Tracking the evolution of epialleles during neural differentiation and brain development: D- aspartate oxidase as a model gene

Full Te ms & Condi ions o access and use can be ound a h p://www. and online.com/ac ion/jou nalIn o ma ion?jou nalCode=kepi20 Download by: [Tampe e Uni e si y] Da e: 03 Decembe 2017, A : 23:01 Epigene ics ISSN: 1559-2294 (P in ) 1559-2308 (Online) Jou nal homepage: h p://www. and online.com/loi/kepi20 T acking he e olu ion o epialleles du ing neu al di e en ia ion and b ain de elopmen : D- Aspa a e oxidase as a model gene E manno Flo io, Simona Kelle , Lo ena Co e i, O nella A ini o, Gio anni Scala, F ancesco E ico, Annalisa Fico, F ancesca Boscia, Ma ia Josè Sisalli, Ma alda Gio anna Reccia, Genna o Miele, An onella Mon icelli, An onella Sco ziello, F ancesca Lembo, Luca Colucci-D'Ama o, Gab iella Minchio i, Vi o io En ico A edimen o, Alessand o Usiello, Se gio Cocozza & Lo enzo Chia io i To ci e his a icle: E manno Flo io, Simona Kelle , Lo ena Co e i, O nella A ini o, Gio anni Scala, F ancesco E ico, Annalisa Fico, F ancesca Boscia, Ma ia Josè Sisalli, Ma alda Gio anna Reccia, Genna o Miele, An onella Mon icelli, An onella Sco ziello, F ancesca Lembo, Luca Colucci- D'Ama o, Gab iella Minchio i, Vi o io En ico A edimen o, Alessand o Usiello, Se gio Cocozza & Lo enzo Chia io i (2017) T acking he e olu ion o epialleles du ing neu al di e en ia ion and b ain de elopmen : D-Aspa a e oxidase as a model gene, Epigene ics, 12:1, 41-54, DOI: 10.1080/15592294.2016.1260211 To link o his a icle: h ps://doi.o g/10.1080/15592294.2016.1260211 © 2017 The Au ho (s). Published wi h license by Taylo & F ancis G oup, LLC© E manno Flo io, Simona Kelle , Lo ena Co e i, O nella A ini o, Gio anni Scala, F ancesco E ico, Annalisa Fico, F ancesca Boscia , Ma ia José Sisalli, Ma alda Gio anna Reccia, Genna o Miele, An onella Mon icelli, An onella Sco ziello, F ancesca Lembo, Luca Colucci-D'Ama o, Gab iella Minchio i, Vi o io En ico A edimen o, Alessand o Usiello, Se gio Cocozza, and Lo enzo Chia io i View supplemen a y ma e ial Accep ed au ho e sion pos ed online: 18 No 2016. Published online: 18 No 2016. Submi you a icle o his jou nal A icle iews: 662 View ela ed a icles View C ossma k da a RESEARCH PAPER T acking he e olu ion o epialleles du ing neu al di e en ia ion and b ain de elopmen : D-Aspa a e oxidase as a model gene E manno Flo io a , b , Simona Kelle a , b , Lo ena Co e i a , b , O nella A fini o a , b , Gio anni Scala c , F ancesco E ico a , d , Annalisa Fico e , F ancesca Boscia , Ma ia Jos e Sisalli , Ma alda Gio anna Reccia g , Genna o Miele c , An onella Mon icelli b , An onella Sco ziello , F ancesca Lembo h , Luca Colucci-D’Ama o g , Gab iella Minchio i e , Vi o io En ico A edimen o a , b , Alessand o Usiello d , g , Se gio Cocozza a , and Lo enzo Chia io i a , b a Dipa imen o di Medicina Molecola e e Bio ecnologie Mediche, Uni e si  a degli S udi di Napoli ‘Fede ico II’, Naples, I aly; b Is i u o di Endoc inologia ed Oncologia Spe imen ale, IEOS, Consiglio Nazionale delle Rice che, Naples, I aly; c Dipa imen o di Fisica, Uni e si  a degli S udi di Napoli “Fede ico II”and Is i u o Nazionale di Fisica Nuclea e, Sezione di Napoli, Naples, I aly; d CEINGE Bio ecnologie A anza e, Naples, I aly; e Ins i u e o Gene ics and Biophysics ‘A. Buzza i-T a e so’, Consiglio Nazionale delle Rice che, Naples, I aly; Depa men o Neu oscience, Rep oduc i e and Den is y Sciences, Uni e si  a degli S udi di Napoli ‘Fede ico II’, Naples, I aly; g Depa men o En i onmen al, Biological and Pha maceu ical Science and Technologies, Second Uni e si y o Naples, Case a, I aly; h Dipa imen o di Fa macia, Uni e si  a degli S udi di Napoli ‘Fede ico II’, Naples, I aly ARTICLE HISTORY Recei ed 26 Augus 2016 Re ised 31 Oc obe 2016 Accep ed 8 No embe 2016 ABSTRACT We pe o med ul a-deep me hyla ion analysis a single molecule le el o he p omo e egion o de elopmen ally egula ed D-Aspa a e oxidase (Ddo), as a model gene, du ing b ain de elopmen and emb yonic s em cell neu al di e en ia ion. Single