54 France. 75248 ai,728France. 75248 Paris, Teeatoscnrbtdeulyt hswork this to equally contributed authors *These France. Paris, 75006 PSL, h eieti eeohoai tt uigtecl cycle cell Boyarchuk the Ekaterina by during controlled state is heterochromatin centromeres pericentric of the composition variant histone The ARTICLE RESEARCH eevd2 eebr21;Acpe 9My2014 May 19 Accepted 2013; December 28 Received ` the and 1 formation, kinetochore of site contribute domains the (PHC) 2011). heterochromatin pericentric with surrounding as centric Cheeseman, The domains serves features. chromatin and chromatin domain and adjacent properties Gascoigne functional distinct two in 2003; (reviewed comprise composition al., Centromeres variant et histone the unique Cleveland its because landmark chromosomal histone on remodeled, this depend how of or function The address and transmitted 2010). propagation to identity, is Henikoff, model and composition attractive Talbert variant an 2013; offers al., al., centromere et et DNA (Soria Szenker on repair impact 2012; and which transcription states, important recombination, chromatin is replication, of variants specification histone the of for distribution genomic proper The INTRODUCTION Cell CENP-A, cycle H2A.Z, Heterochromatin, Centromere, WORDS: centromere. KEY the timing at deposition variants the of histone regulate of to distinct stoichiometry signal modes and highlighting provide potential variant, a cycle H2A as different other integrity cell the PHC or the two one during replenish to PHC The opportunities the the chromatin. of at level nucleosomal rearrangement of structure centromeric the chromatin at amount dynamics within higher-order influences the domain the deposition. pericentric that its of increasing conclude upon timing domain. the We impacts changing chromatin, the without also CENP-A at centromeric centric H2A.Z integrity of PHC neighboring accumulation compromising are the features Interestingly, of heterochromatin to replication- This when H2A.Z. a leading waves lost for in is altered, phase phase discrete restriction G1 cycle S during two cell late and G1 to H2A reveal mid for manner during we deposited dependent – cycle, PHC in are at H2A.Z cell Whereas deposition and the (PHC). H2A throughout synthesized heterochromatin newly pericentric is euchromatin variants histone at H2A.Z and address H2A controlled we canonical chromatin Here, of vicinity. deposition unique their the how in a and centromeres requires at environment segregation chromosome Correct ABSTRACT Genevie uhrfrcrepnec ([email protected]) correspondence for Author ntttCre eted ehrh,Prs 54 France. 75248 Paris, Recherche, de Centre Curie, Institut 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,34–39doi:10.1242/jcs.148189 3347–3359 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. v Almouzni `ve 4 PC M36,Prs 54 France. 75248 Paris, UMR3664, UPMC, 3 qieLabellise Equipe 1,2,3,4,5, 1,2,3,4,5, ´ iu otel acr M36,Paris, UMR3664, Cancer, le contre Ligue e ` ,DnFilipescu Dan *, 5 obneUniversity, Sorbonne 2 NS UMR3664, CNRS, 1,2,3,4,5, ,Iael Vassias Isabelle *, 42m3 swl sadpeino ctltdhsoe and histones acetylated non-coding- of a depletion in (HP1) 1 a protein heterochromatin as of accumulation well as constitutive for H4K20me3, and H3K9me2/3 al., enrichment methylation, DNA an et as such display Boyarchuk are marks heterochromatin also in domains but several (reviewed PHC and CenH3, H2A.Z 2011). H3.3 al., including H3, lack canonical variants, et of domains H2A Sullivan presence and the PHC 2012; by H3K4me2 surrounding characterized al., carrying The et 2004). (Bergmann nucleosomes marks H3 is H3K36me2/3 which vertebrates), with in (CENP-A by CenH3 interspersed marked variant is chromatin H3 Bernard Centric histone 2004). 2010; the al., al., et et Guenatri (Alonso 2001; chromatids al., et sister of cohesion the to ako n hlly 95 iuae l,20) asn an raising given a 2006), at al., rate maintenance stable et rapid their Kimura regarding a question 1985; important Chalkley, at unknown. and exchange remain G1 Jackson in constantly (Bo centromeres nucleus early reviewed the variants at throughout deposition 2007; and the H2A variants CENP-A, al., H2A telophase Notably, to et contrast of human in In Jansen dynamics 2011). In occurs al., 2008; far. et al., it Boyarchuk so et studied lines, (Hemmerich metazoans cell all 2009)]. cultured in centromeric al., replication from et uncoupled Foltz DNA is 2009; deposition junction al., CENP-A [Holliday et Importantly, (Dunleavy HJURP state protein involves chaperone, chromatin recognition in that CENP-A-specific changes a process progression, and cycle multistep cell controlled by regulation a revealed regions variants regulated. these of of is localization the mechanisms which by the mechanism to the important understand elucidate is it fully function, kinetochore to (Carr and al., Swaminathan propagation Therefore, centromere et 2013; Krogan 2005). PHC al., 2010; et al., al., Sharma in et et 2004; Hou