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Genomics 91 (200

Cell cycle-dependent nucleosome occupancy at cohesin bindingsites in yeast chromosomes

Jie Liu a, Daniel M. Czajkowsky a, Shoudan Liang b, Zhifeng Shao a,⁎

a Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, P.O. Box 800736, Charlottesville, VA 22908, USAb Department of Bioinformatics and Computational Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA

Received 9 July 2007; accepted 14 November 2007

Abstract

In the budding yeast, cohesin is loaded onto the chromosome during the late G1 phase, establishes sister chromatid cohesion concomitant withDNA replication, and dissociates by the telophase. Here, using oligonucleotide tiling arrays, we show that, at the anaphase, nearly all of thecohesin binding sites contain nucleosome-free regions. The majority of these sites remain nucleosome-free throughout the cell cycle, consistentwith the suggestion of a DNA-binding anchoring protein present at these sites, although such a region could also serve as part of a marker for thebinding of cohesin in the next cell cycle. However, a third of these sites are remodeled in the G1 phase, being reoccupied by nucleosomes by theG1/S boundary, though their subsequent removal in the S phase appears to be independent of DNA replication. Whether this difference is a resultof other functions of cohesin or of the chromatin remains to be elucidated.© 2007 Elsevier Inc. All rights reserved.

Keywords: Nucleosome; Tiling; Microarray; Chromatin; Cohesion; Yeast

Cohesin is a multisubunit, ring-shaped protein complex thatis the principal component of the structure holding sisterchromatids together during mitosis and meiosis [1–3], though ithas also been implicated in a number of other chromosomalfunctions [4]. In the budding yeast, the cohesin complex, con-sisting of four subunits, Scc1, Scc3, Smc1, and Smc3 [5], firstbecomes associated with the chromosome during the late G1phase [6], establishes sister chromatid cohesion concomitantwith DNA replication during the S phase [7,8] and, followingthe cleavage of its subunit Scc1 at the anaphase, finally dis-sociates from the chromosomes by the telophase [9]. Eventhough this general order of events is now well characterized, itis not yet fully understood how cohesin mediates this robustphysical connection between sister chromatids [2]. So far,several models have been proposed, which include a singlecohesin ring encircling both sister chromatids [1], two inter-locking rings with each ring also directly binding the DNA ofone chromatid [3], and a single cohesin ring encircling one

⁎ Corresponding author. Fax: +1 434 982 1616.E-mail address: zs9q@virginia.edu (Z. Shao).

0888-7543/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.ygeno.2007.11.006

chromatid but attached to the other chromatid via an anchoringprotein [10].

These different models, each based on well-documentedexperimental observations [3,10–12], would imply differentpredictions on the local chromatin structure at the cohesinbinding sites, which has previously been suggested [13] toplay a role in the specific localization of cohesin along thechromosome [6,9,13–15]. For example, since the ring struc-ture formed by the cohesin complex is sufficiently large toencompass two-nucleosome-packaged chromatin [5], a modelof simple entrapment of the two sister chromatids would notnecessarily require an alteration of the chromatin structure atthe nucleosomal level. On the other hand, for those modelsthat include direct DNA binding, whether by the cohesincomplex itself or through an anchoring protein, the nucleo-somes at the cohesin binding sites may be removed to accom-modate this interaction, similar to the now well-understoodcase of transcription factor binding sites at which nucleosomesare generally depleted under most conditions [16–20]. Todetermine whether the local chromatin has been altered at thecohesin binding sites, we have investigated the cell-cycle-dependent nucleosome occupancy at each of the previously

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identified cohesin binding sites across more than three full-length chromosomes of the budding yeast Saccharomycescerevisiae using high-resolution oligonucleotide tiling arrays[6]. Strikingly, nearly all of the cohesin binding sites containnucleosome-free regions at the anaphase. Among these reg-ions, only about one-third undergo chromatin remodeling inthe G1 phase, while the remaining are nucleosome-free throu-ghout the cell cycle. These findings support the notion that thelocal chromatin structure is an integral part of a cohesin bind-ing site and could play an essential role in the demarcation andmaintenance of chromosomal cohesion.