molecule me hyla ion analysis enabled us o es ablish he e ec i e epiallele composi ion wi hin mixed o pu e b ain cell popula ions. In his amewo k, an epiallele is defined as a specific combina ion o me hyla ed CpG wi hin Ddo locus and can ep esen he epigene ic haplo ype e ealing a cell- o-cell me hyla ion he e ogenei y. Using his app oach, we ound a high deg ee o polymo phism o me hyla ed alleles (epipolymo phism) e ol ing in a ema kably conse ed ashion du ing b ain de elopmen . The di e en se s o epialleles ma k s age, b ain a eas, and cell ype and un a el he possible ole o specific CpGs in a o ing o inhibi ing local me hyla ion. Undi e en ia ed emb yonic s em cells showed non-o ganized dis ibu ion o epialleles ha appa en ly o igina ed by s ochas ic me hyla ion e en s on indi idual CpGs. Upon neu al di e en ia ion, despi e de ec ing no changes in a e age me hyla ion, we obse ed ha he epiallele dis ibu ion was p o oundly di e en , g adually shi ing owa d o ganized pa e ns specific o he glial o neu onal cell ypes. Ou findings p o ide a deep iew o gene me hyla ion he e ogenei y in b ain cell popula ions p omising o u nish inno a i e ways o un a el mechanisms unde lying me hyla ion pa e ns gene a ion and al e a ion in b ain diseases. KEYWORDS B ain DNA me hyla ion; b ain cells; epipolymo phism; epialleles; epigene ic dynamics In oduc ion Epigene ic p ofiles a e sculp ed du ing de elopmen ; in pa - icula , he DNA me hyla ion landscape o mammalian b ain cells is dynamically econfigu ed h ough de elop- men . 1-3 Such econfigu a ion occu s a b ain specific genes p e alen ly du ing he la e s ages o emb yogenesis and ea ly pos -na al de elopmen al pe iod, leading o c i ical changes in he gene exp ession p og am ha , once he p o- cess comes o an end, is hough o be main ained h ough- ou li e. A p og essi e shaping o DNA me hyla ion pa e ns occu s in a egula ed manne , bo h a CpG and CpH si es, du ing de elopmen and fi s weeks o yea s o li e, possibly p o iding an epigene ic memo y specific o each ype o b ain cells. 3 Such phenomenon, c ucial o he comple ion o emb yonic de elopmen , is he esul o a dynamic in e play be ween DNA me hyla ion and deme h- yla ion e en s assis ed by di e en DNA me hyl ans e ases (DNMT1, DNMT3a, and DNMT3b), which may con e cy osine o me hylcy osine, 4-7 o by Ten-11 ansloca ion enzymes (TETs), which p omo e 5mC deme hyla ion. 8 A he genomic le el, as a esul o hese p ocesses, each ype o b ain cells acqui es a cell ype-specific genomic DNA me hyla ion landscape ha may p o ide an ‘iden i y ca d’ o di e en b ain cells and go e n and s abilize an elec ed gene exp ession p og am. A ecen s udy 9 showed ha di - e en ypes o glial cells and neu ons display e y dis inc- i e me hyla ion signa u es, sugges ing ha he epigenomic landscape eflec s neu onal di e si y. App op ia e pa e ns o DNA me hyla ion in he b ain play an impo an ole in neu ode elopmen and neu opsychia ic condi ions, 10-20 CONTACT Lo enzo Chia io i [email p o ec ed] Dipa imen o di Medicina Molecola e e Bio ecnologie Mediche, Uni e si  a degli S udi di Napoli ‘Fede ico II’, Via S. Pansini, 5, 80131 Naples, I aly; Alessand o Usiello [email p o ec ed] Supplemen al da a o his a icle can be accessed on he publishe ’s websi e. Published wi h license by Taylo & F ancis G oup, LLC © E manno Flo io, Simona Kelle , Lo ena Co e i, O nella A fini o, Gio anni Scala, F ancesco E ico, Annalisa Fico, F ancesca Boscia , Ma ia Jos e Sisalli, Ma alda Gio anna Reccia, Genna o Miele, An onella Mon icelli, An onella Sco ziello, F ancesca Lembo, Luca Colucci-D’Ama o, Gab iella Minchio i, Vi o io En ico A edimen o, Ales- sand o Usiello, Se gio Cocozza, and Lo enzo Chia io i. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion-Non-Comme cial License (h p://c ea i ecommons.o g/licenses/by-nc/3.0/), which pe mi s un e- s ic ed non-comme cial 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. The mo al igh s o he named au ho (s) ha e been asse ed. EPIGENETICS 2017, VOL. 12, NO. 1, 41–54 h p://dx.doi.o g/10.1080/15592294.2016.1260211 Downloaded by [Tampe e Uni e si y] a 23:01 03 Decembe 2017 poin ing ou o he impo ance o co ec o ma ion and p ese a ion o DNA me hyla ion pa e ns in b ain cells. Howe e , he as majo i y o hese s udies, ega dless o he echniques employed, ook in conside a ion he a e age amoun o CpG me hyla ion in specific genomic egions o hei genome-wide dis ibu ion wi h only ela i e high eso- lu ion. In p inciple, each cell may bea a specificcombina- ion o me hyla ed CpGs a specificloci ha may eflec he o igin o he cell and/o he unc ional s a e o a gi en gene (allele) in ha cell. This in oduces he concep o “epipoly- mo phism,”which goes a beyond he simple iden ifica ion o di e en ially me hyla ed egions, by means ha b ain cells may be conside ed a popula ion o epigene ically he - e ogeneous cells in which each combina ion o me hyl CpGs a a gi en locus ep esen s a specific epiallele. Such in o ma ionislos when hea e ageme hyla ion,e ena single CpG si es, is e alua ed. Using genome-wide app oaches, ecen s udies based on he compa ison o he me hyla ion o ew adjacen CpGs ha e add essed he issue o he e ogeneous me hyla ion, accoun ing o cell- o-cell me hyla ion a iabili y in li e , 21 leukemias, 22 and immo - alized fib oblas s, 23 desc ibing he s ochas ic and clonal e olu ion o epialleles du ing ca cinogenesis. In his wo k, we in es iga ed whe he cell- o-cell me hyla- ion a iabili y exis s in b ain and i his a iabili y ep esen s he esul o me hyla ion and deme hyla ion e en s occu ing s ochas ically a indi idual CpG si es o de i es om a well o ches a ed p ocess. In o de o gain insigh in o his ma e , we pe o med ul a-deep me hyla ion single molecule analysis o a selec ed locus, he D-Aspa a e oxidase (Ddo) gene, 24,25 in di e en b ain de elopmen al s ages, a eas, cell ypes, and emb yonic s em cells (ESCs) neu al di e en ia ion p ocess. The choice o Ddo as a model gene was mainly based on he ac s ha his gene is de elopmen ally egula ed in b ain by DNA me hyla ion changes, 25 he a e age me hyla ion le els ange be ween 60 and 30% in di e en s ages, and he p omo e is el- a i ely poo in CpG con en , 25 which we conside ed ideal con- di ions o s udy a ia ion o in e media e epiallele composi ion a di e en imes du ing de elopmen and in di e en b ain cell ypes. Resul s Epiallele analysis p inciples Amplicon bisulfi e sequencing o a gi en genomic egion enables us o de e mine whe he each included CpG dinu- cleo ide, in each single molecule, is me hyla ed o unme hy- la ed. Upon high-co e age bisulfi e sequencing, i is possible o de e mine, wi h high p ecision, ei he he a e age me h- yla ion a each CpG si e o he asse o me hyla ed and unme hyla ed CpG si es p esen in each amplicon-de i ed sequence. As an example, a egion ha includes 4 CpG si es may gi e o igin o 16 possible combina ions ha may be po en ially ound in a mixed popula ion o cells (Fig. 1). These di e en combina ions will be he e e e ed o as “epialleles;” he numbe o di e en exhibi ed epialleles p o- ides a measu e o he le el o “epipolymo phism”(see Fig. 1 o de ails and examples). We applied his kind o me hyla ion analysis o in es iga e he epiallele combina- ions and e olu ion o he Ddo gene, as example o a locus unde going me hyla ion changes du ing b ain de elopmen . 25 A e aged me hyla ion and epiallele equency dis ibu ion analysis o a 3 kb egion in he Ddo gene in whole b ain du ing de elopmen Using mouse b ain a s ages emb yonic day 15 (E15), pos -na al day 0 (P0), and pos -na al day 30 (P30), we pe o med high- co e age a ge ed bisulfi e sequencing including 7 amplicons (R1-R7) co e ing an ex ended la ge genomic egion (abou 3 kb) su ounding he Ddo ansc ip ional s a si e (TSS) (Fig. 2A). P ime s used in his s udy a e epo ed in Table 1. No e ha we p e iously epo ed a e aged quan i a i e me hyla ion da a o egions R4 and R5, 25 which we ha e he e in eg a ed in o Fig. 2B in o de o p o ide a comp ehensi e iew o a e aged me hyla ion changes a he ex ended Ddo locus. Then, we pe o med single molecule analysis o he R4 egion by de e mina ion o epiallele