al., 2007; et changes Rangasamy al., 2004; et structural Greaves 1994; al., and et cohesion of loss formation, centromere chromosome heterochromatin to improper leads defects, loss segregation its because centromeres, the 2003; severe functional al., of for et in Goshima activation 1999; and al., results Re et assembly (Buchwitz CenH3 kinetochore checkpoint progression, spindle of mitotic of in impairment of delay all depletion an a determinant In defects, 2003). the epigenetic segregation al., chromosome the et tested, also Cleveland in species but (reviewed 2012; 2013) al., identity et centromere al., Rop De centromere functional et in and in (reviewed Szenker structural kinetochore involved the the be for only foundation to not provides reported CenH3 been function. both have H2A.Z 2011; 2009). al., al., et et Probst 2010; Boyarchuk al., et in Maison (reviewed manner RNA-dependent ´ rvossuisadesn EPApoaaina centric at propagation CENP-A addressing studies Previous and CenH3 centromeres, at localized variants histone Among ne ta. 05 tlre l,19) 2. sas necessary also is H2A.Z 1995). al., et Stoler 2005; al., et gnier 1,2,3,4,5 yvi Cantaloube Sylvain , nshe l,21;Hgsie l,2007; al., et Higashi 2012; al., et ¨nisch 1,2,3,4,5 and 3347

Journal of Cell Science hra t eesaecntn tteprcnrcdomain of pericentric form specific the that a suggest requires at observations centromeres recent at compartments. constant These maintenance 2012). H2A.Z are al., genomic of et levels amount (Nekrasov mitosis, different total during its domain the centric whereas the dynamics cells, in broader at increases trophoblast its transiently a H2A.Z mouse distinct CENP-A, in displays than Interestingly, be H2A.Z genome the that might across given localization Furthermore, locus. ARTICLE RESEARCH 3348 monitored we histones variants, synthesized newly histone of of deposition PHC the dynamics at the deposition H2A.Z characterize and To H2A of dynamics Different RESULTS variant histone the centromere. regulate steady-state dynamic the cycle that at cell controlling the composition propose during we PHC by Thus, in on changes CENP-A. domain, influence of state. its levels steady centric exerts centromeric also at state neighboring PHC PHC the the the that at the reveal to we that H2A.Z lead Finally, and endogenous show H2A, of of in further not and but enrichment H2A.Z, We extent rearrangements of the incorporation phases. affect of structural state timing during G1 heterochromatin PHC in major and alterations at induced S of variants in H2A periods heterochromatin, new distinct of In euchromatin, deposition two at PHC. the incorporation at uniform report H2A.Z and we constant and a H2A to canonical contrast the of time, first dynamics the for deposition characterize, we histones, synthesized newly cycle. cell the during regulation ee yepotn en odsigihbtenprna and parental between distinguish to means exploiting by Here, nvivo in yexploiting by lblaayi splmnaymtra i.SC,adconsistent and our S1C), with Fig. microscopy material agreement (supplementary In using analysis S1D). global Fig. basis material cell-by-cell supplementary 1; a (Fig. on histones incorporated S1C). dynamics Fig. deposition material and al., and (supplementary H2A–SNAP synthesis et level, similar global (Ray-Gallet a exhibit electrophoresis on H2A.Z–SNAP that, gel conclude could after deposition corresponding we H2A 2011), bands a and pulse-labeled H2A.Z out to fluorescently By carried 1A). the model (Fig. analyzing approach our we ‘quench-chase-pulse’ the in following globally, newlyassay levels 2004), examine histones steady-state To al., S1A,B). synthesized at neither Fig. et and material PHC Rangasamy (supplementary distribution system Dryhurst the 2007; nuclear similar at 2006; al., a accumulated et al., showed Greaves variants et both 2009; (Bulynko Consistent al., epitope. observations et SNAP the previous with tagged with H2A.Z or H2A either ta. 06,wti h eeto ag formto both newly method of our deposition of extensive range comparable detection a the Kimura showed within 1985; variants Chalkley, 2006), and al., (Bo Jackson 2007; cells et recovery al., human et (fluorescence Higashi in 2012; FRAP analyses and photobleaching) after biochemical previous with ta. 01.Teetg a edtce hog h s of Flp-In use two the through correspondingly. generated tags, CLIP detected or We fluorophore, SNAP by be TMR-Star bound covalently the can are for which to tags effective coupled Ray-Gallet be These substrates distinguishes 2007; cell-permeable to 2011). al., proven et al., that (Jansen has et deposition technology and variant H3 histones CLIP-tag analyzing new and and parental SNAP-tag the et eaaye h ula itiuino newly of distribution nuclear the analyzed we Next, ora fCl cec 21)17 3735 doi:10.1242/jcs.148189 3347–3359 127, (2014) Science Cell of Journal condition). experiments; 2.