (A) Yeast lysates were incubated with BglII restriction enzyme as indicated. Minichromosomes were co-immunoprecipitated with Scc1-Myc18. DNA was prepared by phenol/chloroform extraction and separated on a 1% agarose gel with ethidium bromide. Southern blot probed with a <i>TRP1</i>-specific probe is shown. Nicked (N), linear (L), and closed circular (C) forms of the minichromosome are indicated. (B) Minichromosomes were immunoprecipitated with anti-HA antibody. Minichromosomes from <i>SCC3-HA6</i> but not <i>SCC3-HA6</i>-degron strains could be co-immunoprecipitated with Scc3 indicating the efficient depletion of Scc3 from the minichromosomes in the <i>SCC3-HA6</i>-degron strain. Since Pds5 association with minichromosomes is very salt-sensitive, they could not be co-immunoprecipitated with Pds5-HA6 in either the wild type or <i>PDS5-HA6</i>-degron strains under our experimental conditions.</p