H4K20me1 and H3K27me3 are concurrently loaded onto the inactive X chromosome but dispensabe for inducing gene silencing

A icle H4K20me1and H3K27me3a e concu en ly loaded on o he inac i e X ch omosome bu dispensable o inducing gene silencing Sjoe d J D Tjalsma 1,† , Mayako Ho i 2,† , Yuko Sa o 2,3,† , Au elie Bousa d 1 , Aki o Ohi 2 , Ana Cl audia Raposo 4 , Julia Roensch 1 , Agnes Le Saux 1 , Jumpei Nogami 5 , Kazumi su Maeha a 5 , Tomoya Kuji ai 6 , Te suya Handa 3 , Sand a Bag es-A nal 7 , Yasuyuki Ohkawa 5 , Hi oshi Ku umizaka 6 , Sim~ ao Teixei a da Rocha 4 , Jan J ˙ Zylicz 1,7,8,* , Hi oshi Kimu a 2,3,** & Edi h Hea d 9,10,*** Abs ac Du ing X ch omosome inac i a ion (XCI), in emale placen al mammals, gene silencing is ini ia ed by he Xis long non-coding RNA. Xis accumula ion a he X leads o en ichmen o speci ic ch o- ma in ma ks, including PRC2-dependen H3K27me3and SETD8- dependen H4K20me1. Howe e , he dynamics o his p ocess in ela ion o Xis RNA accumula ion emains unknown as is he in ol emen o H4K20me1in ini ia ing gene silencing. To ollow XCI dynamics in li ing cells, we de eloped a gene ically encoded, H3K27me3-speci ic in acellula an ibody o H3K27me3-min body. By combining li e-cell imaging o H3K27me3,H4K20me1, heXch o- mosome and Xis RNA, wi h ChIP-seq analysis we unco e concu en accumula ion o bo h ma ks du ing XCI, albei wi h dis inc genomic dis ibu ions. Fu he mo e, using a Xis BandC epea mu an , which s ill shows gene silencing on he X bu no H3K27me3deposi- ion, we also ind a comple e lack o H4K20me1en ichmen . This demons a es ha H4K20me1is dispensable o he ini ia ion o gene silencing, al hough i may ha e a ole in he ch oma in compac ion ha cha ac e ises acul a i e he e och oma in. Keywo ds emb yonic s em cells; H4K20me1; he e och oma in; polycomb; X inac i a ion Subjec Ca ego y Ch oma in, T ansc ip ion & Genomics DOI 10.15252/emb .202051989 | Recei ed 31 Oc obe 2020 | Re ised 22 Decembe 2020 | Accep ed 7Janua y 2021 EMBO Repo s (2021)e51989 In oduc ion Dynamic changes o he ch oma in landscape allow o imely execu ion o de elopmen al and di e en ia ion p og ammes. Indeed, ch oma in modi ie s o en ein o ce signalling cues o ini i- a e and/o main ain an exac ansc ip ional ou come (S ase ich e al, 2014; Zylicz e al, 2015; Jambheka e al, 2019). One powe ul model, whe e such egula ion akes place, is X ch omosome inac i- a ion (XCI) in emale mammals. Al hough much is known abou he in ol emen o ce ain ch oma in ma ks in ini ia ing XCI ( e- iewed in Zylicz & Hea d, 2020), i was hus a impossible o ack ch oma in ea angemen s and he inac i e X ch omosome (Xi) in li ing cells. Wha is mo e, he ole and dynamics o some his one modi ica ions du ing XCI emains enigma ic. In eu he ian emale mammals, XCI is ini ia ed a a ound he ime o implan a ion, when each cell andomly inac i a es one o he wo X ch omosomes (Lyon, 1962). This p ocess can be modelled in i o by di e en ia ing emale mouse emb yonic s em cells (ESC) (Ras an & Robe son, 1985). Bo h in i o and in i o, XCI depends on he coa ing o he Xi by a long non-coding RNA called Xis (X-inac i e- speci ic ansc ip ) (Penny e al, 1996). Xis RNA accumula ion along he Xi induces no only he silencing o o e a 1,000 genes bu also a cascade o ch oma in al e a ions ( e iewed in Zylicz & Hea d, 1Mammalian De elopmen al Epigene ics G oup, Ins i u Cu ie, CNRS UMR3215, INSERM U934, PSL Uni e si y, Pa is, F ance 2G adua e School o Bioscience and Bio echnology, Tokyo Ins i u e o Technology, Yokohama, Japan 3Cell Biology Cen e , Ins i u e o Inno a i e Resea ch, Tokyo Ins i u e o Technology, Yokohama, Japan 4Faculdade de Medicina, Ins i u o de Medicina Molecula , Jo~ ao Lobo An unes, Uni e sidade de Lisboa, Lisboa, Po ugal 5Di ision o T ansc ip omics, Medical Ins i u e o Bio egula ion, Kyushu Uni e si y, Fukuoka, Japan 6Ins i u e o Quan i a i e Biosciences, The Uni e si y o Tokyo, Tokyo, Japan 7The No o No disk Founda ion Cen e o S em Cell Biology, Copenhagen, Denma k 8Depa men o Physiology, De elopmen and Neu oscience, Uni e si y o Camb idge, Camb idge, UK 9EMBL Heidelbe g, Heidelbe g, Ge many 10 Coll ege de F ance, Pa is, F ance *Co esponding au ho . Tel: +45 23839889; E-mail: jan.zy[email p o ec ed] **Co esponding au ho . Tel: +81 45 924 5742; E-mail: [email p o ec ed].jp ***Co esponding au ho . Tel: +49 6221 3878 201; E-mail: [email p o ec ed] † These au ho s con ibu ed equally o his wo k ª2021 The Au ho s. Published unde he e ms o he CC BY 4.0license EMBO epo s e51989 |2021 1o 17 2020). Xis is a modula non-coding RNA wi h speci ic egions play- ing dis inc oles. I s 50A- epea egion is i al o he induc ion o gene silencing (Wu z e al, 2002). I does so by ec ui ing SPEN, a key RNA-binding p o ein, which in eg a es many ep essi e complexes including NCOR/SMRT and HDAC3 (McHugh e al, 2015; Zylicz e al, 2019; Dossin e al, 2020). On he o he hand, he B and o a lesse ex en C epea egions a e i al o he ec ui men o Polycomb-g oup ep essi e complexes (PRC) (Bousa d e al, 2019; Cologno i e al, 2019; Nes e o a e al, 2019). Indeed, upon Xis RNA coa ing Xi becomes apidly en iched wi h he PRC1-depen- den H2AK119Ub and only subsequen ly wi h PRC2-dependen H3K27me3 (Pla h e al, 2003; Sil a e al, 2003; de Napoles e al, 2004; Zylicz e al, 2019). Recen da a indica e ha he B and C epea egion o Xis RNA di ec ly ec ui s hnRNPK, which in u n binds non-canonical PRC1 allowing o apid H2AK119Ub deposi- ion (Almeida e al, 2017; Pin acuda e al, 2017; Bousa d e al, 2019; Cologno i e al, 2019). H2AK119Ub could po en ially be ecognised by PRC2 