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Heterochromatin formation involves changes in histone modifications over multiple cell generations

2005; Springer Nature; Volume: 24; Issue: 12 Linguagem: Inglês

10.1038/sj.emboj.7600692

1460-2075

Yael Katan‐Khaykovich, Kevin Struhl,

Epigenetics and DNA Methylation

Article26 May 2005free access Heterochromatin formation involves changes in histone modifications over multiple cell generations Yael Katan-Khaykovich Yael Katan-Khaykovich Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Kevin Struhl Corresponding Author Kevin Struhl Search for more papers by this author Yael Katan-Khaykovich Yael Katan-Khaykovich Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Kevin Struhl Corresponding Author Kevin Struhl Search for more papers by this author Author Information Yael Katan-Khaykovich1 and Kevin Struhl 1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA *Corresponding author. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. Tel.: +1 617 432 2104; Fax: +1 617 432 2529; E-mail: [email protected] The EMBO Journal (2005)24:2138-2149https://doi.org/10.1038/sj.emboj.7600692 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Stable, epigenetic inactivation of gene expression by silencing complexes involves a specialized heterochromatin structure, but the kinetics and pathway by which euchromatin is converted to the stable heterochromatin state are poorly understood. Induction of heterochromatin in Saccharomyces cerevisiae by expression of the silencing protein Sir3 results in rapid loss of histone acetylation, whereas removal of euchromatic histone methylation occurs gradually through several cell generations. Unexpectedly, Sir3 binding and the degree of transcriptional repression gradually increase for 3–5 cell generations, even though the intracellular level of Sir3 remains constant. Strains lacking Sas2 histone acetylase or the histone methylases that modify lysines 4 (Set1) or 79 (Dot1) of H3 display accelerated Sir3 accumulation at HMR or its spreading away from the telomere, suggesting that these histone modifications exert distinct inhibitory effects on heterochromatin formation. These findings suggest an ordered pathway of heterochromatin assembly, consisting of an early phase, driven by active enzymatic removal of histone acetylation and resulting in incomplete transcriptional silencing, followed by a slower maturation phase, in which gradual loss of histone methylation enhances Sir association and silencing. Thus, the transition between euchromatin and heterochromatin is gradual and requires multiple cell division cycles. Introduction Epigenetically inheritable patterns of gene expression control important aspects of cell physiology, differentiation, and development. Eukaryotic genomes are composed of stable domains of euchromatin and heterochromatin that, respectively, are transcriptionally competent and silent. Heterochromatin accounts for diverse epigenetic phenomena, such as position effect variegation in Drosophila, X-chromosome inactivation in mammals, and telomeric and mating-type silencing in yeast. Despite notable difference among organisms and silencing systems, many functional and molecular aspects of heterochromatin are highly conserved (Moazed, 2001; Grewal and Moazed, 2003). Silent chromatin domains are compact, relatively inaccessible, and characterized by histone hypoacetylation and hypomethylation of lysines 4 and 79 of histone H3 (H3-K4 and H3-K79). The different proteins that mediate heterochromatin formation often possess enzymatic activities that covalently modify histones, and they interact with the modified histones, polymerize, and spread across large genomic regions. Spreading of heterochromatin is thus thought to occur through cycles of histone modification and binding, in which silencing complexes interact with the product of their own enzymatic activity (Moazed, 2001; Grewal and Moazed, 2003). In Saccharomyces cerevisiae, heterochromatin is formed at the HMR and HML mating type loci and telomeric regions by products of the silent information regulator genes SIR2, SIR3, and SIR4, which form the Sir complex (Rusche et al, 2003). Heterochromatin formation is initiated at silencers that are composed of binding sites for sequence-specific DNA-binding proteins. These DNA-binding proteins (together with Sir1 at the mating type loci) recruit the Sir complex, which then spreads across the entire locus. Sir2 is an evolutionarily conserved NAD-dependent histone deacetylase (HDAC), whose enzymatic activity is important for silencing (Moazed, 2001). Histones at silenced loci are hypoacetylated at all tested lysine residues, and the Sir complex binds preferentially to hypoacetylated histones (Grunstein, 1998; Suka et al, 2001). Of particular importance for silencing is lysine 16 of histone H4 (H4-K16), which is a direct target for Sir2-mediated deacetylation (Grunstein, 1998; Suka et al, 2002; Rusche et al, 2003). Histone deacetylation by Sir2 is presumed to promote silencing by creating high-affinity binding sites for the spreading Sir complex. Telomeric heterochromatin spreads from the chromosome end to a distance of several kb, and this domain can be enlarged by overexpression of Sir3 (Renauld et al, 1993; Hecht et al, 1996). The boundaries of telomeric heterochromatin domains are determined by several factors that limit Sir binding. Sas2, which acetylates the critical H4-K16, counteracts the histone deacetylation activity of Sir2 and thus blocks the spreading of heterochromatin (Kimura et al, 2002; Suka et al, 2002). H3 acetylases and other euchromatic components, the bromodomain protein Bdf1 and the histone variant H2A.Z, have similar effects (Kristjuhan et al, 2003; Ladurner et al, 2003; Meneghini et al, 2003). Methylated H3-K4 and H3-K79 are also marks associated with active chromatin (Bernstein et al, 2002; Ng et al, 2003a), and strains lacking the corresponding enzymes (Set1 and Dot1) compromise Sir binding and silencing at heterochromatin regions (Briggs et al, 2001; Ng et al, 2002; van Leeuwen et al, 2002). These histone methylases are thought to promote silencing indirectly, by preventing promiscuous binding of Sir proteins throughout the genome, thus concentrating the Sir proteins at their normal sites of action (van Leeuwen and Gottschling, 2002; van Leeuwen et al, 2002; Ng et al, 2003a; Santos-Rosa et al, 2004). While all these euchromatic factors display 'antisilencing' properties, it is largely unknown how they impact the process of heterochromatin assembly. Histone lysine methylation has emerged in recent years as an important mark associated with stable and transient transcriptional states, affecting both activation and silencing. For example, methylation of H3-K9 promotes heterochromatin formation in Schizosaccharomyces pombe and metazoans, while di-methylated H3-K4 and H3-K79 are universally associated with potentially active chromatin domains. Upon transcriptional induction, H3-K4 becomes tri-methylated at the active gene (Santos-Rosa et al, 2002; Ng et al, 2003b). While histone acetylation is highly dynamic and can be rapidly reversed by HDACs (Waterborg, 2001; Katan-Khaykovich and Struhl, 2002), histone methylation is stable in bulk chromatin, and transcriptionally induced H3-K4 tri-methylation persists to mark recently active genes after a transcriptional response has ended (Ng et al, 2003b). The stability of histone methylation marks renders them particularly suitable for the propagation and inheritance of epigenetic states. Several mechanisms have been proposed to address the fate of such marks during transitions between epigenetic states, where histone methylation associated with the initial transcriptional state might counteract establishment of the new state. First, upon removal of a histone methylase, the modified histones can be slowly eliminated by dilution through replication cycles. Second, methylated histones can be removed through replication-independent histone exchange, as occurs during the act of transcriptional elongation that disrupts, and possibly evicts, histones from the DNA template (Ahmad and Henikoff, 2002; Saccani and Natoli, 2002; Ghosh and Harter, 2003; Janicki et al, 2004; Lee et al, 2004; Schwabish and Struhl, 2004). Third, histone methylation could be rapidly eliminated by cleavage of the histone tails (Jenuwein and Allis, 2001; Bannister et al, 2002) or active demethylation of specific lysine residues (Shi et al, 2004). Fourth, methylation marks may persist but no longer perform their function, due to removal of their interacting proteins or occurrence of additional modifications, such as phosphorylation, on nearby residues (Bannister et al, 2002; Fischle et al, 2003). The particular mechanism that counteracts histone methylation may depend on the specific methylation mark and circumstance, and it is likely to impact the nature of the transcriptional transition. The intrinsic stability of epigenetic transcriptional states is important for the long-term maintenance of gene expression patterns; yet, transitions between such states can occur during development and cellular differentiation (Lyko and Paro, 1999; Heard, 2004; Su et al, 2004). In S. cerevisiae, subtelomeric silent chromatin is partially disrupted during the DNA damage response and reestablished following recovery (Martin et al, 1999; Mills et al, 1999), and its extent can be modulated in response to environmental conditions (Ai et al, 2002). Under normal growth conditions, even the relatively stable HML silencing is occasionally disrupted and re-established. At subtelomeric regions, where silencing is semistable, switches between silencing and activation occur more frequently (Pillus and Rine, 1989; Gottschling et al, 1990). The notion of heterochromatin domain formation through