Mitotic progression, arrest, exit or death relies on centromere structural integrity, rather than de novo transcription

*Fo co espondence: maia [email protected] (HM); c is ina. e [email protected] (CF) Compe ing in e es s: The au ho s decla e ha no compe ing in e es s exis . Funding: See page 23 Recei ed: 23 Ma ch 2018 Accep ed: 03 Augus 2018 Published: 06 Augus 2018 Re iewing edi o : Jon Pines, The Gu don Ins i u e, Uni ed Kingdom Copy igh No ais-C uz e al. This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use and edis ibu ion p o ided ha he o iginal au ho and sou ce a e c edi ed. Mi o ic p og ession, a es , exi o dea h elies on cen ome e s uc u al in eg i y, a he han de no o ansc ip ion Ma co No ais-C uz 1,2 , Ma ia Alba Abad 3 , Wil ed FJ an IJcken 4 , Niels Galja 5 , A A ockia Jeyap akash 3 , Helde Maia o 1,2,6 *, C is ina Fe a ´s 1,2 * 1 Ch omosome Ins abili y & Dynamics Labo a o y, Ins i u o de Biologia Molecula e Celula , Uni e sidade do Po o, Po o, Po ugal; 2 Ins i u o de In es igac¸a ˜o e Ino ac¸a ˜o em Sau ´de (i3S), Uni e sidade do Po o, Po o, Po ugal; 3 Wellcome T us Cen e o Cell Biology, Uni e si y o Edinbu gh, Edinbu gh, Uni ed Kingdom; 4 Cen e o Biomics, E asmus Medical Cen e , Ro e dam, Ne he lands; 5 Depa men o Cell Biology, E asmus Medical Cen e , Ro e dam, Ne he lands; 6 Cell Di ision G oup, Expe imen al Biology Uni , Depa men o Biomedicine, Faculdade de Medicina, Uni e sidade do Po o, Po o, Po ugal Abs ac Recen s udies ha e challenged he p e ailing dogma ha ansc ip ion is ep essed du ing mi osis. T ansc ip ion was also p oposed o sus ain a obus spindle assembly checkpoin (SAC) esponse. He e, we used li e-cell imaging o human cells, RNA-seq and qPCR o in es iga e he equi emen o de no o ansc ip ion du ing mi osis. Unde condi ions o pe sis en ly una ached kine ocho es, ansc ip ion inhibi ion wi h ac inomycin D, o ea men wi h o he DNA- in e cala ing d ugs, delocalized he ch omosomal passenge complex (CPC) p o ein Au o a B om cen ome es, comp omising SAC signaling and cell a e. Howe e , we we e unable o de ec signi ican changes in mi o ic ansc ip le els. Mo eo e , inhibi ion o ansc ip ion independen ly o DNA in e cala ion had no e ec on Au o a B cen ome ic localiza ion, SAC esponse, mi o ic p og ession, exi o dea h. Mechanis ically, we show ha DNA in e cala ing agen s educe he in e ac ion o he CPC wi h nucleosomes. Thus, mi o ic p og ession, a es , exi o dea h is de e mined by cen ome e s uc u al in eg i y, a he han de no o ansc ip ion. DOI: h ps://doi.o g/10.7554/eLi e.36898.001 In oduc ion When cells commi o mi osis he nuclea en elope disassembles and ch oma in o ganizes in o highly condensed ch omosomes. This causes he displacemen o se e al ansc ip ion ac o s om DNA and he inac i a ion o he ansc ip ion machine y in a cell-cycle-dependen manne (Go es eld and Fo bes, 1997;Kim e al., 1997;Kon ad, 1963;Ma ı´nez-Balba ´s e al., 1995; Mu phy and A a di, 1973;Pa sons and Spence , 1997;P esco and Bende , 1962; Rizkallah and Hu , 2009;Segil e al., 1996;She moen and O’Fa ell, 1991;Spence e al., 2000; Taylo , 1960). Because he hal -li e o mos ansc ip s exceeds he no mal du a ion o mi osis ( ypically ~30 min in human cells), he p e ailing dogma was ha ansc ip ion is la gely ep essed and dispensable du ing mi osis in highe euka yo es (Go es eld and Fo bes, 1997;Kon ad, 1963; Ma ı´nez-Balba ´s e al., 1995;Mu phy and A a di, 1973;P esco and Bende , 1962; Rizkallah and Hu , 2009;Spence e al., 2000;Taylo , 1960). Se e al ecen s udies ha e challenged his no ion and p o ided e idence ha he le el o ch o- ma in compac ion on mi o ic ch omosomes is highly he e ogeneous (Nishino e al., 2012), allowing No ais-C uz e al. eLi e 2018;7:e36898. DOI: h ps://doi.o g/10.7554/eLi e.36898 1 o 27 RESEARCH ARTICLE accessibili y o some ansc ip ion ac o s and ch oma in-modi ying enzymes (Bu ke e al., 2005; Chen e al., 2005;Dey e al., 2009;Egli e al., 2008;Gau hie -Rou ie ` e e al., 1991; Michelo i e al., 1997;Segil e al., 1996;Yan e al., 2006). No ewo hy, a pos - ansla ionally modi ied o m o RNA Polyme ase II ha is no mally associa ed wi h ansc ip ion elonga ion was ound o bind mi o ic ch omosomes a hei cen ome es (Chan e al., 2012;Di ks and Snaa , 1999;Liu e al., 2015;Molina e al., 2016), sugges ing ha some ansc ip ion is s ill aking place du ing mi osis. T ansc ip ion migh also play an ins umen al ole du ing a p olonged mi osis due o incapaci y o sa is y he spindle assembly checkpoin (SAC), a signaling mechanism ha can delay mi osis up o se e al hou s in he p esence o una ached kine ocho es (Musacchio, 2015b). In line wi h his hypo hesis, he mas e mi o ic egula o Cyclin B1 was p oposed o be ac i ely ansc ibed du ing mi osis and o be equi ed o sus ain a obus SAC esponse (Mena e al., 2010;Scio ino e al., 2001). Cen ome e assembly, which unde lies he ca aly ic mechanism behind he SAC, has also been ecen ly p oposed o depend on ansc ip ion o cen ome ic a-sa elli e DNA and o be egula ed by non-coding RNAs (Blowe , 2016;Ca one e al., 2009;Chan e al., 2012;Du e al., 2010; G en ell e al., 2016;Li e al., 2008;Liu e al., 2015;Nakano e al., 2003;Peze and Uga ko ic ´, 2008;Ros ˇic ´e al., 2014;Topp e al., 2004;Wong e al., 2007;Zhang e al., 2005). In pa icula , he cen ome ic localiza ion and ac i a ion o Au o a B, he ca aly ic subuni o he ch omosomal passenge complex (CPC) equi ed o SAC esponse and e o co ec ion du ing mi osis (Ca mena e al., 2012;San aguida e al., 2011) was shown o be dependen on cen ome ic an- sc ip ion (Blowe , 2016;G en ell e al., 2016). Howe e , whe he cen ome ic ansc ip ion pe sis s du ing mi osis emains deba able. Finally, a ecen s udy using pulse-labeling o nascen ansc ip s and RNA-seq o mi o ic-en iched cell popula ions epo ed ha o e 8000 ansc ip s a e exp essed de no o du ing mi osis and mi o ic exi o p epa e cells o he subsequen in e phase (Palozola e al., 2017). Al hough some le el o ansc ip ion migh s ill ake place du ing mi osis, he expe imen s sup- po ing his conclusion we e based on he analysis o synch onized, ye impu e, mi o ic cell popula- ions, ixed ma e ial and a di e se ange o ansc ip ion inhibi o s wi h imp ecise empo al con ol o ansc ip ional ep ession. Mo eo e , i emains unclea whe he ansc ip ion is equi ed o no - mal mi o ic p og ession and exi . Fo hese easons, we sough o in es iga e he equi emen o de no o ansc ip ion du ing mi osis using di ec li e-cell imaging and a wide ange o ansc ip ion inhibi o s. We ound ha he capaci y o human cells o p og ess, sus ain, exi o die in mi osis is independen o de no o ansc ip ion. In ag eemen , quan i a i e RNA-seq and qPCR analysis ailed o un eil po en ial ansc ip s p oduced de no o du ing mi osis. Finally, we unco e ed ha DNA- in e cala ing agen s, which include well-es ablished ansc ip ion inhibi o s, such as ac inomycin D, educe he in e ac ion o he CPC wi h nucleosomes and signi ican ly comp omise cen ome e s uc- u al in eg i y and unc ion in a ansc ip ion-independen manne . Ou indings aise awa eness abou he use o some ansc ip ion inhibi o s in li ing cells and shed ligh on a long-las ing con o e sy. Resul s Ac inomycin D comp omises spindle assembly checkpoin obus ness in a ansc ip ion-independen manne We easoned ha i de no o ansc ip ion akes place du ing a no mal mi osis, i s ole would be exace ba ed du ing a p olonged mi osis. To es his, we used li e-cell imaging o quan i y he du a- ion o mi osis in human HeLa cells ea ed wi h he mic o ubule depolyme izing d