Heterochromatin formation involves changes in histone modifications in multiple cell cycles
Heterochromatin formation involves changes
in histone modi?cations over multiple cell generations
Yael Katan-Khaykovich and Kevin Struhl*
Department of Biological Chemistry and Molecular Pharmacology,Harvard Medical School,Boston,MA,USA
Stable,epigenetic inactivation of gene expression by silen-cing complexes involves a specialized heterochromatin structure,but the kinetics and pathway by which euchro-matin is converted to the stable heterochromatin state are poorly understood.Induction of heterochromatin in Saccharomyces cerevisiae by expression of the silencing protein Sir3results in rapid loss of histone acetylation,whereas removal of euchromatic histone methylation occurs gradually through several cell generations.Unexpectedly,Sir3binding and the degree of transcrip-tional repression gradually increase for 3–5cell genera-tions,even though the intracellular level of Sir3remains constant.Strains lacking Sas2histone acetylase or the histone methylases that modify lysines 4(Set1)or 79(Dot1)of H3display accelerated Sir3accumulation at HMR or its spreading away from the telomere,suggesting that these histone modi?cations exert distinct inhibitory effects on heterochromatin formation.These ?ndings sug-gest 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 en-hances Sir association and silencing.Thus,the transition between euchromatin and heterochromatin is gradual and requires multiple cell division cycles.
The EMBO Journal (2005)24,2138–2149.doi:10.1038/sj.emboj.7600692;Published online 26May 2005Subject Categories :chromatin &transcription
Keywords :epigenetic transition;heterochromatin;histone methylation;Sir proteins;transcriptional silencing
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,respec-tively,are transcriptionally competent and silent.Hetero-chromatin 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 organ-isms 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 4and 79of histone H3(H3-K4and H3-K79).The different proteins that mediate heterochromatin formation often pos-sess enzymatic activities that covalently modify histones,and they interact with the modi?ed histones,polymerize,and spread across large genomic regions.Spreading of hetero-chromatin is thus thought to occur through cycles of histone modi?cation 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-speci?c DNA-binding proteins.These DNA-binding proteins (together with Sir1at the mating type loci)recruit the Sir complex,which then spreads across the entire locus.Sir2is 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 16of 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 Sir2is presumed to promote silencing by creating high-af?nity binding sites for the spreading Sir complex.
T elomeric 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 heterochro-matin domains are determined by several factors that limit Sir binding.Sas2,which acetylates the critical H4-K16,counter-acts the histone deacetylation activity of Sir2and thus blocks the spreading of heterochromatin (Kimura et al ,2002;Suka et al ,2002).H3acetylases and other euchromatic compo-nents,the bromodomain protein Bdf1and the histone variant H2A.Z,have similar effects (Kristjuhan et al ,2003;Ladurner et al ,2003;Meneghini et al ,2003).Methylated H3-K4and H3-K79are also marks associated with active chromatin (Bernstein et al ,2002;Ng et al ,2003a),and strains lacking the corresponding enzymes (Set1and Dot1)compromise Sir binding and silencing at heterochromatin regions (Briggs
Received:4February 2005;accepted:29April 2005;published online:26May 2005
*Corresponding author.Department of Biological Chemistry and
Molecular Pharmacology,Harvard Medical School,Boston,MA 02115,USA.T el.:t16174322104;Fax:t16174322529;E-mail:kevin@https://www.wendangku.net/doc/575819155.html,
The EMBO Journal (2005)24,2138–2149|&
2005European Molecular Biology Organization |All Rights Reserved 0261-4189/05
https://www.wendangku.net/doc/575819155.html,
et al,2001;Ng et al,2002;van Leeuwen et al,2002).These histone methylases are thought to promote silencing in-directly,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-K9promotes heterochroma-tin formation in Schizosaccharomyces pombe and metazoans, while di-methylated H3-K4and H3-K79are universally asso-ciated with potentially active chromatin domains.Upon transcriptional induction,H3-K4becomes 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-K4tri-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 establish-ment of the new state.First,upon removal of a histone methylase,the modi?ed 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 speci?c 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 modi?cations,such as phosphorylation,on nearby residues(Bannister et al,2002; Fischle et al,2003).The particular mechanism that counter-acts histone methylation may depend on the speci?c methy-lation 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 re-covery(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 modi?cations in these processes.Our results suggest that histone acetylation and methylation are removed in a temporally and mechanistically distinct man-ner,coinciding with the initiation and enhancement of Sir3 association with chromatin.Both histone modi?cations in-hibit some aspect of heterochromatin formation,in that they control the rate of Sir3association and spreading.These ?ndings support a two-phase mechanism for the assembly of silent chromatin,driven by sequential changes in distinct histone modi?cations that limit Sir3association. Unexpectedly,the transition between stable epigenetic states is gradual,taking3–5cell division cycles to complete. Results
Complete transcriptional silencing and Sir3association take several generations after Sir3induction
T o induce the formation of silent chromatin,we used a yeast strain in which HA-tagged Sir3is expressed from the GAL10 promoter.Addition of2%galactose to raf?nose-grown cells causes a substantial induction of Sir3expression after1.5h (Figure1A),with no cytotoxic effect.Sir3levels increase mildly(about two-to three-fold)up to4.5h,and show no obvious change afterwards.Transcriptional repression of HMRa1is already evident after1.5h,and it is approximately 8-fold at3h(Figure1B).At the3-h time-point,the cells have undergone one cell division cycle,and,in this regard,ef?-cient de novo silencing of HMRa1requires 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,reaching71-and217-fold repression after7.5and15h,respectively,even though Sir3 protein levels are unchanged.This continued decrease in RNA levels could re?ect a decreasing,small subpopulation of cells that fail to initiate heterochromatin,or a gradual process of transcriptional inactivation over the whole popu-lation that takes several generations for complete silencing.
