ugs nuatcptdrl fYT rti nchromatin in on clues organisms. protein mechanistic multicellular in time, YETI development first control proteins the of BCNT findings how for our role provide, the Overall, and regulate unanticipated organization sites. H2A.V- to of an chromatin subunit acts with at suggest new and H2A.V interacts a of complex, as YETI accumulation remodeling or DOM/Tip60 chaperone that a the propose as machinery, we DOM/Tip60 exchanging results, the the as of these of YETI target identify downstream subunit we and a Furthermore, HP1a ATPase complex. H2A.V, remodeling the variant chromatin histone (DOM-A), the with Domino-A BCNT that and interacts conserved find its and histones through also domain chromosomes We polytene nucleosomal to chromosomes. binds H2A.V, polytene YETI with histone marks of epigenetic association in impairment higher- severe the in by evidenced defects as and organization, pupation chromatin before order lethality causes YETI of loss the of role ainaUniversita Sapienza al,Germany. Halle, Ato o orsodne([email protected]) correspondence for *Author Italy. Roma, 00185 Roma, ivniMessina Giovanni organization chromatin for required protein Yeti ARTICLE RESEARCH ß eevd2 aur 04 cetd5Mrh2014 March 5 Accepted 2014; January 20 Received 6 1 ATP-dependent the that of the component indicate functions; a is literature cellular Swc5, protein, important the BCNT perform yeast in might information proteins Sparse BCNT the 2003). (Iwashita however, unknown al., largely plants, remain et Despite family and protein 1). animals this (Fig. of in end functions C-terminal distribution the widespread at located its stretch acids a amino to domain, 80 BCNT yeast so-called of from the by conserved characterized family, is humans, protein (BCNT) Bucentaur The INTRODUCTION Yeti Chromosome, remodeling, Chromatin WORDS: KEY Using unknown. its yet largely plants, and remains animal melanogaster role in distribution biological proteins widespread (BCNT) a Bucentaur of exhibits family conserved evolutionarily The ABSTRACT Dimitri Patrizio and etrfrBoehooy epeUiest,Piaepi,P 92,USA. 19122, PA Philadelphia, University, Temple Biotechnology, for Center ai Italy. Bari, rnec oaaMariotti Romana Francesca ai Picchioni Daria iatmnod ilga Universita Biologia, di Dipartimento Universita Sapienza Darwin’, ‘Charles Biotecnologie e Biologia di Dipartimento 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,27–58doi:10.1242/jcs.150243 2577–2588 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. nessential an , 5 broIsiuefrCne eerhadMlclrMdcn and Medicine Molecular and Research Cancer for Institute Sbarro Drosophila 4 samdlogns,w netgtdthe investigated we organism, model a as iatmnod ilga Universita Biologia, di Dipartimento ` iRm,015Rm,Italy. Roma, 00185 Roma, di 1,2 oet Moschetti Roberta , 2 1,2 CTmme aldYT.W eotthat report We YETI. called member BCNT siuoPserFnain Cenci-Bolognetti, Fondazione Pasteur Istituto 1,2, lsbtaDamia Elisabetta , * ` Drosophila eeioI,814Npl,Italy. Napoli, 80134 II, Federico 1,2 ai aml Accardo Carmela Maria , rspiamelanogaster Drosophila O-.O h ai of basis the On DOM-A. 3 niueo eeis D-06120 Genetics, of Insitute ` el td iBr,70121 Bari, di Studi degli 4 ugeoCaizzi Ruggiero , 1,2 ar Fanti Laura , Drosophila Drosophila nvivo in ` , di 1 ai eeaAtterrato Teresa Maria , 1,2 4 ui Piacentini Lucia , atisWalther Matthias , 00.Adtoa tde aelne h CTpoen to proteins al., BCNT et the (Ohta linked chicken have in studies kinetochores Additional with been 2010). have asociate they to and 2011), and shown Naoki, cytoplasmic and both (Iwashita in found fractions nuclear 2003). been have al., proteins of et BCNT subunits (Wisniewski Related both kinesin-I bind motor to The microtubule-based able the protein 2004). within kinesin-binding al., a H2A as et defined canonical Mizuguchi the 2004; melanogaster with al., Drosophila et H2A.Z (Kobor variant euchromatin histone exchange the the in involved of SWR1, complex remodeling chromatin xhnigmcieyadpriiae ntese fH2A.V of step the in H2A.V- the participates with deposition. and evidence and HP1a provide machinery with results exchanging interacts organization our YETI that chromatin addition, suggesting In and/or development. chromosome during higher-order for melanogaster the of craniofacial – 2011). symptoms Bayarsaihan, others and Williams–Beuren among (Makeyev for – abnormalities candidates causes primary which be disease, by encoded to are turn suggested in genes which factors, transcription TFII-I of target BCNT Zebrafish osteodifferentiation the Third, induced 2011). called gene, al., of experimentally et upregulation neural 2008); (Bustos-Valenzuela after in al., gene et PAX3-encoding observed (Wu the cells of crest expression persistent to downregulation mouse, in al., the Second, et of 2002). (Diekwisch al., development et bone Diekwisch and 1999; functions teeth cellular for limited essential required BCNT plays is mouse and CFDP1, though or the CP27 First, called osteogenesis, available. protein, is and understanding mechanistic development craniofacial culgn ttemlclrlvl esree o P-element- denoted and insertions, for lethal non-complementing screened two complement we recovered to the level, failed identify that molecular To mutations 2003a). the induced al., at et gene (Cenci meiosis actual and mitosis organization chromosome both proper for in required is 2007), al., et Rossi melanogaster Drosophila that found previously We of gene vital the Associating RESULTS rhsta ilgv iet h rnoailsrcue (Thisse, structures craniofacial the human to the rise Finally, give 2004). will that arches nti ok ehv are u ndphfntoa studies functional in-depth out carried have we work, this In Drosophila LP2 Cfdp1 Yeti seMtrasadMtos.Mlclraayi showed analysis Molecular Methods). and Materials (see eeadisecddpout EI in YETI, product, encoded its and gene Rltpr eecreae ihteidcino lf aaedue palate cleft of induction the with correlates gene eso htteYT rti srequired is protein YETI the that show We . sdvlpetlyepesdi h branchial the in expressed developmentally is , 1 ivniCenci Giovanni , ee noe a encodes gene, 3 imet Vernı Fiammetta , 1,2 l(2)41Aa CTpoen EI a originally was YETI, protein, BCNT Hlie,17;Dmtie l,2003; al., et Dimitri 1976; (Hilliker, mneeCelauro Emanuele , Cfdp1 l(2)41Aa ia eeohoai eeof gene heterochromatin vital a , eehsbe hw ob a be to shown been has gene ihthe with 1,5 ni Giordano Ennio , ` 1 l(2)41Aa , Yeti Cfdp1 1,2 gene Drosophila , lee and alleles a also was 2577 LP1 6

