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

Abstract

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.

Figure 1.

Pericentromeric Mcd1 and Scc2 recruitment is defective in an ndc10-42 mutant. Scc2-3Flag Mcd1-6HA ndc10-42 cells (1406-43C) were staged in G1 with α-factor and then released into fresh medium containing nocodazole. Cultures were split following release from G1, and aliquots were cultured at 23°C (black squares) or 37°C (gray circles). Following mitotic arrest, cells were fixed and processed for ChIP using anti-HA or anti-Flag serum to detect Mcd1 (cohesin) and Scc2 (loader) distributions, respectively. Quantitation of DNA in the ChIPs, expressed as the percentage of the input DNA, is plotted as a function of the locations of the midpoints of the DNA fragments based on Saccharomyces cerevisiae Genome Database (SGD) coordinates. Centromere positions are indicated by black ovals (not drawn to scale). Association profiles for Mcd1 (A) and Scc2 (B) are shown throughout the CHRIII pericentromeric region.

Figure 2.

Mcd1 and Scc2 are recruited de novo to neocentromere-flanking regions. Cells containing the CHRIII ectopic centromere (RMY26) and isogenic wild-type cells (PMY656) were staged in G1 with α-factor and then released into fresh medium containing nocodazole. Cells were fixed following mitotic arrest and processed for ChIP using anti-HA and anti-Flag antibodies to determine the distributions of Mcd1 and Scc2, respectively. The Mcd1-binding profile (A) and Scc2-binding profile (B) at the ectopic location on endogenous CHRIII (gray circles) and in the presence of the ectopic centromere (black squares) are shown. The location of the ectopic centromere in RMY26 is shown with black ovals (not drawn to scale).

Figure 3.

Scc2 colocalizes with pericentromeric CARs. Mcd1-6HA Scc2-3Flag cdc16 cells (PMY615) were staged in G1 and then released into fresh prewarmed (37°C) media containing nocodazole. Cells were fixed and processed for ChIP using HA and Flag antisera following mitotic arrest. The profiles of Mcd1 (A,B) and Scc2 (C,D) association in the pericentromeric regions of CHRIII (left) and CHRXIV (right) are shown. Centromere positions are indicated with black ovals (not drawn to scale). Vertical lines, representing the locations of Mcd1 peaks, are included for comparison of Mcd1 and Scc2 peak binding locations.

Figure 4.

Scc2 colocalizes with arm CAR sites. Mcd1-6HA Scc2-3Flag cells (PMY615) were treated as described in the legend for Figure 3. The profiles of Mcd1 (A–C) and Scc2 (D–F) association at arm CAR locations on CHRXII (left and middle) and on CHRIII (right) are shown. Data are plotted on scales similar to those of pericentromeric regions for comparison.

Figure 5.

Chromosome biorientation reduces pericentromeric Mcd1 and Scc2 associations. Scc2-3Flag Mcd1-6HA cdc16 cells (PMY615) were staged in G1 with α-factor and then released into fresh media at 37°C containing either DMSO alone (vehicle control) or nocodazole in DMSO. Upon mitotic arrest (∼3 h), cells were fixed and processed for ChIP using anti-HA and anti-Flag sera. The profiles of Mcd1 (A) and Scc2 (B) association in the CHRXIV pericentromeric region in the presence (DMSO, gray circles) and absence (nocodazole, black squares) of bipolar kinetochore–microtubule attachments are shown. The positions of centromeres are indicated with black ovals. The data obtained from nocodazole-treated cells are the same data shown in Figure 3 and are replotted here for comparison.

Figure 6.

The affinity of Scc2 for chromosomal association is cell cycle-dependent. (A) Scc2-3Flag cdc16 cells (PMY615) and untagged cdc16 control cells (1847-22C) were staged in G1 with α-factor, or released from G1 into fresh media at 37°C. Aliquots of cells arrested in G1 or M phase were fixed and processed for anti-Flag ChIPs. The levels of sequences corresponding to CHRIII pericentromeric regions present in Scc2 ChIPs are shown in G1-arrested cells in the presence (black squares) or absence (light-gray circles) of the Scc2-3Flag epitope tag, and in Scc2-3Flag-tagged metaphase-arrested cells (darker-gray diamonds). The position of the centromeric DNA (SGD ∼115 kb) is indicated with an asterisk. (B) Scc2 protein levels were determined by Western blot. Strain PMY615 was synchronized in α-factor and then released into fresh media. Aliquots of cells were taken in the α-factor arrest at the time of release (0 min) and at 10-min intervals, as indicated. An asynchronous population of untagged Scc2 Mcd1-6HA cells (1847-22C, first lane) was processed as a specificity control for the Scc2-3Flag blot. The PVDF membrane was divided after transfer of proteins and probed with anti-Flag (Scc2), anti-HA (Mcd1), and anti-glucose-6-phosphate dehydrogenase antibodies, as a loading control.

Figure 7.

Scc2 localization to CARs is independent of functional cohesin complexes. Scc2-3Flag cells containing mcd1-1 (RMY31) or smc1-2 (1408-35D) were synchronized in α-factor and then released into 37°C prewarmed media containing nocodazole or into the same media at 23°C. After reaching metaphase arrest, cells were fixed and processed for Scc2 ChIP. The levels of Scc2 in the CHRIII pericentromeric region are shown in mcd1-1 (A) and smc1-2 (B) mutants grown at 23°C (black circles) or 37°C (gray squares). The position of the centromere is indicated with a black oval.