Symmetry, asymmetry, and kinetics of silencingestablishment in Saccharomyces cerevisiaerevealed by single-cell optical assaysErin A. Osbornea, Yasushi Hiraokab,c, and Jasper Rined,1

aDepartment of Plant and Microbial Biology, University of California, Berkeley, CA 94720; bGraduate School of Frontier Biosciences, Osaka University, Suita565-0871, Japan; cKobe Advanced Information and Communication Technology Research Center, National Institute of Information and CommunicationsTechnology, Kobe, Japan; and dDepartment of Molecular and Cell Biology and California Institute for Quantitative Biosciences, University of California,Berkeley, CA 94720-3220

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2008.

Contributed by Jasper Rine, December 21, 2010 (sent for review September 27, 2010)

In Saccharomyces cerevisiae, silent chromatin inhibits the expres-sion of genes at the HML, HMR, and telomeric loci. When silentchromatin forms de novo, the rate of its establishment is influ-enced by different chromatin states. In particular, loss of the en-zyme Dot1, an H3 K79 methyltransferase, leads to rapid silencingestablishment. We tested whether silencing establishment wasantagonized by H3 K79 methylation or by the Dot1 protein itselfcompeting with Sir3 for binding sites on nucleosomes. To do so,we monitored fluorescence activity in cells containing a GFP genewithin the HML locus during silencing establishment in a series ofdot1 and histone mutant backgrounds. Silencing establishmentrate was correlated with Dot1’s enzymatic function rather thanwith the Dot1 protein itself. In addition, histone mutants thatmimicked the conformation of unmethylated H3 K79 increasedthe rate of silencing establishment, indicating that the H3 K79residue affected silencing independently of Dot1 abundance. Us-ing fluorophore-based reporters, we confirmed that mother anddaughter cells often silence in concert, but in instances whereasymmetric silencing occurs, daughter cells established silencingearlier than their mothers. This noninvasive technique enabledus to demonstrate an asymmetry in silencing establishment ofa key regulatory locus controlling cell fate.

silent information regulator 3 | disruptor of telomeric silencing 1 |lysine methyltransferase 4

The transcriptional output of a gene must integrate both ac-tivating and repressing forces. To turn off transcription,

repressors and silencing proteins must override activating signalsand vice versa. In the yeast Saccharomyces cerevisiae, transcrip-tional silencing of the cryptic mating genes at HML and HMRrequires the localization of silent information regulatory (Sir)proteins across the silenced loci (1–4). HML and HMR loci areconstitutively repressed in wild-type cells. However, conditionalalleles of the SIR genes have led to a greater understanding ofheterochromatin establishment, loss, and maintenance (5–9).Investigating the dynamics of transitions between heterochro-matin and euchromatin in mutants with altered chromatinstructure should reveal how chromatin modifications impact theformation of these two states.Silencing establishment is thought to occur in multiple steps

with Sir proteins initially recruited to the silencers flanking HMLandHMR and subsequently enriched throughout the silenced loci(3, 8, 10). The apparent spreading of Sir proteins from silencersrequires Sir2-dependent deacetylation of the critical histone res-idue H4 K16 and possibly other acetylated lysines as well (3, 11–14). Because nucleosomes composed of unacetylated histoneshave a higher affinity for Sir protein complexes, histone deacety-lation promotes the localization of Sir proteins throughout HMLandHMR, leading to loss of transcription at those loci (9, 15, 16).Unlike transcriptional activation that can occur on the order

of a few minutes, transcriptional silencing at the HML and HMR

loci is a longer process that requires multiple generations for fullpopulation-wide completion. Many events occur concurrentlywith silencing establishment such as DNA replication, cell-cycleprogression, movement of the silent locus from an internal nu-clear space to a more peripheral nuclear space, and an alterationof the chromatin status across the silenced region. Interestingly,DNA replication and nuclear localization are not required forsilencing establishment (17–19). Cell-cycle progression throughM-phase, however, is required for silencing establishment, butprogression through S-phase is required only at HMR and not atHML (5, 20, 21). Further, chromatin modifications influence thespeed of the silencing establishment process.Previously, we showed that when Sir3 protein is first in-

troduced to sir3Δ cells, typically two cell divisions are requiredbefore HML is silenced (22). In that study, we used a phenotypicoutput, the sensitivity of matΔ cells to α-factor, to report func-tional silencing at HML in a dividing population. The expressionstatus of the HML locus was assayed by monitoring a cell’s abilityto adopt the a-cell mating type and respond to α-factor, a featthat is possible only when HMLα is silenced. For this currentstudy, we monitored the progress of silencing using a destabilizedGFP reporter at HML (23). This approach complemented ourprevious technique that could evaluate a cell’s expression onlyduring G1 of the cell cycle, when the cells are competent to re-spond to α-factor. In contrast, the expression of the GFP can bemonitored continuously throughout one or more cell cycles andcan, in principle, provide a graded signal rather than a binaryresponse. Further, the GFP technique is noninvasive as it doesnot require micromanipulations or α-factor exposure.Yeast euchromatin is enriched for nucleosomes that contain

acetylation and methylation marks, whereas silent chromatinlacks these modifications (24, 25). When silent chromatin isformed, the levels of H4 K16 acetylation decline across the HMRlocus followed soon after by a decline in H3 K4 methylation andH3 K79 methylation (8). Interestingly, the removal of methyla-tion at these positions by deletion of the genes encoding theirmethyltransferases allows silencing to establish more rapidly asmeasured either by the association of Sir proteins with chromatinor by phenotypic changes (8, 22). Thus, the removal of methylmarks seems to be an integral step in silent chromatin formationrather than a passive consequence of silencing. By inference, H3K79 and H3 K4 methyl marks antagonize silent chromatin for-mation in some way.

