The Scc2/Scc4 cohesin loader determines the distribution of cohesin on budding yeast chromosomes

Cohesins mediate sister chromatid cohesion and DNA repair and also function in gene regulation. Chromosomal cohesins are distributed nonrandomly, and their deposition requires the heterodimeric Scc2/Scc4 loader. Whether Scc2/Scc4 establishes nonrandom cohesin distributions on chromosomes is poorly characterized, however. To better understand the spatial regulation of cohesin association, we mapped budding yeast Scc2 and Scc4 chromosomal distributions. We find that Scc2/Scc4 resides at previously mapped cohesin-associated regions (CARs) in pericentromeric and arm regions, and that Scc2/Scc4–cohesin colocalization persists after the initial deposition of cohesins in G1/S phase. Pericentromeric Scc2/Scc4 enrichment is kinetochore-dependent, and both Scc2/Scc4 and cohesin associations are coordinately reduced in these regions following chromosome biorientation. Thus, these characteristics of Scc2/Scc4 binding closely recapitulate those of cohesin. Although present in G1, Scc2/Scc4 initially has a poor affinity for CARs, but its affinity increases as cells traverse the cell cycle. Scc2/Scc4 association with CARs is independent of cohesin, however. Taken together, these observations are inconsistent with a previous suggestion that cohesins are relocated by translocating RNA polymerases from separate loading sites to intergenic regions between convergently transcribed genes. Rather, our findings suggest that budding yeast cohesins are targeted to CARs largely by Scc2/Scc4 loader association at these locations

Genome-wide mapping of the cohesin complex in the yeast Saccharomyces cerevisiae.

In eukaryotic cells, cohesin holds sister chromatids together until they separate into daughter cells during mitosis. We have used chromatin immunoprecipitation coupled with microarray analysis (ChIP chip) to produce a genome-wide description of cohesin binding to meiotic and mitotic chromosomes of Saccharomyces cerevisiae. A computer program, PeakFinder, enables flexible, automated identification and annotation of cohesin binding peaks in ChIP chip data. Cohesin sites are highly conserved in meiosis and mitosis, suggesting that chromosomes share a common underlying structure during different developmental programs. These sites occur with a semiperiodic spacing of 11 kb that correlates with AT content. The number of sites correlates with chromosome size; however, binding to neighboring sites does not appear to be cooperative. We observed a very strong correlation between cohesin sites and regions between convergent transcription units. The apparent incompatibility between transcription and cohesin binding exists in both meiosis and mitosis. Further experiments reveal that transcript elongation into a cohesin-binding site removes cohesin. A negative correlation between cohesin sites and meiotic recombination sites suggests meiotic exchange is sensitive to the chromosome structure provided by cohesin. The genome-wide view of mitotic and meiotic cohesin binding provides an important framework for the exploration of cohesins and cohesion in other genomes

Mcd1p Binding Profiles in Centromere-Proximal and -Distal Regions

<p>Cells containing Mcd1-6HAp were first staged in G1 using αF, and then released from G1 into medium containing nocodazole to arrest the cells in mitosis. For the centromere excision experiments (B–D), the cultures were divided in half after G1 arrest, and one half of each culture was treated with galactose for 2 h to induce centromere excision (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020260#s4" target="_blank">Materials and Methods</a>). Both the induced (acentric) and uninduced (centric) control cultures were then released from the G1 arrest into fresh medium and rearrested in mitosis. Once arrested in mitosis, cells were fixed in formaldehyde and then processed for ChIP using antiserum against epitope-tagged Mcd1p (Mcd1-6HAp) as an indicator of the cohesin complex. DNA isolated from the ChIPs and diluted input DNA not subject to immunoprecipitation were then subjected to PCR analysis using oligonucleotide primer pairs that amplify approximately 300-bp fragments within the indicated regions. Quantitation of DNA in the Mcd1p ChIPs, expressed as a percentage of the input DNA, is plotted as a function of the locations of the midpoints of those DNA fragments based on the SGD coordinates. Centromere position is indicated by an oval (not drawn to scale). (A) The Mcd1p association profile for the CHRI pericentric region in strain 1377A1-4B is shown. Mcd1p binding adjacent to <i>CEN1</i> is difficult to assess fully because of the presence of a moderately repetitive Ty element in the region from approximately 160 to 166 kb, indicated with the dashed line. Similarly, the Mcd1p binding profiles in the pericentric regions of CHRIII (B) and CHRXIV (C) are shown in the presence (black squares) and absence (gray circles) of <i>CEN3</i> and <i>CEN14</i> using strains PMY185 and PMY206, respectively. (D) The Mcd1p binding profiles for a centromere-distal region of CHRIII are shown for comparison in the presence (black squares) and absence (gray circles) of <i>CEN3</i>.</p

