Heritable capture of heterochromatin dynamics in Saccharomyces cerevisiae
Heterochromatin is a heritable , condensed chromatin structure that silences the expression of most genes within or near it . Phenomena such as clonal inheritance of inactivated X-chromosomes in female mammals , as well as position-effect variegation in Drosophila and yeast , demonstrate the remarkable ability of cells to propagate heterochromatic repression through mitosis . As an epigenetic state , heterochromatic gene repression provides a means for genetically identical cells to differentiate into stable , distinct cell types . However , despite its significance , little is known about the dynamics of heterochromatic repression and which factors contribute to or disrupt its stability . In Saccharomyces cerevisiae , heterochromatin forms at the silent mating-type loci , HML and HMR , through the recruitment and subsequent spreading of Sir proteins ( Grunstein and Gasser , 2013 ) . DNA elements known as the E and I silencers flank each locus and nucleate complexes of Sir2 , Sir3 and Sir4 . Sir complexes then deacetylate histones and bind nucleosomes throughout the region , thereby rendering HML and HMR transcriptionally silenced and largely inaccessible to DNA-interacting proteins . Since each locus contains either a or α mating-type information , as does the MAT locus , heterochromatic repression of HML and HMR ensures that the MAT genotype is the only determinant of whether haploids mate as a or α cells . Following its initial establishment , Sir-mediated heterochromatin can be maintained through the G1 , G2 and M phases and inherited through S phase . Sir2 , Sir3 and Sir4 are essential for all aspects of silencing ( Rine and Herskowitz , 1987 ) . Thus , mutants lacking any of these proteins express HML and HMR to the level of the transcriptionally active MAT locus . In contrast , mutants lacking Sir1 exhibit a bistable silencing phenotype ( Pillus and Rine , 1989; Xu et al . , 2006 ) . Within a population of sir1 cells , HML and HMR exist in one of two phenotypic states: silenced or expressed . Each state is heritable for multiple cell divisions , demonstrating the epigenetic nature of Sir-mediated heterochromatin and inspiring the notion that Sir1 functions in the establishment of silencing , but not the maintenance or inheritance thereafter . Notably , rare switches occur between the two expression states of HML and HMR in sir1 mutants , during which silencing is either lost or established . If Sir1 functioned exclusively in establishment , then losses of silencing should also occur in wild-type cells , yet no such event has been detected . Wild-type expression levels of genes at the HML and HMR loci are 1000-fold lower than the expression levels of the same genes when at the MAT locus , and efforts to detect expression of HML and HMR by any molecular method have shown the expression signal is indistinguishable from background noise . Moreover , 100% of MATa cells respond to α-factor , and diploids homozygous at the MAT locus are completely unable to sporulate . Thus , by all previous molecular criteria , the silent mating-type loci are transcriptionally inert . However , heterochromatin undergoes regular exchange of at least some of its structural components with newly synthesized molecules of the same proteins ( Cheng and Gartenberg , 2000; Festenstein et al . , 2003; Cheutin et al . , 2003; Ficz et al . , 2005 ) and is subject to perturbations , such as its replication in S phase . These fluctuations in heterochromatin structure imply that either the mechanism of silencing compensates for these changes and flawlessly reassembles each cell cycle , or that there are rare , as yet undetected losses of silencing resulting from heterochromatin dynamics . To address whether RNA polymerase ever succeeds in transcribing silent chromatin at HML and HMR , we designed an assay capable of detecting short-lived gene expression with single-cell resolution . By capturing the consequences of transcription with a permanent , heritable mark , we detected transient losses of silencing at HML and HMR in wild-type cells , characterized the nature of these losses , and identified genetic determinants of heterochromatin stability .
After plating cells with the cre gene at either HML or HMR on non-selective medium , we imaged the fluorescence of the resulting colonies . We first tested whether the expression of cre from the α2 promoter would produce enough recombinase to cause an efficient RFP-to-GFP switch by adding the Sir2 inhibitor nicotinamide ( NAM ) to the medium . The nicotinamide-induced derepression of either HMLα::cre or HMRα::cre resulted in entirely green colonies ( Figure 2A and Figure 2—figure supplement 1 ) . In contrast , cells that contained the RFP-GFP cassette but lacked any source of the cre gene grew into colonies that were entirely red ( Figure 2A and Figure 2—figure supplement 1 ) . These observations were consistent with the quantification of GFP fluorescence by flow cytometry ( Figure 2B ) . Therefore , expression of the cre gene at either HML or HMR was both necessary and sufficient for the switch from RFP to GFP expression . We then plated HMLα::cre and HMRα::cre cells on medium lacking nicotinamide to determine the stability of gene silencing under normal conditions . The resulting colonies were predominantly red , indicating the majority of cells maintained the repression of cre throughout growth of the colony ( Figure 2A ) . Within these colonies , however , we observed discrete green sectors , each of which represented a loss-of-silencing event that occurred in a cell at the vertex of the sector ( Figure 2A and Figure 2—figure supplement 1 ) . Although the switch to GFP expression was irreversible , some of the smaller sectors did not extend to the edge of the colony , likely due to a combination of genetic drift ( Hallatschek et al . , 2007 ) and the nature of three-dimensional growth of the colony . GFP expression was also detectable by flow cytometry in a fraction of HMLα::cre and HMRα::cre cells ( Figure 2B ) . Thus , silent chromatin at HML and HMR was transcriptionally dynamic , exhibiting losses of silencing below the level of detection by all previous assays . 10 . 7554/eLife . 05007 . 004Figure 2 . Transient transcription of HML and HMR captured by the Cre-based assay . ( A ) Colonies derived from single cells containing the RFP-GFP cassette in addition to either HMLα::cre , HMRα::cre , or no source of the cre gene ( No cre ) . Colonies of each genotype were grown in either the presence or absence of 5 mM nicotinamide ( NAM ) . Additional colonies are shown in Figure 2—figure supplement 1 . Scale bar , 2 mm . ( B ) Flow cytometry measurements of GFP fluorescence intensity in individual cells from within a single colony . Cells containing the RFP-GFP cassette and either HMLα::cre , HMRα::cre or no cre gene were plated on Complete Supplement Mixture ( CSM ) −Trp in the presence ( red ) or absence ( blue ) of 5 mM NAM . On day 6 of growth , representative colonies of each genotype and condition were resuspended in synthetic complete ( SC ) medium and grown to early log phase for analysis by flow cytometry . Each distribution represents approximately 105 cells . The percentage of GFP-positive cells shown for each sample was determined by a gate ( dotted line ) that was set based on the intensity profile of the HMLα::cre ( +NAM ) sample . ( C ) Quantitative RT-PCR measurements of HMLα::cre transcription in red and green sectors . To determine whether silencing loss was transient , cre and GFP mRNA levels were quantified in the GFP-expressing ( green ) and RFP-expressing ( red ) regions of a colony of cells containing HMLα::cre and the RFP-GFP cassette ( JRY9628 ) . Three independent experiments were performed , each on a different colony with a large green sector . Cells from the red and green regions of each colony were grown to log phase in YPD and harvested for RNA isolation . As a positive control for HMLα::cre expression , mRNA levels were also measured in a sir2Δ mutant ( JRY9633 ) . All mRNA values were normalized to ACT1 mRNA values . Expression levels are shown relative to the corresponding sir2Δ values ( set to 1 ) ( orange dotted line ) . The fold repression of HMLα::cre did not significantly differ between the red and green samples ( p = 0 . 43; Student's t test ) . Data are means ± SD ( n = 3 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 05007 . 00410 . 7554/eLife . 05007 . 005Figure 2—figure supplement 1 . GFP fluorescence of additional colonies with either HMLα::cre , HMRα::cre , or No cre grown in the presence or absence of 5 mM nicotinamide ( NAM ) . Scale bar , 2 mm . DOI: http://dx . doi . org/10 . 7554/eLife . 05007 . 