co ac o s allowing o de no o deposi ion o H3K27me3 (Blackledge e al, 2014). Impo an ly, Polycomb accumula ion is dispensable o he ini ia ion o gene silencing, as Xis mu an s lack- ing he B and C epea egion can s ill induce XCI albei wi h sligh ly lowe e iciency (Bousa d e al, 2019; Nes e o a e al, 2019). Ins ead, PRC2 enables s able main enance o he ep essed s a e, pa icula ly in he con ex o ex a-emb yonic lineages (Kalan y e al, 2006). The dynamics o accumula ion o PcG ma ks ha e been s udied using ChIP-seq, al hough i emains unclea how p ecisely his co ela es wi h Xis RNA coa ing o he Xi. Indeed, he ime ha lapses be ween Xis RNA up egula ion and H3K27me3 deposi ion is no known. No how his ela es o o he ep essi e his one ma ks. Ano he modi ica ion ha apidly accumula es on he Xi is H4K20me1 (Kohlmaie e al, 2004), al hough i s ole in XCI is no well unde s ood. H4K20me1 is deposi ed by SETD8 (also called P - Se 7) (Nishioka e al, 2002) and is hough o egula e a a ie y o p ocesses ela ing o ansc ip ion, ch omosome condensa ion, DNA eplica ion and he DNA damage esponse ( e iewed in an Nuland & Gozani, 2016). Consis en wi h he in ol emen o H4K20me1 in cell di ision, i ansien ly accumula es du ing G2 and mi osis, whe eas H4K20me2 and -me3 do no show simila luc ua ions du ing he cell cycle (Wu e al, 2010). This dynamics is achie ed by s able exp ession o SETD8 du ing G2/M when i s phospho yla ion p e en s he in e ac ion wi h he anaphase-p omo ing complex (APC). Du ing la e mi osis, CDC14 dephospho yla es SETD8 hus s imula ing i s deg ada ion by APC. On he o he hand, PHF8, H4K20me1 deme hylase, is deg aded by APC du ing ea ly G2 (Lim e al, 2013) oge he allowing o e icien deposi ion o H4K20me1 and success ul p og ession h ough mi osis. Fu he mo e, H4K20me1 accumula es a cen ome es whe e i p omo es kine o- cho e assembly and hus co ec ch omosome seg ega ion (Ho i e al, 2014). The egula ion o H4K20me1 le els could also be achie ed by modula ing he e iciency o u he me hyla ion by SUV420H1/2 o deme hyla ing H4K20me2/3 by speci ic enzymes. DPY-21 media es he la e a eac ion in Caeno habdi is elegans, and i s wo mouse o hologs (RSBN1/RSBN1L) e ain speci ic deme hy- lase ac i i y in i o (B ejc e al, 2017). Howe e , he biological unc- ion o hese enzymes emains unknown. The s udy o H4K20me1 du ing XCI has been hampe ed by he ac ha SETD8 is absolu ely equi ed o he p og ession h ough mi osis and emb yos lacking i die be o e he onse o XCI (Oda e al, 2009; Shika a e al, 2020). While he genomic dis ibu ion o H4K20me1 a a s ably inac i a ed X was p e iously desc ibed (Calab ese e al, 2012), he dynamics o H4K20me1 accumula ion du ing XCI ini ia ion in ela ion o Xis RNA and H3K27me3 deposi ion emains unclea . Fu he mo e, he p ecise dis ibu ion o i s H4K20me1 en ichmen along he X and he molecula mechanisms unde lying i s dynamic accumula ion on he Xi ha e no p e iously been in es iga ed. To add ess hese ques ions, i is impo an o ollow he spa io- empo al dynamics o his one modi ica ions as hey accumula e on he X ch omosome in li ing cells. To his end, we ha e p e iously de eloped gene ically encoded, modi ica ion-speci ic in acellula an ibodies o min bodies, by using a single-chain a iable agmen (scF ) o he speci ic an ibody wi h a luo escen p o ein (Sa o e al, 2013; Sa o e al, 2016). The exp ession o min bodies enables acking o changes in endogenous his one modi ica ion le els wi hou a ec ing cell cycle p og ession and de elopmen al p ocesses in such model o ganisms as ission yeas , nema ode, d osophila and zeb a ish (Sa o e al,2013;A aie al, 2017). He e, o ollow XCI dynamics in li ing cells, we de eloped a min body speci ic o he PRC2-dependen H3K27me3. In addi ion, we implemen ed sgRNA-dCas9 sys em o isualise bo h X ch omosomes in li ing cells (Chen e al, 2013; An on e al, 2014). We used hese no el ools in combina ion wi h an H4K20me1-speci ic min body (Sa o e al, 2016; Sa o e al, 2018) and a me hod o isualise endogenous Xis RNA in li e cells (Masui e al, 2018; Dossin e al, 2020). This allowed us o show ha he e a e s ik- ing simila i ies in he accumula ion dynamics o bo h H3K27me3 and H4K20me1 ma ks on he Xi. Fu he ChIP-seq analysis con i med concu en accumula ion o H4K20me1 and H3K27me3 du ing XCI albei wi h a he dis inc genomic dis ibu ions and co ela ions wi h gene silencing dynamics. By using cells exp essing mu an Xis RNA, we demons a e ha H4K20me1 accumula ion, jus like ha o PcG- dependen H3K27me3, elies on he Xis RNA B and C epea s. This also e eals ha bo h ma ks a e dispensable o he ini ia ion o gene silencing. Taken oge he , ou analysis unco e s ha he H4K20me1 and H3K27me3 his one ma ks accumula e a he Xi wi h compa able dynamics bu wi h a he di e en dis ibu ions. Reliance o bo h ma ks on he Xis -BC egion o hei en ichmen sugges s a mechanis- ic link be ween Polycomb and H4K20me1 accumula ion du ing acul- a i e he e och oma in o ma ion on he X ch omosome. These obse a ions also imply ha he gene al unc ion o H4K20me1 may be in ch oma in compac ion ha cha ac e ises acul a i e he e och o- ma in a he han in he ini ia ion o gene silencing. Resul s H3K27me3min body (2E12LI) speci ically acks H3K27me3in li ing cells Me hods o acking polycomb-dependen his one ma ks in li ing cells ha e been lacking up un il now. Such ools would be hugely bene icial o he s udy o dynamic epigene ic p ocesses such as XCI. To add ess his, we decided o gene a e an H3K27me3-speci ic min - body. Upon sc eening o mouse hyb idomas, we selec ed a 2E12 clone, which exp esses an H3K27me3-speci