Journal of Cell Science lmn a on ob netdwti h rdce tr codon start predicted P- the within of The inserted ATG be AABU01002199. to scaffold found was in element located gene 5 967-bp-long the the search with identity 3 to used the and using sequenced melanogaster PCR, Drosophila inverse by recovered EERHARTICLE RESEARCH 2578 ( size expected the of transcript a indicate revealed results these together, Taken the lacking 1). that protein (Fig. truncated region TAA a terminal a producing into changed likely is , most Lys for codon, AAA stop codon where 106, position of the EMS-31 allele of lethal for sequencing homozygous larvae a and from DNA genomic amplification using region PCR performed we locus genetic the between those than of smaller glands. 40% nuclei salivary wild-type about in are and present glands chromosomes salivary homozygous displayed polytene disorganized cells degrees. Moreover, different polytene at and albeit examined morphology, chromosome aberrant decondensed all such 2B); hemocoel revealed (Fig. salivary chromosomes their larvae DAPI-stained from in hemizygous of masses cells examination melanotic imaginal gland before Cytological with reduced 2A). lethality brains, severely (Fig. by and exhibited larvae discs followed Mutant development pupation. larval prolonged that 0 ftepoenicuigteeouinr osre C- conserved evolutionary the including protein the of 60% otenaayi sn h 0.9-kb the using analysis Northern the of sequences flanking The LP1 l(2)41Aa saPisrinlall ht hnhmzgu,rslsin results homozygous, when that, allele P-insertional a is are igebs armtto fo oT at T) to A (from mutation pair base single a carried Yeti 9 tpsto 2 ofrhrvrf h connection the verify further To +2. position at n lnigsqecsof sequences flanking end ia eecrepnst the to corresponds gene vital l(2)41Aa 9 n einof region end LP1/LP1 l(2)41Aa eoedtbs.BATanalysis BLAST database. genome euneiseto eeldthat revealed inspection Sequence . ooyosand homozygous CG40218 LP1 n the and Yeti netoa leewere allele insertional , LP1 Yeti localled also , . b nlra of larvae in kb) 0.9 DAa probe a as cDNA Yeti eeld100% revealed eesequence, gene LP1/Df(2R)B gene. LP1/LP1 EMS-31 Yeti Yeti ,a , h xrsino h EIpoenin assess protein To YETI Methods). the and of Materials expression in the details S1A; Fig. with morphology material chromosome associated the and full-length lethality phenotypes larval the the both indeed carrying flies using transgene transgenic the was generated Yeti that whether we assess To case, mutants. in observed phenotypes ave ua,aut n vra N Fg D.T further To 2D). (Fig. the RNA ovarian whether and confirm adults embryos, in pupae, development throughout larvae, in detected absent was transcript was The that indicating which 2C) strain, (Fig. homozygotes wild-type Oregon-R the ltig ige3-D adwsdtce netat from extracts in detected was band 32-kDa LP1/LP1 western single by extracts A protein blotting. analyzed (see and protein YETI Methods) the and of Materials portion C-terminal conserved the against tub-GAL4 exogenous with l(2)41Aa If in the morphology rescues chromosome protein YETI expressed Exogenously hoooeognzto togyrsml hs originally those resemble in strongly characterized of organization defects in these Remarkably, chromosome cells. appearance mock-treated in loose recorded level and fuzzy abnormally , with displayed interference cells chromosomes RNAi-treated (dsRNA)-mediated condensed The 2E). RNA (Fig. (RNAi) double-stranded the using depleted we organization, chromosome 0 feaie eahss( metaphases examined of 70% Yeti DA vrxrsino h A-eiM7transgene UAS-Yeti-M17 the of Overexpression cDNA. ora fCl cec 21)17 5728 doi:10.1242/jcs.150243 2577–2588 127, (2014) Science Cell of Journal culycrepnst h ia eeohoai gene heterochromatin vital the to corresponds actually hnreconstituting then , ; UAS-Yeti-M17 ave epoue oylnlatbde directed antibodies polyclonal produced we larvae, h at8 eiuso h -emnled nthe In end. C-terminal of the allele of lethal in residues similarity) 80 45% last (about the sequence protein of conservation hne oaTAso oo.Telsn eiu changed residue lysine The is codon. lysine in stop for TAA AAA a codon where to 316, changed position at found was T) humans, from musculus sequences proteins BCNT proteins. other BCNT The 1. Fig. EMS31 tub-GAL4 l(41A) Yeti , eepoutsol ecetedefective the rescue should product gene leei oae tpsto 0 seteasterisk). the (see 106 position at located is allele .thaliana A. uat Cnie l,2003a). al., et (Cenci mutants Yeti / tub-GAL4 Yeti oidc h A rmtrrescued promoter UAS the induce to igebs arsbttto fo to A (from substitution pair base single a , rdc srqie o proper for required is product LP1 Yeti LP1 and n 5 mutants ulalls(supplementary alleles null 5) esste3 baseline 3% the versus 457), ooyosmtn strains mutant homozygous ave(i.2) u o in not but 2F), (Fig. larvae .cerevisiae S. LP1 ls lgmnso EIand YETI of alignments Blast LP1/LP1 sanl leeof allele null a is Yeti oetehigh the Note . ; eei 2cells S2 in gene UAS-Yeti-M17 Mus LP1/LP1 EMS-31 Yeti / .