spreading and blocking of silencing proteins suggests a competition-based process, but a temporal dynamic view of heterochromatin formation is unknown. Here we investigate the molecular events associated with heterochromatin assembly and spreading in S. cerevisiae, and the roles of histone modifications in these processes. Our results suggest that histone acetylation and methylation are removed in a temporally and mechanistically distinct manner, coinciding with the initiation and enhancement of Sir3 association with chromatin. Both histone modifications inhibit some aspect of heterochromatin formation, in that they control the rate of Sir3 association and spreading. These findings support a two-phase mechanism for the assembly of silent chromatin, driven by sequential changes in distinct histone modifications that limit Sir3 association. Unexpectedly, the transition between stable epigenetic states is gradual, taking 3–5 cell division cycles to complete. Results Complete transcriptional silencing and Sir3 association take several generations after Sir3 induction To induce the formation of silent chromatin, we used a yeast strain in which HA-tagged Sir3 is expressed from the GAL10 promoter. Addition of 2% galactose to raffinose-grown cells causes a substantial induction of Sir3 expression after 1.5 h (Figure 1A), with no cytotoxic effect. Sir3 levels increase mildly (about two- to three-fold) up to 4.5 h, and show no obvious change afterwards. Transcriptional repression of HMRa1 is already evident after 1.5 h, and it is approximately 8-fold at 3 h (Figure 1B). At the 3-h time-point, the cells have undergone one cell division cycle, and, in this regard, efficient de novo silencing of HMRa1 requires passage through S-phase (Miller and Nasmyth, 1984) and a later M-phase event (Lau et al, 2002). Interestingly, RNA levels continue to decline throughout the time-course, reaching 71- and 217-fold repression after 7.5 and 15 h, respectively, even though Sir3 protein levels are unchanged. This continued decrease in RNA levels could reflect a decreasing, small subpopulation of cells that fail to initiate heterochromatin, or a gradual process of transcriptional inactivation over the whole population that takes several generations for complete silencing. Figure 1.Transcriptional inactivation of HMRa1 following Sir3 induction. Expression of HA-tagged Sir3 from the GAL10 promoter was induced by treating raffinose-grown THC70 cells with 2% galactose for the indicated times. The average cell doubling time was 3.1 h. (A) Sir3 levels were monitored by Western blot analysis with an HA antibody. TBP served as a loading control. (B) HMRa1 RNA levels normalized to the DED1 control, averaged from two independent experiments. Download figure Download PowerPoint To study the molecular events associated with heterochromatin formation, we used chromatin immunoprecipitation, focusing on two loci that are subject to silencing, the HMR mating type locus and the subtelomeric region of chromosome VI-R. At HMR, the a1 and a2 divergently transcribed genes are flanked by two silencers, E and I, of which E is the stronger (Figure 2A). The telomeric silencer is defined by tandemly repeated Rap1 sites at the end of the chromosome, with the heterochromatic domain extending several kb away from the telomere. Figure 2.Sir3 association with silenced loci during heterochromatin assembly. (A) A diagram of the HMR locus, containing the divergently transcribed a1 and a2 genes, flanked by the E and I silencers. The positions of PCR products are shown above. (B) Sir3 association with the indicated genomic regions (TEL primer pairs are centered around 0.7 and 1 kb from the end of chromosome VI-R) in THC70 cells treated with 2% galactose for the indicated times. The level of Sir3 association at HMRa2 at 7.5 h was set as 10, and the average of two independent experiments is shown. Download figure Download PowerPoint Sir3 binding (monitored with the HA-1 antibody) was detected at all loci at 1.5 h, whereas it is not detected prior to galactose induction (Figure 2B). Surprisingly, levels of Sir3 at all these heterochromatin loci increase throughout the entire 15-h time-course. This gradual increase in Sir3 association with heterochromatic loci occurs over several generations, even though intracellular Sir3 levels are essentially constant after the first generation following galactose induction. This observation suggests that the bulk of the population undergoes a gradual change in heterochromatin structure throughout the time-course. Furthermore, the continuous increase in Sir3 association up to 15 h roughly mirrors the continuous decline in transcription (Figure 1B), suggesting that incomplete transcriptional silencing is due to an intermediate state of heterochromatin. Distinct kinetics of loss of H3 acetylation, H3-K79 methylation, and H3-K4 methylation during heterochromatin formation Heterochromatic