ic an ibody. We de e - mined he cDNA sequence o he IgG hea y and ligh chains in 2E12 by deep sequencing (Kuniyoshi e al, 2016) and cloned he a iable agmen s by PCR o cons uc a min body exp ession ec o (Figs 1A 2o 17 EMBO epo s e51989 |2021 ª2021 The Au ho s EMBO epo s Sjoe d JD Tjalsma e al and EV1A). To alida e ha he min body p ope ly localises o H3K27me3-en iched ch oma in in li ing cells, we used mouse e a o- ca cinoma cell line MC12 (Abe e al, 1988). MC12 is a mix u e o diploid and e aploid emale cells ha bou ing one o wo Xi, on which he min body is expec ed o be concen a ed. When exp essed in MC12, he supe olde (s ) GFP e sion o 2E12 min body (2E12- s GFP) localised mos ly in cy oplasm and ended o o m agg ega es (Fig EV1B, le ), sugges ing ha 2E12-s GFP was no able o unc ion in cells, p obably due o a ypical p oblem in olding and/o s uc- u al s abili y associa ed wi h he in acellula exp ession o scF (Ca aneo & Biocca, 1999; Ewe e al, 2004). To p e en agg ega e o ma ion, we pe o med PCR-based andom mu agenesis and an- sien ans ec ion in o MC12 cells. One mu an ha localised p e e en- ially in he nucleus wi h ocal en ichmen (p esumably Xi) had Me 86 o Leu subs i u ion in he hea y chain (M86L) (Fig EV1B, middle). Changes in localisa ion sugges ed ha M86L mu a ion imp o ed he min body unc ion o bind o H3K27me3. To gain insigh in o he con ibu ion o M86L mu a ion in scF s uc u e, we looked in o he a omic models buil based on he X- ay s uc u e o he mos simila scF (Fig EV1C, le ). The modelling indica ed ha Me 86 is loca ed wi hin a hyd ophobic co e o he hea y chain, and i s subs i u ion o Leu appea s o ill he space in he co e be e han he o iginal Me o s eng hen hyd ophobic in e ac ions. We ha e p e iously obse ed ha he hyd ophobici y o an amino acid in a hyd ophobic co e is impo an o he unc ionali y o a H4K20me1-min body (Sa o e al, 2016). Howe e , he 2E12M86L-s GFP was s ill no exclusi ely loca ed in he nucleus and we we e unable o ob ain cells s ably exp essing i . Thus, we decided o u he s eng hen scF olding wi h an addi ional mu a ion a Me 158 in he ligh chain’s hyd opho- bic co e (Fig EV1C, igh ). We cons uc ed h ee mu an s in which Me 158 was subs i u ed o Ile, Leu and Val in 2E12M86L. Among he h ee mu a ions, M185I mu an was he mos en iched in he nucleus wi h e iden oci (Fig EV1B, igh ). To con i m ha nuclea oci o 2E12M86L, M185I (2E12LI)-s GFP ep esen H3K27me3-en iched Xi, we employed immuno luo escence (IF) on ansien ly exp essing cells using p e iously cha ac e ised speci ic an ibodies di ec ed agains H3K27me3 and H3K9me3 (CMA327 and CMA318, espec- i ely (Chand a e al, 2012)). The nuclea oci we e colocalised wi h a H3K27me3-speci ic an ibody, bu no wi h H3K9me3-speci ic an i- body ha is concen a ed in Hoechs -dense pe icen ome ic he e- och oma in (Fig 1B). These da a a e consis en wi h he min body oci ep esen ing Xi in li ing cells. The u ili y o 2E12LI min body was u he demons a ed by ime-lapse imaging o s ably exp essing MC12 cells, which allowed o he acking o Xi du ing in e phase and mi osis (Fig 1C and Mo ie EV1). To u he alida e he speci ici y o 2E12LI o H3K27me3, we pe o med bo h biochemical and cell-based assays. We i s e alua ed he binding speci ici y o bac e ially exp essed and pu i ied 2E12LI- s GFP in i o using a modi ied his one pep ide a ay (Fig EV2A). H3K27me3-con aining pep ides we e highligh ed o e o he pep ides, ega dless o he neighbou ing R26 modi ica ions (Fig EV2B). The 2E12LI-s GFP binding was, howe e , occluded by S28 phospho yla- ion (S28ph), as commonly obse ed o me hyl-speci ic an ibodies (Kimu a e al, 2008; Hayashi-Takanaka e al,2011;Kimu a,2013). Nex , we assessed whe he he pu i ied 2E12LI-s GFP can selec i ely bind o H3K27me3 in cells by manipula ing he le el o speci ic me hyla ion. HeLa cells we e ans ec ed wi h HaloTag- agged lysine deme hylases KDM6B and KDM4D and ixed o s aining wi h he speci ic an ibodies o 2E12LI-s GFP. Consis en ly wi h he subs a e speci ici y o hese KDMs, IF indica ed ha HeLa cells o e exp essing Halo-KDM6B and Halo-KDM4D exhibi ed d as ic dec ease o H3K27me3 and H3K9me3, espec i ely (Fig EV2C and D). The esul o s aining wi h pu i ied 2E12LI-scF was simila o H3K27me3-speci- ic an ibody, showing dec eased le els o H3K27me3 by Halo-KDM6B o e exp ession and no changes in H3K27me3 by Halo-KDM4D (Fig EV2E and F). Taken oge he wi h he immuno luo escence pa e n and biochemical analysis, we concluded ha 2E12LI-s GFP selec i ely binds o H3K27me3 o e he o he modi ica ions including H3K9me3. Fo con enience, we now call 2E12LI used wi h a luo escen p o ein as H3K27me3-min body. Simul aneous acking o X ch omosome loci, H3K27me3and H4K20me1du ing XCI In o de o isualise he dynamics o his one modi ica ions du ing XCI, we decided o de elop a me hod o iden i y he X ch omosome min body VHVLFluo escen p o ein scF A B H3K27me3-min body (2E12LI) H3K9me3 (IF)H3K27me3 (IF) Hoechs 33342 25:0024:45 25:15 25:4525:30 26:00 VH VL FP H3K27me3 min body H3K27me3 (IF) H3K9me3 (IF) min body C min body Figu e 1. Es ablishing H3K27me3min body o isualise H3K27me3in li ing cells. A Schema ic ep esen a ion o he min body. H3K27me3-speci ic single-chain a iable agmen (scF ) is gene ically used wi h a luo escen p o ein (FP). B Immuno luo escence (IF) alida ion o min body speci ici y. Mouse MC12 cells, which s ably exp ess H3K27me3-min body (s GFP), a e labelled wi h an ibodies speci ic o H3K27me3(Cy5) and H3K9me3(Cy3). DNA is s ained wi h Hoechs 33342. Single con ocal sec ions a e shown. A ows ma k Xi. Scale ba =10 μm. C