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Article |
Address correspondence to James E. Haber, Rosenstiel Center and Dept. of Biology, Brandeis University, Waltham, MA 02454-9910. Tel.: (781) 736-2462. Fax: (781) 736-2405. email: haber{at}brandeis.edu
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Abstract |
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Key Words: Saccharomyces cerevisiae; mating-type switching; donor preference; deconvolution fluorescence microscopy; chromosome dynamics
Abbreviations used in this paper: 3D, three-dimensional; DSB, double-strand break; LacI, lactose repressor; lacO, lactose operator; MAT, mating-type gene; MSD, mean-squared change in distance; RE, recombination enhancer; tetO, tetracycline operator; TetR, tetracycline repressor; YPD, yeast extract-peptone-dextrose.
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Introduction |
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Donor preference during MAT switching is controlled by the recombination enhancer (RE), a small cis-acting locus control-type region, which acts at a distance to promote recombination along the entire left arm of chromosome III. In MAT cells, the Mcm1pMat
2p repressor complex binds to and inactivates the RE (Tanaka et al., 1984; Szeto and Broach, 1997; Szeto et al., 1997; Weiss and Simpson, 1997). Loss of Mat
2p binding causes MAT
cells to appear almost MATa-like with respect to donor preference (Wu et al., 1998). Deletion of the RE abolishes donor preference in MATa cells such that HML usage is reduced from 8590% to only 10% (Wu and Haber, 1996). Thus, the left arm of chromosome III exists in a constitutively recombinationally inaccessible state against which the RE works to activate HML as a donor in MATa cells.
Recent work by Sun et al. (2002) has demonstrated that RE-mediated donor preference during MAT switching is also regulated by the interaction of the forkhead protein Fkh1p with the RE. Donor preference in MATa cells is significantly reduced by deletion of FKH1 or by mutation of the Fkh1p/Fkh2p-binding sites within a subdomain of the RE. The fkh1 mutation also eliminates the mating typedependent activation of spontaneous heteroallelic leu2 recombination observed in MATa cells, such that the frequency of Leu+ is similar in MATa fkh1
and MAT
cells (Sun et al., 2002). However, the mechanism by which the RE, through interaction with this and other protein factors, controls recombination along an entire chromosome arm remains unclear.
One model to explain donor preference is that the left arm of chromosome III is stimulated for recombination in MATa cells and as a result, HML is more able to pair frequently with MAT in these cells than is HMR. In contrast, pairing of HML with MAT may be prevented in MAT cells. This region appears to be rendered inaccessible by an undefined mechanism.
Recent work from several labs in yeast, Drosophila, and mammalian cells has demonstrated that the dynamics of nuclear architecture are a key component of cellular processes such as replication and transcription (Belmont, 2001). Chromosomal loci move within the interphase nucleus, yet their motion is constrained within small territories. Centromeric and telomeric regions of S. cerevisiae chromosomes are more constrained than origins in the G1 phase, but all sites exhibit spatial constraint in S phase (Heun et al., 2001). Perhaps nuclear architecture, with respect to chromosome arrangement and dynamics, plays a role in donor preference during MAT switching in S. cerevisiae.
Here, we tested the hypothesis that donor preference reflects a mating typespecific difference in the mobility of opposite arms of chromosome III. Cytological analysis of GFP-tagged chromosomes in the nuclei of living cells was performed to visualize movement of the HML and HMR donor loci in G1, when MAT switching normally occurs. We observed rapid diffusive motion of all tagged loci, regardless of mating-type. However, this motion was constrained to small volumes within the nucleus. Interestingly, the HML locus exhibited a striking mating typedependent difference in the frequency of allelic pairing in diploid strains. Moreover, in MATa/mat cells, the left arm of chromosome III is less constrained than the right arm or in MAT
/mat
cells. Our analysis of re
strains demonstrates that the increased pairing and mobility of HML in MATa/mat
cells is dependent on the presence of the RE.
