Msc1 links dynamic Swi6/HP1 binding to cell fate determination

Abstract

Eukaryotic genomes can be organized into distinct domains of heterochromatin or euchromatin. In the fission yeast Schizosaccharomyces pombe, assembly of heterochromatin at the silent mating-type region is critical for cell fate determination in the form of mating-type switching. Here, we report that the ubiquitin ligase, Msc1, is a critical factor required for proper cell fate determination in S. pombe. In the absence of Msc1, the in vivo mobility of Swi6 at heterochromatic foci is compromised, and centromere heterochromatin becomes hyperenriched with the heterochromatin binding protein Swi6/HP1. However, at the mating-type locus, Swi6 recruitment is defective in the absence of Msc1. Therefore, Msc1 links maintaining dynamic heterochromatin with proper heterochromatin assembly and cell fate determination. These findings have implications for understanding mechanisms of differentiation in other organisms.

Emil Heitz introduced the terms “euchromatin” and “heterochromatin” to describe cytological observations of eukaryotic chromosomes (1). Euchromatin describes the portion of chromatin that decondenses at telophase, whereas heterochromatin describes the portion that remains visibly condensed after the completion of mitosis.

The role of heterochromatin in regulation of epigenetic phenomena (heritable changes in gene expression that do not result from altered nucleotide sequence), such as dosage compensation and imprinting, is well established and has been recognized in a broad range of eukaryotic species including animals, fungi, and plants (2, 3). Cell fate determination via mating-type switching in the fission yeast Schizosaccharomyces pombe is a well-studied epigenetic phenomenon that requires the formation of heterochromatin throughout the 20-kb mating-type locus (4). This heterochromatin domain is critical for long-range directed recombination of sequences from silent donor loci to an expressed locus that specifies mating-type (5).

Central to the formation of heterochromatin is the posttranslational modification of histones. The Su(var)3–9 family KMTs (KMT1, formerly Clr4, in S. pombe) can di- or tri-methylate histone H3 at lysine 9 (H3K9me2/3) (6). This heterochromatic histone mark serves as a binding site for the chromodomain of Swi6/HP1, which can self-associate via its chromoshadow domain; thus, condensing chromatin (6). Swi6 can also be directly recruited by DNA binding proteins such as Atf1/Pcr1 or Taz1 (7, 8). Interestingly, Swi6/HP1 has recently been shown to be highly mobile, and binds heterochromatin only transiently; however, what role this mobility has in heterochromatin formation is unknown (9–11).

We sought to investigate the role of the ubiquitin ligase, Msc1, in heterochromatin assembly in S. pombe (12). Msc1 was originally identified as an Rb binding protein 2 homolog that acts as a multicopy suppressor of DNA damage checkpoints, and has been shown to interact genetically and molecularly with the ubiquitin conjugating enzyme Rhp6, the histone H2A variant Pht1, as well as histone deacetylase activity (13, 14). Intriguingly, we demonstrate that loss of Msc1 leads to distinct effects at centromere and mating-type heterochromatin. Centromeres become hyperenriched with Swi6/HP1, whereas Swi6/HP1 recruitment to the mating-type locus is impaired, leading to defects in mating-type switching. Also, our results link cell fate determination via mating-type switching with dynamic binding of Swi6/HP1 to heterochromatin.

Results

Msc1 Antagonizes Swi6 Occupancy at Centromeres.