ug nocodazole, which gene a es pe sis en ly una ached kine ocho es ha p e en SAC sa is ac ion. A e 3 h in nocodazole, we di ec ly moni o ed cells ha became commi ed o mi osis, as de e mined by ypical cell ounding, and inhibi ed ansc ip ion wi h he well-es ablished DNA-in e cala ing d ug ac inomy- cin D (Figu e 1A). Consis en wi h p e ious obse a ions in ixed cells (Becke e al., 2010), we ound ha li e cells ea ed wi h nocodazole emained in mi osis o 19.2 ±7.0 h (median ±SD, n = 386 cells), whe eas cells ea ed wi h nocodazole ollowed by ac inomycin D pe sis ed in mi osis o only 16.1 ±5.6 h (n = 301 cells, p<0.0001, Mann-Whi ney Rank Sum Tes , Figu e 1B,C and No ais-C uz e al. eLi e 2018;7:e36898. DOI: h ps://doi.o g/10.7554/eLi e.36898 2 o 27 Resea ch a icle Cell Biology Cyclin B1-Venus Phase Con as A F -00:15 00:00 03:00 DMSO Ac D -00:15 00:00 02:30 -00:15 00:00 Ac D + MG132 03:00 NOC + Ac D NOC NOC + Ac D + MG132 19:00 14:15 24:45 0 100 200 300 400 0.8 0.9 1.0 1.1 Cyclin B1-Venus NOC NOC + MG132 NOC + Ac D NOC + Ac D + MG132 **** ** * NOC NOC + MG132 NOC + Mps1i NOC + Mps1i + MG132 NOC + Ac D NOC + Ac D + MG132 0 20 40 60 80 100 Cell a e (%) Dea h Mi o ic slippage C D 0 1000 2000 3000 4000 5000 Time in mi osis (min) **** **** n.s. **** ** **** ** NOC NOC + MG132 NOC + Mps1i NOC + Mps1i + MG132 NOC + Ac D NOC + Ac D + MG132 E Li e-cell imaging Time T ansc ip ion inhibi ion (Ac D) Mi o ic a es (NOC) 03:15 06:30 03:15 06:30 03:15 06:30 03:15 06:30 00:00 00:00 NOC + Ac D 00:00 00:00 NOC Cyclin B1-Venus Phase Con as Rela i e luo escence in enis y o o al Cyclin B1-Venus Time in mi osis (min) B Figu e 1. Ac inomycin D comp omises SAC esponse. (A) Schema ic ep esen a ion o he expe imen al p o ocol used o s udy he e ec o ac inomycin D (Ac D) on mi o ic cells by li e cell imaging. (B) Selec ed ime ames om phase con as mic oscopy o HeLa cells ea ed wi h nocodazole and ei he DMSO (NOC), Ac D (NOC + Ac D) o Ac D wi h MG132 (NOC + Ac D + MG132). Images we e acqui ed e e y 15 min. A ows highligh examples o con ol (NOC) and Ac D wi h MG132 (NOC + Ac D + MG132) cells en e ing and dying in mi osis a e a p olonged a es . In cells Figu e 1 con inued on nex page No ais-C uz e al. eLi e 2018;7:e36898. DOI: h ps://doi.o g/10.7554/eLi e.36898 3 o 27 Resea ch a icle Cell Biology Video 1). In e es ingly, app oxima ely 90% o nocodazole- ea ed cells died a e a p olonged mi o ic delay, whe eas 54% o he cells ea ed wi h nocodazole and ac inomycin D unde wen mi o ic slippage (Riede and Maia o, 2004) (Figu e 1B,D and Video 1). Thus, ea men wi h ac ino- mycin D comp omises he capaci y o cells o sus ain a p olonged mi o ic delay and in e e es wi h hei a e. The ela ionship be ween he capaci y o sus ain a obus SAC esponse and he subsequen cell a e a e a mi o ic delay has been shown o depend on he kine ics o anaphase-p omo ing complex (APC)-media ed Cyclin B1 deg ada ion, wi h slowe deg ada ion p omo ing mi o ic cell dea h, and as e deg ada ion p omo ing mi o ic slippage (B i o and Riede , 2006;Gascoigne and Taylo , 2008). We he e o e es ed whe he inhibi ion o Cyclin B1 p o eolysis wi h he p o easome inhibi o MG132 could escue he as e exi and di e en a e o cells ea ed wi h nocodazole and ac inomy- cin D. As con ols, we modula ed mi o ic du a ion ei he by inhibi ion o he SAC kinase Mps1 ( o accele a e mi o ic exi ), o inhibi ion o he p o easome wi h MG132 ( o delay mi o ic exi ) a e nocodazole ea men . Acco dingly, cells ea ed wi h nocodazole and MG132 inc eased he mi o ic delay ela i e o cells ea ed only wi h nocodazole ( om 19.2 ±7.0 h o 30.1 ±14.7 h , p<0.0001, Mann-Whi ney Rank Sum Tes , Figu e 1C and Video 1), and caused cell dea h in mi osis in 100% o he cases (Figu e 1D and Video 1). Con e sely, acu e SAC inac i a ion h ough Mps1 inhibi ion a e nocodazole ea men signi ican ly dec eased he mi o ic delay ela i e o nocodazole- ea ed cells ( om 19.2 ±7.0 h o 5.4 ±2.6 h , p<0.0001, Mann-Whi ney Rank Sum Tes , Figu e 1C and Video 1), and caused almos immedia e mi o ic slippage in 100% o he cells (Figu e 1D and Video 1). P o ea- some inhibi ion wi h MG132 e e ed he Mps1 inhibi ion pheno ype, inc easing mi o ic du a ion and swi ching cell a e om mi o ic slippage o dea h (p<0.0001, Mann-Whi ney Rank Sum Tes , Figu e 1C,D and Video 1). Mos impo an , he abili y o ac inomycin D o educe he mi o ic delay and induce slippage in cells ea ed wi h nocodazole was escued a e inhibi ion o p o- easome ac i i y wi h MG132 (p0.01, Mann- Whi ney Rank Sum Tes , Figu e 1C,D and Video 1). O e all, hese expe imen s sugges ha ac inomycin D comp omises SAC esponse and he capaci y o sus ain a p olonged mi o ic delay, in a p o easome-dependen manne . The conclusions abo e a e a odds wi h mod- els in which SAC esponse depends on de no o ansc ip ion o Cyclin B1 du ing mi osis (Mena e al., 2010;Scio ino e al., 2001). To cla i y his issue, we used quan i a i e ime-lapse Figu e 1 con inued ea ed wi h Ac D (NOC + Ac D), a ow highligh s an example o a cell en e ing mi osis and hen exi ing mi osis h ough mi o ic slippage. Scale ba = 10 mm. Time = h :min. (C) Sca e ed plo showing he du a ion o he mi o ic a es o HeLa cells ea ed wi h nocodazole and ei he Mps1 inhibi o (NOC + Mps1 i) o Ac D (NOC + Ac D) wi h o wi hou MG132. The ed line ep esen s he mean and he e o ba s ep esen he s anda d de ia ion om a pool o a leas h ee independen expe imen s (NOC, 19.2 ±7.0 h , n = 386; NOC + MG132, 30.1 ±14.7 h , n = 108; NOC + Mps1 i, 5.4 ±2.6 h , n = 154; NOC + Mps1 i+MG132, 30.9 ±18.1 h , n = 150; NOC + Ac D, 16.1 ±5.6 h , n = 301; NOC + Ac D + MG132, 20.3 ±9.4 h , n = 65; median ±SD; **p0.01, ****p0.0001, Mann-Whi ney Rank Sum Tes ). (D) Cell a e o mi o ic HeLa cells ea ed wi h he same d ugs as in 1C. (E) Selec ed ime ames om phase con as and luo escence mic oscopy o Cyclin B1-Venus HeLa cells ea ed wi h DMSO o Ac D in he p esence o nocodazole. Images we e acqui ed e e y 15 min. Fo Ac D- ea ed cells (NOC + Ac D) a ows highligh a mi o ic cell showing educ ion o Cyclin B1 o e ime. Scale ba = 10 mm. Time = h :min. (F) Cyclin B1 deg ada ion cu es o con ol and Ac D- ea ed Cyclin B1-Venus HeLa cells a e nocodazole ea men wi h o wi hou MG132. Fluo escence in ensi ies we e no malized o he le el a ime = 0. The cu es depic mean Cyclin B1-Venus luo escen in ensi y om all analyzed cells pe condi ion (NOC n = 12; NOC + MG132 n = 10; NOC + Ac D n = 9; NOC + Ac D + MG132 n = 10; om ime = 0 o 26 ime ames), and e o ba s ep esen he s anda d de ia ion (*p0.05, **p0.01, ****p0.0001 ela i e o con ol, Analysis o co a iance). DOI: h ps://doi.o g/10.7554/eLi e.36898.002 Video 1. Phase con as mic oscopy o HeLa cells ea ed wi h DMSO, Mps1 o Ac D a e nocodazole (NOC) ea men wi h (+) o wi hou (-) MG132. Images we e acqui ed e e y 15 min. Time = h:min. DOI: h ps://doi.o g/10.7554/eLi e.36898.003 No ais-C uz e al. eLi e 2018;7:e36898. DOI: h ps://doi.o g/10.7554/eLi e.36898 4 o 27 Resea ch a icle Cell Biology luo escence mic oscopy o moni o he le els and espec i e kine ics o deg ada ion o Cyclin B1 agged a i s endogenous genomic locus wi h Venus (Collin e al., 2013) (Figu e 1E). In ag eemen wi h p e ious epo s, Cyclin B1-Venus luo escence decayed e y slowly in he p esence o nocoda- zole, likely due o esidual APC ac i i y (Figu e 1F) (B i o and Riede , 2006). T ea men o nocoda- zole-a es ed cells wi h ac inomycin D caused a as e decline in Cyclin B1-Venus (p<0.0001, Analysis o Co a iance (ANCOVA), Figu e 1F). In e es ingly, Cyclin B1-Venus