Results and discussion

Nucleosome occupancy at cohesin binding sites at the anaphase

To determine the nucleosome distribution along S. cerevisiaechromosomes at the anaphase, mononucleosomal DNA waspurified from cells released from α factor synchronizationand hybridized to oligonucleotide tiling arrays. The quality ofthe synchronization, as well as the stage in the cell cycle, wasdetermined using FACS and confirmed with optical microscopyby DNA staining before harvesting (see Supplementary Fig. S1).DNA bound to mononucleosomes after micrococcal nuclease(MNase) digestion was purified, labeled, and hybridized to thetiling arrays, which cover chromosomes III, IV, and V and theright arm of chromosome VI at an average probe distance of27 bp. After normalization to the reference with total genomicDNA, a chromosome-wide map of nucleosome occupancy isproduced. Since gene transcription is largely repressed at theanaphase [21], there should be minimal interference from trans-binding factors on the nuclease digestion assay. Furthermore,since the Scc1 subunit of the cohesin complex is already cleavedat the anaphase [22], any steric occluding effect on the ac-cessibility of the nuclease owing to the tight linkage between thesister chromatids should also be minimal. To improve the signal-to-noise ratio of the dataset, a Gaussian averaging (σ=75 bp) isapplied. Nucleosome-free regions are then identified from thesedata using 1 standard deviation as the threshold (see SupplementaryData). Owing to variations from array to array, the standard devia-tion was calculated separately for each experiment.

As shown with chromosome III (Supplementary Fig. S2),there is a high degree of general agreement between these dataand those published by Yuan et al. [18] in terms of nucleosomeoccupancy, although the precise positions of individual nu-cleosomes are not always discernable owing to residual cross-hybridizations present with these arrays [23,24]. Overall, morethan 80% of the chromosomes are occupied by nucleosomes asexpected [18]. Despite the somewhat lower signal-to-noise ratioof these short oligonucleotide tiling arrays, the results are suf-ficiently reliable to identify nucleosome-free regions along thechromosomes, albeit at a lower spatial resolution compared withthat found by Yuan et al. [18].

For the budding yeast, Glynn et al. [13] identified a total of1095 cohesin binding sites with a genome-wide chromatinimmunoprecipitation (ChIP-on-chip) experiment. Similar re-sults were also identified in [6], as a number of authors have

also found [1,2,25–29]. Among these sites, a total of 222 arerepresented on our tiling array. Using these as a reference, weexamined the nucleosomal occupation state centered at thesecoordinates within a window of ±500 bp. It is noted that in theChIP-on-chip experiment [13], the average size of the chroma-tin fragments used in the immunoprecipitation was on the orderof 200–1000 bp and the hybridization was performed on anarray of long probes, with which the data were further processedwith a larger rolling window (see Supplementary Data). There-fore, the criterion chosen here should be appropriate.

Unexpectedly, we find that virtually all of the identifiedcohesin binding sites (216 of the total 222; N97%) are associatedwith at least one nucleosome-free region (Fig. 1A). The spatialextent of these nucleosome-free regions varies somewhat in arange from 150 bp to 1 kb, equivalent to about one to fivenucleosomes of packaged chromatin (including the linker DNAand 146 bp of DNA for the nucleosome core particle [30]). Usingthe maximum signal position in each nucleosome-free region atthese sites as the coordinate to compare with the coordinate ofthe cohesin binding sites, the (absolute) mean difference inposition is surprisingly small, only ∼160 bp or about a singlenucleosome. This is a much better overall accordance than whatmight be expected from a cursory examination of the raw data.However, since there are many more nucleosome-free regionsthan the number of the cohesin binding sites along the chromo-somes (Fig. 1B and Supplementary Fig. S2), it is important todetermine that this apparent association is statistically signifi-cant. Therefore, we estimated the probability with which thesecohesin binding sites would coincide with nucleosome-freeregions simply by chance. Using randomized 1-kb segmentsalong the chromosomes (see Supplementary Data), the occur-rence of nucleosome-free regions present in these segments canbe easily computed. With these calculations, we found that thep value is less than 10−5 for the complete dataset. Therefore, thecorrelation of nucleosome-free regions with the cohesin bindingsites is highly statistically significant.

