Función de la sumoilación en el desarrollo del sistema nervioso central de vertebrados

! ! ! Función!de!la!SUMOilación!en!el!desarrollo! del!sistema!nervioso!central!de!vertebrados! ! Function!of!SUMOylation!during!development! of!the!central!nervous!system!in!vertebrates! ! ! Trabajo!de!tesis!para!el!acceso!al!título!de! Doctor!en!Biología!Molecular!y!Biomedicina! por!la!Universidad!de!Sevilla.! En!Sevilla,!a!…!de!………!de!2017.! ! ! !!!!!!!!!Director!del!trabajo:! ! ! ! ! !Tutor!del!trabajo:! ! Fdo:!Mario!García!Domínguez! ! ! ! !!!!Fdo:!Andrés!Aguilera!López! ! ! ! ! ! ! !Autor!del!trabajo:! ! ! ! ! !!!!Fdo:!Francisco!de!Paula!Juárez!Vicente! ! ! 2! ! ! ! ! 3! Table!of!Contents! ! 1!Resumen!..................................................................................................!7! 2!Abstract!...................................................................................................!9! 3!Introduction!............................................................................................!11! 3.1!The!Central!Nervous!System!(CNS)!in!vertebrates!.................................................!11! 3.1.1!Morphogenesis!of!the!central!nervous!system!...................................................!12! 3.1.2!Neurogenesis!in!the!central!nervous!system!......................................................!19! 3.2!SUMO!(Small!Ubiquitin-like!MOdifier)!..................................................................!26! 3.2.1!The!SUMOylation!pathway!..................................................................................!28! 3.2.2!Role!of!SUMOylation!in!the!cell!...........................................................................!35! 3.3!Objectives!............................................................................................................!39! 4!Results!....................................................................................................!41! 4.1!Study!of!the!role!of!SUMO!during!hindbrain!formation.!.......................................!42! 4.1.1!Krox20!interacts!with!Ubc9.!................................................................................!42! 4.1.2!Krox20!functions!as!a!SUMO!ligase.!....................................................................!44! 4.1.3!Nab!mediates!SUMO!recruitment!to!the!chromatin.!..........................................!52! 4.1.4!Nab!SUMOylation!modulates!Krox20!activity!in!vivo.!.........................................!53! 4.2!Study!of!the!components!of!the!SUMOylation!pathway!in!the!process!of!neuronal! differentiation.!.............................................................................................................!58! 4.2.1!Retinoic!acid-mediated!neuronal!differentiation!results!in!free!SUMO!pool! changes.!...........................................................................................................................!58! 4.2.2!SUMO!overexpression!impairs!neuronal!differentiation.!....................................!60! 4.2.3!Neuronal!differentiation!proceeds!without!significant!changes!in!gene! expression!of!SUMO!ligases!and!basic!pathway!components.!........................................!65! 4.2.4!The!SUMO!proteases!Senp5!and!Senp7!are!upregulated!during!neuronal! differentiation.!................................................................................................................!67! 4.2.5!Senp7!is!required!for!proper!neuronal!differentiation.!.......................................!69! 4.2.6!The!Senp7!promoter!is!transiently!activated!following!induction!of!neuronal! differentiation.!................................................................................................................!73! ! ! 4! 4.3!Identification!of!proteins!that!exhibit!changes!in!their!SUMOylation!state!during! P19!neuronal!differentiation.!........................................................................................!77! 4.3.1!P19!cells!undergo!neuronal!differentiation!when!grown!under!SILAC! conditions.!.......................................................................................................................!77! 4.3.2!SUMO!immunoprecipitation!and!peptide!elution!effectively!enriches!P19!cell! extracts!with!endogenously!SUMOylated!proteins.!........................................................!81! 4.3.3!Several!proteins!are!identified!as!differentially!SUMOylated!targets!between! proliferative!and!differentiating!P19!cells.!......................................................................!84! 4.3.4!SUMOylation!analysis!by!SDS-PAGE!of!identified!proteins!validates!the!SILAC! protocol!on!proliferating!and!differentiating!P19!cells.!..................................................!91! 5!Discussion!...............................................................................................!93! 5.1!The!transcription!factor!Krox20!as!an!E3!SUMO!ligase.!.........................................!93! 5.2!Regulation!of!the!hindbrain!development!by!SUMO.!............................................!94! 5.3!SUMOylation!as!a!regulated!process!during!neurogenesis.!...................................!96! 5.4!Senp7!accounting!for!proper!neuronal!differentiation.!.........................................!98! 5.5!Cellular!functions!of!SUMO!targets!in!the!process!of!neuronal!differentiation!....!101! 6!Conclusions!...........................................................................................!108! 7!Materials!and!Methods!.........................................................................!111! 7.1!Materials!............................................................................................................!111! 7.1.1!Vectors!...............................................................................................................!111! 7.1.2!Oligonucleotides!................................................................................................!114! 7.1.3!Antibodies!and!peptides!....................................................................................!116! 7.1.4!Organisms!..........................................................................................................!118! 7.2!Methods!.............................................................................................................!119! 7.2.1!Culture!conditions!of!employed!organisms!.......................................................!119! 7.2.2!DNA/RNA!techniques!........................................................................................!120! 7.2.3!Model!organisms!...............................................................................................!128! 7.2.4!Protein!techniques!............................................................................................!131! 7.2.5!Other!techniques!...............................................................................................!140! 7.2.6!Data!processing!.................................................................................................!141! ! ! 5! 8!Abbreviations!........................................................................................!143! 9!Appendix!...............................................................................................!148! 10!Bibliography!........................................................................................!150! 11!Agradecimientos!.................................................................................!172! ! ! 6! ! ! ! ! 7! 1! Resumen! ! ! La! formación! del! Sistema! Nervioso! Central! (SNC)! y! la! diferenciación! de! neuronas,! células!gliales!y!otros!tipos!celulares!que!lo!conforman,!son!procesos!esenciales!que!implican! un! gran! número! de! eventos! celulares! que! tienen! lugar! durante! el! desarrollo! en! los! eucariotas!superiores.!Estos!eventos!incluyen!la!activación!de!varias!rutas!de!señalización!o! el!control!transcripcional!de!un!gran!número!de!genes,!siendo!todos!ellos!mecanismos!que! tienen!que!ser!regulados!de!una!forma!muy!precisa!en!la!célula.!Muchos!son!los!procesos! celulares!en!los!que!la!modificación!post-traduccional!por!SUMO!se!ha!visto!implicada,!pero! todavía!no!se!ha!establecido!una!función!clara!de!la!SUMOilación!en!el!desarrollo!neural,! constituyendo!el!establecimiento!de!esta!función!el!objetivo!principal!de!esta!tesis.! ! En!una!primera!parte!de!este!trabajo,!nuestros!estudios!se!han!centrado!en!el!factor! de!transcripción!Krox20,!el!cual!regula!la!formación!del!rombencéfalo!o!cerebro!posterior!y! cuya!interacción!con!la!enzima!de!conjugación!de!SUMO!Ubc9!ha!sido!previamente!descrita.! Los! resultados! obtenidos! a! partir! de! experimentos! realizados! in# vitro!e! in# vivo! nos! han! permitido! determinar! la! función! de! Krox20! como! ligasa! E3! en! la! SUMOilación! de! sus! correpresores,! las! proteínas! Nab.! Experimentos! posteriores! nos! han! permitido! establecer! una!función!para!la!SUMOilación!de!Nab!in#vivo,!reprimiendo!la!regulación!transcripcional! que!Krox20!ejerce!sobre!varios!genes!y!sobre!sí!mismo.! ! En!una!segunda!aproximación!hemos!detectado!una!mayor!acumulación! de! SUMO! libre!en!diferenciación!neuronal,!asociada!a!un!menor!nivel!de!SUMOilación!de!proteínas,!lo! que!nos!ha!llevado!a!estudiar!la!regulación!de!la!ruta!de!SUMOilación!en!este!proceso!de! diferenciación.!Tras!un!análisis!de!la!expresión!de!los!distintos!componentes!de!la!ruta!de! SUMOilación!en!neurogénesis,!nuestros!resultados!muestran!un!aumento!en!la!expresión!de! las! proteasas! específicas! de! SUMO! Senp5!y! Senp7! en! células! que! se! están! diferenciando.! Experimentos! adicional! han! puesto! de! manifiesto! que,! de! hecho,! este! aumento! en! la! expresión!de!Senp7!es!necesario!al!comienzo!de!la!diferenciación!neuronal!para!su!correcta! progresión.! ! Una!vez!hemos!establecido!cómo!la!ruta!de!SUMOilación!se!regula!en!neurogénesis,! hemos!querido!determinar!si!existe!un! cambio! en!el!patrón!de!SUMOilación! de! proteínas! diana! entre! condiciones! de! proliferación! y! de! diferenciación! neuronal.! Con! este! objetivo,! ! ! 8! hemos! utilizado! la! técnica! del! SILAC! (Stable! Isotope! Labelling! with! Amino! acids! in! Cell! culture)! para! posteriormente! purificar! proteínas! SUMOiladas! en! estas! dos! condiciones,! empleando! un! protocolo! de! inmuno-precipitación! que! utiliza! péptidos! correspondientes! a! los! epítopos! reconocidos! por! los! anticuerpos! empleados,! permitiendo! así! la! elución! específica! de! proteínas! que! han! sido! modificadas! por! SUMO! de! forma! endógena.! Tras! la! posterior! identificación! de! proteínas! por! espectrometría! de! masas! (MS),! el! análisis! y! validación!de!los!datos!obtenidos!entre!condiciones!de!proliferación!y!diferenciación!nos!ha! permitido!detectar!cambios!en!el!estado!de!SUMOilación!de!un!gran!número!de!proteínas! que,! de! forma! interesante,! corresponden! en! gran! medida! con! factores! que! presentan! funciones!relacionadas!con!la!transcripción!o!su!regulación.! ! Por!tanto,!el!uso!de!distintas!aproximaciones!durante!el!desarrollo!de!este!trabajo! nos!ha!permitido!demostrar!que!la!SUMOilación!es!fundamental!para!el!desarrollo!neural,! ejerciendo!funciones!críticas!y!actuando!a!distintos!niveles!durante!este!proceso.!! ! ! 9! 2! Abstract! ! ! Morphogenesis!of!the!Central!Nervous!System!(CNS),!as!well!as!differentiation!of!the! neurons,!glia!and!other!cell!types!that!comprise!it,!is!an!essential!process!involving!a!great! number!of!cellular!events!that!take!place!during!development!of!higher!eukaryotes.!These! events!include!activation!of!a!variety!of!signalling!pathways!and!the!transcriptional!control! of! several! genes,! all! comprising! mechanisms! that! need! to! be! tightly! regulated! in! the! cell.! Many!are!the!cellular!processes!regulated!by!the!post-translational!modification!of!proteins! by!SUMO,!but!a!role!for!SUMOylation!in!neuronal!development!remains!unestablished!and,! therefore,!constitutes!the!aim!of!this!thesis.! ! In!the!first!part!of!this!work,!we!have!centered!our!investigations!on!the!transcription! factor! Krox20,! which! regulates! hindbrain! formation! and! was! previously! shown! to! interact! with! the! SUMO! conjugating! enzyme! Ubc9.! Results! obtained! in! experiments! performed! in# vitro!and!in#vivo!have!evidenced!that!this!interaction!is!due!to!the!role!of!Krox20!as!an!E3! ligase! for! SUMOylation! of! its! coregulators,! the! Nab! proteins.! Further! experiments! have! permitted! to! us! the! establishment! of! a! functional! role! for! Nab! SUMOylation! in# vivo,! repressing!the!transcriptional!activity!of!Krox20,!which!in!a!latter!manner!reverts!in!Krox20! own!repression.! ! In!a!second!scientific!approach,!we!have!encountered!that!neuronal!differentiation! proceeds!with!net!SUMO!deconjugation!in!the!cell,!which!has!prompted!us!to!study!how!the! SUMO! modification! pathway! is! regulated! during! this! differentiation! process.! After! a! wide! expression! analysis! of! the! different! components! from! the! SUMOylation! pathway! during! neurogenesis,! our! results! show! how! the! SUMO-specific! proteases! Senp5! and! Senp7! are! upregulated! in! differentiating! cells.! Further! experiments! have! elucidated! that! Senp7! upregulation! at! the! onset! of! neuronal! differentiation! is! indeed! required! for! proper! progression!of!this!process.! ! Once!establishing!how!the!SUMOylation!pathway!is!regulated!during!neurogenesis,! we! also! wanted! to! determine! whether! there! is! a! change! in! the! SUMOylation! pattern! of! target!proteins!between!proliferative!and!neuronal!differentiating!cells.!For!this!purpouse,! we! have! performed! a! Stable! Isotope! Labelling! with! Amino! acids! in! Cell! culture! (SILAC)! experiment! to! purify! SUMOylated! proteins! in! these! two! conditions,! employing! an! Introduction! ! ! 16! 3.1.1.1 Hindbrain!formation!and!Krox20!regulation! ! ! As! previously! mentioned,! neuron! territory! patterning! is! very! complex! during! development! of! the! neural! tube! in! those! parts! that! will! give! rise! to! the! brain! and! the! cerebellum!(forebrain,!midbrain!and!hindbrain).!The!transcription!factor!Krox20!has!a!critical! role! in! hindbrain! segmentation! during! neural! development! of! vertebrates.! This! segmentation! process! consists! on! the! formation! of! 7! to! 8! segments! denominated! rhombomeres!that!distribute!along!the!rostral-caudal!axis!of!the!embryo!(Lumsden,!A!and! Krumlauf,!R,!1996).!This!division!permits!the!differentiation!of!neural!progenitors!located!in! the!hindbrain!into!different!subtypes!of!neurons,!organization!of!the!brachial!nerves!and!the! establishment!of!the!neural!crest.!Given!the!hindbrain!complexity,!its!formation!depends!on! a!really!strict!transcriptional!control!and!gene!expression!pattern!associated!to!each!of!the! rhombomeres,!which!present!unique!characteristics!that!vary!through!the!rostral-caudal!axis! (Figure!I-4).!A!high!number!of!genes!are!implicated!in!this!segmentation,!showing!complex! expression!patterns!whose!regulation!has!not!been!completely!elucidated.! ! ! ! Figure! I-4:! territory! expression! pattern! of! Krox20!and! related! genes.!During! formation! of! the! hindbrain,! the! transcription! factor! Krox20!is! expressed! in! r3! and! r5,! together! with! its! corregulators! Nab1!and! Nab2! (Nab)! and! EphA4.! The! transcription! factor! Hoxb1!is! expressed! in! r4! and!all!along!r6,!r7!and!r8.! ! ! ! ! ! ! ! ! ! Krox20,!also!known!as!Egr2,!is!a!transcription!factor!belonging!to!the!family!of!Early! Growth!Response!(Egr)!factors!and!is!expressed!in!the!rhombomeres!3!(r3)!and!5!(r5),!being! essential! for! the! establishment! and! maintenance! of! these! segments! during! hindbrain! formation! (Figure! I-4)! (Schneider-Maunoury,! S# et# al.,! 1993).! The! Egr! family! of! proteins! is! md# r1# r2# r3# r4# r5# r6# r7# r8# sc# Krox20' Nab' EphA4' Hoxb1' ! ! Introduction! ! 17! comprised! by! four! factors,! firstly! described! by! their! association! with! cellular! proliferation! response! to! different! growth! factors! (Chavrier,! P# et# al.,! 1988).! A! common! feature! of! this! family!of!proteins!is!the!presence!of!three!zinc-finger!domains!to!bind!DNA,!located!to!the! C-terminal!part!of!the!proteins!(Figure!I-5).!Each!of!these!zinc-finger!domains!allows!for!the! recognition! of! nine! base! pairs,! also! called! Egr-response! elements,! which! localize! in! the! promoter!regions!of!Egr-regulated!genes.!Most!of!the!proteins!from!this!family!also!have!an! R1! repressor! domain! upstream! to! the! zinc-finger! domains! (Figure! I-5).! The! R1! domain! is! responsible!for!Krox20!interaction!with!NGFI-A/Egr1-binding!(Nab)!proteins,!Nab1!and!Nab2,! which!were!initially!identified!as!corepressors!of!Egr!factors!(Russo,!MW#et#al.,!1995).!To!this! point,!the!I268F!single!mutation!in!this!domain!has!been!reported!to!abrogate!interaction! with!Nab!proteins!(Svaren,!J#et#al.,!1998).!Additionally,!two!acidic!regions!located!towards! the!N-terminal!part!of!the!protein!and!an!intermediate!region!between!R1!and!one!of!these! acidic!domains!(Figure!I-5)!have!been!described!to!mediate!transcriptional!regulation!from!a! number! of! target! genes;! as! an! example,! the! intermediate! domain! between! both! acidic! domains!and!R1!has!been!associated!to!transcriptional!activation!of!EphA4!and!repression!of! follistatin!(Desmazieres,!A#et#al.,!2009).! ! Nab!proteins!have!a!critical!role!over!the!control!of!different!inflammatory!processes! and!during!development!of!the!embryo,!especially!on!CNS!formation!and!myelinisation!(Le,! N#et#al.,!2005;!Mager,!GM#et#al.,!2008).!Regarding!their!relationship!with!Krox20,!a!mainly! corepressive! function! has! been! established,! though! they! have! also! been! occasionally! described! as! transcriptional! coactivators! (Russo,! MW# et# al.,! 1995;! Svaren,! J# et# al.,! 1996).! Nab1!and!Nab2!show!NLS!sequences!and!share!a!high!sequence!identity,!showing!in!their!N- terminal! part! two! Nab! corepressor! domains! (NCD),! each! one! being! related! with! specific! functions!(Figure!I-5).!As!an!example,!the!NCD1!has!been!associated!to!the!interaction!with! Egr! factors! and! also! to! homo-! and! heterodimerization! between! Nab! proteins.! In! fact,! the! double! mutation! Q64R/H95Q! has! been! shown! to! disrupt! the! interaction! with! Krox20! through!this!domain!(Svaren,!J#et#al.,!1998).!On!the!other!hand,!the!NCD2,!together!with!the! C-terminal! part! of! the! protein! (CID),! has! been! implicated! in! the! transcriptional! repressor! activity!(Figure!I-5).!This!C-terminal!domain!interacts!with!the!chromodomain!helicase!DNA- binding!protein!4!(CHD4)!subunit!of!the!chromatin!remodelling!complex!NuRD,!resulting!in! transcriptional! repression.! Nevertheless,! interaction!with! CHD4! seems!not! to! be! sufficient! for!the!full!repressive!activity!associated!to!Nab!(Srinivasan,!R#et#al.,!2006).! Introduction! ! ! 18! ! ! ! ! Figure!I-5:!structural!domains!from!Krox20!and!Nab2!proteins.!Krox20!structural!domains!comprise! two! acidic! domains! (AC1! and! AC2)! and! an! intermediate! region! (Int)! involved! in! transcriptional! regulation!of!various!target!genes,!a!repressor!R1!motif!that!also!binds!to!Nab!proteins!and!three! zinc-finger!domains!for!DNA!binding.!Structural!domains!of!Nab2!are!also!represented,!showing!an! NLS! domain,! together! with! the! NCD1! domain! for! interaction! with! Krox20! and! the! NCD2! and! CID! motifs,!which!have!been!associated!to!repressor!activity.! ! ! ! As! previously! mentioned,! some! of! the! structural! domains! present! in! Krox20! have! been! described! to! control! in! a! positive! or! a! negative! way! the! expression! of! some! genes! during!the!establishment!of!rhombomeres!3!and!5.!This!group!of!genes!include!Krox20!itself,! the! genes! for! Nab1! and! Nab2!corepressors! and! also! Hox! genes! associated! to! the! establishment!of!the!rostral-caudal!axis!of!the!embryo,!the!follistatin!glycoprotein!that!has! been!implicated!in!rhombomere-to-rhombomere!signalling,!and!adhesion!factors!from!the! Eph! family! of! proteins! that! are! necessary! for! the! control! of! cellular! segregation! and! the! establishment!of!frontiers!between!adjacent!rhombomeres!((Chomette,!D#et#al.,!2006;!Theil,! T#et#al.,!1998)!and!reviewed!in!(Mellitzer,!G#et#al.,!2000)).!It!has!been!specifically!described! how!Krox20!directly!controls!in!a!positive!sense!the!expression!of!EphA4!in!r3!and!r5!(Figure! I-6)! (Desmazieres,! A# et# al.,! 2009),! while! Hoxb1! is! indirectly! controlled! in! a! negative! sense! (Garcia-Dominguez,! M# et# al.,! 2006).! Finally,! Nab1! and! Nab2! genes! are! also! activated! by! Krox20!in!r3!and!r5,!thus!indicating!the!presence!of!a!negative!regulatory!feedback!by!which! Krox20!activates!the!expression!of!its!own!corepressors!(Figure!I-6)!(Mechta-Grigoriou,!F#et# al.,!2000).! ! ! ! Krox20' Nab2' 470' 525' NCD1' NCD2' NLS' CID' Zinc'Finger'R1'AC1' AC2' Int' ! ! Introduction! ! 19! ! ! Figure! I-6:! Krox20! regulation! in! r3! and! r5! specification! and!maintenance! during! hindbrain! development.!Expression!of!Krox20!is!initially!activated!by!various!factors!(X).!Then,!the!transcription! factor! activates! its! own! expression! together! with! the! EphA4!gene.! Krox20! also! activates! the! expression!of!genes!encoding!its!Nab!corepressors,!thus!establishing!a!negative!feedback!in!which! Nab!proteins!repress!the!transcriptional!activity!of!Krox20,!eventually!leading!to!its!own!inactivation.! Coloured!arrows!indicate!protein!biosynthesis,!black!arrows!indicate!transcriptional!activation!and! flat-end!arrow!indicates!corepressor!activity.! ! ! 3.1.2 Neurogenesis!in!the!central!nervous!system! ! ! During! morphogenesis! of! the! CNS,! neuroepithelial! cells! under! nuclear! interkinetic! migration!undergo!proliferation!while!they!remain!in!the!ventricular!zone!of!the!neural!tube.! As! previously! mentioned,! under! certain! circumstances,! they! enter! in! cell-cycle! arrest! and! start!migrating!to!the!mantle!layer!while!get!differentiated!into!neuron!or!glial!cells.!Taking! into!consideration!that!differentiation!of!neural!progenitors!requires!cell-cycle!arrest!after! G1-phase,! the! interaction! and! proper! coordination! between! neurogenic! factors! and! cell!cycle! progression! machinery! is! critical! for! the! establishment! of! functional! neuronal! populations!((Willardsen,!MI!and!Link,!BA,!2011)!and!reviewed!in!(Swiss,!VA!and!Casaccia,!P,! 2010)).! ! Another!key!feature!during!CNS!development!is!the!balance!between!proliferation!of! neural!progenitors!and!differentiation!to!neurons,!which!needs!to!be!tightly!maintained.!For! this,!three!different!types!of!cellular!divisions!have!been!described:!symmetric!proliferative! Krox20' Nab' X' EphA4& Krox20& Nab& Introduction! ! ! 20! (producing! two! new! proliferative! progenitors),! asymmetric! neurogenic! (producing! one! proliferative!progenitor!and!one!postmitotic!cell)!and!symmetric!neurogenic!(producing!two! postmitotic!cells).! The! best-studied! cellular! feature! known! to! affect! neurogenesis! during! these!divisions!is!the!partitioning!of!determinants!at!cytokinesis,!where!certain!factors!are! distributed! equally! or! unequally! in! daughter! cells,! thus! creating! either! symmetric! or! asymmetric!divisions,!respectively!