Recent data have demonstrated that the ubiquitin ligase Msc1 is required for incorporation of the histone variant CENP-A (Cnp1) into centromere cores, and that msc1 can suppress a cnp1–1 mutation (12, 13). However, the effect of msc1Δ on heterochromatin flanking the CENP-A core has not been investigated. We sought to extend these findings by determining the consequence of the absence of Msc1 on the occupancy of heterochromatin components throughout centromeres by using ChIP. Surrounding the S. pombe centromere cores are inverted repeats denoted ImrR/L (innermost repeat right/left); immediately surrounding the Imr are the outer dg/dh repeats, where heterochromatin assembles (Fig. 1A). We mapped Swi6, H3K9me2, and H3K9me3 at high resolution by using a set of qPCR primers every 200–300 bp throughout the dg/dh repeats; these primers amplify products from all 3 centromeres. We also used primers specific for centromere 1; thus, assembled the data from ChIP experiments corresponding to centromere 1 (Fig. 1 A–D). In msc1Δ cells, we found an overall increase in Swi6 occupancy throughout centromere heterochromatin, as compared with wild type [Fig. 1B and supporting information (SI) Fig. S1A]. Interestingly, we observe an increase in enrichment of H3K9me2 throughout centromere 1 in msc1Δ, but a loss of enrichment of H3K9me3 (Fig. 1 C and D and Fig. S1A). The mechanism of changes in H3K9 methylation patterns in msc1Δ is enigmatic, but could be due to the increased occupancy of Swi6 binding to H3K9 methylated tails inhibiting Kmt1 from completing the reaction from H3K9 di- to tri-methylation. Similar effects on Swi6, H3K9me2, and H3K9me3 were also found at the subtelomere of chromosome 1 (Fig. S1B and data not shown). No overall changes in enrichment of histone H3, H2A, H2B, or the histone variant Pht1/Htz1 were detected in msc1Δ compared with wild type, suggesting that the observed msc1Δ phenotypes are not due to changes in overall histone occupancy or changes in histone variant incorporation (Fig. S2 and data not shown).

Fig. 1.

Msc1 antagonizes Swi6 occupancy throughout centromere 1. (A–D) ChIP analysis using qPCR primers throughout centromere 1 (see schematic representation below individual graphs) in wild type and msc1Δ mutants. ImrL/R, innermost repeats left/right; DG and DH, outer repeats. Fold enrichment for Swi6, H3K9me2, or H3K9me3 was calculated as the ratio of ChIP for each primer set versus the euchromatic locus gpd3⁺. (E–G) At the top, a schematic representation of centromere 1 with the indicated qPCR primers corresponding to 4 loci in centromere 1. At the bottom, ChIP analysis in wild type and msc1Δ mutants treated with the histone deacetylase inhibitor TSA. For simplicity, control strains not treated with TSA are indicated in A–D. (H) Western blot analysis of Swi6 protein levels in wild-type or msc1Δ cells, using histone H3 as a loading control. (I) ChIP analysis for HA-Msc1 throughout centromere 1. A negative control ChIP using the HA antibody in a wild-type strain lacking HA-Msc1 is also shown (gray line). Fold enrichment was calculated as the ratio of ChIP for each primer set versus a primer set for the rDNA ETS. Error bars indicate SEM.

Recent data have shown that Swi6 binds with equal affinity to H3K9me2 and H3K9me3. Therefore, the increase in Swi6 binding in msc1Δ is most likely the result of some other mechanism and not merely due to changes in H3K9 methylation (15). Western blot analysis of msc1Δ and wild-type protein extracts revealed Swi6 levels were similar in both, excluding the possibility that msc1Δ cells simply have more Swi6 available to bind centromeres (Fig. 1H). We could not rule out changes in H3K9 methylation in msc1Δ contributed to the increase in Swi6 enrichment at the centromere; therefore, to equalize the levels of H3K9me2 and H3K9me3 between wild-type and msc1Δ cells, we used the histone deacetylase inhibitor trichostatin A (TSA). Treatment of S. pombe cultures with TSA results in an global loss of enrichment of H3K9 methylation and Swi6 occupancy, because histone deacetylases act upstream of the H3K9 methyltransferase, Kmt1 (6). We found an overall reduction of both H3K9me2 and H3K9me3 in wild-type and msc1Δ cells treated with TSA. Importantly, on TSA treatment, H3K9me2 enrichment was reduced to similar levels in wild type and msc1Δ, as was H3K9me3 (Fig. 1 F and G). Strikingly, although TSA treatment nearly eliminates Swi6 occupancy at 4 loci throughout the centromere in wild-type cells (8.1-, 4.3-, 6.8-, and 13.1-fold decreases in enrichment versus untreated cells for primers A, B, C, and D, respectively), TSA-treated msc1Δ cultures still retain significantly more Swi6 (2.3-, 1.7-, 1.6-, and 2.6-fold decreases in enrichment versus untreated cells for primers A, B, C, and D, respectively) (Fig. 1E).