Author contributions: E.A.O. designed research; E.A.O. performed research; Y.H. contrib-uted new reagents/analytic tools; E.A.O., Y.H., and J.R. analyzed data; and E.A.O. and J.R.wrote the paper.

The authors declare no conflict of interest.1To whom correspondence should be addressed. E-mail: jrine@berkeley.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1018742108/-/DCSupplemental.

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Among the sites of histone methylation, H3 K79 methylation(catalyzed by the Dot1 methyltransferase) has the largest an-tagonistic effect on silent chromatin formation (26–29). Recentbiochemical evidence suggests that Dot1 influences silencing intwo ways. First, Dot1 catalyzes the mono-, di-, and trimethylationstates of H3 K79 (27, 28, 30, 31). This particular lysine is locatedon the loss of rDNA silencing (LRS) face of H3, a surface whoseelectrostatic properties are important for association betweenthe nucleosome and the BAH domain of Sir3 (32–36). H3 K79methylation, therefore, interferes with the nucleosome’s abilityto adequately bind Sir3 (9). Dot1 can impact silencing formationin a second manner. Dot1 and Sir3 also compete directly forbinding to a basic patch on the N-terminal tail of histone H4. Sir3proteins at the telomeres can reduce Dot1 enrichment there bythis competition (37, 38).We are interested in determining the extent to which Dot1

impacts the kinetics of silent chromatin formation either in-directly through H3 K79 methylation or directly via competitionwith Sir3. To determine which possible mechanism of Dot1exerts a greater impact, we combined the tools developed tomonitor silencing dynamics continuously and at the single-celllevel (23) with classics of the genetics toolkit including DOT1overexpression vectors (37) and mutant alleles of histone genes(27). In addition, to determine whether cell-cycle dynamics orhistory within a pedigree affected silencing, we continuouslymonitored dividing populations of cells that encoded a destabi-lized GFP allele at HML, building on the foundation laid by Xuand Broach (23) for combining fluorescent reporters and flowcytometry in studies of transcriptional silencing.

ResultsKinetics of Silent Chromatin Formation. A reporter gene encodinga fast-folding, high-turnover GFP protein at the HML locusallows real-time monitoring of silencing-dependent expressionpatterns (23). The stability of fluorophores likeGFP can limit theiruse in the detection of dynamic changes. By fusing a Cln2 PESTdegradation tag to the carboxyl terminus of GFP, the half-lifeof GFP was significantly reduced. Cln2 undergoes constant 26S-proteosome-dependent degradation throughout the cell cycle due,in part, to its PEST sequence (39–41). It is estimated that the fold-ing time of the degron-tagged GFP is on the order of 20 min,whereas its half-life estimates range from 20 to 50 min (23, 40).Because a trade-off in reducing fluorophore stability is lower signalaccumulation, a nuclear localization sequence (NLS) was fused toGFP to concentrate GFP to the nucleus, thereby improving de-tection. All strains generated for this study are listed in Table S1.When placed within the HML locus, the GFP::PEST::NLS

construct is subject to Sir-protein–mediated silencing.We verifiedthat GFP fluorescence intensity was not detected unless silencingwas disrupted by either mutation of one of the Sir proteins (23) orchemical inhibition of Sir2 using nicotinamide (NAM) (Fig. 1).The expression output of GFP in unsilenced conditions was vi-sualized and quantified by both flow cytometry (Fig. 1 B–D) andquantitative microscopy (Fig. 1 E and F). In both instances, cellswith silenced HML loci exhibited minimal fluorescence, whereasthose with expressed HML had a 3-fold increase in fluorescenceintensity. This fold difference is narrower than molecular meas-urements of silencing such as qRT-PCR of the a1 mRNA (1,000-fold dynamic range) or microscopy experiments with a stableGFP reporter (10-fold dynamic range). However, the high turn-over rate of GFP is optimized to assay for dynamic sensitivityat the cost of signal strength. Despite the more restricted signalintensity, measurements of fluorescence intensity yielded nar-row 95% confidence intervals and were reproducible across rep-licates (Fig. S1).To determine whether our reporter strains were capable of

visualizing dynamic changes in expression status, we used flowcytometry to measure the fluorescence intensity of hml::GFP::PEST::NLS strains after silencing was disrupted by nicotinamideand then reestablished (Fig. 2 and Fig. S1). To do so, we grew cellsin nicotinamide to produce a silencing-compromised condition in