–NPcl ie.Dt hwthe show mean Data and lines. H2A–SNAP cell in H2A.Z–SNAP PHC at variants synthesized H2A newly of of deposition percentage exhibiting the cells of analysis PHC. Quantitative and (C) TMR euchromatin on in based intensity variants, signal H2A new deposition of observed patterns three the of definition oan.Saebr 10 bar: Scale domains. DAPI-dense as identified were chromatin (chromocenters) are pericentric planes of Single Clusters area. shown. selected the of images after vivo (green) in H2A–SNAP incorporated visualization newly panel, of Middle step.) pulse. quenching P, the quench; of Q, cell efficiency the the of and characterization lines for S1 Fig. material supplementary also (See chromatin- histones. only incorporated visualize to X-100 fixation Triton before with treated are Cells H2A.Z– SNAP. and H2A–SNAP incorporated newly the for quench- assay the chase-pulse of Upper scheme (B) experimental time. panel, chase newly the are during that synthesized histones of visualization enables specific This substrates. (labeling) fluorescent or (quenching) nonfluorescent cell- permeable with covalently reacts SNAP polypeptide The histones. SNAP-tagged quench-chase-pulse using the assay of PHC. principle at The H2A.Z (A) and of H2A deposition synthesized of newly dynamics Different 1. 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Journal of Cell Science aallwt lal itnusal noprto fH2A detectable of of incorporation lack in distinguishable domain, the clearly this at to with histones TMR-Star corresponds synthesized parallel of newly PHC of Exclusion incorporation even 1B). from exclusion, (Fig. – enrichment signal patterns be distinct or could three euchromatin distribution of to one relative into situation distribution the histones categorized where the synthesized domains, PHC newly However, the of at different euchromatin. be to in proved histones synthesized ARTICLE RESEARCH nldsmdfctosi itn ak n rnin but latter transient The a mitosis. and HP1 fork during marks of replication second, histone loss to and in 2010; substantial due al., 2004), modifications et phase, its al., includes Maison S during et 2009; first, late Almouzni, Quivy and cycle; to (Corpet cell mid progression the in during in replication two windows at deposition described time been constitutive Indeed, have specific PHC deposition. in variant to PHC H2A rearrangements with of characteristic H2A dynamics contrast associated the properties synthesized influence in local might newly that PHC suggests the euchromatin at of deposition variants non-uniform The different H2A PHC become synthesized at newly can show of deposition H2A Replication-dependent new H2A.Z at only domain. the and and dynamics at PHC, enriched significantly at deposition H2A in with kinetics that, indistinguishable cells conclude deposition regions, detect we their Thus, not euchromatin PHC. could to at we variant contrast and this pattern, of of that enrichment even deposition over in an contrast, PHC observed By showed PHC at at 1C). H2A.Z H2A (Fig. synthesized newly synthesized euchromatin of at newly Deposition observed H2A.Z. the ( and half of H2A population, enrichment in this for only occurred within h domains PHC 2 and, at PHC of H2A at synthesized time histones newly chase synthesized a newly with the of interval. time deposition given a in difference during a deposition indicate of likely but rate of domain, the deposition the reflect at two both variants euchromatin, H2A histone to other the respect signal with H2A The TMR-Star PHC the the of of heterochromatin. at distribution even a deposition and and enrichment suggests the euchromatin patterns, pattern regulating at this of variants Importantly, mode euchromatin. different at variants piioi n bevdals facmlto fnewly in of arrested cells accumulation the in of even with 2B), latter loss (Fig. DNA PHC the a at to inhibited H2A coupled observed we synthesized is possibility, and deposition this its aphidicolin test that mid suggests To in PHC phase Fig. synthesis. at material S H2A (supplementary late synthesized cycle cycle newly to cell of from cell enrichment the excluded The of the S2A). mostly By point to 2004). this was al., at corresponds H2A.Z et PHC synthesized which (Quivy replicated newly phase, is contrast, domain S cells this late that when were found window to PHC we at mid H2A strategy, synthesized Crosio this in newly 2011; of Using al., enrichment 2A). an et (Fig. exhibiting (Ray-Gallet 2002) B al., Aurora et deoxyribonucleotide marker cycle the cell-cycle-dependent cell using the a sites with in replication 5-ethynyl-2 strategy analog of quench-chase-pulse vary labeling the could coupled the thus PHC We synthesized at in manner. newly of consider 1999). variants deposition to al., the crucial H2A et whether thus Murzina investigate were 2005; to windows order al., time et particular Hirota These 2005; al., et (Fischle ete uniidtefato fclsta exhibited that cells of fraction the quantified then We 9 doyrdn EU n muoeeto of immunodetection and (EdU) -deoxyuridine a oho hc r etrdi G1 in restored are which of both , , 5 fttl ipae an displayed