(reviewed!in!(Willardsen,!MI!and!Link,!BA,!2011)).!At!this! point,!proteins!like!epidermal!growth!factor!receptor!(EGFR)!or!TRIM32!proteins!have!been! unequally! detected! in! both! offspring! cells! after! asymmetric! neurogenic! divisions! (Schwamborn,!JC#et#al.,!2009;!Sun,!Y#et#al.,!2005).!Tissue!specificity!of!the!factors!involved!in! controlling! these! divisions! has! also! been! described,! as! neuroepithelial! cortical! cells! that! undergo!mitosis!and!receive!the!new!centrosome!are!prone!to!migrate!and!differentiate!into! neurons,! while! cells! with! the! former! centrosome! remain! located! in! the! ventricular! zone! (Wang,!X#et#al.,!2009).!Another!factor!controlling!proliferative!or!differentiating!state!of!the! neural!progenitors!is!the!G1-phase!duration,!as!the!specifying!action!of!determinants!after! mitosis! would! need! a! certain! duration! time! of! the! G1-phase.! To! this! point,! it! has! been! described! how! shortening! G1-phase! reverts! in! delayed! neurogenesis,! with! the! resulting! increased!generation!of!neural!progenitors!(Lange,!C#et#al.,!2009).! ! A!major!regulator!of!the!neural!progenitors!fate!is!the!Notch!signalling!pathway.!In! this!pathway,!receptor!and!ligand!are!both!transmembrane!proteins!that!confer!specificity! to!neighbouring!cells!(reviewed!in!(Bray,!SJ,!2006)).!Notch!ligands!are!represented!by!Delta! and!Serrate/Jagged!transmembrane!factors!that!belong!to!the!DSL!(Delta,!Serrate!and!LAG- 2)! family! of! proteins.! DSL! is! an! extracellular! domain! present! in! these! proteins! that! is! essential!to!interact!with!the!Notch!receptor.!The!Notch!receptor!contains!an!extracellular! domain!comprising!a!variable!number!of!epidermal!growth!factor!(EGF)!repeats!and!a!linker! region! that! connects! to! the! transmembrane! domain! and! the! Notch! intracellular! domain! (Nicd).!This!way,!in!a!group!of!neuroepithelial!cells,!when!the!Notch!receptor!from!one!of! the! cells! interacts! with! a! DSL! ligand! from! other! cell,! the! Nicd! domain! is! cleavaged! and! translocated!to!the!nucleus,!where!it!binds!to!the!DNA-binding!protein!CSL!(CBF1,!Su(H)!and! LAG-1)! and,! together! with! Mastermind! (Mam),! induces! CSL-mediated! transcriptional! activation!of!the!E(spl)/HES!class!of!genes!(Hes/Her/Esr)!to!maintain!the!proliferative!state!of! neural!progenitors!(reviewed!in!(Bray,!SJ,!2006)).!Regulation!of!the!Notch!pathway!is!related! to!the!fate!establishment!of!neural!progenitors.!An!interesting!regulator!of!this!process!is! ! ! Introduction! ! 21! Numb,! a! well-characterized! inhibitor! of! the! Notch! pathway! that! induces! Notch! receptor! endocytosis.! It! has! been! described! how! Numb! is! unequally! distributed! into! neural! progenitors! from! Drosophila,! leading! to! a! misbalance! of! the! Notch! pathway! between! the! two! newly! generated! neural! progenitors! (Berdnik,! D# et# al.,! 2002).! Interplay! between! the! Notch! signalling! pathway! and! some! basic! helix-loop-helix! (bHLH)! proteins! has! been! established!to!occur!during!both!invertebrate!and!vertebrate!neural!development.! ! Genes! encoding! bHLH! transcription! factors! were! originally! described! in! Drosophila# melanogaster! and! related! genes! were! further! discovered! in! vertebrates! (reviewed! in! (Bertrand,!N#et#al.,!2002;!Guillemot,!F,!1999)).!These!genes!have!a!common!bHLH!structural! motif!that!is!present!in!many!transcription!factors,!which!is!characterized!by!two!α-helices! separated!by!a!loop.!The!helices!have!been!associated!to!dimerization,!while!the!upstream! basic!region!is!required!for!binding!to!specific!DNA!sites!with!the!sequence!CANNTG,!also! named! as! E-box.! Various! families! of! Class! II! bHLH! genes! have! been! established! in! vertebrates,! showing! similarities! with! Drosophila! bHLH! genes! and! specificities! between! them.!Thus,!bHLH!vertebrate!genes!with!similarities!with!Drosophila#ato!(atonal)!would!be! grouped!in!the!Atonal!family!(Math1!and!Math5),!the!Neurogenin!family!(Ngn1,#Ngn2#and# Ngn3),! the! NeuroD! family! (NeuroD,! NeuroD2,!Math2# and! NeuroM)! and! the! Olig! family! (Olig1,!Olig2!and!Olig3).!Genes!related!to!Drosophila!asc!(achaete-scute)!are!classified!in!the! Achaete-Scute!family!(Mash1!and!Mash2)!(Table!I-1).!These!previously!mentioned!proteins! form!heterodimers!with!ubiquitously!expressed!Class!I!bHLH!proteins!or!E!proteins,!encoded! by! E2A! (with! its! two! alternative! products! E12! and! E47),! HEB! and! E2-2! in! vertebrates! (Johnson,!JE#et#al.,!1992;!Massari,!ME!and!Murre,!C,!2000).!When!heterodimers!are!formed,! they! bind! to! the! previously! mentioned! E-boxes! to! activate! transcription! of! target! genes.! Most! bHLH! proteins! act! as! transcriptional! activators,! with! only! few! exceptions! like! Olig2! (Mizuguchi,! R# et# al.,! 2001;! Novitch,! BG# et# al.,! 2001).! As! their! action! depends! on! heterodimerization,!a!regulation!system!for!bHLH!proteins!has!been!identified,!consisting!on!HLH- containing!proteins!that!compete!for!binding!to!E!proteins.!These!competitors!lack!a!basic! region! to! interact! with! DNA,! thus! preventing! transcriptional! activation.! To! this! point,! the! inhibitor!of!differentiation!(Id)!group!of!proteins!have!been!described!to!act!as!regulators!of! bHLH! proteins! in! vertebrates! (reviewed! in! (Massari,! ME! and! Murre,! C,! 2000;! Yokota,! Y,! 2001)).! ! Introduction! ! ! 22! ! Table! I-1:! Neural! development-associated! bHLH! (basic! helix-loop-helix)! proteins.!bHLH!proteins! described!in!invertebrates!(yellow)!and!vertebrates!(green),!grouped!in!distinct!families!according!to! sequence!similarities!in!their!bHLH!domains.! ! ! ! ! ! bHLH! proteins! are! functionally! heterogeneous,! playing! distinct! roles! during! invertebrate!and!vertebrate!neural!differentiation!pathways,!in!a!fashion!that!often!depends! on! the! cellular! lineage! and! localization.! Studies! of! the! function! of! these! genes! have,! in! general,!supported!the!division!of!bHLH!genes!into!early!acting!determination!genes!and!late! acting!differentiation!genes.!In!vertebrates,!early!acting!determination!genes!or!proneural! genes!are!expressed!in!proliferative!neuroepithelial!cells!and!are!necessary!and!sufficient!to! promote! the! generation! of! neural! progenitors! in! the! ventricular! zone! of! the! neural! tube! (Figure!I-7).! Atonal'family' Math1& Math5& Atonal& Amos& Cato& Lin32& Olig'family' Olig1& Olig2& Olig3& Beta3& Beta4& E'proteins' E12& Daughterless& NeuroD'family' Math2& Math3&/&NeuroM& NeuroD& NeuroD2& Neurogenin' family' amphioxNgn& Ngn1& Ngn2& Ngn3& Biparous& Achaete-Scute' family' Mash1& Mash2& Xash3& Cash4& CnASH& Asense& Achaete& Scute& Lethal&of&Scute& Nato'family' Nato3& Nato3& Nscl'family' Nscl1& Nscl2& ! ! Introduction! ! 23! ! Figure! I-7:! Neural! progenitors! commitment! and! differentiation.! In! vertebrates,! neuroepithelial! cells! are! already! specified! for! a! neural!fate!while!they!proliferate.!Activation! of!proneural!bHLH!genes!in!these!cells!leads! to!their!commitment!into!neural!progenitors! with!limited!mitotic!potential!that,!following! subsequent! activation! of! other! bHLH! genes,! will! finally! differentiate! into! glial! cells! or! neurons! (adapted! from! (Bertrand,! N#et# al.,! 2002)).! ! ! ! ! ! ! ! ! ! Proneural! genes! comprise! the! Achaete-Scute! genes! Mash1! together! with! the! Neurogenin!genes!Ngn1!and!Ngn2!and!the!Atonal!gene!Math1!(reviewed!in!(Bertrand,!N#et# al.,! 2002)).! An! additional! characteristic! of! proneural! genes! is! their! ability! to! induce! the! expression! of! Notch! ligands! Delta! and! Serrate/Jagged,! as! well! as! activating! the! Notch! signalling!pathway!(Figure!I-8)!(reviewed!in!(Bertrand,!N#et#al.,!2002)).!Proneural!genes!are! subjected!to!repression!by!inhibitors!Hes,!Her!and!Esr,!previously!mentioned!to!be!under!the! control!of!Notch!signalling!pathway!(Figure!I-8)!(Davis,!RL!and!Turner,!DL,!2001;!Kageyama,!R! and!Nakanishi,!S,!1997).!It!is!also!interesting!to!mention!that!the!cell!cycle!controlling!cyclin! D1! has! been! related! to! regulation! of! proneural! genes! (Lukaszewicz,! AI! and! Anderson,! DJ,! 2011)!and!that!expression!of!Cdk!(cyclin-dependent!kinases)!inhibitors!such!as!p27!has!been! reported! following! induction! of! bHLH! proteins! in! mouse! cells! (Farah,! MH# et# al.,! 2000),! to! induce!cell-cycle!arrest!(Figure!I-8).! ! Neuroepithelium, Proneural,func2on, Neural,progenitor, Neuron, Glia, Introduction! ! ! 24! ! ! Figure! I-8:! regulatory! pathways! controlling! neural! progenitors! differentiation.!Through! lateral! inhibition,! a! regulatory! loop! between!two! neuroepithelial! cells! takes! place,! resulting! in! upregulation! of! Delta!expression! by! proneural! gene! products! and! downregulation! of! proneural! gene! expression! by! activation! of! the! Notch! signalling!pathway.!As!a!result,!a!slight!increased! expression!of!proneural!genes!in!one!of!the!cells,! which!will!further!become!a!neural!progenitor,!will!revert!in!repression!of!the!proneural!function!in! the! neighbouring! cell! and! further! increase! of! proneural! gene! expression! in! the! future! neural! progenitor.!In!neural!progenitors,!the!highly!expressed!proneural!genes!ensure!the!cell-cycle!arrest! by!activation!of!Cdk!(cyclin-dependent!kinases)!inhibitors!and!promote!the!initiation!of!the!neuronal! differentiation! programme!by! induction! of! other! bHLH! differentiation! genes,! such! as! NeuroD!in! vertebrates.! Neuronal! differentiation! continues! with! the! subsequent! activation! of! later! differentiation! genes! like! Math2!or! NeuroD2!and! the! final! establishment! of! neuronal! lineages! (adapted!from!(Bertrand,!N#et#al.,!2002)).! ! ! ! Late! acting! differentiation! genes! encode! bHLH! proteins,! among! others,! that! have! been!associated!to!differentiation!of!the!committed!neural!progenitors!and!are!expressed!in! early!post-mitotic!cells!migrating!toward!the!mantle!layer,!where!proneural!genes!like!Ngn! no!longer!maintain!appreciable!expression!levels.!These!late!differentiating!bHLH!genes!are! shown! to! be! under! the! direct! control! of! proneural! genes.! This! way,! gain-of-function! experiments!in!chicken!embryos!have!shown!how!overexpression!of!Ngn!proneural!genes! cease!progenitor!division!and!promote!ectopic!neuronal!differentiation!through!activation!of! the!differentiation!genes!NeuroD!and!NeuroM,!thus!placing!expression!of!these!genes!in!a! later!stage!during!neuronal!differentiation!(Figure!I-8)!(Blader,!P#et#al.,!1997;!Ma,!Q#et#al.,! Delta& Notch& Proneural&gene& Delta& Notch& Cdk& inhibitors& NeuroM& Nscl1/2& NeuroD& Math2& NeuroD2& Ebf3& Cell-cycle& arrest& Neuronal& differen@a@on& Proneural&gene& ! ! Introduction! ! 25! 1996;!Mizuguchi,!R#et#al.,!2001).!NeuroD!and!NeuroM,!as!well!as!some!proneural!genes!like! Ngn2!and!Mash1,!have!been!implicated!in!the!specification!of!neuronal!or!glial!fate!of!neural! progenitors! in! defined! regions! from! the! CNS! (Nieto,! M# et# al.,! 2001;! Sun,! Y# et# al.,! 2001;! Tomita,!K#et#al.,!2000).!Interestingly,!the!overexpression!of!late!differentiating!proteins!also! induces! the! ectopic! activation! of! proneural! genes,! thus! suggesting! an! effect! for! these! proteins!in!triggering!the!complete!differentiation!programme!(Garcia-Dominguez,!M#et#al.,! 2003).! ! Activation!of!bHLH!genes!also!occurs!in!even!further!stages,!thus!also!accounting!for! late!acting!differentiation!genes.!In!this!manner,!the!use!of!knockout!mice!has!permitted!to! describe! how! expression! of! NeuroD2! is! necessary! for! the! proper! development! of! hippocampus,! cortex,! dentate! gyrus! and! cerebellum.! It! expression! is! described! to! be! regulated! by! Ngn1! and! NeuroD,! situating! NeuroD2! in! a! latter! step! during! neuronal! differentiation!(Figure!I-8)!(Lee,!JE,!1997;!Lin,!CH#et#al.,!2004;!Olson,!JM#et#al.,!2001).!Other! genes! encoding! non-bHLH! proteins! also! coordinate! differentiation! of! neural! progenitors.! Examples!of!this!are!Ebf!genes,!which!encode!for!proteins!with!an!atypical!helix-loop-helix! (HLH)!domain.!Ebf3,!for!instance,!is!expressed!in!an!extended!fashion!during!cell-cycle!exit! and!differentiation!of!neural!progenitors!in!the!chick!hindbrain!and!spinal!cord!regions!from! the!neural!tube!(Garcia-Dominguez,!M#et#al.,!2003).! ! Finally,!the!neuronal-subtype!specification!is!another!key!step!in!the!differentiation! process.!Proneural!genes!have!been!associated!to!the!establishment!of!certain!populations! of!neurons,!assigning!them!a!role!not!only!in!the!initial!commitment!of!neural!progenitors! but!also!in!the!final!determination!of!neuron!specificity.!In!this!manner,!a!role!for!Mash1!in! the!specification!of!noradrenergic!neurons!has!been!well!established!(Goridis,!C!and!Brunet,! JF,! 1999).! Genes! encoding! for! bHLH! proteins! with! no! proneural! function! have! also! been! implicated! in! the! specification! of! neuronal! identity,! for! example! in! retina! (reviewed! in! (Cepko,! CL,! 1999)).! An! interesting! feature! of! neuron! specification! is! the! necessity! of! interaction!between!bHLH!and!homeobox!proteins.!In!this!way,!it!has!been!described!how! amacrine!cells!establishment!requires!the!coexpression!of!NeuroD!or!Math3!together!with! the! homeobox! gene! Chx10! or! how! bipolar! cells! are! generated! after! NeuroD!or! Math3! coexpression!together!with!Pax6!(Figure!I-5C)!((Hatakeyama,!J#et#al.,!2001;!Inoue,!T#et#al.,! 2002)!and!reviewed!in!(Bertrand,!N#et#al.,!2002)).! ! Introduction! ! ! 32! located!in! PIAS! ligases!has! not! been! related! with!a! SUMO! ligase! function! and,! indeed,! its! deletion!on!PIASxα!has!been!described!to!stimulate!its!ligase!activity!(Kotaja,!N#et#al.,!2002).! It!is!also!worth!noting!that!specificity!for!SUMO!paralogs!has!been!poorly!described!for!these! ligases.! Nevertheless,! one! example! is! PIAS4! whose! PINIT! domain! has! been! related! to! specificity!for!SUMO1!(Wong,!KA#et#al.,!2004).! ! ! ! ! Figure! I-10:! E3! SUMO! ligases! and! PIAS1! structure! domains.! (A)!Structural! domains! of! human! PIAS1! E3! ligase.! The! SAP! domain!has!been!related!to!sequence-specific!DNA!binding.!A! PINIT!domain!has!been!associated!to!the!nuclear!localization! of!the!protein.!The!following!SP-RING!domain!is!described!to! be!responsible!for!binding!to!the!E2!conjugating!enzyme!Ubc9.! A! SIM! and! a! C-terminal! region! enriched! in! serine! and! threonine!residues!(S/T)!are!also!represented.!(B)!The!listed!proteins!show!SUMO!E3!ligase!activity! and!have!been!deeply!characterised.! ! ! ! As! previously! mentioned,! RanBP2! (also! known! as! Nup358)! sums! to! the! list! of! E3! SUMO!ligases!that!have!been!widely!studied!(Figure!I-10B),!but!does!not!resembles!to!E3! ubiquitin!ligases,!as!it!does!not!contain!neither!HECT!nor!RING!domains,!typical!features!of! many!ubiquitin!ligases!(Pichler,!A#et#al.,!2004).!RanBP2!is!a!large!protein!of!358!kDa!located! in! the! Nuclear! Pore! Complex! (NPC).! Its! in# vitro! activity! requires! only! one! of! two! closely! spaced!amino!acid!repeat!regions!(IR1!and!IR2)!that,!combined!with!a!short!spacing!region! (M),!can!bind!to!the!Ubc9-SUMO!thioester!bond!(Saitoh,!H#et#al.,!2002;!Tatham,!MH#et#al.,! 2005).! In# vivo,! together! with! SUMOylated! Ran!GTPase-activating! protein! 1! (RanGAP1)! and! Ubc9,!forms!an!E3!SUMO!ligase!complex!capable!of!interacting!with!multiple!proteins!that! are! prone! for! nucleocytoplasmic! transport! through! the! NPC! and! could! be! possible! SUMOylation!targets!(Hamada,!M#et#al.,!2011;!Werner,!A#et#al.,!2012).! ! Other! proteins! whose! E3! SUMO! ligase! activity! has! been! also! studied! comprise! the! human! polycomb! protein! Pc2! (Kagey,! MH# et# al.,! 2003),! the! class! II! histone! deacetylases! (HDACs!II)!and!the!Topors!proteins!(Gregoire,!S!and!Yang,!XJ,!2005;!Hammer,!E#et#al.,!2007;! SAP$ PINIT$ SP-RING$ SIM$ S$/$T$ 651$PIAS1$(H.#sapiens)$ A" PIAS1& Topors& PIAS2&(x)& HDAC&II& PIAS3& Pc2& PIAS4&(γ)& PHD&domain& RanBP2& B" ! ! Introduction! ! 33! Weger,! S# et# al.,! 2005).! In! addition,! the! PHD! domains! present! in! some! plant! and! mammal! proteins!have!also!been!described!to!exhibit!E3!SUMO!ligase!activity!(Figure!I-10B)!(Garcia- Dominguez,!M#et#al.,!2008;!Ivanov,!AV#et#al.,!2007).! ! 3.2.1.2 SUMO!isopeptidases! ! ! In!eukaryote! cells,!SUMO-specific! peptidases! are! responsible! of!three! mayor!tasks:! performing! the! initial! proteolytic! cleavage! for! SUMO! maturation,! removing! single! SUMO! molecules! from! modified! proteins! and! depolymerising! SUMO! chains.! Contrary! to! what! is! described!for!ubiquitin,!where!a!large!number!of!specific!isopeptidases!have!been!identified! and! grouped! in! various! classes! of! proteases! (Reyes-Turcu,! FE# et# al.,! 2009;! Wilkinson,! KD,! 1997),!a!small!number!of!SUMO!proteases!have!been!identified!up!to!date,!belonging!to!the! group!of!cysteine!proteases.!In!this!regard,!the!most!well!studied!SUMO!proteases!comprise! two! ubiquitin-like! protein-specific! proteases! (Ulp1! and! Ulp2)! that! have! been! identified! in! yeast! and! six! sentrin-specific! proteases! (SENPs)! (SENP1-3! and! SENP5-7),! identified! in! humans.!The!first!identified!SUMO!isopeptidase!was!yeast!Ulp1!(Li,!SJ!and!Hochstrasser,!M,! 1999).! SENP! proteins! were! then! identified! in! other! organisms! by! sequence! identity-based! studies!(reviewed!in!(Hay,!RT,!2007;!Hickey,!CM#et#al.,!2012)).!The!structural!analysis!of!Ulp1! (Mossessova,!E!and!Lima,!CD,!2000)!and!further!analysis!of!SENP1!(Reverter,!D!and!Lima,!CD,! 2006;!Shen,!L#et#al.,!2006;!Shen,!LN#et#al.,!2006)!revealed!that!these!proteins!belong!to!the! family!of!cysteine!proteases,!showing!a!characteristic!His-Asp-Cys!catalytic!triad!present!in! the!adenovirus!processing!protease!(AVP)!(Li,!SJ!and!Hochstrasser,!M,!1999).!The!active!site! of!Ulp1!is!comprised!by!a!ß-sheet!in!addition!to!two!helixes,!creating!a!central!cleft!in!the! protein! with! the! approximate! length! of! a! lysine! side! chain.! The! cysteine! included! in! the! catalytic!triad!is!located!at!the!N-terminus!of!the!central!helix,!whereas!histidine!and!aspartic! acid! residues! from! the! active! site! are! both! located! in! the! ß-sheet.! The! C-terminal! Gly-Gly! motif!of!SUMO!is!located!to!this!cleft!and!orientates!in!a!cis#configuration,!establishing!a!turn! in! the! C-terminal! end! of! immature! SUMO! precursors! or! in! the! isopeptide! bond! of! SUMOylated! proteins.! Primary! structures! of! Ulp1,! SENP1! and! SENP2! show! how! their! catalytic!domain,!similar!between!them,!is!located!to!their!C-terminal!end!(Figure!I-11).! ! Introduction! ! ! 34! ! ! ! Figure! I-11:! structure! domains! in! SUMO-specific! isopeptidases.!Structure! domains! and! length! (in! amino! acids)! of! SUMO-specific! isopeptidases! present! in! yeast! and! vertebrates,! showing! the! N-terminal!domains!involved!in!subcellular!localization!(green)!and!the!C-terminal!catalytic!domain! (yellow),!indicating!the!key!histidine!(H)!and!cysteine!(C)!residues!from!the!active!sites.!Putative!SIM! sequences!have!been!indicated!with!asterisks.!Human!SENP1-7!proteins!are!represented,!while!for! deSUMOylating!isopeptidase!1!(DESI1)!and!DESI2,!the!mouse!proteins!are!shown.!Human!Ubiquitinspecific!protease-like!1!(USPL1)!is!also!represented.! ! ! ! Catalytic!domains!of!SENP6,!SENP7!and!Ulp2!have!also!been!described!to!date!and! have!less!than!30%!of!sequence!identity!with!the!previous!ones,!comprising!a!new!subgroup! of!SUMO-specific!proteases!(Li,!SJ!and!Hochstrasser,!M,!1999).!This!last!group!of!proteases! present!four!conserved!loop!insertions!in!their!catalytic!domain!that!could!be!related!with! SUMO!paralog!specificity,!as!SUMO2/3!recognition!by!the!loop1!has!been!described!to!affect! the!higher!activity!that!these!proteases!present!towards!SUMO2/3!(Alegre,!KO!and!Reverter,! D,!2011;!Lima,!CD!and!Reverter,!D,!2008).!Specificity!of!the!C-terminal!catalytic!domain!from! these! proteins! for! conjugated! SUMO! isoforms! has! been! shown! not! to! be! strong! in# vitro! though! some! of! them,! as! the! previously! mentioned! SENP6! and! SENP7,! exhibit! increased! 621$ 1034$ 643$ 589$ 574$ 755$ 1112$ 984$ 168$ 194$ *$ *$ *$ *$ *$*$ *$ *$ *$ *$ *$ I$ I$ I$ I$ I$ I$ I$ I$ I$ I$ I$ I$ I$ I$ I$ I$ I$ I$ I$ I$ C580$ C602$ C548$ C532$ C713$ C1030$ C926$ C108$ C108$ H514$ H533$ H478$ H465$ H646$ H765$ H794$ H38$ H30$ C624$H531$ Ulp1$ Ulp2$ SENP1$ SENP2$ SENP3$ SENP5$ SENP6$ SENP7$ DESI1$ DESI2$ USLP1$ H456$C236$ I$ I$ 1092$ ! ! Introduction! ! 35! activity! towards! one! or! another! paralog.! Interestingly,! greater! paralogue! specificity! is! sometimes!observed#in#vivo!but!the!mechanisms!implicated!are!not!completely!understood.! Nevertheless,!high!paralog!specificity!of!the!C-terminal!catalytic!domain!for!immature!SUMO! precursors!has!been!detailed!(Di!Bacco,!A#et#al.,!2006;!Shen,!LN#et#al.,!2006;!Xu,!Z!and!Au,! SW,!2005).!On!the!contrary,!the!N-terminal!part!of!Ubl!and!SENP!proteases!has!been!shown! to!regulate!the!specificity!for!SUMO!substrates!in#vivo!by!promoting!interaction!with!specific! targets!and!controlling!subcellular!localization!of!the!proteases.!To!this!point,!it!is!described! that!SENP1!and!SENP2!localize!in!the!nuclear!pore,!nuclear!bodies!and!nucleoplasm.!SENP3! and! SENP5! localize! to! the! nucleolus! and! SENP6! and! SENP7! localize! to! the! nucleoplasm! (reviewed!in!(Hickey,!CM#et#al.,!2012)).!In!contrary!to!the!C-terminus,!the!N-terminal!regions! are!poorly! conserved!(Figure! I-11).!The! presence!of! putative!SIMs! in!non-catalytic! regions! has!also!been!described!for!Ulp2!and!most!of!SENP!proteases.!Though!SUMO!is!bound!to!the! catalytic! domain! by! other! surfaces,! these! SIMs! could! increase! the! affinity! for! SUMO! or! SUMO!chains,!or!could!account!for!a!correct!orientation!of!the!SUMOylated!protein!in!the! protease.! ! Recently,! three! new! SUMO-specific! proteases! have! been! described! in! humans,! deSUMOylating!isopeptidase!1!(DESI1),!DESI2!(Shin,!EJ#et#al.,!2012)!and!the!ubiquitin-specific! protease-like! 1! (USPL1)! (Schulz,! S# et# al.,! 2012).! DESI1! and! DESI2! proteins! share! little! sequence!identity!with!Ulp!and!SENP!proteases!and!belong!to!the!PPPDE!(permuted!papain! fold! peptidases! of! double-stranded! RNA! viruses! and! eukaryotes)! class! of! proteases.! Interestingly,! its! catalytic! activity! is! based! on! their! ability! to! dimerise,! thus! comprising! a! surface! groove! between! the! two! monomers! that! contains! the! active! sites! of! the! dimeric! protease!(Suh,!HY#et#al.,!2012).! ! 3.2.2 Role!of!SUMOylation!in!the!cell! ! !Post-translational!modification!by!SUMO!has!been!associated!with!multiple!functions! in!the!eukaryotic!cell!and!this!is!understandable,!taking!into!consideration!the!high!number! of!SUMO!substrates!identified!so!far!and!the!basis!of!the!modification,!in!which!the!addition! of!a!polypeptide! to!a! given! protein!adds! new! interacting!surfaces! or! either!blocks! certain! domains!present!in!the!target!substrate.! Introduction! ! ! 