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Results |
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Mating typedependent associations of the left arms of chromosome III in diploid cells are dependent on the RE
Previous studies have demonstrated that deletion of the RE in MATa cells abolishes both HML donor preference in MAT switching and increases spontaneous heteroallelic recombination when one of the alleles is located on the left arm of chromosome III (Wu and Haber, 1995, 1996). To examine the potential role of the RE in the mobility of the left arm of chromosome III, we analyzed strains homozygous for a deletion of the RE by time-lapse microscopy as above. Similar to the other strains examined, the GFP-tagged loci in the re strains exhibited dynamic movement throughout the time courses (Fig. 2 C). However, in contrast to observations from MATa/mat
RE+ cells, the homologous chromosome arms are much less often close to one another in MATa/mat
re
/re
cells (Fig. 2 C; and Fig. 3 A). In fact, the loci are observed to be within 0.5 µm in only 8% (32/406) of the images from MATa/mat
re
/re
nuclei, compared with 23% (111/473) for the MATa/mat
RE+ strain (Fig. 3 A). Although on average the HML-proximal loci in MATa/mat
re
/re
cells are closer than in MAT
/mat
cells (1.17 ± 0.02 µm vs. 1.55 ± 0.02 µm, respectively; Fig. 3 A), the distribution of distances for the MATa/mat
re
/re
cells suggests that the range of motion is relatively more limited in these cells than in MAT
/mat
cells. Given that nearly 60% of images contain foci between 1 µm and 1.5 µm apart (Fig. 3 A), we propose that deletion of the RE in MATa/mat
cells results in tethering of the left arm of chromosome III. The RE is not active in MAT
cells, and, as expected, deletion of the RE in MAT
/mat
cells did not affect the behavior of the left arms of chromosome III (unpublished data). These data clearly demonstrate a role for the RE in the observed mobility of the left arm of chromosome III in MATa/mat
cells.
Pairing between allelic sites is not a general feature of homologous chromosomal loci
MAT cells preferentially use the right arm as a donor during MAT switching (Strathern and Herskowitz, 1979). MATa cells in which HML has been deleted switch efficiently using HMR as a donor (Klar et al., 1982), suggesting that HMRa is not inaccessible in MATa strains. We asked whether donor preference in MAT
cells reflects a difference in mobility or tethering of the right arm. Diploid cells of both mating-types homozygous for a lacO array insertion adjacent to the HMR locus on the right arm of chromosome III (Fig. 1 A) were imaged as described in Figs. 1 and 2. Similarly to the loci on the left arm of chromosome III, the GFP-tagged loci on the right arm were quite dynamic throughout each time course (unpublished data). Unlike the left arms of chromosome III, the tagged loci on the right arm were rarely observed in close proximity in diploid cells of either mating-type (Fig. 3 B). The right arms of chromosome III were within 0.5 µm in 7% (21/287) of images of MAT
/mat
nuclei and <2% (5/369) of images of MATa/mat
nuclei (Fig. 3 B), compared with 23% for HML-associated cases in MATa/mat
cells. However, there was a significant mating typedependent difference in the mean distance between the HMR-distal loci. In MATa/mat
cells, the loci on average were 1.49 ± 0.02 µm apart, whereas they were 1.06 ± 0.02 µm apart in MAT
/mat
cells. In addition, the range of the distance distribution in MAT
/mat
cells is quite narrow, with 63% of images containing foci between 0.75 and 1.25 µm apart (Fig. 3 B). This difference in proximity of the right arms may be reflected as a change in mobility of the right arm of chromosome III in MAT
cells, and along with the constraint of HML, could account for the preferred usage of the right arm of chromosome III during MAT switching in MAT
cells (Strathern and Herskowitz, 1979).
MATa cells deleted for the RE switch efficiently using HMR on the right arm of chromosome III as a donor (Wu and Haber, 1996). To examine the possibility that deletion of the RE in MATa/ cells results in a change in mobility of the right arm, we examined the relative movement of lacO arrays inserted adjacent to the HMR loci in MATa/mat
re
/re
cells. Unlike the HML-adjacent GFP tag, deletion of the RE in MATa/mat
cells does not have a profound effect on the behavior of GFP-tagged right arms of chromosome III. The distribution of distances between HMR-distal loci in MATa/mat
re
/re
cells resembles that for MATa/mat
RE+/RE+ cells (Fig. 3 B). Moreover, the right arm loci are rarely observed in close proximity in MATa/mat
re
/re
cells and are within 0.5 µm in only 5% (17/368) of images (Fig. 3 B), similar to the results from MAT
/mat
and MATa/mat
cells. These observations are consistent with donor preference data in which the right arm donor can be used both during MAT switching in haploid MATa RE+ cells in which the left arm donor has been deleted and in MATa re
cells where the left arm donor is inactivated by deletion of the RE (Wu and Haber, 1996). However, these data do not account for the relative increase in HMR usage in haploid MAT
cells compared with MATa RE+ cells undergoing MAT switching. Therefore, we conclude that there is a mating typedependent control on the mobility of allelic sites near HMR on the right arm of chromosome III, but propose that other factors, such as the constraint on HML in re
cells, also influence the association of HMR with MAT during homothallic switching.