Journal of Cell Science EERHARTICLE RESEARCH ltasy fdntrdpoenetat Fg F,cudntreact not could 2F), (Fig. extracts protein western denatured in of well assays performing blot although staining, antibody, clear the any reveal that to suggesting failed analyses cytological immunostaining but in salivary assays, might anti-YETI in using protein YETI chromosomes the localize polytene how to gland of sought we understanding function, chromatin this in more perform protein gain YETI To the implicate organization. far thus reported with results work previous binds Our and nucleus the chromosomes to polytene localizes protein fusion YETI–GFP A strains. mutant in organization, defective chromosome highly proper is for which required protein a encodes the express pro disordered from intrinsically these extracts of properties physical the to 2011). owing Naoki, possibly and proteins, BCNT (Iwashita bovine structure for three-dimensional reported also stable as kDa), 27 (about predicted the lacking deletion the of analyses molecular and Cytological 2. Fig. xrcsfo aiaygad n risof brains and glands salivary from extracts of stage; knockdown pupal RNAi-mediated residual larval/early (E) a 5, that ovaries. shows lane 9, RNAi larvae; with lane third-instar combined females; treatment 4, adult lane 8, (15 embryos; lane RNA 18–21-hour-old males; stranded 3, adult lane 7, embryos; lane 9–12-hour-old pupae; 2, lane embryos: 3-hour-old in in detected organization was Chromosome and Methods). panel) and (left Materials wild-type Oregon-R of chromosomes opeet h ia eeohoai gene heterochromatin vital the that the demonstrate complements therefore results These from or ciainwt MIT ln ) EIwsntdtce netat rmnnatvtdB2 el ln ) adof ho band 4 A 2). after (lane cells cells bacterial BL21 BL21 non-activated chromatids of from sister extracts extracts of total in morphology in detected disorganized antibody not highly was anti-YETI the YETI by and 1). recognized (lane comp chromatin is panel) IPTG condensing protein (right mM of YETI cells 1 appearance The RNAi-treated with loose antibodies. in activation and organization anti-YETI fuzzy chromosome with the defective blotting highly note Western a panel); revealed (left panel) controls (bottom RNAi analysis Cytological control. as used of blots Northern (D) LP1/Df M17 LP1 LP1/LP1 ulhmzgtslcigtefunctional the lacking hemizygotes null m rngn nteasneo the of absence the in transgene Yeti )adto oS elclue togyrdcdtelvl fedgnu RAatr7 or ssonb TPR DNase RT-PCR. by shown as hours 72 after mRNA endogenous of levels the reduced strongly cultures cell S2 to addition g) ooyoe.Saebr 10 bar: Scale homozygotes. Yeti eergo seMtrasadMtos.Temgaino EIpoen(paetmlclrms f3 D)i oehtsoe than slower somewhat is kDa) 32 of mass molecular (apparent protein YETI of migration The Methods). and Materials (see region gene oydnltdRAfo ifrn eeomna tgs The stages. developmental different from RNA polyadenylated ; UAS-Yeti-M17/+ l(2)41Aa Cnie l,20a n the and 2003a) al., et (Cenci LP1/LP1 m .()Nrhr ltaayi of analysis blot Northern (C) m. ave hc onot do which larvae, Yeti ; Yeti Yeti UAS-Yeti-M17 Df(2R)B/LP1 l(2)41Aa Yeti tub-GAL4 seii mlfe adi u ognmcDA(o ae) TPRapiiainof amplification RT-PCR panel). (top DNA genomic to due is band amplified -specific gene. ee()fully (1) gene uatgntpsi lal etre.Asmlryanra oyeecrmsm organization chromosome polytene abnormal similarly A perturbed. clearly is genotypes mutant (A) Yeti n (2) and , eiyosmtn ave(ih panel). (right larvae mutant hemizygous Yeti tbGL ave n sasn in absent is and larvae, /tub-GAL4 driver, gene. uatlra hwn eaoi assi hi eool B AIsandpolytene DAPI-stained (B) hemocoel. their in masses melanotic showing larvae mutant Yeti ave xrsigthe GAL4 expressing larvae, carrying larvae of glands salivary from extracts the protein protein fusion total YETI–GFP in the detected specifically antibody GFP ulibtnndtcal ncrmsms(supplementary chromosomes in on Moreover, S2). detectable Fig. non that material ganglia in with but present neural was of overlaps staining nuclei anti-GFP cells that larvae, mitotic third-instar from pattern On isolated staining DAPI-staining. arms by is a chromosome and produced polytene yielding nuclei along all 3A), sites in (Fig. of present hundreds is to show protein recruited larvae fusion these YETI–GFP in the assays that immunofluorescence GFP 3B). (Fig. salivary in the expression using its brains induced and and glands Methods) and Materials ihtepoeni t aiecnomto nwoecls To cells. transgenic whole generated in conformation we melanogaster native problem, its this in circumvent protein the with oydnltdRAfo idtp ln )and 1) (lane wild-type from RNA polyadenylated UAS-Yeti-GFP rp49 ora fCl cec 21)17 5728 doi:10.1242/jcs.150243 2577–2588 127, (2014) Science Cell of Journal rvr eceo bratpltn chromosome polytene aberrant of rescue a driver, iooa rti eewsue salaigcnrl ae1 0– 1, Lane control. loading a as used was gene protein ribosomal ie ariga carrying lines Yeti Yeti in rngn ciae by activated transgene nl uatlarvae. mutant -null Df(2R)B rspiamelanogaster Drosophila Yeti-GFP elav-GAL4 al ocmlmn the complement to fails , UAS-Yeti-GFP P/P;tub-GAL4 LP1/LP1; 2kai nydtcal nprotein in detectable only is kDa 32 rngn ciae by activated transgene f2h,Df(2R)MS41A1 Df(2Rh), rvr(i.3) Anti- 3A). (Fig. driver LP1/LP1 2cells. S2 elav-GAL4 barren rngn (see transgene LP1 ave(ae2). (lane larvae en akn a lacking teins Yeti Drosophila rdt non- to ared ae6 late 6, lane / lee(see allele RAwas mRNA Yeti-GFP r of urs double- driver (F) . 2579 ,a tub-