36! ! SUMOylation!has!been!implicated!in!the!subcellular!localization!of!proteins,!with!a! predominant!role!in!nuclear!localization.!A!paradigmatic!example!for!this!function!would!be! RanGAP1!modification!by!SUMO1,!which!translocates!the!target!protein!from!the!cytoplasm! to!the!NPC!(Mahajan,!R#et#al.,!1997;!Matunis,!MJ#et#al.,!1996).!SUMOylation!has!been!shown! to!be!also!necessary!to!target!RanGAP1!to!the!mitotic!spindle!during!mitosis!(Joseph,!J#et#al.,! 2002).!Nevertheless,!it!should!be!mentioned!that,!though!SUMOylated!proteins!have!been! identified! in! the! cell! cytoplasm,! nuclear! targeting! is! required! for! most! of! them! to! be! modified!by!SUMO!in#vivo!(Rodriguez,!MS#et#al.,!2001).! ! Several!works!also!establish!an!association!for!SUMO!with!DNA!repair.!SUMOylation,! together! with! ubiquitylation,! has! been! shown! to! orchestrate! the! cellular! responses! to! a! variety!of!DNA!aberrations,!due!either!to!exogenous!damage!or!to!natural!occurring!DNA! metabolism,! such! as! postreplication! repair,! base-excision! repair! and! the! repair! of! most! cytotoxic!double-strand!breaks!(reviewed!in! (Jackson,! SP!and!Durocher,!D,!2013)).! Several! works! in! yeast! and! higher! eukaryotes! have! established! genetic! associations! with! chromosome!condensation,!cohesion!and!segregation!during!mitosis!and!meiosis!for!every! component! of! the! SUMOylation! pathway! (reviewed! in! (Seeler,! JS! and! Dejean,! A,! 2003)),! adding!modification!by!SUMO!to!the!factors!that!maintain!genetic!stability!in!the!eukaryotic! cell.! ! SUMOylation! has! shown! to! be! necessary! for! regulation! and! stabilization! of! some! proteins,! as! it! has! been! studied! how! the! same! lysine! residues! can! be! modified! in! certain! circumstances!either!by!SUMOylation,!ubiquitylation,!acetylation!or!phosphorylation.!A!wellknown!example!of!this!competition!mechanism!is!Iκbα,!where!SUMOylation!of!lysine!21!is! shown! to! inhibit! phosphorylation-induced! ubiquitylation! and! subsequent! proteasomal! degradation!(Desterro,!JM#et#al.,!1998).! ! One!of!the!most!important!functions!attributed!to!SUMOylation!is!the!modification! of! transcription! factors! in! order! to! control! gene! expression.! This! way,! modification! by! SUMO1! has! been! typically! related! to! transcriptional! repression.! Certain! exceptions! are! known,! like! the! increase! in! target! gene! expression! mediated! by! heat! shock! transcription! factors!HSF1!and!HSF2!when!modified!by!SUMO1!(Goodson,!ML#et#al.,!2001;!Hong,!Y#et#al.,! 2001).!But!as!said,!many!are!the!examples!where!repressor!domains!present!in!transcription! factors! display! their! repressor! activity! when! at! least! one! lysine! residue! included! in! the! repressor!domain!is!modified!by!SUMO1!(reviewed!in!(Garcia-Dominguez,!M!and!Reyes,!JC,! ! ! Introduction! ! 37! 2009)).!SUMO2/3!though,!has!been!mostly!shown!not!to!be!conjugated!to!proteins!under! normal!conditions,!but!to!rapidly!modify!a!variety!of!proteins!during!the!cellular!response!to! various! stresses! like! hypoxia! (Yang,! W# et# al.,! 2012)! or! heat! stress! (Castoralova,! M# et# al.,! 2012).! ! Finally,!though!not!being!a!cellular!function!per#se,!it!is!important!to!mention!how! evolution!has!made!possible!for!pathogens!to!take!advantage!of!the!SUMOylation!pathway! during!infection.!As!the!SUMO!modification!pathway!only!comprises!one!single!E1!and!E2! enzyme,!any!interference! with!these! enzymes! has!remarkable! effects.! In!fact,! it! has!been! described!how!certain!viruses!and!bacteria!enhance!their!infection!by!targeting!host!E1!or!E2! enzymes! for! proteasome-mediated! degradation! (reviewed! in! (Flotho,! A! and! Melchior,! F,! 2013)).! ! 3.2.2.1 SUMOylation!in!neurons! ! ! A!number!of!functions!for!SUMO!in!adult!neurons!have!been!encountered!(reviewed! in! (Martin,! S# et# al.,! 2007)).! As! an! example,! SUMOylation! of! the! postsynaptic! scaffolding! protein! gephyrin! has! been! described! in! neurons! and! related! to! GABAergic! transmission! (Ghosh,!H#et#al.,!2016).!In!addition!to!this,!previous!works!suggested!a!role!for!SUMOylation! in!synaptic!plasticity!and!trafficking.!To!this!point,!a!regulatory!role!has!been!established!for! SUMO!in!the!synapse!formation!of!cerebellar!granule!neurons,!where!the!myocyte!enhancer! factor! 2! (MEF2)! turns! to! be! a! transcriptional! repressor! or! an! activator! depending! on! its! SUMOylated!or!not!SUMOylated!state,!respectively!(reviewed!in!(Beg,!AA!and!Scheiffele,!P,! 2006)).!In!addition!to!this,!not!only!substrates!but!also!enzymes!of!the!SUMO!modification! pathway! like! SAE1,! Ubc9,! SENP1! and! SENP6,! have! been! detected! in! rat! synaptosomes! (reviewed!in!(Luo,!J#et#al.,!2013)).! ! The!implication!of!SUMO!with!neurodegenerative!diseases!that!generate!due!to!the! accumulation! of! aberrant! proteins,! like! Alzheimer! and! Parkinson! diseases,! has! also! been! described.! In! these! cases,! SUMOylation! seems! to! prevent! degradation! of! these! aberrant! products!by!competition!with!ubiquitylation!and!proteasome-mediated!degradation.!In!the! case! of! Alzheimer,! pathological! progression! has! been! related! to! synaptic! failure! and! later! accumulation! of! insoluble! amyloid-ß! and! microtubule-associated! protein! tau! aggregates.! Introduction! ! ! 38! Both! amyloid-ß! and! tau! proteins! have! been! shown! to! be! SUMOylated! and! alterations! in! enzymes!of!the!SUMO!pathway,!like!SAE2!or!SENP3,!have!been!reported!in!affected!tissues! (reviewed!in!(Lee,!L#et#al.,!2013)).!Parkinson!disease!is!not!defined!by!a!single!pathology,!but! rather! comprises! a! variety! of! syndromes.! Though! little! is! known! about! the! causes! of! the! disease,! most! of! the! cases! are! characterized! by! the! presence! of! Lewy! bodies! containing! fibrillar! α-synuclein.! DJ-1! and! parkin! are! proteins! also! related! with! the! disease.! SUMO! conjugation!has!been!described!for!the!three!proteins!and!been!associated!to!their!solubility! and!aggregation!properties,!though!no!clear!relationship!with!disease!progression!has!been! established!(reviewed!in!(Eckermann,!K,!2013)).! ! SUMOylation! is! associated! with! transcriptional! regulation,! a! critical! feature! in! neuronal!development.!Up!to!date,!few!reports!have!linked!SUMOylation!with!the!control!of! this! process.! Thus,! SUMOylation! of! the! transcription! factor! SoxN! has! been! proposed! to! repress!its!transcriptional!activity,!which!is!necessary!for!the!proper!formation!of!the!CNS!in! Drosophila!(Savare,!J#et#al.,!2005).!On!the!other!hand,!SUMOylation!of!Braf35,!a!subunit!of! the! LSD1-CoREST! histone! demethylase! complex! has! been! shown! to! be! necessary! for! the! maintenance!of!the!undifferentiated!state!of!neuronal!precursors!(Ceballos-Chavez,!M#et#al.,! 2012).! Despite! these! findings,! the! establishment! of! clear! implication! of! additional! SUMO! targets!and!of!components!of!the!SUMOylation!pathway!in!orchestrating!different!aspects!of! neural!development!remains!elusive.!Thus,!it!is!mandatory!to!address!research!in!the!field! through!new!and!complementary!approaches.! ! ! ! ! Introduction! ! 39! 3.3 Objectives! ! ! Taking!into!consideration!the!previously!mentioned!works,!enough!evidences!point! to!a!connection!between!the!SUMO!modification!pathway!and!neuronal!development.!Thus,! three!main!objectives!have!been!projected!in!this!work!to!study!this!connection:! ! 1) To! analyse! the! function! of! the! interaction! between! Krox20! and! Ubc9! during! hindbrain!development.! ! 2) To! study! the! regulation! of! the! SUMOylation! pathway! and! its! components! in! the! context!of!neurogenesis.! ! 3) To! identify! SUMO! targets! differentially! modified! under! proliferation! and! neuronal! differentiation!conditions.! ! ! ! ! ! Results! ! 41! 4! Results! ! ! Understanding! the! mechanisms! involved! in! the! neural! development! is! an! arduous! task! since! it! involves! the! comprehension! of! numerous! pathways! and! signalling! systems! implicated! in! the! tight! regulation! and! control! of! this! process.! Many! of! these! mechanisms! remain!unclear.! ! A!signalling!system!that!is!acquiring!more!relevance!regarding!transcriptional!control! is! the! post-translational! modification! by! SUMO.! SUMOylation! is! essential! in! vertebrates.! Adding! this! consideration! to! its! relevance! in! transcriptional! control! and! many! other! functions! in! the! cell,! it! is! likely! to! think! that! this! post-translational! modification! might! be! implicated! in! the! neuronal! differentiation! process.! In! this! work,! we! analyse! the! role! of! SUMOylation! in! three! different! scenarios! of! neural! development,! to! shed! light! on! the! mechanisms!involved!in!this!complex!process.! ! The!first!scenario!focuses!on!the!formation!of!the!hindbrain!in!vertebrates,!where!an! intricate! and! highly! regulated! compartmentalizing! process! takes! place! during! the! development! of! this! region! of! the! embryonic! central! nervous! system.! Starting! from! preliminary! results! obtained! from! a! two-hybrid! experiment,! we! have! described! a! system! where!SUMOylation!plays!a!regulatory!role!during!hindbrain!formation.! ! The!second!scenario!turns!into!the!neural!tube.!The!development!of!this!structure!is! simpler!than!that!of!the!hindbrain!and!we!have!taken!advantage!of!a!variety!of!tools!and! models! to! analyse! how! does! the! SUMOylation! machinery! behave! during! the! neuronal! differentiation!processes!that!take!place!during!its!formation.! ! Once! having! investigated! the! behaviour! of! the! SUMO! modification! pathway! in! neurogenesis,!the!last!scenario!focuses!on!determine!which!target!proteins!have!a!role!in! neuronal! differentiation! through! regulation! by! SUMO.! For! this! purpose,! a! Stable! Isotope! Labelling! with! Amino! acids! in! Cell! culture! (SILAC)! experiment! comparing! the! SUMO! proteome! of! proliferative! and! differentiating! P19! cells! has! been! done! to! initially! identify! proteins! that! change! their! SUMOylation! state! during! this! process,! without! changing! their! expression!levels.!The!results!obtained!could!open!new!ways!on!understanding!the!role!of! the!SUMO!modification!pathway!in!neural!development.!! Results! ! ! 48! ! Overexpression! of! the! protein! inhibitor! of! activated! STAT! (PIAS)! proteins! was! also! tested,!not!significantly!modifying!Nab!SUMOylation!in!the!presence!of!Krox20!(Figure!R-6B).! To! check! whether! the! two! consensus! sites! identified! in! Nab! were! responsible! of! its! SUMOylation,!we!designed!a!double!mutant!of!the!putative!target!lysines,!K379R!and!K517R! (KR2).!To!test!that!Nab!cellular!properties!were!not!altered!by!these!two!mutations,!cellular! localization!was!analysed!on!one!hand!by!immunofluorescence!and!compared!to!the!wild! type!Nab,!showing!in!both!cases!a!nuclear!localization!as!revealed!by!DAPI!counterstaining! (Figure!R-7A).!On!the!other!hand,!Nab-Krox20!interaction!capabilities!were!not!altered!by! these! mutations! as! shown! by! quantification! of! ß-galactosidase! activity! in! two-hybrid! experiments! (Figure! R-7B).! As! expected,! this! mutant! was! not! SUMOylated! after! cotransfection!of!293T!cells!with!Krox20!and!His-SUMO1!(Figure!R-7C).! ! We! next! investigated! the! involvement! of! Krox20-Nab! interaction! in! Nab! SUMOylation,!as!would!be!expected!for!a!SUMO!E3!ligase,!by!transfection!of!293T!cells.!The! single! mutation! I268F! in! Krox20! and! the! double! mutation! Q64RH95Q! in! Nab! have! been! shown! to! abrogate! Krox20-Nab! interaction! (Svaren,! J# et# al.,! 1998).! We! found! that! both! mutations!prevented!SUMOylation!(Figure!R-8A).!By!contrast,!neither!Krox20!nor!the!I268F! mutant! showed! any! effect! in! general! SUMOylation,! as! monitored! by! modification! of! the! carboxyl! terminal! part! of! RanGAP1! (RanGAP1-C-ter),! a! well-known! SUMO1! substrate! (Figure!R-8B).! ! To! shed! light! on! the! endogenous! mechanisms! that! involve! SUMO! ligase! action! of! Krox20!in!the!cell,!we!turned!to!the!P19!cell!line,!as!these!cells!express!both!Krox20!and!Nab.! Quantitative! PCR! analysis! of! non-treated! and! serum! stimulated! cells! were! carried! out! to! observe!that,!though!Nab!presented!a!basal!expression!prior!to!serum!stimulation,!P19!cells! showed!an!increase!in!the!expression!levels!of!both!Nab!and!Krox20!after!serum!stimulation! (see!Materials!and!Methods)!(Figure!R-9A).!When!analysing!Nab!endogenous!SUMOylation! at!the!protein!level,!we!were!not!able!to!observe!Nab!modification!by!endogenous!SUMO1.! It!has!been!described!how!SUMOylation!of!some!proteins!is!difficult!to!observe!by!Western! blot,!as!the!amount!of!modified!protein!is!very!limited!in!comparison!with!the!unmodified! form! (Hay,! RT,! 2005).! Thus,! we! decided! to! transfect! P19! cells! with! low! amounts! of! the! His-SUMO1!expression!vector!to!analyse!Nab!SUMOylation!after!pull-down!of!the!His-tagged! products.! As! shown! by! Western! blot,! although! SUMOylated! Nab! forms! could! not! be! detected!in!the!input!(1.5%!of!the!total),!they!were!observed!in!the!precipitates.!Moreover,! Results! ! 49! the!presence!of!these!bands!was!strongly!reinforced!after!serum!stimulation;!that!is,!upon! induction! of! Krox20! (Figure! R-9B).! SUMOylation! of! endogenous! Nab! was! also! observed! in! pull-down! experiments! after! transfection! of! a! Krox20! expression! vector! without! serum! stimulation! (Figure! R-9C).! Finally,! knockdown! of! induced! Krox20! by! a! combination! of! two! short!interfering!RNA!(siRNA)!molecules!that!were!cotransfected!with!His-SUMO1!prior!to! serum!stimulation!prevented!serum-induced!SUMOylation!of!endogenous!Nab!as!shown!by! Western!blot!after!pull-down!(Figure!R-9D).! ! ! ! ! ! Figure! R-8:! Physical! interaction! between! Nab! and! Krox20! is! necessary! for! Nab! SUMOylation.! SUMOylation! assays! were! performed!by!transfection!of!293T!with! the!indicated!expression!constructs.! Cells! were! harvested! 24h! after! transfection!and!denaturing!protein! extracts!were!analysed!by!Western! blot.!(A)!SUMOylation! of! Nab! and! Krox20! non-interacting! mutants! was!analysed!by!Western!blot!using! Flag! antibodies! against! the! Flagtagged! Nab.! The! absence! of! SUMOylated! Nab! bands! in! the! presence! of! the! mutants! shows! how! Krox20! and! Nab! need! to! interact! for! proper! SUMO! ligase! and! target! function,! respectively.! Blots!with!HA!antibodies!are!shown! as! controls! for! the! expression! of! Krox20! constructs.! (B)!Analysis! of! Flag-tagged! RanGAP1-Cter! (Ran)! SUMOylation! was! conducted! on! transfected!293T!cells!to!discard!an!indirect!effect!of!Krox20IF!mutant!on!overall!SUMO!modification! in!the!cell.!Western!blot!with!Flag!antibodies!showed!how!Ran!was!equally!modified!by!SUMO!in!the! presence! of! either! wild! type! Krox20! or! I268F! mutant.! HA! antibodies! were! used! to! check! the! expression!levels!of!wild!type!Krox20!and!I268F!mutant.! ! ! 31# 66# 45# 45# kDa# –#–# HA-Krox20# –# +# His-SUMO1# +#–# +# +# +#–# HA-Krox20IF# –# –# Flag-Ran# HA-Krox20# HA-Krox20IF# B" –# +#+# HA-Krox20#–# –# Flag-NabQRHQ#–# +#–# –# +# –# –#–# HA-Krox20IF# +# –# Flag-Nab#+# –#+# +# –# ANab# NabQRHQ# HA-Krox20# HA-Krox20IF# 45# Flag-Nab# Flag-NabQRHQ# 66# kDa# 97# 116# His-SUMO1#+#+# +# +#+# ############## Results! ! ! 50! ! ! ! Figure!R-9:!Endogenous!Nab!is!SUMOylated!in!P19!cells!after!serum-induced!Krox20!expression.!(A)! Expression!levels!of!Nab2!and!Krox20!were!analysed!by!quantitative!PCR!in!proliferating!P19!cells! after!RNA!isolation!(grey!bars).!Expression!levels!of!absent!(GFP)!and!well-expressed!genes!(Oct4)!in! this!condition!are!also!shown!as!controls.!Nab2!and!Krox20!expression!was!also!analysed!after!serum! stimulation! (black! bars).! Means! of! arbitrary! units! of! three! independent! experiments! ±! s.d.! are! represented.!(B-D)!SUMOylation!of!endogenous!Nab!was!analysed!by!pull-down!experiments!against! the!polyhistidine!tag!of!SUMO1!and!Western!blot!using!the!indicated!antibodies.!Upper!blots!show! pull-down!of!SUMOylated!endogenous!Nab!revealed!with!Nab!antibodies!while!the!two!lower!blots! show! 1.5%! input! of! the! indicated! proteins.! Note! that!Krox20! antibodies! also! reveal!a!non-specific! upper!band!(*).!(B)!Endogenous!Nab!SUMOylation!was!visualized!after!transfection!of!His-SUMO1! and!induction!of!Krox20!expression!by!serum!stimulation.!Blot!using!antibodies!against!endogenous! Krox20! is! shown! as! a! control! of! Krox20! expression! induction.! Endogenous! Nab! expression! is!also! shown.! (C)!SUMOylation! of! endogenous! Nab! was! also! observed! when! P19! cells! were! transfected! with! HA-tagged! Krox20! under! normal! growth! conditions.! (D)!Knock-down! experiments! show! how! SUMOylation!of!endogenous!Nab!was!impaired!when!transfecting!a!small!interfering!RNA!(siRNA;!si)! against!Krox20!prior!to!serum!induction,!but!not!with!a!control!siRNA!against!the!GFP.! ! HA-Krox20* Nab* +* HA-Krox20*–* C" 66* 97* 66* kDa* 97* 116* 45* His-SUMO1* Krox20* Nab* Serum* –*+* +* +*+*–* B" 66* 97* 116* 66* kDa* 97* 116* 45* 90* 10* 8* 6* 4* 2* Arbitrary*units* 80* Nab2% Oct-4% 0* 100* Krox20% GFP% Serum*inducGon* No*inducGon* A" Krox20* Nab* si*Krox20* –*+* +* si*GFP* –* D" 66* 97* 66* kDa* 97* 116* 45* His-SUMO1*+*+*His-SUMO1*+*+* ** ** Results! ! 51! ! To! definitively! demonstrate! that! Krox20! functions! as! a! SUMO! ligase! for! Nab,! we! performed!in#vitro!SUMOylation!assays!with!proteins!purified!from!E.#coli!(Figure!R-10A!and! R-10B).! As! illustrated! in! Figure! R-10C,! SUMOylation! of! Nab! took! place! in! vitro! and! was! indeed! dependent! on! the! presence! of! Krox20.! Furthermore,! chemiluminescence! measurement!from!the!bands!detected!by!Western!blot!and!comparison!with!the!amount!of! loaded! protein! revealed! that! 0.3x10-12! mol! of! Krox20! were! sufficient! to! modify! at! least! 2.78x10-12!mol!of!Nab.!These!results!suggest!a!catalytic!and!not!stoichiometric!function!of! Krox20,!as!would!be!expected!for!a!SUMO!ligase.! ! ! ! Figure! R-10:! In) vitro!SUMOylation! assay! to! assess! the! SUMO! ligase! role! of! Krox20! in! Nab! modification!by!SUMO1.!(A,!B)!Coomassie!blue!staining!of!recombinant!fusion!proteins!purified!from! E.#coli.!Mouse!E1!monomers!(red!arrows),!Ubc9,!Krox20!and!human!matured!SUMO1!(SUMO1GG)! were! purified! as! GST-fusion! proteins! and! SUMO1GG! was! later! excised! from! GST.! Nab-Flag! was! purified!as!a!His-tagged!version!using!a!nickel!affinity!matrix.!(C)!In#vitro!SUMOylation!assay!with!the! previously! shown! purified! proteins! to! test!modification! by! SUMO1GG! of! 300! ng! of! Nab! in! the! presence!of!15!ng!of!Krox20!by!Western!blot!using!Flag!antibodies.!SUMOylated!products!correspond! to! 52.96%! ±! 2.74%! (means! ±! s.d.)! of! total! loaded! protein! when! chemiluminescence! of! three! independent!experiments!was!measured.! kDa$E1$ 97$ 66$ 31$ 45$ 21$ 14$ SUMO1GG$ Ubc9$ A" 66$ kDa$ 97$ 116$ Krox20$ –$ +$–$ +$ SUMO1GG$ –$ +$ +$–$ His-Nab-Flag$ C" kDa$ 97$ 66$ 45$ GST-Krox20$ His-Nab-Flag$ B" Results! ! ! 52! 4.1.3 Nab!mediates!SUMO!recruitment!to!the!chromatin.! ! ! SUMOylation! has! been! related! to! many! roles! in! the! cell,! being! modification! by! SUMO1!mostly!connected!to!transcriptional!regulation!(Seeler,!JS!and!Dejean,!A,!2003).!As! Nab! SUMOylation! by! Krox20! has! been! shown! to! be! specific! for! SUMO1! (Figure! R-5C),! we! next! investigated! whether! SUMO1! can! be! recruited! to! a! Nab! regulated! sequence! in! the! context!of!chromatin.!We!chose!to!examine!the!Id4!promoter!because!it!has!been!previously! shown!to!be!regulated!by!the!Krox20–Nab!complex!(Mager,!GM#et#al.,!2008).!In!P19!cells! growing! under! serum! deprivation,! chromatin! immunoprecipitation! (ChIP)! experiments! analysing!SUMO1!incorporation!revealed!that!serum!addition!led!to!a!threefold!increase!in! SUMO! levels! associated! to! the! Id4! promoter! (Figure! R-11).! To! investigate! whether! Nab! SUMOylation! was! involved,! cells! were! transfected! with! wild! type! Nab! or! the! KR2! mutant! immediately!after!serum!deprivation.!Whereas!in!the!presence!of!wild!type!Nab!the!increase! of! SUMO! levels! was! similar! to! those! observed! in! the! absence! of! transfection,! serummediated!accumulation!of!SUMO!on!the!Id4!promoter!was!impaired!with!the!KR2!mutant,! presumably!due!to!competition!with!the!endogenous!Nab!(Figure!R-11).! ! ! ! ! ! Figure!R-11:!Serum!induction!triggers!SUMO1! accumulation!to!a!Nab-Krox20!regulated!DNA! sequence.!SUMO1!incorporation!levels!to!the! Id4!promoter,! analysed! by! chromatin! immunoprecipitation! (ChIP)! assays! and! quantitative!PCR!of!P19!cells!transfected!with! Flag-tagged! wild! type! Nab! or! with! the! KR2! mutant.!Untransfected!P19!were!also!included! in! the! experiments! (–).! SUMO1! incorporation! was! measured! under! normal! growth! conditions! (white! bars)! or! after! serum! stimulation! (black! bars).! Levels! of! SUMO1! incorporation! were! normalized! to! the! values! of! untransfected!and!untreated!cells.!Flag!incorporation!was!also!measured!as!a!control!(grey!bars)!and! values!were!normalized!to!untreated!cells!transfected!with!wild!type!Nab.! ! 3.5$ 2.5$ 2.0$ 1.5$ 1.0$ 0.5$ Fold$increase$ 3.0$$ Nab$ KR2$ –$ No$induc9on$ Serum$induc9on$ 0$ 4.5$ 4.0$$ an9-Flag$ $$$$$$$$$$$$$$ Results! ! 53! ! It!has!been!reported!that!CHD4!interacts!with!Nab!proteins!and!participates!in,!but! does!not!account!for,!full!repression!activity!associated!with!Nab!(Srinivasan,!R#et#al.,!2006),! which!indicates!the!existence!of!additional!mechanisms!involved!in!the!repressive!function! of! Nab.! To! this! point,! it! is! worth! nothing! to! mention! that,! among! the! numerous! roles! attributed! to! SUMO! in! the! eukaryotic! cell,! SUMOylation! has! been! mostly! linked! to! transcriptional!repression!(Garcia-Dominguez,!M!and!Reyes,!JC,!2009).!Thus,!we!wanted!to! analyse! how! Nab! SUMOylation! correlates! with! its! interaction! with! CHD4.! After! cotransfection! of! P19! cells! with! CHD4! and! the! wild! type! Nab! or! the! KR2! mutant,! immunoprecipitation!experiments!showed!how!CHD4!is!equally!bound!to!both!wild!type!Nab! and!KR2!(Figure!12),!suggesting!that!SUMOylation!is!not!involved!in!CHD4!recruitment.! ! ! Figure! R-12:! Nab! interaction! with! CHD4! is! not! altered! by! mutation! of! Nab! SUMOylation! sites.! 293T! cells! were! transfected! with! expression! vectors! of! a! HA-tagged! version! of! the! Nabinteracting! domain! from! CHD4! and! with! Flagtagged! versions! of! Nab! and! the! KR2! mutant.! Cell! extracts! were! subjected! to! immunoprecipitation! with! Flag! antibodies! and! the! precipitates! were! analysed!by!Western!blot!with!HA!antibodies.!10%!of!input!proteins!are!also!shown.! ! ! 4.1.4 Nab!SUMOylation!modulates!Krox20!activity!in!vivo.! ! ! To! evaluate! whether! Nab! SUMOylation! has! an! impact! on! Krox20! transcriptional! control,!we!took!advantage!of!a!lacZ-based!reporter!under!the!control!of!an!EphA4!element! (Garcia-Dominguez,!M#et#al.,!2006),!known!to!be!under!the!positive!control!of!Krox20!