Cohesin association with yeast and human chromosomes has been studied4,9-15
, but the defining characteristics of association sites, and how cohesin gets to these sites, remained unclear. We analysed cohesin binding to chromosome VI of the budding yeast Saccharomyces cerevisiae
by chromatin immunoprecipitation (ChIP) followed by hybridisation to a high-density oligonucleotide array16
. The pattern of association in metaphase was similar for all cohesin subunits analysed, Scc1, Scc3, Smc3, and Pds5 (, and Supplementary Figure S1
). It was also similar before the establishment of sister chromatid cohesion, in cells arrested with the replication inhibitor hydroxyurea (Ref. 9
, and Supplementary Figure S1
). Cohesin bound 28 distinct sites, each spanning 1-4 kilobases (kb) in width. The intensity of association varied, with the strongest peaks found around the centromere, consistent with previous analyses9-11
. The distance between neighbouring cohesin association sites ranged from 2 to 35 kb. Almost all cohesin association sites were centred in intergenic regions where genes from opposite strands converged (), as previously suggested17
. Using an additional high-density array, we also mapped the association of Scc1 with chromosomes III, IV, and V (Supplementary Table 1 and Figure S2
). 91% (276 of 304) cohesin association sites identified lie at intergenic regions between converging genes, and of 328 convergent intergene regions 84% were bound by cohesin.
Figure 1 Cohesin localises to convergent intergene regions along budding yeast chromosome VI. Cells containing Smc3-Flag3 or Scc1-HA6 were arrested in metaphase by nocodazole treatment and processed for ChIP against the epitope tagged subunits. Enrichment in the (more ...)
We searched for shared motifs in the nucleotide sequences at cohesin binding sites, but failed to identify any. We therefore wondered whether the process of transcription itself was responsible for positioning cohesin towards converging intergenes. We tested this at the strong cohesin association site between STE2, a highly expressed gene on chromosome VI that is non-essential for vegetative growth, and BST1. Deletion of the STE2 promoter largely abolished cohesin association at this site (). Transcription therefore appears to direct the accumulation of cohesin downstream of the gene, and not the nucleotide sequence at this site that remained unchanged.
Figure 2 Cohesin is moved towards 3′-ends of genes by their transcription. a, Localisation of Scc1-HA6 around STE2 on chromosome VI in a nocodazole-arrested wild-type strain (top), and after deletion of the STE2 promoter (bottom). b, Localisation of Scc1-HA (more ...)
Deletion of the STE2
promoter may have introduced unwanted changes to chromosomal architecture, so we addressed whether physiologic changes in transcription would have similar effects on cohesin localisation. The MSH4
gene, close to the centromere of chromosome VI, is not expressed during vegetative growth, but upregulated upon entry into meiosis18
. The gene is covered by cohesin in mitosis. During meiosis, cohesin over MSH4
shifted towards the 3′-end of the gene, suggesting that MSH4
transcription causes cohesin relocalisation (). This further suggests that even the abundant cohesin around centromeres is positioned by transcription. We visualised meiotic cohesin by ChIP against Rec8, a meiosis-specific Scc1 homolog. Rec8 colocalised with Scc1 that also remained detectable in meiosis (Supplementary Figure S3
). Further differences compared to the mitotic cohesin pattern were all correlating with known transcriptional alterations in meiosis18
. E. g. YFR022w expression is downregulated in meiosis, and the locus now was covered by cohesin extending from the neighbouring PES4
/YFR024c association site (Supplementary Figure S3
). Thus, decreased transcription allowed cohesin to occupy otherwise inaccessible space along the gene.