which GFP was expressed from the HML locus. By washing outthe nicotinamide and resuspending cells in fresh media, we couldobserve the loss of GFP signal as a proxy for the loss of tran-scription occurring at HML. The kinetics of silencing establish-ment measured using this approach were consistent with previousstudies (22, 42) showing that silencing is reestablished within 6 hfollowing restoration of Sir protein function. Interestingly, be-tween the first and the second time points taken after washing,a small elevation of GFP fluorescence was observed before a dropof signal intensity. Although the reason for this initial elevation insignal intensity was unknown, it is possible that fresh media itselfmay be stimulatory or that nicotinamide may have off-target ef-fects that dampen transcriptional activity or growth in general.We also observed slight differences in the starting signal in-tensities of some replicates. Some starting point signal intensitieswere slightly variable across replicates whereas others appearedcharacteristic of a given strain (Fig. S1).Our flow-cytometry–based assays of silencing establishment

confirmedearlier observations that the loss of chromatin-modifyingenzymes alters the rate of silencing establishment (8, 22). Cells

Fig. 1. The expression status of HML visualized using fluorescent markers.(A) A gene encoding a nuclear-localized, destabilized version of GFPexpressed from the URA3 promoter was integrated into the HML locusreplacing α1 and α2 genes. (B) Fluorescence intensity of cells containing thehml::GFP reporter in either SIR3 or sir3Δ backgrounds was measured usinga flow cytometer. Fluorescence intensity is plotted against forward scatter,a measurement proportional to cell size. (C) Fluorescence intensity meas-urements of cells containing an hml::GFP reporter are shown as a scaledhistogram. Both sir3Δ and 5-mM nicotinamide profiles are shown. (D) Thesame information in C is here illustrated as “violin plots,” which are sym-metrized kernel density plots with a small box plot illustrated inside them. Inthese plots, the wider the violin shape is at a given fluorescence intensity,the more cells are associated with that value. The white dot indicates thegeometric mean of the full population and the thick and thin lines are a boxplot. (E) Cells containing the hml::GFP reporter imaged by microscopy. (F)Signal intensities tabulated by quantitative microscopy for hml::GFP cells inthree cultures.

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deficient in the H3 K79 methyltransferase encoded by DOT1 es-tablish silencing more rapidly than wild-type cells. In the flow cyto-metry experiments, dot1Δ cells reached half-maximal signal intensityby an average of 86.3min after release from nicotinamide comparedwith 104.7 min inDOT1 cells (Fig. 2 and Fig. S2). Whereas previousstudies measured silencing using mating-type-dependent α-factorsensitivity, a binary readout, these new data indicated that the rapidrate of silencing establishment in dot1Δ cells was also evidencedby a graded output (GFP intensity) and was free from potentialcomplications ofα-factor treatment. By 240min, the dot1Δ andwild-type strains were indistinguishable with respect to the extentof silencing.

Mechanisms of Dot1 Antagonism on Silencing. dot1Δ cells establishsilencing up to one cell division faster than DOT1 cells as mea-sured by phenotypic outputs in single cells (22), 1–2 h faster asmeasured by Sir3 occupancy in ChIP studies (8), and up to 20min faster in the GFP-reporter experiments (Fig. 2 and Fig. S2).One explanation for the accelerated silencing observed in dot1cells may involve H3 K79 methylation impeding Sir3 assocationwith histone H3. Alternatively, Dot1 protein may directly slowsilencing by competing with Sir3 for association with a basicpatch on histone H4 (37). Both mechanisms impact Sir3 proteinbinding in telomeric regions of the genome, but their impact onsilencing at HML and HMR is not known, and their impact onthe rate of silencing establishment has never been investigated. Totest which mechanisms are responsible for Dot1’s ability to an-tagonize silencing establishment, we evaluated dot1Δ cells with anoverexpression plasmid containing either a functional copy of theDOT1 gene or a dot1 point mutant (Fig. 3 A and B, Table S2).This dot1 mutant allele replaced a single amino acid critical forcatalytic function but still allowed stable association with histone

H4 (37). If Dot1 catalytic activity were required to antagonizesilencing, then the catalytically inactive mutant should phenocopythe accelerated kinetics of silencing establishment observed indot1Δ cells. Conversely, if Dot1 protein were impeding the kineticsof silencing establishment due to its competition with Sir3 forhistone H4, the overexpression of Dot1—catalytically active ornot—should impede silencing establishment.The overexpression of a catalytically inactive version of dot1

accelerated silencing establishment to the same extent as thedot1Δ mutation (Fig. 3 C and D and Fig. S3A). In contrast, over-expression of functional DOT1 slowed silencing establishmentand altered levels of silencing at both the start and the finish ofthe time course (Fig. 3 C and D and Fig. S3A). Therefore, it wasthe modification status of H3 K79 that affected silencing and not

Fig. 2. The establishment of silencing visualized by flow cytometry. (A)Isogenic cultures of genotypes DOT1 (JRY9101), dot1Δ (JRY9104), and sir3Δ(JRY9103) were grown in 5 mM nicotinamide to derepress hml::GFP::PEST::NLS. Silencing establishment was measured in cultures washed of nicotin-amide. The mean fluorescence intensity in relative fluorescence units (RFIunits) is shown plotted against time for the three cultures. Ninety-five per-cent confidence intervals for these datasets are tabulated in Fig. S1. (B) Vi-olin plots of the same data in A are illustrated to show the spread of thedata. (C) The time to half-maximal signal intensity was calculated for eachstrain in each replicate as illustrated in Fig. S2. These values were averagedover the three replicates and SDs were calculated and tabulated.