Time-lapse imaging o a di iding MC12 cell s ably exp essing H3K27me3-min body (s GFP). P ojec ion images o 7con ocal sec ions wi h 2μm in e als a e shown wi h elapsed ime (hh:mm). Scale ba =10 μm. ª2021 The Au ho s EMBO epo s e51989 |2021 3o 17 Sjoe d JD Tjalsma e al EMBO epo s in li e cells. We used an sgRNA-dCas9 (single guide RNA-nuclease dead Cas9) sys em o label X-linked loci in li ing cells (An on e al, 2014; Ma e al, 2015). We sc eened epe i i e sequences loca ed speci ically on he mouse X ch omosome and chose 32 mic osa el- li e sequences as a ge candida es. The exp ession ec o s o sgRNAs ha bind o he sequences we e cons uc ed and co ans- ec ed wi h an exp ession ec o o EGFP- agged dCas9 in o MC12 cells. Among hem, 4 sgRNAs (mX2, mX8, mX18 and mX26) enabled isualising X-linked loci in li ing cells (Fig EV3). We selec ed mX8 (nea Xis locus) and mX26 (Dxz4 locus) o label X AB XIC Xis Cen Dxz4 mouse Ch . X mX26 mX8 76Mb 103Mb C dCas9/mX8+26 dCas9/mX8+26 Min Body (H3K27me3) -LIF, 3 days +LIF Min Body (H3K27me3) dCas9/mX8+26 Min Body (H3K27me3) Min Body (H4K20me1) dCas9/mX8+26 Min Body (H3K27me3) dCas9/mX8+26 Min Body (H4K20me1) 0:00 h s 1:00 h s 2:00 h s 3:00 h s 4:00 h s 5:00 h s 6:00 h s D Min Body-mChe y H3K27me3 -LIF Xi Xa H3K27me3 dCas9 3 s GFP sgRNA mX26 mX8 Female ESC Figu e 2. Simul aneous isualisa ion o his one ma ks and X ch omosome loci in li ing cells. A Schema ic diag am o CRISPR/dCas9-3×s GFP a ge ing loci on mouse X ch omosome. sgRNA mX26 and mX8 a ge mic osa elli e epea s on Dxz4and Xis loci, espec i ely. B Expe imen al design: emale ESCs a e enginee ed o s ably exp ess wo sgRNAs, dCas9-3xs GFP and a nuclea localisa ion signal (NLS)- used H3K27me3-min body (mChe y). Upon ESC di e en ia ion by LIF wi hd awal H3K27me3accumula ion occu s a he inac i a ing X (Xi) bu no a he ac i e X (Xa). G een oci allow ma king o wo X-linked loci (Dxz4and Xis ). C Li e imaging o emale mESCs as in (B) in undi e en ia ed condi ions (+LIF) o upon 3days o LIF wi hd awal. Double a owheads ma k Xa and single a owhead ma ks Xi. Shown a e maximum in ensi y p ojec ions o 11 z-plane con ocal sec ions. Scale ba =10 μm. D ESCs as in (B) we e enginee ed o exp ess H3K27me3-min body (SNAP/JF646) and H4K20me1-min body (mChe y). Cells we e cul u ed in he absence o LIF o 2days. Time-lapse images o 12 z-plane con ocal s ack we e acqui ed e e y 1h. Maximum in ensi y p ojec ion images a e shown wi h elapse ime (hh:mm). A owheads ma k Xi. Scale ba =10 μm. 4o 17 EMBO epo s e51989 |2021 ª2021 The Au ho s EMBO epo s Sjoe d JD Tjalsma e al ch omosomes and used 3×s GFP- agged dCas9 (3×s GFP-dCas9), o ampli y he signal o u he analyses using ESCs. To ack H3K27me3 du ing X inac i a ion, we es ablished emale mouse ESCs (PGK12.1) exp essing wo sgRNA (mX8 and mX26), 3×s GFP-dCas9 and H3K27me3-min body (mChe y e sion) (Fig 2 A and B). In undi e en ia ed ESCs, X ch omosome loci we e iden i- ied as 2 pai s o 3×s GFP-dCas9 spo s and H3K27me3-min body was no en iched in ei he allele (Fig 2C, uppe panels). In con as , 3 days a e wi hd awal o leukaemia inhibi o y ac o (LIF), H3K27me3-min body accumula ed a ound one pai o 3×s GFP- dCas9 spo s (Fig 2C, lowe panels). This esul demons a ed ha H3K27me3-min body oge he wi h he sgRNA-dCas9 sys em allows o acking he dynamics o his one modi ica ion in li ing and di - e en ia ing cells. Since H4K20me1, unlike H4K20me2/3, also becomes en iched on Xi (Fig EV4A; Kohlmaie e al, 2004), we sough o e eal he ela i e accumula ion kine ics o he H3K27me3 and H4K20me1 his one modi ica ions du ing XCI. We es ablished ESCs exp essing he wo sgRNAs, 3×s GFP-dCas9, H3K27me3-min - body (SNAP-Tag e sion, isualised wi h JF646) and H4K20me1- min body (mChe y e sion, (Sa o e al, 2016)) and induced di - e en ia ion (3d LIF wi hd awal). Time-lapse imaging e ealed ha whene e H3K27me3 accumula ion was isible a he Xi, some le el o H4K20me1 en ichmen was also disce nible (Fig 2D), sugges ing ha bo h ep essi e ma ks accumula e concu en ly du ing XCI. This ini ial analysis in di e en ia ing emale ESCs con i med he success ul use o bo h H3K27me3- and H4K20me1-min bodies o ollow en ichmen o hese wo ma ks on he X ch omosome. Howe e , gi en he asynch onous na u e o andom XCI in di e en- ia ing ESCs and he a he weak signal om he agged X-linked loci, i was challenging o dis inguish he Xi om he ac i e X (Xa) p io o a signi ican accumula ion o bo h ep essi e ch oma in ma ks on he Xi. To amelio a e ou analysis, we he e o e decided o ollow Xis RNA i sel in li ing cells alongside H3K27me3 o H4K20me1. H3K27me3accumula es concu en ly wi h H4K20me1a he Xi In o de o ack he ela i e dynamics o H3K27me3, H4K20me1 and Xis RNA du ing XCI, we combined he use o min bodies wi h he inducible Xis -Bgl sys em (Masui e al, 2018; Dossin e al, 2020; Fig 3A). The la e model is based on he hyb id (Mus musculus cas aneus x C57BL/6) TX1072 emale ESCs line allowing o doxycy- cline (DOX) induc ion o he endogenous Xis gene om C57BL/6 (B6) allele (Schulz e al, 2014). By adding DOX, we can induce Xis A DOX inducible p omo e Xis BglSL Xis BglG-FP Xis RNA min body-FP H4K20me1/H3K27me3 +DOX Xis Xi Xa H3K27me3/ H4K20me1 01 23 45 1 1.2 ime since Xis accumula ion [h s] 1.4 1.6 No malised en ichmen Me geBglG (Xis ) min body (H3K27me3) Me geBglG (Xis ) min body (H4K20me1) 0:00h s1:00h s2:00h s3:00h s4:00h s5:00h s6:00h s 0:00h s1:00h s2:00h s3:00h s4:00h s5:00h s6:00h s B C H3K27me3 H4K20me1 emale ESC 2i+LIF * * * Figu e 3. Simul aneous isualisa ion o his one ma ks and Xis RNA in li ing cells. A Schema ic ep esen