le els in nocodazole- ea ed cells emained cons an o se e al hou s a e inhibi ion o he p o easome wi h MG132, ega dless o he p esence o ac inomycin D (p0.05, ANCOVA, Figu e 1F). We concluded ha ac inomycin D comp omises SAC obus ness and he capaci y o p e en APC-media ed Cyclin B1 deg ada ion, independen ly o de no o ansc ip ion. Ac inomycin D comp omises he localiza ion o Au o a B and Mad1 a cen ome es/kine ocho es To de e mine how ac inomycin D comp omises SAC obus ness du ing a p olonged mi osis, we in es iga ed he localiza ion o he SAC- ela ed p o eins Au o a B (a cen ome es) and Mad1 (a una ached kine ocho es). Acco dingly, we added ac inomycin D o 8 h in cells p e- ea ed wi h nocodazole, which was kep h oughou he en i e du a ion o he expe imen . To assis in he iden i- ica ion o mi o ic cells, while assessing Au o a B ac i i y on ch omosomes, we also in es iga ed he phospho yla ion o His one H3 a Se ine 10 [pH3S10; (C osio e al., 2002)]. In con as wi h nocoda- zole- ea men in which all mi o ic cells showed high le els o pH3S10, addi ion o ac inomycin D educed o abolished pH3S10 accumula ion in app oxima ely 17% o he nocodazole- ea ed cells (Figu e 2A,B). Impo an ly, hese cells s ill showed signs o ch omosome condensa ion (as de e - mined by DAPI s aining o DNA) ha we e indis inguishable om neighbou ing cells wi h no mal le - els o pH3S10 (Figu e 2A). Mo eo e , he obse ed educ ion/abolishmen o pH3S10 was no associa ed wi h a p e-apop o ic s a e, as hose cells we e nega i e o a clea ed o m o caspase 3 (Figu e 2A). In line wi h he obse ed educ ion/abolishmen o pH3S10, hese cells also showed abno mally low le els o o al Au o a B and i s ac i e phospho yla ed o m on Th eonine 232 (Yasui e al., 2004). No ewo hy, e en in hose nocodazole- and ac inomycin D- ea ed cells ha showed high le els o pH3S10, Au o a B, including i s ac i e o m, was ound displaced along he ch omosome a ms (Figu e 2A). Simila indings we e ound in non- ans o med RPE1 cells (Fig- u e 2— igu e supplemen 1A–B). Thus, ac inomycin D comp omises no mal Au o a B localiza ion and ac i i y. In o de o ge spa io empo al insigh in o he e ec o ac inomycin D on Au o a B localiza ion, we moni o ed GFP-Au o a B by quan i a i e luo escence li e-cell mic oscopy in nocodazole- ea ed cells. Addi ionally, we u he es ed whe he Au o a B localiza ion was dependen on mi o ic ansla- ion and on i s own kinase ac i i y by inhibi ing p o ein syn hesis wi h cycloheximide and ea ing cells wi h he Au o a B inhibi o ZM447439, espec i ely. We ound ha while Au o a B cen ome ic le els emained cons an o e a 5-h pe iod in cells ea ed ei he wi h nocodazole alone o nocoda- zole and cycloheximide, ac inomycin D caused he displacemen o Au o a B om cen ome es on o ch omosome a ms 2 h a e addi ion o ac inomycin D, leading o a 30% educ ion o cen ome ic luo escence a e 4 h (p<0.0001, ANCOVA, Figu e 2— igu e supplemen 2A,B). Cu iously, Au o a B inhibi ion led o an en ichmen o i s own le els a cen ome es, wi hou any de ec able displace- men on o ch omosome a ms (Figu e 2— igu e supplemen 2A,B). These obse a ions we e con- i med by immuno luo escence analysis in ixed cells (Figu e 2C,D and Figu e 2— igu e supplemen 3A–C). These da a di ec ly demons a e ha ac inomycin D displaces Au o a B om cen ome es. We hen es ed whe he ac inomycin D ea men comp omised he localiza ion o Mad1 a una - ached kine ocho es. We ound ha addi ion o ac inomycin D o 4 h o nocodazole- ea ed cells led o a 45% educ ion o Mad1 a he ou e kine ocho e (Figu e 2C,E). Simila indings ha e p e i- ously been epo ed o he SAC p o eins Bub1 and BubR1 (Becke e al., 2010). Impo an ly, ac i- nomycin D ea men o 4 h did no a ec he kine ocho e localiza ion o ac i e Mps1 (Mps1 pT676; (Jelluma e al., 2008)) (Figu e 2— igu e supplemen 4A,B) o he phospho yla ion o his- one H3 a h eonine 3 (H3T3p), a p oxy o Haspin ac i i y p e iously implica ed in Au o a B ec ui - men o cen ome es (Kelly e al., 2010;Wang e al., 2010) (Figu e 2— igu e supplemen 4C,D). Taken oge he , hese expe imen s show ha ac inomycin D comp omises he no mal localiza ion o No ais-C uz e al. eLi e 2018;7:e36898. DOI: h ps://doi.o g/10.7554/eLi e.36898 5 o 27 Resea ch a icle Cell Biology Au B pT232H3-pS10 me ge w/ DAPI NOC Au B pT232H3-pS10 me ge w/ DAPI To al Au BH3-pS10 me ge w/ DAPI H3-pS10 me ge w/ DAPI DAPI DAPI DAPI DAPI Clea ed Casp 3 NOC + Ac D A ACA ACA Au B pT232 Au B pT232 ACA ACA C Au B pT232H3-pS10ACA me ge w/ DAPI Au B pT232H3-pS10ACA me ge w/ DAPI NOC + Ac D NOC Au B pT232ACA me ge w/ DAPI Mad1Au B pT232ACA me ge w/ DAPI NOC + Ac D NOC Mad1 ACA ACA Mad1 ACA ACA Mad1 B D E NOC NOC + Ac D 0 1 2 3**** NOC NOC + Ac D 0 1 2 3 4 Fuo escence in ensi y Cen ome ic Au B pT232/ACA **** Fuo escence in ensi y Mad1/ACA NOC NOC + Ac D ** 0 5 10 15 20 25 H3-pS10 nega i e mi o ic cells Figu e 2. Ac inomycin D comp omises he localiza ion o Au o a B and Mad1 a cen ome es/kine ocho es. (A) Rep esen a i e immuno luo escence images o HeLa cells ea ed wi h DMSO (NOC) o Ac D o 8 h (NOC + Ac D) a e nocodazole ea men wi h he indica ed an ibodies. In he NOC + Ac D condi ion o ange a ows highligh a mi o ic cell wi h educed le els o phospho yla ed His one H3 (H3–pS10) and de oid o he ac i e o m o Au o a B (Au B pT232). Yellow a ows highligh a mi o ic cell wi h abolished le els o phospho H3 (H3–pS10) and de oid o o al Au o a B. Scale Figu e 2 con inued on nex page No ais-C uz e al. eLi e 2018;7:e36898. DOI: h ps://doi.o g/10.7554/eLi e.36898 6 o 27 Resea ch a icle Cell Biology Au o a B a cen ome es and SAC p o eins o una ached kine ocho es, independen ly o an e ec o e Mps1 and Haspin kinase ac i i y. Ac inomycin D o Au o a B inhibi ion dis u bs he kine ocho e ec ui men o Knl1 and Mad1 Nex , we in es iga ed whe he he educ ion o Mad1 a una ached kine ocho es in he p esence o ac inomycin D was induced by he pe u ba ion o Au o a B localiza ion and ac i i y a cen ome es. To do so, we s a ed by e alua ing he s a us o known Au o a B subs a es wi hin he KMN ne wo k (Welbu n e al., 2010) a e ac inomycin D ea men . Consis en wi h an a enua ion o Au o a B ac i i y a cen ome es, we ound ha addi ion o ac inomycin D o 4 h o nocodazole- ea ed cells led o a 22% and 43% educ ion on he phospho yla ion le els o Knl1 and Dsn1 (a membe o he Mis12 complex), espec i ely (Figu e 3A–D). In e es ingly, we also ound a 29% educ ion in o al Knl1 (Figu e 3E,F), whe eas o al Dsn1, Hec1 (a membe o he Ndc80 complex) and CENP-A we e una ec ed by ac inomycin D ea men (Figu e 3G,H,Figu e 3— igu e supplemen 1A–D). Thus, pe u ba ion o Au o a B cen ome ic localiza ion by ac inomycin D ansla es in o a local educ ion o ac i i y and he consequen dec ease in phospho yla ion o key Au o a B subs a es a he kine o- cho e. Mo eo e , while ac inomycin D ea men o 4 h does no pe u b he no mal localiza ion o CENP-A and membe s o he Ndc80 and Mis12 complex, i comp omises he ec ui men o Knl1 o una ached kine ocho es. Because Knl1 is known o ec ui se e al SAC p o eins (including Mad1) o kine ocho es (Musacchio, 2015a), we es ed whe he Knl1 and Mad1 ec ui men o kine ocho es depends on Au o a B ac i i y a cen ome es. We ound ha , simila o ac inomycin D ea men , Au o a B inhibi- ion in nocodazole- ea ed cells caused a 38% and 28% educ ion o Mad1 and Knl1 a una ached kine ocho es, espec i ely (Figu e 3— igu e supplemen 2A–D). Impo an ly, i has p e iously been shown ha cons i u i e a ge ing o Au o a B o cen ome es by exp essing a Cenp-B-INCENP usion p o ein