Fig. 3. Overexpression of catalytically active and inactive Dot1. Cells carryingthe overexpression construct pGAL1::DOT1 (labeled DOT1-ox) or the catalyti-cally inactive overexpression construct pGAL1::dot1G401Rwere tested for theirkinetics of silencing establishment. dot1Δ cells and DOT1 cells were used ascontrols in these assays. (A) Strains grown in galactose mediumwere tested forglobal H3 K79 me3 levels by protein immunoblot with α-H3 K79 me3 antibody.Due to the overexpression of Dot1 proteins, the wild-type levels of Dot1 cannotbe markedly observed at these exposure levels. However, the presence ofwild-type Dot1 is confirmed by its enzymatic property, H3 K79 trimethylation.(B) H3 K79 me3 signals were quantified and normalized to H3 levels. Dot1 andH3 signalswerequantifiedandnormalized to PGK1. (C) Silencing establishmenttime-course assays of strains were performed under inducing (galactose) con-ditions. Mean fluorescence intensities are plotted against time after releasefrom nicotinamide. (D) Violin plots indicate the distribution of the data from C.(E) The time to half-maximal signal intensity was calculated for each strain ineach replicate and averaged over the three replicates.

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the overall concentration of Dot1. Together, these results in-dicated that Dot1 antagonized silencing via H3 K79 methylation.To provide an independent test of whether H3 K79 methyla-

tion was the critical feature affecting the kinetics of silencing, weperformed silencing establishment time-course assays in yeaststrains containing mutated histone proteins. Dimethylation (andby inference, trimethylation) of lysine at residue H3 K79 causesthe lysine side chain to assume an alternative conformation thatalters the local electrostatic potential by partially uncovering H4V70 and H3 L82 (43). The replacement of lysine with arginineprevents this electrostatic alteration, thereby mimicking theunmethylated conformation. If H3 K79 methylation were criticalin antagonizing silencing, then the H3 K79R mutant would beexpected to phenocopy dot1Δ cells with respect to the kinetics ofsilencing. For comparison, mutating H3 K79 to alanine abro-gates silencing potentially by disrupting the charge-based in-teraction between either H3 and Sir3 or H3 and H4 (32, 44). Wereplaced plasmids carrying HHT2::HHF2 alleles with plasmids car-rying the appropriate K79R mutation (hht2K79R::HHF2). Con-firming the dot1 mutant analysis above, the H3 K79R mutant cellsphenocopied the dot1Δ strains by accelerating the establishment ofsilencing (Fig. 4 and Fig. S3). Because Dot1 itself was unchanged inthese experiments, H3 K79 methylation status was the main featurethat correlated with silencing establishment efficacy.

Silencing Establishment in Dividing Populations of Cells. An advan-tage of the GFP reporter strain is its ability to report expressionfrom the HML locus continuously throughout the cell cyclewhereas earlier studies evaluated the expression status of HMLonly at G1. To determine whether quantitative microscopy couldbe used to image HML expression dynamics in dividing pop-ulations of cells, we measured the fluorescence intensity in sir3Δhml::GFP::PEST::NLS cells using quantitative, time-lapse mi-croscopy. This technique is distinct from flow cytometry in thatcell-cycle, cell history, and pedigree information can be de-termined in addition to GFP reporter intensity measurements.To visualize the range of GFP expression over time in the ab-sence of silencing, we quantified the GFP fluorescence of 10–20

cells and all their progeny using an automated system. The totalGFP intensities of each cell were graphed over time as a series ofline traces (Fig. 5) that illustrate the variation in fluorescenceintensity over time.The level of GFP fluorescence in a given cell integrates the

rates of GFP transcription, translation, folding, and degradation.Because the rates of GFP protein folding and degradation havebeen shown to remain constant throughout the cell cycle and areroutinely used as a negative control for proteins with altered cell-cycle folding or degradation (23, 40, 45), we assumed that theimpact of translation, folding, and degradation should be con-stant and that altered transcription would have the biggest im-pact on fluorescence intensity variation. Still, we measured thehalf-life of our GFP proteins over time by tracking the decline offluorescence intensity in cells treated with 2.5 μg/mL cyclohexi-mide, an inhibitor of translation. Indeed, GFP signal intensitydropped precipitously in response to cycloheximide addition(Fig. 5, Lower), reaching a half-maximal level after just 50 min.Cells exposed to cycloheximide showed synchronous loss offluorescence intensity even though they were grown asynchro-nously, further confirming that cell-cycle impacts on degradationrate were minimal.To determine whether quantitative microscopy could be used