Mcd1p Binding within the Endogenous CHRIII Pericentric Region after Centromere Excision or Centromere Movement

<p>The Mcd1p binding profiles in the endogenous CHRIII pericentric region are shown in cells in which the centromere is absent, either because of centromere excision (gray circles, PMY185) or because of the movement of the centromere to an ectopic location on the right arm of CHRIII (black triangles, PMY318). PMY185 and PMY318 are highly related strains; PMY185 was one of the parental strains used to generate PMY318. In the centromere excision strain, Mcd1p binding was examined in the same cell cycle in which the centromere was lost, whereas in the ectopic centromere strain, Mcd1p association was determined many generations after centromere relocation (see text for further discussion). Mcd1p binding data from the centromere excision experiment are the same as those shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020260#pbio-0020260-g002" target="_blank">Figure 2</a>B, but are replotted here for clarity. <i>CEN3</i> normally occupies the interval between SGD coordinates 114382-114498.</p

A Functional Centromere–Kinetochore Complex Is Essential for Enhanced Pericentric Cohesin Association

<p>Cultures of isogenic wild-type (1846-15A) and <i>ndc10-42</i> mutant (1846-15C) cells were arrested in αF at 23 °C and then released into fresh medium containing nocodazole at 37 °C. After the cells arrested in mitosis (approximately 3 h), the cultures were crosslinked with formaldehyde and processed for ChIP using a monoclonal antiserum against epitope-tagged Mcd1p (Mcd1-6HAp) as an indicator of the cohesin complex. The cohesin association profiles in the pericentric regions of CHRIII (A) and CHRI (B) are shown for <i>NDC10</i> (black squares) and <i>ndc10-42</i> (gray circles) cultures. The positions of the centromeres are indicated by ovals (not drawn to scale). The dashed line in (B) indicates a region containing a Ty element. (C) To identify chromosomal regions depleted for cohesin binding in the absence of a functional kinetochore, the Mcd1p-ChIP-to-input fluorescence ratio obtained for each ORF and intergenic region in genomic microarray analyses of CHRV, CHRVI, and CHRIX in <i>ndc10-42</i> cells was divided by the ratio obtained for <i>NDC10</i> cells and plotted on a map of the chromosomes. Regions that demonstrated 2.5-fold or greater reduction in Mcd1p binding in the <i>ndc10-42</i> mutant are shaded dark green, while lighter green hues represent further fold reductions in Mcd1p binding. Regions where the magnitude of Mcd1p binding was similar in <i>NDC10</i> and <i>ndc10-42</i> cells are shown in gray. Gaps in the chromosomal maps are genomic regions not represented on the microarrays, while regions shaded blue were present on the arrays but gave no data during hybridizations for reasons described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020260#s4" target="_blank">Materials and Methods</a>. The location of the centromere on each chromosome is indicated by an asterisk.</p

Centromere Excision

<div><p>(A) <i>CEN1</i> on CHRI was replaced with a <i>CEN3-URA3</i> cassette flanked by head-to-tail-oriented site-specific recombination target sites (red arrows) for the R recombinase from <i>Zygosaccharomyces rouxii,</i> as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020260#s4" target="_blank">Materials and Methods</a>. This strain (1824-23B) contained the R recombinase under the control of a galactose-inducible promoter. Genomic DNA samples, taken prior to the addition of galactose to the culture medium (0) and at 0.5-h intervals for 4.5 h after the galactose addition, were digested to completion with PvuII (black arrows) and analyzed by Southern blot analysis using a 1.25-kb probe corresponding to CHRI SGD coordinates 151823 to 153080.</p> <p>(B) The percentage of centromere excision was determined for the timecourse shown in (A). Briefly, a phosphorimage of the Southern blot and ImageQuant software were used to determine the pixel intensities of the unexcised and excised bands (top and bottom bands, respectively). The percent excision was then calculated as the pixel intensity present in the excised band divided by the total pixel intensities of both bands at each timepoint.</p> <p>(C) A Southern blot analysis of centromere excision from CHRIII. The endogenous <i>CEN3</i> on CHRIII was replaced by R-recombinase target-site-flanked <i>CEN3</i> in strain 1829-15B, as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020260#s4" target="_blank">Materials and Methods</a>. The efficiency of centromere excision from CHRIII was determined by Southern blot analysis in two independent experiments using genomic DNA samples digested with SnaBI and a probe corresponding to CHRIII SGD coordinates 113799-114336. Lanes 1 and 3 represent uninduced controls, and lanes 2 and 4 represent the extent of centromere excision after 2 h of recombinase induction. The percent excision was determined as in (B).</p></div