We also analysed HSP30
on chromosome III, a gene strongly induced after heat-shock19
. In a metaphase arrested culture at 23°C cohesin covered much of HSP30
, but after shift of the culture to 37°C for 15 min HSP30
was free of cohesin and an increased signal was observed downstream of HSP30
at the neighbouring MAK32
site (, and Supplementary Figure S4
). This indicates that even once cohesin is already loaded onto chromosomes, and sister chromatid cohesion is established, transcription has the ability to relocate cohesin. Another gene on chromosome III, SRO9
, is downregulated in response to heat-shock19
. Correspondingly, we found that cohesin covered SRO9
after, but not before, shift to 37°C (). These results can be explained if cohesin rings encircle and are free to slide along chromatin. The elongating transcription machinery may push cohesin towards the 3′ end of transcriptional units, and recurring transcription may be required to maintain cohesin at these sites. Alternatively an event correlating with transcription, e. g. a certain type of chromatin modification, may be responsible for positioning cohesin.
We next addressed where cohesin is first loaded onto chromosomes after its synthesis in late G1. Cohesin interacts with, and its loading onto chromosomes depends on the Scc2/Scc4 complex8,20,21
. Cytological studies suggested that cohesin does not colocalise with Scc2/Scc4 on chromosomes, which made the molecular function of this complex difficult to rationalise. We analysed the localisation of Scc2/Scc4 along chromosome VI in cells arrested in early S-phase with the replication inhibitor hydroxyurea (). Scc2 and Scc4 showed an identical pattern of localisation, next to both telomeres, at the centromere, as well as at numerous places along chromosome arms. Association sites differed in width and, as expected, they were distinct from the sites occupied by cohesin. A similar pattern of Scc4 localisation was seen in G1 and metaphase arrested cells (Supplementary Figure S5
). The strong Scc2/Scc4 binding site at the centromere, most prominent in S-phase cells, may be responsible for loading of abundant cohesin around this locus. Comparison of Scc2/Scc4 localisation to transcriptional activity along chromosome VI revealed that at some places Scc2/Scc4 binding coincided with strong transcription (). Statistical tests suggest transcriptional activity may correlate with the binding of Scc2/Scc4 (Supplementary Note 1
). This for the first time describes the chromosomal distribution of the Scc2/Scc4 complex.
Figure 3 Cohesin loading at, and movement away from sites of Scc2/Scc4 binding. a, Localisation of Scc4-HA6 and Scc2-HA6 along chromosome VI in hydroxyurea-arrested cells. See the legend to chromosomal features in . b, Transcriptional activity along chromosome (more ...)
To investigate the role of the Scc2/Scc4 complex in cohesin loading, we analysed cohesin association in scc2-4
mutant cells. We were surprised to find cohesin now at the sites normally occupied by the Scc2/Scc4 complex (, and Supplementary Figure S6
). This suggests that an early step in the loading of cohesin onto DNA, before the step disrupted by the scc2-4
mutation, takes place at Scc2/Scc4 binding sites. Cohesin association under these conditions may be weak, which could explain why other methods failed to reveal it8
. We next analysed early events of cohesin loading in wild-type cells traversing synchronously through G1. Cells were arrested with the mating pheromone α-factor and released at 16°C to slow progression through G1. The first detectable association of cohesin with chromosomes, 10 min after release, overlapped with Scc2/Scc4 binding sites (, and Supplementary Figure S6
). After 30 min, cohesin became detectable also at the expected converging intergenic sites. This is consistent with the idea that cohesin is loaded onto chromosomes at Scc2/Scc4 binding sites, and thereafter relocates to more permanent places.
To see whether cohesin localisation by converging transcription was a general feature of eukaryotic chromosomes, we performed ChIP of cohesin in the fission yeast Schizosaccharomyces pombe
, followed by hybridisation to a high-density oligonucleotide array covering its chromosomes 2 and 3. Compared to budding yeast, genome organisation and chromatin biology in fission yeast resembles more closely the situation in higher eukaryotes. We found that the fission yeast Scc1 homolog, Rad21, preferentially localised to regions of convergent transcription, even though the peaks of association often extended over larger regions (, and Supplementary Note 2
). Therefore, cohesin distribution in response to transcription appears to be a conserved mechanism.