Journal of Cell Science EERHARTICLE RESEARCH 2580 chromatin transgenic for different required three is melanogaster constructed YETI test of we To domain unknown. 80 Bucentaur binding, is BCNT the about which of the of of function portion whether the stretch 1), C-terminal (Fig. conserved the family highly in protein located a chromatin acids is to amino binding domain YETI BCNT for essential The is domain BCNT to The binds is and in protein protein found phenotype wild-type fusion chromosomal YETI mutants. aberrant the the YETI–GFP rescue of to absence able the was the pattern in that staining chromatin shows the and result in 3C) found that (Fig. to similar seen was morphology ie arigdiverse carrying lines lvGL;Yeti-GFP elav-GAL4; Yeti -3 6 LGtasee:(1) transgenes: FLAG le Fg C.This 3C). (Fig. flies Drosophila Yeti hoooe,w on F loecn inl nlarvae in signals fluorescent GFP found we polytene immunostained (see we nucleus when chromosomes, However, the 3E). enter Fig. to in inserts able the are YETI–GFP, like larvae proteins, truncated in both YETI that anti-FLAG showing transgenes, using three the observed of each was expressing staining nuclei clear gland A salivary 3E). of the (Fig. antibodies using anti-FLAG using by assays glands blot salivary in transgene elav-GAL4 each of expression induced next the We Methods). and Materials see 3D,E; (Fig. 159–238) akn h 3 the lacking 5 the for the deleted and 145–241), residues (containing rm fthe of frame the Yeti-Nter Yeti ora fCl cec 21)17 5728 doi:10.1242/jcs.150243 2577–2588 127, (2014) Science Cell of Journal -3 6 rvradpromdbt muotiigadwestern and immunostaining both performed and driver LGtaseebaigacmlt pnreading open complete a bearing transgene FLAG 3 9 6 n of end LGtasee(otiigrsde –6)and 1–166) residues (containing transgene FLAG Yeti LGtge rtisi aiaygad specifically glands salivary YETI- in deleted proteins or tagged in full-length FLAG shown of is Expression 159–238) (E) acids, green. amino (80 domain BCNT YETI-Cter3 red; in YETI-Nter3 underscored: are proteins the and transgene salivary in cells. proteins gland YETI–GFP of Expression 3. Fig. rgtpnl osnot. does panel) (right YETI-Nter3 whereas chromosomes, polytene YETI-Cter3 and gland YETI-3 salivary Full-length of (inserts). staining nuclei anti-FLAG as the nuclei by gland indicated salivary enter proteins truncated YETI-3 YETI-Cter3 Full-length and (green). (blue) anti-FLAG DAPI with fixed with and of stained detail sequentially the glands show salivary anti- inserts with The and (green). (blue) FLAG DAPI chromosomes with and polytene stained 3 show sequentially 1, panels (lanes lower expression The driver 5). of absence the in not under occurs cd) otospeeti h rnae YETI-3 truncated the in amino present (241 portions sequence acids); acid The amino (D) YETI panel). full-length (right bands chromosome -intermediate fluorescent or antibody DAPI-intense The the panels). decorates middle chromosome and of (left rescue morphology with its associated and is chromosomes expression polytene to binds YETI-GFP (green) (green). and anti-GFP (blue) with DAPI immunostained with stained were larvae Polytene from protein. chromosomes YETI–GFP of by 2) rescue (lane morphology absence or 1) (lane elav-GAL4 gland presence salivary the in in protein cells of YETI–GFP specificity for the antibody showing GFP assay blotting panel). Western (right (B) and arms panel) chromosomes (left polytene nuclei binds gland salivary in YETI–GFP accumulates (green): anti-GFP with immunostained Yeti-GFP from chromosomes Polytene ee 2 the (2) gene, Yeti hc nldsteBN oan(at domain BCNT the includes which , avewr tie ihDP bu)and (blue) DAPI with stained were larvae rvrepeso.()Chromosome (C) expression. driver A w ie arigadifferent a carrying lines Two (A) elav-GAL4 6 6 LG(idepnl id to binds panel) (middle FLAG YETI-Nter3 and FLAG 6 elav-GAL4 w LG(4–4)i le The blue. in (145–241) FLAG P/P;tub-GAL4 LP1/LP1; Yeti-Cter rvr(ae ,4ad6 but 6) and 4 2, (lanes driver lvGL;Yeti-GFP/ elav-GAL4; w 3 rvrwr examined. were driver 6 6 LGtransgene FLAG LG(etpanel) (left FLAG 6 6 LG(1–166) FLAG 9 / Yeti-GFP FLAG n,ad(3) and end, 6 Yeti-GFP 6 6 FLAG FLAG FLAG

Journal of Cell Science hto h otos(i.4A,C). (Fig. controls to the comparable were of chromocenter that the in H3K4me3 indeed H3K9me3, of were in marks reduced strains. modification strongly mutant histone H3K9ac, H3K4me3 and of levels H3K9me3, wild-type that showed in of experiments Immunofluorescence methylation, level histone e.g. and function, overall and acetylation organization the chromatin and of establishment assessed the maintenance for we required marks epigenetic chromosomes, chromosomal polytene in itnsadhsoemdfcto marks modification histone nucleosomal and variant, histones H2A.V histone of accumulation osof for Loss essential is YETI of domain BCNT binding. chromatin the that show therefore, Nter Cter full-length both expressing ARTICLE RESEARCH uat eecmaal otoeo idtp tan;however, strains; wild-type of those to polytene comparable in Histone in were HOAP incorporation mutants and ISWI their of for and levels chromosomal 2004), et The Cenci al., chromosomes. 