equency (Fig. 3A). Six y- ou (2 6 ) possible epialleles we e expec ed. Fig. 3B shows he esul s om he analysis o whole b ains (n D3) a E15 s age. Se e al in e es ing aspec s we e wo h o no e. Fi s , we ound, wi h di e en equencies, he occu ence o almos all he he- o e ically p edic ed epialleles. A E15, ully me hyla ed mole- cules ep esen ed abou 13% ( he a e age me hyla ion o each si e was e alua ed a abou 56%), he unme hyla ed molecules o aled abou 10%, while he emaining 77% o he cells ha - bo ed one o he 62 in e media e epialleles, which, al hough wi h di e en equencies, we e all ep esen ed in he b ain cell mix u e. Howe e , we obse ed ha some specific epialleles we e ep esen ed wi h highe han expec ed equency. Such equency dis ibu ion was s ikingly conse ed among indi id- uals. These findings highligh ed ei he a high deg ee o epipoly- mo phism o he Ddo p omo e in he b ain o a high in e indi idual conse a ion o epiallele equency dis ibu ion. The in e indi idual conse a ion o epialleles pa e ns was s ikingly e ained in b ains a each o he analyzed de elopmen al s ages om E15 o P30 (0.786 Pea son R0.992, P<0.001) (Fig. 4,Table 2). Simula ion app oach confi med he non- andomness o he obse ed epialleles dis ibu ion showing a consis en de ia ion om he simula ed equency o each o he epialleles (Supple- men al Fig. S1). Thus, he obse ed phenomenon appea s o be a consequence o a p ecise mechanism go e ning he o - ma ion and main enance o p ede e mined me hyla ion p o- files in each o he di e en b ain cells. The same analyses we e pe o med on o he egions o he Ddo gene (R1-R7). Al hough each o he analyzed egions showed a iable numbe s o CpGs and di e en me hyla ion a e ages ( ang- ing om 96 o 20%) and a ia ion o e de elopmen al s ages, he da a clea ly confi med he exis ence o a s ong in e indi idual conse a ion o all he egions (Supplemen- al Fig. S2 and da a no shown). Howe e , lowe epiallele polymo phism and no changes o e ime we e obse ed in he ups eam egions R1 and R2, as expec ed by he con- s an high a e o a e age me hyla ion in hese egions (85–95%) (Supplemen al Fig. S2). 42 E. FLORIO ET AL. Downloaded by [Tampe e Uni e si y] a 23:01 03 Decembe 2017 Di e en b ain a eas display dis inc epiallele configu a ions The e en uali y ha dis inc i e epiallele pa e ns we e ecog- nizable in di e en b ain a eas was in es iga ed. Me hyla ion a e age and epiallele equency we e analyzed in hippocam- pus, co ex, p e on al co ex (PFC), ce ebellum, and s ia um a R4 in he Ddo p omo e egion. By a e aged me hyla ion analysis he highes me hyla ion le els (abou 50%) we e obse ed in s ia um (Fig. 5A and Fig. 5B). Con e sely, in hip- pocampus, he lowes me hyla ion le el (abou 15%) was p es- en . Ce ebellum, co ex, and PFC showed in e media e le els (abou 20–25%) ha esembled hose obse ed in whole b ain. We analyzed he equency o in e media e epialleles Figu e 1. P inciple o epipolymo phism and epiallele analysis. A e aged me hyla ion deg ee, e en a single base esolu ion, does no gi e any in o ma ion on cell- o-cell me hyla ion a iabili y. In he example epo ed in he Figu e, which shows analysis o 4 adjacen CpG si es, 50% me hyla ion deg ee may co espond o comple ely di - e en me hyla ion scena ios. These ange om he lowes deg ee o epipolymo phism (bo om le ) o he highes le el (bo om igh ). Howe e , eliable analysis o he ela i e equency o each epiallele mus ely on a high numbe o analyzed sequences. Compa ed o genomic app oaches p e iously used o measu e he gene al epipo- lymo phism deg ee, he he e adop ed amplicon bisulfi e sequencing, al hough limi ed o a ge ed genomic egions, allowed us o magni y he de ails o cell- o-cell epial- lele a iabili y a single loci, hanks o he e y high sequencing co e age (abou 210,000 in his s udy) and o comp ehensi ely es ablish he me hyla ion pa e n o se e al adjacen CpG si es o longe egions (up o 600–1000 bp) in which all CpGs wi hin indi idual eads a e e ec i ely phased and may ep esen he epigene ic haplo- ype. TSS: ansc ip ion s a si e. The pie cha s ep esen he pe cen age o ully