To control for differences in mobility due to the position of the GFP-tagged sites along the chromosome, lacO arrays were inserted on the left arms of chromosome V in an analogous position (equivalent distance from the telomere) to that on chromosome III (Fig. 1 A). As visualized by LacI-GFP bound to these arrays, the movement and mean distance between the loci on the left arms of chromosome V resembled those of HML on the left arm of chromosome III in MAT/mat
cells (Fig. 3 C). The two GFP-tagged homologous loci were rarely observed within 0.5 µm (<10% of images) in cells of either mating-type (Fig. 3 C). The mean distance between the left arms of chromosome V was similar in both MATa/mat
(1.32 ± 0.02 µm) and MAT
/mat
(1.39 ± 0.02 µm) cells.
These data indicate that the high frequency of association between the left arms of chromosome III in diploid MATa/mat cells is not seen at other chromosomal loci, but rather is a unique mating typedependent feature of this particular chromosomal region.
Chromosomal locus confinement is mating-type dependent
Previous studies have shown that a given chromosomal locus does not freely explore the entire space of the nucleus; rather, movement is constrained (Marshall et al., 1997; Heun et al., 2001). The relative constraint on each of the three loci examined can be determined from the mean-squared change in distance (MSD) over increasing time intervals for each pair of loci as described previously (Marshall et al., 1997). At relatively large time intervals, the curves represent the degree of confinement, which can be estimated from the height of the plateau. The height of the plateau is proportional to the radius of confinement (or inversely proportional to the degree of constraint).
The MSD curves reveal significant differences between the strains and the loci examined. Mobility of the left arm of chromosome III is relatively less constrained in MATa/mat versus MAT
/mat
cells (Fig. 4 A). However, when the RE is deleted in MATa/mat
cells, the loci on the left arm of chromosome III become constrained to an even greater extent than in MAT
/mat
cells (Fig. 4 A). This is consistent with donor preference data which reveal that left arm usage during MAT switching is even lower in MATa re
cells versus MAT
RE+ cells (Wu and Haber, 1996). The right arm of chromosome III does not show a mating typedependent difference in the radius of confinement, and resembles the left arm of chromosome III in MAT
/mat
cells (Fig. 4 A). In addition, the calculated radius of confinement for the left arms of chromosome V is similar to that for chromosome III in MAT
/mat
cells, independent of mating-type (Fig. 4 B). Therefore, the left arms of chromosome III in MATa/mat
cells exhibit a cell typespecific increase in mobility relative to the other GFP-tagged chromosomal regions examined. It appears that, specifically in MATa/mat
RE+ cells, the left arm of the chromosome is less confined relative to other loci on the same chromosome and to the analogous region on a different chromosome.
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As above, the proportion of cells in which the two GFP spots are seen as a single focus is 10%, both in MATa and in MAT
cells. In cells that have two visible GFP spots, the mean distance between the two GFP-tagged loci is 0.75 ± 0.04 µm and 0.89 ± 0.05 µm in MATa and MAT
cells, respectively (Fig. 6 B, top). There is a higher proportion of images in which the GFP spots are within 0.5 µm of one another in MATa cells (26%) than in MAT
cells (10%; Fig. 6 A). In fact, 35% of MAT
cells examined contained GFP spots separated by at least 1 µm, whereas this was the case in only 16% of MATa cells. A t test of the distance data reveals that the observed difference in distance between HML and MAT in MATa versus MAT
cells is statistically significant, with p-values of 0.016 (1-tailed test) and 0.03 (2-tailed test). As a control to test the effect of fixation on the relative positioning of the nuclear foci, the mean distance between the GFP-tagged loci was determined from live cells (Fig. 6 B, bottom). The relative mean distance between each pairwise combination of loci remained the same as for fixed cells, despite the overall mild decrease in distance values. This discrepancy is likely due to rapid motion of foci during imaging, as observed upon examination of individual image sections compared with images obtained by quick projections of entire stacks. Nevertheless, although donor preference is not determined by prealignment of MAT with HML, it appears that proximity between these loci or their ability to interact plays some role in donor selection during MAT switching.