a ion o he expe imen al design. Female mouse ESC line was used, in which Xis can be induced om one allele (TX1072) and his allele also ha bou s an a ay o 18 Bgl s em loops (BglSL) inse ed in o he 7 h exon o Xis gene. BglG used o a luo escen p o ein (GFP o mChe y) de ec s Xis RNA as i binds o BglSL. Cells also s ably exp ess a min body allowing he de ec ion o H3K27me3(GFP) o H4K20me1(mChe y). B Cells we e ea ed wi h DOX o induce Xis exp ession and ime-lapse imaging was pe o med wi h images acqui ed e e y 15 min. Maximum in ensi y p ojec ion images a e shown wi h elapse ime (hh:mm). A owheads ma k Xi accumula ing Xis RNA. Scale ba =5μm. C Li e imaging analysis o a e age H3K27me3( ed) and H4K20me1(blue) accumula ion a he Xi. A e age no malised min body en ichmen is shown wi h shading ep esen ing 25 and 75 qua iles. Signal was calcula ed s a ing om he i s accumula ion o Xis RNA. A leas 30 cells we e analysed. *unpai ed - es P- alue <0.05. ª2021 The Au ho s EMBO epo s e51989 |2021 5o 17 Sjoe d JD Tjalsma e al EMBO epo s exp ession and hus educe he le el o he e ogenei y obse ed du ing XCI in di e en ia ing ESCs. In addi ion, 18 Bgl RNA s em loops (BglSL) we e knocked in o he 7 h exon o DOX-inducible Xis (Fig EV4B) (Dossin e al, 2020). To ack Xis RNA accumula ion, BglSL a e isualised by a BglG p o ein used o ei he EGFP o mChe y and exp essed om he Rosa26 o TIGRE locus (Fig EV4C and D). The cell lines enabling Xis RNA isualisa ion a e named TX-Xis -EGFP and TX-Xis -mChe y. These cell lines we e used o gene a e s ably exp essing PiggyBac ansgenes o he H3K27me3- o H4K20me1-min bodies (Fig EV4C and D). Two ESC lines, TX- Xis -EGFP; H4K20me1-mChe y and TX-Xis -mChe y; H3K27me3- s GFP we e used o subsequen analyses. Impo an ly, he use o Xis -BglSL o he min bodies did no a ec he e iciency o gene silencing du ing XCI (Fig EV4E). In o de o isualise he ea lies e en s ollowing Xis RNA accu- mula ion, we s a ed li e imaging o ESCs 5 min a e DOX induc- ion (Fig 3B, Mo ies EV2–EV3). Imaging was pe o med o a leas 6 h in 15-min in e als allowing o single-cell acking and de ailed quan i a i e analysis. Xis RNA domains appea ed a e 2–6h o DOX induc ion (Fig EV4F) and we e segmen ed using he Xis - EGFP/mChe y signal. The min body en ichmen wi hin such domains was measu ed and ollowed in indi idual cells h oughou he leng h o he expe imen (Fig 3C). O no e, unlike H3K27me3- min body, H4K20me1-min body shows inc eased nuclea signal du ing G2/M phase o he cell cycle, hus acking he oscilla ions in H4K20me1 le els (Sa o e al, 2016). We hus excluded mi o ic cells om he analysis due o e y high o e all le els o H4K20me1. Nex , we ollowed he en ichmen o H3K27me3/H4K20me1 signal in a leas 30 nuclei indi idually synch onised o he ime poin when Xis RNA en ichmen was i s obse ed (i.e., ypically 2–6h a e DOX addi ion). A signi ican en ichmen o bo h his one ma ks was obse ed wi hin he Xis RNA domain abou 45 min a e Xis RNA appea ance (adj. P- alue <0.05; Fig 3B and C). Ini ial accu- mula ion o H3K27me3 and H4K20me1 ollowed e y simila dynamics. Howe e , a e 2.5 h he wo ma ks signi ican ly di e ged (Fig 3C). Indeed, H3K27me3 con inued o apidly accumula e, while he inc ease in H4K20me1 en ichmen signi ican ly slowed down (Fig 3C). Thus, we ha e success ully pe o med simul aneous ack- ing o Xis RNA and ep essi e his one ma ks. In summa y, he en ichmen o bo h H3K27me3 and H4K20me1 is delayed compa ed o Xis RNA accumula ion by abou 45 min. H3K27me3 shows a con inued accumula ion o e ime while ha o H4K20me1 e en u- ally slows down. This e eals ha he wo ma ks sha e simila dynamics o en ichmen bu only a he ea ly s ages o XCI. Allele-speci ic na i e ChIP-seq e eals simila i ies be ween H3K27me3and H4K20me1accumula ion dynamics Gi en he abo e esul s indica ing ha al hough H3K27me3 and H4K20me1 a e bo h ini ially en iched soon a e Xis RNA accumula- ion, hei pa e ns di e ge a e a ew hou s, we decided o in es iga e he molecula dis ibu ions o bo h hese ma ks along he Xi. To his end, we pe o med allele-speci ic na i e ChIP-seq (nChIP-seq) o H4K20me1 in he hyb id emale cell line TX1072 (Schulz e al, 2014). Thanks o an inducible Xis gene on he B6 ch omosome, DOX addi ion leads o apid gene silencing and ch oma in ma k al e a ions ha can be measu ed allelically hanks o he high a e o polymo phism be ween he B6 and Cas X ch omosomes (Zylicz e al, 2019). Following DOX ea men , we acked H4K20me1 accumula ion on he Xis RNA- coa ed B6 allele ac oss i e ime poin s a up o 4-h esolu ion on biolog- ical duplica es (Fig 4A). We compa ed all ou esul s wi h a ma ched published da ase o H3K27me3 (Zylicz e al, 2019). Impo an ly, we alida ed he speci ici y o H4K20me1 an ibody using a pep ide a ay (Fig EV5A) and con olled o Xis RNA induc ion e iciency (Fig EV5B). Upon sequencing, eads we e spli acco ding o con en o allele-speci ic single nucleo ide polymo phisms (SNPs: B6 mapping o Xi; Cas mapping o Xa). Such allelic in o ma ion was analysed, e eal- ing p og essi e en ichmen o B6-speci ic eads (o igina ing om Xi) upon Xis induc ion o H4K20me1 as is he case o H3K27me3 (Fig EV5C). We analysed ela i e B6- ead en ichmen wi hin 10-kb windows ac oss he whole X ch omosome no malised o =0 h (Fig 4B). This e ealed H4K20me1 accumula ion a e 8 h o DOX induc ion, a ime poin when H3K27me3 also s a s o acc ue bu signi ican ly la e han ini ial H2AK119Ub en ichmen (Zylicz e al, 2019). The le els o en ich- men o H4K20me1 a e lowe han o H3K27me3 and seem o each hei pla eau ea lie . To be e isualise he iming o accumula ion, we no malised