005 At least two lines of evidence indicated that these losses of silencing were transient and did not arise from mutations in SIR genes . First , cre and GFP mRNA levels were measured in both red and green regions from within the same colony . If the loss-of-silencing event that resulted in the green sector were temporary , then the level of HMLα::cre repression would be indistinguishable between red and green sectors . Alternatively , if the loss of silencing were permanent , then cre mRNA would be detectable in cells from the green sector . Consistent with the efficiency of heterochromatin formation , which occurs de novo in approximately 1–2 cell divisions ( Osborne et al . , 2009 ) , HMLα::cre repression was fully restored in the descendants of a cell that experienced a loss of silencing ( Figure 2C ) . The slight difference in GFP expression between wild-type cells from the green sector and sir2Δ cells may have been due to the constitutive presence of Cre protein in the sir2Δ mutant , which could have potentially disrupted transcription by binding the loxP site between the promoter and the GFP gene . In addition , the GFP mRNA values in the red population likely reflected a low level of GFP expression since colonies were grown in the absence of G418 selection and therefore RFP-to-GFP switches occurred at a low rate . As secondary confirmation that losses of silencing were transient , we have never detected green sectors in colonies of a wild-type strain containing the GFP gene under control of the α2 promoter at HMLα ( unpublished observations ) . The pattern of colony fluorescence revealed the history of individual lapses in silencing , with the size of each green sector corresponding to how early the silencing loss occurred during colony growth . Colonies that were half red/half green reflected a loss of cre repression in either the mother or daughter cell of the first division that gave rise to the colony ( Figure 3A ) . Therefore , the frequency of half-sectored colonies equaled the rate of RFP-to-GFP switches per cell division . In the HMLα::cre strain , the frequency of half-sectored colonies was 1 . 6 × 10−3 ( Table 1 and Figure 3B ) . Thus , for every thousand cells , one to two cells temporarily failed to repress HMLα . Silencing of HMRα was slightly more stable than HMLα , with a lower rate of 7 × 10−4 losses per division ( p = 0 . 003; Student's t test ) ( Table 1 and Figure 3B ) . 10 . 7554/eLife . 05007 . 006Figure 3 . Rates of silencing loss as measured by half-sector frequency . ( A ) Example of a half-sectored colony derived from a single cell containing HMLα::cre and the RFP-GFP cassette . Scale bar , 1 mm . ( B ) Frequency of half-sectored colonies . The half-sector frequency of a strain containing the cre gene at HMLα ( JRY9628 ) was significantly different ( p = 0 . 003; Student's t test ) from the half-sector frequency of a strain containing the cre gene at HMRα ( JRY9629 ) . Data are means ± SD ( n = 3 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 05007 . 00610 . 7554/eLife . 05007 . 007Table 1 . Frequency of half-sectored colonies for each strain that showed a sectoring phenotypeDOI: http://dx . doi . org/10 . 7554/eLife . 05007 . 00710 . 7554/eLife . 05007 . 008Table 1—source data 1 . Half-sector frequencies from three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 05007 . 008FigureStrainRelevant genotypeHalf-sector frequency3JRY9628matΔ HMLα::cre0 . 00158 ± 0 . 00007JRY9629matΔ HMRα::cre0 . 0007 ± 0 . 00025JRY9739MATa HMLα::cre sir1Δ0 . 055 ± 0 . 007JRY9740MATa HMRα::cre sir1Δ0 . 061 ± 0 . 0026JRY9731MATα/matΔ HMLα/HMLα::cre0 . 00037 ± 0 . 00004JRY9732MATα/matΔ HMLα/HMLα::cre sir1Δ/SIR10 . 0007 ± 0 . 0002JRY9734MATα/matΔ HMLα/HMLα::cre sir3Δ/SIR30 . 0016 ± 0 . 0005JRY9735MATα/matΔ HMLα/HMLα::cre sir4Δ/SIR40 . 0013 ± 0 . 00037JRY9636matΔ HMLα::cre hst3Δ0 . 0111 ± 0 . 0005JRY9639MATa HMLα::cre hht1-hhf1Δ hht2-hhf2Δ [HHT2-HHF2]0 . 0033 ± 0 . 0004JRY9640MATa HMLα::cre hht1-hhf1Δ hht2-hhf2Δ [hht2K56R-HHF2]0 . 0111 ± 0 . 0008JRY9641MATa HMLα::cre hht1-hhf1Δ hht2-hhf2Δ [hht2K56Q-HHF2]0 . 009 ± 0 . 002JRY9736MATa HMLα::cre hst3Δ hht1-hhf1Δ hht2-hhf2Δ [HHT2-HHF2]0 . 0096 ± 0 . 0008JRY9737MATa HMLα::cre hst3Δ hht1-hhf1Δ hht2-hhf2Δ [hht2K56R-HHF2]0 . 009 ± 0 . 001JRY9738MATa HMLα::cre hst3Δ hht1-hhf1Δ hht2-hhf2Δ [hht2K56Q-HHF2]0 . 0105 ± 0 . 0004All values represent the mean of three independent experiments ± standard deviation . Full dataset is shown in Table 1—source data 1 . All strains contained a copy of the RFP-GFP cassette . See Supplementary file 1 for full genotypes . In principle , the rates of RFP-to-GFP switches could be compatible with at least three possible distributions of cre expression . In one model , cre is transcribed at a low level in many cells , but the resulting level of Cre protein is sufficient to catalyze recombination of the RFP-GFP cassette in only a subset of those cells . Alternatively , cre transcription could be completely absent in most cells , but in a low fraction of cells , occur at the same level as in a sir- mutant . Finally , cre transcription may be absent in most cells , and limited even in the small fraction of cells that switch from RFP to GFP expression . Thus far , efforts to detect transcription at HML and HMR have relied on quantitative RT-PCR and other population-based assays that report the average level of RNA for all the cells in a sample . However , advances in RNA imaging now enable the quantification of individual transcripts in single cells ( Itzkovitz and van Oudenaarden , 2011 ) . Using a version of fluorescence in situ hybridization ( FISH ) with single-molecule sensitivity ( Raj et al . , 2008 ) , we hybridized fixed cells with two sets of probes distinguished by their fluorophore label ( Figure 4A ) . One probe set was specific to the cre RNA expressed from HMLα::cre . As an internal control to account for sample variation , the other probe set was specific to KAP104 RNA , which encodes a protein involved in nuclear transport . We measured cre and KAP104 transcripts by imaging and quantifying fluorescent spots , each representative of a single RNA molecule . 10 . 7554/eLife . 05007 . 009Figure 4 . Single-molecule imaging of HMLα::cre transcription . ( A ) Schematic of the cre and KAP104 coding sequences ( black arrows ) , with colored lines representing sites targeted by the 20-nucleotide FISH probes ( see Supplementary file 4 for probe sequences ) . To detect both RNA sequences in the same cell , the cre-specific and KAP104-specific probes were differentially labeled . ( B ) Single-molecule RNA FISH of cre ( yellow ) and KAP104 ( red ) transcripts shown as maximum-intensity projections of z-stacks . DNA was stained with 4′ , 6-diamidino-2-phenylindole ( DAPI ) ( blue ) . Merged images were superimposed on the corresponding brightfield ( BF ) micrographs . Larger fields are shown in Figure 4—figure supplement 1 . Scale bar , 5 μm . ( C ) Distributions of cre and KAP104 RNA abundance determined by FISH . Each violin plot was normalized so that maximum bin width was the same across all samples . Data shown are from a representative replicate . DOI: http://dx . doi . org/10 . 7554/eLife . 05007 . 00910 . 7554/eLife . 05007 . 010Figure 4—figure supplement 1 . Imaging of HMLα::cre and KAP104 transcription . Larger fields of the RNA FISH , as described in Figure 4 . Maximum-intensity projections of cre RNA ( yellow ) , KAP104 RNA ( red ) and 4′ , 6-diamidino-2-phenylindole ( DAPI ) ( blue ) were merged . Scale bar , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 05007 . 01010 . 7554/eLife . 05007 . 011Figure 4—figure supplement 2 . Boxplots of cell widths measured using brightfield images from the RNA FISH replicate shown in Figure 4C . The widths of 1000s of hand-drawn cell boundaries were quantified using Matlab ( MathWorks ) . Red lines represent the median values . The whiskers ( dashed lines ) extend to the values within 1 . 5 times the interquartile range ( box ) . Outliers are shown as red points . DOI: http://dx . doi . org/10 . 7554/eLife . 05007 . 011 As a positive control , we examined cre transcription in a mutant lacking Sir4 , a protein integral to heterochromatin structure . As expected , HML was transcriptionally active in the sir4Δ mutant ( Figure 4B , C and Figure 4—figure supplement 1 ) , with a mean of 12 cre transcripts per cell ( ±2 standard error of the mean [SEM] calculated from three independent experiments ) , and 98% ( ±1 SEM ) of cells containing at least one RNA molecule ( Table 2 ) . Transcripts of the constitutively active KAP104 gene were present in 99 . 0% ( ±0 . 3 SEM ) of cells ( Table 2 , Figure 4B , C and Figure 4—figure supplement 1 ) . In concordance with previous studies ( Zenklusen et al . , 2008; Gandhi et al . , 2011 ) , KAP104 expression approximated a Poisson distribution , suggesting that most of the cell-to-cell variation in KAP104 RNA levels could be explained by stochastic , constitutive transcription . In contrast , the variance of cre RNA abundance was large in relation to the mean ( Table 2 ) , perhaps reflecting an alternative mode of transcription . 10 . 7554/eLife . 05007 . 012Table 2 . Summary of single-molecule RNA FISH statisticsDOI: http://dx . doi . org/10 . 