T o study the molecular events associated with heterochro-matin formation,we used chromatin immunoprecipitation, focusing on two loci that are subject to silencing,the HMR mating type locus and the subtelomeric region of chromo-some VI-R.At HMR,the a1and a2divergently transcribed genes are?anked by two silencers,E and I,of which E is the stronger(Figure2A).The telomeric silencer is de?ned by tandemly repeated Rap1sites at the end of the chromosome, with the heterochromatic domain extending several kb away from the telomere.
Sir3binding(monitored with the HA-1antibody)was detected at all loci at1.5h,whereas it is not detected prior to galactose induction(Figure2B).Surprisingly,levels of Sir3 at all these heterochromatin loci increase throughout the entire15-h time-course.This gradual increase in Sir3associa-tion with heterochromatic loci occurs over several genera-tions,even though intracellular Sir3levels are essentially
Dynamics of heterochromatin formation
Y Katan-Khaykovich and K Struhl
constant after the ?rst generation following galactose induc-tion.This observation suggests that the bulk of the popula-tion undergoes a gradual change in heterochromatin structure throughout the time-course.Furthermore,the continuous increase in Sir3association up to 15h 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 H3acetylation,H3-K79methylation,and H3-K4methylation during heterochromatin formation
Heterochromatic loci display low levels of H3acetylation and methylation at H3-K4and H3-K79(see Supplementary Figure 1for regional pro?les of these modi?cations with different time-points).Upon Sir3induction,H3acetylation decreases substantially (two-fold)after 1.5h,and it is largely eliminated by 3–4.5h (Figure 3A).This rapid decrease in H3acetylation is almost certainly due to histone deacetylation,with Sir2histone deacetylase presumed to be the major enzymatic activity that is responsible.
In contrast to the rapid deacetylation of H3,levels of H3-K79di-methylation decrease much more slowly.Levels of H3-K79di-methylation are essentially unchanged 1.5h after induction,and they decline gradually throughout the time-course,with an average half-life of 2.9h at the exponen-tially declining phase of each curve (Figure 3A).As the average cell-doubling time in these experiments is around 3.1h,levels of di-methylated H3-K79decrease 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-K79being removed primarily by two-fold dilutions through replication cycles.
Loss of H3-K4di-methylation during heterochromatin for-mation occurs with kinetics that are distinct from those of both H3acetylation and di-methylated H3-K79(Figure 3A).At the HMR silencers and subtelomeric regions (but not the HMRa1/a2region;see below),di-methylated H3-K4declines exponentially at a fairly constant rate throughout the time-course,long after H3is completely deacetylated.However,loss of di-methylated H3-K4is more rapid than loss of di-methylated H3-K79,with the average half-life of H3-K4di-methylation being 1.7h,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-K4and H3-K79
methylation
Figure 1Transcriptional inactivation of HMRa1following Sir3induction.Expression of HA-tagged Sir3from the GAL10promoter was induced by treating raf?nose-grown THC70cells with 2%galactose for the indicated times.The average cell doubling time was 3.1h.(A )Sir3levels were monitored by Western blot analysis with an HA antibody.TBP served as a loading control.(B )HMRa1RNA levels normalized to the DED1control,averaged from two independent
experiments.
Figure 2Sir3association with silenced loci during heterochroma-tin assembly.(A )A diagram of the HMR locus,containing the divergently transcribed a1and a2genes,?anked by the E and I silencers.The positions of PCR products are shown above.(B )Sir3association with the indicated genomic regions (TEL primer pairs are centered around 0.7and 1kb from the end of chromosome VI-R)in THC70cells treated with 2%galactose for the indicated times.The level of Sir3association at HMRa2at 7.5h was set as 10,and the average of two independent experiments is shown.
Dynamics of heterochromatin formation Y Katan-Khaykovich and K Struhl
marks strongly suggest a mechanistic difference in their removal from chromatin.Whereas replication-mediated dilu-tion can largely account for the loss of di-methylated H3-K79,the more rapid removal of H3-K4methylation requires a replication-independent component that is speci?c for H3-K4modi?cation.
In contrast to the results at the HMR silencer and sub-telomeric regions,a 2.4-fold increase in di-methylated H3-K4is observed around the HMRa1/a2genes at the early time-points of Sir3induction,after which methylation levels gradually decline throughout the time-course.T o explore the basis for this unexpected initial increase in H3-K4di-methylation,we examined H3-K4mono-and tri-methylation.Tri-methylation of H3-K4is maximal prior to induction and then displays a gradual,continuous decline throughout the entire time-course,with an average half-life of 1.4h,and a 4.6-fold decrease per cell cycle (Figure 3B).In contrast,mono-methylation of H3-K4keeps increasing until
later
Figure 3Dynamics of H3acetylation and methylation during heterochromatin assembly.Levels of H3acetylation (AcH3),H3-K79di-methylation (diMeH3-K79)and H3-K4di-(diMeH3-K4),tri-,and mono-methylation after induction of Sir3expression.For each histone modi?cation,the initial (A )or maximal (B )level was set to 100.The exponentially declining phase of each H3methylation graph was used to calculate the half-life of histone methylation on chromatin (t 1/2)and the modi?cation’s decline per 3.1h replication cycle.The results represent the average of three (A)or two (B)independent experiments.
Dynamics of heterochromatin formation
Y Katan-Khaykovich and K Struhl
times (around 6h),reaching low levels again only at 15h.The time-course of the three H3-K4methylation marks,showing consecutive peaks of tri-,di-,mono-,and ?nally 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 modi?cations
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).T o address the mechanism by which histone methyla-tion marks are removed during heterochromatin assembly,and speci?cally 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 ?anked 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 ?rst inducing ring forma-tion and then expressing a Gal4–Sir1fusion protein,hetero-chromatin assembles at an extrachromosomal HMR locus that is stable throughout the cell cycle but does not replicate (Figure 4B).