total) of 15% , 0 ftecells the of 50% , 0 fcells of 30% 2) nti ae eoiino 2. tPCwsvsbei the in visible was Fig. PHC ( at material cells H2A.Z the of supplementary of majority deposition 3B; after case, (Fig. this h In G1 2.5 S2B). that early time-point histones a to block, supplementary synthesized corresponds mitotic monastrol-induced 3A; emphasized newly a to (Fig. further from able labeled release was G1 deposition cells we in G1 of when This were majority S2A). PHC The Fig. material at 2A). (Fig. at H2A.Z deposition stage deposit according H2A.Z cycle cells of only cell proliferating timing their in asynchronously the classified observed down we PHC narrow PHC, was at To deposition and 1C). H2A.Z (Fig. constant pool, not euchromatic the was to contrast In occurs PHC at G1 H2A.Z during synthesized newly of Deposition aeilFg 2) h leain nPCi ohmdlsystems model both in PHC in alterations (supplementary The repeats S2E). CpG Fig. satellite in material major reduction drastic pericentric at a at (over 5-methylcytosine in methylation existing resulting h the replication, 48 by that for diluted ensure 1980), was to Taylor, cycles) methylases cell and chromatin 5 two DNA Jones least inactivates 1982; with that al., analog other et and nucleoside inhibited (Creusot methylated a be several fibroblasts was 5-Aza, cannot applied 3T3 that methylation We NIH and (5-Aza). (ii) DNA deoxycytidine and lost which PHC at in is altered in are 2001), (i) al., modifications H3K9me3 state: et (Peters PHC (dnMEFs) which models disrupt fibroblasts embryonic different to mouse two means with used independent Suv39h cells We two in H2A PHC. the providing and monitored altered H2A.Z we synthesized PHC, experimentally newly at actually of H2A.Z could of deposition state incorporation heterochromatin the in regulate changes whether PHC at test deposition To and this H2A.Z extent of the timing restricting affect the state heterochromatin deposition, in Alterations H2A.Z further G1. limit to deposition a H2A.Z its to as once work promote hypothesis, would barrier heterochromatin this changes re-established, could to fully these is According structure domain. restoration that the hypothesize at and deposition to disruption tempting newly G2–M–G1 involving was into H2A, the it Taking during to G1. transition, early rearrangements contrast in heterochromatin PHC in at account that, deposited time is of given conclude H2A.Z the proportion synthesized in we PHC the at Thus, deposition for H2A.Z window. account show not monastrol could is do (supplementary the that which from mitosis cells it from in S2B), still release Fig. rather, were result after cells material h the in exit, of 2 cannot 20% note, difference mitotic about block, Of H2A.Z after PHC. the for and synthesis that specific protein H2A indicates differential which between S2C), (supplementary deposition analyses gel Fig. incorporation in similar and material were synthesis variants both of for levels rate the Importantly, minimal. tPC etu ocueta el yteie 2 is H2A synthesized newly manner. that replication-coupled a conclude in thus PHC at We deposited H2A.Z H2A.Z heterochromatin. PHC. towards switch on deposition compensatory at H2A and a of effect cause loss necessarily the not no euchromatin does that suggesting had PHC, at at replication incorporation deposition inhibiting no regulation surrounding Furthermore, had the H2A the between treatment at difference this H2A of the Notably, of underlining deposition 2C). euchromatin, the (Fig. on phase impact S significant late to mid ora fCl cec 21)17 3735 doi:10.1242/jcs.148189 3347–3359 127, (2014) Science Cell of Journal obenl ( double-null , 0,Fg B,weesHAdpsto was deposition H2A whereas 3B), Fig. 80%, Suv39h1 2 / 2 and Suv39h2 2 / 2 , immortalized ) 0 fcells of 50% 9 aa2 -aza 3349 9 -

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(Fig. 3350 cells control in detected rarely very we in euchromatin to respect PHC with at enriched affected, became H2A.Z not synthesized was newly deposition that detected H2A we of extent the Although 4A). labeling treated quench-chase-pulse performing (Fig. before were h that 48 for H2A.Z–SNAP 5-Aza with or H2A–SNAP expressing cells to state. response PHC acute in cell an changes show several fibroblasts during in 5-Aza-treated perturbations generations, heterochromatin than the pronounced to cells adapted more whereas untreated that with note to to 2010), relative al., 5-Aza-treated Suv39h et in Sugimura cases both changes 2003; Lehnertz in 2009; al., increased al., occurred et et (Eymery Furthermore, repeats S2F) Fig. satellite material PHC S2E). (supplementary major at methylation of Fig. DNA transcription in material decrease but 2001) S2D) striking al., (supplementary a et Fig. Taddei display 2010; material not al., et did (supplementary Sugimura HP1 2001; and al., marks et H3K9me3 (Peters these heterochromatic of lost enrichment typical an dnMEFs retained as such cells marks, 5-Aza-treated Importantly, S2D. Fig. whereas