7554/eLife . 05007 . 01210 . 7554/eLife . 05007 . 013Table 2—source data 1 . Single-molecule RNA FISH values from three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 05007 . 013RNAGenotypeMean spots per cellVariance% Cells with >0 spots% Cells with >1 spotcreHMLα::cre , sir4Δ12 ± 253 ± 998 ± 195 ± 2HMLα::cre , sir1Δ5 ± 158 ± 1346 ± 741 ± 7HMLα::cre , SIR+<0 . 005–0 . 42 ± 0 . 070 . 04 ± 0 . 01No cre , SIR+<0 . 003–0 . 3 ± 0 . 10KAP104HMLα::cre , sir4Δ7 . 3 ± 0 . 411 ± 199 . 0 ± 0 . 397 . 2 ± 0 . 8HMLα::cre , sir1Δ7 . 1 ± 0 . 210 . 8 ± 0 . 999 . 28 ± 0 . 0497 . 5 ± 0 . 1HMLα::cre , SIR+6 . 1 ± 0 . 27 . 8 ± 0 . 399 . 1 ± 0 . 296 . 5 ± 0 . 8No cre , SIR+5 . 9 ± 0 . 57 . 1 ± 0 . 599 . 0 ± 0 . 796 ± 2All values represent the mean of three independent experiments ± standard error of the mean . Full dataset is shown in Table 2—source data 1 . In a wild-type strain with HMLα::cre , cre transcripts were absent in nearly every cell , whereas KAP104 transcripts were present at a level comparable to that in sir4Δ cells ( Figure 4B , C and Figure 4—figure supplement 1 ) . The distribution of KAP104 expression in wild-type cells was slightly lower than the distribution in the sir4Δ mutant , which may be explained by our observation that sir4Δ cells were slightly larger than wild type ( Figure 4—figure supplement 2 ) , as noted before for sir2Δ mutants ( Moretto et al . , 2013 ) . Therefore , HML was completely silent in the vast majority of cells , indicating the Cre-based assay was sensitive to rare events of transcription . Although we detected an apparent cre signal in 4 . 2 × 10−3 ( ±0 . 7 × 10−3 SEM ) wild-type cells , we also detected an apparent cre signal at a frequency of 3 × 10−3 ( ±1 × 10−3 SEM ) in cells that lacked the cre gene , which were imaged to control for hybridization specificity ( Table 2 , Figure 4B , C and Figure 4—figure supplement 1 ) . The wild-type strain and the negative control strain were distinguishable , however , by the frequency of cells containing more than one spot of cre signal ( Table 2 ) . We observed five wild-type cells across all three replicates ( 13 , 695 wild-type cells total ) that contained more than one cre spot , ranging from 2 to 4 spots per cell , whereas we never detected more than one cre spot per cell in the negative control strain ( 13 , 722 No-cre cells total ) . Together , the RNA FISH analysis and the Cre-based assay converged on the same striking conclusion: the low rate of RFP-to-GFP switches reflected the complete absence of HMLα::cre transcription in the vast majority of cells . There was no evidence of any wild-type cells containing cre RNA levels typical of sir4Δ cells , suggesting that in the rare cells that lost silencing , the duration of transcription was short , presumably due to continuous nucleation of silent chromatin by Sir1 and other factors . Silencing in individual sir1 cells was previously measured by indirect bioassays ( Pillus and Rine , 1989; Xu et al . , 2006 ) , and with respect to transcription , the expression levels of HML and HMR underlying the two phenotypically distinct cell types in a sir1 population have never been established . RNA FISH allowed us to evaluate sir1Δ cells at the level of transcription on a per cell basis to determine whether these two states were molecularly equivalent to the levels of HML expression seen in sir4Δ and wild-type cells . In the absence of Sir1 , 46% ( ±7 SEM ) of cells expressed HML ( Table 2 ) , exhibiting a range in cre RNA levels that resembled the sir4Δ mutant ( Figure 4B , C and Figure 4—figure supplement 1 ) . The remaining 54% of sir1Δ cells lacked detectable cre RNA molecules , but expressed KAP104 at normal levels ( Figure 4B , C and Figure 4—figure supplement 1 ) . Therefore , in approximately half of all cells lacking Sir1 , HML was fully repressed , whereas the other half expressed HML at levels indistinguishable from those in a sir4Δ mutant . Although Sir1 was defined by its role in the establishment of silencing , and is not required for maintenance or inheritance , the possibility that it contributes to these other processes has never been directly tested . Such a test would require a comparison of the rate of silencing loss in wild-type cells to the rate of silencing loss in the population of sir1 cells in which HML was silenced . Whereas past measurements in wild-type cells were confounded by the role of Sir1 in establishment ( Xu et al . , 2006 ) , the Cre-based assay is sensitive enough to record losses of silencing before re-establishment occurs . To determine whether Sir1 contributes to the maintenance or inheritance of silencing , we sporulated a sir1 hemizygote containing HMLα::cre and the RFP-GFP cassette and then imaged the fluorescence of colonies derived from the meiotic products . If the rate of silencing loss were unaffected by the absence of Sir1 , then the sectoring patterns would be indistinguishable between colonies arising from SIR1 cells and colonies arising from sir1Δ cells in which HMLα::cre was repressed . However , green sectors were notably more abundant in the colonies of spores lacking Sir1 ( Figure 5A , Figure 5—figure supplement 1 ) . Moreover , the frequency of half-sectored colonies in a sir1Δ haploid was 35-fold above the frequency of half-sectored colonies in a wild-type haploid ( Table 1 and Figure 5B ) , indicating that cells lost silencing at HML at a strikingly higher rate when Sir1 was absent . In strains containing HMRα::cre instead of HMLα::cre , sir1Δ colonies showed elevated sectoring and a higher abundance of half sectors in comparison to wild-type colonies , as well ( Table 1 and Figure 5 ) . Thus , beyond its role in establishment , Sir1 contributed to either the maintenance or inheritance of silencing at both HMLα and HMRα . Consistent with the RNA FISH measurements , which revealed a large population of sir1Δ cells that lacked detectable cre transcripts ( Table 2 and Figure 4C ) , many of the cells within the sir1Δ colonies remained red ( Figure 5A ) . Therefore , sir1Δ cells that were in the silenced state exhibited the same level of repression as wild-type cells , but transitioned to a transcriptionally active state more often than wild-type cells . 10 . 7554/eLife . 05007 . 014Figure 5 . Increased silencing loss in cells lacking Sir1 . ( A ) Fluorescence of colonies derived from SIR1 and sir1Δ cells containing the RFP-GFP cassette and either HMLα::cre or HMRα::cre . All colonies shown were grown from MATa spores of JRY9729 and JRY9730 tetrad dissections . Additional colonies are shown in Figure 5—figure supplement 1 . Scale bar , 1 mm . ( B ) Frequencies of half-sectored colonies for haploid strains carrying the sir1Δ mutation and either HMLα::cre ( JRY9739 ) or HMRα::cre ( JRY9740 ) . For both HMLα::cre- and HMRα::cre-containing strains , the half-sector frequency of the sir1Δ mutant was significantly higher ( p = 2 × 10−4 for HMLα::cre , p = 7 × 10−7 for HMRα::cre; Student's t test ) than the half-sector frequency of wild type ( red dotted lines represent the rates shown in Figure 3B ) . Data are means ± SD ( n = 3 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 05007 . 01410 . 7554/eLife . 05007 . 015Figure 5—figure supplement 1 . GFP fluorescence of additional SIR1 and sir1Δ colonies carrying the RFP-GFP cassette and either HMLα::cre or HMRα::cre . Scale bar , 1 mm . DOI: http://dx . doi . org/10 . 7554/eLife . 05007 . 015 Sir1 facilitates the recruitment of Sir2 , Sir3 and Sir4 and therefore helps maintain a local concentration of heterochromatin components at HML and HMR . To test whether the dynamics of silencing were sensitive to the availability of Sir proteins , HMLα::cre expression was monitored in diploids hemizygous for individual SIR genes . While measuring silencing loss in diploids , we noticed that a wild-type diploid with one copy of HMLα::cre and the RFP-GFP cassette showed considerably reduced colony sectoring and a fourfold decrease ( p = 1 × 10−5; Student's t test ) in the frequency of half-sectored colonies in comparison to an isogenic haploid ( Table 1 ) , indicating a ploidy effect on the rate of RFP-to-GFP switching . Compared to the wild-type diploid , colonies of the sir1 , sir3 and sir4 hemizygotes showed a modest yet consistent increase in the number of green sectors ( Figure 6A and Figure 6—figure supplement 1 ) and a higher occurrence of half sectors ( Table 1 and Figure 6B ) . Therefore , reducing the dose of the genes encoding Sir3 or Sir4 , two structural components of heterochromatin , or Sir1 , a protein that recruits these components , rendered silencing at HML less stable . Altered gene dosage of SIR1 and SIR4 also affects the silencing of a sensitized HMR::ADE2 allele ( Sussel et al . , 1993 ) . Hemizygosity for SIR2 had no perceivable impact on colony sectoring ( Figure 6A and Figure 6—figure supplement 1 ) . 10 . 7554/eLife . 05007 . 