Since chromosomes are still largely condensed at the ana-phase [31], a potential concern is whether the lack of nucleo-some occupancy in at least some of these regions could be aresult of the higher order folding of the chromatin. Such fold-ings could limit the access of MNase to the linker DNA, espe-cially under crosslinked conditions, resulting in a lower efficiencyof digestion. Since only those DNA fragments with a lengthconsistent with amononucleosomewere used in the hybridization(i.e., Fig. 1), regions with higher order structures could thenmistakenly appear as nucleosome-free, owing to the exclusion oflonger DNA from hybridization. To assess the effect of thispossibility, we purified the total DNA that remained after MNasedigestion under identical conditions without further length selec-tion, so that regions that have a poor MNase digestion efficiencyshould still produce a sufficient signal on the tiling array. Theresults from this experiment are in excellent agreement with theresults using only mononucleosomal sized DNA, with the samenucleosome-free regions identified in the two cases (Supplemen-tary Fig. S3). These results, therefore, support the conclusion thatthese nucleosome-free regions are indeed correlated with thecohesin binding sites that were previously identified.

Fig. 1. Nucleosome occupancy at the cohesin binding sites in yeast chromosomes at the anaphase. (A) The nucleosome occupancy at all 222 cohesin binding sites onchromosomes III, IV, and Vand part of VI is shown. Each block spans 1 kb DNA in length, centered at the documented coordinates of the cohesin binding sites. Thecohesin binding sites on the respective chromosomes are shown on the left (drawn in proportion). Regions not represented on the tiling array are coded in blue (ND)and the scale bar is in log2 with 1 σ as the unit (σ: standard deviation of the respective dataset). Here, 1 standard deviation from the mean is used as the threshold todetermine nucleosome occupancy. Clearly, most of these 1-kb segments contained well-resolved nucleosome-free regions (green). (B) As a comparison, a continuous31-kb region on chromosome III is shown, in which the majority of the chromosome is occupied by nucleosomes (yellow), consistent with previous findings [18].Arrowheads point to the cohesin binding sites within this chromosome region. Color coding: occupied regions in shades of yellow and unoccupied regions in shades ofgreen (see scale bar).

276 J. Liu et al. / Genomics 91 (2008) 274–280

It should be noted that the cohesin complex appears to extendover 20–50 kb of pericentric sequences on the budding yeastchromosomes, showing up as higher and broader peaks comparedto those on the arms in genome-wide ChIP-on-chip analysis[13,32]. All of these peaks correlated well with nucleosome-freeregions in our dataset. However, the width of the nucleosome-freeregions did not have a clear correlation with the width of thecohesin peaks found in the ChIP-on-chip experiments [13]. Thesignificance of this difference, if any, is not clear at present.

Loss of nucleosome at cohesin binding sites is not coupled toDNA replication

It is now well documented that sister chromatid cohesion isestablished concomitant with DNA replication [7,8,33]. This directcoupling with DNA replication raises the important question ofwhether the nucleosome-free regions at the cohesin binding sitesare generated when chromatid cohesion is established at the time ofDNA replication, or whether these nucleosomes are removedmuch

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later, in conjunction with the cleavage of the cohesin subunit Scc1.Therefore, we further examined the nucleosome occupancy at thecohesin binding sites at three different time points in the cell cycle:early in the S phase, when only a small fraction of the DNAcompleted replication, and late in the S phase, when themajority ofthe DNA was already replicated, as well as at the G2/M phase,when DNA replication was complete (Fig. 2A and SupplementaryFig. S4). Somewhat surprisingly, the data clearly show that theoverwhelming majority (N90%) of the cohesin binding sites arenucleosome-free at each of these three cell cycle stages. Therefore,DNA replication is not a prerequisite for the formation ofnucleosome-free regions at the cohesin binding sites and thecleavage of Scc1 that is known to trigger chromosome separationat the anaphase is likewise not correlated with the removal ofnucleosomes at these sites. Of course, whether these sites could beoccupied by other, as yet unidentified, DNA binding proteins [2],which could have a dependence on DNA replication, cannot bedetermined with the present assay and remains an open questionfor further examination.

Although for the majority of the cohesin binding sites, theoccupational pattern of nucleosomes does not significantlychange from the early S phase through G2/M to the anaphase,there is a small (b10%), but significant, fraction of the cohesinbinding sites that do exhibit a dynamic occupancy with a generaltrend of nucleosome loss with the progression from the S phaseto G2/M (Fig. 2B). A comparative analysis with the DNA rep-lication timing profiles [34] at these 18 sites failed to identify aclear temporal correlation with the loss of the nucleosome in theseregions though. Therefore, overall, these data are consistent withthe conclusion that the absence of the nucleosome at the cohesinbinding sites is not directly coupled to DNA replication.