unme hyla ed epialleles (U Dwhi e), ully me hyla ed epialleles (F Dblack), and he 62 emaining possible me hyl CpG combina ion o in e media e epialleles (I Dg ay g adien ). EPIGENETICS 43 Downloaded by [Tampe e Uni e si y] a 23:01 03 Decembe 2017 and obse ed high epiallele he e ogenei y wi hin each a ea (Fig. 5C). In e es ingly, al hough he p ofiles we e clea ly dis- inguishable among he di e en b ain a eas, hese mainly di - e ed in he ela i e abundance o a ew majo p e e en ial CpG combina ions (see peaks in he bo om g aph in Fig. 5C). Mo eo e , i is wo h no ing ha e en in highly me hyla ed s ia um (a e age me hyla ion le el o abou 50%), mo e han 30% o he cells bea ully unme hyla ed alleles, agains 18% o cells bea ing ully me hyla ed ones. Specific epiallele pa e ns dis inguish di e en pu ified b ain cell popula ions The high deg ee o epipolymo phism ound bo h in o al b ain and sepa a ed b ain a eas could be po en ially explained by cellula he e ogenei y. The e o e, we decided o in es iga e whe he di e en pu ified b ain cells, namely neu ons, oligodend ocy es, as ocy es, and mic oglial cells, displayed cell ype-specific me hyla ion ea u es. Fi s , we pe o med quan i a i e me hyla ion analysis and showed ha di e en cell ypes exhibi ed di e en a e age me hyla- ion deg ee, wi h as ocy es being he less me hyla ed and mic oglial cells he mos me hyla ed (Fig. 6A). Analysis o epiallele equency dis ibu ion indica ed ha , as was also obse ed in sepa a ed b ain a eas, ha highe a e age me h- yla ion le els we e associa ed wi h highe pe cen age o hype me hyla ed epihaplo ypes [see, as an example, mic o- glia (g een line) s. as ocy es (pu ple line) in Fig. 6B]. Con e sely, lowe a e age me hyla ion was associa ed wi h highe equency o specificmono-anddi-me hyla edcon- o ma ions. Howe e , specific epialleles, al hough en iched a di e en deg ees, showed peaks ha we e clea ly coinciden ac oss cell ypes and, o he mos pa , also wi h peaks obse ed in whole b ain issue samples (compa e Figs. 4 and 5). These obse a ions again poin o he exis- ence o a hie a chy among epialleles ha is highly con- se ed ac oss di e en b ain de i ed cells. In summa y, we confi med ha mos o he me hyla ion ules obse ed in b ain issues applied also o isola ed b ain cells, including he high pe cen age o in e media e epialleles, high deg ee o epipolymo phism, and high conse a ion o epialleles e- quency among he same ype o cells among indi iduals. Ou da a s ongly sugges ha , wi hin a cell popula ion, ei he in whole b ain, sepa a ed b ain a eas, o isola ed p i- ma y cells, he epiallele equency dis ibu ion occu s in a non-s ochas ic manne . In pa icula , we obse ed a spa ial specifici y by means o some CpG si es (¡363, ¡330, ¡242) being p one and some (¡318, ¡125, ¡175) being mo e esis an o me hyla ion. Howe e , in a subse o cells o each lineage, in a p opo ion consis en wi h he le el o a e age me hyla ion, me hyla ion sp ead om suscep ible si es by ec ui ing nea by CpGs, inc easing he numbe o molecules in which esis an CpGs became me hyla ed. In he u u e, i would be pa icula ly in iguing o explo e he po en ial con ibu ion o epiallele dis ibu ion analysis o he eliable e alua ion o cell unc ion and cell ype-specific en ichmen in gi en a eas, du ing li e ime and/o in pa ho- logical condi ions. P ima y s. immo alized neu onal cells In e es ingly, when we analyzed epiallele equencies in A1 cells, c-myc immo alized neu ons a di e en cul u e passages (Fig. 6C), despi e obse ing an a e age me hyla ion simila o he one obse ed in p ima y neu ons (see Fig. 6A and pies in 6B Figu e 2. Quan i a i e me hyla ion analysis o a 3 kb genomic egion o Ddo gene. A) S uc u e o he pu a i e mouse Ddo gene p omo e . Blue a ow indica es he an- sc ip ion s a si e (C1). Whi e box ep esen s he pu a i e egula o y ups eam egion. Black box ep esen s exon 1. G ay box ep esen s fi s in on. Posi ions o CpG si es a e indica ed as ela i e o TSS. The analyzed 3 kb genomic po ion is di ided in 7 egions (R1-R2-R3-R4-R5-R6-R7) co esponding o di e en amplicons. B) G aph ep e- sen s me hyla ion a e age a each CpG si e analyzed by Illumina MiSeq Sequence . Each colo line ep esen s he a e age o 3 mice a a gi en de elopmen al s age. Blue DE15; ed DP0; g een DP30. E o ba s show s anda d de ia ions. Fo each CpG si e, he poin s labeled wi h di e en le e s on op a e significan ly di e en (P<0.05) based on pos -hoc ANOVA analysis (Tukey es , pe o med on each CpG si e). Whe e wo de elopmen al imes we e conside ed, S uden es was applied wi h  P<0.05,  P<0.01,  P<0.001. 