Expression of Gal4–Sir1in the absence of FLP causes a decrease in H3acetylation (two-fold),di-methylated H3-K79(two-fold),and tri-methylated H3-K4(three-fold),whereas levels of di-methylated H3-K4initially increase and then slightly decrease (Figure 4C).All these effects are similar to those observed at the early times of heterochromatin forma-tion via Sir3induction (Figure 3).The smaller decreases in euchromatic histone modi?cations upon Gal4–Sir1induction are consistent with the reduced silencing capacity of the Gal4-based silencer as compared to more ef?cient natural silencers (Kirchmaier and Rine,2001;Li et al ,2001).
Induction of FLP resulted in ef?cient excision and ring formation,as veri?ed by PCR (data not shown).At the ring-borne HMR ,H3acetylation levels decline upon Gal4–Sir1induction to a level comparable to that of the chromosomal locus (Figure 4C),con?rming that loss of H3acetylation is independent of DNA replication.By contrast,di-methylated H3-K79levels are signi?cantly higher at the ring-borne HMR ,as compared to the chromosomal locus.The ring-borne HMR
shows no reduction at 4.5h,and only a 15–20%reduction at 6h,whereas the chromosomal locus shows a two-fold de-crease at 4.5h.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-K79marks during heterochromatin formation.
The dynamics of di-methylated H3-K4also differ between the chromosomal and extrachromosomal HMR
locus
Figure 4Replication-dependent and -independent changes in his-tone modi?cations during heterochromatin assembly via a synthetic silencer.(A )A synthetic derivative of the HMR silencer (contains Rap1and Abf1sites and four copies of the Gal4-binding site)directs heterochromatin formation upon expression of a Gal4–Sir1fusion.T wo FLP target sites ?ank 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 a1and a2genes.(B )JRY7131cells were grown in raf?nose (control)or galactose to induce FLP,resulting in HMR excision and ring forma-tion.Both cultures were subsequently washed and grown in raf?-nose media lacking methionine to induce Gal4–Sir1.(C )Changes in histone modi?cations at the replicating chromosomal (chromo-some)and nonreplicating ring-borne (circle)HMR locus following Gal4–Sir1induction.The chromosomal modi?cation level at time 0was set to 100,and the average of three independent experiments is shown.
Dynamics of heterochromatin formation Y Katan-Khaykovich and K Struhl
(Figure 4C).In both cases,methylation increased upon Gal4–Sir1induction,yet a higher increase occurred at the ring-borne HMR ,and was followed by a substantial decrease.The levels of tri-methylated H3-K4declined signi?cantly at both the chromosomal and the extrachromosomal locus,indicat-ing replication-independent removal.The higher tri-methy-lated H3-K4levels at the ring-borne HMR following induction may also suggest a role for replication in tri-methylated H3-K4removal.Altogether,these experiments suggest that di-methylated H3-K79is removed primarily through replication,while H3-K4methylation loss is mediated by replication-dependent and -independent processes,consistent with the distinct dynamics of these methylation marks upon Sir3expression.
H3methylation delays Sir3accumulation at HMR
T o address whether and how the process of heterochromatin assembly is impacted by the relatively persistent histone methylation marks,we examined the kinetics of Sir3associa-tion in strains lacking Dot1(H3-K79methylase),Set1(H3-K4methylase),or Sas2(H4-K16acetylase that counteracts Sir2-mediated deacetylation and the spreading of heterochroma-tin).The strains grew at comparable rates during a 6-h galactose induction,and had roughly comparable levels of Sir3expression (Figure 5B;there is perhaps a small decrease in the set1strain at later times and a slightly higher level in the sas2strain at certain times).As expected,transcriptional analysis of a natural heterochromatic gene (Yfr055W ,located B 5kb from the end of chromosome 6R)shows weakened
g
n i d n i b 3r i S
g
n i d n i b 3r i S g
n i d n i b 3r i S
g
n i d n i b 3r i S Sir3 expression (h)
g
n i d n i b 3r i S
Sir3 expression (h)
g
n i d n i b 3r i S Sir3 expression (h)
g
n i d n i b 3r i S g
n i d n i b 3r i S g
n i d n i b 3r i S Sir3(HA)
TBP
Induction:
2 h
4 h
6 h 15 h W
s
d
t
W
s d
t
W
s
d
t
W
s
d
t
W
s
d
t
A
B Figure 5Effects of histone-modifying enzymes on Sir3association at HMR .(A )Sir3association at the indicated loci in wild-type (THC70;W),
sas2(s),dot1(d),or set1(t)cells induced for Sir3expression for the indicated times.The level of Sir3binding in a wild-type strain at TEL 0.27at 15h (see Figure 7)was set as 5.The average of three independent experiments is shown.(B )Western blot analysis of HA-Sir3levels,using TBP as a loading control.
Dynamics of heterochromatin formation
Y Katan-Khaykovich and K Struhl
silencing in the dot1strain,enhanced silencing in the sas2strain,and marginally increased transcription in the set1strain (Supplementary Figure 2).
As shown above,Sir3binding at HMRa1/a2in the wild-type strain is relatively low at the 2h time-point,and increases substantially afterwards (Figure 5A).At the HMRE silencer,the delay in Sir3binding is smaller.Deletion of SAS2does not relieve the delay in Sir3association or enhance Sir3binding at HMR ,but rather causes a slight decrease in Sir3association (Figure 5A,left panels).By contrast,Sir3accu-mulation at HMR is signi?cantly faster in the dot1strain (Figure 5A,middle panels),with a three-to four-fold en-hancement being evident at HMRa1/a22h after induction.A similar enhancing effect,albeit less pronounced,is observed in the set1deletion strain (three-fold increase in HMRa1/a2binding at 2h,Figure 5A,right panels).These results suggest that persistent euchromatic histone methylation marks,gen-erated by Dot1and Set1,delay the accumulation of silencing proteins at HMR .