loci display low levels of H3 acetylation and methylation at H3-K4 and H3-K79 (see Supplementary Figure 1 for regional profiles of these modifications with different time-points). Upon Sir3 induction, H3 acetylation decreases substantially (two-fold) after 1.5 h, and it is largely eliminated by 3–4.5 h (Figure 3A). This rapid decrease in H3 acetylation is almost certainly due to histone deacetylation, with Sir2 histone deacetylase presumed to be the major enzymatic activity that is responsible. Figure 3.Dynamics of H3 acetylation and methylation during heterochromatin assembly. Levels of H3 acetylation (AcH3), H3-K79 di-methylation (diMeH3-K79) and H3-K4 di- (diMeH3-K4), tri-, and mono-methylation after induction of Sir3 expression. For each histone modification, the initial (A) or maximal (B) level was set to 100. The exponentially declining phase of each H3 methylation graph was used to calculate the half-life of histone methylation on chromatin (t1/2) and the modification's decline per 3.1 h replication cycle. The results represent the average of three (A) or two (B) independent experiments. Download figure Download PowerPoint In contrast to the rapid deacetylation of H3, levels of H3-K79 di-methylation decrease much more slowly. Levels of H3-K79 di-methylation are essentially unchanged 1.5 h after induction, and they decline gradually throughout the time-course, with an average half-life of 2.9 h at the exponentially declining phase of each curve (Figure 3A). As the average cell-doubling time in these experiments is around 3.1 h, levels of di-methylated H3-K79 decrease on average 2.2-fold per cell cycle (ranging between 1.9- and 2.6-fold depending on the locus). These results are consistent with di-methylated H3-K79 being removed primarily by two-fold dilutions through replication cycles. Loss of H3-K4 di-methylation during heterochromatin formation occurs with kinetics that are distinct from those of both H3 acetylation and di-methylated H3-K79 (Figure 3A). At the HMR silencers and subtelomeric regions (but not the HMRa1/a2 region; see below), di-methylated H3-K4 declines exponentially at a fairly constant rate throughout the time-course, long after H3 is completely deacetylated. However, loss of di-methylated H3-K4 is more rapid than loss of di-methylated H3-K79, with the average half-life of H3-K4 di-methylation being 1.7 h, which corresponds to a 3.6-fold decrease per cell cycle (range between 3.2- and 4.2-fold). The different persistence times of H3-K4 and H3-K79 methylation marks strongly suggest a mechanistic difference in their removal from chromatin. Whereas replication-mediated dilution can largely account for the loss of di-methylated H3-K79, the more rapid removal of H3-K4 methylation requires a replication-independent component that is specific for H3-K4 modification. In contrast to the results at the HMR silencer and subtelomeric regions, a 2.4-fold increase in di-methylated H3-K4 is observed around the HMRa1/a2 genes at the early time-points of Sir3 induction, after which methylation levels gradually decline throughout the time-course. To explore the basis for this unexpected initial increase in H3-K4 di-methylation, we examined H3-K4 mono- and tri-methylation. Tri-methylation of H3-K4 is maximal prior to induction and then displays a gradual, continuous decline throughout the entire time-course, with an average half-life of 1.4 h, and a 4.6-fold decrease per cell cycle (Figure 3B). In contrast, mono-methylation of H3-K4 keeps increasing until later times (around 6 h), reaching low levels again only at 15 h. The time-course of the three H3-K4 methylation marks, showing consecutive peaks of tri-, di-, mono-, and finally no methylation, supports the notion that heterochromatin is assembled through a gradual process that takes multiple cell divisions. Replication-dependent and -independent removal of euchromatic histone modifications Heterochromatin formation in S. cerevisiae depends on an unknown S-phase event that is not DNA replication (Miller and Nasmyth, 1984; Kirchmaier and Rine, 2001; Li et al, 2001). To address the mechanism by which histone methylation marks are removed during heterochromatin assembly, and specifically the role of DNA replication, we used an experimental system that uncouples the progression through S-phase from replication (Kirchmaier and Rine, 2001). In the strain used, a derivative of HMR containing a synthetic silencer with Gal4-binding sites is flanked by two target sites for the FLP recombinase. FLP induction results in the formation of an extrachromosomal ring that lacks any origin of DNA replication (Figure 4A). By first inducing ring formation and then expressing a Gal4–Sir1 fusion protein, heterochromatin assembles at an extrachromosomal HMR locus that is stable throughout the cell cycle but does not replicate (Figure 4B). Figure 4.Replication-dependent and -independent changes in