016Figure 6 . Stability of silencing at HML in diploids hemizygous for individual SIR genes . ( A ) Fluorescence of colonies derived from single diploid cells containing one copy of HMLα::cre , one copy of the RFP-GFP cassette , and the indicated SIR genotypes . Additional colonies are shown in Figure 6—figure supplement 1 . Scale bar , 2 mm . ( B ) Frequencies of half-sectored colonies for a wild-type diploid and different sir hemizygotes . The half-sector frequencies of the sir1 , sir3 and sir4 hemizygotes were significantly higher ( *p < 0 . 05 , **p < 0 . 01; Student's t test ) than the half-sector frequency of the wild-type diploid . Data are means ± SD ( n = 3 ) . The frequency of half-sectored colonies was not determined for the sir2 hemizygote . DOI: http://dx . doi . org/10 . 7554/eLife . 05007 . 01610 . 7554/eLife . 05007 . 017Figure 6—figure supplement 1 . GFP fluorescence of additional colonies derived from single diploid cells hemizygous for individual SIR genes . All strains contained one copy of HMLα::cre and one copy of the RFP-GFP cassette . Scale bar , 2 mm . DOI: http://dx . doi . org/10 . 7554/eLife . 05007 . 017 Sir2 is the only member of the sirtuin family of NAD+-dependent deacetylases previously shown to have a role in silencing at HML or HMR . As measured by mating efficiency , silencing is indistinguishable between wild type and mutants lacking any of the four HST genes ( Brachmann et al . , 1995; Yang et al . , 2008 ) . However , Hst3 and Hst4 have been shown to contribute to the silencing of subtelomeric genes ( Brachmann et al . , 1995; Yang et al . , 2008 ) , as well as silencing of a URA3 reporter in a plasmid-borne HMR cassette ( Grünweller and Ehrenhofer-Murray , 2002 ) . Since nicotinamide inhibits all five sirtuins in yeast ( Imai et al . , 2000; Landry et al . , 2000a , 2000b; Smith et al . , 2000; Tanner et al . , 2000; Tanny and Moazed , 2001 ) , we tested whether other sirtuins , in addition to Sir2 , were involved in the nicotinamide-induced loss of silencing observed in Figure 2 . As expected , colonies of cells with HMLα::cre and the RFP-GFP cassette turned completely green in the absence of Sir2 , a protein essential for the nucleation and maintenance of heterochromatin ( Figure 7A and Figure 7—figure supplement 1A ) . The colonies of hst1Δ , hst2Δ and hst4Δ mutants exhibited sectoring patterns that were indistinguishable from wild-type colonies ( Figure 7A and Figure 7—figure supplement 1A ) . In contrast , the hst3Δ mutant showed a striking increase in colony sectoring and a sevenfold greater frequency of half-sectored colonies compared to wild type ( Table 1 , Figure 7A , B and Figure 7—figure supplement 1A ) . Thus , the Hst3 deacetylase contributed to the stability of silenced chromatin at HML . Whereas silencing defects have not been detected at subtelomeric genes in either the hst3Δ or hst4Δ single mutant , the hst3Δ hst4Δ double mutant shows a measurable phenotype ( Brachmann et al . , 1995; Yang et al . , 2008 ) . Therefore , it is likely that the deletion of HST4 would enhance the sectoring phenotype of the hst3Δ mutant shown in Figure 7A . 10 . 7554/eLife . 05007 . 018Figure 7 . Effects of sirtuin gene deletions and histone H3 K56R or K56Q substitutions on the stability of silencing at HML . ( A ) Fluorescence of colonies derived from single cells containing HMLα::cre , the RFP-GFP cassette , and the indicated deletions . Additional colonies are shown in Figure 7—figure supplement 1A . Scale bar , 2 mm . ( B ) Frequency of half-sectored colonies for the hst3Δ mutant shown in panel A . The half-sector frequency of the hst3Δ mutant was significantly greater ( p = 6 × 10−6; Student's t test ) than the half-sector frequency of wild type ( red dotted line represents the rate shown in Figure 3B ) . Data are means ± SD ( n = 3 ) . The frequency of half-sectored colonies was not determined for the hst1Δ , hst2Δ or hst4Δ mutant . ( C ) Fluorescence of colonies derived from single cells containing HMLα::cre , the RFP-GFP cassette , hht1-hhf1Δ and hht2-hhf2Δ mutations , and a plasmid-borne copy of either HHT2-HHF2 , hht2K56R-HHF2 or hht2K56Q-HHF2 . Additional colonies are shown in Figure 7—figure supplement 1B . Scale bar , 2 mm . ( D ) Frequency of half-sectored colonies for the genotypes shown in panel C . The half-sector frequency of the wild-type strain containing only one copy of the histone H3–H4 gene pair was significantly higher ( p = 0 . 001; Student's t test ) than the half-sector frequency of the strain shown in Figure 3B containing both copies of the histone H3–H4 gene pair ( red dotted line ) ( see Figure 7—figure supplement 2 ) . Data are means ± SD ( n = 3 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 05007 . 01810 . 7554/eLife . 05007 . 019Figure 7—figure supplement 1 . Additional colonies showing the effects of sirtuin gene deletions and histone H3 K56 substitutions on the stability of silencing at HML . ( A ) GFP fluorescence of additional colonies lacking individual sirtuins . All strains contained HMLα::cre and the RFP-GFP cassette . Scale bar , 2 mm . ( B ) GFP fluorescence of additional HST3 and hst3Δ colonies carrying the indicated histone H3 K56 alleles . All strains contained HMLα::cre and the RFP-GFP cassette . Scale bar , 2 mm . DOI: http://dx . doi . org/10 . 7554/eLife . 05007 . 01910 . 7554/eLife . 05007 . 020Figure 7—figure supplement 2 . Histone H3–H4 gene dosage effect on silencing at HML . GFP fluorescence of colonies derived from single cells containing HMLα::cre , the RFP-GFP cassette , and either the native histone H3–H4 gene pairs ( JRY9628 ) , or deletions of both histone H3–H4 gene pairs and a plasmid-borne copy of HHT2–HHF2 ( JRY9639 ) . Scale bar , 2 mm . DOI: http://dx . doi . org/10 . 7554/eLife . 05007 . 020 Hst3 regulates the deacetylation of acetylated lysine 56 on histone H3 ( H3 K56-ac ) ( Celic et al . , 2006; Maas et al . , 2006; Yang et al . , 2008 ) . To determine whether Hst3 stabilized silencing through deacetylation of H3 K56-ac or through deacetylation of yet undiscovered substrates , we measured the effects of amino acid substitutions that either mimic ( K56Q ) or prevent ( K56R ) the acetylation of H3 K56 . Although these histone gene mutations were previously analyzed for their effects on silencing , the outcome varies depending on the locus and the assay ( Hyland et al . , 2005; Xu et al . , 2007; Miller et al . , 2008; Yu et al . , 2011 ) . In our strains , with cre under the native α2 promoter at HML , colonies of the K56Q mutant showed a dramatic increase in sectoring , whereas the K56R mutant was similar to wild type ( Figure 7C and Figure 7—figure supplement 1B ) . Consistent with the possibility that the hst3Δ silencing defects were due to an increase in acetylated histone H3 K56 , the sectoring phenotypes suggested that neutralizing the positive charge at this position on histone H3 impaired the stability of repression . By half-sector analysis , however , both the K56Q and K56R mutants showed a significant increase ( p = 3 × 10−3 and p = 9 × 10−5 , respectively; Student's t test ) in the rate of silencing loss compared to wild type ( Table 1 and Figure 7D ) . The relatively high frequency of half-sectored colonies in the histone H3 K56R mutant was unexpected due to the similarity in sectoring patterns between histone H3 K56R mutant colonies and wild-type colonies ( Figure 7C and Figure 7—figure supplement 1B ) . However , the histone H3 K56R substitution has previously been shown to decrease silencing under various conditions ( Xu et al . , 2007; Yu et al . , 2011 ) . Differences between sectoring patterns and half-sector frequencies are considered further in the ‘Discussion’ . Cells expressing the H3 K56Q mutant produced similar patterns of colony sectoring regardless of whether Hst3 was present ( Figure 7C and Figure 7—figure supplement 1B ) . This phenotype was slightly less severe than that of hst3Δ cells expressing wild-type H3 K56 ( Figure 7C and Figure 7—figure supplement 1B ) , perhaps because glutamine was not quite as disruptive to silencing as acetylated lysine at this residue . In addition , the H3 K56R substitution in hst3Δ cells restored silencing to wild-type levels , as determined by overall colony sectoring ( Figure 7C and Figure 7—figure supplement 1B ) . Therefore , blocking the acetylation of residue 56 suppressed the hst3Δ sectoring phenotype . Collectively , the sectoring patterns suggested that Hst3 promoted the stability of silencing through the deacetylation of histone H3 K56-ac . Whereas the deletion of HST3 caused a threefold increase in the half-sector frequency of wild type ( p = 3 × 10−4; Student's t test ) , it did not significantly affect the half-sector frequency of the H3 K56Q mutant ( p = 0 . 3; Student's t test ) or the surprisingly high half-sector frequency of the H3 K56R mutant ( p = 0 . 