to monitor silencing establishment, we conducted time-courseexperiments in which SIR3 hml::GFP::PEST::NLS-containingcells were grown overnight in 5 mM nicotinamide, rinsed ofnicotinamide, and mounted on a microscope slide for observa-tions of silencing reestablishment. Cells grown continuously in5 mM nicotinamide retained relatively high GFP signal in-tensities (Fig. 6A) similar to those of sir3Δ cells (Fig. 5). Con-sistent with previous experiments, GFP signal intensity decreasedover time when nicotinamide was washed from the media (Fig.6B). The loss of Dot1 function accelerated silencing establish-ment at the HML reporter (Fig. 6C), confirming that assays using

Fig. 4. Dot1 antagonized silencing through H3 K79 methylation. (A) Wild-type cells (JRY9121), dot1Δ cells (JRY9123), cells containing H3 K79A histonemutations (JRY9125), and cells containing H3 K79R histone mutations(JRY9127) were grown in 5 mM nicotinamide and then rinsed of nicotin-amide at the start of a time-course assay. The mean fluorescence intensity isplotted against time after release from nicotniamide. (B) Violin plots areillustrated to show the distributions. (C) The time to half-maximal signalintensity was calculated for each strain in each replicate and averaged overthe three replicates.

Fig. 5. Transcriptional variation and GFP degradation. (Upper) To measurefluorescent variability over time, cells of the genotype hml::GFP::NLS::PESTsir3Δ (JRY9103) were measured for fluorescence intensity using quantitativemicroscopy. Traces of the fluorescence intensity of each cell are displayed asline plots where each line represents the intensity of a given cell over the timecourse. Three replicates are depicted. (Lower) To determine the degradationrate of the GFP fluorophore, hml::GFP::NLS::PEST sir3Δ cells (JRY9103) wereexposed to 2.5 μg/mL cycloheximide 10 min before the start of a similar timecourse. The loss of fluorescence intensity over time is shown for three repli-cates. Fluorescence intensities are plotted in pixel intensity units.

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quantitative microscopy of an HML-encoded destabilized GFPreporter were consistent with previous techniques (8, 22).Moreover, this technique was uniquely able to report fluctua-tions in signal intensity in individual cells over time. The highlyvariable or “bumpy” quality of the signal traces during expressionand establishment was noteworthy.

Cell History Impacted Silencing Establishment. Previously, we ob-served a slight bias in the rate of silencing establishment such thatdaughter cells seemed slightly more likely to establish silencingbefore their mother cells. However, in the earlier study, a relianceon a G1-specific response to α-factor pheromone or microma-nipulation itself may have, in somemanner, caused or affected thebias and thus complicated its interpretation. Therefore, to testwhether mother and daughter cells establish silencing with thesame probability, we surveyed roughly 40 mother/daughter pairsfrom the silencing establishment assays in Fig. 6 for their relativepatterns of silencing establishment (Fig. 7 and Fig. S4). In thesegraphs, the mother cell’s trace (blue) began earlier and thedaughter cell’s trace (red) arose after the daughter cell had grownto>326 pixels in size. Depending on the concordance between themother and daughter fluorescence intensity traces, we estimatedwhether the mother cell had greater signal than the daughter orvice versa. If the traces overlapped, we called the pair “synchro-nous.”Of the 39 traces, 24 had near perfect synchronicity betweenthe mother and daughter fluorescence intensity traces. Twelvepairs showed a slight bias in silencing dynamics in which thedaughter cell silencedHML before the mother (daughter silencesfaster). And in three cases, the mother cell silenced HML beforethe daughter (mother silences faster). The data mirrored andextended the trends previously documented in single-cell pedi-grees (22), independently confirming that mother and daughtercells usually established silencing in the same cell division but incases of asymmetry, the daughter cells established silencing morerapidly than the mothers.

HML Expression Variation Was Independent of Cell-Cycle Phase. Itwas possible that the observed fluctuations in HML transcriptionand silencing might be cell-cycle dependent. Indeed the URA3promoter used to drive expression of the GFP reporter gene atHML has been reported to be more easily expressed withinheterochromatin when cells are at G2/M than at other phases ofthe cell cycle (46). To test whether this variability correlated withcell-cycle phase, we used the data from the sir3Δ time courses(Fig. 5) and manually annotated the time of bud emergence (anapproximation of S-phase) and of mother/daughter separation(an approximation of G1). By normalizing and stacking the tracesof these different cells about the point of bud emergence, wecould potentially capture phase-dependent rises or falls in ex-pression. However, when these stacked traces were viewed en

masse or averaged, we detected no dependence of bud emer-gence on fluorescence intensity. The same was true for the pointof mother/daughter separation (Fig. S5). In short, there was noobvious dependence on cell cycle for the GFP expression trendsin these assays. The variability in expression could instead beattributed to stochastic aspects of gene expression or to micro-environmental factors beyond our control.

