Requirement of Heterochromatin for Cohesion at Centromeres Pascal Bernard, Jean-François Maure, Janet F. Partridge, Sylvie Genier, Jean-Paul Javerzat, Robin C. Allshire

To address whether the lack of Rad21 at centromeres in cells lacking Swi6 was due to an alteration of the cell cycle, ChIP experiments were performed on a homogenous cell population blocked in early mitosis. At the restrictive temperature (18°C), nda3-KM311 cells fail to assemble a mitotic spindle and arrest in a prometaphase-like stage due to spindle checkpoint activation (1, 2). Cohesion must be preserved under these conditions and indeed, Rad21-3xHA is highly enriched at both imr and otr regions of cen1 (Web. fig. 2A). However, this enrichment is clearly reduced in the absence of Swi6, as is the case in cycling cells. Thus, the effect of Swi6 on association of Rad21 with cen1 does not appear to be caused by perturbation of the cell cycle. This result is consistent with recent observations indicating that the level of Rad21 bound to chromatin does not vary throughout the cell cycle (3). However, Rad21-3xHA enrichment is two- to threefold higher in nda3-KM311 arrested cells than in cycling cells (compare Fig. 3A and Web fig. 2A). An increase in cohesin density at the centromere has also been observed in S. cerevisiae upon mitotic spindle disruption by nocodazole treatment (4). Because cohesin must be initially loaded on chromosomes during S-phase, this enhancement suggests the existence of a de novo loading process, perhaps triggered by spindle checkpoint activation.

Association of the Psc3/Scc3 Cohesin Component with Centromeres Also Requires Swi6

Next, we asked whether Swi6's influence on sister-chromatid cohesion was restricted to the presence of Rad21 at centromeres or whether it also affects other cohesin factors. For this, Psc3, the fission yeast counterpart of the cohesin subunit Scc3 (3), was tagged with green fluorescent protein (GFP) (5), and its chromatin association was determined by chromatin immunoprecipitation (Web fig. 2B). Psc3-GFP is clearly enriched at cen1 and this enrichment is abolished in the absence of Swi6. Thus, Swi6 is also required for Psc3 association with cen1. Because cohesin functions as a complex, these data suggest that Swi6 may be required for centromere association of all cohesin subunits.

Dissociation of Rad21 from Centromeric ura4 Is Not Due to Transcription in the Absence of Swi6.

Rad21 association with a centromeric marker gene is dependent on Swi6 (Fig. 3). Swi6 is required for silencing of the marker (6), so transcription of the ura4 marker in swi6 cells may contribute to Rad21 dissociation. To address this, the promoter of the centromeric ura4 marker was deleted to generate the transcriptionally incompetent P ura4 (7), and association of Rad21 with this sequence was tested in a swi6 background (Web fig. 3). Rad21 association with P ura4 was still lost in swi6 cells even in the absence of transcription of these sequences. In conclusion, it is not centromeric transcription in a swi6 background that promotes dissociation of Rad21. Instead, Rad21 association with centromeres is likely dependent on a physical link with Swi6-heterochromatin.

References and Notes

1. Y. Hiraoka, T. Toda, M. Yanagida, Cell 39, 349 (1984).

2. P. Bernard, K. Hardwick, J. P. Javerzat, J. Cell Biol. 143, 1775 (1998).

3. T. Tomonaga et al., Genes Dev. 14, 2757 (2000).

4. S. Laloraya, V. Guacci, D. Koshland, J. Cell Biol. 151, 1047 (2000).

5. A fragment corresponding to the last 410 bp of psc3⁺ was produced by PCR. This fragment contains a Pst I site at the 5' end and Bam HI site instead of the stop codon at the 3' end. This PCR product was cut by Pst I and Bam HI and cloned into pREP41-EGFPC (8) to create pPSC3-GFP in which the nmt1 promotor is replaced by the psc3 410-bp ORF fragment now fused in frame with GFP. pPSC3-GFP was cut at the unique Nde I site present within psc3 sequence and transformed into wild type cells. Integration at the psc3 locus was confirmed by PCR.