bo h ma ks o hei e ec i e dynamic ange, i.e. o a e age accumula ion a e 24 h o DOX induc ion (Fig 4C). This con i med ha a e 8 h o DOX ea men , he e is concu en ini ia ion o H4K20me1 and H3K27me3 accumula ion. To quan i y his u he , we plo ed he B6- eaden ichmen ela i e o =0 h o each 10-kb window as a unc- ion o ime and i ed a sigmoidal cu e (see Ma e ials and Me hods). To ex ac he in o ma ion abou ela i e iming o his one ma k accu- mula ion, we ob ained he ime when each cu e eaches i s maximum slope (e ec i e dose 50%, ED50). ED50 analysis e ealed ha H4K20me1 eaches i s mos e icien accumula ion p io o H3K27me3 (Fig 4D) bu la e han H2AK119Ub (Zylicz e al, 2019). This is in line wi h H4K20me1 achie ing i s pla eau signi ican ly be o e H3K27me3. Thus, ou analysis con i med li e imaging obse a ions ha H4K20me1 accumula es concu en ly wi h H3K27me3 bu quickly eaches maxi- mum en ichmen (Fig 3C). We nex examined he deg ee o which he dis ibu ions o he wo ma ks o e lapped ac oss he Xi. We ound ha H4K20me1 becomes p e e en ially en iched a loci in p oximi y o he Xis gene (g een ba ) as well as a egions ha Xis RNA i s in e ac s wi h (so called “en y si es”, black ba s (Pin e e al, 2012); Fig 4E). The ini ial en ichmen o H4K20me1 on he Xis -coa ed X ollows he same pa e n p e iously de ec ed o no only H3K27me3 bu also o PRC1-dependen H2AK119Ub (Fig 4E; Zylicz e al, 2019). To con i m his obse a ion, we in es iga ed ch omosome-wide co ela ion be ween H3K27me3 and H4K20me1 accumula ion wi hin di e en genomic windows (Fig 4F). Consis en ly wi h ou ini ial obse a ion, we ound s iking co ela ion in he accumula ion o bo h ma ks ac oss in e genic windows as well as bodies o silen genes. In con as , he bodies o genes ha we e ac i e p io o DOX ea men (i.e. ini ially ac i e genes) showed a much lowe Pea son’s co ela- ion wi h ρ=0.2, implying ha bo h ma ks di e in hei co ela ion wi h ansc ip ion (see below). Wi h he excep ion o ini ially ac i e genes, nChIP-seq o H4K20me1 e ealed ha i s accumula ion ollows a s ikingly simila pa e n and dynamics o H3K27me3. H4K20me1accumula es in e genically and is dispensable o XCI ini ia ion The abo e nChIP-seq analysis e ealed simila i ies and di e ences be ween H4K20me1 and H3K27me3 accumula ion on he X du ing 6o 17 EMBO epo s e51989 |2021 ª2021 The Au ho s EMBO epo s Sjoe d JD Tjalsma e al XCI. While in es iga ing genes ha a e ini ially ac i e and hen become silenced ollowing Xis induc ion, we obse ed ha H4K20me1 is s ongly biallelically en iched (p ema ked) a an- sc ibed gene bodies, p io o silencing (Fig 5A). This con as s s ikingly wi h he dis ibu ion o H3K27me3, which ne e p ema ks ini ially ac i e genes (Zylicz e al, 2019). We con i med ha his H4K20me1 en ichmen a ansc ibed genes is a gene al ea u e bo h on he X ch omosome (Fig 5B) and au osomes (Fig EV5D). This is in line wi h p e ious epo s indica ing ha H4K20me1 co ela es wi h ansc ip ional elonga ion and is en iched a ac i e gene bodies (Beck e al, 2012; Kapoo -Vazi ani & Ve ino, 2014; Veloso e al, 2014). Upon Xis induc ion (+DOX), we ound a signi ican accumula ion o H4K20me1 on he Xi a ini ially ac i e p omo e s and in e genic egions (Fig 5A and B). This esembles he pa e n obse ed o H3K27me3, wi h he in iguing ca ea ha H4K20me1 does no seem o u he accumu- la e a bodies o ini ially ac i e genes, a he , H4K20me1 le els emain cons an . Thus, H4K20me1 and H3K27me3 accumula ion pa e ns a e s ikingly simila wi hin in e genic egions bu di e in he bodies o ini ially ac i e genes (Fig 4F). Nex , o e alua e he ela ionship be ween H4K20me1 accumula ion and he p ocess o gene silencing, we sepa a ely analysed genes inac i a ed ea ly and la e upon DOX ea men (Fig 5C). This e ealed ha while H4K20me1 de no o accumula ion ollowing Xis induc ion is es ic ed o in e genic and p omo e egions, i occu s mo e e i- cien ly in he p oximi y o apidly silenced genes e.g. o Rn 12 (Fig 5A). All in all, hese indings sugges ha p io o XCI, H4K20me1 co ela es wi h ac i e ansc ip ion (in gene bodies), bu ollowing Xis induc ion H4K20me1 becomes en iched de no o a egions su ounding genes ha a e apidly silenced. To u he explo e he po en ial ela ionship o H4K20me1 en ichmen and H3K27me3 deposi ion du ing XCI, we decided o in es iga e ch oma in s a es in Xis mu an cell lines. Cu en models sugges ha H3K27me3 is deposi ed hanks o a complex H3K27me3 H4K20me1 0.0 0.2 0.4 0 4 8 12162024 ime [h s] B6 (Xi) ead accumula ion el. o =0h B 0.0 0.5 1.0 1.5 04812162024 ime [h s] no malised B6 (Xi) ead accumula ion C H3K27me3 H4K20me1 n=9069 n=4971 0 8 16 24 H3K27me3 H4K20me1 D ED50 [h s] DOX inducible p omo e Xis Xis B6 Cas ESC 2i+LIF (TX1072) B6 Xi Cas Xa H4K20me1 allele-speci ic nChIP-seq DOX ea men 0, 4, 8, 12, 24h A 2 1 0 B6 (Xi) ead accumula ion el. o =0h 1 0 0 50 100 150 H3K27me3 - 12h DOX H4K20me1- 12h DOX E X ch omosome posi ion [Mb] * 0.0 -0.1 0.1 0.0 0.1 0.2 Ini ially ac i e gene bodies ρ = 0.2 0.0 0.1 0.2 0.3 0.0 0.1 Inac i e gene bodies ρ = 0.656 0.1 0.20.0 0.1 In e genic egions ρ = 0.828 0.0 H3K27me3 accumula ion 24h s [Lo g 2 scale] H4K20me1 accumula ion 24h s [Log2 scale] H3K27me3 accumula ion 24h s [Lo g 2 scale] H3K27me3 accumula ion 24h s [Lo g 2 scale] F Figu e 4. Na i e ChIP-seq e eals H4K20me1and H3K27me3co-accumula ion du ing XCI. A Schema ic ep esen a ion o he expe imen al design. The hyb id TX1072 mouse emale ESC line was used, in which Xis can be induced om he endogenous B6 allele. Time cou se nChiP-seq o H4K20me1was pe o med and compa ed o a published H3K27me3da ase (GSE116480) (Zylicz e