DiscussionSingle-cell–based assays have provided insight into variation,stochasticity, and dynamics of gene expression within populationsof cells. Quantitative real-time microscopy has revealed dynamicaspects of cell and molecular biology. In this study, we combinedthese two approaches to illuminate the pattern and kinetics ofsilencing establishment as a function of chromatin modification,cell history, and the cell cycle.

Dot1 Antagonized Silencing Through H3 K79 Methyl Status. Thisstudy evaluated how different mechanisms of Dot1 action impactsilencing establishment kinetics and provided independent sup-port of our past results indicating that dot1Δ cells establish si-lencing faster thanDOT1 cells.Moreover, we were able to test twocompeting models for how Dot1 antagonizes Sir-protein–medi-ated silencing. Our data indicated that Dot1 impacted silencingestablishment through H3 K79 methyl status and not throughdirect competition of the Dot1 protein with Sir3 for binding siteson nucleosomes. Both the catalytically dead Dot1 protein and thewild-type Dot1 protein bind histone H4 to the same extent (37).However, cells overexpressing a catalytically inactive Dot1 pro-tein phenocopied cells lacking the Dot1 protein altogether, in

Fig. 6. Establishment of silencing over time visualized continuously by quantitative microscopy. DOT1 (JRY9101) or dot1Δ (JRY9104) cells were grown in5 mM nicotinamide to derepress the hml::GFP::PEST::NLS reporter gene, and silencing establishment was then initiated by washing nicotinamide out of themedia. As a control, DOT1 cells were maintained in 5 mM nicotinamide media for the duration of the time course. The fluorescence intensity of each cell isillustrated as a line plot. Each graph represents ∼10–20 starting cells and all their subsequent descendants.

Fig. 7. Silencing establishment in mother–daughter pairs. Silencing estab-lishment between mother and daughter cells was investigated using datafrom Fig. 6B. (A) The fluorescence intensity for mother–daughter pairs isshown in Fig. S4 and bar plots of the data are shown here. Binomial testsperformed on different categories yielded the following scores: synchronousvs. mother, P value = 4.9 × 10−5; synchronous vs. daughter, P value = 0.065;and mother vs. daughter, P value = 0.035.

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both cases speeding the establishment of silencing. Furthermore,cells containing H3 K79R, an unmethylated lysine mimic, phe-nocopied dot1Δ cells in their silencing establishment dynamics,supporting the notion that Dot1’s catalytic activity opposes si-lencing establishment through the methylation status of H3 K79.H3 K79 methylation is important for silencing, for cell-cycle–

regulated gene expression, and for meiotic checkpoint control.Because Dot1 is a distributive enzyme and because mono-, di-,and trimethylation of H3 K79 disrupt telomeric silencing, thethree states were thought to encode overlapping information (30).However, specific mutants can independently disrupt either di- ortrimethylation of H3 K79, suggesting that the activity of the Dot1enzyme may be alterable (31). Further, a lack of overlap betweendimethyl and trimethyl marks genome-wide supports the notionthat distinct methyl status may inform some processes (31).Both the loss and the overproduction of Dot1 lead to a de-

crease in silencing strength measured by reporters located at twotelomere loci (26). At those markers, the overexpression of Dot1is thought to directly antagonize Sir3 binding to H4 and to de-crease Sir3’s affinity for H3 via H3 K79 methylation. Conversely,the loss of dot1 is thought to decrease silencing indirectly wherebya global depletion of H3 K79 methylation permits inappropriateSir protein binding genome-wide and a resulting dilution of Sirprotein concentrations at silenced regions (27). Given that dot1Δmutants have a decreased ability to silence genes at the telomeres,it may seem paradoxical that the dot1Δmutation increases the rateof silencing establishment at HML and HMR. Although over-expression of DOT1 leads to a reduction of silencing at HML andHMR (22, 36), dot1Δ deletion causes only subtle effects at HMLand HMR that are most obvious in silencing-compromisedmutants. This difference suggests that “dilution effects” may notimpact HML and HMR as strongly as they do reporters at telo-meres. BecauseHML andHMR loci use the combined associationof Abf1, Rap1, the ORC complex, and Sir1 to recruit Sir proteins,the rate of Sir protein recruitment may occur at near wild-typelevels at these loci even when Sir proteins are diluted by dot1 loss.In that case, the rate of silencing establishment would be a func-tion of a near normal rate of Sir protein recruitment at HML andHMR through interactions with ORC, Rap1, and Abf1, combinedwith an accelerated rate of Sir protein accumulation on nucleo-somes unabated by H3 K79 methylation.It is still unclear whether Sir protein binding eventually occurs

despite H3 K79 methylation albeit slowly, whether H3 K79methylation is diluted through replication and/or nucleosomeexchange, or whether H3 K79 methylation is enzymatically re-moved by some heretofore unidentified demethylase. We havenot ruled out the possibility that the accelerated rate of tran-scriptional silencing is due to some deficiency in transcription atthe HML locus. However, because the levels of transcription atHML are not impacted by H3 K79 methylation, this possibilityseems unlikely. Still, our results definitively indicate that H3 K79methylation impacts silent chromatin dynamics at theHML locus.