histone modifications during heterochromatin assembly via a synthetic silencer. (A) A synthetic derivative of the HMR silencer (contains Rap1 and Abf1 sites and four copies of the Gal4-binding site) directs heterochromatin formation upon expression of a Gal4–Sir1 fusion. Two FLP target sites flank HMR, and FLP induction results in excision of HMR from the chromosome, to form a nonreplicating DNA ring. The positions of PCR products are shown above the a1 and a2 genes. (B) JRY7131 cells were grown in raffinose (control) or galactose to induce FLP, resulting in HMR excision and ring formation. Both cultures were subsequently washed and grown in raffinose media lacking methionine to induce Gal4–Sir1. (C) Changes in histone modifications at the replicating chromosomal (chromosome) and nonreplicating ring-borne (circle) HMR locus following Gal4–Sir1 induction. The chromosomal modification level at time 0 was set to 100, and the average of three independent experiments is shown. Download figure Download PowerPoint Expression of Gal4–Sir1 in the absence of FLP causes a decrease in H3 acetylation (two-fold), di-methylated H3-K79 (two-fold), and tri-methylated H3-K4 (three-fold), whereas levels of di-methylated H3-K4 initially increase and then slightly decrease (Figure 4C). All these effects are similar to those observed at the early times of heterochromatin formation via Sir3 induction (Figure 3). The smaller decreases in euchromatic histone modifications upon Gal4–Sir1 induction are consistent with the reduced silencing capacity of the Gal4-based silencer as compared to more efficient natural silencers (Kirchmaier and Rine, 2001; Li et al, 2001). Induction of FLP resulted in efficient excision and ring formation, as verified by PCR (data not shown). At the ring-borne HMR, H3 acetylation levels decline upon Gal4–Sir1 induction to a level comparable to that of the chromosomal locus (Figure 4C), confirming that loss of H3 acetylation is independent of DNA replication. By contrast, di-methylated H3-K79 levels are significantly higher at the ring-borne HMR, as compared to the chromosomal locus. The ring-borne HMR shows no reduction at 4.5 h, and only a 15–20% reduction at 6 h, whereas the chromosomal locus shows a two-fold decrease at 4.5 h. These results are consistent with the kinetic analysis (Figure 3), and they strongly suggest that DNA replication plays a major role in the removal of di-methylated H3-K79 marks during heterochromatin formation. The dynamics of di-methylated H3-K4 also differ between the chromosomal and extrachromosomal HMR locus (Figure 4C). In both cases, methylation increased upon Gal4–Sir1 induction, yet a higher increase occurred at the ring-borne HMR, and was followed by a substantial decrease. The levels of tri-methylated H3-K4 declined significantly at both the chromosomal and the extrachromosomal locus, indicating replication-independent removal. The higher tri-methylated H3-K4 levels at the ring-borne HMR following induction may also suggest a role for replication in tri-methylated H3-K4 removal. Altogether, these experiments suggest that di-methylated H3-K79 is removed primarily through replication, while H3-K4 methylation loss is mediated by replication-dependent and -independent processes, consistent with the distinct dynamics of these methylation marks upon Sir3 expression. H3 methylation delays Sir3 accumulation at HMR To address whether and how the process of heterochromatin assembly is impacted by the relatively persistent histone methylation marks, we examined the kinetics of Sir3 association in strains lacking Dot1 (H3-K79 methylase), Set1 (H3-K4 methylase), or Sas2 (H4-K16 acetylase that counteracts Sir2-mediated deacetylation and the spreading of heterochromatin). The strains grew at comparable rates during a 6-h galactose induction, and had roughly comparable levels of Sir3 expression (Figure 5B; there is perhaps a small decrease in the set1 strain at later times and a slightly higher level in the sas2 strain at certain times). As expected, transcriptional analysis of a natural heterochromatic gene (Yfr055W, located ∼5 kb from the end of chromosome 6R) shows weakened silencing in the dot1 strain, enhanced silencing in the sas2 strain, and marginally increased transcription in the set1 strain (Supplementary Figure 2). Figure 5.Effects of histone-modifying enzymes on Sir3 association at HMR. (A) Sir3 association at the indicated loci in wild-type (THC70; W), sas2 (s), dot1 (d), or set1 (t) cells induced for Sir3 expression for the indicated times. The level of Sir3 binding in a wild-type strain at TEL 0.27 at 15 h (see Figure 7) was set as 5. The average of three independent experiments is shown. (B) Western blot analysis of HA-Sir3 levels, using TBP as a loading control. Download figure Download PowerPoint As shown above, Sir3 binding at HMRa1/a2 in the wild-type strain is relatively low at the 2 h time-point, and