Histone modi?cations affect the kinetics of Sir3spreading to subtelomeric regions
T o study the effects of histone modi?cations on Sir3spread-ing away from a silencer,we ?rst determined the Sir3-binding pro?les 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.Sir3binding remained relatively high up to 10kb,and then signi?cantly dropped around 15–17kb.Sir3associa-tion in this strain extends further than in strains expressing SIR3from its own promoter (Hecht et al ,1996),probably due to higher induced Sir3levels.As expected from the role of Sas2in limiting the spread of telomeric silencing (Kimura et al ,2002;Suka et al ,2002),Sir3binding in the sas2strain is enhanced at positions more than 5kb from the telomere.Also,as expected (Ng et al ,2002;van Leeuwen et al ,2002),loss of Dot1does not affect Sir3binding at the telomere,but it does reduce Sir3association at telomere-distal positions.In our strain,loss of Set1has a minimal effect on Sir3asso-ciation at telomeric loci.
Analysis of Sir3-binding kinetics in the wild-type strain shows a dramatic difference between different subtelomeric positions (Figure 6B).Near the telomere,signi?cant Sir3binding occurs early on,reaching 75%of the ?nal level by 4h.As the distance from the telomere increases,Sir3associa-tion is progressively slower.At 5.5and 10kb,binding is modest during the ?rst 6h,reaching only B 10%of the ?nal level.Substantial Sir3association with these telomere-distal regions thus required more than two generations.
In the sas2strain,Sir3association is dramatically enhanced at genomic regions 4–10kb from the telomere between 2–6h after induction,whereas Sir3binding at the telomere (0.27kb)is largely similar to that of the wild-type strain (Figure 7A).T o address whether this effect might be due to the
slightly
Figure 6Sir3association with subtelomeric chromatin in wild-type and mutant strains.(A )Sir3association at the indicated subtelo-meric loci of chromosome VI-R in wild-type (THC70;WT),sas2,dot1,or set1cells treated with 2%galactose for 15h.The level of Sir3binding in a wild-type strain at the telomeric-most position was set as 5.The average of two independent experiments in shown.(B )Sir3association at the indicated subtelomeric region (TEL primer names indicate distances in kb from the end of chromosome VI-R)at the indicated times after Sir3induction.The POL1coding region served as control for nonspeci?c Sir3association with chromatin,and the average of ?ve independent experiments is shown.
Dynamics of heterochromatin formation Y Katan-Khaykovich and K Struhl
increased Sir3expression in the sas2strain,we modi?ed Sir3 expression levels by reducing galactose concentrations such that Sir3levels were comparable between the wild-type and sas2strains and produced a Sir3-binding pro?le resembling that of natural Sir3strains(Supplementary Figure3).Under these conditions,the kinetics of Sir3binding at the0.27kb position is comparable in wild-type and sas2strains,whereas the sas2strain displays markedly higher Sir3association at the 4and6h time-points in regions2.8–10kb from the telomere. Thus,loss of Sas2greatly accelerates Sir3spreading to distal positions,suggesting that Sas2-mediated histone acetylation is a major factor in controlling the rate of Sir3spreading.
g
n
i
d
n
i
b
3
r
i
S
g
n
i
d
n
i
b
3
r
i
S
g
n
i
d
n
i
b
3
r
i
S
g
n
i
d
n
i
b
3
r
i
S
g
n
i
d
n
i
b
3
r
i
S
g
n
i
d
n
i
b
3
r
i
S
Sir3 expression (h)Sir3 expression (h)Sir3 expression (h)Sir3 expression (h)Sir3 expression (h)
Sir3 expression (h)
Sir3 expression (h)
Sir3 expression (h)
Sir3 expression (h)
Sir3 expression (h)
Sir3 expression (h)Sir3 expression (h)Sir3 expression (h)Sir3 expression (h)Sir3 expression (h)
A
B
C
Figure7Effects of Sas2(A),Dot1(B),and Set1(C)on the kinetics of Sir3association with subtelomeric chromatin.ChIP samples from the experiments shown in Figure5were analyzed for Sir3binding 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 POL1coding region served as control for nonspeci?c Sir3association.
Dynamics of heterochromatin formation
Y Katan-Khaykovich and K Struhl
A signi?cant,but less dramatic,increase in Sir3-binding kinetics is observed in the dot1strain(Figure7B).The dot1 effect is evident at2.8–6.3kb and most prominent at4.3and 5.5kb,where the6-h Sir3binding was enhanced?ve-to six-fold.The accelerated kinetics of Sir3spreading in the dot1 strain does not,however,result in higher steady-state bind-ing;indeed,the steady-state level of Sir3binding is lower in dot1strains.In contrast to Sas2and Dot1,loss of Set1does not alter Sir3spreading kinetics signi?cantly(Figure7C). Thus,both Sas2-mediated histone acetylation and Dot1-mediated H3-K79methylation delay Sir3spreading and het-erochromatin formation,with Sas2playing the major role. Discussion
Heterochromatin formation is a gradual process,taking multiple cell generations
The dynamics of heterochromatin formation and the role of various histone modi?cations in the transition from an active to a fully silenced state are poorly understood.Here we follow this process temporally,by inducing heterochromatin rapidly and ef?ciently using a galactose-regulated Sir3gene. Although Sir3overexpression alters the balance between the cellular concentrations of the different silencing proteins, thus possibly affecting some quantitative parameters of het-erochromatin assembly,the basic mechanistic aspects of this process are likely to be the same.In particular,heterochro-matin containing overexpressed Sir3is affected by mutations of histone-modifying enzymes in a manner similar to that of natural strains.
Our results show that the transition from an active to a fully silenced state is surprisingly slow,taking multiple cell generations.Speci?cally,the degree of transcriptional silen-cing and the level of Sir3association progressively increase throughout the15-h time-course,which corresponds to?ve generations.In addition,the kinetics of various forms of H3-K4methylation suggest a gradual loss of Set1action through-
out the process of heterochromatin formation.Thus, although euchromatin and heterochromatin represent stable epigenetic states,the gradual and progressive transition between these stable states suggests the existence of meta-stable,intermediate states of chromatin.As discussed below, our results suggest that the need to remove different histone modi?cations de?nes the dynamic nature of heterochromatin formation.