44 E. FLORIO ET AL. Downloaded by [Tampe e Uni e si y] a 23:01 03 Decembe 2017 and 6C), an opposi e me hyla ion scena io was obse ed in e ms o epiallele equency dis ibu ion. In ac , ew epialleles we e highly p e alen , mos we e absen , and epiallele p ofiles we e disco dan bo h among di e en pla es (Pea son R D0.083) and when compa ed wi h hose de i ed by p ima y cells. These da a sugges ha immo alized neu ons, by con as wi h issue and p ima y cells, show lowe epiallele he e ogenei y (clonal-like beha io ) and a likely s ochas ic epiallele equency dis ibu ion. Figu e 3. All possible me hyl CpG combina ions (epialleles) a he Ddo R4 egion. A) Rep esen a ion o all 64 heo e ically possible epialleles a egion R4 o Ddo p omo e con aining 6 CpG si es. Ci cles ep esen he CpG si es analyzed (¡363, ¡330, ¡318, ¡242, ¡175, and ¡125); black ci cles Dme hyla ed CpG; whi e ci cles Dunme hy- la ed CpG. B) Pe cen equency o each epiallele in whole b ain om 3 mice (M1, M2, and M3) a E15 s age. EPIGENETICS 45 Downloaded by [Tampe e Uni e si y] a 23:01 03 Decembe 2017 Dynamic emodeling o Ddo epiallele p ofiles in ESCs upon induc ion o neu al di e en ia ion E olu ion o epiallele equency dis ibu ion a he Ddo p o- mo e was hen acked in ESCs upon neu al di e en ia ion. Al hough a e age me hyla ion o he 6 CpG si es a he Ddo R4 egion was almos iden ical in undi e en ia ed and di e en i- a ed ESCs (Fig. 7A), and mino di e ences we e obse ed a single CpG si es (Fig. 7B), when analysis o epiallele equency dis ibu ion was applied we obse ed a s iking and ep oduc- ible me hyla ion d i in 3 independen expe imen s (Fig. 7C). Undi e en ia ed ESCs displayed an appa en ly diso ganized, Figu e 4. Epialleles equency dis ibu ion a Ddo R4 egion du ing di e en s ages o mouse de elopmen (E15, P0, P14, P30). The pie cha s ep esen he pe cen age o unme hyla ed epialleles (U Dwhi e), ully me hyla ed epialleles (F Dblack), and all 62 in e media e epialleles (I Dg ay g adien ). The line g aph shows he pe cen age o each o he in e media e epialleles; each colo line iden ifies one mouse. Blue Dmouse 1; ed Dmouse 2, g een Dmouse 3. A he bo om o he las g aph, he specific me hyl CpG combina ions o he 62 in e media e epialleles a e epo ed. 1M iden ifies he g oup o monome hyla ed molecules; 2M Ddime hyla ed; 3M D ime hyla ed; 4M D e ame hyla ed; 5M Dpen ame hyla ed epialleles. Pea son co ela ion R epo ed in Table 2. Table 1. Lis o p ime s used o ampli y Ddo p omo e egions (R1-R7) and he M13 mp18 con ol plasmid. Fo Ddo egions, he posi ions e e o TSS; o M13 mp18 he posi ions e e o sequence en y X02513.1 (NCBI, GenBank). GENE AMPLICON FW PRIMER RV PRIMER Ddo R1 ¡2170/¡1960 T gg a T Taaa TT ga a Aac AAc a aca c cac ccc A Ddo R2 ¡1578/¡1184 Gg ag gg T gTagTT a AAca A ccc caA ccacaa Ddo R3 ¡927/¡499 GT TTaTa g T ggagTT acc ccc AaaaA ca Aa c a Ddo R4 ¡468/¡63 G g g T gaggagg gaTaT Ta262 3 aAc accc cca AA cca Acc Ddo R5 ¡229/C144 GgT gg ggTaagT gaag T g acccc aaaa cccaAaA Aca ac Ddo R6 C267/C671 TT ag g aaT a agagT g gg AA acaa ccc c AcaacaAAca Ddo R7 C801/C1201 Gagggag gggTa ggagTaTaTa a Aac c aAAAaAcaAacacaAaAA c M13 mp18 5946 / 6294 Gg gaaggg aa ag g g ccaa accaaac aca acc The capi al le e s in he p ime s sequences indica e he o iginal C o G. 46 E. FLORIO ET AL. Downloaded by [Tampe e Uni e si y] a 23:01 03 Decembe 2017 a he sca e ed, dis ibu ion o he possible 62 combina ions, wi h a sligh p e alence o monome hyla ed molecules. Upon neu al di e en ia ion owa d neu al cells, we obse ed a spe- cific and highly ep oducible ea angemen o he epiallele