06; Student's t test ) ( Table 1 and Figure 7D ) . Thus , the state of histone H3 K56 affected the stability of silencing at HML , and amino acid substitutions of this residue were epistatic to the hst3Δ mutation . Colonies derived from cells in which the only source of histone H3–H4 genes was a plasmid-borne copy of HHT2-HHF2 exhibited more sectoring than colonies of cells containing both HHT1-HHF1 and HHT2-HHF2 at their native chromosomal loci ( Figure 7—figure supplement 2 ) . Assuming the decrease in gene copy number led to a decrease in protein abundance , silencing was likely affected by levels of histones H3 and H4 that were either limiting or improperly balanced with the levels of histones H2A and H2B . Consistent with these possibilities , histone dosage has previously been shown to affect various functions of heterochromatin ( Moore et al . , 1979; Moore et al . , 1983; Venditti et al . , 1999 ) . In principle , the effect of histone H3–H4 gene dosage should sensitize silencing in all genetic backgrounds . Whereas the reduction from two copies of the histone H3–H4 gene pair to one copy did enhance the sectoring phenotype of hst3Δ colonies ( compare panels A and C in Figure 7 and panels A and B in Figure 7—figure supplement 1 ) , it did not increase the half-sector frequency in the hst3Δ background as it did in the wild-type background ( Table 1 and Figure 7B , D ) . Therefore , the degree to which histone H3–H4 gene dosage affected silencing , at least during the first cell division of colony growth , depended on the genetic background . Whereas colony sectors revealed the history of heterochromatin dynamics , we sought to also capture these events in real time at single-cell resolution to determine whether observable losses of silencing exhibited a fixed pattern or were more stochastic . Therefore , we monitored the fluorescence of cells containing HMLα::cre and the RFP-GFP cassette as they divided in a chamber supplied with fresh medium . Most cells remained red throughout the duration of the time course . Consistent with the sectors observed within colonies , rare RFP-to-GFP switches occurred in a subset of the lineages ( see Video 1 for an example ) . The rate of switching observed with live-cell imaging was within 10% of the rate of switching determined by the half-sector assay . 10 . 7554/eLife . 05007 . 021Video 1 . RFP-to-GFP switch captured in real time . Time-lapse imaging of wild-type cells containing HMLα::cre and the RFP-GFP cassette . Images were taken every 10 min for 13 hr . DOI: http://dx . doi . org/10 . 7554/eLife . 05007 . 021 The time lag between the expression of cre and the ultimate maturation of GFP limits the temporal resolution from such analyses . Nonetheless , a survey of the pattern of switch events allowed some useful inferences . For each RFP-to-GFP switch , we determined whether cells were unbudded , small-budded , or large-budded at the moment when GFP expression was initially detected . We observed a fluorescent switch in 24 different cells , nine of which were unbudded and thus in the G1 phase during the onset of GFP expression ( Figure 8A , D ) . Multiple occurrences of both patterns 1 and 2 ( Figure 8D ) indicated that silencing loss followed by recombination was not restricted to either the mother or daughter cell ( Given that pattern 3 was supported by only one example , we refrained from offering an interpretation of this event ) . 10 . 7554/eLife . 05007 . 022Figure 8 . Patterns of RFP-to-GFP switches observed in real time . ( A–C ) Brightfield ( BF ) and GFP fluorescence montages ( shown in 20-min intervals , 140 min total ) showing different switching patterns of wild-type cells containing HMLα::cre and the RFP-GFP cassette . Labeled arrows track the mother ( m ) and daughter ( d ) cells of interest . ( A ) GFP expression first detected in an unbudded daughter cell . ( B ) GFP expression first detected in a small-budded cell . Both the mother ( m ) and daughter ( d2 ) cells remained GFP-positive ( pattern 6 ) . *A second RFP-to-GFP switch occurred in the daughter ( d1 ) of the preceding cell division ( see Figure 8—figure supplement 1 , pedigree B ) . ( C ) GFP expression first detected in a large-budded cell . Only the mother ( m ) cell remained GFP-positive . ( D ) Distribution of different switch patterns . RFP-to-GFP switches were initially categorized by bud morphology at the time that GFP expression was first detected ( depicted as yellow cells in the cartoons since RFP was still present ) . Events in each bud category were then classified according to whether the mother cell , daughter cell , or both underwent recombination of the RFP–GFP cassette , as determined by whether they continued to express GFP ( depicted as green cells in the last phase of the pedigree cartoons ) . Asterisks denote the number of events within a particular category that were associated with a second switch in a directly related cell ( see Figure 8—figure supplement 1 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 05007 . 02210 . 7554/eLife . 05007 . 023Figure 8—figure supplement 1 . Cartoons of lineages that showed two RFP-to-GFP switches in directly related cells . Switches are labeled with the numbers 1 and 2 . In pedigree A , onset of GFP expression ( depicted as yellow ) was detected in a large-budded cell ( 1 ) . At the same time , GFP also appeared in the daughter cell that arose from the previous cell division ( 2 ) . Due to the assymetry of switch 1 ( only the mother cell remained green , implying that recombination occurred after DNA replication ) , switches 1 and 2 must have arisen from independent recombination events . It is unknown , however , whether the two recombination events resulted from two losses of silencing , or rather one loss of silencing that occurred when the two cells shared a cytoplasm ( earliest stage shown for the pedigree ) . In pedigree B ( montage shown in Figure 8B ) , GFP fluorescence was first detected in a small-budded cell that gave rise to two cells that remained green ( 1 ) . Approximately 40 min later , the daughter cell of the preceding cell division also began to show GFP fluorescence as a small-budded cell . In pedigree C , GFP expression was first detected in a mother-daughter pair subsequent to cytokinesis , but prior to both cells budding . Whereas the mother cell became green , however , the daughter cell developed a large bud without ever reaching a similar level of GFP expression , at which point the experiment ended . This pattern could be explained by two switching events , one early asymmetric switch where only the mother inherited a recombined RFP–GFP cassette and the daughter cell inherited GFP molecules along with an intact RFP–GFP cassette , followed by a later switch that occurred in the daughter cell . Alternatively , there could have been only one early switch where the RFP–GFP cassette inherited by the daughter cell either did not undergo recombination and GFP perdurance was especially long , or the cassette did undergo recombination , but GFP expression was weak , perhaps due to an error in recombination . DOI: http://dx . doi . org/10 . 7554/eLife . 05007 . 023 Of the remaining cells that switched , three cells had small buds when GFP fluorescence was first detected and 12 had large buds ( Figure 8D ) . All three small-budded cells gave rise to mothers and daughters that both remained green ( pattern 6 ) ( Figure 8B , D ) . Since the bud emerges during early S phase , and there were no cases of only the mother or daughter cell remaining green following the onset of GFP expression in a small-budded cell ( patterns 4 and 5 ) , it was likely that recombination of the loxP sites occurred prior to DNA replication . Alternatively , recombination may have occurred shortly after DNA replication at both newly synthesized copies of the RFP-GFP cassette . In contrast , 8 of the 12 cells that expressed detectable GFP as large-budded cells gave rise to mother-daughter pairs in which only the mother or daughter remained green ( patterns 7 and 8 ) ( Figure 8C , D ) , indicating that Cre catalyzed recombination after S phase and acted on only one of the two RFP-GFP cassettes . Switches that followed patterns 7 and 8 also demonstrated that the time between recombination , which occurred some point after DNA replication , and GFP detection , which occurred when a large bud was present , spanned only a fraction of a complete cell cycle . For three of the RFP-to-GFP switches , we observed a second switch in the daughter cell that arose from the immediately preceding cell division ( Figure 8B , D and Figure 8—figure supplement 1 ) . These paired switches , which occurred either simultaneously or within 40 min of each other , could have reflected either two loss-of-silencing events in directly related cells , or rather one loss-of-silencing event that happened when the mother cell and its daughter still shared a cytoplasm , assuming there was perdurance of the Cre protein in both cells and a delay in recombination . These patterns are considered further in Figure 8—figure supplement 1 and the ‘Discussion’ .