(Theil,! T#et#al.,!1998).!This!reporter!was!transfected!in!P19!cells!together!with!Krox20,!SUMO1!and! Nab! expression! constructs.! The! results! showed! how! the! reporter! was! activated! when! transfecting!the! Krox20!expression! construct! but! downregulated! when! also! cotransfecting! Nab,!which!could!be!modified!by!the!endogenous!SUMO1.!A!more!severe!downregulation!is! observed! when! SUMO1! is! also! cotransfected,! as! a! high! amount! of! free! SUMO1! would! be! available!in!this!condition.!The!sole!transfection!of!Krox20!with!SUMO1!showed!no!effect,!as! –" Flag-Nab2" –" +" HA-CHD4" +" +" +" –" Flag-KR2"–" +" Input"HA-CHD4" HA-CHD4" IP:"an:-flag" WB:"an:-HA" Input" Flag-Nab" Flag-KR2" Results! ! ! 54! the!amount!of!Nab!to!be!SUMOylated!was!not!changed.!Interestingly,!the!cotransfection!of! the! KR2! mutant! with! Krox20! resulted! in! an! increased! reporter! activity,! suggesting! a! dominant-negative!effect!of!the!KR2!mutant!(Figure!R-13).! ! ! ! ! ! ! Figure! R-13:! Nab! SUMOylation! represses! Krox20! transcriptional!activity!in!P19!cells.!A!LacZ!reporter! under!the!control!of!an!EphA4!element,!responsive! to! Krox20,! was! cotransfected! with! the! indicated! expression!constructs!to!analyse!the!transcriptional! activity!of!Krox20!in! P19! cells.!Relative! units!of!ßgalactosidase! activity! were! normalized! to! the! values! obtained! in! cells! only! cotransfected! with! Krox20.! Values! represent! means!±! s.d.! of! three! independent!experiments.! ! ! ! To!shed!light!on!how!SUMOylated!Nab!modulates!Krox20!activity!in!vivo,!we!decided! to! perform! a! functional! analysis! in! the! hindbrain.! In! this! embryonic! structure,! Krox20! regulates!various!genes!during!the!formation!of!rhombomeres!3!and!5!(r3!and!r5),!including! itself!and!Nab!genes!(Chomette,!D#et#al.,!2006;!Desmazieres,!A#et#al.,!2009;!Giudicelli,!F#et# al.,! 2001;! Mechta-Grigoriou,! F# et# al.,! 2000).! Prior! to! the! functional! analysis,! we! thought! appropriate!to!check!the!expression!of!Ubc9!in!the!same!territories!where!Krox20!and!Nab! express!during!hindbrain!development.!In!fact,!it!has!been!described!that!Ubc9!expression!is! regulated!during!nervous!system!development!in!rats!(Watanabe,!M#et#al.,!2008).!To!analyse! the! expression! pattern! of! Ubc9! we! turned! to! the! chick! embryo! and! performed! in# situ! hybridization!experiments!using!specific!riboprobes!(see!Materials!and!Methods).!As!shown! in!Figure!R-14A,!Ubc9!is!widely!expressed!in!the!whole!hindbrain,!but!higher!expression!can! be!observed!in!the!intersections!between!rhombomeres.!Double!in#situ!hybridizations!were! also!performed!including!riboprobes!against!Krox20,!which!expression!is!restricted!to!r3!and! r5.!Coexpression!of!both!markers!can!be!observed!in!Figure!R-14B.!To!conform!this!pattern,! immunofluorescence! experiments! were! conducted! to! analyse! the! expression! of! Ubc9! 140$ 100$ 80$ 60$ 40$ 20$ 0$ Rela,ve$units$(%)$ Krox20$ +$–$ +$ +$ Nab$ –$ –$ +$+$ KR2$ –$ –$ –$ –$ SUMO1$ –$ –$ –$ +$ +$ +$ –$ –$ –$ +$ +$ –$ 120$ +$ –$ +$ +$ 150$ Results! ! 55! together!with!EphA4!(Figure!R-14C),!expressed!in!r3!and!r5!and!under!the!direct!control!of! Krox20!(Theil,!T#et#al.,!1998),!and!the!neuron!specific!marker!ßIII-tubulin!(Figure!R-14D).!This! last!marker!reveals!progenitors!that!have!differentiated!into!neurons!and!that,!in!the!studied! developmental! stages,! mostly! appear! in! even! numbered! rhombomeres.! These! results! confirmed! that! Ubc9! is! expressed! in! rhombomeres! 3! and! 5.! A! detailed! analysis! of! Ubc9! expression! revealed! that,! as! shown! in! the! previous! in# situ! hybridization! experiments,! a! higher! accumulation! of! the! protein! was! observed! in! the! frontiers! between! rhombomeres! (Figure!R-14E).! ! ! ! ! Figure! R-14:! Expression! of! Ubc9! in! the! hindbrain.! Flat! mounts! from! the! hindbrain! of! HH13-14! chick! embryos! processed! to! analyse! the! expression! of! various! markers! in! rhombomeres!3!to!5.!(A,!B)!In#situ!hybridization! using! antisense! RNA! probes! against! Ubc9! (purple)! or! against! Ubc9! (orange)! and! Krox20! (purple).! (C,! D)!Immunofluorescence! using! antibodies!against!Ubc9!(red)!and!EphA4!(green)!(C)!or!against!Ubc9!(blue)!and!the!neural!marker! ßIII-tubulin! (red)! (D).! (E)!Detail! of! an! immuno-fluorescence! experiment! as! described! in! (C).! Rhombomere! 4! (r4)! marks! the! limits! of! EphA4! expression! (green)! in! r3! and! r5.! Higher! expression! levels!of!Ubc9!(red)!can!be!observed!in!the!intersections!between!rhombomeres!(white!arrows).! ! ! ! Electroporation! of! chick! embryos! allows! gain-of-function! analysis! in! the! hindbrain! (see!Materials!and!Methods).!This!technic!allows!the!transfection!of!the!neural!progenitors! from!one!side!of!the!hindbrain,!so!that!the!other!side!can!be!used!as!a!control!(Itasaki,!N#et# al.,!1999).!Using!this!technic,!we!analysed!the!effect!of!Nab!and!SUMO1!ectopic!expression! over!the!expression!levels!of!the!marker!EphA4!(Figure!R-15).!Limited!repression!of!EphA4! Ubc9% A" r3# r5# Ubc9%%%Krox20% B" r4# Ubc9###EphA4# C" r4# Ubc9###ßIII-tub# D" r3# r5# merge% Ubc9%%%EphA4% r3% r5% E" r4% Results! ! ! 56! after!electroporation!of!Nab!expression!vectors!has!been!previously!reported!(Desmazieres,! A# et# al.,! 2009)! and! was! confirmed! in! our! experiments! (Figure!R-15A,! 15B! and! 15I).! Interestingly,! we! observed! that! coelectroporation! of! Nab! expression! vectors! with! SUMO! expression!vectors!led!to!more!severe!repression!(Figure!R-15C,!15D!and!15I)!while!the!sole! electroporation!of!SUMO1!had!no!effect!(Figure!R-15E,!15F!and!15I).! ! ! ! ! ! ! Figure! R-15:! Nab! SUMOylation! represses! Krox20! transcriptional!activity!in!the!chick!hindbrain.!(A-H)!Flat!mount! of! the! hindbrain! from! HH13-14! chick! embryos! electroporated! on!the!left!side!with!the!indicated!expression!constructs.!24h! later,! embryos! were! harvested! and! immunofluorescence! experiments! were! conducted! to! analyse! the! transcriptional! activity!of!Krox20!using!EphA4!antibodies!(red).!To!monitor!the! electroporated!side!of!the! hindbrain,!a!GFP!expression!vector! was! included! in! all! the! experiments! (green).! (I)!Fluorescence! intensities! of! EphA4! were! measured! in! A,! C,! E! and! G! with! MetaMorph! software! (see! Materials! and! Methods).! For! this,! regions! of! the! same! area! in! r3! and! r5! were! defined! on! both! electroporated! and! control! sides! and! intensity! values! were! normalized!to!the!control!side!(–).!Values!are!means!±!s.d.!of!5-8!samples!from!three!independent! experiments.!Statistical!significance!was!analysed!using!the!Student’s!t-test.! ! KR2$ EphA4$$$$$$GFP$ Nab$ A B $$Nab$+$S1$ C D G H S1$ E F I" EphA4&fluorescence& –& A& C& E& G& *& ***& **& 2.0& 1.5& 1.0& 0.5& 0.0& 2.5& &&&&&&&&&&&&&&&& Results! ! 57! ! To!check!that!these!results!were!due!to!Nab!SUMOylation!and!not!to!simultaneous! overexpression! of! Nab! and! SUMO1,! the! SUMOylation! defective! mutant! KR2! was! tested,! showing!a!very!interesting!result!as!EphA4!expression!was!not!reduced!but!increased!in!the! electroporated!side!(Figure!R-15G,!15H!and!15I).!This!result!supports!a!dominant-negative! effect!of!the!KR2!mutant!that!would!compete!with!wild!type!Nab!for!interacting!with!Krox20.! Taking! these! results! into! consideration,! we! decided! to! further! investigate! the! effects! of! electroporating!the!KR2!mutant.!Expression!analysis!of!the!r4!marker!Hoxb1!together!with! EphA4!by!double!in#situ!hybridization!showed!that!electroporation!of!the!KR2!mutant!also! resulted! in! reduced! r4! size! (Figure! R-16A! and! 16B),! possibly! associated! with!aberrant! expansion!of!r3!and/or!r5.!Finally,!we!analysed!the!effects!of!electroporating!the!KR2!mutant! over!Krox20!expression,!as!it!is!described!to!be!self-regulated!in!a!positive!way!(Chomette,!D# et# al.,! 2006;! Schneider-Maunoury,! S# et# al.,! 1993).! Expression! of! Krox20! itself! appeared! upregulated!after!KR2!electroporation,!similarly!to!EphA4!(Figure!R-16A!and!16B).!Together,! these!data!support!an!involvement!of!Nab!SUMOylation!in!the!repression!of!Krox20!activity! during!hindbrain!development.! ! ! Figure! R-16:! Nab! modification! by! SUMO1! affects! the! expansion! of! r3! and! r5.!(A)! Flat! mounts!from!the!hindbrain!of!HH13-14!chick! embryos! electroporated! on! the! left! side! (*)! with! the! Nab! KR2! expression! construct.! Embryos! were! harvested! 24h! after! electroporation! and!processed! for! in# situ! hybridization.! Specific! antisense! RNA! probes! against! EphA4!(orange),! Hoxb1!and! Krox20! (purple)! were!utilised!to!analyse!these!genes! expression!and!determine!the!extension!of!r4! territory!(white!arrows).!(B)!The!size!of!r4!was! measured! as! the! extension! of! Hoxb1! expression! with! ImageJ! software! and! the! intensity!of!Krox20!expression!was!measured! with!MetaMorph!software!(see!materials!and! Methods).!In!both!cases,!signal!values!of!the! electroporated!sides!were!normalized!to!their! control! sides.! Values! are! means! ±! s.d.! of! 5-8! samples! from! three! independent! experiments.!Statistical!significance!was!analysed!using!the!Student’s!t-test.! Hoxb1&(r4$size)$ Controls$ Krox20$$ (r3/r5$intensi2es)$ **$ *$ 2.0$ 1.5$ 1.0$ 0.5$ 0.0$ 2.5$ Rela2ve$levels$ B" A" Krox20& KR2$ EphA4&&&Hoxb1& *$*$ Results! ! ! 64! ! Analysis!of!Neurofilaments!also!confirmed!these!results!(Figures!R-21A!and!21C).!To! investigate! whether! SUMO! overexpression! in! fact! interferes! with! neuronal! differentiation! rather!than!simply!delaying!it,!we!also!analysed!effects!of!SUMO!overexpression!40!h!after! electroporation.!Results!were!similar!to!those!obtained!at!30!h!(Figure!R-22),!supporting!an! impairment!of! the! process! rather! than! a! delay.! These! results! confirm!in! an! animal! model! that!overexpressing!SUMO!interferes!with!neuronal!differentiation.! ! ! ! ! ! Figure!R-22:!SUMO!overexpression!impairs!neuronal!differentiation!rather!than!delaying!it.!(A)!The! neural!tube!of!chick!embryos!at!stage!HH17!was!electroporated!on!the!right!side!as!previously!shown! in! Figure! R-21.! Neurogenesis! was! analysed! 40h! after! electroporation! by! immunofluorescence! on! transverse!sections!of!the!neural!tube!using!ßIII-tubulin!antibodies! (red).!DNA!was!counterstained! with!DAPI!(blue).!Electroporated!cells!that!expressed!GFP!and!were!differentiating!into!neurons!were! identified! by! localization! to! the! mantle! layer! (ML)! and! expression! of! the! neuron! specific! marker! ßIII-tubulin.!Scale!bar!50!μm.!(B)!Quantification!of!neurogenesis!in!(A)!obtained!as!the!percentage!of! GFP!positive!cells!(green)!that!have!migrated!from!the!proliferative!compartment!or!ventricular!zone! (VZ)!to!the!differentiated!compartment!(ML)!and!that!express!ßIII-tubulin.!Values!are!means!±!s.d.! obtained! by! quantifying! cells! of! a! total! of! 6! sections! from! 2! independent! experiments.! Statistical! analysis!was!performed!using!the!Student’s!t-test.!! A" 40#h# ßIII-tubulin# Ngn2# Empty# SUMO1# SUMO2# Empty# GFP# GFP# DAPI# 100# 50# 0# %#neurogenesis# (ßIII-tubulin)# –# Ngn# Ngn#+#S1# Ngn#+#S2# B" ***# ***# Results! ! 65! 4.2.3 Neuronal!differentiation!proceeds!without!significant!changes!in!gene!expression!of! SUMO!ligases!and!basic!pathway!components.! ! ! As! the! previous! results! showed! that! SUMOylation! is! altered! during! neuronal! differentiation,! it! seemed! appropriate! to! investigate! the! components! of! its! modification! pathway!during!this!process.!For!this!purpose,!we!undertook!a!gene!expression!analysis!of! the!basic!components!of!the!SUMOylation!pathway!and!SUMO!ligases!of!the!PIAS!family.! ! The!RA!differentiation!protocol!of!P19!cells!involves!two!periods!of!two!days!in!which! cells!are!treated!with!RA!in!non-adherent!dishes,!where!they!form!embryonic!bodies,!plus!a! period!of!three!days!in!which!cells!are!disaggregated!and!cultured!in!adherence!without!RA.! At! this! point,! cells! have! reached! an! advanced! differentiation! state! and! have! extended! dendrite!and!axon-like!processes.!Thus,!0,!2,!4!and!7!days!appeared!as!relevant!points!for! the!analysis!of!neuronal!differentiation!(Figure!R-23).! ! ! ! ! Figure! R-23:! RA! treatment! to! induce! neural! differentiation! of! P19! cells.!P19!cells!are!cultured!in! suspension!with!low!serum!and!RA!supplemented!medium!to!induce!the!initial!response!(1).!Cells!are! grown! in! these! conditions! during! four! days! (with! RA! replacement! at! day! 2)! in! which! they! form! embryoid!bodies!and!the!initial!differentiation!takes!place!(2).!Finally,!P19!cells!are!disaggregated! and! plated! in! adherence! and! grown! in! complete! medium! without! RA! to! reach! the! terminal! differentiation,!in!which!axon!and!dendrite!like!processes!start!to!extend!(3).! ! ! ! After!designing!oligonucleotides!coding!regions!of!genes!corresponding!to!different! components!of!the!SUMO!modification!pathway!from!mouse!(see!Table!M-6)!real-time!PCR! analysis!showed!in!Figure!R-24!(see!Materials!and!Methods)!indicated!that!genes!coding!for! Sumo1,!Sumo2!and!Sumo3!were!not!significantly!altered!along!the!differentiation!protocol.! Those!coding!for!other!basic!components!of!the!SUMOylation!pathway,!notably!monomers! 3" 1" 2" Suspension" Adherence" RA" Days" RA" RA" Results! ! ! 66! of!the!E1!heterodimer!Sae1/Uba2!and!the!conjugating!enzyme!Ubc9!similarly!did!not!show! significant!differences!(Figure!R-24).!Regarding!genes!coding!for!PIAS!ligases,!some!of!them! showed!a!progressive!increase!along!differentiation,!with!maximum!levels!at!7!days,!and!not! exceeding!a!3-fold!increase!in!any!case.!As!a!control!of!the!cellular!differentiation!process,! the! pluripotency! gene! Oct4! appeared! drastically! downregulated! at! day! 2,! while! high! expression!levels!of!the!neuronal!ßIII-tubulin!gene!(Tubb3)!were!detected!from!day!4!(Figure! R-24).!Hence,!gene!expression!analysis!indicates!that!genes!coding!for!SUMO!molecules!and! basic!components!of!the!SUMOylation!pathway!are!not!significantly!subjected!to!regulation! along!the!neuronal!differentiation!protocol!in!P19!cells,!while!some!PIAS!ligases!genes!are! moderately!upregulated.! ! ! ! ! Figure! R-24:! Expression! analysis! of! PIAS! ligases! and! basic! components! of! the! SUMOylation! machinery! during! P19! neuronal! differentiation! with! RA! treatment.! Real-Time! PCR! determined! relative! expression! levels! of! the! indicated! genes.! P19! cells! were! harvested! after! the! following! conditions:!2!or!4!days!(d)!of!RA!treatment!in!suspension,!and!4!days!of!RA!treatment!in!suspension! following! growth! on! adherent! dishes! for! 3! more! days! (7).! Proliferative! cells! grown! in! adherence! were!also!harvested!as!t!=!0!(0).!Expression!levels!were!normalized!to!the!levels!at!0!days.!Values!are! means!±!s.d.!of!three!independent!experiments!in!duplicate.!Note!that!Y-axis!for!Oct4!and!Tubb3!are! different!from!the!rest!of!genes.!Statistical!significance!of!changes!in!gene!expression!was!analysed! by!the!Student's!t-test,!and!P-values!for!Pias2,!Pias3,!Pias4!and!Tubb3!upregulation,!as!well!as!for! Oct4!downregulation,!were!indicated.! ! ! 3" 0" Rela(eve"levels" 0" RA"(d)" 2"4"7" 3" 0" Rela(ve"levels" 0"RA"(d)" 2"4"7" 3" 0" 0"2"4"7" 3" 0" 0"2"4"7" 3" 0" 0"2"4"7" 3" 0" 0"2"4"7" 3" 0" 0"2"4"7" 3" 0" 0"2"4"7" 3" 0" 0"2"4"7" 3" 0" 0"2"4"7" 1" 0" 0"2"4"7" 20" 0" 0"2"4"7" Sumo1& ***" ***" *" **" ***" Sumo2& Sumo3& Sae1& Uba2& Ubc9& Pias3& Pias4& Oct4& Tubb3& Pias2& Pias1& Results! ! 67! 4.2.4 The! SUMO! proteases! Senp5! and! Senp7! are! upregulated! during! neuronal! differentiation.! ! ! We! continued! our! gene! expression! analysis! by! looking! at! genes! coding! for! SUMO! proteases.!For!that!purpose,!we!investigated!the!six!members!of!the!SENP!family!(Senp1-3,! 5-7)!and!one!member!of!the!more!recently!described!DeSI!family!of!proteases,!Desi1!(Shin,! EJ# et# al.,! 2012).! Results! indicated! that! expression! levels! of! most! of! the! analysed! genes! remained!quite!unaltered!along!the!differentiation!protocol,!with!the!surprising!exception!of! Senp5!and!Senp7.!Senp5!expression!progressively!augmented!to!reach!a!9-fold!increase!at! day!7!(Figure!R-25A).!In!contrast,!Senp7!levels!picked!at!day!4,!displaying!a!16-fold!increase!if! compared! with! day! 0,! and! dropping! slightly! at! day! 7! (Figure! R-25A).! As! Senp7!gene! expression! displayed! the! highest! variation! following! induction! of! neuronal! differentiation,! we!chose!to!further!investigate!it.! ! We! first! studied! whether! increased! gene! expression! correlated! with! increased! protein!levels.!Western!blot!experiments!demonstrated!a!4.3-!fold!increase!in!protein!levels! at! day! 4,! which! decreased! to! 2.8-fold! by! day! 7! (Figures! R-25B! and! 25C).! As! controls,! we! confirmed!downregulation!of!Oct4,!upregulation!of!ßIII-tubulin!and!no!changes!in!α-tubulin! (Figures!R-25B!and!25C).!To!exclude!that!the!obtained!results!were!due!to!a!direct!effect!of! RA!rather!than!the!process!of!neuronal!differentiation,!we!confirmed!upregulation!of!Senp7! also! when! applying! the! neuronal! differentiation! protocol! involving! transfection! of! the! neurogenic! factor! NeuroD2! and! its! cofactor! E12! (Figure! R-26).! As! an! additional! control,! Senp3!levels! were!also! checked! and!no! differences!were! observed!between! days! 0!and! 4! (Figure!R-26).!Altogether,!these!results!show!that!both!mRNA!and!protein!levels!of!Senp7! are!upregulated!following!induction!of!neuronal!differentiation!in!P19!cells.! ! ! ! ! ! ! ! ! Results! ! ! 68! ! ! ! ! Figure! R-25:! Senp5! and! Senp7! are! upregulated! during! P19! neuronal! differentiation! by! RA! treatment.!(A)!Real-Time!PCR!determined!relative!expression!levels!of!the!indicated!SUMO!proteases! genes.! P19! cells! were! treated! as! previously! indicated! in! Figure! R-24.! Expression! levels! were! normalized! to! the! levels! at! 0! days.! Values! are! means! ±! s.d.! of! three! independent! experiments! in! duplicate.!Statistical!significance!of!changes!in!gene!expression!was!analysed!by!the!Student's!t-test,! and! P-values! for! Senp5!and! Senp7!upregulation! were! indicated.! (B)!Protein! levels! of! Senp7! were! analysed!by!Western!blot!along!the!differentiation!protocol!applied!in!(A).!Levels!of!Oct4!and!ßIII- tubulin! were! determined! as! controls! for! neurogenesis.! Blots! for! α-tubulin! are! also! included! as! a! loading!control.!20!μg!of!total!protein!were!loaded!per!lane.!(C)!Relative!levels!of!Senp7!protein!as! quantified!in!(B).!Protein!levels!were!normalized!to!the!level!at!0!days.!Values!are!means!±!s.d.!from! four! independent! experiments.! Statistical! significance! of! this! experiment! was! analysed! by! the! Student's!t-test!and!P-value!for!the!increase!at!day!4!with!respect!to!day!0!was!indicated.! ! ! ! 0" 2" 4" 7" RA"(d)" Senp7" Oct4" ßIII-tubulin" α-tubulin" 130" 45" 55" 55" kDa" B" 0"RA"(d)" 2" 4" 7" 0" 1" 2" 3" 4" 5" Niveles"relaDvos" Senp7" α-tubulin" ***" C" 20" 20" 0" RelaDve"levels" 0"RA"(d)" 2" 4" 7" Senp1& 20" 0" 0" 2" 4" 7" Senp2& 0" 0" 2" 4" 7" Senp3& 20" 0" 0" 2" 4" 7" ***" Senp5& 20" 0" 0" 2" 4" 7" Desi1& 20" 0" 0" 2" 4" 7" ***" Senp7& 20" 0" 0" 2" 4"7" Senp6& RelaDve"levels" RA"(d)" A" Results! ! 69! ! ! ! ! ! ! Figure! R-26:! Both! RA! treatment! and! overexpression! of! neurogenic! factor! NeuroD2! upregulate! Senp7.!!Senp7,! Senp3,! Oct4,! ßIII- tubulin! and! α-tubulin! protein! levels! were! analysed! by! Western! blot! in! P19! cells! treated! with! RA! for! 0! or! 4! days! (d)! (left),! or! in! P19! cells! transfected! with! empty! vector! (−)! or! expression! constructs!for!NeuroD2!and!E12!(ND)!for!3!days!(right).!20!μg!of!total!protein!were!loaded!per!lane.! ! ! 4.2.5 Senp7!is!required!for!proper!neuronal!differentiation.! ! ! To!functionally!investigate!the!role!of!Senp7!in!neuronal!differentiation,!we!opted!for! a!loss!of-function!approach.!Thus,!we!generated!expression!constructs!for!short!hairpin!RNA! (shRNA)! molecules! in! order! to! knockdown! Senp7! by! RNA! interference! in! P19! cells.! We! generated!two!different!molecules!targeting!Senp7!and!both!performed!equally!in!knocking! down!the!SUMO!protease!and!in!the!first!experiments!of!the!functional!characterization,!so! results! presented! correspond! to! one! of! these! molecules! (shRNA! mouse! Senp7#1)! (see! Table!M-4).!In!P19!cells!under!proliferation,!Senp7!showed!basal!expression!levels!that!were! lessened!to!a!33%!in!cells!transfected!with!the!shRNA!molecule!against!Senp7,!compared!to! a! control! shRNA! (Figures!R-27A! and! 27C).! As! Senp7! upregulation! was! detected! after! induction! of! neuronal! differentiation,! we! decided! to! also! check! Senp7!knocking! down! by! cotransfection! of! the! shRNA! against! Senp7! in! addition! to! NeuroD2! and! E12.! It! was! under! these! neuronal! differentiation! conditions! that! a! 10-fold! reduction! in! the! SUMO! protease! expression! was! observed! when! compared! to! cotransfection! of! a! Control! shRNA! construct! and!the!neurogenic!factors!(Figures!R-27B!and!27D).!We!then!turned!to!analyse!the!possible! effects! that! Senp7!knocking! down! could! have! over! SUMOylated! products! accumulation! under!neuronal!differentiation!conditions.!While!no!appreciable!differences!were!observed! in!free!and!conjugated!SUMO1!levels!when!comparing!effects!of!Control!and!Senp7!shRNA! molecules!under!neuronal!differentiation!conditions,!a!clear!effect!on!SUMO2/3!conjugation! RA#(d)# 0# 4# Transfec1on# –# ND#kDa# 130# 45# 70# 55# 55# Senp7# Senp3# Oct4# ßIII-tubulin# α-tubulin# ################ Results! ! ! 70! was!observed.!Thus,!under!neuronal!differentiation!conditions,!Senp7!shRNA!preserved!high! molecular! mass! products! from! deconjugation! in! comparison! with! Control! shRNA,! which! correlated!with!no!increase!in!free!SUMO2/3!(Figure!R-28).! ! ! ! Figure! R-27:! Senp7! knockdown! during! P19! neuronal! differentiation.!(A)!P19! cells! growing! under! proliferative! conditions! were! transfected! with! either! control! shRNA! (shC)! or! Senp7! shRNA! (sh7)! expression! constructs,! and! Senp7! protein! levels! were! analysed! 72h! after! transfection! by! western! blot.! (B)!P19! cells! were! transfected! with! NeuroD2! and! E12! expression! constructs! or! with! the! corresponding!empty!vector!(–).!Cells!transfected!with!NeuroD2!and!E12!expression!constructs!were! cotransfected!with!either!control!shRNA!(shC)!or!Senp7!shRNA!(sh7)!expression!constructs,!or!with! the!corresponding!empty!vector!(–).!Senp7!levels!were!analysed!72h!after!transfection!by!Western! blot.!(A,!B)!α-tubulin!was!revealed!as!a!loading!control.!20!μg!of!total!protein!were!loaded!per!lane.! (C,!D)!Quantification!of!relative!protein!levels!determined!in!(A)!and!(B),!respectively.!Protein!levels! in!(C)!were!normalized!to!the!levels!of!cells!transfected!with!the!control!shRNA!construct.!Protein! levels!in!(D)!were!normalized!to!the!levels!of!cells!transfected!with!empty!vectors.!Values!are!means! ±! s.d.! of! two!independent! experiments.! Statistical! significance! of! Senp7! knockdown! by! the! Senp7! shRNA!with!respect!to!control!shRNA!in!(C)!and!with!respect!to!Control!shRNA!or!empty!vector!in!(D)! was!analysed!by!the!Student’s!t-test.! ! ! ! ! 130$ 55$ Senp7$ α-tubulin$ shC$sh7$kDa$ A" 130$ 55$ Senp7$ α-tubulin$ shC$sh7$–$–$ NeuroD2/E12$ kDa$ B" Senp7$ α-tubulin$ 1$ 0,5$ 0$ shC$sh7$ RelaAve$levels$ C" ***$ Senp7$ α-tubulin$ 5$ 3$ 0$ 1$ 4$ 2$ shC$sh7$–$–$ NeuroD2/E12$ RelaAve$levels$ D" ***$ **$ Results! ! 