To rule out indirect effects of msc1Δ mutants, we analyzed expression of 17 genes known to be involved in heterochromatin formation, and found no significant changes in their expression (Table S1). Also, HA-Msc1 is enriched throughout centromere 1,consistent with a direct role for Msc1 in heterochromatin regulation at centromeres (Fig. 1I).

msc1Δ Results in Enhanced Silencing of a Reporter Gene Integrated at Centromere Heterochromatin.

Expression of reporter genes inserted into the heterochromatic centromere repeats in S. pombe (referred to as dg or dh repeats) is repressed by heterochromatin spreading into the reporter (16). The ura4⁺ reporter is commonly used to study this activity, because both active and repressed states can easily be monitored. When ura4⁺ is expressed, cells have the ability to grow on media lacking uracil. When ura4⁺ is repressed, cells have the ability to grow on media containing the counterselective toxin 5-fluoroorotic acid (5-FOA). Wild-type strains with a ura4⁺ reporter gene integrated into the dg/dh repeats of centromere 1 (otr1::ura4⁺) grow on both media supplemented with FOA and media lacking uracil, demonstrating that ura4⁺ repression is unstable, or “leaky” (Fig. 2A). Intriguingly, deletion of msc1 results in enhanced repression of otr1::ura4⁺, as shown by the loss of growth on medium lacking uracil (Fig. 2A). Quantitative real-time RT-PCR demonstrated a decrease in steady-state ura4⁺ transcripts in an msc1Δ strain, as compared with wild type (Fig. 2B). ChIP revealed an increased enrichment of H3K9me2 and Swi6 at the ura4⁺ reporter in msc1Δ strains relative to wild type, consistent with enhanced repression (Fig. 2C), whereas ChIP using a kmt1Δ strain as a control demonstrated an expected loss of histone H3K9me2 and a loss of Swi6 (Fig. 2C). Enhanced reporter gene silencing in msc1Δ is consistent with the above results, showing increased recruitment of Swi6 throughout centromeres in msc1Δ (Fig. 1B). We also tested whether overexpression of msc1⁺ results in a loss of silencing at otr1::ura4⁺; however, strong overexpression of msc1⁺ led to retarded growth that precluded silencing assays (Fig. S3).

Fig. 2.

msc1Δ enhances silencing of reporter genes integrated at centromere heterochromatin. (A) Schematic representation of S. pombe centromere 1, detailing the integration site of the ura4⁺ reporter (otr1::ura4⁺), Core (centromeric core). (Lower) Serial dilution assay of mutants harboring otr1::ura4⁺. NS, no selection; −URA, medium lacking uracil; FOA, medium supplemented with 5-FOA. (B) Quantitative RT-PCR of ura4⁺ transcripts in wild-type and msc1Δ strains, normalized to the wild-type control. Error bars reflect SD. (C) ChIP analysis at otr1::ura4⁺ using qPCR primers specific for ura4⁺, normalized to wild-type levels of enrichment (set as 1.0), and calculated as the ratio of enrichment of each ChIP for ura4⁺ versus the euchromatic locus gpd3⁺. Error bars reflect SEM.

Cell Fate Determination via Mating-Type Switching Is Modulated by Msc1.