6. R. C. Allshire, E. R. Nimmo, K. Ekwall, J. P. Javerzat, G. Cranston, Genes Dev. 9, 218 (1995).

7. Promoter deleted version of ura4 was generated by PCR to remove bases -410 to -3 relative to the ATG of ura4. This PCR fragment was used to delete the promoter of centromeric ura4 located at cen1 otr1R (Sph I) in a chp1 background (9), and FOA resistant transformants were screened by PCR. P ura was outcrossed into Rad21-HA and Rad21-HA swi6 backgrounds. Association of Rad21 with P ura was assessed by ChIP as described, and transcription of both ura4 DS/E and P ura was monitored by PCR amplification of oligo dT primed cDNA.

8. R. A. Craven, D. J. Griffiths, K. S. Sheldrick, R. E. Randall, I. M. Hagan, A. M. Carr, Gene 221, 59 (1998).

9. J. F. Partridge, B. Borgstrom, R. C. Allshire, Genes Dev. 14, 783 (2000).

10. To identify regions of Rad21-3xHA association along chromosome arms, primer pairs were designed that spanned several intergenic regions on cosmids SPAC8C9 and SPAC56F8. All primer pairs had identical G/C:A/T content and a similar predicted Tm to those used to amplify ura4 and ura4-DS/E. Enrichment of sites was assessed in anti-Rad21-3xHA chromatin immunoprecipitates, relative to precipitates from untagged control cells. All ChIP assays were performed on identical numbers of cells. PCR products were quantified by phosphorimaging after -³²P incorporation.

Supplemental Figure 1. PCR within linear range of amplification was designed to identify arm cohesion sites (10). Amplification of the ura4 gene is shown.

Medium version | Full size version

Supplemental Figure 2. Swi6 affects Rad21 and Psc3 association with centromeres, independently of the cell cycle. (A) Cells were cultured overnight at 18°C to induce the nda3-KM311 mitotic arrest and ChIP was performed using antibodies to HA. T, total extract; IP, immunoprecipitated sample. Competitive PCR assay for enrichment of ura4 within cen1 (upper band) over the ura4-DS/E minigene allele (lower band). (B) Psc3-GFP association with cen1 in cycling cells was determined by ChIP using antibodies to GFP. Psc3-GFP immunoprecipates were analyzed for the levels of the imr1/otr1 junction PCR products (imr1/otr1) relative to the fbp1 control (fbp). Duplicate experiments are shown.

Medium version | Full size version

Supplemental Figure 3. Association of Rad21 with non-transcribed centromeric ura4 marker is dependent on Swi6. Upper panel, Rad21-3xHA association with a promoter-deleted version of the ura4⁺ marker (P ura4) inserted within cen1 was determined by ChIP using antibodies to HA. Competitive PCR assay for enrichment of P ura4 within cen1 (upper band) over the ura4-DS/E minigene (lower band). Lower panel, the promoter-deleted P ura4 marker is not transcribed. Transcription of both ura4 DS/E and P ura was monitored by PCR amplification of oligo dT primed cDNA. A fully transcribed ura4⁺ gene inserted at an ectopic location (ura4⁺ R-INT) was used as a control.

Medium version | Full size version

Supplemental Table 1. Distribution of Rad21 along chromosome 1 in swi6+ and swi6 cells. Brackets indicated the location of the cen1 site examined or the coordinates of the intergenic region analyzed by PCR after chromatin immunoprecipation using antibodies to HA . See (10) for details
Site	IP/T
	wt (no tag)	rad21-HA	rad21-HA swi6
cen1 (otr 1L (Hindlll))	0.8 ± 0.05	6.2 ± 2.48	0.7 ± 0.009
c8C9 (4589-5144)	0.05 ± 0.05	0.23 ± 0.03	0.21 ± 0.11
c8C9 (7034-7522)	0.04 ± 0.04	0.19 ± 0.03	0.27 ± 0.10
c8C9 (12457-13018)	0.001 ± 0.001	0.14 ± 0.02	0.22 ± 0.12
c56F8 (1919-2567)	0.25 ± 0.09	0.18 ± 0.03	0.22 ± 0.05
c56F8 (24758-25223)	0.06 ± 0.01	0.47 ± 0.10	0.53 ± 0.19
c56F8 (26417-27011)	0.07 ± 0.01	0.26 ± 0.07	0.23 ± 0.12
c56F8 (27978-28486)	0.19 ± 0.03	0.15 ± 0.05	0.27 ± 0.12
c56F8 (34349-34868)	0.04 ± 0.01	0.53 ± 0.16	0.46 ± 0.16
c56F8 (34848-35387)	0.06 ± 0.01	0.75 ± 0.29	0.79 ± 0.23