material supplementary in summarized are in defined (as PHC at mean H2A.Z–SNAP the and show H2A–SNAP Data deposited treatment. newly (Aph) of aphidicolin enrichment without exhibiting or cells with of 1B), quantification Fig. panel, Lower experiment. H2A pulse of incorporation of patterns panel, Lower pulse. P, After quench; phase. Q, S late; late L, to late; mid to in mid cells ML, in early; PHC E, at scheme. H2A.Z–SNAP experimental and panel, PHC. Upper at cycle. H2A cell of the deposition Replication-dependent 2. Fig. efrteaie h eoiindnmc fHAvrat in variants H2A of dynamics deposition the examined first We nEsrltv owl-yeMF.I sas important also is It MEFs. wild-type to relative dnMEFs Suv39h nEsrpeetasse hthas that system a represent dnMEFs , 0 fcls eoiinpattern deposition a cells, of 30% m m. 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(Fig. 2006; and al., euchromatin (Bulynko observations et at previous distribution with consistent displayed even variants heterochromatin, with H2A both localization, state, PHC similar staining unaltered in antibody an alterations By with H2A.Z. cells into in total translated of PHC distribution extensive altered steady-state wanted deposition the with H2A.Z we unscheduled cells new Therefore, in in in domain. perturbations observed the the whether result at determine variant to PHC and this deposition of major in H2A.Z a deposition of as extent perturbations and acts timing state that the heterochromatin both the of that regulator suggest data Our H2A.Z of PHC localization at the domain. regulates the state at Heterochromatin deposition We H2A.Z 4D). of the (Fig. both timing to G2 perturb the state in and led PHC and extent of also alterations G1 S that alone during concluded during H2A.Z treatment therefore PHC new 5-Aza at of Moreover, H2A.Z enrichment new 4E). (Fig. not of did enrichment phase this DNA robust but inhibited the cells, 5-Aza-treated we prevent in coupled, aphidicolin with of replication mode synthesis was unscheduled this deposition whether determine H2A.Z To mean 4C). the (Fig. shown phase Data deposition). even as 1B depositi Fig. the S2B,C. in synthesized exhibiting Fig. (defined cells Newly material block of block. supplementary mitotic quantification mitotic from panel, Pre the left release treatment. from Lower after monastrol released pulse. h P, by then distributi 2.5 quench; mitosis were the Q, at in for cells experiments; release. PHC arrested different S2A the after at Fig. were and h Aurora H2A.Z–SNAP material Cells monastrol, 2 and or supplementary scheme. at of EdU See SNAP experimental SNAP–TMR-Star presence using planes. with panel, the single cycle labeled left in as cell were Upper shown quenched the histones (B) are were in B) cycle. 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PHC steady of results state’ ‘heterochromatin at at the state PHC that concluded heterochromatin at H2A.Z variant and in this synthesized of perturbations extensive accumulation to newly contrast, order due in of By domain replication, deposition density. DNA nucleosomal unscheduled during diluted maintain are to H2A– that replenish to dimers deposition H2A H2B extensive enrichment the Such reflect PHC. to not likely at does is H2A replication total DNA of during treatment accumulation the PHC into 5-Aza translate that at note H2A after to new important state of is enrichment steady It S3D). at Fig. material PHC H2A–SNAP,(supplementary at not accumulated but also H2A.Z–SNAP, total Furthermore, S3B,C). and 5-Aza- in cells PHC at treated accumulated eH2A, not anti-HA but with eH2A.Z, staining that histones, showed endogenous for of obtained agreement data In transfection. with following distribution h 48 of the treatment during 5-Aza fibroblasts 3T3 correct by followed eH2A.Z), and (eH2A variants the H2A wild-type assessing transfected have transiently MEFs, we might variants, from histone recognition the endogenous antigen us exclude 5-Aza in To after prevented differences S3A). 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Journal of Cell Science EERHARTICLE RESEARCH 3352 Following PHC HP1 6A). at (Fig. accumulation 2009) and H2A.Z al., of loss methylation et expression, Loyola Myc–SUV39H1 2003; DNA local al., the et H3K9me3, in (Lehnertz results of PHC at reaccumulation binding transfected whose we Myc–SUV39H1, status, with PHC restore To dnMEFs. survival state cell long-term with PHC (Ju affected agreement In treatment normal domain. 5-Aza a the studies, at previous of levels H2A.Z re-establishment restore also the would whether tested we 4. Fig. temn ta. 94,adteeoew oue on focused we therefore and 1994), al., et ¨ttermann e etpg o legend. for page next See Suv39h dnMEFs Suv39h a y–U3H ihn6has ugssta natv histone active an that suggests also expressing h cells 6 the of within majority the Myc–SUV39H1 the Intriguingly, in maintenance. accumulation H2A.Z H2A.Z of dictates loss locus heterochromatin the the protein that of H2A.Z conclude status total we the Therefore, after in 6D). changes cycle (Fig. no h cell levels were one 6 least there at and after 6B,C), is, by that (Fig. h, PHC 24 at cells after H2A.Z maintained of stably level transfected was low relatively H3K9me3 the Importantly, of of 6B,C). reaccumulation (Fig. the majority with coincided and the transfection, in occurred ora fCl cec 21)17 3735 doi:10.1242/jcs.148189 3347–3359 127, (2014) Science Cell of Journal