al,2019). B Quan i ica ion o a e age H4K20me1(blue) and H3K27me3( ed) en ichmen a he Xi (B6allele) compa ed wi h =0hin10-kb windows spanning he whole ch omosome. Shading is he in e qua ile ange. C As in (B) bu shown is no malised B6en ichmen o he a e age accumula ion a 24 h. D Pai wise compa ison o H4K20me1and H3K27me3accumula ion dynamics (ED50) a he X ch omosome. All 10-kb windows wi h ED50 <24 h a e plo ed. * ai ed Wilcoxon ank-sum es P- alue <0.05. EH4K20me1(blue) and H3K27me3( ed) accumula ion ac oss he Xi a e 12 h o DOX ea men . The black line is a locally es ima ed sca e plo smoo hing (LOESS) eg ession on all 10-kb windows (do s). Below each plo shown is he Xis locus (g een ba ) and Xis en y si es (black ba s). F Co ela ion be ween H3K27me3and H4K20me1accumula ion a e 24 h o DOX ea men a ini ially ac i e gene bodies (le ), inac i e gene bodies (middle) and 10- kb in e genic windows spanning he X ch omosome ( igh ). All scales a e loga i hmic. All co ela ions (ρ) a e wi h P<0.01 om Pea son’s co ela ion es . ª2021 The Au ho s EMBO epo s e51989 |2021 7o 17 Sjoe d JD Tjalsma e al EMBO epo s machine y, in ol ing ini ial PRC1 ec ui men by he B and C epea s o Xis and subsequen PRC2 ec ui men hanks o H2AK119Ub en ichmen (Almeida e al, 2017; Pin acuda e al, 2017; Bousa d e al, 2019; Cologno i e al, 2019; Nes e o a e al, 2019). We he e o e es ed whe he H4K20me1 en ichmen a he Xi is also dependen on he B and C egions o Xis RNA. To his end, we employed p e iously es ablished male ESC lines ha bou ing a DOX- inducible, endogenous Xis in ei he i s ull leng h o m (Xis FL)o lacking he B and C epea egions (Xis ΔBC) (Fig 6A), in which H3K27me3 changes on he Xi had al eady been mapped (Bousa d e al, 2019). This sys em ecapi ula es hallma ks o XCI, namely ch omosome-wide Xis coa ing, X-linked gene silencing and he e- och oma in o ma ion (Bousa d e al, 2019). A e DOX ea men , we pe o med IF/RNA FISH expe imen s o quan i y he e iciency o H4K20me1 en ichmen a he Xis RNA domains (Fig 6B). While he global le el o H4K20me1 was compa able in bo h cell lines, i showed, as p edic ed, s iking a iabili y be ween cells due o di - e en cell cycle s ages. Ne e heless, we obse ed comple e loss o H4K20me1 en ichmen om he Xis RNA domains when Xis ΔBC was exp essed; a esul eminiscen o wha was p e iously obse ed o H3K27me3 (Fig 6C) (Bousa d e al, 2019). These da a indica e ha H4K20me1 en ichmen depends on he same egions o Xis RNA ha a e in ol ed in ec ui ing PRC1 and lead o subse- quen PRC2 ec ui men . Which ac o s di ec ly allow o H4K20me1 en ichmen a he Xi s ill emains unclea , howe e . Ano he impo an conclusion om his esul pe ains o gene silencing. Indeed, he Xis ΔBC RNA is able o ini ia e gene silencing, albei a sligh ly lowe e iciency (Bousa d e al, 2019). This implies ha he de no o accumula ion o H4K20me1 is la gely dispensable o he ini ia ion o gene silencing, simila ly o bo h Polycomb-asso- cia ed H3K27me3 and H2AK119Ub. Discussion We epo he spa io- empo al dynamics o wo ea ly ch oma in changes, H4K20me1 and H3K27me3 du ing he o ma ion o acul a- i e he e och oma in in XCI. Using a unique combina ion o li e-cell imaging and ch oma in p o iling in he same cell sys ems, we e eal he ela i e iming and dis ibu ions o ch oma in en ichmen o hese ma ks du ing X inac i a ion. Ou s udy p o ides insigh s in o he p ocess o XCI and new ools o he s udy o epigene ic p ocesses in gene al. Indeed, we use he powe ul echnology o min bodies, gene ically encoded luo escen p obes ha can de ec speci ic p o eins and hei modi ica ions. In his way, we isualise he dis ibu ion and le els o speci ic his one modi ica ions, in longi- udinal single-cell analyses o he epigene ic p ocess o XCI. We p e iously epo ed on he de elopmen o min bodies speci ic o H4K20me1 and H3K9ac (Sa o e al, 2013; Sa o e al, 2016). The H3K27me3 min body de eloped he e will enable he s udy o PRC2- dependen epigene ic mechanisms beyond XCI. Ou esul s e eal ha i s de ec able en ichmen o bo h H3K27me3 and H4K20me1 on he Xis -coa ed X ch omosome occu s abou 45 min ollowing Xis RNA coa ing, hus ~3–7 h a e DOX ea men . This sugges s ha bo h ma ks become en iched on he Xi in a Xis -dependen bu likely indi ec mechanism. I should be no ed ha p o eins such as SPEN which a e ec ui ed di ec ly by Xis RNA (Chu e al, 2015; McHugh e al, 2015; Minajigi e al, 2015) ARn 12 10kb 0h 4h 8h 12h 24h 0h 4h 8h 12h 24h 3- 3- 3- 3- 3- 7- 7- 7- 7- 7- H3K27me3 H4K20me1 Pgk1 10kb 0h 4h 8h 12h 24h 0h 4h 8h 12h 24h 3- 3- 3- 3- 3- 7- 7- 7- 7- 7- H3K27me3 H4K20me1 B6 (Xi) Cas (Xa) O e lap (Xi&Xa) -30kb 30kbTSS TES -30kb 30kbTSS TES 0.01 0.02 0.01 0.02 0.01 0.02 H4K20me1 ini ially ac i e genes inac i e genes mean B6 (Xi) en ichmen -30kb 30kbTSS TES -30kb 30kbTSS TES 0.01 0.02 H3K27me3 ini ially ac i e genes inac i e genes 0h 4h 8h 12h 24h 0h 4h 8h 12h 24h B C -30kb 30kbTSS TES -30kb 30kbTSS TES 0.1 0.3 0.5 0.1 0.3 0.5 0h 4h 8h 12h 24h H4K20me1 ea ly silenced genes la e silenced genes mean B6 (Xi) en ichmen Figu e 5.H4K20me1accumula es in e genically. A Genome b owse acks showing H3K27me3( op) and H4K20me1(bo om) accumula ion a a gene silenced apidly (Rn 12) o mo e slowly (Pgk1). Allele-speci ic acks we e o e laid (B6in ed and Cas in blue). No e s ong H4K20me1bi-allelic p ema king a gene bodies. B A e age H3K27me3( op) and H4K20me1(bo om) en ichmen a he B6allele o e ini ially ac i e o inac i e genes 30 kb a he X ch omosome. Shown a e da a o all ime poin s. C A e age H4K20me1en ichmen a he B6allele o e ea ly and la e silenced genes 30 kb a he X ch omosome. Shown