was able o escue SAC esponse in ac inomycin D- ea ed cells (Becke e al., 2010). O e all, hese da a sugges ha pe u ba ion o Au o a B localiza ion and consequen dec ease in ac i i y a cen ome es is he p ima y e ec o Ac inomycin D ea men and his likely accoun s o he weakened SAC esponse due o a downs eam e ec o e Knl1 and Mad1 ec ui men . Figu e 2 con inued ba = 5 mm. (B) Pe cen age o mi o ic phospho H3-nega i e cells in con ol (NOC) and 8 h ac inomycin D (NOC + Ac D)- ea ed HeLa cells. The ba g aph ep esen s he mean and he s anda d de ia ion om h ee independen expe imen s (NOC 0.0 ±0.0, n = 600; NOC + Ac D, 16.1 ±4.5, n = 600; **p0.01 ela i e o con ol, es ) (C) Rep esen a i e immuno luo escence images o Hela cells ea ed wi h DMSO (NOC) o Ac D o 4 h (NOC + Ac D) a e nocodazole ea men wi h he indica ed an ibodies. Scale ba = 5 mm. 10x magni ica ion o a pai o kine ocho es a e shown in he igh . Dashed ci cle encompasses a single kine ocho e and he a ow indica es he posi ion o cen ome ic Au o a B. Scale ba = 0.5 mm. (D) No malized a io o pAu o a B/ACA luo escence signal a inne cen ome e o NOC and NOC + Ac D. Each do ep esen s an indi idual kine ocho e. The ed line ep esen s he mean o all quan i ied kine ocho es and he e o ba s ep esen he s anda d de ia ion om a pool o a leas wo independen expe imen s. (NOC, 1.00 ±0.48, n = 474; NOC + Ac D, 0.63 ±0.23, n = 309; ****p0.0001 ela i e o con ol, Mann-Whi ney Rank Sum Tes ). (E) No malized a io o Mad1/ACA luo escence signal a ou e kine ocho es o NOC and NOC + Ac D cells. Each do ep esen s an indi idual kine ocho e. The ed line ep esen s he mean o all quan i ied kine ocho es and he e o ba s ep esen he s anda d de ia ion om a pool o a leas wo independen expe imen s. (NOC, 1.00 ±0.39, n = 261; NOC + Ac D, 0.55 ±0.39, n = 258; ****p0.0001 ela i e o con ol, Mann-Whi ney Rank Sum Tes ). DOI: h ps://doi.o g/10.7554/eLi e.36898.004 The ollowing igu e supplemen s a e a ailable o igu e 2: Figu e supplemen 1. Ac inomycin D comp omise Au o a B localiza ion in RPE1 cells. DOI: h ps://doi.o g/10.7554/eLi e.36898.005 Figu e supplemen 2. Ac inomycin D causes Au o a B displacemen om cen ome es on o ch omosome a ms. DOI: h ps://doi.o g/10.7554/eLi e.36898.006 Figu e supplemen 3. Inhibi ion o mi o ic ansla ion o Au o a B kinase ac i i y du ing p ome aphase does no a ec Au o a B localiza ion. DOI: h ps://doi.o g/10.7554/eLi e.36898.007 Figu e supplemen 4. Ac inomycin D does no comp omise pMPS1 localiza ion a kine ocho es and pH3T3 le els. DOI: h ps://doi.o g/10.7554/eLi e.36898.008 No ais-C uz e al. eLi e 2018;7:e36898. DOI: h ps://doi.o g/10.7554/eLi e.36898 7 o 27 Resea ch a icle Cell Biology pKNL1ACA me ge w/ DAPI ACA me ge w/ DAPI NOC + Ac D NOC To al Au B pKNL1 To al Au B A B NOC NOC + Ac D 0.0 0.5 1.0 1.5 2.0 2.5 Fluo escence in ensi y pKNL1/ACA **** DSN1ACA me ge w/ DAPI NOC ACA me ge w/ DAPI NOC + Ac D DSN1 Au B pT232 Au B pT232 G H NOC NOC + Ac D 0.0 0.5 1.0 1.5 2.0 2.5 Fluo escence in ensi y DSN1/ACA n.s. pDSN1ACA me ge w/ DAPI NOC To al Au B ACA me ge w/ DAPI NOC + Ac D pDSN1To al Au B C D NOC NOC + Ac D 0.0 0.5 1.0 1.5 2.0 2.5 Fluo escence in ensi y pDSN1/ACA **** KNL1ACA me ge w/ DAPI ACA me ge w/ DAPI NOC + Ac D NOC KNL1 To al Au B To al Au B E F NOC NOC + Ac D 0.0 0.5 1.0 1.5 2.0 2.5 **** Fluo escence in ensi y KNL1/ACA Figu e 3. Ac inomycin D a ec ec ui men o KNL1 and pDSN1 o kine ocho es. (A) Rep esen a i e immuno luo escence images o HeLa cells ea ed wi h DMSO (NOC) o Ac D o 4 h (NOC + Ac D) a e nocodazole ea men wi h he indica ed an ibodies. Scale ba = 5 mm. (B) No malized a io o pKNL1/ACA luo escence signal a ou e kine ocho es o NOC and NOC + Ac D cells. Each do ep esen s an indi idual kine ocho e. The ed line ep esen s he mean o all quan i ied kine ocho es and he e o ba s ep esen he s anda d de ia ion om a pool o wo echnical eplica es (NOC, Figu e 3 con inued on nex page No ais-C uz e al. eLi e 2018;7:e36898. DOI: h ps://doi.o g/10.7554/eLi e.36898 8 o 27 Resea ch a icle Cell Biology Ac inomycin D does no a ec o al CPC p o ein le els no he le els o mi o ic ansc ip s Wes e n blo analysis e ealed ha Au o a B, as well as he o al p o ein le els o he CPC egula o y subuni s Su i in and Bo ealin emained unchanged ela i e o con ols (including inhibi ion o ans- la ion du ing mi osis wi h cycloheximide o Au o a B inhibi ion wi h ZM447439) a e 4 h ea men wi h ac inomycin D (Figu e 4A–C). To es whe he Au o a B was egula ed by an e ec o ac inomy- cin D o e mi o ic ansc ip ion, we pe o med a compa a i e genome-wide RNA-seq analysis be ween cells ea ed ei he wi h nocodazole alone o nocodazole and ac inomycin D. Impo an ly, only mi o ic cells we e analyzed, as hey we e ob ained by shake-o a e nocodazole ea men and subsequen ly ans e ed o new cul u e lasks be o e addi ion o ac inomycin D (Figu e 4A). Ou quan i a i e ansc ip ome analysis did no e eal any signi ican changes ei he in anno a ed o no el assembled ansc ip s (including all bio ypes) upon ac inomycin D ea men , uling ou di e - en ial gene exp ession a e ansc ip ion inhibi ion du ing mi osis. Impo an ly, we con i med ha Cyclin B1, as well as all key mi o ic egula o s, including Au o a B, we e no de no o ansc ibed e en du ing a p olonged mi osis (Figu e 4D; see Ma e ials and me hods). Due o he low mappabili y o he cen ome e, RNA-seq eads could no be p ope ly aligned o hese egions. Consequen ly, he ole o cen ome ic ansc ip ion, namely o egula o y non-coding RNAs, could no be p ope ly assessed in he p e ious expe imen . Ne e heless, we we e able o di ec ly measu e by qPCR he exp ession o cen ome ic a-sa elli e RNA ela i e o a con ol house- keeping gene (GAPDH) in cells ha had been a es ed in mi osis wi h nocodazole, in he p esence o absence o ac inomycin D. We ound ha ac inomycin D ea men did no educe he le el o cen ome ic a-sa elli e RNA (Figu e 4E,F). Taken oge he , ou esul s demons a e ha inhibi ion o ansc ip ion wi h ac inomycin D du ing a p olonged mi osis caused by pe sis en ly una ached kine - ocho es does no impai no mal ansc ip le els, including cen ome ic a-sa elli e RNA, no he no - mal le els o CPC p o eins. Inhibi ion o ansc ip ion independen ly o DNA-in e cala ion does no a ec spindle assembly checkpoin obus ness no he capaci y o igge mi o ic cell dea h So a , ou esul s indica ed ha ac inomycin D comp omises SAC esponse and Au o a B cen o- me ic localiza ion independen ly o de no o ansc ip ion. The e o e, we easoned ha ac inomycin D could displace Au o a B om cen ome es, no by inhibi ing mi o ic ansc ip ion, bu h ough i s Figu e 3 con inued 1.00 ±0.40, n = 126; NOC + Ac D, 0.79 ±0.35, n = 126; ****p0.0001 ela i e o con ol, es ). (C) Rep esen a i e immuno luo escence images o Hela cells ea ed wi h DMSO (NOC) o Ac D o 4 h (NOC + Ac D) a e nocodazole ea men wi h he indica ed an ibodies. Scale ba = 5 mm. (D) No malized a io o pDNS1/ACA luo escence signal a ou e kine ocho es o NOC and NOC + Ac D cells. Each do ep esen s an indi idual kine ocho e. The ed line ep esen s he mean o all quan i ied kine ocho es and he e o ba s ep esen he s anda d de ia ion om a pool o wo echnical eplica es (NOC, 1.00 ±0.37, n = 116; NOC + Ac D, 0.57 ±0.29, n = 116; ****p0.0001 ela i e o con ol, Mann-Whi ney Rank Sum Tes ). (E) Rep esen a i e immuno luo escence images o HeLa cells ea ed wi h DMSO (NOC) o Ac D o 4 h (NOC + Ac D) a e nocodazole ea men wi h he indica ed an ibodies. Scale ba = 5 mm. (F) No malized a io o KNL1/ACA luo escence signal a ou e kine ocho es