At a low rate , the heterochromatic loci HML and HMR underwent losses of silencing that had escaped detection by all previous efforts , due to the combination of their rarity and transience . Independent observations indicated that silencing was promptly re-established following its loss . First , HMLα::cre was fully silenced in the descendants of cells that switched to GFP expression , showing that cre expression was only temporary . Secondly , single-molecule RNA FISH did not detect any wild-type cells expressing HMLα::cre to the average level of sir4Δ cells , implying that when silencing was lost in wild-type cells , it was restored before cre transcription could reach that level . These data , taken together with the inability of less sensitive assays to capture expression of HML or HMR , suggested that losses of silencing were rare and short-lived . The detection of silencing loss in a subset of wild-type cells supported a model where Sir proteins and the transcriptional machinery constantly compete for access to the silent mating-type loci . Such a concept is consistent with the dynamic exchange of structural components in heterochromatin , the continuous recruitment of Sir proteins by the silencers ( Cheng and Gartenberg , 2000 ) , and the ability of increased levels of a transcriptional activator to overcome the silenced state ( Aparicio and Gottschling , 1994; Ahmad and Henikoff , 2001; Xu et al . , 2006 ) . This model could explain why losses of silencing were more frequent when cells contained only half the normal gene dosage of SIR1 , SIR3 or SIR4 . Perhaps losses of silencing at HML and HMR resulted from stochastic fluctuations in the local concentration of silencing factors . Alternatively , specific perturbations such as DNA replication may have disrupted the binding of Sir proteins to nucleosomes , thereby creating an opportunity for RNA polymerase to access heterochromatin . The contribution of Sir1 to silencing in wild-type cells was previously underappreciated . Heritability of the repressed state in a subset of sir1Δ cells showed that Sir1 was not absolutely required for the maintenance or inheritance of silenced chromatin , and suggested that Sir1 primarily functioned in establishment ( Pillus and Rine , 1989 ) . However , the presumed role of Sir1 seemed paradoxical under the assumption that wild-type cells never lose silencing and thus do not undergo events requiring re-establishment . This point of confusion can now be resolved by two key findings . First , the Cre-based assay revealed that transient transcription of HML and HMR occurred at a low rate in wild-type cells . Therefore , Sir1 may serve a purpose in the recovery of silencing on the rare occasion that it is lost . Secondly , losses of silencing occurred more often in cells lacking Sir1 , indicating that Sir1 contributed to either the maintenance or inheritance of silencing , or both . Previous measurements of silencing established that two phenotypic states exist within a population of sir1 cells ( Pillus and Rine , 1989; Xu et al . , 2006 ) , yet the levels of expression in each of the two subpopulations remained unresolved . For example , in sir1Δ cells containing a fluorescent reporter under control of the URA3 promoter at HML , the fluorescence intensity profile of each subpopulation shifted in relation to the profiles of wild-type cells and sir3Δ cells depending on whether the URA3 trans-activator Ppr1 was present ( Xu et al . , 2006 ) . By directly measuring the transcription of a gene from the native α2 promoter at HML with single-cell and single-molecule resolution , we showed that the silenced subset of sir1Δ cells was fully repressed , whereas the expressed subset of sir1Δ cells transcribed cre to the level of sir4Δ cells . Together , the Cre-based assay and the RNA FISH measurements resolved two separable aspects of silencing: the level of repression and the rate at which it is lost . Deletion of SIR1 affected the rate of silencing loss , but not the level of repression since approximately half of all sir1 cells completely lacked cre transcripts . Moreover , the persistence of RFP expression within regions of sir1Δ colonies containing either HMLα::cre or HMRα::cre and the RFP-GFP cassette underscored the remarkable ability of heterochromatin to template its own replication for multiple cell divisions . In principle , the dynamic nature of silenced chromatin could result from stochastic processes intrinsic to , for example , the binding constants of Sir proteins for their nucleosomes and serve no useful role . Alternatively , the stability of silencing at HML and HMR could be tuned high enough to achieve cell-type specificity , yet just low enough to allow for events such as DNA replication or mating-type interconversion . Haploid cells of a homothallic strain can switch mating types through repair of a double-stranded DNA break at MAT using HML or HMR as a template . Therefore , successful switching requires that the invading MAT strand , along with the accessory proteins involved in recombinational repair , gain access to the heterochromatic donor sequence . We speculate that the dynamics of silenced chromatin may contribute to the efficiency of mating-type interconversion . The stability of silencing at HML depended in part on Hst3 and the deacetylation of H3 K56-ac . H3 K56 localizes to the DNA entry/exit region of the nucleosome ( Luger et al . , 1997 ) , and in the acetylated state it is thought to promote transient DNA ‘breathing’ , during which the DNA partially unwraps from the nucleosome ( Neumann et al . , 2009; Simon et al . , 2011; North et al . , 2012 ) . Furthermore , H3 K56-ac is important for transcriptional activation ( Williams et al . , 2008; Värv et al . , 2010 ) , and telomeric heterochromatin is more accessible to the bacterial dam DNA methylase in various H3 K56 mutants ( Xu et al . , 2007 ) . Collectively , these observations suggest that H3 K56-ac destabilizes the nucleosome conformation and thereby renders the DNA more accessible to RNA polymerase . H3 K56 acetylation and Hst3 expression are both regulated by the cell cycle . Whereas H3 K56 acetylation peaks during S phase ( Masumoto et al . , 2005 ) , Hst3 expression does not peak until G2/M phase ( Spellman et al . , 1998; Celic et al . , 2006; Maas et al . , 2006 ) . Therefore , heterochromatin may be especially susceptible to transcription during the time after K56-acetylated histone H3 is deposited into chromatin , but before Hst3 removes the modification . As previous measurements of HML silencing were unable to distinguish hst3 mutants from wild type , our detection of a stability phenotype in this mutant underscored the importance of measuring fluctuations in repression for a fuller understanding of heterochromatin . The ability of this assay to capture the existence of transient events through their conversion to permanent , heritable marks can now allow a comprehensive evaluation of the contribution of any and all genes to heterochromatin dynamics . By preserving a historical record of transcription , the Cre-based assay also provides the opportunity to measure the effects of transient environmental or cellular stresses on silencing subsequent to their occurrence . Such analyses will inform our understanding of the mechanisms by which cells maintain integrity of the epigenome . Measuring the frequency of half-sectored colonies allowed for quantification of the rate at which silencing was lost . In general , half-sector frequencies were consistent with the overall patterns of colony sectoring . However , strains bearing the histone H3 K56R substitution , for example , were indistinguishable from wild type by sectoring pattern , yet showed a significant increase in the frequency of half-sectored colonies . While we do not fully understand why silencing in histone H3 K56R mutants appeared to be less stable at the two-cell stage of colony growth than at later stages , the complexities of colony development may offer a simple explanation . The physiology of cells during the first cell division of colony growth , at which point silencing was measured by half-sector analysis , would be expected to differ from the physiology of cells during later divisions . Therefore , it is possible that the histone H3 K56R mutation rendered heterochromatin more sensitive to the physiological state of cells . Overall , both assays were informative , and sectoring patterns in particular provided the opportunity to survey heterochromatin dynamics throughout the various microenvironments and metabolic states of colonies . A direct comparison could not be made between the rate of RFP-to-GFP switching , as determined by half-sector analysis , and the frequency of wild-type cells expressing HMLα::cre , as determined by FISH . Whereas half-sector analysis measured events throughout the duration of an entire cell cycle , the FISH method measured expression in a snapshot of time . Furthermore , the background levels of FISH signal in the negative control strain , though very low , limited quantitative interpretation of the low cre RNA levels in wild-type cells . The FISH measurements did show , however , that any detectable transcription of HMLα::cre in wild-type cells was both rare and restricted to relatively low levels . These observations implied that a small number of cre transcripts produced enough recombinase to catalyze the RFP-to-GFP switch . Further experiments would be necessary to determine the number of cre RNAs sufficient for recombination . The live-cell imaging of pedigrees of wild-type cells endowed with the Cre-based assay reflected our first efforts to visualize in real time the silencing loss events whose history is written in the number and size of green colony sectors . In principle , RNA FISH by itself could provide better time resolution , but morphological classification of fixed spheroplasts using brightfield images was unreliable , especially with the high density of cells needed to capture rare events . Despite the time lag between loss-of-silencing events and the production of the GFP chromophore , several points were clear . First , there was no obvious mother-daughter bias as to which cell contained the more labile HML locus . Secondly , the expression of cre and the subsequent switches from red to green could occur within the same cell cycle . Overall , the variety of patterns we observed were consistent with the possibility that losses of silencing occur at multiple points during the cell cycle , but such conclusions rely on assumptions for which we have no independent verification . Knowing the extent of variation in the time lag between HMLα::cre expression and recombination would help solidify further interpretation . Intriguingly , there were three pedigrees in which a red daughter , born from a mother that switched to green , itself switched to green . Given the low probability of a switch , two switches in such closely related cells implied there was either perdurance of the Cre protein following a single loss-of-silencing event , or some ‘heritable’ instability shared between these two cells , as has been previously suggested in a different context ( Kaufmann et al . , 2007 ) . Future experiments such as live-cell imaging of transcription should reveal the exact timing of silencing loss and address whether the history of a lineage affects stability .
The strains , plasmids , and oligonucleotides used in this study are listed in Supplementary files 1 , 2 and 3 , respectively . All strains were derived from the W303 background . Deletions were made using one-step integration of gene disruption cassettes ( Longtine et al . , 1998; Goldstein and McCusker , 1999; Gueldener et al . , 2002 ) and confirmed by PCR of the 5′ and 3′ junctions . For analysis of HHT2 ( histone H3 ) mutations , the URA3-marked plasmid ( pJR2657 ) in JRY9638 was replaced with a TRP1-marked plasmid ( either pJR2759 , pJR3212 or pJR3213 ) by plasmid shuffle to produce JRY9639 , JRY9640 and JRY9641 . Using the hst3Δ::K . l . LEU2 fwd/rev primers and pUG73 ( Gueldener et al . , 2002 ) , HST3 was deleted in each of these strains to create JRY9736 , JRY9737 and JRY9738 . The cre gene from bacteriophage P1 was integrated at HML by transformation of a sir4Δ strain containing HMLα::K . l . URA3 and counter-selection on medium containing 5-fluoroorotic acid ( 5-FOA ) . First , the K . l . URA3 gene was amplified by PCR from pUG72 ( Gueldener et al . , 2002 ) using the HMLα::K . l . URA3 fwd/rev primers and integrated at HMLα , replacing the coding sequence of the α2 gene . Then , the cre gene was amplified from pSH47 ( Güldener et al . , 1996 ) using the HMLα::cre fwd/rev primers , and the resulting PCR product was transformed into the strain containing HMLα::K . l . URA3 . 5-FOA was used to select for HMLα::cre cells , which no longer contained K . l . URA3 . Integration of cre was confirmed by sequencing . The resulting strain was mated with a SIR4 strain and sporulated to obtain a haploid containing both SIR4 and the HMLα::cre allele . To construct the HMRα::cre allele , the K . l . URA3 gene was amplified by PCR from pUG72 ( Gueldener et al . , 2002 ) using the HMRa::K . l . URA3 fwd/rev primers and integrated at HMRa in a sir4Δ strain , thus replacing the coding sequence of the a1 gene with K . l . URA3 . Then , the HMRα::cre fwd/rev primers were used to amplify a fragment of HMLα::cre from JRY9628 genomic DNA that spanned from the X region to the Z1 region ( see Figure 1A ) , and the resulting PCR product was transformed into the strain containing HMRa::K . l . URA3 . 5-FOA was used to select for HMRα::cre cells , which no longer contained K . l . URA3 . Integration of cre was confirmed by sequencing . The resulting strain was mated with a SIR4 strain and sporulated to obtain a haploid containing both SIR4 and the HMRα::cre allele . For integration of the RFP-GFP cassette at the URA3 locus , the PGPD-loxP-yEmRFP-TCYC1-kanMX-loxP-yEGFP-TADH1 sequence in pJR3214 was amplified by PCR using the ura3Δ::RFP-GFP fwd/rev primers . pJR3214 contains the following features: the ampR-containing fragment of pUG73 ( Gueldener et al . , 2002 ) resulting from digestion with restriction enzymes NdeI and NotI ( 2301 bp ) ; the GPD promoter from p413-GPD ( Mumberg et al . , 1995 ) ; a loxP site ( 5′-ATAACTTCGTATAGCATACATTATACGAAGTTAT-3′ ) , separated from PGPD by a SalI restriction site ( GTCGAC ) ; immediately downstream of the first loxP site is the yEmRFP gene ( Keppler-Ross et al . , 2008 ) , followed by a BglII restriction site ( AGATCT ) and the CYC1 terminator from pSH63 ( Gueldener et al . , 2002 ) ; the kanMX cassette from pUG6 ( Güldener et al . , 1996 ) , separated from TCYC1 by a StuI restriction site ( AGGCCT ) ; adjacent to the kanMX cassette is a second loxP site ( 5′-ATAACTTCGTATAGCATACATTATACGAAGTTAT-3′ ) ; and immediately following the second loxP site is an EcoRV restriction site ( GATATC ) and the yEGFP-TADH1 sequence from pKT127 ( Sheff and Thorn , 2004 ) . The version of the RFP-GFP cassette marked with hphMX instead of kanMX ( in strains JRY9729 and JRY9730 ) was constructed by transforming strains containing the kanMX-marked RFP-GFP cassette with PCR product amplified from pAG32 ( Goldstein and McCusker , 1999 ) using the PTEF fwd and TTEF rev primers . All colonies were imaged using a Zeiss Axio Zoom . V16 microscope equipped with ZEN software ( Zeiss , Jena , Germany ) , a Zeiss AxioCam MRm camera and a PlanApo Z 0 . 5× objective . The colonies shown in Figures 2 , 6 and 7 ( and the associated figure supplements ) were imaged on day 6 of growth , whereas the colonies shown in Figures 3 and 5 ( and the associated figure supplements ) were imaged on day 3 of growth . Images were assembled using Photoshop ( Adobe Systems , San Jose , CA ) . In preparation for colony analysis , red cells were selected using medium containing G418 ( Geneticin ) ( Life technologies , Grand Island , NY ) . Due to the complete loss of silencing in sir2Δ cells , JRY9633 was unable to grow in the presence of G418 and was therefore grown on nonselective medium . Cells were then grown to log phase in Complete Supplement Mixture ( CSM ) −Trp ( Sunrise Science Products , San Diego , CA ) , and serial dilutions were performed to spread approximately 10 cells/plate ( CSM −Trp , 1% agar ) . To inhibit sirtuin activity , 200 μl of 0 . 5 M nicotinamide was spread onto CSM −Trp , 1% agar plates prior to plating cells . For Figure 5 and Figure 5—figure supplement 1 , red cells of JRY9729 and JRY9730 were selected using medium containing both G418 ( Life technologies ) and Hygromycin B ( Sigma–Aldrich , St . Louis , MO ) , and then sporulated for 4–5 days at room temperature on 1% potassium acetate . The resulting tetrads were dissected on CSM −Trp plates and grown into colonies for imaging . For half-sector analysis , red cells were selected using G418 and then grown to log phase in CSM −Trp . Serial dilutions were performed to plate approximately 100 cells/plate ( CSM −Trp ) . On day 3 of growth , plates were scanned , face up , with a Typhoon Trio ( GE Healthcare Life Sciences , Buckinghamshire , England ) using the 488-nm laser and 520-nm emission filter to detect GFP fluorescence . Since colonies of cells containing the H3 K56R substitution grew notably slower than colonies of other genotypes , plates were scanned on day 4 of growth for JRY9640 and JRY9737 . Colonies that were half green or completely green were manually counted . The total number of colonies was determined with Matlab ( MathWorks , Natick , MA ) by applying a threshold and converting scans to a binary image ( code available upon request ) . Then , the following equation was used to calculate the half-sector frequency: # of half-sectored colonies/ ( # of total colonies–# of fully green colonies ) Three independent experiments were performed for each genotype . See Table 1—source data 1 for the total number of colonies , as well as the number of half-sectored colonies and fully green colonies determined for every experiment . Colonies were grown as described above , scraped off the agar surface and resuspended in synthetic complete ( SC ) medium ( Sunrise Science Products ) . After growth overnight , cultures were diluted back to 0 . 