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To extend the analysis of the relative position of MAT and its two donors, we used strains bearing LacI-GFPbound lacO arrays of different sizes at HML and HMR loci as described previously (Fig. 5 A) and a TetR-(CFP)3tagged MAT locus (Fig. 7, A and B). We determined the 3D configuration of the MAT, HML, and HMR loci in nuclei of fixed intact G1 (unbudded) MATa and MAT cells. There is a mating typedependent difference in the 3D nuclear arrangement of these loci. In MAT
cells, the mean distance between MAT and HMR (0.58 ± 0.06 µm) is significantly shorter than that between HML and either of these loci (HML-MAT: 0.84 ± 0.06 µm; HML-HMR: 0.98 ± 0.08 µm; Fig. 7 C). This correlates with our findings in MAT
cells tagged at MAT and either HML or HMR described previously (Fig. 6 B), and supports a model in which HML is tethered and thereby excluded from interaction with MAT
. In contrast, we found MAT, HML, and HMR in MATa cells to be equidistant (0.8 ± 0.1 µm; Fig. 7 C). It is unlikely that the mating typedependent differences we observed between distance measurements are due to variations in the size of the fluorescent signal because data from strains in which the size of the TetR-(CFP)3 signal near the MAT locus is large correlate with data from strains in which the TetR-GFP signal is relatively smaller. Although our data do not enable us to propose a detailed configuration of the left arm of chromosome III, they do support a role for mating-type in regulating chromosome conformation.
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Discussion |
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In MATa/mat re
/re
cells, the region of the left arm containing HML is more severely constrained than either HMR or a site on chromosome V that is similarly close to its telomere; consequently, in a diploid, two lacO arrays at this position only rarely come close enough so that the two LacI-GFP spots cannot be distinguished. In contrast, in MATa/mat
RE+/RE+ cells, the lacO arrays become exceptionally mobile, with frequent pairings of the GFP spots. In fact, the freedom of movement of the lacO arrays on the left arm of chromosome III, as determined from MSD plots (Fig. 4), is significantly greater than that observed at HMR or on chromosome V. Thus, the RE exerts a profound influence on the localization and mobility of the left arm of chromosome III. Recently, we showed that the activity of RE depends on the binding of the transcription activator Fkh1p and that an array of Fkh1p binding sites is sufficient to activate HML (Sun et al., 2002). We suggest that Fkh1p interacts with other sites on the left arm of chromosome III; these sites would act to tether the left arm, perhaps to the nuclear envelope, and constrain its movement. Fkh1p interaction with these sites would then free the left arm to be more mobile within the nucleus.
It should be noted that all chromosome regions are constrained in their motion (Marshall et al., 1997; Heun et al., 2001). The left arm of chromosome III is exceptional because it goes from being more severely constrained (in MATa/mat re
/re
) to becoming more mobile (in MATa/mat
RE+/RE+) than the other loci that we and others have examined (Fig. 4). In contrast, deletion of the RE in diploid MATa/mat
cells did not significantly affect the behavior of the GFP-tagged HMR-distal loci. It is important to point out that in haploid cells undergoing MAT switching, the right arm donor is used
90% of the time in both MAT
cells and MATa re
cells. Interestingly, we observe a mating-type dependence in the distribution of distances between the tagged allelic loci near HMR, which would be consistent with an increased mobility of HMR to pair with MAT in MAT
cells (Fig. 3 B). Thus, although the primary control of MAT donor preference lies with the way RE controls the accessibility of the left-arm donor, there may also be a mating typedependent, but RE-independent, control on the movement and/or positioning of the right arm of chromosome III.
Perhaps reflecting the changes in constraint of the left arm of chromosome III, we also find significant mating typedependent differences in the relative positions of HML, HMR, and MAT within the nucleus (Fig. 7). This conclusion is somewhat different from that reached by Dekker et al. (2002), using a PCR-based technique to determine chromosome structure by the frequency with which various sites are close enough together to be cross-linked. In that paper, there was no apparent difference in the chromosome conformation of MATa and MAT cells, and the MAT-HML distance was
1.31.5 times that of MAT-HMR and HML-HMR (Dekker et al., 2002; Dekker, J., personal communication). In our case, in MATa cells, all three loci are equidistant, and in MAT
, the shortest distance is between MAT and HMR and the longest is between MAT and HML. The differences between our results may be attributable to the different methods and also to the locations of the lacO and tetO arrays relative to the pairs of PCR primers used by Dekker et al. (2002). In addition, Dekker et al. (2002) examined MATa cells that were arrested by
factor in the G1 phase whereas we used nonarrested G1 cells sampled from an exponential population.
We note one other difference between our work and that of Dekker et al. (2002). Using a direct microscopic measurement of the distances separating GFP- and CFP-tagged locations on chromosome III, we find distances that are four- to fivefold greater than those estimated by Dekker et al. (2002), who made assumptions about the length of chromosome III based on the size of a theoretical 30-nm fiber of 300 kb. We suggest that the yeast chromosome is likely to be more extended, so that the 100-kb distance between MAT and HMR, for example, is between 0.6 and 0.8 µm.