o NOC and NOC + Ac D cells. Each do ep esen s an indi idual kine ocho e. The ed line ep esen s he mean o all quan i ied kine ocho es and he e o ba s ep esen he s anda d de ia ion om a pool o wo echnical eplica es (NOC, 1.00 ±0.34, n = 160; NOC + Ac D, 0.71 ±0.34, n = 160; ****p0.0001 ela i e o con ol, Mann-Whi ney Rank Sum Tes ). (G) Rep esen a i e immuno luo escence images o HeLa cells ea ed wi h DMSO (NOC) o Ac D o 4 h (NOC + Ac D) a e nocodazole ea men wi h he indica ed an ibodies. Scale ba = 5 mm. (H) No malized a io o DSN1/ACA luo escence signal a ou e kine ocho es o NOC and NOC + Ac D cells. Each do ep esen s an indi idual kine ocho e. The ed line ep esen s he mean o all quan i ied kine ocho es and he e o ba s ep esen he s anda d de ia ion om a pool o wo echnical eplica es (NOC, 1.00 ±0.34, n = 110; NOC + Ac D, 0.96 ±0.32, n = 110; n.s. p>0.05 ela i e o con ol, es ). DOI: h ps://doi.o g/10.7554/eLi e.36898.009 The ollowing igu e supplemen s a e a ailable o igu e 3: Figu e supplemen 1. Ac inomycin D does no a ec le els o HEC1 and CENP-A (A) Rep esen a i e immuno luo escence images o HeLa cells ea ed wi h DMSO (NOC) o Ac D o 4 h (NOC + Ac D) a e nocodazole ea men wi h he indica ed an ibodies. DOI: h ps://doi.o g/10.7554/eLi e.36898.010 Figu e supplemen 2. Au o a B inhibi ion a ec s ec ui men o Mad1 and KNL1 o kine ocho es. DOI: h ps://doi.o g/10.7554/eLi e.36898.011 No ais-C uz e al. eLi e 2018;7:e36898. DOI: h ps://doi.o g/10.7554/eLi e.36898 9 o 27 Resea ch a icle Cell Biology isola ed 40 min a e nocodazole washou migh ha e de i ed om cells ha had al eady exi ed mi osis and eached ea ly G1 s age whe e ansc ip ion is expec ed o be pe missi e (Hsiung e al., 2016). Thus, he use o synch onized, ye impu e, cell popula ions, as opposed o moni o ing an- sc ip ion inhibi ion only a e cells commi o mi osis by di ec li e-cell imaging, migh accoun o he di e ences obse ed be ween s udies. Ne e heless, i emains possible ha , as cells p og ess h ough mi osis and en e anaphase, de no o ansc ip ion s a s o be pe missi e, despi e i s dis- pensabili y o he comple ion o and exi om mi osis. Consis en wi h a global shu -down o ansc ip ion du ing p ome aphase ( he physiological equi alen o nocodazole-a es ed cells), RNA Polyme ase II is gene ally ound displaced om A 00:00 00:20 01:00 00:00 01:20 02:00 00:00 00:40 01:20 Ac D DMSOT ip olide C D DMSO T ip olide 0 20 40 60 80 100 Cell a e (%) No mal di ision Mul ipola di ision Mi o ic slippage Ci okinesis ailu e Mi o ic dea h Ac D 0 50 100 150 200 250 300 350 NOC washou - AO (min) **** **** n.s. DMSO T ip olide Ac D 3h 2h Time Mi o ic a es (NOC) T ansc ip ion inhibi ion (Ac D o T ip olide) Mi o ic elease (NOC Washou ) Li e-cell imaging NOC washou B Figu e 7. Mi o ic p og ession and cy okinesis a e nocodazole washou does no equi e de no o ansc ip ion. (A) Schema ic ep esen a ion o he expe imen al p o ocol used o s udy he e ec o he ansc ip ion inhibi o s Ac D and ip olide on mi o ic p og ession. (B) Selec ed ime ames om phase con as mic oscopy o HeLa cells ea ed a e he NOC washou wi h DMSO, Ac D o T ip olide. Images we e acqui ed e e y 20 min. Fo con ol (DMSO) and T ip olide (T ip olide), a ows highligh a no mal mi o ic p og ession a e nocodazole washou . Fo Ac D- ea ed cells (Ac D), a ows highligh a cell ha ails o di ide a e elease om nocodazole a es . Scale ba = 10 mm. Time = h :min. (C) Sca e ed plo showing he mi o ic du a ion be ween nocodazole washou and anaphase onse in con ol (DMSO), Ac D and T ip olide ea ed HeLa cells (DMSO, 79.1 ±28.8 min, n = 116; Ac D, 140.5 ±55.7 h , n = 79; T ip olide, 86.2 ±31.9 h , n = 113, median ±SD om a pool o wo echnical eplica es, n.s. p>0.05, ****p0.0001, Mann- Whi ney Rank Sum Tes ). (D) Cell a e o mi o ic HeLa cells ea ed wi h he same d ugs as in 7B. DOI: h ps://doi.o g/10.7554/eLi e.36898.017 The ollowing igu e supplemen is a ailable o igu e 7: Figu e supplemen 1. Ac inomycin D p omo es cy okinesis ailu e. DOI: h ps://doi.o g/10.7554/eLi e.36898.018 No ais-C uz e al. eLi e 2018;7:e36898. DOI: h ps://doi.o g/10.7554/eLi e.36898 16 o 27 Resea ch a icle Cell Biology ch oma in (Hsiung e al., 2016), wi h he no o i- ous excep ion o he cen ome ic egion (Chan e al., 2012). Because ecen s udies ha e sugges ed ha non-coding RNAs associa e wi h cen ome e and kine ocho e p o eins, including CENP-A, CENP-C and Au o a B (Blowe , 2016; Ca one e al., 2009;Du e al., 2010;Fe i e al., 2009;Molina e al., 2017, 2016;Ros ˇic ´e al., 2014;Wong e al., 2007), i is possible ha an- sc ip ion o non-coding RNAs plays a ole in cen- ome e assembly and unc ion. Howe e , whe he his occu s du ing mi osis o du ing G1, when cen ome e assembly akes place in mam- malian cells (Jansen e al., 2007) emains unclea . Cen ome ic ansc ip ion is in ol ed in nucleosome disassembly du ing in e phase (Boege e al., 2003) o acili a e he eplacemen o his one H3 by CENP-A (McKi ick e al., 2004;Tagami e al., 2004). In iguingly, he obse a ion ha he elonga ing o m o RNA Polyme - ase II is p esen a cen ome es du ing mi osis (Chan e al., 2012) lea e open he possibili y ha some le el o local ansc ip ion o non-coding RNAs migh s ill ake place. Howe e , i should be no ed ha a ecen s udy in S. pombe indica ed ha RNA Polyme ase II s alls a cen ome ic DNA and he le el o s alling is di ec ly p opo ional o he le el o cnp1p (cen ome e-speci ic his one H3) nucleosome assembly (Ca ania e al., 2015). While we we e unable o expe imen ally exclude ha non-coding RNAs a e being ansc ibed om he cen ome e du ing a p olonged mi osis, we ailed o de ec any signi ican change in he o al le els o a-sa elli e RNA a e ansc ip ion inhibi ion, in ag eemen wi h p e ious epo s (Liu e al., 2015). Indeed, expe imen al inc ease o a-sa elli e RNA le els we e shown o educe he binding o Sgo1 o nucleosomes (Liu e al., 2015) and led o p oblems in ch omosome seg ega ion (Chan e al., 2017;Zhu e al., 2011), sugges ing ha he le els o a-sa elli e RNA mus be igh ly egula ed o ensu e a ai h ul mi osis. O e all, ou esul s demons a e ha , ega dless o ou capaci y o de ec esidual ansc ip ion ha migh s ill ake place on speci ic ch omosomal loci du ing mi osis, his is no equi ed o human cells o sus ain, p og ess, exi o die in mi osis. Ma e ials and me hods Key esou ces able Reagen ype Designa ion Sou ce o e e ence Iden i ie s Addi ional in o ma ion Cell line (human) HeLa pa en al P o ided by Y. Mimo i-Kiyosue Cell line (human) GFP-Au o a B HeLa P o ided by M. Lampson Cell line (human) Cyclin B1- Venus HeLa P o ided by J. Pines An ibody an i-Au o a B pT232 (Rabbi Polyclonal) Rockland Immuno chemicals Ca #600-401-677 IF: (1:500) An ibody an i-Aim1 (Mouse Monoclonal) BD Biosciences Ca #611083 IF: (1:500) WB: (1:1,000) An ibody an i-Mad1 (Mouse Monoclonal) Me ck Millipo e Ca #MABE867 IF: (1:500) An ibody an i-phospho- His one H3 Se 10 (Mouse Monoclonal) Abcam Ca #ab14955 IF: (1:100,000) Con inued on nex page Video 3. Phase con as mic oscopy o HeLa cells ea ed wi h DMSO, Ac D o T ip olide a e NOC washou . Images we e acqui ed e e y 20 min. Time = h :min. DOI: h ps://doi.o g/10.7554/eLi e.36898.019 No ais-C uz e al. eLi e 2018;7:e36898. DOI: h ps://doi.o g/10.7554/eLi e.36898 17 o 27 Resea ch a icle Cell Biology Con inued Reagen ype Designa ion Sou ce o e e ence Iden i ie s Addi ional in o ma ion An ibody an i-Clea ed Caspase-3 (Rabbi Polyclonal) Cell Signaling Technology Ca #9661 IF: (1:1,000) An ibody an i-Cen ome e an ibody (Human) Fi zge ald Ca #90C-CS1058 IF: (1:500) An ibody an i-phospho- Mps1 Th 676 (Rabbi Polyclonal) a gi om G. Kops IF: (1:2,000) An ibody an i-phospho- His one H3 Th 3 (Mouse Monoclonal) a gi om J. Higgins IF: (1:1,000) An ibody an i-HEC1 (Mouse Monoclonal) a gi om R. Gassmann IF: (1:2,000) An ibody an i-KNL1 (Rabbi Polyclonal) a gi om R. Gassmann IF: (1:500) An ibody an i-phosho DSN1 (Rabbi Polyclonal) a gi om I. Cheeseman IF: (1:1,000) An ibody an i-phospho KNL1 (Rabbi Polyclonal) a gi om I. Cheeseman IF: (1:1,000) An ibody an i-CENPA (Mouse Monoclonal) a gi om L. Jansen IF: (1:200) An ibody an i-DSN1 (Mouse Monoclonal) a gi om A. Musacchio IF: (1:200) An ibody Alexa 488- o 568-o 647 seconda ies In i ogen IF: (1:1,000) An ibody an i-Su i in (Rabbi Polyclonal) No us Biologicals Ca #NB500-201 WB: (1:1,000) An ibody an i-Bo ealin (Rabbi Polyclonal) a gi om R. Gassmann WB: (1:1,000) An ibody an i-a- ubulin clone B-512 (Mouse Monoclonal) Sigma-Ald ich Ca #T5168 WB: (1:5,000) An ibody an i-RNA Pol II S2 (Rabbi Polyclonal) Abcam Ca #ab5095 WB: (1:1,000) An ibody an i-mouse o an i- abbi Jackson Immuno Resea ch WB: (1:5,000) Chemical compound, d ug Nocodazole Sigma-Ald ich Ca #M1404 1 mM Chemical compound, d ug MG132 EMD Millipo e Ca #133407-82-6 5 mM Con inued on nex page No ais-C uz e al. eLi e 2018;7:e36898. DOI: h ps://doi.o g/10.7554/eLi e.36898 18 o 27 Resea ch a icle Cell Biology Con inued Reagen ype Designa ion Sou ce o e e ence Iden i ie s Addi ional in o ma ion Chemical compound, d ug Mps1-IN-1 P o ided by N. G ay 10 mM Chemical compound, d ug RO3306 Roche Ca #SML0569 10 mM Chemical compound, d ug Ac inomycin D Sigma-Ald ich Ca #A9415 8 mM Chemical compound, d ug a-amani in Sigma-Ald ich Ca #A2263 2 mM Chemical compound, d ug T ip olide Sigma-Ald ich Ca #T3652 1 mM Chemical compound, d ug Cycloheximide Sigma-Ald ich Ca #01810 35.5 mM Chemical compound, d ug E hidium B omide Sigma-Ald ich Ca #E8751 25 mM Chemical compound, d ug ZM447439 (Au o a B inhibi o ) Selleckchem.com Ca #S1103 3.3 mM Cell cul u e and eagen s GFP-Au o a B exp essing HeLa cells and Cyclin B1-Venus exp essing HeLa cells we e p o ided by M. Lampson and J. Pines, espec i ely. All cell lines including pa en al HeLa cells we e g own in DMEM o L15 medium supplemen ed wi h 10% FBS (In i ogen) and penicillin/s ep omycin (100 IU/ml and 100 mg/ml) in a 37˚C incuba o wi h 5% CO2. Mic o ubule depolyme iza ion was induced by nocoda- zole (Sigma-Ald ich) a 1 mM o 2–16 h , acco ding o he expe imen . To inhibi he p o easome, induce a me aphase a es , and p e en exi due o a comp omised SAC, cells we e ea ed wi h 5 mM MG132 (EMD Millipo e). Fo Mps1 inhibi ion, cells we e ea ed wi h 10 mM Mps1-IN-1 (p o ided by N. G ay). Fo Au o a B inhibi ion, cells we e ea ed wi h 3.3 mM ZM447439 o 4 h p io o immuno luo escence. G2-en iched ex ac s we e de i ed om cells incuba ed o 16 h wi h he Cdk1 inhibi o RO3306 (Roche) a 10 mM, whe eas mi o ic ex ac s we e ob ained by shake-o upon nocodazole ea men . Fo ansc ip ion inhibi ion, ac inomycin D, a-amani in and ip olide (all om Sigma-Ald ich) we e added o inal concen a ion o 8 mM, 2 mM and 1 mM, espec i ely. To inhibi ansla ion, cells we e ea ed wi h 35.5 mM cycloheximide (Sigma-Ald ich). E hidium b omide (Sigma-Ald ich) was used a inal concen a ion o 25 mM. The cell lines we e ou inely es ed nega- i e o mycoplasma con amina ion. Li e cell imaging Con ol and ansc ip ionally ep essed HeLa cells we e imaged wi h phase-con as mic oscopy (Axi- o e 200M; Ca l Zeiss; 20 objec i e lens; A-Plan Ph1; 0.3 NA) equipped wi h a CCD came a (Cool- SNAP HQ2; Pho ome ics) a 37˚C in DMEM supplemen ed wi h 10% FBS. To de e mine mi o ic iming, images we e cap u ed e e y 15 min o 72 h using he Mic o-Manage 1.3 so wa e (www. mic o-manage .o g). In he washou expe imen , images we e cap u ed e e y 20 min o 24 h . HeLa cells s ably exp essing GFP-Au o a B (LAP-Au o a B) we e imaged wi h an in e ed mic oscope (TE2000U; Nikon; 20 objec i e lens; LWD; 0.4 NA) equipped wi h an elec on-mul iplying cha ge- coupled de ice (CCD) came a (iXonEM+; Ando Technology) a 37˚C in DMEM phenol ed ee medium (In i ogen) supplemen ed wi h 10% FBS and 25 mM HEPES. Ele en 1 mm sepa a ed z-planes co e ing he en i e olume o he mi o ic spindle we e collec ed e e y hou o 6 h using he NIS-Elemen s Viewe so wa e (Nikon). Cyclin B1-Venus we e imaged e e y 15 min o 7 h using an IN Cell Analyze 2000 mic oscope (GE Heal hca e) a 37˚C in phenol ed DMEM ee medium sup- plemen ed wi h 10% FBS. The du a ion o all d ug ea men s in li e-cell expe imen s a e schema i- cally illus a ed in each espec i e igu e. All images we e analyzed wi h open sou ce image analysis so wa e ImageJ and cell p o ile . No ais-C uz e al. eLi e 2018;7:e36898. DOI: h ps://doi.o g/10.7554/eLi e.36898 19 o 27 Resea ch a icle Cell Biology Immuno luo escence mic oscopy HeLa cells we e ixed wi h 4% pa a o maldehyde o 10 min and subsequen ly ex ac ed in 0.3% T i- on X-100 in 1 x PBS o 10 min. A e sho washes in PBS wi h 0.1% T i on X-100 and blocking wi h 10% FBS in PBS wi h 0.1% T i on X-100, all p ima y an ibodies we e incuba ed a 4˚C o e nigh . Then, he cells we e washed wi h PBS con aining 0.1% T i on X-100 and incuba ed wi h he espec- i e seconda y an ibodies o 1 h a oom empe a u e. DNA was coun e s ained wi h DAPI (1 mg/ ml; Sigma-Ald ich) be o e co e slips we e moun ed in 90% glyce ol +10% T is, pH 8.5,+0.5% N-p o- pyl galla e on glass slides. Rabbi an i-Au o a B pT232 (1:500; Rockland Immunochemicals Inc.), mouse an i-Aim1 (1:500; BD Biosciences), mouse an i-Mad1 (1:500; Me ck Millipo e), mouse an i- phospho-His one H3 Se 10 (1:100,000; Abcam), abbi an i-Clea ed Caspase-3 (1:1,000; Cell Signal- ing Technology), human an i-cen ome e an ibody (ACA; 1:500, Fi zge ald Indus ies In e na ional), abbi an i-phospho-Mps1 Th 676 (1:2000, a gi om G. Kops), mouse an i-phospho-His one H3 Th 3 (1:1000, a gi om J. Higgins), mouse an i-HEC1 (1:2000, a gi om R. Gassmann), abbi an i- KNL1 (1:500, a gi om R. Gassmann), abbi an i-phosho-DSN1 and an i-phospho-KNL1 (1:1000, a gi om I. Cheeseman), mouse an i-CENPA (1:200, a gi om L. Jansen) and mouse DSN1 (1:200, a gi om A. Musacchio) we e used as p ima y an ibodies, and Alexa Fluo 488, 568, and 647 (In i o- gen) we e used as seconda y an ibodies (1:1,000). Images we e acqui ed on an AxioImage Z1 (63, Plan oil di e en ial in e e ence con as objec i e lens, 1.4 NA; all om Ca l Zeiss) equipped wi h a cha ge-coupled de ice (CCD) came a (ORCA-R2; Hamama su Pho onics) using he Zen so wa e (Ca l Zeiss) and blind decon ol ed using Au oquan X (Media Cybe ne ics). Fo y-one 0.2 mm sepa- a ed z-planes co e ing he en i e mi o ic cell we e collec ed. Images we e analyzed in ImageJ, p oc- essed in Pho oshop CS4 (Adobe) and ep esen ed wi h a maximum in ensi y p ojec ion o a decon ol ed z s ack. Fluo escence quan i ica ion Fo quan i a i e measu emen s, all images compa ed we e acqui ed using iden ical acquisi ion se - ings. Image analysis was pe o med using ImageJ. B ie ly, indi idual kine ocho es we e iden i ied by ACA s aining and ma ked by a egion o in e es (ROI). The a e age luo escence in ensi y o signals (pixel g ay le els) o pAu o a B a he inne cen ome e and Mad1 a kine ocho es we e measu ed on he ocused z plan, and he backg ound luo escence was measu ed ou side he ROI and sub- ac ed. Fluo escence in ensi y measu emen s we e no malized o he ACA signals. The esul s a e epo ed a e no malized o ela i e luo escence le els in con ol samples. App