71! ! ! Figure! R-28:! Senp7! knockdown!prevents! deconjugation! of! SUMO2/3! during! P19! neuronal! differentiation.!Effect!of!Senp7!shRNA!compared!to!control!shRNA!on!SUMO1!(left)!and!SUMO2/3! (right)! conjugation! was! verified! by! Western! blot! of! denaturing! protein! extracts! from! P19! cells! transfected! as! indicated! in! Figure! R-27B.! α-tubulin! was! revealed! as! a! loading! control.! Arrowhead! indicates!SUMOylated!RanGAP1!(Ran)!on!SUMO1!blot.!30!μg!of!total!protein!were!loaded!per!lane.! ! ! ! We!then!performed!a!neurogenesis!analysis!similar!to!the!one!previously!described! (Figures!R-19!and!R-21).!Thus,!NeuroD2/E12-induced!neurogenesis!in!P19!cells!corresponded! to! 61%! of! transfected! cells! and! interestingly! dropped! to! 27%! when! cotransfecting! an! expression! construct! for! Senp7! shRNA,! while! no! significant! effects! were! observed! when! using!a!Control!shRNA!expression!construct!(58%!of!neurogenesis)!(Figures!R-29A!and!29B).! We! also! developed! two! different! molecules! targeting! the! chick! Senp7!sequence.! Both! molecules! also! performed! equally! in! the! loss-of-function! analysis! so! results! presented! correspond!to!one!of!these!molecules!(shRNA!chick!Senp7#1)!(see!Table!M-4).!The!obtained! results! indicated! that! when! electroporating! chick! Senp7! shRNA,! Neurogenin2-promoted! differentiation!was!significantly!impaired,!while!a!Control!shRNA!molecule!had!no!effect.!The! use!of!Senp7!shRNA!lessened!neurogenesis!to!40%,!while!almost!80%!was!observed!in!the! presence! of! Control! shRNA! (Figures! R-29C! and! 29D).! Thus,! induced! neurogenesis! by! overexpression! of! neurogenic! factors,! both! in! P19! cells! and! in! chick! embryos,! is! severely! compromised!by!Senp7!knock-down.! ! ! kDa$ 250$ 130$ 100$ 70$ 55$ 15$ shC$sh7$–$–$ NeuroD2/E12$ shC$sh7$–$–$ NeuroD2/E12$ Free$ SUMO1$ Free$ SUMO2/3$ α-tubulin$ α-tubulin$ Conjugated$ SUMO1$ Conjugated$ SUMO2/3$ SUMO1-Ran$ Results! ! ! 72! ! ! ! ! ! Figure!R-29:!Senp7!knockdown!impairs!neuronal!differentiation!in!both!P19!cells!and!chick!neural! tube.!(A)!P19!cells!were!transfected!with!empty!vector!or!expression!vectors!for!NeuroD2!and!E12!to! induce!neuronal!differentiation,!together!with!expression!constructs!for!Senp7!shRNA!(sh!Senp7)!or! control! shRNA! (sh! Control)! molecules,! or! the! corresponding! empty! vector.! Transfected! cells! were! monitored!by!expression!of!a!GFP!expression!vector!(green)!and!those!differentiating!into!neurons! were! revealed! by! expression! of! the! neuron! specific! marker! ßIII-tubulin! (red).! DNA! was! counterstained!with!DAPI!(blue).!Scale!bar!20!μm.!(B)!Quantification!of!the!%!of!neurogenesis!of!the! analysis!in!(A)!as!the!proportion!of!GFP!positive!cells!(green)!expressing!the!ßIII-tubulin!marker!(red).! Values! are! means±! s.d.! obtained! by! counting! cells! from! a! total! of! 30! areas! of! 3! independent! experiments.! Statistical! significance! of! the! effect! of! the! Senp7! shRNA! construct! on! neuronal! differentiation!with!respect!to!Control!shRNA!or!empty!vector!was!analysed!by!the!Student's!t-test.! (C)!The!neural!tube!of!chick!embryos!at!stage!HH17!was!electroporated!on!the!right!side!with!empty! vector!or!an!expression!vector!for!Neurogenin2!(Ngn2)!to!induce!neuronal!differentiation,!together! with! expression! constructs! for! Senp7! shRNA,! control! shRNA! or! the! corresponding! empty! vector.! Neurogenesis!was!evaluated!by!immunofluorescence!after!30h!on!transverse!sections!of!the!spinal! cord.! Electroporated! cells! were! monitored! by! expression! of! a! GFP! reporter! (green)! and! those! differentiating!into!neurons!were!identified!by!localization!to!the!ML!and!expression!of!the!neuronal! ßIII-tubulin* C" 30*h* NeuroD2/E12* Empty* sh*Control* sh*Senp7* ßIII-tubulin* A" 100* 50* 0* %*neurogenesis* (P19*cells)* **** **** –* ND* ND*+*shC* ND*+*sh7* D Empty* GFP* Ngn2* Empty* sh*Control* sh*Senp7* Empty* GFP* GFP* DAPI* GFP* DAPI* 100* 50* 0* %*neurogenesis* (neural*tube)* **** **** –* Ngn2* Ngn2*+*sh7* B" Ngn2*+*shC* Results! ! 73! marker! ßIII-tubulin! (red).! DNA! was! counterstained! with! DAPI! (blue).! Scale! bar! 50! μm.! (D)! Quantification!of!the!%!of!neurogenesis!of!the!analysis!in!(C)!as!the!percentage!of!the!GFP!positive! cells!(green)!that!have!migrated!to!the!ML!and!express!the!neuronal!marker!ßIII-tubulin!(red).!Values! are!means!±!s.d.!obtained!by!counting!cells!in!a!total!of!6!sections!from!2!independent!experiments.! Statistical!significance!of!the!effect!of!the!Senp7!shRNA!construct!on!neuronal!differentiation!with! respect!to!Control!shRNA!was!analysed!by!the!Student's!t-test.! ! ! 4.2.6 The! Senp7! promoter! is! transiently! activated! following! induction! of! neuronal! differentiation.! ! ! To!get!insights!into!the!way!Senp7!responds!to!induction!of!neuronal!differentiation,! we!performed!an!in#vivo!reporter!analysis!in!the!developing!neural!tube.!Thus,!we!decided! to!analyse! reporter! activation!of! a! GFP!construct! driven!by!the! 5ʹ!upstream!region! of!the! Senp7!gene,!encompassing!the!Senp7!promoter!(S7p).!To!this!end,!we!compared!expression! of! this! construct! (S7p-GFP)! with! that! of! a! GFP! driven! by! the! constitutive! promoter! CMV! (CMV-GFP)!in! electroporation!experiments! on! the!chick! neural!tube.! We!initially! used!the! human! S7p! sequence,! as! it! was! available! in! our! laboratory.! In! all! the! experiments,! transfected! cells! were! monitored! by! expression! of! the! mCherry! protein! driven! by! a! CMV! promoter! (CMV-mCherry).! This! expression! construct! was! coelectroporated! with! the! GFP! constructs!(Figure!R-30)!and!analysed!10-20!h!after!electroporation.! ! ! ! ! Figure!R-30:!Schematic!representation!of!the!reporter!constructs!used!to!analyse!the!activation!of! Senp7!expression! during! neurogenesis.!GFP!or!monomer!Cherry!reporters!were!placed!under!the! control!of!the!constitutive!CMV!promoter!(CMV)!and!GFP!reporter!was!also!placed!under!the!control! of!the!promoter!region!of!the!human!Senp7!gene!(S7p).! ! ! CMV$ CMV$ S7p$ Cherry$ GFP$ GFP$ CMV-Cherry$ CMV-GFP$ S7p-GFP$ Results! ! ! 80! be! obtained! in! a! single! experiment! comparing! two! different! labelled! conditions.! These! considerations! made! us! to! work! with! a! high! number! of! cells! in! the! final! large-scale! experiment! (see! Materials! and! Methods),! detailed! in! the! workflow! chart! shown! in! Figure!R-35.! ! ! ! ! Figure!R-35:!Large-scale! workflow!for!SILAC!experiment! and! further! SUMO!immunoprecipitation! (IP)!of!proliferating!and!differentiating!P19!cells.!The!cells!for!both!direct!and!reverse!SILAC!sets!of! experiments!were!cultured!in!SILAC!Lys!and!Arg-free!media!and!harvested!as!represented:!the!first! set!(Exp.!1)!was!cultured!with!D4!Lysine!and!13C!Arginine!heavy!labelled!amino!acids!(heavy!AA)!and! non-labelled!amino!acids!(light!AA)!in!proliferating!(Prolif.)!and!differentiating!(Dif.!RA)!conditions,! respectively.! In! the! second! set! (Exp.! 2),! light! AA! and! heavy! labelled! AA! were! employed! in! proliferation! and! differentiation! conditions,! respectively.! After! harvesting,! lysing! and! mixing! proliferative! and! differentiating! cells! from! each! experiment! as! indicated,! three! SUMO! IPs!per! experiment! were! performed! using! specific! antibodies! against! SUMO1,! SUMO2/3! and! control! IgG.! Inputs!and!eluates!from!the!SUMO!IP!were!sent!for!mass!spectrometry!(MS)!analysis.!! ! ! Exp.%1%Direct% Prolif.% Prolif.% Prolif.% Dif.%RA% Heavy%AA% Light%AA% SUMO%IP% (SUMO1;%SUMO2/3;%IgG)% MS%Analysis% Medium%w/o%Arg/Lys% Exp.%2%Reverse% Prolif.% Prolif.% Prolif.% Dif.%RA% SUMO%IP% (SUMO1;%SUMO2/3;%IgG)% MS%Analysis% Medium%w/o%Arg/Lys% Heavy%AA%Light%AA% Heavy%AA% Light%AA% Heavy%AA%Light%AA% Results! ! 81! 4.3.2 SUMO! immunoprecipitation! and! peptide! elution! effectively! enriches! P19! cell! extracts!with!endogenously!SUMOylated!proteins.! ! ! To! immunoprecipitate! SUMO! conjugates,! the! use! of! an! affinity! matrix! coupled! to! SUMO! antibodies! is! needed.! In! this! work,! SUMO1! and! two! different! batches! of! SUMO2! antibodies!were!obtained!from!hybridoma!21C7!and!8A2!cells!(The!Developmental!Studies! Hybridoma!Bank!at!the!University!of!Iowa),!respectively,!to!be!coupled!to!the!agarose!beads! that! form! the! affinity! matrix.! Control! beads! were! also! prepared! with! commercial! normal! mouse! IgG.! We! checked! the! coupling! efficiency! comparing! the! release! of! heavy! and! light! chains! of! the! antibodies! prior! and! after! crosslink! treatment! to! the! agarose! beads! by! SDS-PAGE!(Figure!R-36).! ! ! ! ! ! ! Figure! R-36:! Coomassie! staining! of! agarose! beads! coupled! to! SUMO1! and! two! batches! of!SUMO2! antibodies! as! well! as! control! IgG.!The! beads! incubated!with!each!SUMO! antibody! or! IgG! were! crosslinked,! taking! control! samples! before! (Non! X-link)!and!after!(X-link)!the! crosslink!treatment.!To!test! crosslinking! efficiency,! bead! supernatants! from! the! control! samples! were! subsequently! incubated! in! the! presence! of! SDS! at! 37°!C! or! boiled! with! SDS!+!DTT! at! 96°! C! and! separated! by! SDS-PAGE.! Coomassie! staining! shows! how! denatured! light! and! heavy!chains!from!the!antibodies!are!almost!absent!from!supernatants!of!the!crosslinked!fractions! while!they!are!strongly!present!(arrows)!in!supernatants!of!the!non-crosslinked!fractions.! 37# 96# (°C)# 37# 96# 37# 96# 37# 96# 37# 96# 37# 96# 37# 96# 37# 96# Non#X-link# Non#X-link# Non#X-link# Non#X-link# X-link# X-link# X-link# X-link# Control#IgG# αSUMO1# αSUMO2.1# αSUMO2.2# (°C)# 200# 120# 100# 70# 50# 40# 30# 25# 200# 120# 100# 70# 50# 40# 30# 25# kDa# kDa# Results! ! ! 82! ! As!expected,!when!analysing!bead!pellets!and!supernatants,!non-crosslinked!samples! showed!a!high!amount!of!heavy!and!light!chains!from!the!antibodies,!both!in!bead!pellets! and! supernatants,! indicating! the! successful! binding! of! antibodies! to! the! beads.! When! analysing! the! crosslinked! samples,! no! antibody! chains! were! observed! in! the! supernatants! and! only! a! small! fraction! appeared! in! the! beads,! indicating! that,! though! being! incubated! with! DTT,! crosslinking! was! efficient! and! most! of! the! antibodies! remained! attached! to! the! beads.!These!satisfactory!results!were!obtained!for!control!and!SUMO1!beads,!as!well!as!for! the!second!batch!of!SUMO2!beads.! ! After!harvesting!all!cellular!samples!of!both!sets!of!SILAC!experiments!according!to! the!workflow!chart!in!Figure!R-35!(see!Materials!and!Methods),!cell!lysates!were!made!in! radioimmunoprecipitation! assay! (RIPA)! conditions! according! to! the! protocol! detailed! in! (Barysch!et!al.!2014).!For!each!set!of!experiments!the!protein!concentration!was!determined! for!proliferative! and! differentiating! samples,!both! samples! of! each!set! were! mixed!to! the! same! protein! amount! and! inputs! were! taken.! Once! again,! amino! acid! incorporation! efficiency!was!determined!to!be!98%!and!the!Arg!to!Pro!conversion!was!minimum.!Then,! immunoprecipitation!(IP)!of!SUMO1,!SUMO2!and!control!IgG!was!performed!for!each!set!of! lysates! using! the! previously! synthesized! beads! and,! after! elution! with! specific! peptides! corresponding! to! the! epitopes! recognized! by! the! antibodies,! SUMO! conjugates! were! efficiently! purified! as! shown! by! Western! blot! (Figure! R-37).! Finally,! for! each! set! of! experiments,! inputs! and! eluted! IgG,! SUMO1! and! SUMO2! samples! were! sent! to! the! Core! Facility! for! Mass! Spectrometry! and! Proteomics! (CFMP)! at! the! ZMBH! for! MS! analysis! and! identification! of! proteins! that! appear! to! be! SUMOylated! in! one! of! the! proliferating! or! differentiating!conditions!and!get!deSUMOylated!in!the!contrary!condition.! ! ! ! ! ! ! ! ! ! Results! ! 83! ! ! ! ! ! Figure!R-37:!Purification!of!endogenously!SUMOylated!products!in!proliferative!and!differentiating! P19!cells.!P19!cells!cultured!for!SILAC!under!proliferation!and!differentiation!(see!figure!R-35)!were! harvested! and! lysated! in! denaturing! conditions.! After! SUMO! IP! (see! Materials! and! Methods),! Western!blot!analysis!showed!purified!endogenously!SUMOylated!products!when!comparing!IgG!and! SUMO1!or!SUMO2!eluted!proteins.!0.05%!of!the!eluates!(Eluted)!and!flow-throughs!(Flowthr.)!were! loaded!and!revealed!with!SUMO1!(A)!and!SUMO2!(B)!antibodies!for!each!set!of!SILAC!experiments.! 0.02%! of! inputs! relative! to! the! lysates! volume! were! also! loaded.! Flow-through! lanes! show! how! SUMOylated!proteins!are!efficiently!bound!to!the!matrix!when!comparing!SUMO1!and!SUMO2!with! the!control!IgG.! Input& Flowthr.& Exp.&1& Exp.&2& IgG& SUMO1& Eluted& IgG& SUMO1& Input& Flowthr.& IgG& SUMO1& Eluted& IgG& SUMO1& SUMO1-Ran& Conjugated& SUMO1& A" 200& 120& 100& 70& 50& kDa& 40& Exp.%1% Exp.%2% Input% Flowthr.% IgG% SUMO2% Eluted% IgG% SUMO2% Input% Flowthr.% IgG% SUMO2% Eluted% IgG% SUMO2% Free% SUMO2/3% Conjugated% SUMO2/3% B" 200% 120% 100% 70% 50% kDa% 40% 30% 25% 20% Results! ! ! 84! 4.3.3 Several! proteins! are! identified! as! differentially! SUMOylated! targets! between! proliferative!and!differentiating!P19!cells.! ! ! Mass! spectrometry! data! analysis! was! conducted! in! the! group! of! Prof.! Frauke! Melchior,!at!the!ZMBH!in!Heidelberg.!For!this,!data!of!identified!Razor!and!unique!peptides,! as! well! as! intensities,! were! considered! from! inputs! and! from! IgG-IP,! SUMO1-IP! and! SUMO2-IP!in!both!sets!of!experiments.! ! After! a! first! statistical! analysis! of! the! data,! only! 2240! proteins! from! the! total! identified! were! considered! as! true! SUMO! targets! (see! Apendix).! Then,! ratios! for! each! light/heavy! labelling! intensities! from! SUMO1-IP! and! SUMO2-IP! fractions! were! considered.! Targets! that! showed! inverse! ratios! between! both! experiments! for! at! least! one! of! the! SUMO-IP! fractions! (either! SUMO1-IP! or! SUMO2-IP)! were! considered! as! SILAC! candidates,! including!in!this!category!a!total!of!318!proteins!(Figure!R-38).!These!ratio!changes!could!be! essentially! due! to! a! remarkable! change! in! the! SUMOylation! state! of! target! proteins,! but! changes!could!also!be!due!to!a!significant!alteration!of!the!target!expression!levels!during! the! differentiation! process.! To! look! for! proteins! that! adjust! to! this! first! option,! a! further! selection!step!was!necessary,!following!these!criteria:! ! • Proteins! were! only! considered! if! they! showed! an! intensity! ratio! for! SUMO1-IP! or! SUMO2-IP!in!both!sets!of!experiments!and!these!ratios!were!≥2!in!one!experiment! and!≤0.5!in!the!reverse.! ! • SUMO1-IP!or!SUMO2-IP!ratios!from!both!sets!of!experiments!needed!to!have!at!least! a!2-fold!difference!with!the!control!ratios!(either!inputs!or!IgG-IP),!according!to!the! following!statements:! ! o When!both!input!ratios!were!present,!they!were!compared!to!the!ratios!of!SUMO1-IP! or! SUMO2-IP.! In! the! absence! of! one! of! the! inputs! data,! both! IgG-IP! ratios! were! considered! to! compare! with! the! IP! fractions.! Proteins! were! also! included! if! they! presented!input!ratios!from!a!set!of!experiments!and!IgG-IP!ratios!from!the!reverse.! Results! ! 85! o When!proteins!only!showed!control!ratios!from!one!set!of!experiments,!they!were! considered!only!if!there!was!at!least!a!2-fold!difference!with!the!ratios!from!SUMO1- IP!or!SUMO2-IP!in!the!same!experiment.! ! • A!final!consideration!included!proteins!without!any!control!ratio!that!showed!at!least! a!2-fold!difference!between!their!SUMO1-IP!and!SUMO2-IP!ratios!since!this!indicates! that,!at!least,! one! of!the! ratios! is!due! to! real!changes!in! their!SUMOylation!levels,! assuming!that!the!other!SUMO-IP!may!be!considered!as!a!control.! ! !Figure! R-38! shows! the! number! of! identified! proteins! that,! in! accordance! to! the! previously!mentioned!criteria,!are!finally!considered!as!targets!that!presumably!change!their! SUMO1-IP!or!SUMO2-IP!ratios!due!to!relevant!changes!in!their!SUMOylation!state!(relative)! without!relevant!changes!in!their!expression!levels.!It!is!important!to!remark!that!selection! criteria!do!not!exclude!proteins!moderately!changing!their!expression!levels.! ! ! Figure!R-38:!Venn!diagram!of!SUMO!targets!identified!by!MS!from!the!SILAC!experiment.!!Target! proteins!that!changed!their!SILAC!signal!when!P19!cells!got!differentiated,!due!to!differential!SUMO! modification!or!to!great!differences!in!their!expression!levels,!are!represented!(SUMO!SILAC;!left).! Numbers!of!proteins!modified!by!SUMO1,!SUMO2/3!or!both!paralogs,!are!indicated!above.!The!total! amount!of!proteins!considered!in!this!case!is!indicated!below.!Target!proteins!whose!SILAC!signal! only!changed!due!to!differential!SUMO!modification!but!are!similarly!expressed!in!both!conditions! are! also! represented! (SUMO! relative;! right).! Again,! numbers! of! proteins! modified! by! SUMO1,! SUMO2/3! or! both! paralogs,! are! indicated! above.! The! total! amount! of! proteins! considered! is! indicated!below.! ! ! 89#96#133# 222#229# SUMO1# SILAC# SUMO2/3# SILAC# 42#30#13# 55#43# SUMO1# rela8ve# SUMO2/3# rela8ve# 318#85# Differen8al#SUMOyla8on# Similar#target#expression# Results! ! ! 86! ! For!a!first!work,!some!of!these!proteins!were!selected!due!to!outstanding!changes!in! their! SUMO1-IP! or! SUMO2-IP! ratios! or! to! previously! described! roles! showing! a! potential! implication!in!the!neuronal!differentiation!process!(Tables!R-1,!R-2!and!R-3).!At!this!point,!it! is!worth!noting!that!many!of!these!selected!proteins!correspond!to!transcription!factors!and! that!some!of!them!show!to!be!preferentially!modified!by!SUMO1!or!SUMO2/3,!though!some! others!do!not!show!this!paralog!specificity.! ! Results' ' Table&R-1:&Selection&of&targets&differentially&modified&by&SUMO1.'Relative'SILAC'labelling'shows'proteins'that'get'SUMOylated'in'proliferation'(green)'or' neuronal'differentiation'(red)'of'P19'cells'in'the'first'experiment'(Exp.1).'The'reverse'labelling'is'shown'for'the'second'experiment'(Exp.'2).' ' ' ' IgG:'elution'from'control'immunoprecipitation'(IP);'S1:'elution'from'SUMO1'IP;'S2:'elution'from'SUMO2'IP;'NaN:'not'a'number.' ' ' IgG Input S1 *S2 IgG Input S1 S2 IgG Input S1 S2 IgG Input S1 S2 Ftl1 Ferritin*light*chain*1*and*2 3 4 16 3 6 8 15 24,33 3,00 9,49 1,93 0,13 0,12 0,05 NaN Tet1 Methylcytosine*dioxygenase*TET1 0 0 11 11 0 0 19 5 NaN NaN 8,12 3,10 NaN NaN 0,14 0,22 Pml Protein*PML 0 3 22 27 4 1 19 25 NaN 2,68 7,95 6,38 0,34 NaN 0,22 0,27 Morc3 Microrchidia*family*CW-type*zinc-finger*3* 10 543 50 9 3 41 45 3,04 1,82 4,71 3,35 0,65 0,61 0,25 0,45 Sall2 Sal-like*protein*2 0 5 8 7 1 5 5 5 NaN 1,52 3,32 2,59 NaN 0,62 0,26 0,46 Zfp518b Zinc*finger*protein*518B 1 0 7 12 0 0 9 8 NaN NaN 3,01 11,22 NaN NaN 0,38 0,14 Nacc1 Nucleus*accumbens-associated*protein*1 0 4 10 9 1 1 6 8 NaN 0,93 1,92 1,04 1,59 NaN 0,47 1,12 Gon4l GON-4-like*protein 0 0 10 13 0 1 17 8 NaN NaN 1,05 0,40 NaN NaN 1,38 5,67 Kctd1 BTB/POZ*domain-containing*protein*KCTD1 0 1 8 11 10916 NaN NaN 0,57 0,27 NaN NaN 1,84 4,30 Kctd15 BTB/POZ*domain-containing*protein*KCTD15 0 2 3 8 2 0 5 11 NaN NaN 0,48 0,08 NaN NaN 4,06 17,65 Trim24 Transcription*intermediary*factor*1-alpha 2 3 25 30 4 1 24 29 NaN NaN 0,47 0,20 9,23 NaN 2,24 5,68 Ncbp2 Nuclear*cap-binding*protein*subunit*2 0 2 4 0 1 3 3 1 NaN 1,49 0,42 NaN NaN 0,69 2,48 NaN Cotl1 Coactosin-like*protein 0 4 7 0 1 7 3 0 NaN 0,60 0,19 NaN NaN 2,01 4,53 NaN Nrip1 Nuclear*receptor-interacting*protein*1 0 0 12 21 10925 NaN NaN 0,18 0,16 NaN NaN 2,83 9,44 SUMO1*SILAC*relative Razor*+*unique*peptides Heavy/Light*labelling*ratios Exp.*1 Exp.*2 Exp.*1*(Prolif/Dif) Exp.*2*(Dif/Prolif) Gene Protein Results' ' ' 88' Table&R-2:&Selection&of&targets&differentially&modified&by&SUMO2/3.'As'in'the'previous'table,'modified'proteins'in'proliferation'(green)'or'differentiation' (red)'of'P19'cells'are'shown'for'the'first'experiment'(Exp.'1).'The'reverse'labelling'is'shown'for'the'second'experiment'(Exp.'2).' ' ' ' IgG:'elution'from'control'immunoprecipitation'(IP);'S1:'elution'from'SUMO1'IP;'S2:'elution'from'SUMO2'IP;'NaN:'not'a'number.' ' ' IgG Input S1 *S2 IgG Input S1 S2 IgG Input S1 S2 IgG Input S1 S2 Tcof1 Treacle*protein 312 14 17 817 13 20 8,77 3,83 5,19 7,94 0,27 0,29 0,14 0,11 Nolc1 Nucleolar*and*coiled-*body*phosphoprotein*1 116 7 7 8 17 9 8 NaN 1,86 3,42 4,05 0,52 0,54 0,30 0,28 Utp3 Something*about*silencing*protein*10 0 0 5 6 1 5 5 6 NaN NaN 2,61 3,99 NaN 0,78 0,43 0,27 Krr1 KRR1*small*subunit*processome*component* homolog 0 6 1 4 0 5 1 4 NaN 1,52 NaN 3,90 NaN 0,79 0,36 0,31 Nop58 Nucleolar*protein*58 819 26 33 14 25 26 27 1,53 1,38 2,15 3,78 0,69 1,00 0,49 0,30 Cdca8 Borealin 0 2 7 14 2 0 5 12 NaN 1,17 2,04 3,09 0,34 NaN 0,35 0,19 Bptf Bromodomain*PHD-finger*Transcription* Factor 0 3 13 17 0 2 13 8 NaN 0,85 1,16 1,78 NaN NaN 0,95 0,47 H2afz Histone*H2A,*V*and*Z 1 4 4 4 3 4 4 4 NaN 0,45 0,59 0,91 1,86 2,08 1,67 1,01 Nup214 Nuclear*pore*complex*protein*Nup214 020 17 17 122 17 19 NaN 1,17 0,92 0,41 NaN 0,86 1,93 3,60 Trim33 E3*ubiquitin-protein*ligase*TRIM33 2 7 25 26 2 4 25 24 NaN 0,92 0,85 0,28 NaN 1,24 1,29 4,17 Dnajc9 DnaJ*homolog*subfamily*C*member*9 111 0 3 1 2 3 5 NaN 0,43 NaN 0,20 NaN 1,83 1,87 17,43 Sec22b Vesicle-trafficking*protein*SEC22b 1 6 5 9 4 1 5 7 NaN 0,35 0,54 0,17 1,56 NaN 1,60 13,02 Etv6 Transcription*factor*ETV6 0 2 2 7 0 0 1 8 NaN 0,45 NaN 0,15 NaN NaN NaN 10,25 Nxn Nucleoredoxin 314 12 7 6 6 12 10 NaN 0,08 0,21 0,09 7,11 6,43 4,20 49,20 9 12 8 14 6 10 Razor*+*unique*peptides Gene Protein Undifferentiated*embryonic*cell*transcription* factor*1 Utf1 1 11 0,13 0,15 0,05 SUMO2*SILAC*relative NaN 13,92 7,84 94,62 0,11 Heavy/Light*labelling*ratios Exp.*1 Exp.*2 Exp.*1*(Prolif/Dif) Exp.*2*(Dif/Prolif) Results' ' 89' Table&R-3:&Selection&of&targets&indistinctly&modified&by&SUMO1&and&SUMO2/3&during&P19&cells&neuronal&differentiation.'Modified'proteins'in'proli-feration' (green)'or'differentiation'(red)'are'shown'for'the'first'experiment'(Exp.'1).'The'reverse'labelling'is'shown'for'the'second'experiment'(Exp.'2).' ' ' ' IgG:'elution'from'control'immunoprecipitation'(IP);'S1:'elution'from'SUMO1'IP;'S2:'elution'from'SUMO2'IP;'NaN:'not'a'number.' ' ' IgG Input S1 *S2 IgG Input S1 S2 IgG Input S1 S2 IgG Input S1 S2 Sall4 Sal-like*protein*4 112 31 26 5 9 27 27 NaN 6,60 ### 19,35 NaN 0,24 0,05 0,07 Utp14a U3*small*nucleolar*RNA-associated*protein*14* homolog*A 0 9 15 8 3 5 13 12 NaN 1,73 4,53 6,31 0,46 0,59 0,22 0,19 Dnttip2 Deoxynucleotidyltransferase*terminal- interacting*protein*2 0 5 23 6 2 6 20 11 NaN 1,14 4,03 3,67 0,70 0,87 0,25 0,30 Mphosph10 U3*small*nucleolar*ribonucleoprotein*protein* MPP10 0 0 8 7 0 2 7 12 NaN NaN 3,83 3,70 NaN 0,71 0,33 0,22 Atrx Transcriptional*regulator*ATRX 15 15 48 55 311 45 36 3,54 0,83 2,93 2,77 0,67 1,09 0,32 0,39 Incenp Inner*centromere*protein 0 2 3 3 0 2 4 4 NaN 0,85 2,51 5,66 NaN 0,84 0,27 0,20 Suz12 Polycomb*protein*Suz12 1 1 8 12 0 7 10 17 NaN NaN 1,61 1,78 NaN 1,38 0,69 0,51 Gtf2i General*transcription*factor*II-I 239 48 73 21 35 43 60 NaN 0,60 0,33 0,15 3,50 1,65 3,79 6,93 Prox1 Prospero*homeobox*protein*1 0 0 16 14 50920 NaN NaN 0,31 0,58 1,10 NaN 6,02 12,52 Ranbp2 E3*SUMO-protein*ligase*RanBP2 52 40 121 160 28 39 123 115 0,10 1,16 0,23 0,05 11,37 0,75 6,37 6,05 Razor*+*unique*peptides Heavy/Light*labelling*ratios Exp.