Nonrandom nuclear organization may account in part for the frequency of pairing of the HML-proximal loci in MATa/mat RE+/RE+ cells and the mating typedependent differences in the configuration of MAT, HML, and HMR in haploid cells. Support for the idea that certain loci tend to be close together due to the nonrandom organization of the nucleus stems from recent advances in the visualization of chromosome dynamics and positioning in living cells. Clustering of yeast telomeres near the nuclear periphery has been correlated with telomeric end maintenance and silencing (Gasser, 2001), and in Drosophila embryos the kinetics of somatic pairing are strongly influenced by nuclear position (Fung et al., 1998). In addition, recent studies of loci found to be rearranged in malignant cells were observed to be nonrandomly close together in normal interphase mammalian cells (Marshall, 2002). It is unlikely that the observed transient associations of the left arms of chromosome III in MATa/mat
RE+/RE+ cells represent actual recombination events, but rather, reflect a state which facilitates recombination between these loci and sites of DSBs, such as the MAT locus during MAT switching. More importantly, the data presented in this paper reveal both mating type and RE-dependent components to nuclear organization in yeast.
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Materials and methods |
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Fluorescence microscopy of fixed cells
To obtain accurate distance measurements between nuclear foci in intact cells, cells from exponentially growing cultures in yeast extract-peptone-dextrose (YPD, rich medium) were fixed by direct addition of PFA (2% final concentration) to 1-ml samples of cultures for 10 min at RT, pelleted, washed for 10 min in 0.1 M potassium phosphate, pH 6.6, pelleted again, and resuspended in 50 µl of fresh potassium phosphate. Fixed cells were maintained at 4°C before imaging at RT (25°C). Cell size was consistent among strains imaged, as determined by the ability of cells to fit within an imaging window of fixed dimensions.
Images of fixed cells were acquired with a CoolSnapHQ (Photometrics) camera mounted onto a DeltaVisionTM (Applied Precision) optical sectioning microscope on a TE200 base (PlanApo 100X, 1.4 NA objective lens; Nikon; He et al., 2001; Muhlemann et al., 2001; Rines et al., 2002). Data sets were obtained as 1620 optical sections per wavelength spaced 0.2 µm apart along the Z-axis using Chroma 86002 JP4 (CFP) and/or 41017 (GFP) filters. Out of focus information was removed using a constrained iterative deconvolution algorithm (Agard et al., 1989; softWoRxTM; Applied Precision). Distance measurements between nuclear foci were calculated from 3D coordinates of the center of intensity of each focus using the FindPoints and Distance components of the Image Visualization Environment Priism software program (Chen et al., 1996).
For MAT switching experiments, yeast strains were cultured in YPD (rich medium) at 30°C for 1012 h, resuspended in fresh yeast extract-peptone medium containing 2% raffinose at 1 x 106 cells/ml, and grown overnight at 30°C to a density of 12 x 107 cells/ml. A sample of uninduced cells was removed and galactose was added (2% final concentration) to the remaining culture to induce the HO endonuclease. Glucose was added (2% final concentration) to turn off expression of HO after 30 min. Samples of uninduced and induced cells were fixed in PFA. The frequency of cells with one or two visible GFP spots was scored from 50 nuclei per strain for each time point using an BX41 fluorescence microscope (UplanFl 100X, 1.3 NA objective lens; Olympus) with a Chroma 41017 (GFP Bandpass) filter.
Live-cell fluorescence microscopy
Live-cell imaging was performed using a DeltaVisionTM deconvolution microscopy system as described for fixed cells as well as on an IX70 base (PlanApo 100X, 1.4 NA objective lens; Olympus) with a Quantix (Photometrics) camera and a FITC filter. Yeast strains were cultured in YPD (rich medium) to mid-log phase, resuspended in fresh medium, and spotted onto depression slides containing 1.2% agarose in synthetic complete dextrose medium supplemented with a complete mixture of amino acids, 20 µg/ml of additional adenine, and 2% dextrose as described previously (He et al., 2001; Rines et al., 2002). Data sets were acquired at RT (25°C) as 1620 optical sections (0.2 µm apart) every 30 s for a total of 20 min per nucleus. Viability of strains under these conditions was confirmed by the cells' ability to undergo successful mitotic division. A total of at least 328 images were obtained from 817 independent nuclei per strain. Images were deconvolved and distance measurements obtained as described for fixed cells. MSD values were obtained from distance data using a statistics program (model 10.51 Xtra; MiniTab Inc.).
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Acknowledgments |
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This work was supported by National Institutes of Health grant GM20056 to J.E. Haber and the American Cancer Society Virginia Cochary Award for Excellence in Breast Cancer Research to D.A. Bressan.
Submitted: 12 November 2003
Accepted: 22 December 2003
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