oxima ely 1,300 KT pai s om 140 cells we e analyzed o pAu o a B and o Mad1, app oxima ely 500 KT pai s om 50 cells we e analyzed. The mean luo escence in ensi y o LAP-Au o a B signals (pixel g ay le els) om he li e cell imaging was measu ed on he sum p ojec ion and he backg ound luo escence was measu ed ou side he ROI and sub ac ed. Fluo escence in ensi ies we e no malized o he le el a ime = 0 and ep esen ed as a unc ion o ime. App oxima ely eigh cells we e analyzed pe condi ion. Wes e n blo HeLa cells we e essuspended and lysed in NP-40 lysis bu e (20 mM HEPES-KOH pH 7.9, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 0.5% ( / ) NP-40, 20% ( / ) Glyce ol, 2 mM DTT, 1 mM PMSF) supplemen ed wi h p o ease and phospha ase inhibi o s. The p o ein concen a ion was measu ed using he B ad o d p o ein assay and 50 mg o p o eins we e esuspended in P o ein Loading Bu e . P o eins we e sepa a ed in o a 4–20% SDS-PAGE and ans e ed o a ni ocellulose Hybond-C memb ane using an iBlo Gel T ans e De ice (The mo Scien i ic). The memb anes we e blocked wi h 5% milk in PBS wi h 0.1% Tween-20 (PBST) a oom empe a u e du ing 1 h , and all p ima y an ibodies we e incuba ed a 4˚C o e nigh . A e i e washes in PBST, he memb anes we e incu- ba ed wi h he seconda y an ibodies du ing 1 h a oom empe a u e. The memb anes we e washed in he same condi ions han p e iously and he de ec ion was pe o med wi h Cla i y Wes e n ECL Subs a e (Bio-Rad). The ollowing an ibodies we e used o wes e n blo : abbi an i-Su i in (1:1000; No us Biologicals), mouse an i-Aim1 (1:1000; BD Biosciences), abbi an i-Bo ealin (1:1000; p o ided by R. Gassmann), mouse an i-a- ubulin (clone B-512, 1:5,000; Sigma-Ald ich) and abbi RNA Pol II S2 (1:1000; Abcam) we e used as p ima y an ibodies, and an i– abbi and an i–mouse No ais-C uz e al. eLi e 2018;7:e36898. DOI: h ps://doi.o g/10.7554/eLi e.36898 20 o 27 Resea ch a icle Cell Biology an ibodies we e used as seconda y an ibodies (1:5,000; Jackson ImmunoResea ch Labo a o ies, Inc.). RNA ex ac ion and ansc ip ion analysis by RT-qPCR To al RNA was isola ed om in e phase and mi o ic HeLa cells (shake-o ) using izol (Li e echnolo- gies). Re e se ansc ip ase eac ion was pe o med wi h 500 ng o o al RNA wi h isc ip (Bio- ad) using he andom p ime s and oligo(dTs) supplied, ollowing he manu ac u e ’s ins uc ions. All p ime sequences a e in Supplemen al Ma e ial. Fo each analysis, gapdh was used o no maliza- ion. RT-qPCRs we e pe o med in he iCycle iQ5 Real-Time PCR De ec ion Sys em (Bio-Rad Labo- a o ies). The da a ob ained we e analyzed using he Bio-Rad iQ5 Op ical Sys em So wa e 2.1 (BioRad Labo a o ies). The ampli ied p oduc s we e addi ionally analyzed on aga ose gel elec opho esis. RNA lib a y p epa a ion o RNAome sequencing Quan i y and o al RNA in eg i y was checked ollowing isola ion using an Agilen Technologies 2100 Bioanalyze . Sample wi h RNA In eg i y Numbe (RIN) alue g ea e han eigh we e selec ed. The RNA samples we e p epa ed as desc ibed be o e in (De ks e al., 2015) wi h he ollowing modi ica ions. Fi e mic og ams o o al RNA we e deple ed acco ding o he Illumina Ribo-ze o magne ic p o ocol (www.illumina.com). The Ribo-ze o- ea ed RNA was pu i ied using Agencou RNAClean XP Beads. RNA was agmen ed on a Co a is S220. One mic oli e o he shea ed RNA deple ed RNA was loaded on an Agilen Technologies 21000 Bioanalyze RNA Pico chip o de e - mine success ul ibosomal RNA deple ion. The shea ed RNA sample was concen a ed in a The mo scien i ic SPD100 speed ac o 5 ml and a sequencing lib a y was p epa ed acco ding o he Illumina T uSeq Small RNA p o ocol (www.illumina.com). In sho , adap e s a e subsequen ly liga ed o he 3’end and he 5’end o he RNA. The RNA is e e se ansc ibed, ampli ied by PCR and un on SDS- PAGE gel. RNA agmen s be ween 140 and500 bp a e cu ou o he gel and pu i ied. One mic oli- e was loaded on an Agilen Technologies 2100 Bioanalyze using a DNA 1000 assay o de e mine he lib a y concen a ion and quali y. B idge ampli ica ion and sequencing by syn hesis Clus e gene a ion was pe o med acco ding o he Illumina T uSeq SR Clus e ki 2 (cBo ) Reagen s P epa a ion Guide (www.illumina.com). B ie ly, six RNAome lib a ies we e pooled oge he o ge a s ock o 2 nM. Fi e mic oli e o he 2 nM s ock was dena u a ed wi h NaOH, dilu ed o 11 pM and hyb idized on o he lowcell. The hyb idized p oduc s we e sequen ially ampli ied, linea ized and end-blocked acco ding o he Illumina Single Read Mul iplex Sequencing use guide. A e hyb idiza- ion o he sequencing p ime , sequencing-by-syn hesis was pe o med using he HiSeq 2000 wi h a 36-cycle p o ocol. The sequenced agmen s we e dena u a ed wi h NaOH using he HiSeq 2000 and he index-p ime was hyb idized on o he agmen s. The index was sequenced wi h a six-cycle p o ocol. Bioin o ma ics analysis Raw FASTQ iles we e aligned o he human genome (NCBI/build37.1) and anno a ed using Gen- code e sion 25. De no o ansc ip ome assembly was pe o med using Gencode anno a ion and he S ingTie assemble (Pe ea e al., 2015). Mapping o sequence eads was pe o med using STAR aligne and ansc ip de ec ion and quan i ica ion was pe o med using Cu links. Gene/ an- sc ip le els we e compa ed be ween con ol and ac inomycin D- ea ed samples using he ballgown p og am (F azee e al., 2014). Da a deposi ion P ocessed RNA-seq ha e been deposi ed and can be consul ed a A ayExp ess unde accession numbe E-MTAB-6661 (h p://www.ebi.ac.uk/ g/anno a e/). CPC-CTM exp ession and pu i ica ion Full leng h ( .l.) Su i in was cloned as a 3C-clea able His-GFP agged p o ein in a pRSET ec o (The mo Fishe Scien i ic), .l. Bo ealin was cloned as a TEV-clea able His- agged p o ein in a pETM No ais-C uz e al. eLi e 2018;7:e36898. DOI: h ps://doi.o g/10.7554/eLi e.36898 21 o 27 Resea ch a icle Cell Biology ec o (gi om C. Romie , IGBMC, S asbou g), and INCENP 1-58 was cloned as an un agged p o- ein in a pMCNcs ec o . The complex o .l. Su i in, .l. Bo ealin and INCENP 1-58 was p epa ed by co-exp essing he subuni s in E. Coli s ain BL21 pLysS wi h an o e nigh induc ion a 18˚C. Cells we e lysed in lysis bu e con aining 25 mM Hepes pH 7.5, 500 mM NaCl, 25 mM Imidazole, 2 mM b-me cap oe hanol (Bme). The complex was pu i ied by a ini y ch oma og aphy using a HisT ap HP column (GE Heal hca e). The p o ein-bound column was washed wi h lysis bu e , ollowed by 25 mM Hepes pH 7.5, 1 M NaCl, 50 mM KCl, 10 mM MgCl, 25 mM Imidazole, 2 mM ATP, 2 mM Bme and p o eins we e elu ed using 25 mM Hepes pH 7.5, 500 mM NaCl, 500 mM Imidazole, 2 mM Bme. Tags we e clea ed du ing an o e nigh incuba ion wi h 3C and TEV while dialysing agains 25 mM Hepes pH 7.5, 150 mM NaCl, 4 mM di hio h ei ol (DTT) a 4˚C. The complex was u he pu i- ied by a ca ion exchange ch oma og aphy (HiT ap SP, GE Heal hca e) ollowed by gel il a ion using a Supe dex 200 inc ease 10/300 column (GE Heal hca e) equilib a ed wi h 25 mM Hepes pH 8, 200 mM NaCl, 4 mM DTT. Exp ession and pu i ica ion o ecombinan his ones and e olding o his one oc ame s Human H2A and H2B and Xenopus lae is H3 and H4 we e pu i ied as desc ibed be o e (Luge e al., 1999, Me hods Mol Biol) wi h mino changes. LB media was used ins ead o 2X TY-AC media o exp ession o H2A, H2B and H3 in E. coli BL21 (DE3) pLysS cells and H4 in E. coli BL21 cells. The p epa a ion o inclusion bodies was pe o med using a Dounce glass/glass homogenize . A e solubilisa ion o he inclusion bodies and he h ee-s ep dialysis agains U ea dialysis bu e (7 M U ea, 100 mM