equency dis ibu ion p ofile (Pea son R D0.492, P<0.001) (Fig. 7C and Fig. 7D) ha s ongly suppo ed he exis ence o a p og ammed combina o ial code and a me hyla ion d i wi hin he cell popula ion. In pa icula , di e en ia ed ESCs showed sha p o ganized peaks co esponding o cons an en ichmen o ew specific in e media e epialleles and concom- i an impo e ishmen o mos o he o he s and hei de i a- i es (e.g., molecules con aining me hyla ed ¡242 and/o ¡125). No iceably, 7 (ou o 62) in e media e epialleles ha bloomed upon di e en ia ion (¡330; ¡363; ¡330/¡318; ¡363/¡330; ¡363/¡330/¡318; 363/¡330/¡318/¡242, 363/ ¡330/¡318/¡242/¡175) mos ly co esponded o he majo peaks obse ed in de eloping whole b ain as well as neu ons and glial cells, wi h di e en ela i e abundance. In ac , we pe o med an analysis o he epigene ic co ela ion based ei he on he ype o displayed epialleles and on he ela i e amoun o each possible me hyl CpG a angemen ound in each analyzed sample. This was pe o med by p incipal com- ponen analysis (PCA). Resul s epo ed in Fig. 8A and Fig. 8B confi med he ep oducibili y o he Ddo epigene ic d i be ween undi e en ia ed and di e en ia ed s a e and showed ha di e en ia ed ESCs d ama ically shi ed owa d ma u e neu al and glial pa e ns. Taken oge he , hese da a a e com- pa ible wi h a model in which in he undi e en ia ed s a e he CpG me hylabili y a he Ddo p omo e mainly depends on he p ima y sequence and occu s in a andom manne a one o mo e CpG si es. Con e sely, in he di e en ia ed s a e, possibly due o newly gene a ed s uc u ed ch oma in con o ma ion cons ain s, a coo dina ed in e play be ween con iguous CpGs di e ing in me hyla ion suscep ibili y unde lies specificepial- lele equency and dynamics in a spa ial-specific manne , whe e he me hyla ed s a e o mo e suscep ible si es (¡330 and ¡363) inc eases he likelihood ha adjacen esis an si es become me hyla ed. These basic ules a e hen e ained in he ma u e glia and neu ons. In suppo o his model, Fig. 8C shows he ela i e con ibu ion o each CpG o he o ma ion o di e en epialleles. Discussion In his s udy we ha e pe o med an ul a-deep me hyla ion analysis o s udy he me hyla ion p ofiles o single genomic egions (su ounding he Ddo gene) aking in o accoun cell- o-cell he e ogenei y and quan i ying he equency o each me hyla ion pa e n (epiallele) p esen in mixed and pu e pop- ula ions o b ain cells du ing de elopmen and neu al di e en- ia ion. The esul s subs an ia e well-app ecia ed gene al phenomena: i) me hyla ion is highly polymo phic and possibly unde goes a con inuous u no e ; ii) such di e si y is de e min- is ic and no s ochas ic; and iii) he di e si y con e ges in he cou se o neu al de elopmen . Fu he mo e, an epigene ic d i a he Ddo gene, clea ly app ecia ed in e ms o epiallele p ofiles and ully unp edic able by con en ional me hyla ion de e mi- na ion, ma ked ESCs neu al di e en ia ion p ocess. O e all, he findings suppo he hypo hesis ha he me hyla ion s a e o each CpG in single cells is no s able; a he , i is subjec o pe iodic fluc ua ions. This appea s o occu in a spa ial-specific manne , whe e he me hyla ed s a e o mo e suscep ible si es (e.g., ¡330 and ¡363 in he R4 egion) a o s me hyla ion o adjacen o he wise pa ially esis an si es. These ules a e e ained in ma u e b ain whe e he di e en ial a e age me hyl- a ion in each cell ype mainly de i es om he ela i e pe cen - age o ully me hyla ed and unme hyla ed molecules and om he ela i e abundance o specific peaks, wi hin a p e alen fixed se o in e media e epialleles. Whe he hese ules may apply o di e en genomic egions o a e limi ed o some spe- cific egions (e.g., CpG-poo p omo e ) emains o be de e - mined. Howe e , i is in iguing o hypo hesize ha specific CpGs lying ups eam he TSS may se e as a “co e”and “seed” by means ha hei me hyla ion s a e may di ec ly influence he me hyla ion s a e o he adjacen egions, gi ing o igin, in a con inuous dynamic manne , o specific epiallele p ofiles dis i- bu ions. Al hough a limi o his app oach is ha i does no allow he di ec ma ch o cell-by-cell