2003; 1996; et HP1a al., al., et Kusch Shareef et 2003b; 2001; al., Caravaggio), Rubertis al. et De Ruhf 1999; 1989; al., as et al., Corona et (James known chromatin telomeric RPD3 major Deacetylase HOAP (also remodelers, known DOM-A of and protein panel ISWI Kingston, as a and such Simon tested factors, 2012; therefore al., We other et 2013). for and (Hopfner responsible ATPase proteins subunits remodeling chromatin chromatin more a or modification, one expression. histone gene contain neighboring complexes of the repression These turn, or ensure in activation leading, either states, that and closed to or complexes open by in chromatin multiprotein of recognized organization different are with histones interact modified in Post-translationally factors chromatin of Distribution H2A. and H2A.V of These 4A,C). accumulation (Fig. chromosomal of H2A loss and also that H2A.V are indicate of results species those H4 to variant and compared H3 extent of H2A.V H2B, chromosomes the polytene the on of affected to levels The similar 4A,C). in (Fig. reduced chromosomes, dramatically indeed, polytene H2A, was that, H2A of found mutant Histone We distribution of histones. To the abundance chromosomal mutant. the studied H4 the and also in H3 we affected H2B, question also assess that to are us types address faint prompted histone finding rare other This with 4A,C). whether (Fig. fluorescence, banding of background signs residual 2000). only al., instead et intensity consistent (Leach LP1/LP1 chromocenter, fluorescence reports the in previous varying and with of arms chromosome sites the along H2A.V multiple show 4A,C, of Fig. pattern and the wild-type examined of chromosomes Yeti therefore polytene in (Cenci We variant function paper). histone H2A.V this YETI chromosome/ 2003a; of in al., absence role defects et the a observed in Such the 2005). organization al., with chromatin et consistent Wu 2004; be al., (Kobor would et chromatin Mizuguchi into 2004; Htz1 required al., histone is et variant of and recruitment complex of the component remodeling for a is ATP-dependent YETI, SWR1 of ortholog the yeast the protein, SWC5 The sls of loss As 3 3 uatsrisb muoloecnemtos ssonin shown As methods. immunofluorescence by strains mutant 6 6 LGtasee u o nlra xrsigthe experiments, expressing these of results larvae The 3E). (Fig. in transgene FLAG not but FLAG-transgene, Yeti ooyoe and homozygotes rspiamelanogaster Drosophila ucinipispltn chromosome polytene impairs function Yeti fet h bnac fncesmlhistones nucleosomal of abundance the affects Yeti uat Fg A.Hwvr h levels the However, 4A). (Fig. mutants Yeti Yeti LP1/Df(2R)B -3 ucinpeeetal mar the impairs preferentially function 6 LGtaseeadthe and transgene FLAG Yeti Yeti idtp aiayglands salivary wild-type uat,btt lesser a to but mutants, eiyoe]showed hemizygotes] mutants Yeti uat [both mutants Yeti- Yeti- Yeti Yeti uat;H1 n P3as hwdrdcdpoenlvl in in levels histones protein both reduced modified of showed also specifically amount Yeti RPD3 protein and and HP1a overall H2A mutants; reduced and a H2A.V depicted 5A) (Fig. cuuaino ohHAVadHA n fsm histone some of and H2A, chromosomes of polytene and along factors, chromatin H2A.V and modifications both of accumulation 4B,C). (Fig. chromocenter controls at the of levels those HP1a to comparable Histone the were and However, HP1a RPD3. of abundance Deacetylase chromosomal reduced by found reduced were we 4B,C) (Fig. DOM-A of those noigHAV ulooa itns PaadRD is RPD3 and HP1a genes of histones, transcripts of their nucleosomal level H2A.V, the whether H2A.V, that in show test encoding encoding next analysis perturbed to this of We genes is RPD3, results abundance and 2007). for HP1a transcript al., assays histones, et chromosome nuclesomal qRT-PCR higher-order in (Corona in found performed regulation defects those to to transcriptional leads similar ISWI, structure as such and genes In products protein-encoding of chromatin abundance from reduced transcripts yields YETI of Loss i.6,,GTH1,btntGTaoe rcpttsYETI in precipitates shown extracts. alone, As gland GST salivary HP1a. larval not and YETI–GFP-expressing but YETI from experiments GST–HP1a, between pulldown 6C,D, interaction GST Fig. the performed test also to not we (data was immunoprecipitate Thus, the YETI–GFP HP1a shown). same in the However, in found detected in 6B). not (Fig. was shown immunoprecipitate H2A.V Oregon- As YETI–GFP Reciprocally, immunoprecipitate control from the immunoprecipitate. H2A.V). absent antibodies in was R the visualize but found extracts, using selectively (to H2A.V–GFP analyzed was from GFP YETI blotting then 6A, or Fig. and western glands YETI H2A.V–GFP salivary against by of expressing extracts immunoprecipitates larvae from DOM-A protein immunoprecipitated of and we fusion system, HP1a GFP-TRAP H2A.V–GFP H2A.V, the with Using interact proteins. might YETI whether 2013). Becker, and (Baldi nucleosomes in latter incorporation facilitating the thus of H2A.V, proposed with also is H2A and back 2004) exchange al., to et (Kusch of damage DNA complex of during repair H2A remodeling the with H2A.V phosphorylated Tip60 of exchange the the catalyzes of the subunit in ATPase Drosophila before masses the DOM- melanotic Notably, is lethality 2006). large A Steward, of and by (Minakhina presence hemocoel the larval followed by and development by pupation characterized larval phenotype lethal similar prolonged a have all DOM-A, and other in 2005). HP1a among and show, al., H2A.V of both chromosomes findings of et polytene content present poor (Swaminathan