NaCl, 10 mM T is pH 8, 1 mM EDTA, 5 mM Bme), he sample was applied o a HiT ap Q anion exchange column and hen o a HiT ap SP ca ion exchange column (GE Heal hca e). The his ones we e elu ed om he HiT ap SP column using a linea g adien om 100 mM o 1 M NaCl in 7 M U ea, 10 mM T is pH 8, 1 mM EDTA and 1 mM DTT. A e he ion exchange s ep, pu i- ied ecombinan his ones we e dialyzed agains wa e wi h 5 mM Bme, lyophilized and s o ed a 80˚C. Re olding o his one oc ame s was pe o med as p e iously desc ibed (Luge e al., 1999, Me hods Mol Biol). B ie ly, lyophilized co e his ones we e esuspended in un olding bu e (7M Gua- nidine HCl, 20 mM T is pH 7.5, 10 mM DTT) and mixed o equimola a ios. The his one mix was hen dialyzed agains h ee changes o 500 ml e olding bu e (10 mM T is pH 8, 2M NaCl, 1 mM EDTA, 5 mM Bme) and he oc ame s we e pu i ied by size exclusion ch oma og aphy using a Supe - dex 200 inc ease 10/300 column (GE Heal hca e) equilib a ed wi h e olding bu e . Pooled ac ions wi h equimola a ios o his ones we e s o ed a 80˚C. Nucleosome co e pa icle econs i u ion and d ug ea men The 147 bp 601 Widom posi ioning sequence was ampli ied om a pBS-601Widom ec o wi h 5’ IR700 labelled p ime s. Mononucleosome econs i u ion was ca ied ou using he sal g adien dialy- sis me hod (Luge e al., 1999, Me hods Mol Biol). His one oc ame s we e added o DNA o a 0.9 mola a io o oc ame o DNA a e adjus ing he sal concen a ion o 2 M NaCl. The mix u e was hen dialysed agains TE bu e (10 mM T is pH 8, 1 mM EDTA, 50 mM NaCl) by g adually dec eas- ing he ionic s eng h om 2 M o 50 mM NaCl o e a pe iod o 20 h using a pe is al ic pump. Recons i u ed NCPs we e ea ed wi h DMSO, 80 mm Ac inomycin D, 10 mm T ip olide o 25 mm E hidium B omide o 1 h a 4˚C be o e incuba ing hem wi h he CPC-CTM ecombinan p o ein. Elec opho e ic mobili y shi assay Elec opho e ic mobili y assay (EMSA) was used o de ec speci ic in e ac ion be ween CPC and he nucleosome co e pa icles (NCPs). 25 and 50 nM ecombinan CPC-CTM was added o 5 nM IR700- labelled NCPs in eac ion bu e (25 mM Hepes pH 7.5, 100 mM NaCl, 1 mM DTT, 10% Glyce ol). Reac ions we e incuba ed 30 min a 4˚C and esol ed in a 0.5% aga ose gel in 1X T is-CAPS bu e (60 mM T is, 40 mM CAPS pH 9.3). The luo escen bound and unbound NCPs we e de ec ed wi h Odyssey CLx In a ed Imaging Sys em (LI-COR Biosciences). The a io o CPC-bound NCPs o ee NCPs was quan i ied using he Image S udio so wa e (LI-COR Biosciences). Signal a he 0 nM CPC lane was sub ac ed om all alues o accoun o any e ec on NCPs des abiliza ion upon d ug ea men (E B - ea ed nucleosomes showed a ain smea abo e he unbound-NCP band indica ing a sligh NCP des abiliza ion upon E B ea men ). No ais-C uz e al. eLi e 2018;7:e36898. DOI: h ps://doi.o g/10.7554/eLi e.36898 22 o 27 Resea ch a icle Cell Biology S a is ical analysis Fo s a is ical analysis, we i s pe o med a p opo ion es o unpai ed S uden ’s - es o con i m using p alues ha we ha e sampled a su icien numbe o cells o kine ocho es o concluding on he epo ed di e ences. Second, we measu ed s anda d e o o he mean (SEM) ac oss expe imen- al epea s o ac oss cells and con i med ha he epo ed di e ences we e no only based on di e - ences in mean alues, bu also he sp ead o he mean alues be ween expe imen s, cells o kine ocho es. SD alues we e ob ained ac oss expe imen s o cells as indica ed in he igu e legend. S a is ical analysis was pe o med using G aphpad P ism e sion 5. p-Values we e calcula ed wi h a S uden ’s es . Fo da a ha did no ollow a no mal dis ibu ion, s a is ical analysis was pe o med using a Mann-Whi ney Rank Sum es . Acknowledgemen s We hank Jona han Pines (ICR, London, UK), Ben Black and Michael Lampson (Uni e si y o Pennsyl- ania, Pennsyl ania, USA) o p o iding he cyclin B1- enus and LAP-Au o a B HeLa cells, espec- i ely, Re o Gassmann (i3S, Po o, Po ugal), Iain Cheeseman (MIT, Camb idge, USA), And ea Musacchio (MIP, Do mund, Ge many), Jona han Higgins (Ins i u e o Cell and Molecula Bioscien- ces, Newcas le, UK), La s Jansen (IGC, Oei as, Po ugal) and Gee Kops (Hub ech Ins i u e, U ech , The Ne he lands) o he gene ous gi o an ibodies, Na hanael G ay (Ha a d Uni e si y, USA) o he gi o Mps1-IN-1, Paula Sampaio (i3S, Po o, Po ugal) and And e´ Maia (i3S, Po o, Po ugal) o assis ance wi h mic oscopy, Pa icia Oli ei a and Ped o Fe ei a om Bioin 2Bio o bioin o ma ics and da a analysis, and Be na d O o he c i ical eading o he manusc ip and help ul sugges ions. We also hank Jose´ Sil a o inspi ing discussions and CID Lab membe s o eedback du ing he cou se o his p ojec . The au ho s decla e no con lic ing inancial in e es s. MC holds a doc o al el- lowship (SFRH/BD/117063/2016) and CF has an In es iga o s a ing g an (IF/00765/2014) om Fun- dac¸a ˜o pa a a Cieˆncia e a Tecnologia (FCT) o Po ugal. This p ojec has been unded by No e-01– 0145-FEDER-000029 and No e-07–0124-FEDER-000003 om No e 2020 and EXPL/IF/00765/2014/ CP1241/CT0003 om FCT. Wo k in he labo a o y o HM is unded by he Eu opean Resea ch Coun- cil (ERC) unde he Eu opean Union’s Ho izon 2020 esea ch and inno a ion p og amme (g an ag eemen No 681443) and FLAD Li e Science 2020. Addi ional in o ma ion Funding Funde G an e e ence numbe Au ho Eu opean Regional De elop- men Fund COMPETE 2020: No e-01- 0145-FEDER-000029 C is ina Fe a ´s Eu opean Regional De elop- men Fund COMPETE 2020: No e-07- 0124-FEDER-000003 C is ina Fe a ´s Fundac¸a ˜o pa a a Cie ˆncia e a Tecnologia EXPL/IF/00765/2014/ CP1241/CT0003 C is ina Fe a ´s Fundac¸a ˜o pa a a Cie ˆncia e a Tecnologia FCT In es iga o g an IF/ 00765/2014 C is ina Fe a ´s Fundac¸a ˜o pa a a Cie ˆncia e a Tecnologia FCT PhD g an SFRH/BD/ 117063/2016 Ma co No ais-C uz Eu opean Resea ch Council Ho izon 2020 esea ch and inno a ion p og amme: 681443 Helde Maia o Fundac¸a ˜o Luso-Ame icana pa a o Desen ol imen o FLAD Li e Science 2020 Helde Maia o The unde s had no ole in s udy design, da a collec ion and in e p e a ion, o he decision o submi he wo k o publica ion. No ais-C uz e al. eLi e 2018;7:e36898. DOI: h ps://doi.o g/10.7554/eLi e.36898 23 o 27 Resea ch a icle Cell Biology Au ho con ibu ions Ma co No ais-C uz, In es iga ion, Me hodology, W i ing—o iginal d a ; Ma ia Alba Abad, In es iga- ion; Wil ed FJ an IJcken, Me hodology; Niels Galja , A A ockia Jeyap akash, In es iga ion, Me h- odology; Helde Maia o, Concep ualiza ion, Resou ces, Supe ision, Funding acquisi ion, In es iga ion, W i ing— e iew and edi ing; C is ina Fe a´s, Concep ualiza ion, Fo mal analysis, Supe ision, Funding acquisi ion, In es iga ion, Me hodology, W i ing—o iginal d a , W i ing— e iew and edi ing Au ho ORCIDs Wil ed FJ an IJcken h p://o cid.o g/0000-0002-0421-8301 Helde Maia o h ps://o cid.o g/0000-0002-6200-9997 C is ina Fe a´s h ps://o cid.o g/0000-0003-1134-7387 Decision le e and Au ho esponse Decision le e h ps://doi.o g/10.7554/eLi e.36898.024 Au ho esponse h ps://doi.o g/10.7554/eLi e.36898.025 Addi ional iles Supplemen a y iles .T anspa en epo ing o m DOI: h ps://doi.o g/10.7554/eLi e.36898.020 Da a a ailabili y P ocessed RNA-seq ha e been deposi ed and can be consul ed a h ps://www.ebi.ac.uk/a ayex- p ess/expe imen s/E-MTAB-6661 The ollowing da ase was gene a ed: Au ho (s) Yea Da ase i le Da ase URL Da abase, license, and accessibili y in o ma ion Fe a ´s C 2018 RNA seq Mi o ic cells h ps://www.ebi.ac.uk/a - ayexp ess/expe imen s/ E-MTAB-6661 Publicly a ailable a A ayExp ess (accession no. 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