Early and late events in heterochromatin assembly involving distinct histone modi?cations
Our results suggest that the pathway of heterochromatin assembly consists of two phases,controlled by distinct histone modi?cations(Figure8).The‘initiation’phase is characterized by moderate association of Sir proteins,rapid histone deacetylation by Sir2(and possibly other HDACs), and a minimal change in histone methylation.This initial state of‘partial heterochromatin’results in signi?cant,but incomplete,transcriptional silencing.We suggest that this initial phase of heterochromatin formation limits the access or activity of histone methylases.In the subsequent‘matura-tion’phase,H3-K79and H3-K4methylation is slowly lost throughout the time-course(at least three generations)by silencing-independent mechanisms(including DNA replica-tion),thereby allowing progressive enhancements of Sir3binding.This increased association of Sir proteins is required for the?nal heterochromatic state in which transcription in the region is completely silenced.
The correlations between changes in histone modi?cations and heterochromatin formation,as de?ned by Sir3binding and transcriptional silencing,suggest that the former may be controlling the latter.This idea is strongly supported by our observations that euchromatic histone modi?cations exert distinct inhibitory effects on the rate of heterochromatin formation.Loss of Dot1or Set1,but not Sas2,accelerates the accumulation of Sir3at HMR.Loss of Sas2,and to a lesser extent Dot1,dramatically accelerates Sir3spreading to telo-mere-distal regions.Collectively,our results suggest that the sequential loss of multiple euchromatic marks may be a driving force in the formation of silent chromatin.Although the stability of the silenced state has not been directly assayed here,we speculate that this notable characteristic of hetero-chromatin develops during the‘maturation’phase,with the progressive hypomethylation of histones.As a possible ana-logy,transcriptional inactivation of certain mammalian genes is initially reversible,and becomes epigenetically silenced at later stages of chromatin assembly(Wutz and Jaenisch,2000; Su et al,2004).A combination of histone modi?cations with distinct dynamic properties,which can be sequentially elimi-nated through active and passive mechanisms,may
thus Figure8A two-phase mechanism for the ordered assembly of heterochromatin,driven by sequential changes in histone modi?ca-tions.(A)Euchromatic regions have an open,accessible chromatin structure characterized by histone acetylation,and methylation at H3-K4and H3-K79.(B)During the initiation phase of heterochro-matin assembly,rapid histone deacetylation by Sir2(and possibly other enzymes)generates moderate-af?nity binding sites for the Sir complex,thus promoting the initial association of Sir proteins with chromatin.This generates a heterochromatic-like structure that partially inhibits transcription and the activity of histone methy-lases.This intermediate chromatin state,however,still retains histone methylation marks,due to their stable nature,and these prevent further binding of Sir proteins.(C)During the following maturation phase,the relatively slow and gradual removal of histone methylation allows further accumulation of Sir proteins, resulting in a complete heterochromatic structure that fully silences transcription.
Dynamics of heterochromatin formation Y Katan-Khaykovich and K Struhl
mediate an ordered,gradual transition between stable epi-genetic states.
Potential molecular mechanisms underlying
the dynamics of histone methylation
Unlike the rapid loss of histone acetylation via the action of Sir2and possibly other histone deacetylases,loss of H3-K79 and H3-K4methylation in the course of heterochromatin formation is gradual and relatively slow due to the relative stability of these modi?cations.The dynamics of the different H3-K4methylation marks at HMRa1/a2reveal consecutive peaks of tri-,di-,and mono-methylation,with the latter decreasing substantially only after more than three genera-tions.Importantly,the decline of a given methylation mark correlates with rising levels of the mark with one fewer methyl groups.This observation cannot be explained simply by passive dilution through DNA replication.Instead,the observation suggests that,during heterochromatin assembly, histones are exchanged and Set1action on newly deposited histones is gradually reduced to favor tri-,then di-,then mono-methylation,with complete inhibition occurring at later times.The progressive association of Sir proteins is likely to progressively restrict access of Set1to chromatin, and,in this view,the shift between the different methylated H3-K4’s represents another indication that the transition between euchromatin and heterochromatin is gradual and takes multiple cell generations.An alternative mechanism, which we consider less likely,is direct conversion of tri-to di-to mono-methylation by an unknown histone demethylase. As indicated by the slow and gradual changes in H3-K4 methylation marks,such a hypothetical histone demethylase would be inef?cient in the context of heterochromatin assembly.
Unlike the case for H3-K4methylation,both the kinetic analysis and synthetic silencer experiments suggest that re-plication-coupled histone deposition serves as the major (though not exclusive)removal mechanism for H3-K79 methylation.We consider four possibilities for why H3-K4 and H3-K79methylation is lost with different kinetics during heterochromatin formation.First,replication-independent his-tone exchange might preferentially occur on H3-K4-methy-lated nucleosomes.Second,histone tail cleavage(Jenuwein and Allis,2001)would preferentially remove H3-K4methyla-tion,although such a mechanism would have to be coupled with re-methylation of newly deposited histones to account for the consecutive peaks of different H3-K4methylated forms. Third,the more rapid disappearance of H3-K4methylation might be due to an H3-K4-speci?c histone demethylase,and such an enzyme has been described recently in mammalian cells(Shi et al,2004).Fourth,the extent of histone exchange, inferred from the pattern of H3-K4methylation,might be masked by ef?cient H3-K79methylation of newly deposited histones.In this regard,490%of H3is methylated at K79 (considering all three forms),whereas only35%is methylated at K4(van Leeuwen and Gottschling,2002).