One critical function of heterochromatin in S. pombe is regulation of cell fate determination via mating-type switching. We observe a previously undescribed defect in mating-type switching in msc1Δ cells (Fig. 3). Homothalic strains of S. pombe have the ability to switch mating type from plus (h⁺) to minus (h⁻) and vice versa, whereas heterothallic strains are unable to switch. When grown on medium that promotes mating, switchable homothallic strains (h⁹⁰) are able to conjugate, because both mating types are present. The integrity of switching is commonly verified by staining cells within a colony with iodine vapors that can react with a starch like compound contained within yeast asci. This assay allows identification of asci-containing colonies via dark colony staining, whereas unswitchable heterothallic strains (h⁺or h⁻) or homothallic strains (h⁹⁰) that are defective for mating-type switching exhibit a white (unstained) phenotype. We plated msc1Δ h⁹⁰ cells at low density on mating plates and allowed single cells to form colonies. These colonies were then exposed to iodine vapors to assay for mating competency. Remarkably, this assay revealed the presence of switching defective msc1Δ h⁹⁰ cells (1.5% light staining colonies; see Fig. 3); whereas wild-type h⁹⁰ cells are completely switching competent (100% dark staining colonies). A swi6Δ h⁹⁰ control strain is completely switching defective (100% light staining colonies; see Fig. 3).

Fig. 3.

Mating-type switching defects in msc1Δ. S. pombe mating-type switching assay in wild-type, swi6Δ, or msc1Δ h⁹⁰ switching strains. Cells were plated on sporulation medium, allowed to form single colonies, and then exposed to iodine vapors. Dark staining indicates spores that are switching competent, light staining indicates colonies that are switching defective; 3 light and 1 dark staining msc1Δ h⁹⁰ colonies were picked from sporulation medium, recultured in rich medium, replated on sporulation medium, and exposed to iodine vapors. The schematic representation on the left indicates the area of the sporulation plate the light or dark staining colonies were plated.

We predicted that the alternative switching phenotypes induced by msc1Δ would be mitotically stable. To test this prediction, we picked light-staining msc1Δ switching defective colonies or dark-staining msc1Δ switching competent cells, recultured them in rich growth medium, and replated them on mating plates. The resulting colonies were then exposed to iodine vapors to assay for mating competence. This assay demonstrated that alternative switching phenotypes were indeed mitotically stable (Fig. 3). Importantly, we were unable to detect switching-competent revertants from light-staining colonies, but obtained switching defective colonies from dark-staining switching competent colonies (0.9% light staining colonies).

The heritable alternative switching phenotypes induced by msc1Δ suggests that switching competent or defective phenotypes might reflect alternative epigenetic chromatin states at the mating-type locus. We used ChIP to assay for the enrichment of H3K9me2/3 and Swi6 throughout the mating-type locus in wild-type and msc1Δ light-staining or dark-staining strains. We noted no significant change in H3K9me2 enrichment and a modest increase in H3K9me3 in msc1Δ switching defective strains throughout the mating-type region, as compared with wild type (Fig. S4). However, in contrast to the increase in Swi6 enrichment we noted at centromere 1, in switching defective msc1Δ cells [msc1Δ (light)], we observed a loss of enrichment of Swi6 throughout the mating-type region compared with both wild-type strains and switching competent msc1Δ cells [msc1Δ (dark)] (Fig. 4A and B); this loss was not due to a decrease in Swi6 levels in light staining msc1Δ cells (Fig. 1G, protein extracts were obtained from light staining msc1Δ cells). Intriguingly, although we see a striking effect on mating-type switching in the absence of Msc1, we do not detect any enrichment of Msc1 at the silent mating-type locus (Fig. 4C).

Fig. 4.

Loss of Swi6 at mating-type heterochromatin in msc1Δ is rescued by Swi6 overexpression. (A) Schematic representation of the S. pombe mating-type locus and qPCR primers used in B and C. (B) ChIP analysis of Swi6 in the indicated strains. Fold enrichment was calculated as the ratio of ChIP for each primer set versus the euchromatic locus gpd3⁺. Error bars reflect SEM. (C) ChIP analysis of HA-Msc1 using primer sets for the mating-type region. Fold enrichment was calculated as the ratio of ChIP for each primer set versus the rDNA ETS. Error bars reflect SEM. (D) Schematic representation of the KΔ::ura4⁺ integration at the mating-type locus, which replaces cenH (Upper). Serial dilution assay of a wild-type, stable Ura-Off strain growing on FOA that was crossed to msc1Δ, the resulting msc1Δ strain harboring KΔ::ura4⁺loses silencing as shown by growth on −URA and decreased growth on FOA (Lower). (E) A switching-defective h⁹⁰ msc1Δ strain was transformed directly either with a control pCAT or pNmt1-Swi6 plasmid; 4 individual transformants for each were isolated, plated on sporulation medium, and exposed to iodine vapors.