is da a o all ime poin s. Da a in o ma ion: TSS: ansc ip ion s a si e; TES: ansc ip end si e. 8o 17 EMBO epo s e51989 |2021 ª2021 The Au ho s EMBO epo s Sjoe d JD Tjalsma e al show immedia e colocalisa ion wi h Xis du ing ini ia ion o XCI based on li e-cell imaging using he same ESC sys em (Dossin e al, 2020). While he ini ial en ichmen o bo h H4K20me1 and H3K27me3 ollows simila dynamics, hey soon di e ge wi h H3K27me3 accumula ing mo e e icien ly. Wha is mo e, H4K20me1 shows a unique cell cycle dynamics wi h ansien ly inc eased le els du ing G2/M (Rice e al, 2002). In line wi h hese di e ences, ou nChIP-seq analysis e ealed ha bo h ma ks also show dis inc genomic dis ibu ion especially in he con ex o ini ially ac i e genes. Thus, while accumula ion o bo h ma ks depends on a common Xis -media ed mechanism hei sp eading and long- e m en ichmen is egula ed independen ly. He e, we e eal ha bo h H3K27me3 and H4K20me1 accumula- ion depends on he Xis B+C egion (Bousa d e al, 2019). Howe e , nei he SETD8 no PRC2 co e componen s ha e been iden i ied as di ec Xis RNA-binding p o eins (Chu e al, 2015; McHugh e al, 2015; Minajigi e al, 2015). Ins ead, PRC1 is hough o be ec ui ed by his egion ia he hnRNPK ac o . Fu he mo e, SETD8 does no in e ac wi h any o he i e p o eins, including hnRNPK, ecen ly iden i ied o bind wi h he Xis B+C epea s (Bousa d e al, 2019). I hus emains unclea how SETD8, i a all, is ec ui ed o he Xi and whe he i depends on he non-canonical PRC1 complexes, as p oposed o PRC2 (Almeida e al, 2017; Pin a- cuda e al, 2017). Indeed, PRC2 ec ui men depends on he non- canonical PRC1, which e y apidly deposi s H2AK119Ub. This modi ica ion in u n is p oposed o ec ui PRC2 ia i s co ac o s, e.g. JARID2 (Coope e al, 2016). Whe he a simila mechanism media es H4K20me1 en ichmen emains unclea . A ac o po en- ially in ol ed in indi ec SETD8 ec ui men is a Polycomb-g oup p o ein L3MBTL1, which is no howe e a co ac o o ei he PRC1 o PRC2. L3MBTL1 is a binde o H4K20me1 (Kalakonda e al, 2008) bu i also in e ac s wi h SETD8 and H3K27me1/2 (Kalakonda e al, 2008). Thus, L3MBTL1 may ec ui SETD8, when H3K27 s a s o become me hyla ed by PRC2 bu he H3K27me1/2 en ichmen du ing XCI has no ye been s udied. In line wi h his model, loss o EED, a co e PRC2 componen , has p e iously been epo ed o esul in pa ially educed H4K20me1 en ichmen a he Xi (Schoe ne e al, 2006). Thus, PRC2 con ibu es o H4K20me1 accumula ion bu o he pa hways mus also ope a e. Al e na i ely, i is possible ha SETD8 is no ec ui ed o he Xi; ins ead, i could be he enzymes ca alysing H4K20me2 deme hyla ion o H4K20me1, which become en iched du ing XCI. Indeed, in C. elegans dosage compensa ion, which consis s o a educ ion o X-linked gene ac i i y in XX indi id- uals, is pa ly dependen on an H4K20me2-speci ic deme hylase (B ejc e al, 2017). In his sys em, e icien con e sion o H4K20me2 o H4K20me1 a he X ch omosome by DPY-21 p omo es gene silencing du ing he main enance phase. RSBN1 and RSBN1L a e mouse o hologs o DPY-21 and seem o e ain speci ic his one deme hylase ac i i y (B ejc e al, 2017). Whe he RSBN1/RSBN1L ha e a ole in H4K20 me hyla ion dynamics in mammalian cells and whe he hese ac o s play a ole in XCI me i s u he in es iga ion. Finally, i is also possible ha H4K20me1 en ichmen a he Xi is due o hinde ed con e sion o highe me hyla ion s a es by SUV4- 20H1/2. Iden i ying he B and C epea s o Xis RNA as he key egion o H4K20me1 en ichmen du ing XCI sugges s ha his ma k may only play a mino ole in ini ia ing gene silencing. Indeed, cells exp ess- ing Xis :ΔBC can induce X ch omosome gene silencing du ing XCI albei wi h lowe e iciency (Bousa d e al, 2019). The ac ha H4K20me1 is highly en iched a ansc ibed gene bodies bo h on he X ch omosome (be o e XCI) and au osomes would also sugges ha his ma k is no in ol ed in ansc ip ional ep ession. Indeed, p e ious s udies epo ed a posi i e co ela ion be ween H4K20me1 en ichmen and he a e o ansc ip ional elonga ion (Veloso e al, 2014). SETD8 was also p oposed o acili a e RNA Polyme ase II (RNAPII) elease om p omo e s (Kapoo -Vazi ani & Ve ino, 2014; Nikolaou e al, 2017). The ini ial dis ibu ion o H4K20me1 is in line wi h such indings. Howe e , his ma k also pe sis s a genes which become apidly silenced du ing XCI, e.g. Rn 12 (Fig 5A). Thus, an- sc ip ional silencing does no esul in apid H4K20me1 deple ion a AB Me geXis RNA H4K20me1 Xis FLXis ΔBC C D C BE F A A A A A A DOX inducible p omo e Xis IF+RNA FISH +DOX -LIF 48h s Xis Xi H4K20me1 Xa Male TXY ESC DE F A A A A A A Xis FL Xis ΔBC 0 25 50 75 100 0 25 50 75 100 % o Xis domains wi h en ichmen H4K20me1 H3K27me3 (Bousa d e al., 2019) * * Xis FL Xis ΔBC Xis FL Xis ΔBC Figu e 6.H4K20me1accumula ion elies on Xis B +C epea egion. A Schema ic ep esen a ion o he expe imen al design. By DOX addi ion, male TXY mouse ESC lines allow o exp ession o ull leng h Xis FL o Xis ΔBC. Cells induced o 48 h in he absence o LIF we e used o combined IF and RNA FISH. B Rep esen a i e image o IF/RNA FISH o Xis RNA and H4K20me1in cells exp essing Xis FL o Xis ΔBC. A owheads poin o he Xis RNA domains. Scale ba =5μm. C G aph ep esen s he mean % S De o Xis RNA domains en iched o H4K20me1(le ) o H3K27me3( igh ) in cells exp essing Xis WT o Xis ΔBC. 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Annu Re Biochem 89:255 –282 License: This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s use, dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. ª2021 The Au ho s EMBO epo s e51989 |2021 17 o 17 Sjoe d JD Tjalsma e al EMBO epo s