*1 Exp.*2 Exp.*1*(Prolif/Dif) Exp.*2*(Dif/Prolif) Gene Protein SUMO1/SUMO2*SILAC*relative Discussion' ' ' 96' ' ' ' ' Figure/D-1:/General/model/for/the/regulation/of/Krox20/in/the/hindbrain.'These'schematics'show'the' repressor'effect'that'SUMOylation'of'Nab'mediated'by'Krox20,'together'with'Nab'interaction'with' CHD4,'displays'over'the'transcriptional'activity'of'Krox20.'Domains'in'yellow'correspond'to'Nab'and' domains'in'green'correspond'to'Krox20.'The'black'arrow'indicates'transcriptional'activation'and'the' flat-end' arrow' indicates' corepressor' activity.' Discontinuous' arrows' correspond' to' enzymatic' conjugation.' ' ' 5.3 SUMOylation/as/a/regulated/process/during/neurogenesis./ ' ' The' results' previously' shown' in' this' work' support' a' role' for' SUMO' in' regulating' neural' development.' In' further' experiments' we' have' investigated,' though,' whether' SUMOylation'is'regulated'during'neurogenesis'and,'if'so,'which'mechanisms'are'regulating' it.' Interestingly,' we' have' observed' that' net' SUMO' deconjugation' is' detected' during' neurogenesis.' To' this' point,' in' addition' to' this' decrease' in' the' levels' of' SUMOylated' proteins,' we' have' observed' that' the' free-SUMO' pool' in' P19' cells' doubles' following' induction'of'neuronal'differentiation'with'RA,'while'protein'analysis'under'non-denaturing' conditions'reveals'no'changes'in'the'total'SUMO-pool'along'the'process'(Figure'R-17).'These' results'indicate'that'neuronal'differentiation'is'linked'to'a'net'SUMO'deconjugation'balance' CID$ NCD2$ NCD1$ AC$ Zn$fingers$ R1$ SUMO$ SUMO$ Ubc9$ CHD4$ Nab$ Krox20$ Target$gene$ Discussion' ' 97' and' are' in' agreement' with' previous' works' reporting' that' overall' SUMOylation' decreases' during' brain' development' in' rodents' (Hasegawa,' Y# et# al.,' 2014;' Loriol,' C# et# al.,' 2012;' Watanabe,'M#et#al.,'2008).'Also'in'this'sense,'deSUMOy-lation'of'the'chromatin'associated' factor' Braf35,' which' is' linked' to' neuronal' differentiation,' has' been' reported' (Ceballos- Chavez,'M#et#al.,'2012).' ' The' results' in' this' work' also' show' how' SUMO' overexpression' leads' to' the' impairment' of' neurogenesis' in' P19' cells' and' chicken' neural' tube,' also' pointing' to' the' association' of' enhanced' SUMOylation' with' an' undifferentiated' state.' Interestingly,' overexpression' of' both' SUMO1' and' SUMO2' showed' the' same' effect' on' neurogenesis' (Figures'R-19'and'R-21),'raising'the'question'of'whether'they'work'independently'or'share' overlapping' sets' of' targets' during' the' differentiation' process.' In' this' regard,' though' SUMOylation' has' been' proven' to' be' an' essential' mechanism' in' almost' all' eukaryotes,' SUMO1' has' been' reported' not' to' be' essential' in' mouse,' suggesting' that' most' functions' could' be' fulfilled' by' SUMO2/3.' However,' its' deletion' can' lead' to' severe' phenotypes' that' seem'to'be'background-dependent'(Wang,'L#et#al.,'2014;'Zhang,'FP#et#al.,'2008).'We'have' not' investigated' this' aspect,' but' even' if' SUMO1' and' SUMO2/3' work' independently,' we' cannot'discard'that'when'overexpressed,'specificity'of'target'modification'by'the'different' SUMO'paralogs'is'lost.'Although'these'evidences'point'to'a'net'SUMO'deconjugation'during' neurogenesis,'we'should'not'exclude'that'de'novo'SUMOylation'of'specific'targets'could'be' required'for'this'process.' ' The'analysis'of'the'expression'levels'from'different'components'of'the'SUMOylation' pathway' during' P19' neuronal' differentiation' with' RA' has' delivered' interesting' results.' Through'this'approach,'we'have'observed'how'upregulation'of'Senp'proteases'expression' agrees' with' net' SUMO' deconjugation.' Our' results' also' indicate' that' several' Pias' ligases' genes' are' upregulated' during' this' process,' suggesting' de' novo' SUMOylation' of' certain' targets'(Figure'R-24).'This'possibility'has'to'be'taken'into'account,'given'that'these'ligases' have'been'described'to'regulate'the'Wnt'signalling'pathway,'related'to'embryogenesis'and' cell' fate' specification' (reviewed' in' (Schmidt,' D' and' Muller,' S,' 2003)).' In' addition' to' this,' cellular'functions'of'Pias'proteins'out'of'their'SUMO'ligase'activity'have'also'been'described' (Garcia-Dominguez,'M#et#al.,'2006)'and'the'numerous'post-translational'modifications'and' mechanisms' that' account' for' the' regulation' of' Pias' activity' should' also' be' taken' into' consideration'(review'in'(Rytinki,'MM#et#al.,'2009)).' Discussion' ' ' 98' ' 5.4 Senp7/accounting/for/proper/neuronal/differentiation./ ' ' In' the' previously' mentioned' expression' analyses,' we' have' observed' an' important' upregulation' of' Senp5' and' Senp7' proteases' (Figure' R-25' and' R-26),' which' have' been' associated'with'SUMO2/3'deconjugation'(Di'Bacco,'A#et#al.,'2006;'Gong,'L'and'Yeh,'ET,'2006;' Shen,'LN#et#al.,'2009).'In'this'regard,'as'expected,'our'results'show'that'the'knockdown'of' Senp7' impairs' neurogenesis-mediated' SUMO2/3' deconjugation' during' neuronal' differentiation' of' P19' cells' (Figure' R-28),' while' has' no' appreciable' effects' on' SUMO1' conjugation'levels.'However,'the'fact'that'also'free'SUMO1'increases'during'neurogenesis' suggests' that' all' neurogenesis-mediated' SUMO' deconjugation' cannot' be' exclusively' attributed'to'these' proteases.'Interestingly,'our' results' indicate'that,' while' increased'free' SUMO2/3' during' neurogenesis' correlates' with' a' decrease' of' high' molecular' mass' SUMOylated'products,'increased'free'SUMO1'seems'to'mostly'correlate'with'a'decrease'of' SUMOylated' RanGAP1' levels' (Figure' R-28),' though' additional' experiments' should' be' performed'to'assess'a'specific'decrease'in'the'levels'of'SUMOylated'RanGAP1'and'discard' differentiation-induced'RanGAP1'degradation'(Figure'D-2).'However,'apart'from'the'impact' that'lower'levels'of'SUMOylated'RanGAP1'may'have'on'neuronal'differentiation,'we'cannot' rule' out' that' SUMO1' deconjugation' from' other' targets' has' an' important' function' in' the' process.'Interestingly,'although'not'directly'related'to'neurogenesis,'it'has'been'previously' reported'that'the'SUMO1'associated'protease'Senp1'is'necessary'for'normal'development' of' the' mouse' embryo' (Sharma,' P# et# al.,' 2013).' To' this' point,' we' have' not' observed' significant'changes' on' the' expression' levels'of' Senp1'during' P19' neuronal' differentiation,' but'this'does'not'exclude'that'Senp1'plays'a'role'in'the'process.'Differential'regulation'under' proliferative' and' neuronal' differentiation' conditions' of' Senp' proteins' at' different' levels,' other' than' the' expression' degree,' may' account' for' the' net' balance' of' conjugated' SUMO' during' neurogenesis.' As' an' example,' SENP3' stabilization' against' proteasomal-mediated' degradation'has'been'reported'following'oxidative'stress'(Huang,'C#et#al.,'2009;'Yan,'S#et#al.,' 2010).'We'have'demonstrated'an'increased'expression'of'Senp7'both'at'mRNA'and'protein' levels'following' induction'of' neuronal' differentiation' (Figures' R25' and' R26),' and' knocking' down' experiments' unambiguously' demonstrate' the' involvement' of' Senp7' in' neuronal' Discussion' ' 99' differentiation' (Figures' R-29).' A' scaffolding' function,' essential' for' proper' chromosome' segregation' and' not' directly' related' to' its' SUMO-specific' protease' activity,' has' been' reported'for'Senp7'mediating'HP1'enrichment'at'pericentric'heterochromatin'(Maison,'C#et# al.,'2012;' Romeo,'K# et#al.,' 2015).' We'have' not'investigated' Senp7' enzymatic' activity' but,' regarding' this' aspect,' it' is' interesting' to' remark' that' neurogenesis-mediated' increase' of' Senp7' expression' correlates' with' increased' SUMO' deconjugation,' what' points' to' the' involvement'of'its'protease'activity'in'the'process'(Figure'D-2).' ' ' / / Figure/ D-2:/ General/ model/ for/ changes/ in/ protein/ SUMOylation/ during/ neuronal/ differentiation.' Schematics' showing' how' neuronal' differentiation' proceeds' with' SUMO1' (S1)' deconjugation' from' modified'targets,'being'mostly'appreciable'in'deSUMOylation'of'RanGAP1,'and'with'SUMO2/3'(S2/3)' deconjugation' from' modified' targets' (unidentified' X' and' Y' proteins).' Lower' levels' of' SUMOylated' RanGAP1' detected' under' neuronal' differentiation' could' be' related' to' partial' degradation' of' RanGAP1'following'differentiation'(dashed'box).' ' ' ' As'previously'mentioned,'in'addition'to'Senp7,'Senp5'is'also'upregulated'following' induction'of'neurogenesis'in'P19'cells.'At'this'point,'the'question'about'Senp5'and'Senp7' redundancy' of' function' or' specificity' of' targets' during' this' process' arises.' We' have' performed' preliminary' loss-of-function' analyses' that' reveal' less' dramatic' effects' in' neurogenesis' in' the' absence' of' Senp5' than' those' observed' in' the' absence' of' Senp7' (not' shown).' Interestingly,' Senp5' has' been' recently' involved' in' neutrophil' differentiation' of' myeloid'leukemic'cells,'being'also'upregulated'upon'induction'of'differentiation'in'blast'cells' Y" X" RanGAP1" SENP7" RanGAP1" RanGAP1" RanGAP1" (and%others)% Y" X" S1" S2/3" S1" S1" S2/3" S2/3" S1" S2/3" Neuronal% differen2a2on% Discussion' ' ' 100' from' acute' myeloid' leukemia' patients' (Federzoni,' EA# et# al.,' 2015).' Thus,' it' is' tentative' to' assign'a'role'to'a'subset'of'SUMO'proteases'in'the'onset'of'diverse'differentiation'processes.' ' To' further' investigate' the' way' in' which' Senp7' expression' is' upregulated' during' neuronal' differentiation,' expression' analysis' of' a' GFP' reporter' driven' by' the' Senp7' 5ʹ-upstream' region' encompassing' its' promoter' has' permitted' us' to' monitor' transient' expression'of'the'reporter'upon'induction'of'neuronal'differentiation'in'the'neural'tube'of' chick'embryos.'These'results,'together'with'Senp7'peaking'its'expression'at'day'4'of'the'RA' treatment' to' differentiate' P19' cells' (before' cells' extend' dendritic' and' axonal' processes)' (Figure'R-25),'point'to' a' role'of'Senp7' at' very'early'stages'of'neurogenesis.' As' previously' mentioned'during'the'introduction'of'this'work,'Neurogenins'are'proneural'genes'placed'at' the'earliest'stages'in'the'cascade'of'factors'involved'in'neurogenesis'(Ma,'Q#et#al.,'1999;'Ma,' Q#et#al.,'1996;'Perez,'SE#et#al.,'1999).'Other'proneural'factors'like'NeuroM'are'transiently' expressed'in'neural'progenitors'that'have'just'exited'the'cell'cycle'and'start'migrating'from' the'VZ'to'the'ML'(Roztocil,'T#et#al.,'1997).'This'transient'expression'prefigures'a'“frontier”' region'between'VZ'and'ML,'designated'as'subventricular'zone'(SVZ).'In'our'experiments'with' the'Senp7'expression'report,'when'naturally'occurring'neurogenesis'is'permitted,'we'have' observed' localization' of' reporter' positive' cells' close' to' the' SVZ,' which' may' suggest' that' Senp7' displays' an' expression' pattern' similar' to' that' of' NeuroM.' However,' neither' Neurogenin2'nor'NeuroM'were'able'to'sustain'reporter'expression'over'the'time'when'used' to'induce'neurogenesis,'indicating'that'the'promoter'sequence'is'not'directly'regulated'by' these' factors' (Figures' R-31' and' R-32).' Thus,' other' factors,' transiently' activated' when' triggering' neurogenesis' by' overexpression' of' Neurogenin2' or' NeuroM' in' the' neural' tube,' should' account' for' early' and' transient' activation' of' the' reporter.' When' inducing' neurogenesis'by'overexpression'of'neurogenic'factors,'virtually'all'transfected'cells'migrate' into' the' ML' and' end' up' localizing' to' the' most' distal' part' from' the' VZ.' Accordingly,' upon' induction' of' neurogenesis' following' the' electroporation' of' neurogenic' factors,' although' reporter'was'switched'off'with'time,'electroporated'cells'normally'migrated'to'the'ML,'as' revealed' by' localization' of' cells' expressing' the' CMV-mCherry' construct' that' we' used' to' monitorize'electroporated'cells'(Figure'R-31'and'R-32).' ' In' conclusion,' this' work' describes' for' the' first' time' that' proper' neuronal' differentiation' requires' Senp7,' a' SUMO' specific' protease,' as' a' regulatory' component' of' SUMOylation.'Interfering'with'the'expression'of'Senp7'impairs'neurogenesis'in'a'similar'way' Discussion' ' 101' to' overexpressing' SUMO' molecules.' Senp7' is' transiently' activated' at' early' stages' of' neurogenesis,' correlating' with' significant' and' general' deSUMOylation' of' proteins.' Hence,' Senp7'appears'as'a'key'regulator'of'neuronal'differentiation'and'adds'to'the'list'of'factors' involved'in'this'process.'This'finding'opens'new'and'exciting'research'lines'to'try'to'identify' the'targets'associated'to'Senp7'in'the'context'of'neurogenesis.' ' 5.5 Cellular/functions/of/SUMO/targets/in/the/process/of/neuronal/ differentiation/ ' ' The' identification' of' target' proteins' that' change' their' SUMOylation' state' in' cells' between' proliferative' and' differentiating' conditions' has' always' been' an' attracting' endeavour' during' progression' of' this' work,' focusing' our' efforts' in' the' purification' of' SUMOylated' products' in' both' conditions' and' their' subsequent' identification' by' mass' spectrometry' (MS).' Problems' associated' to' the' highly' dynamic' fashion' of' the' SUMO' conjugation'and'the'low'amount'of'SUMOylated'forms'of'target'proteins'have'hindered'our' progression' to' this' point,' till' the' establishment' of' a' purification' protocol' in' the' group' of' Prof.' Frauke' Melchior' that' allows' for' the' elution' of' SUMOylated' proteins' with' specific' peptides'after'immunoprecipitation'with'SUMO'antibodies'(Barysch,'SV#et#al.,'2014;'Becker,' J#et#al.,'2013).'The'application'of'this'purification'protocol'in'the'analysis'of'proteins'that'get' SUMOylated'or'deSUMOylated'during'neuronal'differentiation'of'P19'cells'has'reverted'in' the'identification'of'hundreds'of'target'proteins.' ' After' the' MS' data' processing,' a' further' analysis' to' get' a' sight' into' the' functional' associations'in'cellular'processes'that'these'proteins'have'was'mandatory.'To'this'purpose,' gene' ontology' (GO)' analyses' were' conducted' over' genes' coding' for' targets' that' were' selected'because'of'remarkable'differences'in'their'SUMOylation'states'compared'to'their' expression' levels' between' the' studied' conditions' (relative' SILAC)' (Figure' R-38).' GO' performed'over'genes'encoding'for'targets'that'are'presumably'modified'by'SUMO1'shows' that'a'high'number'of'them'(34%)'are'related'to'transcriptional'regulation,'with'an'also'high' number' of' genes' implicated' in' general' transcriptional' processes' (28%)' (Table' D-1).' It' is' important' to' mention' that' a' significant' percentage' of' genes' are' also' associated' with' a' variety'of'functions'generally'related'to'negative'regulation'of'distinct'processes'in'the'cell.' Discussion' ' ' 102' According'to'this'point,'it'has'been'previously'discussed'how'transcriptional'regulation'by' SUMO1' is' often' associated' to' a' repressor' activity' ((Ceballos-Chavez,' M# et# al.,' 2012)' and' reviewed'in'(Garcia-Dominguez,'M'and'Reyes,'JC,'2009)).'With'genes'encoding'for'proteins' presumably'modified'by'SUMO2/3,'GO'analysis'shows'that'most'of'them'are'also'associated' to'transcriptional'functions,'mostly'regulatory'(41.5%).'However,'a'high'number'of'functions' attributed' to' chromatin' organization' stand' out' regarding' these' targets' modified' by' SUMO2/3' (Table'D-1),' which' is' interesting,' taking' into' consideration' that' modification' by' SUMO2/3'has'been'classically'associated'to'stress'response.' ' Table/D-1:/Gene/Ontology/analysis/of/SUMO1/and/SUMO2/3/SILAC/relative/targets.'The'number'of' genes' included' in' each' term' is' represented' graphically' by' percentages.' P-values' for' statistical' signification'of'each'term'are'also'indicated.' ' ' ' Regula'on*of*transcrip'on* Transcrip'on* Nega've*regula'on*of*macromolecule*metabolic*process* Nega've*regula'on*of*gene*expression* Nega've*regula'on*of*macromolecule*biosynthe'c*process* Nega've*regula'on*of*cellular*biosynthe'c*process* Nega've*regula'on*of*biosynthe'c*process* Nega've*regula'on*of*transcrip'on* Nega've*regula'on*of*nucleo'de*and*nucleic*acid*metabolic*process* Nega've*regula'on*of*nitrogen*compound*metabolic*process* Maintenance*of*protein*loca'on*in*nucleus* Regula'on*of*transcrip'on* Transcrip'on* Nega've*regula'on*of*macromolecule*metabolic*process* Nega've*regula'on*of*gene*expression* Chroma'n*organiza'on* Chromosome*organiza'on* Chroma'n*modifica'on* M*phase* Cell*cycle*phase* Nega've*regula'on*of*transcrip'on,*DNA-dependent* Nega've*regula'on*of*RNA*metabolic*process* Covalent*chroma'n*modifica'on* Chroma'n*assembly*or*disassembly* Ribosome*biogenesis* Ribonucleoprotein*complex*biogenesis* SUMO1*SILAC*rela've* SUMO2/3*SILAC*rela've* 5.2E-3* 0* 10*20*30*40*(%)* 5.9E-4* 2.0E-3* 1.4E-4* 2.3E-4* 2.6E-4* 3.1E-4* 3.3E-4* 9.2E-4* 1.3E-3* 1.4E-3* 9.2E-3* 6.6E-6* 3.2E-4* 3.8E-3* 9.1E-3* 5.8E-3* 9.7E-3* 7.8E-3* 8.0E-3* 1.9E-3* 2.7E-3* 2.9E-3* 4.5E-5* 1.1E-3* 1.0E-6* (P-value)* Discussion' ' 103' ' It' is' worth' to' further' investigate' whether' SUMO2/3' is' clearly' involved' in' the' remodelling'processes'that'take'place'over'specific'regions'of'the'chromatin'that'need'to'be' silenced'for'cell'cycle'exit'and'neuronal'differentiation'of'cells,'together'with'relaxation'of' the'chromatin'regions'that'need'to'be'accessible'for'the'transcription'machinery'in'order'to' express'neuronal'differentiation'genes.'When'performing'an'additional'GO'analysis'taking' into'consideration'both'sets'of'SILAC'relative'targets'that'are'modified'by'SUMO1'and'by' SUMO2/3,'we'find'that'also'most'of'the'analysed'genes'are'implicated'in'transcription'or' transcriptional'regulation'and'other'functions'in'the'cell'that'might'be'of'interest'regarding' neuronal'differentiation'(Table'D-2).' ' Table/D-2:/Gene/functions/of/SILAC/ relative/SUMO/ targets.'Genes'from'an'additional'GO'analysis' comprising'SILAC'relative'SUMO1' and' SUMO2/3'targets'are' classified'into' five'general'categories.' Note'that'a'given'gene'can'fit'with'more'than'one'category.' ' ' ' ' Ribosome(biogenesis( and(RNA(metabolism( Aa#$ Nop58$ Krr1$ Nup214$ Mphosph10$ Ranbp2$ Ncbp2$ Utp14a$ Mitosis( Cdca8$ Incenp$ Nup214$ Rad21$ Ube2i$ Epigene4cs(and( chroma4n(structure( Atrx$ Hp1bp3$ Baz1b$ Kmt2d$ Bp#$ Rlf$ Dnmt3b$ Suz12$ Ehmt2$ Tet1$ H1fx$ Utp3$ Nuclear( bodies( Morc3$ Pml-like$ Transcrip4on(and(its(regula4on( Ajuba$ Kmt2d$ Suz12$ Atrx$ Nacc1$ Tet1$ Baz1b$ Nrip1$ Trim24$ Bp#$Pml-like$Trim33$ DnOp2$ Prox1$ U#1$ Etv6$ Psip1$ Zfp423$ Gon4l$ Sall2$ Zfp445$ G#2i$ Sall4$ Zfp512$ Hp1bp3$ Sap130$ Zfp518b$ Kctd1$ Sarnp$ Discussion' ' ' 104' ' As' previously' mentioned,' targets' identified' in' the' MS' analysis' change' the' ratio' in' which' they' show' to' be' modified' by' SUMO,' according' to' a' remarkable' change' in' their' SUMOylation'levels'in'comparison'to'their'expression'levels'between'both'conditions'or'due' to'a'high'change'in'their'expression'levels.'To'this'last'point,'it'would'be'important'to'study' why' SUMOylation' of' a' specific' target' is' important' in' one' of' the' analysed' conditions,' and' why'the'target'is'dumped'or'degraded'in'the'opposite'condition.'These'scenarios'have'to'be' taken' into' consideration' to' further' validate' and' investigate' the' identified' targets.' In' this' regard,'it'is'important'to'state'that'some'targets'might'be'mistakenly'excluded'from'the'lists' of' SILAC' relative' candidates,' when' they' indeed' change' their' SUMOylation' levels' in' a' significant'way'compared'to'changes'in'their'expression,'but'not'fulfilling'the'strict'criteria' applied' for' the' relative' candidates' in' a' quantitative' way.' This' has' been' the' case' for' the' Interferon' regulatory' factor-2-binding' protein-1' (IRF2BP1).' Our' validation' experiments' unambiguously' show' that,' although' being' excluded' from' the' list' of' SILAC' relative' candidates,' IRF2BP1' is' indeed' more' efficiently' SUMOylated' following' neuronal' differentiation'than'under'proliferative'conditions,'without'great'changes'in'its'expression' level' (Figure' R-39).' Thus,' we' do' not' exclude' that' additional' proteins,' not' fitting' with' the' established'criteria'to'be'considered'as'SILAC'relative'candidates,'are'indeed'SILAC'relative' candidates.'IRF2BP1'was'initially'described'to'mediate'corepressor'activity,'together'with'its' interaction' partner' Interferon' regulatory' factor-2' (IRF-2),' over' interferon' (IFN)' genes' expression'(Childs,'KS'and'Goodbourn,'S,'2003).'In'later'works,'IRF2BP1'has'been'associated' to' regulation' of' the' transforming' growth' factor' (TGF)' signalling' pathway.' In' this' regard,' IRF2BP1'has' been'shown' to'antagonise' the'repressor' activity' of' the' Transforming' growth' interacting' factor' (TGIF),' thus' promoting' the' TGF-ß-mediated' growth' arrest' and' gene' expression'(Faresse,'N#et#al.,'2008).' ' On' the' other' hand,' we' cannot' rule' out' the' possibility' that' some' of' the' proteins' identified' as' differentially' SUMOylated' are' false' positives.' The' fundament' of' mass' spectrometry,' which' undergoes' peptide' digestion' of' the' protein' samples' prior' to' the' analysis,' permits' the' detection' of' peptides' corresponding' to' proteins' that' are' not' SUMOylated'but'have'been' recognized' by'the'SUMO'antibodies' in' an'unspecific'way'and' therefore'eluted'in'the'immunoprecipitation'protocol.'Validation'of'the'different'candidates,' one'by'one,'by'using'specific'antibodies'will'unambiguously'indicate'which'candidates'are' relevant' to' be' considered' differentially' modified' by' SUMO.' For' instance,' validation' Discussion' ' 