A significant fraction of cohesin binding sites are reoccupiedby nucleosomes in the G1 phase

Excluding the minor fraction of the cohesin binding sites atwhich nucleosome occupancy is dynamic during the S phase(which could be owing to other functions of the cohesin [4]), anattractive possibility is that a constitutively nucleosome-freeregion is required at the cohesin binding site throughout the cellcycle. To determine whether this is indeed the case, we furtherexamined nucleosome occupancy with cells arrested at the G1/Sboundary with α factor. Contrary to the above expectation, athird of the cohesin binding sites are fully occupied by nucleo-somes at this cell cycle stage, while the rest remain nucleosome-free (Fig. 2C and Supplementary Fig. S5). Clearly, during theG1 phase, this subset of the cohesin binding sites has undergonechromatin remodeling to reload the histones back onto the DNA.It is also of interest to note that these G1-phase remodeled siteshave included all of those sites identified to exhibit a dynamicoccupancy during the progression of the S phase (i.e., Fig. 2B).Like the sites with a dynamic occupancy during S phase, none ofthese G1 remodeled locations exhibited similar replication timesor were correlated with any other identifiable functional traits(such as local histone modifications or proximity to tRNA).

Taken together, these results support the hypothesis that thesubstrate chromatin plays an important and integral role in cohesin-

mediated functions. Since the majority of the cohesin binding sitesare nucleosome-free throughout the cell cycle, an attractivehypothesis is that these regions alone or in conjunction withpossible histone modifications [35] in the immediately adjacentchromatin could serve as a marker for the loading and/or thelocalization of the cohesin complex after the dissociation of thecohesin complex at the telophase. Alternatively, these regions couldbe occupied by a specific DNA binding factor that demarks theseregions specifically for sister chromatid cohesion. If such a proteindoes exist, its direct binding toDNAcould exclude the formation ofnucleosomes, similar to those sites that bind transcription factors[16]. Yet if this is indeed the case, then, at least for a third of thecohesin binding sites, such a protein would have to dissociate fromthe DNA to allow the reoccupation of these sites by thenucleosomes. What remodeling complexes are responsible for theloading and the subsequent removal of these nucleosomes arequestions of great interest [10,36–40]. The small fraction of cohesinbinding sites that have an S-phase dependence does not appear tocorrelate with other identifiable functional elements of thechromatin and is scattered throughout the chromosomes. Whetherthe sites that exhibit such a dynamic nucleosome occupancy arerelated to other functions of the cohesin than chromatid cohesionrequires further examination. Clearly, the resolution of thesequestions is essential for an understanding of the mechanism thatgoverns chromosome cohesion.

Materials and methods

Cell culture

S. cerevisiae (derived from W303,MATa ho ade2-1 trp1-1 can1-100 leu2-3,112 his3-11, 15 ssd1, clb1–4tsura::URA3/GPD-TK [41]) was grown in YPD at25 °C and arrested at the G1/S boundary with 10 μg/ml α factor (GenScript,Piscataway, NJ, USA) for 3 h prior to release. To release the cells from the arrest,the cells were first washed to remove the α factor from the medium and thenresuspended in fresh medium supplemented with 50 μg/ml Pronase (Sigma, StLouis, MO, USA). The cells were then processed at 15 (early S phase), 40 (late Sphase), 65 (G2/M), and 95 min (anaphase) after release (see below). The cellcycle stage of the culture was monitored by FACS and optical microscopy byDNA staining (Supplementary Fig. S1). The final concentration of the cellculture was kept below 1.0 at OD600.

DNA purification

Mononucleosome preparation was carried out according to the publishedprotocols [18]. Briefly, cells were first fixed with 2% formaldehyde directly inthe medium for 15 min at 25 °C under constant agitation. The fixation wasterminated with the addition of glycine to the final concentration of 125 mM.The cells were then washed twice with 20 mM Tris–HCl, pH 7.5, 150 mMNaCl and resuspended in 1 M sorbitol, 50 mM Tris, pH 7.4, 10 mM β-mercaptoethanol containing 0.25 mg/ml lyticase (Sigma) to remove the cell wall(30 min at 30 °C). Spheroplasts were pelleted and resuspended in 1 M sorbitol,50 mM NaCl, 10 mM Tris, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, and 0.075%NP-40 (for permeabilization of the cell membrane; Sigma), and MNase(Worthington, Lakewood, NJ, USA) was added (to the final concentration of0.27 unit/μl) and incubated at 37 °C for 45 min. To recover the undigested DNA,proteins were digested with 0.1 mg/ml proteinase K (MP Biomedicals, Solon,OH, USA) and the formaldehyde crosslink was reversed by incubation at 65 °Covernight. After phenol extraction and ethanol precipitation, RNAwas digestedwith an RNase cocktail (25 U/ml RNase A and 1 U/μl RNase T1; Ambion,Austin, TX, USA). Mononucleosomal DNAwas purified by excising the 150-bpband from a 1.5% agarose gel.