Cohesin localisation to convergent intergenic regions is conserved in fission yeast. ChIP was performed against the cohesin subunit Rad21-HA3 from logarithmically growing cells. A 240 kb long stretch from the right arm of chromosome 2 is shown.
Sister chromatid cohesion along chromosome arms is crucial for DNA repair by homologous recombination22
. The prominence of cohesin at converging intergenic sites therefore raises the question whether genes are arranged in a particular order to provide for a regular pattern of cohesin association. When we analysed the succession of left or right facing genes along budding yeast chromosomes III – VI we found that it appeared random. We did not find regions on these chromosomes where the likelihood of orientation of a gene following either a left- or right-facing gene was different from equal (Supplementary Note 3
). Fortuitous clusters of genes in tandem, that have been noted before23
, are expected in this random distribution. The consequent chance distribution of convergence sites agrees with the observed wide range of distances between neighbouring cohesin association sites. Thus, an important structural feature of yeast chromosomes, the sites of cohesin association, and therefore most likely sister chromatid cohesion, are spaced at random. Cohesin can act as a border of transcriptional domains24
. This seems now unlikely to be generally the case, as neighbouring genes in budding yeast show a high probability of co-regulation, irrespective of whether they are oriented in divergent or convergent orientation25
We describe here that cohesin, a critical structural component of the eukaryotic chromosome, does not have a fixed pattern of localisation. The possibility that the ring shaped cohesin complex encircles chromatin offers an intriguing explanation to this finding7
. After DNA is transported into the cohesin ring21,26
, cohesin may be able to slide along chromatin, responding to steric requirements of transcription and maybe other forms of chromosomal metabolism. Cohesin is loaded onto chromosomes next to its loading factor Scc2/Scc4, from where it relocates to places of convergent transcription. Scc2/Scc4 has been suggested to stimulate cohesin's ATPase activity, required to open the ring for loading onto DNA. Once loaded, preventing further Scc2/Scc4 action on cohesin would be vital to secure stability of the ring21
. Moving cohesin away from its loading sites, promoted by strong transcriptional activity found there, would be a safe way to achieve this. But even after sister chromatid cohesion is established cohesin is not stably trapped, and changes in transcriptional activity lead to further redistribution. This suggests cohesin provides chromosomes with an unexpectedly flexible architecture. This scenario does not exclude that places exist where cohesin engages in a more stable contact with chromatin. E. g. fission yeast cohesin's enrichment at centromeric heterochromatin is mediated by a specific interaction with the heterochromatin protein Swi6 (Ref. 13
). Centromeric cohesin is crucial to promote bipolar spindle attachment in mitosis5,27
, but in contrast to budding yeast, centromeres in fission yeast and higher eukaryotes cover large regions devoid of much transcriptional activity. Cohesin cannot therefore be confined there by convergent transcription, and during the evolution of larger centromeres cohesin may have acquired an alternative way to hold on to centromeric chromatin. Stable binding to heterochromatin, versus lateral mobility, may also differentiate cohesin during unloading of a subpool in prophase when chromosomes condense and transcription ceases28
How does transcription locate cohesin towards converging intergenes? Although we cannot exclude that transcriptional termination correlates with, or induces, a specific chromatin state that attracts cohesin, it is unclear how such a feature would be restricted to convergent as opposed to other sites of transcriptional termination. We therefore imagine that the sheer size of the transcription apparatus may be too large to pass through the cohesin ring, thus pushing it while translocating along the DNA. If cohesin is sufficiently mobile, and intergenic regions sufficiently small, cohesin would be passed on along tandem genes until reaching a site of convergence. Transcription through a cohesin ring would be unwanted, as it would leave the transcript trapped. In remarkable contrast, when the replication fork travels along chromosomes during S-phase, cohesion between the replication products might be a consequence of the fork traversing through the cohesin ring. What distinguishes the replication fork from the transcription apparatus so that it does not push cohesin, but instead may slide through it, will be an interesting question to be addressed.