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Journal of Cell Science EERHARTICLE RESEARCH oan hs ete h oa etblzto tef o the nor euchromatin itself, the at deposited destabilization full at actively is local incorporation (which the H2A.Z the of from availability that neither excluded replication, Thus, remain ensure after domain. present would DNA-synthesis- would is H2A.Z histones a but H2A H2A–H2B structure, of of complement the deposition this of During dependent 2008). al., destabilization et Quivy transient 2010; fork al., after et immediately (Maison restored fully progression is and place takes replication 0.5 with treated were CENP-A–CLIP expressing centromeres. Cells at scheme. levels experimental CENP-A panel, regulates Upper state centromeres. PHC The 7. Fig. atrsi 1 aeSadG hssaeson e upeetr aeilFg 4– o elln hrceiain BD cuuaino endogeno of Accumulation (B–D) characterization. cell-line for S4A–C and Fig. depos (WT) material CENP-A–CLIP Wild-type supplementary the thei (B) See resolve for PHC. to shown. perturbed projections) B are with intensity Aurora cells phases (maximum against in G2 stained images centromeres and also Representative at S were CENP-A panel, Cells late Lower CLIP–TMR-Star. G1, with immunofluorescence. in IF, pulsing to patterns pulse; P, prior EdU quench; with Q, labeled stage. were sites Replication labeling. pulse tredfeeteprmns naeaeo 5clsprcniin.()Wsenbo nlsso oa rtiso h el nlzdi .CN- was CENP-A B. in analyzed cells the of control. proteins loading total of a analysis as blot used Western was (D) H4 condition). Histone per antibody. cells corresponding 55 the of with average an experiments; different (three odfn etoee.Fl hne nfursec nest ausrltv otoeo h orsodn oto r hw.Dt hwtemean the show Data shown. are control corresponding the of those wa to signal relative satellites. FISH values DNA minor signal. intensity for antibody 10 fluorescence probes CENP-A bars: in (LNA) of Scale changes intensity acid area. fluorescence Fold selected nucleic of the centromeres. locked quantification of define by using images estimated to FISH enlarged was represent DNA CENP-A Insets by centromere-associated shown. of detected are abundance were projections) intensity domains (maximum Centric patterns CENP-A. Representative against antibodies with stained A ramn ih5Aade o mato h iigo EPAdpsto at deposition CENP-A of timing the on impact not does 5-Aza with Treatment (A) Suv39h P rtisaesbtnilyls n itn ak r highly masks are that phosphorylation marks H3S10 histone transient and exhibiting lost dynamic, substantially dissociate are chromosome. proteins chromocenters each to HP1 corresponding as higher- domains the PHC occurs, individual of into structure rearrangement H2A.Z the global chromatin mitosis, a promote order H2A. window, during not time to but contrast, H2A.Z this By of During sufficient deposition phase. permits PHC S is of in reorganization cycle) PHC at cell deposition the throughout obenl d)MF,cnrlad5AatetdFlp-In 5-Aza-treated and control MEFs, (dn) double-null ora fCl cec 21)17 3735 doi:10.1242/jcs.148189 3347–3359 127, (2014) Science Cell of Journal m -z o 8hbfr n uigquench-chase- during and before h 48 for 5-Aza M m .()Terelative The (C) m. TM 33clswere cells -3T3 elcycle cell r 6 detected s.e.m. used s 3355 ition us

Journal of Cell Science EERHARTICLE RESEARCH 3356 the and phosphorylation H3S10 HP1 of of re-accumulation loss The concomitant is domain. which by state G1, the in PHC organization at characterized domain in the deposition H3K9 of changes H2A.Z restoration and subsequent These promote 2005) 2012). al., possibly al., et could et Hirota 2005; (Ohzeki al., acetylation et (Fischle H3K9me3 HP1 of loss Aurora partial by a H3S10 to of leads phosphorylation (which organization. the B PHC CENP-A acetylation, and of histone H2A dynamics global of cycle mitosis, deposition cell the the of timing and the variants outlining Model 8. Fig. asne l,20)adicroaino elctv 3adH. 