Fig. 2. Cell cycle dependence of nucleosome occupancy at the cohesin binding sites. (A) Shown here is a side-by-side comparison of the nucleosome occupancy at allcohesin binding sites on chromosome IV at four different stages: S1 for early S phase, S2 for late S phase, M for G2/M phase, and A for anaphase. A 1-kb regioncentered at each cohesin binding site is shown. Clearly, the overwhelming majority of the cohesin binding sites is nucleosome-free at each of these cell stages,indicating that at least for these sites, chromatin remodeling is not necessarily involved before or after DNA replication nor after Scc1 cleavage. The full dataset isshown in Supplementary Fig. S4. (B) A small fraction of the cohesin binding sites exhibits an S-phase-dependent nucleosome occupancy. Only18 of such sites areidentified in our dataset and all are shown here, with chromosomal coordinates indicated on the right. In general, these regions exhibit a nucleosome loss from the Sphase to the anaphase. Yet, even for these sites, the decrease in nucleosome occupancy is not clearly correlated with the timing of DNA replication. (C) A sizablefraction of the cohesin binding sites (74 of 222) are reoccupied by nucleosomes during the G1 phase. Shown here is the nucleosome occupancy map of chromosome Vobtained with cells arrested at the G1/S boundary with the α factor (right). As a comparison, the same map but obtained at the anaphase is shown on the left. The fulldataset is shown in Supplementary Fig. S5. These data suggest that chromatin remodeling must be involved in this change in the nucleosome occupancy at these sites.The color coding is the same as in Fig. 1.

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To evaluate the effect of higher order chromatin folding, total DNA afterMNase digestion under the above conditionswas collectedwithout gel purification.The purified DNAwas amplified with random priming (BioPrime DNA LabelingSystem; Invitrogen, Carlsbad, CA, USA) and labeled with 50 μM biotin–ddUTPby terminal transferase (20 U/μl; Roche, Indianapolis, IN, USA). Total genomicDNA was also purified as the reference. To minimize possible bias owing to theamplification procedure, the genomic DNA was first sheared to 100–bp to 1-kbfragments with a Branson Sonifier 450, followed by the same amplificationprocedure by random priming and biotin labeling. Two independent experimentswere performed for each condition, including that with total genomic DNA.

Array hybridization and data analysis

Fifteen micrograms of biotin-labeled DNAwas hybridized to an Affymetrixyeast tiling array (SC3456a520015F; Affymetrix, Santa Clara, CA, USA),following the protocol in Lengronne et al. [6]. The tiling array used in theseexperiments contained 92,812 probe pairs covering a total of 2.55Mb yeast DNAsequence. The data were acquired using a GeneChip Scanner 3000 (Affymetrix).The raw data were first normalized with dCHIP [42]. To improve the quality ofthe data, 8251 probes (b9% of the total) were excluded based on the followingcriteria: (1) they have multiple hits in the S. cerevisiae genome; (2) the signalfrom the genomic DNA hybridization was too weak (a threshold of (PM −MM)/MM ≤ 0.25 was used; PM represents perfect match; MM represents mismatch).The signal intensity (PM − MM) along each chromosome was averaged with aGaussian rolling window (σ=5 bp). Each dataset was normalized by that of thegenomic DNA (two replicates) and log2 ratios were calculated. A region wasassigned as nucleosome-free if the log2 ratio was more than 1 standard deviationbelow the mean. The data discussed in this article have been deposited in NCBI'sGene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and areaccessible through GEO Series Accession No. GSE8130.

Acknowledgments

We thank Kim Nasmyth (University of Oxford, UK) forkindly providing the yeast strain used in this study. We are alsoindebted to Sitong Sheng and Yan Gao of this laboratory fortechnical assistance. Microarray data acquisition was performedin the Biomedical Research Facility at the University of Virginia(partially supported by a NIH Cancer Center Grant) with expertassistance from Yongde Bao and Alyson Prorock. This workwas supported by grants from the National Institutes of Healthto Z.S. (HG003702, GM68729, and HL48807).

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.ygeno.2007.11.006.

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