Spreading kinetics of heterochromatin
In various eukaryotes,the formation of heterochromatin domains involves spreading of silencing proteins away from their nucleation centers.Described in dynamic terms,but experimentally studied in static systems,spreading of bud-ding yeast heterochromatin involves a competition between the opposing enzymatic activities of Sir2and Sas2,which create a gradient of H4-K16acetylation across the subtelo-meric region(Kimura et al,2002;Suka et al,2002).Our kinetic analysis indicates that spreading of Sir3from a silencer is a surprisingly slow process,lasting several cell generations.This slow rate of spreading is not an inherent kinetic property of the Sir proteins,because loss of Sas2(and to a lesser extent Dot1)accelerates the rate of spreading. Instead,the properties of euchromatin,the substrate for heterochromatin formation,determine the rate of Sir spread-ing.Euchromatin may thus restrict the invasion of hetero-chromatin through a kinetic inhibition,where the need to counteract Sas2-mediated acetylation(and to a lesser extent Dot1-mediated methylation)slows down the advancing Sir complex.In a simple model of heterochromatin formation, the extent of a silent domain would be determined by a balance between the spreading rate of individual Sir mole-cules and their stability on chromatin,either of which may be in?uenced by histone modi?cations.In any event,our results suggest a functional link between the spreading kinetics of heterochromatin and the steady-state genomic partition into active and inactive regions.We note that kinetic inhibition on Sir spreading is more likely to be effective at subtelomeric regions,with unde?ned heterochromatin–euchromatin boun-daries,than at the HMR locus,which contains discrete boundary elements(Rusche et al,2003).
Chromatin dynamics in the establishment
and maintenance of epigenetic states
Various euchromatic marks have been implicated in inhibit-ing the binding of silencing proteins;yet,the functional relationship between them and why multiple euchromatic marks are needed have remained unclear.The euchromatic histone modi?cation pattern includes both a dynamic(acet-ylation)and a relatively stable(methylation)component with ‘antisilencing’properties.As a consequence of their dynamic properties,the sequential removal of histone acetylation and methylation seems to promote different phases of hetero-chromatin formation,which together mediate an ordered transition between stable epigenetic states.In particular, this transition involves an intermediate state(s),in which Sir protein association has not reached the level of the?nal state and transcriptional silencing is signi?cant,but incom-plete.The pathway of silent chromatin assembly thus exem-pli?es how the distinct dynamic nature of histone acetylation and methylation can be used to drive and control a complex chromatin process.
Although histone acetylases and methylases both control the genomic distribution of silencing proteins,these enzymes affect heterochromatin in a different manner.Whereas Sas2 limits Sir3accumulation at telomere-distal regions,the most notable effect of Dot1is to increase the steady-state level of Sir3at some of these same regions.Considering these phe-notypes,and the distinct characteristics of the corresponding histone modi?cations,it is possible that the two euchromatic enzymes ful?ll different,partially overlapping roles,which together maintain the proper partition of the genome into active and silent chromatin.Sas2-mediated histone acetyla-tion may be primarily responsible for limiting the linear spreading of silencing proteins from their nucleation centers, whereas histone methylation marks generated by Dot1(and Set1)may function mainly to prevent promiscuous binding
Dynamics of heterochromatin formation
Y Katan-Khaykovich and K Struhl
and titration of Sir proteins throughout the genome.The use of reversible histone acetylation to control the spreading of heterochromatin generates a dynamic boundary that can limit the extent of heterochromatin domains,yet can be overcome to allow silent chromatin assembly at the appro-priate location and circumstance.On the other hand,the more stable histone methylation marks would be ideal for preventing the binding of silencing proteins throughout the euchromatic genome at all times.A combination of reversible and irreversible histone modi?cations may thus provide the required stability to epigenetic chromatin states,while main-taining enough?exibility to allow properly orchestrated epigenetic transitions.
Lastly,the competition between heterochromatic and eu-chromatic states,which de?nes the gradual nature of the transition,is also likely to be affected by ongoing transcrip-tional activity.Signi?cantly,H3-K4tri-methylation occurs around the50end of active genes due to targeting of Set1 methylase(Santos-Rosa et al,2002;Ng et al,2003b).The relative resistance of active genes to the invasion of hetero-chromatin,as observed at HMRa1/a2,may be due to tran-scription-coupled generation of histone methylation marks. In this regard,the?rst few generations of heterochromatin assembly are characterized by reduced,but still ongoing, transcription.The process by which active genes become stably silenced would thus represent a continuous,dynamic competition,where the initial active state counteracts estab-lishment of the new silenced state through the generation of targeted and persistent euchromatic histone modi?cations. Materials and methods
DNAs and yeast strains
THC70was a gift from Mark Gartenberg(Cheng and Gartenberg, 2000).The THC70-derived sas2(YKY7),dot1(YKY10),and set1 (YKY8)strains were constructed by PCR-based gene replacement of the wild-type loci with loxP-LEU2-loxP(Gueldener et al,2002). JRY7131was a gift from Jasper Rine(Kirchmaier and Rine,2001). THC70and its derivatives were grown in YP medium containing2% raf?nose,and Sir3expression was induced by treating the cells with galactose.JRY7131was initially grown in synthetic complete media lacking histidine(for plasmid selection)and containing2% raf?nose and100m M methionine.Induction of FLP and Gal4–Sir1 was carried out by growing cells in media containing2%galactose or lacking methionine,respectively.
Transcriptional analysis
HMRa1mRNA levels were determined with respect to DED1mRNA levels by reverse transcriptase,quantitative PCR in real time,as described(Proft and Struhl,2002).Chromatin immunoprecipitation
Chromatin immunoprecipitation was performed essentially as described(Kuras and Struhl,1999;Aparicio et al,2004),with modi?cations.Insoluble chromatin was pelleted by spinning for 20min in a microfuge,followed by resuspension and sonication. The resulting chromatin solutions were cleared by spinning for 30min in a microfuge.For the experiments shown in Figure4and Supplementary Figure3,crosslinked whole-cell extracts were used, by omitting the above20-min spin.Immunoprecipitations were carried out in150mM NaCl,using antibodies against the following: di-acetylated H3(lysines9and14),di-methylated H3-K79,di-methylated H3-K4(all from Upstate Biotechnology),tri-methylated H3-K4(AbCam),mono-methylated H3-K4(AbCam),and the HA1epitope(F7,Santa Cruz).Quantitative PCR analyses were performed in real time using an Applied Biosystems7700 sequence detector.IP ef?ciency was calculated as the ratio bet-ween the amounts of IP PCR product and input PCR product.For analysis of histone modi?cations,the IP ef?ciency of the tested locus was normalized with respect to that of a control locus(POL1 coding region or ACT1promoter).Error bars represent standard deviations.