The results above (Figs. 1 and 2) demonstrated increased enrichment of Swi6 and enhanced silencing of a reporter gene at centromere heterochromatin in msc1Δ. In contrast, at the mating-type locus, we note a loss of Swi6 enrichment in msc1Δ switching defective cells. Thus, we also tested whether a reporter gene integrated into mating-type heterochromatin loses silencing in msc1Δ. We used a KΔ::ura4⁺ strain, in which a ura4⁺ reporter replaces the cenH at the mating-type locus (Fig. 4D) (17). This strain can be stably propagated in a silenced “Ura-Off” state (Fig. 4D) (17). When the Ura-Off KΔ::ura4⁺ is crossed to msc1Δ, the resulting msc1Δ KΔ::ura4⁺ strain loses silencing, as shown by growth on −URA and a loss of growth on FOA (Fig. 4D), consistent with Msc1 promoting silencing at mating-type heterochromatin, but antagonizing silencing at centromeres.

Rescue of the msc1Δ Mating Defect in the RNAi Mutant dcr1Δ or by Overexpressing Swi6.

The above results suggest that the switching defect observed in msc1Δ mutants is due to failed recruitment of Swi6 to the mating-type locus in msc1Δ. We wondered whether increased levels of Swi6 would lead to increased recruitment of Swi6 to the mating-type locus and rescue of the observed switching defect in the absence of Msc1. To test this idea, we transformed a switching-defective msc1Δ h⁹⁰ strain with a plasmid overexpressing Swi6 (pNmt1-Swi6). Overexpression of Swi6 was sufficient to rescue the mating-defect of msc1Δ h⁹⁰ cells in 4 independent pNmt1-Swi6 transformants, whereas 4 transformants with a control plasmid (pCAT) remain switching defective (Fig. 4E). To further corroborate these findings, we used the RNAi mutant dcr1Δ. In dcr1Δ cells, heterochromatin formation, including H3K9 methylation and Swi6 enrichment, is reduced at centromeres, but heterochromatin formation (and mating-type switching) at the mating-type locus is unaffected (18, 19). Therefore, a dcr1Δ msc1Δ double mutant should rescue the msc1Δ mating defect, because loss of dcr1 should cause release of Swi6 from centromeres and allow it to become available to bind to the mating-type locus. Indeed, we find that this is the case. We analyzed haploid segregants among tetrads from a cross of a switching-defective msc1Δ h⁹⁰ strain crossed to a nonswitching dcr1Δ h⁻ strain (Fig. 5). Remarkably, h⁹⁰ dcr1Δ msc1Δ double mutant segregants recovered from this cross gave rise to switching competent, dark staining colonies (segregant 1d, see Fig. 5; additional h⁹⁰ dcr1Δ msc1Δ segregants not shown), whereas msc1Δ h⁹⁰ segregants remained switching defective (segregants 1c and 2a; see Fig. 5). Stability of the switching defective phenotype observed in msc1Δ h⁹⁰ segregants also demonstrates alternative switching phenotypes induced by msc1Δ are meiotically stable. As expected, wild-type h⁹⁰ (segregant 2d; see Fig. 5) and dcr1Δ h⁹⁰ segregants were switching competent (segregants 3c and 3d; see Fig. 5). Consistent with this observation, ChIP analysis revealed that dcr1Δ msc1Δ double mutants had even greater enrichment of Swi6 throughout the mating-type locus than wild-type and nonswitching msc1Δ strains (Fig. 4B).

Fig. 5.

msc1Δ switching defects are rescued by dcr1Δ. Switching-defective homothallic h⁹⁰ msc1Δ was crossed to a nonswitching heterothallic h⁻ dcr1Δ strain, haploid segregants from 3 tetrads of the cross were replica-plated onto sporulation medium, and exposed to iodine vapors. Genotype for the mat (h⁻ or h⁹⁰), dcr, and msc1 were determined by PCR and are indicated.