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Fan 2002; (Fan al., compaction increased et with the structures facilitate more- secondary and structural fiber of chromatin the formation the a of nucleosomes where folding the as (H2A.Z–H2A.Z) influence context, might Previous homotypic functions 7). heterochromatin hypoacetylated H2A.Z 5, the stable (Figs that in centromeres proposed at component it have to CENP- studies of next amount the in incorporated enrichment increase an an A to to and domain led this status at H2A.Z PHC of in perturbations composition that variant showed histone We and organization interplay PHC the between illustrate dynamics chromatin Centromeric hne nvratdsrbto ol,i un ute affect stability. genome further which and centromere, progression turn, mitotic for the in influence of then context could, potential would organization a distribution These chromatin variant variants. provide higher-order in in histone might (reviewed of changes tumorigenesis and 2013) incorporation or White Lawrence, differential 2005) 2007; al., and al., et Solovei al., Carone et domain 2007; occurs Terranova al., et the et 2009; that (Govin (Probst of differentiation 2005), provides remodeling development plasticity Dalton, mammals, early heterochromatin and centromere, in during maintenance The the evidence both provide at organization. for now composition we means variant and which histone process for the regulate to to barrier CENP-A. of a of overloading restoration and/or as spreading G1, act promiscuous early prevent would in once organization Finally, proceeded phase. PHC has S placeholders during deposition as CENP-A function CENP-A of that dilution variants the H3 following replacement other and of eviction the CENP-A favor with could turn, histone in facilitate which, the its turnover, simply of could of deprived the marks, transiently heterochromatin retention Alternatively, template, repressive 2011). the stable of al., or accessibility et increased HJURP (Boyarchuk targeting chaperone CENP-A including deposited CENP-A deposition, mitosis CENP-A the during of of steps any organization cycle enhance multiple to PHC cell the opportunity of of of the window a loss during provide potentially partial CENP-A could This and the 8). highlight PHC (Fig. and of mammals now in dynamics data crosstalk parallel this Our of 2011). evidence al., provide et in and (Olszak 2009) boundaries al., in heterochromatin et Kagansky demonstrated 2008; been al., et previously pombe has Schizosaccharomyces centric domains between pericentric crosstalk at Such and (thus template). centromeres endogenous established natural at its PHC in alterations by affected compensatory marks. heterochromatin a of repressive absence as canonical the function in structure could heterochromatin maintain H2A.Z to mechanism of enrichment the that sdcae yteitrlybtentomi euaos–cell – domain. regulators site main the this organization. two heterochromatin at between at and variant interplay timing H2A deposition the cycle specific by variant a dictated of is H2A deposition window the for a Therefore, define cycle opportunity cell the organization during of the naturally of occur Thus, that rearrangements 4). PHC dynamic (Fig. of phase the S that late propose to mid we replication-dependent in the H2A affect canonical not of do deposition state PHC in changes such motnl,teitrlybtenPCsaeadcl cycle cell and state PHC between interplay the Importantly, is deposition CENP-A of extent the that found we Intriguingly, ora fCl cec 21)17 3735 doi:10.1242/jcs.148189 3347–3359 127, (2014) Science Cell of Journal nanaı a on v N epae(Folco template DNA ¨ve Drosophila tartificial at

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Journal of Cell Science otoldb otox . ApidPeiin.The Precision). (Applied 5.0 softWoRx1 by controlled enr,P,Mue .F,Prrde .F,Gne,S,Jvra,J .and P. J. Javerzat, S., Genier, F., J. Partridge, F., J. Maure, P., Bernard, M., Nakano, A., Kagansky, M., N. Martins, N., J. Jakubsche, H., J. Bergmann, .-mwdh.W deconvolved (50 We acquisitions width). three-dimensional 0.2-mm allowed stage motorized (Applied camera HQ2 Photometrics a 60 with Precision; equipped microscope IX71 ARTICLE RESEARCH 3358 E. P. Warburton, and F. Cheung, D., Hasson, A., Alonso, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.148189/-/DC1 online available material Supplementary material Supplementary (E Cancer le contre Nationale Ligue La by supported is G.A. of research The the of Funding editing final the to G.A. contributed and G.A. D.F. and E.B., I.V data. D.F., manuscript. the E.B., D.F. paper. analyzed and the G.A. E.B. wrote and imaging. D.F. microscopy E.B., the most of figures. performed part I.V. generated performed and D.F. S.C. E.B., experiments. experiments. the the of designed G.A. and D.F. E.B., contributions Author interests. competing no declare authors The interests Competing Stanford, Medicine, of School reading. University critical (Stanford for (G.A. Attardi USA) Gurard-Levin D. A. Laura and Zachary and discussions Quivy, team) helpful Institute Jean-Pierre for Curie and Facility, team) advice, Imaging (G.A. experimental Tissue Ray-Gallet and Dominique (Cell France), Baccon Paris, Le Patricia thank We Acknowledgements odto R orl ehrh otel acrfellowship. Cancer le contre Recherche la pour Universite Recherche ARC by la la Fondation funded de de was National Nationale D.F. Centre Agence ‘ECenS’. a the project by and funded fellowship was Scientifique E.B. Recherche project cancer’. cancer breast Aviesan-ITMO of the ‘Epigenomics and PSL* ANR-10-IDEX-0001-02]; and number