The statistical signi?cance of the difference between each pair of histone modi?cations in Figure3A was examined by the unpaired t-test.For the relevant time-points where differences are claimed, P-values are typically o0.05for individual time-points and o10à4 for the combination of relevant time-points.H3acetylation levels are lower than the corresponding H3-K79di-methylation levels from1.5to6h,with P-values(from the four time-points combined) of2?10à4for TEL1.0and below10à4for each of the other six loci. H3acetylation levels are lower than the corresponding H3-K4di-methylation levels at HMRE,HMRI,and the TEL regions at1.5h and at HMRE and HMRI at3h,with a P-value(combined)below10à4. H3-K4di-methylation levels are lower than the corresponding H3-K79di-methylation levels from3to7.5h,with P-values(combined) of2?10à4for HMRE and below10à4for HMRI,TEL0.7,and TEL1.0. In the synthetic silencer experiments(Figure4C),the levels of all histone methylation marks are higher at the extrachromosomal than at the chromosomal HMR from3to6h,with P-values(from the three time-points combined)of o10à4for both HMRa1and HMRa2.
Western blotting
Electrophoretically separated proteins(from crosslinked or non-crosslinked cell extracts)were probed with monoclonal anti-HA (12CA5)or polyclonal anti-TBP antibodies.
Supplementary data
Supplementary data are available at The EMBO Journal Online.
Acknowledgements
We thank Marc Gartenberg,Ann Kirchmaier,and Jasper Rine for strains and plasmids,and Huck Hui Ng for fruitful discussions.This work was supported by an EMBO postdoctoral fellowship to YK-K and by research grant GM53720to KS from the National Institutes of Health.
References
Ahmad K,Henikoff S(2002)The histone variant H3.3marks active chromatin by replication-independent nucleosome assembly.Mol Cell9:1191–1200
Ai W,Bertram PG,Tsang CK,Chan TF,Zheng XF(2002)Regulation of subtelomeric silencing during stress response.Mol Cell10: 1295–1305
Aparicio OM,Geisberg JV,Struhl K(2004)Chromatin immunopre-cipitation for determining the association of proteins with speci?c genomic sequences in vivo.In Current Protocols in Molecular Biology,Ausubel FA,Brent R,Kingston RE,Moore DD,Seidman JG,Smith JA,Struhl K(eds),pp21.3.1–21.3.17.New Y ork:John Wiley&Sons Bannister AJ,Schneider R,Kouzarides T(2002)Histone methyla-tion.Dynamic or static?Cell109:801–806
Bernstein BE,Humphrey EL,Erlich RL,Schneider R,Bouman P,Liu JS,Kouzarides T,Schreiber SL(2002)Methylation of histone H3 Lys4in coding regions of active genes.Proc Natl Acad Sci USA99: 8695–8700
Briggs SD,Bryk M,Strahl BD,Cheung WL,Davie JK,Dent SY, Winston F,Allis CD(2001)Histone H3lysine4methylation is mediated by Set1and required for cell growth and rDNA silencing in Saccharomyces cerevisiae.Genes Dev15:3286–3295
Cheng TH,Gartenberg MR(2000)Y east heterochromatin is a dynamic structure that requires silencers continuously.Genes Dev14:452–463
Dynamics of heterochromatin formation Y Katan-Khaykovich and K Struhl
Fischle W,Wang Y,Allis CD(2003)Binary switches and modi?ca-tion cassettes in histone biology and beyond.Nature425: 475–479
Ghosh MK,Harter ML(2003)A viral mechanism for remodeling chromatin structure in G0cells.Mol Cell12:255–260 Gottschling DE,Aparicio OM,Billington BL,Zakian V A(1990) Position effect at S.cerevisiae telomeres:reversible repression of pol II transcription.Cell63:751–762
Grewal SI,Moazed D(2003)Heterochromatin and epigenetic control of gene expression.Science301:798–802
Grunstein M(1998)Yeast heterochromatin:regulation of its assem-bly and inheritance by histones.Cell93:325–328
Gueldener U,Heinisch J,Koehler GJ,Voss D,Hegemann JH(2002) A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast.Nucl Acids Res30:e23 Heard E(2004)Recent advances in X-chromosome inactivation. Curr Opin Cell Biol16:247–255
Hecht A,Strahl-Bolsinger S,Grunstein M(1996)Spreading of transcriptional repressor SIR3from telomeric heterochromatin. Nature383:92–96
Janicki SM,Tsukamoto T,Salghetti SE,T ansey WP,Sachidanandam R,Prasanth KV,Ried T,Shav-T ai Y,Bertrand E,Singer RH,Spector DL(2004)From silencing to gene expression:real-time analysis in single cells.Cell116:683–698
Jenuwein T,Allis CD(2001)Translating the histone code.Science 293:1074–1080
Katan-Khaykovich Y,Struhl K(2002)Dynamics of global histone acetylation and deacetylation in vivo:rapid restoration of normal histone acetylation status upon removal of activators and repres-sors.Genes Dev16:743–752
Kimura A,Umehara T,Horikoshi M(2002)Chromosomal gradient of histone acetylation established by Sas2and Sir2functions as a shield against gene silencing.Nat Genet32:370–377 Kirchmaier AL,Rine J(2001)DNA replication-independent silen-cing in S.cerevisiae.Science291:646–650