Mobility of Swi6 in Heterochromatin Is Compromised in msc1Δ Cells.

Recently, it has been shown that Swi6 is highly mobile and only transiently binds heterochromatin; however, whether this transient association is regulated is not known (9–11). We sought to determine whether the enhanced Swi6 enrichment at centromeres in msc1Δ also correlated with changes in Swi6/HP1 dynamic binding. To test this correlation in vivo, we used fluorescence recovery after photobleaching (FRAP) of GFP-Swi6 foci in wild-type and msc1Δ cells. FRAP allows the determination of the relative mobility of a GFP-tagged protein in live cells. GFP-Swi6 heterochromatin foci are photobleached and the fluorescence recovery at the foci is quantified; thus, reflecting an unbleached mobile population of the protein that exchanges with the bleached population. In agreement with previous reports, GFP-Swi6 within heterochromatin foci is quite mobile in wild-type cells, demonstrated by the short fluorescence recovery halftime after photobleaching (380 ms) (Fig. 6 A and D; see ref. 9). In msc1Δ cells, after photobleaching, fluorescence recovery of GFP-Swi6 slows dramatically (halftime 840 ms, P < 0.002; see Fig. 6 B and D). Also, a comparison of the average recovery curves for GFP-Swi6 in wild-type and msc1Δ cells demonstrates that, although GFP-Swi6 fluorescence recovers to nearly 93% in wild-type cells, GFP-Swi6 fluorescence only recovers to 78% in msc1Δ cells (Fig. 6C). This reduced recovery reflects an increase in the immobile population of GFP-Swi6 at heterochromatin. The decrease in Swi6 mobility observed in msc1Δ suggests that Msc1 promotes the dynamic nature of Swi6 association with heterochromatin.

Fig. 6.

Mobility of Swi6 at heterochromatic foci is compromised in msc1Δ cells. (A and B) Selected images from FRAP analysis of GFP-Swi6 in wild-type and msc1Δ cells. Arrow indicates foci of bleaching. (Right) Pseudocolor. (Left) Zoomed images of the fluorescence. (Scale bar, 1 μm.) (C) Averaged fluorescence recovery curves for wild-type and msc1Δ cells. Data are the average of 21 cells. Error bars indicate SD. (D) Recovery halftime in wild-type and msc1Δ cells. Error bars indicate SD.

Discussion

Our results reveal that in the absence of Msc1 centromere heterochromatin becomes hyperenriched with Swi6, whereas recruitment of Swi6 to the mating-type region is impaired. We find silencing of a reporter gene integrated within heterochromatin at centromere 1 is enhanced, whereas silencing at a reporter gene integrated within the mating-type locus is defective in msc1Δ. Our results also suggest that Msc1 is required for normal cell fate determination via mating-type switching. Enrichment of Msc1 at centromere, but not mating-type sequences, suggests that Msc1 acts at centromeres (Fig. 1H); however, we cannot rule out that Msc1 either associates transiently or weakly with the mating-type locus. Our results suggest that Msc1 may function to antagonize Swi6 binding at centromeres; thus, promote Swi6 recruitment to the silent mating-type loci. Alternatively, it is possible that Msc1 functions by actively recruiting Swi6 to the mating-type locus; however, our results agree with the former possibility, because defects in mating-type switching resulting from loss of Msc1 can be rescued by increased levels of Swi6. Also, because Msc1 acts as a ubiquitin ligase, it could potentially modify the Rik1/Kmt1 H3K9 methyltransferase complex, because we note changes in H3K9me2/3 at centromeres in msc1Δ; or Msc1 could potentially ubiquitinate Swi6 itself, although we have not detected any changes in higher molecular weight species that would indicate ubiquitination in wild-type and msc1Δ immunoblots for Swi6 (Fig. 1G and data not shown).