105' experiments'have'shown'that'the'iron'storage'protein'subunit'ferritin'light'chain'(FLC)'has' resulted'to'be'a'false'positive,'not'being'modified'by'SUMO1'as'was'firstly'indicated'by'the' MS'data'(Figure'R-39).' ' To' assess' the' implication' of' the' SUMO' modification' of' the' selected' targets' in' neuronal' differentiation,' functional' experiments' are' required.' The' identification' of' the' target'lysines'for'SUMO'modification'is'critical'for'each'of'the'selected'proteins.'As'mutation' of' the' identified' target' lysines' to' arginine' for' the' obtainment' of' effective' SUMOylation' mutants'will'contribute'to'get'information'from'functional'analyses,'either'on'P19'cells'or' chicken' embryos.' The' interpretation' of' the' results' will' greatly' depend' on' the' neuronal' differentiation'phenotype'obtained,'thus'arising'a'variety'of'possible'scenarios.'This'way,'a' mutant' protein,' whose' SUMOylation' is' necessary' for' proper' differentiation' of' neural' progenitors,' may' have' an' effect' in' disrupting' or' somehow' impairing' neuronal' differentiation,' but' in' the' case' that' SUMOylation' of' this' protein' is' needed' in' neural' progenitors' for' the' maintenance' of' a' proliferative' state,' the' mutant' may' promote' and' increase'differentiation'upon'induction'of'neurogenesis'with'neurogenic'factors.'It'is'likely' to'think'that'the'obtainment'of'analysable'phenotypes'will'depend'in'a'great'manner'on'the' degree' of' implication' that' this' factors' would' have' in' neuronal' differentiation' and' their' function' on' early' stages' of' the' differentiation' cascade.' A' function' on' later' stages' will' presumably' do' necessary' the' analysis' of' specific' neuron' subtypes' for' the' detection' of' a' given'phenotype.' ' As' has' been' previously' shown,' we' have' currently' validated' SUMOylation' of' some' interesting'proteins' identified' by' MS' (Figure'R-39).' Our'interest' has' been' initially' focused' into' proteins' related' to' transcriptional' regulation' like' TRIM24,' MORC3' or' ATRX,' or' the' previously' mentioned' IRF2BP2,' taking' into' consideration' that' some' of' them' have' never' been' showed' to' be' SUMOylated.' TRIM24' is' a' member' of' a' large' family' of' proteins' that' contain' a' tripartite-motif' (TRIM)' in' their' N-terminal' region' and' is' comprised' by' a' RING' finger,'one'or'two'zinc-binding'motifs'named'B-Box'and'an'associated'coiled-coil'region.'The' C-terminal'region'is'variable'and'has'been'used'to'catalogue'the'TRIM'proteins'into'twelve' subgroups.'TRIM24'is'a'member'of'the'transcriptional'intermediary'factor-1'(TIF1)'subgroup' and' has' been' associated' to' cell' signalling' by' RA' (reviewed' in' (Gudas,' LJ' and' Wagner,' JA,' 2011))' and' to' regulation' of' the' tumour' suppressor' factor' p53' (reviewed' in' (Jain,' AK' and' Barton,'MC,'2009)).'C-terminal'parts'of'TIF1'proteins'share'the'presence'of'a'tandem'PHD' Materials'and'methods' ' ' ' 112' Table/M-2:/yeast'vectors/ Name/ Prom./ Comp./ Res./ Information/ Source/ pDBLeu-Krox20*' ADH' Leu' Km' Two-hybrid'(bait)' Invitrogen' pPC86' ADH' Trp' Amp' Two-hybrid'(prey)' Invitrogen' ' *'Deletion'mutants'in'Figure'R-2'were'obtained'through'standard'PCR'techniques'based'on' this' construct.' Prom:' promoter.' Comp:' auxotrophy' complementation.' Res:' resistance' cassette.' ' ' Table/M-3:/mammalian'vectors'(cell'transfection'and'chick'embryo'electroporation)/ Name/ Prom./ Res./ Information/ Source/ pAdRSV-Sp' RSV' Amp' Cloning'backbone' Dr.'Patrick'Charnay' (Giudicelli,'F#et#al.,'2003)' pAdRSV-Sp- Krox20' RSV' Amp' Krox20' overexpression' Dr.'Mario'Garcia' (Garcia-Dominguez,'M#et#al.,'2006)' pAdRSV-Sp-' HA-Krox20' RSV' Amp' HA-Krox20' overexpression' Dr.'Mario'Garcia' (Garcia-Dominguez,'M#et#al.,'2006)' pAdRSV-Sp-' HA-Krox20IF' RSV' Amp' HA-Krox20'I268F' overexpression' This'work' (Garcia-Gutierrez,'P#et#al.,'2011)' pAdRSV-Sp-' HA-NeuroM' RSV' Amp' HA-NeuroM' overexpression' Dr.'Mario'Garcia' (Garcia-Dominguez,'M#et#al.,'2003)' pAdRSV-Sp-' HA-CHD4' RSV' Amp' HA-'truncated'CHD4' overexpression' This'work' (Garcia-Gutierrez,'P#et#al.,'2011)' pAdRSV-Sp-' Flag-Nab1' RSV' Amp' Flag-Nab1' overexpression' This'work' (Garcia-Gutierrez,'P#et#al.,'2011)' pAdRSV-Sp-' Flag-Nab2' RSV' Amp' Flag-Nab2' overexpression' This'work' (Garcia-Gutierrez,'P#et#al.,'2011)' pAdRSV-Sp-' Flag-Nab2QRHQ' RSV' Amp' Flag-Nab2' Q64RH95Q' overexpression' This'work' (Garcia-Gutierrez,'P#et#al.,'2011)' pAdRSV-Sp-' Flag-Nab2KR2' RSV' Amp' Flag-Nab2' K379R/K517R' overexpression' This'work' (Garcia-Gutierrez,'P#et#al.,'2011)' pAdRSV-Sp-Flag- RanGAP1Cter' RSV' Amp' Flag-RanGAP1'Cter' overexpression' This'work' (Garcia-Gutierrez,'P#et#al.,'2011)' pAdRSV-Sp-' Flag-Ubc9C93S' RSV' Amp' Flag-Ubc9'C93S' overexpression' This'work' (Garcia-Gutierrez,'P#et#al.,'2011)' pAdRSV-Sp- Neurogenin2' RSV' Amp' Neurogenin2' overexpression' Dr.'Mario'Garcia' (Garcia-Gutierrez,'P#et#al.,'2011)' pAdRSV-Sp-' His-SUMO1' RSV' Amp' His-SUMO1' overexpression' This'work' (Garcia-Gutierrez,'P#et#al.,'2011)' Materials'and'methods' ' 113' pAdRSV-Sp-' His-SUMO2' RSV' Amp' His-SUMO2' overexpression' This'work' (Garcia-Gutierrez,'P#et#al.,'2011)' pBS-KS-EphA4e- LacZ' Minimal' ß-globin' Amp' LacZ'reporter,'EphA4' enhancer-driven' Dr.'Mario'Garcia' (Garcia-Dominguez,'M#et#al.,'2006)' pGL4.51' CMV' Amp' Constitutive' Luciferase'reporter' Promega' pSuper*' H1' Amp' Cloning'backbone'for' shRNA'constructs' Oligoengine' pCS2-MT- NeuroD2' CMV' Amp' NeuroD2' overexpression' Dr.'Kristen'L.'Kroll' (Seo,'S#et#al.,'2005)' pCS2-MT-E12' CMV' Amp' E12'overexpression' Dr.'Kristen'L.'Kroll' (Seo,'S#et#al.,'2005)' pmCherry-N1' CMV' Km' Constitutive'mCherry' reporter' Clontech' pEGFP-N1' CMV' Km' Constitutive'GFP' reporter' Clontech' pEGFP-C2' CMV' Km' Cloning'backbone'for' GFP'fussions' Clontech' pEGFP-C2- SUMO1' CMV' Km' GFP-SUMO1' overexpression' This'work' (Garcia-Gutierrez,'P#et#al.,'2011)' phS7P-GFP' SENP7p' Km' GFP'reporter,'SENP7' (h)'promoter-driven' This'work' (Juarez-Vicente,'F#et#al.,'2016)' pchS7P-GFP' Senp7p' Km' GFP'reporter,'Senp7' (ch)'promoter-driven' This'work' (Juarez-Vicente,'F#et#al.,'2016)' / */ All'shRNA'constructs'were'cloned'in'the'pSuper'vector'using'annealed'oligonucleotides.' Prom:'promoter.'Res:'resistance'cassette.' / / / Materials'and'methods' ' ' ' 114' 7.1.2 Oligonucleotides/ / Table/M-4:/RNA'interference'sequences/ Name/ Sequence/5’-3’/ siRNA'mouse'Krox20#1' CGCCAAGGCCGUAGACAAA' siRNA'mouse'Krox20#2' GCCCUUCCAGUGUCGGAUC' siRNA'GFP'(control)' GGCACAAGCUGGAGUACAA' shRNA'mouse'Senp7#1' TTACAGCCTCCTCATGAGA' shRNA'mouse'Senp7#2' TCAGACTCATTGCCTTCGA' shRNA'chicken'Senp7#1' GGATTCTGTTGCTCAGACA' shRNA'chicken'Senp7#2' GAAGAAAGCTGGAGAAGAA' shRNA'Control' CCATCAAGACTCATAGATG' / / Table/M-6:/oligonucleotides'for'qPCR/ Name/ Forward/5’-3’/ Reverse/5’-3’/ Nab2# AGGAAGAGGAGATCCGGAAG' GTGTTGTCCCTCATGCAGAA' Krox20# CAGGAGTGACGAAAGGAAGC' GACCAGAGGCTGAAGACTGG' GAPDH# AACTTTGGCATTGTGGAAGG' GGATGCAGGGATGATGTTCT' Id4'promoter' GCGCGGCTCTACAAATACTGC' AACCGCGCCTCCCAGCTCAAC' GFP# CAAGATCCGCCACAACATCG' GTCCATGCCGAGAGTGATCC' Sumo1# GGCGATAAGAAGGAAGGAGAA' CATTGGAACTCCCTGTCTTTG' Sumo2# GGACAGGATGGTTCTGTGGT' CGGAATCTGATCTGCCTCA' Sumo3# ATTCCGGTTTGATGGACAAC' GAGGCTGATCCTCCTGTCTG' Sae1# GCTGGACCACGAACAGGTAT' CCCACAGACCCAGTCTGAAT' Uba2# GCATCGTATGGGCCAAGTAT' GCTTCAGCCTCTGTTGGTTC' Ubc9# GTCCCAACAAAGAACCCTGA' GGTGGTGAGGACGGATAGTC' Materials'and'methods' ' 115' Pias1# TCACCTCACTTGTCCGATTG' CCCTTTGCTCGTAACCTCTG' Pias2# AGTCCAACCAAAGGGGTTCT' CGTGTGGTGGAATGGTACTG' Pias3# AGTTTCGATGCTGCCCTTTA' TCATCACAATCCGAACAGGA' Pias4# CACTGAACTGGTCCCACAGA' CTGCACAGCTTTCACTCCAG' Senp1# CAGAGGCGACATTTCAGCAC' AGAACTGCTTCCCTGTGACC' Senp2# AAGGTTCTCGGCACCATTCTT' TTTGGCTGGGATCTCATCAGT' Senp3# TTTGACTCCCAGCGAACTCT' TTTGACTGCCTCTGCCTGTA' Senp5# GGCGAGCTGATAATGGATGC' ACAAGTCCACCTTCTTCGTCC' Senp6# TGGAACTGTGGTTCATGGGAG' ACCTTGGCCATGACTTAGCA' Senp7# ACAGTTCCAGGGTCAGCAGT' ATGGCCGCTTACACATTTTC' Desi1# GCGGTATTTCCAGCTGTACC' CAGGGAGGACAGGTATTCCA' Pou5f1#(Oct4)' CCAATCAGCTTGGGCTAGAG' CTGGGAAAGGTGTCCCTGTA' Tubb3# TGGAGCGCATCAGCGTATAC' GCCCTGGGCACATACTTGTG' Rn18S# GTAACCCGTTGAACCCCATT' CCATCCAATCGGTAGTAGCG' ' / Table/M-5:/oligonucleotides'for'cloning'strategies/ / Name/ Forward/5’-3’/ Reverse/5’-3’/ hSenp7#promoter' GATCACGAGGTCAGGAGATCG' ATGTTCAGCCCTTCTCTGACC' chSenp7#promoter/ GAAGTTCCAGCAATACATATGCG' TAAGGCTAGCAATTTGTATTTCATTT ATTCAGAAGG' / ' ' ' ' ' ' ' Materials'and'methods' ' ' ' 116' 7.1.3 Antibodies/and/peptides/ / Table/M-7:/primary'antibodies/ Name/ Antigen/ Host/ Source/ Dilution/ (M2)'Anti-FLAG' FLAG'peptide' (DYKDDDDK)' Mouse,' monoclonal' Sigma-Aldrich' (F1804)' 1:200'(IF)' 1:2000'(WB)' 2/250'µg'(ChIP)' (M2)'Anti-FLAG' affinity'gel' FLAG'peptide' (DYKDDDDK)' Mouse,' monoclonal' Sigma-Aldrich' (A2220)' –' Anti-HA' HA-tag'(YPYDVPDYA)' from'human'influenza' virus'HA'(hemaggltinin)' Rat,' monoclonal' Roche'(11-867- 431-001)' 1:2000'(WB)' Anti-HA' KLH'conjugated'HA-tag' (YPYDVPDYA)'of'human' influenza'virus'HA' Rabbit,' polyclonal' Sigma-Aldrich' (H6908)' 1:2000'(WB)' Anti-Krox20' Amino'acids'8'to'95'from' human'Krox20'fused'to' GST' Rabbit,' polyclonal' Covance' (PRB-236P)' 1:1000'(WB)' (1C4)' Anti-Nab2' Recombinant'human' Nab2' Mouse,' monoclonal' Santa'Cruz'Btg.' (sc-23867)' 1:1000'(WB)' (D-11)' Anti-SUMO1' Amino'acids'1'to'101' from'human'SUMO1' Mouse,' monoclonal' Santa'Cruz'Btg.' (sc-5308)' 2/250'µg'of' protein'(ChIP)' (21C7)' Anti-SUMO1' Recombinant'6x'Histagged'human'SUMO1' Mouse,' monoclonal' DSHB,'Iowa,' IA,'USA' 1:1000'(WB)' (8A2)' Anti-SUMO2/3' Recombinant'human' SUMO2' Mouse,' monoclonal' DSHB,'Iowa,' IA,'USA' 1:1000'(WB)' Anti-Fluorescein' Fluorescein' Sheep,' polyclonal' Roche' (11426338910)' 1:2000'(ISH)' Anti-Digoxigenin' Digoxigenin' Sheep,' polyclonal' Roche' (11093274910)' 1:2000'(ISH)' Anti-SENP7' Internal'peptide'from' human'SENP7' Rabbit,' polyclonal' Abcam' (ab58422)' 1:1000'(WB)' (P18)' Anti-SENP3' Internal'peptide'from' human'SENP3' Goat,' polyclonal' Santa'Cruz'Btg.' (sc-46640)' 1:1000'(WB)' (DM1A)' Anti-α-tubulin' Microtubules'from' chicken'embryo'brain' Mouse,' monoclonal' Sigma-Aldrich' (T9026)' 1:5000'(WB)' (H-134)' Anti-Oct3/4' Amino'acids'1'to'134' from'human'Oct-4' Rabbit,' polyclonal' Santa'Cruz'Btg.' (sc-9081)' 1:1000'(WB)' Materials'and'methods' ' 117' Anti-'neuron' specific' βIII-tubulin' KLH-conjugated'amino' acids'350'to'C-terminus' from'human'βIII-tubulin' Rabbit,' polyclonal' Abcam' (ab18207)' 1:2000'(WB)' 1:500'(IF)' (3A10)'Antineurofilaments' Chicken'neurofilaments' Mouse,' monoclonal' DSHB,'Iowa,' IA,'USA' 1:200'(IF)' Anti-Morc3' Internal'peptide'from' human'MORC3'protein' Rabbit,' polyclonal' Santa'Cruz'Btg.' (sc-83730)' 1:500'(WB)' (H-300)' Anti-ATRX' Amino'acids'2193'to'2492' from'human'ATRX' Rabbit,' polyclonal' Santa'Cruz'Btg.' (sc-15408)' 1:1000'(WB)' Anti-Trim24' Recombinant'6x'Histagged'human'TRIM24' Rabbit,' polyclonal' Proteintech' (14208-1-AP)' 1:2000'(WB)' Anti-Irf2bp1' Human'IRF2BP1'protein' (Accession'NM_182972)' Rabbit,' polyclonal' Proteintech' (18847-1-AP)' 1:1000'(WB)' Anti-Ubc9' Amino'acids'1'to'81'from' human'Ubc9' Rabbit,' polyclonal' Santa'Cruz'Btg.' (sc-'10759)' 1:1000'(WB)' ' ' Table/M-8:/secondary'antibodies' Name/ Conjugate/ Host/ Source/ Dilution/ Anti-mouse'IgG' HRP' Goat,' polyclonal' Sigma-Aldrich'(A4416)' 1:10000'(WB)' Anti-rabbit'IgG' HRP' Goat,' polyclonal' Sigma-Aldrich'(A6154)' 1:10000'(WB)' Anti-rat'IgG' HRP' Goat,' polyclonal' Sigma-Aldrich'(A9037)' 1:10000'(WB)' Anti-goat'IgG' HRP' Donkey,' polyclonal' Bethyl'(A50-201P)' 1:10000'(WB)' Anti-rabbit'IgG' DyLight'549' Donkey,' polyclonal' Jackson'Immunoresearch' (711-505-152)' 1:800'(IF)' Anti-rabbit'IgG' DyLight'488' Donkey,' polyclonal' Jackson'Immunoresearch' (711-485-152)' 1:800'(IF)' Anti-mouse'IgG' DyLight'649' Donkey,' polyclonal' Jackson'Immunoresearch' (715-495-151)' 1:800'(IF)' Anti-mouse'IgG' DyLight'Cy3' Donkey,' polyclonal' Jackson'Immunoresearch' (715-165-150)' 1:800'(IF)' / Materials'and'methods' ' ' ' 118' Table/M-9:'elution'peptides'for'SUMO'immunoprecipitation./ Peptide/ Sequence/ SUMO1' VPMNSLRFLFE' SUMO2/3' IRFRFDGQPI' / / 7.1.4 Organisms/ / Table/M-10:/bacterial'or'yeast'strains'and'mammal'cell'lines'employed'in'this'work.' Organism/ Strain/Line/ Information/ Bacteria' DH5α' Cloning'strategies' BL21' Heterologous'protein'expression' Yeast' MaV203' Two-hybrid'(His-,'Leu-,'Trp-,'LacZ+)' Mammals' HEK-293T' Transformed'human'embryonic'kidney'cells'for'protein' expression' P19' Mouse'teratocarcinoma'for'RA-'or'transfection-mediated' neuronal'differentiation' / / / Materials'and'methods' ' 119' 7.2 Methods/ / 7.2.1 Culture/conditions/of/employed/organisms/ / § Bacterial'culture'and'maintenance' ' DH5α'and'BL21'strains'from'E.#coli'were'grown'at'37°'C'in'constant'agitation'at'200' rpm.'The'culture'medium'employed'was'LB'(Luria-Bertani)'medium'consisting'on'0.5%'(w/v)' yeast'extract,'1%'(w/v)'bacto-tryptone,'1%'(w/v)'NaCl,'pH'7.0.'For'agar'plates,'LB'medium' was'supplemented'with'2%'(w/v)'agar'prior'to'sterilization.' ' § Yeast'culture'and'maintenance' ' The'yeast'strain'MaV203'from'S.#cerevisiae'was'grown'at'30°'C'in'constant'agitation' at' 250' rpm.' The' culture' medium' employed' was' YPD' consisting' on' 10' g/l' yeast' extract,' 20'g/l'of'tryptone'and'20'g/l'of'glucose.'For'growing'restrictions,'essential'minimal'medium' (Q-BIOgene)' commercially' available' was' employed,' being' supplemented' with' 0.8' mM' leucine,'0.64'mM'histidine'and'0.32'mM'triptophane,'when'required.' ' § Mammal'cell'lines'culture'and'maintenance' ' HEK' 293T' cells' were' cultured' in' DMEM' (Dulbecco’s# Modified# Eagle’s# Medium)' commercially' supplemented' with' 4.5' g/l' D-glucose,' L-glutamine,' sodium' pyruvate' and' sodium'bicarbonate'(Sigma-Aldrich).'500'ml'of'medium'were'supplemented'with'10%'(v/v)' fetal' bovine' serum' (FBS)' and' 10' ml' of' a' solution' consisting' on' 100' U/ml' penicillin' and' 100'µl/ml' streptomycin' (P/S).' The' cells' were' subcultured' for' weekly' maintenance' at' 1/10' dilution'from'an'almost'confluent'plate'washing'the'cells'with'PBS,'detaching'with'Trypsine- EDTA'and'diluting'them'in'fresh'medium.' ' P19' cells' were' cultured' in' αMEM' (alpha# modification# Minimum# Eagle’s# Medium)' commercially'supplemented'with'4.5'g/l'D-Glucose,'L-glutamine,'sodium'pyruvate,'sodium' bicarbonate,' ribonucleosides' and' deoxyribonucleosides' (HyClon).' 500' ml' of' medium' were' supplemented'with'7.5%'(v/v)'newborn'calf'serum'(NCS),'2.5%'(v/v)'FBS'and'10'ml'of'a'P/S' solution.'The'cells'were'subcultured'for'weekly'maintenance'at'1/20'dilution'from'an'almost' confluent'plate'washing'the'cells'with'PBS,'detaching'with'Trypsine-EDTA'and'diluting'them' in'fresh'medium.' Materials'and'methods' ' ' ' 120' ' All' the' cell' lines' were' cultured' in' HEPA# Class# 100' (Thermo' Fisher)' incubators' with' humid'atmosphere'at'37°'C'and'5%'CO2.'10'cm'ø'plates,'in'addition'to'6x'and'12x'well'plates' (Nunc)'were'used'to'culture'the'cells'in'adherent'conditions.' / 7.2.2 DNA/RNA/techniques/ / 7.2.2.1 DNA/RNA/manipulation/and/analysis/ / § PCR'(Polymerase'Chain'Reaction)' ' The'polymerase'chain'reaction'was'employed'when'amplification'of'DNA'fragments' was' necessary.' It' was' performed' using' the' Expand# High# Fidelity' (Roche)' kit' and' a# TC3000'(Techne)'thermal'cycler.'Each'PCR'reaction'was'conducted'in'a'total'volume'of'50'µl,' containing' 20-50' ng' of' template' DNA,' 1' µM' of' each' primer,' 2' mM' of' each' deoxyribonucleotide,'1'mM'MgCl2'and'2.5'units'of'DNA'polymerase'in'1x'supplied'PCR'buffer.' ' The'PCR'program'consisted'on'an'initial'denaturing'step'at'94°'C'for'5'min,'followed' by'30'amplification'cycles'and'a'final'elongation'step'at'72°'C'for'10'min.'Each'amplification' cycle'was'initiated'by'a'denaturing'step'at'94°'C'for'1'min,'followed'by'a'DNA'hybridization' step'at'variable'temperature'(depending'on'the'Tm'for'each'primer)'for'1'min'and'a'final' polymerization' step' at' 72°' C' for' a' time' dependant' on' the' elongated' fragment' size' (1'min/Kb).' / § Enzymatic'manipulation'of'DNA'fragments' ' For'DNA'fragments'digestion,'diverse'restriction'enzymes'(Fermentas,'Takara,'New' England' Biolabs)' were' employed' following' the' manufacturer’s' instructions,' in' addition' to' the' appropriate' restriction' buffer' and' digestion' temperature.' For' blunt' end' production' starting' from' cohesive' 5’' end' fragments,' the' Klenow#fragment' from' the# E.# coli'DNA' polymerase'I'(Promega)'was'used.'For'cloning'purposes,'1'µg'of'plasmid'DNA'or'1/10'from' the'total'volume'of'a'PCR'was'digested'with'5-10'units'of'restriction'enzyme'for'2'h'at'the' required'temperature.' ' Prior'to'DNA'ligation,'dephosphorylation'of'the'required'vector'was'performed'using' the' calf' intestinal' alkaline' phosphatase' (Promega)' according' to' the' manufacturer’s' instructions.' Materials'and'methods' ' 121' ' For' the' ligation' of' DNA' fragments,' the' T4' DNA' ligase' (Promega)' was' employed,' following' the' manufacturer’s' instructions.' Ligations' were' performed' over' night' at' 15°' C,' following'transformation'of'DNA'in'competent'E.#coli'DH5α.' ' § Synthesis'of'RNA'probes' ' Antisense' RNA' probes' were' synthesized' for' in# situ' hybridization' experiments.' For' this,' cDNA' constructs' for' EphA4,# HoxB1# and# Krox20' were' linearized' with' NotI,' XbaI' and' HindIII'restriction'enzymes'(Fermentas),'respectively,'analysing'the'restriction'efficiency'by' electrophoresis'in'0.8%'(w/v)'agarose'gels,'as'further'explained.'RNA'probes'were'obtained' by' transcription' of' the' linearized' plasmids,' using' the' RNA' T3' or' T7' polymerases' with' a' mixture' of' fluorescein-' or' digoxigenin-labelled' ribonucleotides' (Roche).' Transcription' reactions'were'conducted'in'the'presence'of'RNAse'inhibitors'(Invitrogen)'for'2'h'at'37°'C.' Synthesized'probes'were'then'recovered'by'phenol-chloroform'extraction'and'precipitated' with'2'vol'of'ethanol'+'1/10'vol'3'M'of'lithium'chloride'(LiCl)'for'30'min'at'-20°'C.'Integrity'of' the'probes'was'checked'by'electrophoresis'in'0.8%'(w/v)'agarose'gels,'as'further'described.' / § RNA'isolation' ' Total' RNA' isolation' from' mammal' cells' was' performed' with' the' RNeasy®# mini# Kit' (QIAGEN),'according'to'the'manufacturer’s'instructions.'For'RNA'quality'monitoring,'1'µl'of' each' RNA' sample' was' supplemented' with' 1' µl' of' 10x' loading' buffer' in' a' total' volume' of' 10'µl,' filled' with' DEPC' H2O.' Integrity' of' the' samples' was' checked' by' electrophoresis' in' 1%'(w/v)'agarose'gels,'as'further'described.' ' § Retro-transcription'(RT)'for'cDNA'obtainment' ' Prior' to' cDNA' synthesis,' contaminant' DNA' was' erased' with' the' RQ1# RNase-Free# DNase'(Promega)'kit,'according'to'the'manufacturer’s'instructions.'For'these'cases,'1'µg'of' RNA'was'treated'with'1'unit'of'DNase.'For'the'obtainment'of'cDNA,'the'SuperScript®#First- Strand# Synthesis# System' (Invitrogen)' kit' was' employed,' following' the' manufacturer’s' instructions' for' the' use' of' random' hexamers' as' reaction' primers.' Each' PCR' reaction' was' performed'in'a'total'volume'of'20'µl'using'0.5-1'µg'of'RNA.' ' ' Materials'and'methods' ' ' ' 128' solution'supplemented'with'1:100'vol'of'the'required'RNA'probes,'over'night'at'65°'C'under' agitation.' After' three' washes' of' 20' min' each' with' 2x' SSC' +' 0.1%' (v/v)' CHAPS' and' three' additional' washes' with' 0.2x' SSC' +' 0.1' (v/v)' CHAPS,' hindbrains' were' equilibrated' in' KTBT' solution' (50'mM' Tris-HCl' pH' 7.5,' 150' mM' NaCl,' 20' mM' KCl,' 0.1%' (v/v)' Triton' X-100)' for' 10'min'at'room'temperature.' ' For' hybridization' with' specific' antibodies' for' digoxigenin' or' fluorescein,' hindbrains' were'previously'incubated'with'blocking'solution'(20%'(v/v)'FBS'in'KTBT'solution)'for'2'h'at' room'temperature'and'then'incubated'with'digoxigenin'or'fluorescein'antibodies'(Roche)'at' a'1:2000'dilution'in'blocking'solution'over'night'at'4°'C'under'rotation.'Next,'nine'washes'of' 20'min'each'were'performed'with'KTBT'at'room'temperature.' ' Revelation' of' hybridized' probes' was' conducted' by' equilibrating' the' hindbrains' in' NTMTL'solution'(0.1'M'Tris-HCl'pH'9.5,'50'mM'MgCl2,'0.1'M'NaCl,'0.1%'(v/v)'Triton'X-100' and'0.5'mg/ml'Levamisole'(Sequoia'Research'Products'Ltd)),'following'incubation'with'fresh' NTMTL' supplemented' with' NBT' or' BCIP' substrate' (Roche)' for' digoxigenin' or' fluorescein' staining,'respectively,'in'darkness'at'room'temperature'till'tissue'staining'was'appreciable.' Revelation'was'stopped' with'subsequent' washes'with'PBTr' 0.1'and'then' hindbrains'were' mounted'on'cover'slides'for'image'acquisition'with'a'epifluorescence'DM6000'microscope' (Leica'Microsystems'GmbH).' ' In' the' cases' where' double' in# situ' RNA' hybridization' was' necessary,' sequential' hybridization'was'performed,'revealing'in'the'first'place'the'fluorescein'antibody.' ' 7.2.3 Model/organisms/ / 7.2.3.1 Cell/culture/techniques/ ' § Transient'transfection'of'mammal'cells' ' To' perform' transient' transfection' of' P19' growing' in' αMEM' +' 7.5%' (v/v)' NCS' +' 2.5%'(v/v)'FBS'+'P/S'or'HEK-293T'cells'growing'in'DMEM'+'10%'(v/v)'FBS'+'P/S,'a'number'of' cells'were'seeded'in'adherent'10'cm'ø'plates,'6x'or'12x'well'plates'and'transfected'after' 24'h'with'1'µg,'2'µg'or'10'µg'of'DNA'respectively.'For'this'purpose,'the'Lipofectamine®#2000' (Invitrogen)'reagent' and' the' OptiMEM®' (Gibco)' medium' were'employed' according'to' the' manufacturers'instructions.'Cells'were'harvested'24-48'h'after'transfection.' Materials'and'methods' ' 129' ' Transient' transfection' of' small' interfering' RNA' (siRNA)' molecules' was' performed' using'the'Oligofectamine®#2000#(Invitrogen)'transfection'reagent'and'the'OptiMEM®'(Gibco)' medium,'according'to'the'manufacturers'instructions.'The'transfected'cells'were'harvested' 48-72'h'after'transfection,'depending'on'the'experiment.' ' § P19'neuronal'differentiation'with'RA' ' To'obtain'neuron'differentiated'P19'cells,'5·105'cells'were'seeded'in'non-adherent' sterile'10'cm'ø'plates'(Monolab)'and'grown'in'suspension'with'αMEM'+'5%'(v/v)'FBS'+'P/S'+' 2'µM'RA'during'4'd,'refreshing'the'medium'the'second'day.'The'fourth'day,'when'embryonic' bodies' were' properly' formed,' they' were' harvested' into' 15' mL' tubes' and' centrifuged' at' 1,500x' g' for' 5' min' and' media' was' removed,' resuspending' the' embryonic' bodies' in' fresh' αMEM' +' 7.5%' NCS' +' 2.5%' FBS' +' P/S' without' any' RA.' Embryonic' bodies' were' then' mechanically' disaggregated' with' a' 10' ml' pipette' and' seeded' in' adherent' 10' cm' ø' plates' (Nunc)'for'three'more'days.'During'this'last'period,'axon'and'dendrite-like'processes'start'to' form'during'the'final'differentiation.'The'complete'experiment'took'7'days'but'cells'were' harvested'at'various'time'points'during'the'process.' / § P19'large-scale'SILAC'experiment'for'neuronal'differentiation' ' To' perform' a' Stable' Isotope' Labelling' with' Amino' acids' in' Cell' culture' (SILAC)' experiment'in'proliferating'and'neural'differentiating'P19'cells,'the'cells'were'amplified'for' four'days'in'10'cm'ø'adherent'plates'and'grown'in'light'amino'acids'(AA)'medium'(Silantes' DMEM'(Dulbecco’s#Modified#Eagle’s#Medium)'+'2'mM'L-Glutamine'+'10'U/ml'of'penicillin'+' 10' µg/ml' streptomycin' +' 146'µg/ml' Lys' +' 84' µg/ml' Arg)' supplemented' with' 10%' (v/v)' dialysed'FBS,'or'grown'in'heavy'AA'medium'(Silantes'DMEM'+'2'mM'L-Glutamine'+'10'ml'of' penicillin/streptomycin' +' 146'µg/ml' D4' Lys' +' 84' µg/ml' [13C]Arg)' supplemented' with' 10%' (v/v)' dialysed' FBS.' During' these' four' days,' sufficient' to' achieve' around' six' cell' division' rounds,'the'cells'incorporated'the'SILAC'specific'amino'acids.' ' For' the' first' experiment,' 2·106'of' the' cells' grown' in' the' heavy' AA' condition' were' seeded' in' 45' adherent' plates' for' proliferation,' and' still' grown' in' heavy' AA' medium' supplemented'with'10%'(v/v)'dialysed'FBS'for'two'more'days'before'harvesting'them.'Also,' 106'of'the'cells'grown'in'the'light'AA'condition'were'seeded'in'90'non-adherent'plates'for' differentiation,'and'grown'in'light'AA'medium'supplemented'with'5%'(v/v)'dialysed'FBS'and' Materials'and'methods' ' ' ' 130' 2'µM'RA'during'4'd,'refreshing'the'medium'the'second'day.'The'harvesting'procedure'was' conducted'as'indicated'in'(Barysch,'SV#et#al.,'2014).'Briefly,'a'total'amount'of'approximately' 9·108'proliferative'adherent'cells'were'washed'in'cold'PBS'+'10'mM'NEM'and'then'directly' lysed'on'the'plates'with'170'µl'of'2x'lysis'buffer'(2%'(w/v)'SDS,'10'mM'EDTA,'10'mM'EGTA,' 