ANR-12-BSV5-0022-02] [grant number [grant number ‘CHAPINHIB’ [grant projects 11-LBX-0044], DEEP’ Recherche ‘Labex la ANR-10-BLAN-1326-03], number de [grant Nationale ‘ChromaTin’ Agence the number 2009-AdG_20090506]; [grant GA GA ‘Eccentric’ number Grant [grant Advanced ERC ‘MODHEP’ the project FP7_HEALTH-2010-259743]; FP7-PEOPLE-2008- integrating GA large-scale number and [grant 238176] Initial 4D’ Commission ‘Nucleosome European Network the Training HEALTH-F4-2010-257082]; GA number [grant labellise mgrZ;Cr es)pltdwt eaop otae(Molecular software MetaMorph (Axio with 63 microscope piloted a epifluorescence Zeiss) Devices), an Carl using S3B,C shown. Z1; S1A, obtained is Figs Imager material were plane supplementary 6, middle S4A,C 5, The and Figs 5.0. in shown softWoRx1 images using The cycles) (15 method sn es S 8 ofclmcocp iha63 a with microscope acquired confocal were 780 S4D images Fig. LSM material The Zeiss supplementary Photometrics). and a 7 using HQ2; 4, Figs (CoolSnap in shown camera device coupled betv N .) otoldb E otae(alZis.We Zeiss). (Carl software ZEN by 0.39- controlled 16 1.4), acquired (NA objective otaefrfrhriaepoesn.Alsaebr orsodto correspond bars scale All processing. image 10 further for software nest tcnrmrslbldb N IH eue h 3D-FIED the used we 2012). FISH, al., DNA et fluorescence by (Cantaloube CENP-A labeled macro the quantify centromeres To at of ‘excluded’. intensity remainder as The defined euchromatin. were of deposition cells those not pattern of from did levels a indistinguishable exhibited be that were chromocenters to that the cells deposition’ of ‘even the majority defined the For we whereby ‘enriched’. pattern, of enriched as enrichment an variant display with synthesized chromocenters three newly least the at exhibiting cells defined lsie .C. centromeres. al. R. with et V. compatible Allshire, Larionov, is H., acetylation Masumoto, function. H3K9 and M., histone structure B. kinetochore Turner, engineering: A., Epigenetic D. (2012). Kelly, H., Kimura, eeohoai tfntoa ua neocentromeres. human functional at heterochromatin m .We uniyn h eoiino 2 ainsa H,we PHC, at variants H2A of deposition the quantifying When m. eLge;teErpa omsinNtoko xelneEpiGeneSys Excellence of Network Commission European the Ligue); ´e 6 Science 6 n 100 and 20) eurmn fhtrcrmtnfrchso at cohesion for heterochromatin of Requirement (2001). m A14N i betv esadacildcharge- chilled a and lens objective oil NA PA/1.4 m 294 z 6 sie totmmvxlsz.W sdImageJ used We size. voxel optimum at -slices betvswt Ao .) h eie were devices The 1.4). of NA a with objectives 2539-2542. , .Cl Sci. Cell J. z sakiae yteconservative the by images -stack iree ai ui n a and Curie Marie et Pierre ´ 125 411-421. , pgntc ChromatinEpigenetics 21) act of paucity A (2010). 6 x , immersion z sie of -slices y ´ and quipe 3 ,6. z opt .adAmun,G. Almouzni, and A. Corpet, lvln,D . a,Y n ulvn .F. K. Sullivan, and Y. Mao, W., D. Cleveland, ok .J . uadLvn .A,Vsis .adAmun,G. Almouzni, and I. Vassias, A., Z. Gurard-Levin, L., J. A. Cook, aaoa . atra,M,E aju . eBco,P,Pos,A .and V. A. Probst, P., Baccon, Le F., Marjou, El M., Pasternak, Nurse, M., and J. Casanova, S. Aves, A., G. Phear, J., Hindley, M., S. Dorrington, M., A. Carr, B. J. P. Lawrence, and J. M. D. Quivy, Carone, and G. Almouzni, P., Baccon, Le K., Romeo, S., Cantaloube, Grigoryev, and J. D. Tremethick, W., R. Mason, C., L. Hsing, S. Henikoff, A., and Y. B. Bulynko, M. Roth, L., L. Moore, K., Ahmad, J., B. Buchwitz, B., Wilhelm, C., Sakalar, A., Roguev, M., Durand-Dubief, L., Buchanan, oacu,E,Mne eOa .adAmun,G. Almouzni, and R. Oca, de Montes E., Boyarchuk, rhrt . siah,T,Rs,K . Eirı L., K. Rose, T., Ishibashi, D., Dryhurst, Bo ulay .M,Amun,G n apn .H. G. Karpen, and G. Almouzni, M., E. Dunleavy, Nakamura, D., Ray-Gallet, N., Lacoste, H., Tagami, D., Roche, M., E. Dunleavy, T. R. Kamakaka, and N. Dhillon, S. P. Maddox, and A. Padeganeh, V., Rop, S., De Sen, H., Zhou, Y., Okano, M., Kimura, R., Loury, M., G. Fimia, C., Crosio, acin,K .adCesmn .M. I. Cheeseman, Wood, and A., E. C. E. K. Bassett, III, Gascoigne, R. R., J. Allshire, Yates, O., A. and Bailey, E., T. L. Jansen, Urano, R., D. L., Foltz, A. Pidoux, D., H. Folco, J. D. Tremethick, and K. Luger, D., J. Rangasamy, Y., D. J. Tremethick, Fan, and C. J. Hansen, K., Luger, F., Gordon, Y., J. Fan, Van L., A. Vitte, F., Berger, G., Fourel, M., Atifi-Borel, El B., Horard, A., Eymery, K. J. Christman, and G. Acs, F., Creusot, icl,W,Teg .S,Dran .L,Ubred,B . aca .A., B. Garcia, M., B. Ueberheide, L., H. Dormann, S., B. Tseng, W., Fischle, ic,C,Shedr . Pu K., Schneider, C., nisch, ¨ ieohrs rmeieeist ioi hcpitsignaling. checkpoint mitotic to epigenetics from kinetochores: 1167. pcfcfnto o h itn hprn APt ietn eevi of reservoir a fine-tune to chain. supply NASP histone chaperone the histone in H3-H4 the soluble for function specific 421. eeomn eursasnl-taddnnoigtranscript. noncoding single-stranded a requires development G. 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