Msc1 was originally identified as a multicopy suppressor of DNA damage checkpoint mutations, and its PHD domains have recently been shown to possess ubiquitin ligase activity (12, 14). While this work was under way, Msc1 was shown to require the histone variant Htz1 (Pht1 in S. pombe) for its suppressor of DNA damage checkpoint activity and msc1 suppression of a cnp1–1 mutation requires Pht1 (13). However, we find no change in the occupancy of Pht1 at centromeres in msc1Δ (Fig. S2). Because Msc1 was recently shown to interact genetically with the SWR-C complex, which deposits Pht1, an intriguing possibility is that Msc1 ubiquitinates Pht1 or a component of the SWR-C complex, as was proposed (20).

This work defines a facilitator of Swi6/HP1 mobility and links heterochromatin dynamics to cell fate determination and heterochromatic silencing. It also provides the groundwork for discovering other components of this mechanism. Previous work demonstrated that, in stem cells, HP1 is more mobile than in differentiated cells (21). Potentially, the work presented here can be applied to the observation of “breathing chromatin” in stem cells (22). HP1 may be maintained in a highly mobile state in stem cells to facilitate recruitment to other loci during differentiation; thus, controlling gene expression and cell fate determination.

Materials and Methods

Growth of Yeast Strains.

S. pombe was cultured at 33 °C in nonselective YEA, medium lacking uracil, or medium supplemented with 850 mg/L 5-FOA. When indicated, cells cultured in YEA were supplemented with 15 μg/mL TSA. Strains used are listed in Table S2.

Construction of Yeast Strains and Plasmids.

Deletion mutants and epitope tags were generated by using a 2-step PCR method. Long ≈300-bp “adaptamers” were generated by PCR and then used in a second PCR with vectors that either delete the gene of interest with a kanMX marker (23). GFP-Swi6 and HA-Msc1 was generated by using the same method, with the second round of PCR from a vector harboring the nmt1 promoter driving expression of the GFP or HA fusion (24). Each deletion or tagged strain was confirmed by using PCR with primers outside the genes. The pNmt1-Swi6 plasmid was constructed by PCR amplification of the Swi6 coding sequence and cloning into the pNMT TOPO TA expression kit according to the manufacturers instructions (Invitrogen). The pCAT vector was supplied by the manufacturer (Invitrogen).

ChIP.

ChIP was performed as previously described (19); 3 μL of the following antibodies were used per 500 μL of chromatin: α-H3K9me2 (Upstate Biotechnology no. 07-441), α-H3K9me3 (Upstate Biotechnology no. 07-442), α-Swi6 (Abcam no. 14898), and α −HA (Abcam no. 9110). For detection of HA-Msc1, 10 mM DMA (for 45 min) was used to induce protein–protein cross-links after cross-linking with formaldehyde.

Quantitative PCR.

Quantitative PCR was performed on an MJ Research/BioRad Chromo4 Thermocycler by using Opticon 3.0 software. Fold enrichments and relative expressions were calculated by using the Pfaffl method on GeneEx software (BioRad) (25). Raw Ct values for each primer were input by using either gpd3⁺ (for all ChIPs except HA-Msc1) or the rDNA ETS (for HA-Msc1 ChIPs) as reference genes and normalized to wild-type controls. Essentially, this methodology normalizes the background enrichment from immunoprecipitations, represented by the Ct value obtained for either gpd3⁺or the rDNA relative to the Ct value obtained by immunoprecipitation by using a primer set of interest. This value is then normalized to the ratio of Ct values for the DNA isolated from input samples for the primer set of interest to input samples of the gpd3⁺ or rDNA control. This calculation yields fold enrichment that is simplified as follows and analogous to standard semiquantitative methods (26):

(immunoprecipitated primer set/immunoprecipitated gpd3⁺)/(input primer set/input gpd3⁺)

Primer sets used for ChIP are listed in Table S3.

FRAP.

Strains were grown overnight to saturation in minimal medium supplemented with 5 μM thiamin (EMM), then diluted by adding 500 μL of saturated culture to 5 mL of fresh medium. Strains were grown another 3 h to log phase and then washed with fresh medium, concentrated by centrifugation, and mounted on agar pads in EMM. FRAP was performed as previously described on a Zeiss LSM 510 confocal microscope by using the 488-nm line of an argon laser (9).