a our defects, recruitment chromosomal this, HP1a with in Consistent roles both play DOM-A by and The HP1a found H2A.V, with interacts levels 5A). YETI–GFP Fig. protein 4; (Fig. reduced analyses and western and the apparent immunofluorescence were with marks chromatin correlated and histones for transcripts in affected indeed h eut hsfridct usata eraei the in decrease substantial a indicate far thus results The ae nteeosrain,w eie oinvestigate to decided we observations, these on Based HP1a H2A.V, YETI, for mutants loss-of-function Interestingly, Yeti rspiamelanogaster Drosophila rspiamelanogaster Drosophila mutants. uat.Wsenbotn saso hl rti extracts protein whole of assays blotting Western mutants. ora fCl cec 21)17 5728 doi:10.1242/jcs.150243 2577–2588 127, (2014) Science Cell of Journal Rh ta. 01 uc ta. 04,which 2004), al., et Kusch 2001; al., et (Ruhf LP1 uat Fg B:dcesdlvl of levels decreased 5B): (Fig. mutants Yeti h oso hoai remodelers, chromatin of loss the , itn ain 2. skonto known is H2A.V variant histone uat Fg 4). (Fig. mutants Yeti LP1/LP1 uat n affects and mutants , 0.I addition, In 70%. uat.The mutants. 2581 Yeti

Journal of Cell Science AIadpedclrdi le nioysann a suoooe nylo.()Hsorm hwn h uniiaino hoooa eeso H of levels chromosomal of quantification the showing Histograms (C) proteins. yellow. in chromatin pseudocolored and of was modifications telomeres staining histone on antibody histones, present blue; nucleosomal also in is pseudocolored HP1 and arms. DAPI chromosome euchromatic affects mainly EERHARTICLE RESEARCH 2582 in that H Note Notably, modifiers. in arrowheads. chromatin arrows) by of by indicated Distribution are (marked (B) anti-HOAP chromocenters respectively). by of 72%, stained staining telomeres and significant wild-type; 45% a of (62%, those yield extent to still lesser levels H3K4met3 a comparable and to at reduced H3K9met3 accumulates are HOAP Oregon-R H4 and wild-type and ISWI of H3 mutants chromosomes H2B, polytene of in those proteins whereas chromatin and ( modifications mutants histone histones, nucleosomal H2A.V, and of Levels 4. Fig. ihYT.Tepeetfnig htYT neat ohwith interacts both interacts DOM-A YETI that that findings suggest present The results YETI. these with together, antibody. anti-DOM-A with Taken was immunoprecipitates YETI–GFP 7A, the in Fig. in recovered shown As protein). fusion YETI–GFP of extracts et eimnpeiiae O- rmslvr glands salivary from DOM-A immunoprecipitated we Next, Yeti mutants. LP1 / LP1 lvGl;UAS-Yeti-GFP elav-Gal4; oprdt h idtp rgnR muoloecneaayi eel htlvl fHAVadHAaedatclycompromised, drastically are H2A and H2A.V of levels that reveals analysis Immunofluorescence Oregon-R. wild-type the to compared ) A itiuino 2.,ncesmlhsoe n itn ak.Ntalhsoe n hoai rtistse r qal fetdin affected equally are tested proteins chromatin and histones all Not marks. histone and histones nucleosomal H2A.V, of Distribution (A) ave(xrsigthe (expressing larvae epnil o h erimn fYT ocrmtn othis To chromatin. to YETI of of recruitment recruitment the the for in responsible involved of events upstream in DOM-A proteins. place H2A.V chromatin would and H2A.V, DOM- of of YETI role recruitment known in the with A together H2A.V, with and DOM-A eteeoedrcl se hte O- sactually is DOM-A whether asked directly therefore We Yeti uat mre yarwed) hoooe eesandwith stained were Chromosomes arrowheads). by (marked mutants ora fCl cec 21)17 5728 doi:10.1242/jcs.150243 2577–2588 127, (2014) Science Cell of Journal Yeti uat,sgetn htterrdcdaccumulation reduced their that suggesting mutants, 2A.V, Yeti Yeti P1,

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Journal of Cell Science ugsigadrc oefrBG ntemdlto fHP1 of modulation the in BRG1 complex, for 2009; remodeling role al., ATP-dependent direct chromatin a SWI/SNF et HP1 suggesting other the (Fischer Human of 2009). reported and protein al., been et HP1a has Lavigne between factors remodeling be Association remodeling might chromatin to YETI HP1a regions. that targeting suggests in mediator 6C), important (Fig. al., an assays et pulldown GST similar (Morillo-Huesca in A yeast in function. not histones proposed and both but been 2010). integrity of complex has depletion chromatin DOM/tip60 scenario in disrupting the resulting also by and H2A.V, deposition removed with onto H2A is replaced H2A.V H2A in variant cells, defect homolog the yeast in dramatic loading found his to The like 6A,B; contribute YETI, nucleosomes. 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Df(2R)M41A10 rspiamelanogaster Drosophila eeohoai lmn n the and element heterochromatic , P(w+)19.74.3* ae to males ae,tesnl lmn up all jumps element P single the males, rspiamelanogaster Drosophila , tub-GAL4 sacrmsm arigapartial a carrying chromosome a is Yeti yS/Sco Cy Yeti ae eegnrtdcarrying generated were males cN ln 1)and M17) (line -cDNA Drosophila cN M7adM)under M7) and (M17 -cDNA 9 Df(2R)M41A10/CyS o ahlta mutation, lethal each For . n fCG40218. of end (+1.43C ;delta S; P(w+)19.74.3/Cy rvro the or driver ˚ nsadr cornmeal standard in C l(2)41A hoooecrisa carries chromosome eae orecover to females P(w+)19.74.3 Tub-Gal4 + l(2)41A white mro of embryos le mn the among flies pUAS-Yeti-GFP Df(2R)M41A10 Yeti nrgo h41 region in a eemd as made were Yeti + ee we gene, a phenotypic elav-GAL4 made were -3 lee is alleles and 6 females. ,outof P(w+) FLAG 2585 w delta elav- LP1 Yeti LP1 Yeti 1118 - .