01 OD600 and then grown in SC to approximately 0 . 2 OD600 . Cells were harvested by centrifugation , and then washed and resuspended in PBS , pH 7 . 4 on ice . A FC-500 flow cytometer ( Beckman–Coulter , Brea , CA ) was used to measure the GFP fluorescence intensity of 105 cells/sample . Using FlowJo software ( Tree Star , Ashland , OR ) cells were gated based on forward and side scatter , and the data were exported as FCS files . Data analysis was performed with Matlab ( Mathworks ) . Total RNA was isolated from log-phase cells using hot acidic phenol ( Collart and Oliviero , 2001 ) . RNA was digested with DNase I ( Roche Diagnostics , Indianapolis , IN ) and then purified by phenol-chloroform extraction and precipitation with isopropanol . cDNA was synthesized using the SuperScript III First-Strand Synthesis System for RT-PCR ( Invitrogen , Carlsbad , CA ) and oligo ( dT ) primers . Quantitative PCR of cDNA was performed using the Thermo Scientific DyNAmo HS SYBR Green qPCR Kit ( Fisher Scientific , Chicago , IL ) and a Mx3000P machine ( Stratagene , acquired by Agilent Technologies , Santa Clara , CA ) . Samples were analyzed in technical triplicate for three independent RNA preparations . RNA FISH was performed as described ( Raj et al . , 2008 ) using JRY9630 , JRY9631 , JRY9632 and JRY4012 . All probes ( see Supplementary file 4 for probe sequences ) were designed , synthesized and labeled by Stellaris ( Biosearch Technologies , Novato , CA ) . Probes targeting cre were coupled to Quasar 670 dye ( Biosearch Technologies ) and probes targeting KAP104 were coupled to CAL Fluor Red 590 dye ( Biosearch Technologies ) . Each probe mix ( 5 nmol ) was dissolved in 100 μl of TE buffer , pH 8 . 0 . For hybridization , 1:10 dilutions of the cre and KAP104 probe stocks were made , and 1 μl of each probe dilution was added to 50 μl of hybridization buffer . Cells were grown in CSM to 0 . 2–0 . 3 OD600 and then fixed and hybridized using the protocol as described ( Youk et al . , 2010 ) with additional guidance ( Raj , 2013 ) and the following modification to the spheroplasting procedure: cells were digested with 3 μl of zymolyase ( 2 . 5 mg/ml ) at 30°C and monitored by phase contrast microscopy until approximately 80% of cells appeared to be digested ( 35–50 min ) ( L Teytelman , personal communication , January 2013 ) . The cre and KAP104 probes were hybridized to cells overnight at 30°C in 10% formamide buffer . Samples were then washed , stained with DAPI , washed again and resuspended in 10 μl of glucose-oxygen-scavenging ( GLOX ) solution without enzymes . Prior to imaging , 10 μl of GLOX solution with enzymes ( 1% [vol/vol] catalase [Sigma-Aldrich C3515] , 1% [vol/vol] glucose oxidase [Sigma-Aldrich G2133] , 2 mM Trolox [Sigma-Aldrich 238813] ) was added to the sample . Images were acquired with a DeltaVision RT microscope and softWoRx software ( Applied Precision , Issaquah , WA ) using a 60 × /1 . 40 oil-immersion objective ( Olympus , Tokyo , Japan ) , a CoolSNAP HQ CCD camera ( Photometrics , Tuscon , AZ ) and the following filters from Chroma Technology ( Bellows Falls , VT ) : ET402/15x , ET455/50m ( DAPI ) ; ET555/25x , ET605/52m ( CAL Fluor Red 590 ) ; and ET645/30x , ET705/72m ( Quasar 670 ) . Series of z-stacks were acquired with a step size of 0 . 2 μm . Cell boundaries were hand-drawn using ImageJ software ( NIH , Bethesda , MD ) . Spots were detected and analyzed using Matlab ( MathWorks ) . First , a three-dimensional Laplacian of Gaussian filter ( Raj et al . , 2008 ) was applied to raw images of cre and KAP104 RNA . Then , a fluorescence intensity threshold was selected to identify spots within an expected range of sizes . Given that some sir4Δ and sir1Δ cells contained a relatively large number of cre transcripts , some of which appeared to overlap each other in a maximum-intensity projection of z-stacks , spot detection was performed in three dimensions to resolve individual spots ( code adapted from L Teytelman and available upon request ) . Within each experiment ( three independent experiments were performed ) , the same threshold values ( one for cre RNA images and one for KAP104 RNA images ) were applied across all samples . Cells containing HMLα::cre and the RFP-GFP cassette ( JRY9628 ) were grown to mid-log phase in CSM −Trp , sonicated once for 5 s at a power output of 10% , and then imaged over time using the CellASIC ONIX Microfluidic Platform ( EMD Millipore , Hayward , CA ) . Growth was restricted to a single focal plane within chambers of the Y04C Microfluidic Plate for Haploid Yeast ( EMD Millipore ) . CSM −Trp flowed through the chambers continuously at a pressure of 3 psi . Brightfield and fluorescence images of 46 different fields were taken every 10 min for 13 hr using MetaMorph software ( Molecular Devices , Sunnyvale , CA ) and an Eclipse Ti microscope ( Nikon Instruments , Melville , NY ) equipped with a Clara Interline CCD camera ( Andor Technology , Belfast , Northern Ireland ) and a CFI Apo TIRF 60 × /1 . 49 oil-immersion objective ( Nikon Instruments ) , which was heated to 30°C using an objective heater ( Bioptechs , Butler , PA ) . Exposure times for yEGFP and yEmRFP fluorescence were each 100 ms . Image analysis and video assembly were performed using ImageJ software ( NIH ) .
Heterochromatin exerts a heritable form of eukaryotic gene repression and contributes to chromosome segregation fidelity and genome stability . However , to date there has been no quantitative evaluation of the stability of heterochromatic gene repression . We designed a genetic strategy to capture transient losses of gene silencing in Saccharomyces as permanent , heritable changes in genotype and phenotype . This approach revealed rare transcription within heterochromatin that occurred in approximately 1/1000 cell divisions . In concordance with multiple lines of evidence suggesting these events were rare and transient , single-molecule RNA FISH showed that transcription was limited . The ability to monitor fluctuations in heterochromatic repression uncovered previously unappreciated roles for Sir1 , a silencing establishment factor , in the maintenance and/or inheritance of silencing . In addition , we identified the sirtuin Hst3 and its histone target as contributors to the stability of the silenced state . These approaches revealed dynamics of a heterochromatin function that have been heretofore inaccessible .
A single cell from a plant , an animal or another eukaryote can contain several meters of DNA . In order to fit this length inside the nucleus of the cell , the DNA is wrapped around proteins called histones to form a compact structure known as chromatin . Chromatin exists in two forms: loosely packed chromatin tends to contain the genes that are expressed in cells , whereas highly compacted chromatin ( also called heterochromatin ) silences the expression of most nearby genes . Adding small chemical markings to histone proteins can alter how much the chromatin is compacted , and newly formed cells can inherit these markings whenever a cell divides . This enables the new cell to essentially ‘remember’ which genes were active and which were repressed in the original cell . However , no one has measured how stable heterochromatin is over multiple cell divisions; as such it is unclear if genes that should remain silent are occasionally expressed after a cell has divided a number of times . Budding yeast is a well-established model for studying heterochromatin . The genome of this single-celled organism has distinct regions of highly compacted chromatin , which are established and maintained by a number of different proteins . Dodson and Rine have developed a new technique to detect when gene silencing is lost in individual yeast cells , even if the loss of silencing only occurs for a brief period . This approach revealed that genes in supposedly ‘silent’ heterochromatin were still expressed but only very rarely—in about 1 in every 1000 cell divisions . Dodson and Rine's findings suggest that when silencing is lost , it is promptly re-established with the aid of a protein called Sir1 . In addition , the new technique revealed that Sir1 helps to prevent losses of silencing in the first place . As such , the Sir1 protein appears to have previously unappreciated roles in the maintenance and the inheritance of heterochromatin in dividing yeast cells . Dodson and Rine also discovered that a protein called Hst3—which acts to remove chemical markings from histone proteins—also helps stabilize the silenced state . With this technique in hand , it is now possible to test any molecule , environment , or cellular process for its potential effect on the stability of gene silencing .