me hyla ion, mRNA exp ession, and cell iden i y da a, i may ci cum en he di fi- cul ies and high cos s o single-cell analyses, wi h which i sha es he abili y o add ess cell- o-cell me hyla ion di e ences and unde lying mechanisms in a cell popula ion. We ha e ecen ly de eloped a pipeline, namely amplime hp ophile (h ps://sou ce o ge.ne /p ojec s/amplime hp ofile ), ha en- de s such kind o analysis, pe o med a indi idual genomic loci, easily app oachable by o he esea che s in e es ed in applying hese p inciples o any biological sys em and genomic egions. To ou knowledge, no s udy o da e has add essed he epiallele di e si y in b ain and in b ain-de i ed cells. In he ecen pas , e y ew s udies ingeniously add essed cell- o-cell me hyla ion he e ogenei y, p e alen ly in umo sys ems and a genome-wide le el, 22,23,26-30 suppo ing gene al ules ha a e mos ly consis en wi h he he e p esen ed da a. Landan e al. 23 sugges ed ha a s ochas ic se ies o sub le and p og essi e me hyla ion changes in cance e olu ion leads o de e minis ic me hyla ion p ofiles. Spa ially specific me hyla ion pa e ns eme ged, by means ha some CpGs a e pa icula ly sensi i e o changes in me hyla ion, c ea ing an ini ia ion poin o me hyl- a ion ha hen sp eads o e he egion. Ou da a a e compa i- ble wi h his model, acco ding o which, in subse s o cells wi hin each popula ion, me hyla ion p ofiles ake o igin by he Table 2. Rela ionship among he epialleles dis ibu ion wi hin each s ages g oup. The analyses we e pe o med by co ela ing mice o each de elopmen al s age in wos. Pea son's co ela ion coe ficien R and P- alue a e epo ed. Pea son co ela ion es o epialleles dis ibu ion Ddo R4 egion STAGE Mice Pea son R P- alue E15 M1/M2 0.986067197 1.89637E–48 E15 M1/M3 0.992391755 2.71669E–56 E15 M2/M3 0.9868598 3.30893E–49 P0 M1/M2 0.959438583 1.08587E–34 P0 M1/M3 0.944346038 1.15656E–30 P0 M2/M3 0.88145423 3.27535E–21 P14 M1/M2 0.786309702 3.66001E–14 P14 M1/M3 0.846990179 4.13024E–18 P14 M2/M3 0.981271547 1.26582E–44 P30 M1/M2 0.833321788 4.37257E–17 P30 M1/M3 0.801413887 5.13155E–15 P30 M2/M3 0.984769846 2.69114E–47 EPIGENETICS 47 Downloaded by [Tampe e Uni e si y] a 23:01 03 Decembe 2017 sp eading om suscep ible si es ha , when me hyla ed, become able o influence nea by CpGs. Mo eo e , by he eanalysis o educed ep esen a ion bisulfi e sequencing (RRBS) da a om no mal and cance ous issues, Landan e al. 23 also obse ed ha 2 me hyla ion pa e ns o DNA molecules om he same cell popula ion we e a he di e en om each o he wi h excep ion o H1 ESC and es is, which displayed cohe en and homogeneous me hyla ion p ofiles. By con as , in de eloping b ain and pu ified b ain cells, we ound a s iking conse a ion o epiallele p ofiles a he Ddo gene wi hin he same ype o samples de i ing om di e en mice. Mo eo e , we ound ha in undi e en ia ed ESCs he me hyla ion p ofiles we e a he Figu e 5. Epiallele composi ion analysis o Ddo R4 in sepa a ed a eas o mouse b ain (Hipp: hippocampus; CB: ce ebellum; CX: Co ex; ST: s ia um; PFC: p e on al co ex) and in whole b ain (WB) a P30 s age. A) A e age me hyla ion a he Ddo R4 egion in each sepa a ed b ain a ea. The ba s labeled wi h di e en le e s on op a e signifi- can ly di e en based on pos -hoc ANOVA s a is ical analysis (Tukey es ). B) A e age me hyla ion a each CpG si e (¡363, ¡330, ¡318, ¡242, ¡175, and ¡125). The ba s labeled wi h di e en le e s on op a e significan ly di e en based on pos -hoc ANOVA analysis (Tukey es ) pe o med o each CpG si e. C) Ddo R4 egion epiallele equency dis ibu ion. The pie cha s ep esen he pe cen age o unme hyla ed epialleles (U Dwhi e); ully me hyla ed epialleles (F Dblack); and all 62 in e media e epialleles (I Dg ay g adien ). Each colo line ep esen s he dis ibu ion o 62 in e media e epialleles in one b ain a ea, as indica ed. A he bo om o he g aph, he spe- cific me hyl CpG combina ions o each o he 62 in e media e epialleles a e epo ed. A P- alue 0.05 was conside ed s a is ically significan . 48 E. FLORIO ET AL. Downloaded by [Tampe e Uni e si y] a 23:01 03 Decembe 2017