Individual Cells and Silencing Establishment. Although silencing ofmost cells occurs within two-cell divisions, a substantial decreasein transcripts from HML and HMR has been observed within30 min following Sir3 restoration in cells previously lacking it (8,42), and a minority of cells can even establish silencing withinone cell cycle (22, 23). We used the power of continuous live-cellimaging to examine silencing dynamics within the first cell-cyclephases following the removal of a silencing inhibitor. Althoughthe time required to transcribe, translate, and fold GFP limitsour resolution, several points were clear. First, there was con-siderable cell-to-cell variation in the level of HML expressionmeasured by GFP fluorescence. This variation between cells wasalso evident within individual cells over time. Second, we foundno correlation between variations in fluorescence intensity ateither of two recognizable points in the cell cycle. This analysisargues against models that suggest that low levels of transcriptionin G2 or M allow for easier silencing establishment in thosephases (46).

Asymmetric Silencing Establishment. In our previous work, motherand daughter cells usually established silencing synchronouslywithin the same cell division. However, when mother and daugh-ter cells established silencing asynchronously, the daughter cellswere more likely to silence HML than their mothers. It was con-ceivable that the asymmetry observed in our earlier study wasa consequence of the methods used rather than of the biologybeing studied. Because daughter cells must achieve a certain sizethreshold before budding, a mother cell divides before her daugh-ter cell in time and hencemoves pastG1 (the time when silencing isevaluated using the previous assay) before the daughter cell does.Therefore, were the mother and daughter to silence at the exactlythe same moment in time, the differences in their cell-cycle pro-gression rate could give the illusion of asymmetry.To determine whether the bias toward daughter cell silencing

was real or an artifact of G1 length, we performed an analysis onpairs of mother–daughter cells as they established silencing. Ouranalysis confirmed by this independent method that mother anddaughter cells do show a high degree of expression concordance.Nevertheless, 38% of cell divisions were asymmetric with onecell silencing before the other, and in those cases the daughtercell was significantly more likely to establish silencing than themother. Thus, mother–daughter cell bias could not be explainedby the shorter cell cycle of the mother cell.The mechanism behind the daughter cell bias in silent chro-

matin establishment remains unexplained but would be compat-ible with three or more classes of explanation. Perhaps the easiestto imagine is that asymmetric gene expression causes the accu-mulation of either silencing-promoting proteins in the daughtercell or silencing-inhibitory proteins in the mother cell. Second,an asymmetric inheritance of proteins or cytoplasmic materialbetween the two cells could result in a similar situation. In thisregard, the Sir2-dependent segregation of oxidatively damagedproteins to mothers provides contextual support for such possi-bilities (47). Finally, the phenomenon could be due to a biasedsegregation of sister chromatids into the two cells if a chromatidwith a silenced HML was more likely to segregate into thedaughter. A priori, one would expect such segregation biases tooperate at centromeres rather than at HML. Nevertheless, het-erochromatin has a role in segregation and it is formally possiblethatHMLmay be an indicator of chromatin status at other regionsof that same chromatid. At this point, all models of the mecha-nism of the daughter cell bias in silencing establishment remainspeculative, but previous examples of asymmetric phenomenahave proved to be a rich source of biological insight.

Materials and MethodsStrain Construction. W303-derived yeast strains containing the hml::pURA3::GFP::PEST::NLS were generated from Yex730 (23). The original Yex730strain was created by cloning the first 55 amino acids of the CLN2 PESTdegradation tag onto the carboxy-terminal end of yEGFP (Clontech) (40,48). This construct was transformed into a plasmid to produce the fusiongene URA3promoter::NLS::EGFP::PEST::URA3 terminator. The resulting con-struct was transformed into the HML locus, replacing the α1 and α2 geneswith the GFP construct.

Gene deletions were generated using the one-step integration of knock-out cassettes (49, 50). Strains containing DOT1 and dot1G401R overexpressionplasmids (JRY9108–RY9114) were produced by transforming the plasmidspTCG, pFvL18, and pFvL43 (gifts from F. van Leeuwen, Netherlands Cancer In-stitute, Amsterdam, and D. Gottschling, Fred Hutchinson Cancer ResearchCenter, Seattle) (30) into DOT1 and dot1Δ strains housing the GFP constructat HML (JRY9106 and JRY9107). A yeast strain lacking endogenous histoneH3 and H4 alleles, but carrying HHT2::HHF2 on a plasmid (JRY9119), was in-corporated with the destabilized GFP allele at the HML locus by a cross ofJRY9104 to JRY7989 (51). Toproduce strainswith differentmutations of histoneH3 (JRY9121–JRY9127), we introduced vectors containing mutant versions ofHHT2 (pMP6, pHCL80, and pHCL81, gifts from S. Briggs, Purdue University,West Lafayette, IN) (37, 52) into the strain with the GFP at HML (JRY9119 andJRY9120), thereby replacing the HH2::HHF2 plasmid.