Kristjuhan A,Wittschieben BO,Walker J,Roberts D,Cairns BR, Svejstrup JQ(2003)Spreading of Sir3protein in cells with severe histone H3hypoacetylation.Proc Natl Acad Sci USA100: 7551–7556
Kuras L,Struhl K(1999)Binding of TBP to promoters in vivo is stimulated by activators and requires Pol II holoenzyme.Nature 399:609–612
Ladurner AG,Inouye C,Jain R,Tjian R(2003)Bromodomains mediate an acetyl-histone encoded antisilencing function at het-erochromatin boundaries.Mol Cell11:365–376
Lau A,Blitzblau H,Bell SP(2002)Cell-cycle control of the establish-ment of mating-type silencing in S.cerevisiae.Genes Dev16: 2935–2945
Lee CK,Shibata Y,Rao B,Strahl BD,Lieb JD(2004)Evidence for nucleosome depletion at active regulatory regions genome-wide. Nat Genet36:900–905
Li YC,Cheng TH,Gartenberg MR(2001)Establishment of transcrip-tional silencing in the absence of DNA replication.Science291: 650–653
Lyko F,Paro R(1999)Chromosomal elements conferring epigenetic inheritance.BioEssays21:824–832
Martin SG,Laroche T,Suka N,Grunstein M,Gasser SM(1999) Relocalization of telomeric Ku and SIR proteins in response to DNA strand breaks in yeast.Cell97:621–633
Meneghini MD,Wu M,Madhani HD(2003)Conserved histone variant H2A.Z protects euchromatin from the ectopic spread of heterochromatin.Cell112:725–736
Miller AM,Nasmyth KA(1984)Role of DNA replication in the repression of silent mating type loci in yeast.Nature312:247–251Mills KD,Sinclair DA,Guarente L(1999)MEC1-dependent redis-tribution of the Sir3silencing protein from telomeres to DNA double-strand breaks.Cell97:609–620
Moazed D(2001)Common themes in mechanisms of gene silen-cing.Mol Cell8:489–498
Ng HH,Ciccone DN,Morshead KB,Oettinger MA,Struhl K(2003a) Lysine-79of histone H3is hypomethylated at silenced loci in yeast and mammalian cells:a potential mechanism for position-effect variegation.Proc Natl Acad Sci USA100:1820–1825
Ng HH,Feng Q,Wang H,Erdjument-Bromage H,T empst P,Zhang Y, Struhl K(2002)Lysine methylation within the globular domain of histone H3by Dot1is important for telomeric silencing and Sir protein association.Genes Dev16:1518–1527
Ng HH,Robert F,Y oung RA,Struhl K(2003b)T argeted recruitment of Set1histone methylase by elongating Pol II provides a localized mark and memory of recent transcriptional activity.Mol Cell11: 709–719
Pillus L,Rine J(1989)Epigenetic inheritance of transcriptional states in S.cerevisiae.Cell59:637–647
Proft M,Struhl K(2002)Hog1kinase converts the Sko1-Cyc8-T up1 repressor complex into an activator that recruits SAGA and SWI/SNF in response to osmotic stress.Mol Cells9:1307–1317 Renauld H,Aparicio OM,Zierath PD,Billington BL,Chablani SK, Gottschling DE(1993)Silent domains are assembled continu-ously from the telomere and are de?ned by promoter distance and strength,and by SIR3dosage.Genes Dev7:1133–1145 Rusche LN,Kirchmaier AL,Rine J(2003)The establishment, inheritance,and function of silenced chromatin in Saccharo-myces cerevisiae.Annu Rev Biochem72:481–516
Saccani S,Natoli G(2002)Dynamic changes in histone H3Lys9 methylation occurring at tightly regulated inducible in?ammatory genes.Genes Dev16:2219–2224
Santos-Rosa H,Bannister AJ,Dehe PM,Geli V,Kouzarides T(2004) Methylation of H3lysine4at euchromatin promotes Sir3associa-tion with heterochromatin.J Biol Chem279:47506–47512 Santos-Rosa H,Schneider R,Bannister AJ,Sherriff J,Bernstein BE, Emre NC,Schreiber SL,Mellor J,Kouzarides T(2002)Active genes are tri-methylated at K4of histone H3.Nature419:407–411 Schwabish MA,Struhl K(2004)Evidence for eviction and rapid deposition of histones upon transcriptional elongation by RNA polymerase II.Mol Cell Biol24:10111–10117
Shi Y,Lan F,Matson C,Mulligan P,Whetstine JR,Cole PA,Casero RA,Shi Y(2004)Histone demethylation mediated by the nuclear amine oxidase homolog LSD1.Cell119:941–953
Su RC,Brown KE,Saaber S,Fisher AG,Merkenschlager M,Smale ST(2004)Dynamic assembly of silent chromatin during thymo-cyte maturation.Nat Genet36:502–506
Suka N,Luo K,Grunstein M(2002)Sir2and Sas2opposingly regulate acetylation of yeast histone H4lysine16and spreading of heterochromatin.Nat Genet32:378–383
Suka N,Suka Y,Carmen AA,Wu J,Grunstein M(2001)Highly speci?c antibodies determine histone acetylation in yeast hetero-chromatin and euchromatin.Mol Cell8:473–479
van Leeuwen F,Gafken PR,Gottschling DE(2002)Dot1p modulates silencing in yeast by methylation of the nucleosome core.Cell 109:745–756
van Leeuwen F,Gottschling DE(2002)Genome-wide histone mod-i?cations:gaining speci?city by preventing promiscuity.Curr Opin Cell Biol14:756–762
Waterborg JH(2001)Dynamics of histone acetylation in Saccharomyces cerevisiae.Biochemistry40:2599–2605
Wutz A,Jaenisch R(2000)A shift from reversible to irreversible X inactivation is triggered during ES cell differentiation.Mol Cell5: 695–705
Dynamics of heterochromatin formation
Y Katan-Khaykovich and K Struhl