increases substantially afterwards (Figure 5A). At the HMRE silencer, the delay in Sir3 binding is smaller. Deletion of SAS2 does not relieve the delay in Sir3 association or enhance Sir3 binding at HMR, but rather causes a slight decrease in Sir3 association (Figure 5A, left panels). By contrast, Sir3 accumulation at HMR is significantly faster in the dot1 strain (Figure 5A, middle panels), with a three- to four-fold enhancement being evident at HMRa1/a2 2 h after induction. A similar enhancing effect, albeit less pronounced, is observed in the set1 deletion strain (three-fold increase in HMRa1/a2 binding at 2 h, Figure 5A, right panels). These results suggest that persistent euchromatic histone methylation marks, generated by Dot1 and Set1, delay the accumulation of silencing proteins at HMR. Histone modifications affect the kinetics of Sir3 spreading to subtelomeric regions To study the effects of histone modifications on Sir3 spreading away from a silencer, we first determined the Sir3-binding profiles at the subtelomeric region of chromosome VI-R after a 15-h induction (Figure 6A). In the wild-type strain, binding is maximal near the telomere and gradually decreased over distance. Sir3 binding remained relatively high up to 10 kb, and then significantly dropped around 15–17 kb. Sir3 association in this strain extends further than in strains expressing SIR3 from its own promoter (Hecht et al, 1996), probably due to higher induced Sir3 levels. As expected from the role of Sas2 in limiting the spread of telomeric silencing (Kimura et al, 2002; Suka et al, 2002), Sir3 binding in the sas2 strain is enhanced at positions more than 5 kb from the telomere. Also, as expected (Ng et al, 2002; van Leeuwen et al, 2002), loss of Dot1 does not affect Sir3 binding at the telomere, but it does reduce Sir3 association at telomere-distal positions. In our strain, loss of Set1 has a minimal effect on Sir3 association at telomeric loci. Figure 6.Sir3 association with subtelomeric chromatin in wild-type and mutant strains. (A) Sir3 association at the indicated subtelomeric loci of chromosome VI-R in wild-type (THC70; WT), sas2, dot1, or set1 cells treated with 2% galactose for 15 h. The level of Sir3 binding in a wild-type strain at the telomeric-most position was set as 5. The average of two independent experiments in shown. (B) Sir3 association at the indicated subtelomeric region (TEL primer names indicate distances in kb from the end of chromosome VI-R) at the indicated times after Sir3 induction. The POL1 coding region served as control for nonspecific Sir3 association with chromatin, and the average of five independent experiments is shown. Download figure Download PowerPoint Analysis of Sir3-binding kinetics in the wild-type strain shows a dramatic difference between different subtelomeric positions (Figure 6B). Near the telomere, significant Sir3 binding occurs early on, reaching 75% of the final level by 4 h. As the distance from the telomere increases, Sir3 association is progressively slower. At 5.5 and 10 kb, binding is modest during the first 6 h, reaching only ∼10% of the final level. Substantial Sir3 association with these telomere-distal regions thus required more than two generations. In the sas2 strain, Sir3 association is dramatically enhanced at genomic regions 4–10 kb from the telomere between 2–6 h after induction, whereas Sir3 binding at the telomere (0.27 kb) is largely similar to that of the wild-type strain (Figure 7A). To address whether this effect might be due to the slightly increased Sir3 expression in the sas2 strain, we modified Sir3 expression levels by reducing galactose concentrations such that Sir3 levels were comparable between the wild-type and sas2 strains and produced a Sir3-binding profile resembling that of natural Sir3 strains (Supplementary Figure 3). Under these conditions, the kinetics of Sir3 binding at the 0.27 kb position is comparable in wild-type and sas2 strains, whereas the sas2 strain displays markedly higher Sir3 association at the 4 and 6 h time-points in regions 2.8–10 kb from the telomere. Thus, loss of Sas2 greatly accelerates Sir3 spreading to distal positions, suggesting that Sas2-mediated histone acetylation is a major factor in controlling the rate of Sir3 spreading. Figure 7.Effects of Sas2 (A), Dot1 (B), and Set1 (C) on the kinetics of Sir3 association with subtelomeric chromatin. ChIP samples from the experiments shown in Figure 5 were analyzed for Sir3 binding using primer pairs to different positions within the subtelomeric region of chromosome VI-R. 'TEL' primer names indicate distances in kb from the chromosome end. The POL1 coding region served as control for nonspecific Sir3 association. Download figure Download PowerPoint A significant, but less dramatic, increase in Sir3-binding kinetics is observed in the dot1 strain (Figure 7B). The dot1 effect is evident at 2.8–6.3 kb and most prominent