Flow Cytometry. Cells were grown to 0.1 OD600 in SC medium (53) over twosequential nights of growth at 30 °C, diluting back the culture each day. Cells

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were harvested by centrifugation, fixed in a 4% paraformaldehyde/3.4%sucrose solution for 15 min at room temperature, and then washed andstored in a 1.2-M sorbitol, 0.1-M KPO4 solution, pH 7.5. Cells were stored at4 °C for a maximum of 24 h. GFP expression data were collected for eachsample using the FC-500 (Beckman-Coulter) flow cytometer. A total of100,000 cells were measured per run and gated to identify those that werewithin a specific size and granularity (∼40,000 cells/experiment). We usedFlow-Jo 7.5 analysis software (TreeStar) and the Bioconductor flowCorepackage for R (54, 55) for data analysis and display. For histograms anddensity plots generated in these programs, the Logicle scale is used. This isa scaled transformation that optimizes the display of low signals (56). Violinplots were generated using the Vioplot package for R (54, 57). These plotsillustrate a kernel density plot overlaid with a box plot. The height for thedensity estimator was set to h = 100 in all plots to standardize the images.For samples grown in nicotinamide, cells were grown in SC medium con-taining 5 mM nicotinamide over 2 consecutive night’s growth at 30 °C witha dilution between each night. For silencing establishment time-courseassays, cells were grown in 5 mM nicotinamide in SC medium over 2 nights at30 °C and then collected by centrifugation, washing, and resuspension in SCmedium. Time points were collected during washing (0 min) and at 30, 60,120, 240, and 360 min postwashing.

Microscopy. Fluorescence microscope images were obtained using acomputer-controlled fluorescence microscope system (DeltaVision; AppliedPrecision) based on an inverted fluorescence microscope (IX70; Olympus) withan oil immersion Plan-Apochromat 60× NA 1.4 lens (Olympus) for imaging oflive cells. This system was used in a temperature-controlled room set to 30 °C(58, 59). For time-course assays, cells were grown overnight to a maximum of0.1 OD in 5 mM nicotinamide + SC medium (53) at 30 °C. Live cells wereplaced on a 35-mm glass-bottom culture dish (MatTek) coated with Con A(from a stock of 0.2% Con A, 0.1 M NaCl, 20 mM Tris, 2 mM CaCl2, 0.5 mMMnCl2, pH 6.8), washed with SC medium, and overlaid with a 25-μL 2% low-melt temperature agarose (SC medium) pad. Images were acquired usingSoftWoRx software (Applied Precision) provided as part of the DeltaVisionsystem. A stack of 12 images, separated in the z axis by 0.5-μm incrementsand centered about the focal plane, was recorded every 10 min over a 6-h

time course (37 time points). Image stacks were summed using the fastprojection protocol in the SoftWoRx software. Images were exported toVCell-ID 0.4 (Molecular Sciences Institute) for cell identification, tracking,and quantification (61). VCell-ID data were analyzed using the RCell packagefor R (54, 61, 62). Cells were called accurately by the VCell-ID program ∼93–95% of the time and were manually curated to remove false-positive calls.

Degradation Time. To determine the degradation rate of GFP in our strains,we grew cells containing the hml::GFP::PEST::NLS reporter in the sir3Δbackground (JRY9103) in SC medium overnight at 30 °C to a maximum 0.1OD. Cells were mounted onto glass-bottom culture dishes as describedabove and incubated with either SC medium or 2.5 μg/mL cycloheximide + SCmedium. Quantitative microscopy time-course experiments were performedand analyzed as described above.

Protein Immunoblot. Protein immunoblots were performed using previouslypublished methods and using the AbCam AB2886 anti-H3 K79 me3 antibody,the AbCam AB1791 H3 antibody, the Invitrogen A6457 anti-PGK1 antibody,and a Dot1 antibody generously provided by F. van Leeuwen (NetherlandsCancer Institute, Amsterdam).

ACKNOWLEDGMENTS. This project built upon the pioneering research ofEugenia Xu, Karl Zawadzki, and James Broach who created the original hml::GFP::PEST yeast strains. We thank Fred Van Leeuwen, Dan Gottschling, andScott Briggs for antibodies and plasmids; David Drubin and Voytek Okreglakfor help with microscopy; and Hector Candela for help with the Flow Cytom-eter. Da-Qiao Ding, Yuji Chikashige, Hiromi Maekawa, and especially Haru-hiko Asakawa provided instruction in microscopy, quantification, imagingtechnology, and general life skills in Japan. The Rine laboratory and espe-cially Oliver Zill, Meru Sadhu, and Bilge Ozaydin provided helpful discussionand feedback. Marc Nishimura provided commentary and feedback. Thisresearch was supported by a National Science Foundation Predoctoral Re-search Fellowship (to E.A.O.), a National Science Foundation- and JapanSociety for the Promotion of Science-sponsored East Asian Pacific SummerInstitute Fellowship (to E.A.O.), and National Institutes of Health ResearchGrant GM31105 (to J.R.).

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