Journal of Cell Science on between found Tb Cy M17/T(2;3)ST, olwn C odtos 0mna 95 at the min using 10 and protocol conditions: supplier PCR the following 7300 to ABI according an System, on to PCR performed Real-Time according was (AM1710) RT-qPCR instruction. kit manufacturer’s RETROscript the the with retrotranscribed were oSmae l Smae l,2002). al., et (Somma according al. performed et dsRNA were out of Somma cells Production to RNAi-treated 2000). carried of al., analysis et were cytological (Clemens and al. treatments et heat- Clemens RNAi to 10% according (Sigma-Aldrich). with supplemented FBS MO) inactivated Louis, St. (Sigma-Aldrich, medium gis ytei etd CEEKRSRY oae from located (CFEMEKKLRLSRRPY) peptide synthetic a against against antibodies polyclonal Rabbit Antibodies the Tb of phenotype Cy lethal GAL4/T(2;3)ST, the rescue To ARTICLE RESEARCH 2586 (2 RNA instruction. Tri manufacturer’s the the with to larvae according third-instar (Sigma) wandering Reagent from extracted was RNA Total PCR inverse and RT-PCR Quantitative the using immunodetected BIORAD. were from antibodies bands kit The secondary ECL dilution. rat 1:5000 and at mouse used al., were and Rabbit, et 1:2500. histones Sambrook dilution: 1984; following against al., et Antibodies were (Crowley salivary blotting 2001). protocols western and standard and brains to northern larval and according from preparations, extracted RNA Total were glands. proteins blotting and western RNA and Total blotting northern preparations, RNA treatments Drosophila RNAi and cultures Cell by provided chromatin software and NIS-Elements the modifications using Nikon. of performed histone levels were chromosomal histones, proteins of nucleosomal Quantification camera. H2A.V, microscope CCD (Life epifluorescence analyzed a 50i with were Eclipse antibodies equipped Chromosomes secondary Nikon dilution. computer-controlled dilution; 1:200 antibody a by at 1:500 dilution. used Alexa-Fluor-conjugated at was 1:300 technologies) used Alexa-Fluor- at monoclonal 1:50 were monoclonal used rat antibodies at was rabbit anti-DOM-A used technologies) a Rat were (Life antibody at HOAP antibody secondary used conjugated against a were K9 antibodies as H3met ISWI, dilution; Rabbit against H3K9ac, antibodies dilution. GFP, mouse against 1:100 and antibodies H2A, Rabbit or 2000). H2A.V al., et (Deuring to experiments immunolocalization Drosophila and squashes chromosome Polytene immunostaining and Cytology Universita Biologia Sviluppo, Louis, di Sarah St. dello (Dipartimento USA); in Corona e Albany, University Davide at by Washington Cellulare Rufh; University Biology Marie-Laure provided & of York USA); kindly New Health (Department Disorders were of Elgin Genetic immunostaining) Div. Department protein. Center, HOAP, State the (for (Wadsworth ISWI, of Glazer GFP DOM-A, region Robert HP1a, C-terminal and the H2A.V, RPD3 of against 241 antibodies position Polyclonal to 227 position h neln/xeso epo 60 of temp annealing/extension the the n niH Mlioe,at-Pa niHKm3adanti-H3K9ac (vector). and antibodies anti-GFP-HRP anti-H3K9me3 (Sigma), commercial anti-HP1a, anti-Tubulin Cestra (Abcam), (Millipore), following Gianluca anti-H4 Universita The and Sapienza and respectively. Biotecnologie, Germany); against e Italy), University, Biologia Biotecnologie, Roma, di Halle e (Dipartimento Biologia Genetics, di of (Dipartimento Universita Ciapponi Sapienza Laura Italy); LP1/LP1 Drosophila oyeecrmsmspromda ecie previously described as performed chromosomes polytene 2clswr utrda 25 at cultured were cells S2 ; UAS-Yeti-M17/tub-GAL4 yTb Cy ` rtiswr sd niHA niHB anti-H3 anti-H2B, anti-H2A, used: were proteins and ae.I h rgn fti rs,a21rtowas ratio 2:1 a cross, this of progeny the In males. iRm,Iay;Toa uop (Institute Rudolph Thomas Italy); Roma, di eae eecosdto crossed were females Cy + Tb + rgne,telte orsodn to corresponding latter the progenies, ˚ )1 i t72 at min 10 C) flies. LP1 Drosophila ˚ ,40 C, ` a ˚ nSilsadSn M3 Sang and Shields in C Tblnwr sda the at used were -Tubulin ulallele, null el td iPalermo, di Studi degli 6 1 t95 at s (15 w/Y EIwr raised were YETI ; ˚ .Piesand Primers C. LP1 w/w ˚ ; ,1mnat min 1 C, ; UAS-Yeti- LP1 ; ` tub- c di ) ,5 R, ATCATTATC-3 cmtgn 01.Rltv xrsinwscluae sn the using calculated was (2 expression method Ct Relative comparative 2001). Schmittgen, 5 F, GAAGAAC-3 TAACCGCCAAATCCGTAGAG-3 CAAAC-3 GCTCGGTGCTCTTTTGGTAG-3 GAA-3 CCTCACTGACAGC-3 2-,5 H2B-F, ATTCCATTACG-3 neln eprtrswr sflos 2-,5 H2A-F, follows: as were temperatures annealing n nlzdtedt.PD rt h paper. the wrote experiments P.D. the data. performed the experiments, analyzed the and designed and conceived authors All contributions Author interests. competing no declare authors The interests Competing Molecolare, Patologia e Italy). Biologia Ricerche, di delle Institute thank (Istituto Nazionale CNR also Pathology Consiglio Centre, We and Reference room. Biology Nikon fly Molecular at the of Lavia, with Patrizia support antibodies. and technical of Guarguaglini the gifts Giulia for for Iacueo Ruhf Davide Enzo Marie-Laure Ciapponi, thank and Laura We Glaser Cestra, Robert Gianluca Elgin, helpful to Sarah for grateful Corona, USA) also Seattle, are Center, We Steve Research comments. and Cancer Germany), Hutchinson Munchen, (Fred Institute, Henikoff and Butenandt manuscript Bo Adolf the Kenneth Munich of and University reading Becker critical Peter for and Rufh Washington, suggestions, Marie-Laure of and University Ricerche, Biology, USA) delle of Seattle, Nazionale (Department Consiglio Lavia, Wakimoto Molecolare, Patrizia Barbara Patologia USA), Italy), Piscataway, e Jersey, Biologia Biochemistry, New di and of (Istituto Biology University Molecular State of The (Department Rutgers, Kusch Thomas thank We Acknowledgements mM [20 buffer MgCl lysis mM in 1.5 resuspended 7.9, were pH solution; glands physiological HEPES-KOH salivary in and dissected were brains larvae third-instar Wandering pulldown GST and Co-immunoprecipitation rvosy(gete l,19)addgse sn either were using digested 5 and Fig. 1998) al., in replicates et (Eggert three previously values with The experiments, RpL32. independent of four each. values from average calculated the against 0m a n .%N4) ora 4 at hour 1 NP40), 0.8% with incubated and was KOH, NaF HEPES alone mM (20 mM GST buffer incubation 20 or in brains of was protein 50 mg from extracts Immunodetection fusion 2 protein pulldown, GST–HP1 GST 1:5000. For the BIORAD. at from either kit used ECL the (1:250); all using performed Yeti were (1:2500); antibodies and GFP secondary dilutions: (1:2500) given the against the antibodies H2A.V specific at the proteins using detected following was interest of The proteins (Chromotek). instructions manufacturer’s in to assays GFP-TRAP according hour. performed was 1 for ice on incubated and (Roche)] 2 MNC,3 MNF 5mM 25 NaF, mM 30 NaCl, mM 420 nuain S rtiswr avse n ahdsvrltimes 1 several twice. in least washed at resuspended repeated and were and assays immunoprecipitation harvested buffer were incubation with proteins GST incubation, oooosgn oeso eetdsequences. sequences repeated was or database models flanking gene nonredundant homologous against on performed BLASTN to was company. performed sequencing according MWG and performed 2001) the was al., by et PCR the (Dobie Inverse using procedures ligated (Roche). standard was kit fly a Ligation of Rapid one-fifth approximately to equivalent DNA Na aawr nlzdacrigt ia n cmtgn(ia and (Livak Schmittgen and Livak to according analyzed were Data eoi N rmtot he le a rprda described as prepared was flies three to two from DNA Genomic 3 VO 9 9 -CAAGGACACCAACAGCAAC-3 -TTGGGCTTGCGCCATT-3 4 9 2.-,5 H2A.V-R, ; .%N-0 . MPS,1 PMSF, mM 0.5 NP-40, 0.1% , ora fCl cec 21)17 5728 doi:10.1242/jcs.150243 2577–2588 127, (2014) Science Cell of Journal 9 -TGACACCGGAATTTCGTCGA-3 9 4F 5 H4-F, ; 9 P-,5 HP1-R, ; 9 p3-,5 Rpl32-F, ; 9 2-,5 H2A-R, ; 9 -GTGTGAAGCGCATATCTGGA-3 9 9 -GCCAGCCATTTCTGTTCAAT-3 2.-,5 H2A.V-F, ; a Tbln(:50.Hreaihperoxidase Horseradish (1:2500). -Tubulin 9 -ACCATTTCTGCTTGGTCCAC-3 2 9 DD -GCGCACCAAGCACTTCATC-3 Ct 9 ;mN xrsinwsnormalized was expression mRNA ); . 9 9 -GGCTGCTCCTACTGATCCTG-3 3R 5 H3-R, ; 9 P-,5 HP1-F, ; 2 . 9 0m C,01m DTT, mM 0.1 KCl, mM 10 , P3R 5 RPD3-R, ; b 9 5b nodrt identify to order in bp 25 6 re (Ludwig-Maximilians- ¨rner -CGAAACCGAATTCCGTA- gyeohshts,02mM 0.2 -glycerophosphatase, 9 rtaeIhbtrCocktail Inhibitor Protease -ATGGCTCGTACCAAG- 9 6 ˚ 2-,5 H2B-R, ; ihaiain After agitation. with C 9 -CAAGCGAAAGTCC- aml ufr Co- Buffer. Laemmli 9 -TCGCTGCGGC- 9 -CGGCCAGAT- Hha 9 9 9 -TTGGTTC- 4R 5 H4-R, ; 3F 5 H3-F, ; Drosophila Ior 9 9 RPD3- ; ;Rpl32- Hpa II. 9 9 9 - - ;

Journal of Cell Science iir,P,Craii . os,F,Vernı F., Rossi, N., Corradini, P., Dimitri, E. J. McIntosh, and X. Luan, G., T. Diekwisch, X. Luan, and A. Williams, F., Marches, G., T. Diekwisch, aa,K,Frhsi . aaoo . ksk,K n ioe S. Hirose, and K. Akasaka, T., Yamamoto, H., Furuhashi, K., H. Hanai, A. Lund, and G. G. Galli, M., K., C. Zofall, Fog, C., Rubin, M., Zhou, J., Dhakshnamoorthy, B., Cui, T., Fischer, J., Weko, L., T. H. Saumweber, McGrath, and K. M., Bergemann, V. H., Eggert, Velasco, D., C. Kennedy, W., K. Dobie, T. R. Kamakaka, and N. Dhillon, ordn,N,Rsi . Vernı F., Rossi, N., Corradini, A., S. McClymont, N., Snarskaya, A., J. Armstrong, G., Siriaco, F., D. Corona, M. Gatti, and R. Kellum, D. G. Raffa, G., Siriaco, G., Cenci, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.150243/-/DC1 online available material Supplementary material Supplementary D.). P. 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