20'mM'NEM,'2'mM'Pefabloc'and'2'mg/ml'of'protease'inhibitors'(aprotinin,'leupeptin'and' pepstatin)' in' PBS)' by' scrapping' the' cells' directly' on' the' plates' with' the' lysis' buffer' and' obtaining'a'total'lysate'volume'of'approximately'15'ml.'In'the'case'of'differentiating'cells,'a' total' amount' of' approximately' 9·108' suspended' cells' were' harvested' by' centrifugation' at' 500x'g'for'5'min,'washed'in'cold'PBS'+'10'mM'NEM'and'resuspended'in'a'total'volume'of'16' ml' of' PBS.' Then,' cells' were' lysed' adding' 16' ml' of' 2x' lysis' buffer.' The' cell' lysates' were' snap-freezed'with'liquid'nitrogen'and'stored'at'-80°'C.' ' To'perform'the'reverse'experiment,'2·106'of'the'cells'grown'in'the'light'AA'condition' were' seeded' in' 45' adherent' plates' for' proliferation,' and' still' grown' in' light' AA' medium' supplemented' with' 10%' (v/v)' dialysed' FBS' for' two' more' days' before' harvesting' them.' Additionally,' 106' of' the' cells' grown' in' the' heavy' AA' condition' were' seeded' in' 90' non-adherent'plates'for'differentiation,'and'grown'in'heavy'AA'medium'supplemented'with' 5%'(v/v)'dialysed'FBS'and'2'µM'RA'during'4'd,'also'refreshing'the'medium'the'second'day.' The'harvesting'procedure'was'conducted'as'previously'described'for'the'first'experiment.' ' § P19'serum'stimulation' ' To'induce'protein'expression' dependent' on'serum'stimulation'in' P19' cells,'1.5·105' cells'were'seeded'in'6x'well'plates'and'initially'grown'in'αMEM'+'0.1%'(v/v)'FBS'+'P/S'for' 48'h.' Then,' the' medium' was'replaced' with' fully' supplemented' αMEM' +' 7.5%' (v/v)' NCS' +' 2.5%'(v/v)'FBS'+'P/S'for'2'h'to'trigger'serum-induced'responses'prior'to'cell'harvesting.' ' 7.2.3.2 Chicken/embryos/ / § Electroporation'of'the'chicken'hindbrain'and'neural'tube' ' This' technique' was' used' to' introduce' DNA' expression' vectors' and' silencing' constructs'in'the'neural'progenitors'and'cells'present'in'the'hindbrain'and'neural'tube'of'the' chick'embryo,'as'described'in'(Itasaki,'N#et#al.,'1999)'and'(Giudicelli,'F#et#al.,'2001).' Materials'and'methods' ' 131' ' For'this'purpose,'fertilized'eggs'were'incubated'at'38°'C'in'a'Heraeus'(Thermo'Fisher)' incubator'till'the'desired'developmental'stage'was'reached.'DNA'plasmids'were'suspended' in'10'mM'Tris-HCl'pH'8.0'at'the'required'concentration'and'0.05%'(v/v)'Fast-Green'FCF'was' added'to'visualize'the'DNA'solution'during'the'injections.'A'GFP'expression'vector'was'also' included'in'all'DNA'solutions'at'a'concentration'of'0.3'µg/µl'to'monitor'electroporated'cells' by' immunofluorescence.' The' DNA' solution' was' injected' in' the' lumen' of' the' neural' tube' using'a'glass'capillary'and'the'embryos'were'electroporated'at'the'hindbrain'or'the'neural' tube'level'with'a'BTX820'(Quantum)'electroporator,'employing'4'or'6'pulses,'respectively,'of' 50' ms' at' 25' V' and' 1' Hz' with' 500' ms' between' pulses.' Following' reincubation' for' 24' h' in' hindbrain'analyses'and'for'10'to'40'h'for'spinal'cord'analyses,'the'embryos'were'extracted' from'the'egg'in'PBS'and'fixed'in'a'4%'(w/v)'PFA'solution'for'3'h'at'room'temperature'or'over' night'at'4°'C.'After'four'washes'of'15'min'each'using'PBS'to'remove'traces'of'PFA'solution,' the' electroporated' section' of' the' embryo' was' dissected' by' visualization' of' GFP' in' a' fluorescence'magnifier'(Nikon).'Finally,'the'embryos'were'dehydrated'in'subsequent'washes' of'5'min'using'increasing'concentrations'of'methanol'in'PBS'(25%,'50%,'75%'and'100%'(v/v))' for'long'term'storage'at'-20°'C.' ' 7.2.4 Protein/techniques/ / 7.2.4.1 Protein/induction/and/extraction/ / § In#vitro'protein'translation' 'In#vitro#translation'in'the'presence'of'[35S]Met'was'performed'with'the'TnT®#Quick# Coupled# Transcription/Translation# Systems' (Promega)' kit,' following' the' manufacturer’s' instructions'for'a'standard'reaction.' / § Protein'expression'in'E.#coli' ' Heterologous'expression'of'GST-'or'polyhistidine-tagged'proteins'was'performed'in' E.#coli'by'cloning'the'cDNA'sequence'into'a'pGEX-6P-3'(GE'Healthcare)'vector'for'the'GST' constructs,'or' a' pET28a+' (Novagen)' vector'for' the'polyhistidine-tagged' construct.'For' this' purpose,' the' vectors' were' electroporated' in' E.# coli'BL21'(DE3)' electrocompetent' bacteria' and'15-100'µl'of'this'culture'was'inoculated'to'1-2'l'of'LB'medium'supplemented'with'the' Materials'and'methods' ' ' ' 132' requested'antibiotic.'The'bacterial'culture'was'grown'to'reach'an'OD600'of'about'0.5'and'1' ml'of'bacteria'was'then'harvested'as'non-induced'control.'To'induce'protein'expression,'1' mM' isopropyl-ß-D-1-thiogalactopyranoside' (IPTG)' was' added' to' the' culture,' which' was' grown'at'24°'C'for'4'h,'prior'to'harvest'it'by'centrifugation'for'10'min'at'10000x'g.' ' § Protein'extraction'by'sonication' ' This'technique'was'typically'conducted'to'purify'proteins'expressed'in'E.#coli.'For'this' purpose,'the'cell'pellet'was'resuspended'in'30'ml'of'1'mM'phenylmethylsulfonyl'(PMSF)'in' PBS'to'avoid'protease'action.'Then,'the'cells'were'sonicated'using'a'Digital#Sonifier#S-450D' (Branson)'with'a'3'mm'microtip.'The'parameters'were'set'to'6'pulses'of'30's'each'at'40%' amplitude,'with'a'pause'of'30's'between'each'pulse'and'maintaining'cold'conditions.'Once' sonicated,'the'protein'extract'was'centrifuged'for'10'min'at'15000x'g'and'4°'C,'recovering' the'supernatant'to'remove'cell'debris.' ' § Protein'extraction'with'urea'buffer' ' This' technique' was' performed' in' the' cases' that' denaturing' conditions' had' to' be' achieved' during' protein' extraction' to' preserve' protein' extracts' from' the' action' of' peptidases.'For'this'purpose,'thorough'lysis'of'harvested'cells'was'conducted'in'urea'buffer' (8'M'urea,' 10' mM'Tris-HCl'pH' 8.0),'using'a' 1' ml'syringe'with' a'0.5'mm' ø' needle'(BD)'to' mechanically'break'DNA.'After'protein'quantification'of'the'lysates'and'boiling'in'Laemmli' buffer'(0.1'M'Tris-HCl'pH'6.8,'8%'(v/v)'glycerol,'1.6%'(w/v)'SDS,'4%'(v/v)'2-mercaptoethanol,' 12.5‰'(w/v)'bromophenol'blue)'(Laemmli,'UK,'1970),'samples'were'analysed'by'SDS-PAGE.' ' § Protein'extraction'with'SDS'buffer' ' Alternatively,' for' also' preserving' protein' extracts' from' the' action' of' peptidases,' harvested'cells'were'lysed'in'SDS'buffer'(1%'SDS'wt/v,'5'mM'EDTA,'5'mM'EGTA,'10'mM'N- ethylmaleimide' (NEM),' 1x' cOmpleteTM# Protease# Inhibitor# Cocktail' (Roche)' in' PBS)' and' sonicated'using'a#Digital#Sonifier#S-450D'(Branson)'with'a'3'mm'microtip.'The'parameters' were'set'to'6'pulses'of'30's'each'at'10%'amplitude,'with'a'pause'of'30's'between'each'pulse.' Protein' extracts' were' then' centrifuged' for' 15' min' at' 15000x' g' and' supernatant' was' recovered' to' eliminate' cell' debris.' After' protein' quantification,' samples' were' boiled' in' Laemmli'buffer'and'analysed'by'SDS-PAGE.' Materials'and'methods' ' 133' § Protein'extraction'with'Triton/NP40'buffer' 'When'non-denaturing' conditions'had'to' be' maintained,'harvested'cells'were' lysed' with'Triton'buffer'(50'mM'Tris-HCl'pH'7.5,'150'mM'NaCl,'1%'(v/v)'Triton'X-100,'1'mM'EDTA' and' 1x'cOmpleteTM# Protease# Inhibitor# Cocktail' (Roche))' or' NP-40' buffer' (50' mM' Tris-HCl' pH'8.0,' 150' mM' NaCl,' 1%' (v/v)' Nonidet' P-40,' 0.5%' (w/v)' sodium' deoxycholate' and' 1x'cOmpleteTM#Protease#Inhibitor#Cocktail'(Roche))'as'indicated.'Lysates'were'then'kept'on' ice'for'30'min.'After'centrifugation'and'protein'quantification'of'the'supernatants,'protein' samples' were' boiled' in' Laemmli' buffer' and' analysed' by' SDS-PAGE' or' used' for' other' purposes.' ' 7.2.4.2 Protein/purification,/pull-down/and/immunoprecipitation/ / § Purification'of'GST-tagged'proteins' 'GST-tagged' proteins' were' heterologously' expressed' in' bacteria' by' induction' with' IPTG' and' then' purified' by' glutathione-affinity' chromatography' using' a' Glutathione# SepharoseTM#4B#(GE'Healthcare)'matrix,'following'the'manufacturer’s'instructions.'Then,'the' adsorbed' proteins' were' eluted' with' 10' mM' reduced' glutathione' or' cleavaged' from' GST' using'the'PreScission'(GE'Healthcare)'enzyme'according'to'the'manufacturer’s'instructions.' Protein' purification' was' checked' by' SDS-PAGE' and' staining' with' Coomassie' blue' (0.25%'(w/v)'Coomassie'Brilliant'blue,'40%'(v/v)'methanol,'10%'(v/v)'glacial'acetic'acid).' ' § GST'in#vitro'pull-down' ' For'pulling'down'Krox20'interacting'with'Ubc9,'0.5-1'µg'of'GST'or'GST-Ubc9'bound'to' a'Glutathione#SepharoseTM#4B#(GE'Healthcare)'matrix'were'incubated'with'in#vitro#translated' Krox20'(1/10'of'the'reaction)'in'200'µl'of'binding'buffer'(20'mM'Tris-HCl'pH'7.0,'100'mM' NaCl,'1'mM'EDTA,'10%'glycerol,'0.01%'Nonidet'P40)'and'washed,'first'with'binding'buffer' and' then' with' binding' buffer' supplemented' with' 0.4' M' NaCl.' Samples' were' boiled' in' Laemmli'buffer'and'analysed'by'SDS-PAGE.' ' ' ' ' Materials'and'methods' ' ' ' 134' § Ni2+'affinity'pull-down'for'polyhistidine-tagged'proteins' ' This' protocol' was' used' for' pulling' down' polyhistidine-tagged' proteins,' heterologously' expressed' in' mammal' cells' after' transfection' or' after' protein' purification' from'bacteria'through'expression'induction'with'IPTG.' 'For'pull-down,'the'cell'pellets'were'thoroughly'lysed'in'lysis'buffer'(0.1'M'phosphate' buffer' pH' 8.0,' 8' M' urea)' with' a' 1' ml' syringe' connected' to' a' 0.5' mm' ø' needle' (BD)' to' mechanically'break'DNA.'After'centrifugation'for'10'min'at'16000x'g,'protein'concentration' in' the' supernatant' was' determined' and' inputs' taken.' Then,' 50' µl' of' His-Select®# Nickel# Affinity# Gel# (Sigma-Aldrich)' were' added' and' incubated' for' 2-3' h' under' rotation' at' room' temperature.'Beads'were'centrifuged'for'30's'at'2000x'g'and'washed'three'times'with'1'ml' of'wash'buffer'(0.1'M'phosphate'buffer'pH'6.3,'8'M'urea).'Proteins'were'eluted'from'the' beads'incubating'them'in'Laemmli'buffer'for'20'min'at'room'temperature'and'centrifuging' the'beads'to'recover'the'supernatants.'Inputs'and'precipitated'proteins'were'analysed'by' SDS-PAGE,'following'Coomassie'blue'staining'or'Western'blot'analysis.' ' For'Nab2'purification,'the'same'protocol'was'conducted'using'a'non-denaturing'lysis' buffer'(0.1'M'phosphate'buffer'pH'8.0,'1%'(v/v)'Triton'X-100,'5'M'NaCl'and'1x'cOmpleteTM# Protease# Inhibitor# Cocktail' (Roche))' and' eluting' proteins' from' the' beads' with' increasing' concentrations'of'imidazole,'from'5'mM'to'200'mM.' ' § Immunoprecipitation'of'Flag-tagged'proteins' ' For'this'procedure,'P19'cells'were'harvested'with'cold'PBS'and'centrifuged'for'5'min' at'1500x' g,' following' lysis' in' IP' buffer' (50' mM' Tris-HCl' pH' 8.0,' 150' mM' NaCl,' 1.5'mM'EDTA'pH8.0,'1x'cOmpleteTM#Protease#Inhibitor#Cocktail'(Roche))'+'1%'(v/v)'Triton'X- 100'by'rotating'the'suspension'for'10'min'at'room'temperature.'After'centrifugation'for'10' min'at'10000x'g,'total'protein' in'the'supernatants'was'quantified' and'inputs'were'taken.' Then,' Triton' X-100' concentration' was' lowered' to' 0.33%' (v/v)' adding' IP' buffer' without' detergent.' Flag-tagged' proteins' were' then' immunoprecipitated' using' the' Anti-Flag®# M2' (Sigma-Aldrich)'affinity'gel,'according'to'the'manufacturers'instructions.' ' For' this,' Anti-Flag' beads' were' added' to' the' protein' extracts' and' incubated' for' 2-3'h'at'4°'C,'following'three'washes'with'cold'IP'buffer'+'0.33%'(v/v)'Triton'X-100.'Bound' proteins'were'eluted'boiling'the'beads'at'96°'C'for'4'min'in'the'presence'of'Laemmli'buffer.' Inputs'and'immunoprecipitated'proteins'were'analysed'by'SDS-PAGE.' Materials'and'methods' ' 135' § SUMO'immunoprecipitation'and'peptide'elution' ' For' enrichment' of' SUMOylated' products,' an' immunoprecipitation' protocol' that' involves' specific' peptide' elution' was' employed' (Barysch,' SV# et# al.,' 2014),' allowing' the' purification'of'endogenously'SUMOylated'proteins.' ' Briefly,'denaturing'conditions'were'reached'in'each'lysate'batch'by'addition'of'DTT'at' a'final'concentration'of'50'mM'and'incubating'the'samples'at'60°'C'for'10'min.'Then,'lysates' were'sonicated'using'a'Digital#Sonifier#II#450#(Branson)'with'6-7'cycles.'The'parameters'were' set'to'cycles'of'1'min,'performing'1's'pulses'at'a'45%'of'amplitude,'with'1's'between'pulses.' Then,'RIPA'conditions'were'achieved'by'diluting'each'sample'1/10'with'RIPA'dilution'buffer' (5' mM' EDTA,' 5' mM' EGTA,' 10' mM' NEM,' 1' mM' Pefabloc' and' 1' mg/ml' each' of' protease' inhibitors)' and' adding' 10' mM' NEM.' For' each' set' of' experiments,' proliferative' and' differentiated' cell' lysates' were' then' mixed' in' a' protein' proportion' of' 1:1,' after' protein' quantification,' and' inputs' from' each' set' of' experiments' were' taken.' For' the' first' experiment,'150'ml'of'lysate'from'the'proliferative'condition'at'a'protein'concentration'of' 0.825' mg/ml' were' mixed' with' 160.5' ml' of' lysate' from' the' differentiating' condition' at' a' protein'concentration'of'0.77'mg/ml,'resulting'in'310.5'ml'of'combined'lysate.' ' For' the' second' experiment,' 150' ml' of' lysate' from' the' proliferative' condition' at' a' protein' concentration' of' 1.2' mg/ml' were' mixed' with' 180' ml' of' lysate' from' the' differentiating' condition' at' a' protein' concentration' of' 1.0' mg/ml,' resulting' in' 330' ml' of' combined'lysate.' ' For'the'SUMO'immunoprecipitation,'each'combined'lysate'was'equally'splitted'into' three' batches' and' 1'ml' of' beads,' coupled' with' control' IgG' or' with' SUMO1' or' SUMO2/3' antibodies,' were' added' per' immunoprecipitation' and' incubated' over' night' at' 4°' C' under' rotation.' After' taking' flow-through' samples,' beads' were' washed' three' times' with' RIPA' buffer'(0.1%'(w/v)'SDS,'5'mM'EDTA,'5'mM'EGTA,'10'mM'NEM,'1'mM'Pefabloc'and'1'mg/ml' each' of' aprotinin,' leupeptin' and' pepstatin)' and,' after' a' pre-elution' step' with' pre-elution' buffer' (20'mM'phosphate'buffer' pH' 7.4,' 500' mM' NaCl,' 1%' (v/v)' Triton' X-100,' 0.5%' (w/v)' sodium' deoxycholate,' 0.1%'(w/v)' SDS,' 5' mM' EDTA,' 5' mM' EGTA,' 10' mM' NEM,' 1' mM' Pefabloc' and' 1'mg/ml' of' protease' inhibitors),' elution' of' SUMOylated' proteins' was' performed'in'two'steps,'supplementing'fresh'pre-elution'buffer'with'0.5'mg/ml'of'SUMO1' (VPMNSLRFLFE)'or'SUMO2'(IRFRFDGQPI)'peptides.'For'control'IgG'elution,'a'mixture'of'0.25' mg/ml'of'both'SUMO1'and'SUMO2'peptides'was'used.' Materials'and'methods' ' ' ' 136' ' Eluted'proteins'were'precipitated'with'10%'trichloroacetic'acid'(TCA)'and'washed'in' cold'acetone.'To'check'immunoprecipitation'efficiency,'inputs,'flow-throughs'and'0.05%'of' the' total' eluted' volume' were' analysed' by' SDS-PAGE.' The' remaining' inputs' and' eluted' proteins'were'loaded'into'a'4-12%'NuPAGE®'gel'for'protein'separation'and'MS'analysis.' ' 7.2.4.3 Yeast/two-hybrid/ / ' The' two-hybrid' system' ProQuestTM# Two-Hybrid# System# (Invitrogen)' was' employed' with' a' Krox20' deletion' construct' lacking' its' trans-activation' domains' and' a' cDNA' library' (Invitrogen)'from'8.5'days'whole'mouse'embryo,'to'search'for'protein-protein'interactions' with'Krox20.'The'cDNA'insert'had'an'average'size'of'1.2'kb'and'was'directionally'cloned.' ' In' this' technique,' a' fusion' protein' consisting' on' truncated' Krox20' and' the' DNA' binding'domain'(DB)'of'the'transcription'factor'Gal4'(included'in'the'pDBLeu'vector,'which' allows'growth'in'leucine-free'medium)'is'cloned.'An'additional'fusion'protein'encompassing' the'second'protein'of'interest'coded'in'the'cDNA'library'and'the'transactivation'domain'(AD)' of'the'Gal4'transcription'factor'(in'the'pPC86'vector,'which'allows'growth'in'tryptophan-free' medium)'is'also'obtained.'If'there'is'an'interaction'between'both'proteins'of'interest'in'the' MaV203' strain' of' S.#cerevisiae,' the' chimeric' transcription' factor' is' reconstituted' and' activates' the' expression' of' reporter' genes' HIS3' (that' allows' growth' in' histidine-free' medium)' and' LacZ' (whose' expression' leads' to' colour' appearance' when' treating' with' 5- bromo-4-chloro-3-indolyl-ß-D-galactopyranoside'(X-Gal)).'For'this'experiment,'a'3-amino-1,' 2,'4-triazole'(3AT)'concentration'of'10'mM'was'determined'to'block'basal'activation'of'the' HIS3'reporter'gen.' ' § ß-galactosidase'activity'assay' ' Yeast'to'be'tested'and'their'controls'were'plated'in'10'cm'ø'sterile'plates'(Monolab)' with'minimal'non-selective'medium'and'grown'at'30°'C'for'72'h'till'required'biomass'was' obtained.' Then,' 5' ml' of' 1' M' phosphate' buffer' pH' 7.0' (174,2' g/l' K2HPO4' and' 136,1' g/l' KH2PO4)'and'5'ml'of'agarose'1%'w/v'were'heated'to'50°'C.'This'was'combined'with'100'µl'of' SDS'10%'w/v,'600'µl'of'N,'N’-dimethylformamide'(DMF)'and'100'µl'of'X-Gal'4%'w/v'in'DMF' and'the'mixture'was'poured'over'the'yeast.'Plates'were'dried'for'5'min'and'incubated'at'30°' C'in'darkness.'Results'were'visible'after'30'min-12'h.' Materials'and'methods' ' 137' 7.2.4.4 Protein/analysis/ / § Protein'quantification' ' Protein' extracts' were' quantified' by' Bradford' method.' For' this' purpose,' 3' µl' of' protein'extract'were'mixed'to'1'ml'of'Dye#Reagent#Concentrate'(Bio-Rad).'After'incubating' the' mixture' for' 5' min,' absorbance' was' measured' at' 595' nm' with' a' DU®' 800' spectrophotometer'(Beckman'Coulter)'and'protein'concentration'was'obtained'using'a'calibration' curve' of' bovine' serum' albumin' (BSA).' Alternatively,' purified' proteins' were' quantified' by' comparing'Coomassie'blue'staining'of'serial'1:3'dilutions'of'BSA'in'polyacrylamide'gels.' ' § SDS-PAGE' ' SDS' polyacrylamide' gel' electrophoresis' was' employed' for' molecular' weightdependent' separation' of' proteins' and' conducted' as' described' by' (Laemmli,' UK,' 1970),' following'the'Tris-Glycine'method.' ' For'this'purpose,'discontinuous'7.5'to'15%'mini-gels'were'prepared'using'the'Mini- PROTEAN®#Tetra#Cell'casting'system,'pouring'the'separating'buffer'(0.375'M'Tris-HCl'pH'8.8,' 7.5'or'15%'(v/v)'polyacrylamide'(acrylamide'and'bis-acrylamide'29:1),'0.1%'(w/v)'SDS,'0.1%' APS'and'0.05%'(v/v)'TEMED)'with'an'overlay'of'2-propanol.'Once'the'separating'buffer'was' polymerized' 2-propanol' was' removed,' stacking' buffer' (0.125' M' Tris-HCl' pH' 6.8,' 4%' polyacrylamide'(acrylamide'and'bis-acrylamide'29:1),'0.1%'(w/v)'SDS,'0.1%'(v/v)'ammonium' persulfate' (APS)' and' 0.05%' (v/v)' tetramethylethylenediamine' (TEMED))' added' and' combs' placed.'Once'stacking'buffer'was'polymerized,'gels'were'stored'at'4°'C'under'high'humidity' conditions.' ' Prior'to'protein'migration,'Laemmli'buffer'was'added'to'the'samples'and'they'were' boiled'for'1'min'at'96°'C.'Samples'were'loaded'into'the'gels,'submerged'in'running'buffer' (25'mM'Tris,'192'mM'Glycine'pH'8.3'and'0.1%'(p/v)'SDS).'A'pre-stained'10-170'kDa'marker' (Fermentas,'Thermo'Fisher),'unstained'10-200'kDa'marker'(Thermo'Fisher)'or'unstained'10- 200' kDa' (Biorad)' was' loaded' in' one' of' the' lanes' to' monitorize' the' protein' molecular' weights.'Samples'were'migrated'for'around'45'min'at'190'V.' ' ' ' Abbreviations' ' ' ' 144' HRP' ' Horseradish'peroxidase' IP' ' Immunoprecipitation' IgG' ' Immunoglobuline'G' IPTG' ' Isopropyl-beta-D-thiogalactopyranoside' LB' ' Luria-Bertani' mRNA' ' Messenger'ribonucleic'acid' MS' ' Mass'spectrometry' N-' ' Amino-'(protein)' NCS' ' Newborn'calf'serum' NEM' ' N-ethylmaleimide' PAGE' ' Polyacrylamide'gel'electrophoresis' PBS' ' Phosphate'buffered'saline' PBTr' ' PBS'+'Triton-X100' PBTw' ' PBS'+'Tween'20' PCR' ' Polymerase'chain'reaction' PMSF' ' Phenylmethylsulphonyl'fluoride' RNA' ' Ribonucleic'acid' rRNA' ' Ribosomal'ribonucleic'acid' RSV' ' Human'respiratory'syncytial'virus' S.#cerevisiae# Saccharomyces#cerevisiae# SDS' ' Sodium'dodecyl'sulfate' SILAC' ' Stable'Isotope'Labelling'with'Amino'acids'in'Cell'culture' TBE' ' Tris-borate'+'EDTA' TE' ' Tris'+'EDTA' TEMED'' Tetramethylethylenediamine' Tris' ' Tris(hydroxymethyl)aminomethane' Triton-X100' 4-octylphenol'polyethoxylate' Tween-20' Polyoxylethylene'(20)'sorbitan'monolaurate' '(v/v)' ' Volume'per'volume' (w/v)' ' Weight'per'volume' ' ' Abbreviations' ' 145' § Physical'units' ' A' Ampere' °C'Degree'Celsius' d' Day' g' Gram' x'g' Gravitational'acceleration'on'Earth' h' Hour' l' Liter' m' Meter' M' Molar'(mol/L)' min' Minute' OD' Optical'density' pH' Negative'common'logarithm'for'proton'concentration' s' Second' V' Volt' ' § Prefixes' ' k' Kilo-'103' c' Centi-'10-2' m' Mili-'10-3' μ' Micro-'10-6' n' Nano-'10-9' ' § Nucleic'acids'code' ' A' Adenine' T' Thymine' C' Cytosine' G' Guanine' U' Uracile' ' ' Abbreviations' ' ' ' 146' § Amino'acids'code' ' A' Ala' Alanine' C' Cys' Cysteine' D' Asp' Aspartate' E' Glu' Glutamate' F' Phe' Phenylalanine' G' Gly'Glycine' H' His' Histidine' I' Iso' Isoleucine' K' Lys'Lysine' L' Leu'Leucine' M' Met' Methionine' N' Asn' Asparagine' P' Pro'Proline' Q' Gln' Glutamine' R' Arg'Arginine' S' Ser'Serine' T' Thr'Threonine' V' Val' Valine' W' Trp' Triptophane' Y' Tyr'Tyrosine' ' ' ' ' 147' '/ Appendix' ' ' ' 148' 9/ Appendix/ ' ' The' enclosed' CD' contains' the' data' obtained' from' the' MS' analysis' performed' to' identify'proteins'that'change'their'SUMOylation'levels'between'proliferative'and'neuronal' differentiating'conditions,'comprised'in'the'following'appendix'tables:' ' Table/A-1:/Total/identified/proteins.'Include'the'number'of'Razor'plus'unique'peptides'and' SILAC'intensity'ratios'from'all'proteins'identified'in'this'MS'analysis.' ' Table/ A-2:/ SUMO1/ SILAC/ candidates.'Include'data'from'identified'SUMO1'target'proteins' that'show'a'relevant'SILAC'intensity'ratio.' ' Table/ A-3:/ SUMO2/3/ SILAC/ candidates.' Include' data' from' identified' SUMO2/3' target' proteins'that'show'a'relevant'SILAC'intensity'ratio.' ' Table/ A-4:/ SUMO1/ SILAC/ relative/ candidates.' Include' data' from' identified' SUMO1' target' proteins'with'SILAC'intensity'ratios'due'to'a'remarkable'change'in'their'SUMOylation'state' and'not'to'significant'changes'in'their'expression'levels.' ' Table/ A-5:/ SUMO2/3/ SILAC/ relative/ candidates.' Include' data' from' identified' SUMO2/3' target'proteins'with'SILAC'intensity'ratios'due'to'a'remarkable'change'in'their'SUMOylation' state'and'not'to'significant'changes'in'their'expression'levels.' ' ' 149' ' ' Bibliography' ' ' ' 150' 10/ Bibliography/ ' Alegre' KO' and' Reverter' D' (2011)' Swapping' small' ubiquitin-like' modifier' (SUMO)' isoform' specificity'of'SUMO'proteases'SENP6'and'SENP7.'J'Biol'Chem'286:'36142-36151' Azuma'Y,'Arnaoutov'A'and'Dasso'M'(2003)'SUMO-2/3'regulates'topoisomerase'II'in'mitosis.' J'Cell'Biol'163:'477-487' Azuma'Y,'Tan'SH,'Cavenagh'MM,'Ainsztein'AM,'Saitoh'H'and'Dasso'M'(2001)'Expression'and' regulation'of'the'mammalian'SUMO-1'E1'enzyme.'FASEB'J'15:'1825-1827' Barysch'SV,'Dittner'C,'Flotho'A,'Becker'J'and'Melchior'F'(2014)'Identification'and'analysis'of' endogenous'SUMO1'and'SUMO2/3'targets'in'mammalian'cells'and'tissues'using'monoclonal' antibodies.'Nat'Protoc'9:'896-909' Bayer'P,'Arndt'A,'Metzger'S,'Mahajan'R,'Melchior'F,'Jaenicke'R'and'Becker'J'(1998)'Structure' determination'of'the'small'ubiquitin-related'modifier'SUMO-1.'J'Mol'Biol'280:'275-286' Becker'J,'Barysch'SV,'Karaca'S,'Dittner'C,'Hsiao'HH,'Berriel'Diaz'M,'Herzig'S,'Urlaub'H'and' Melchior'F'(2013)'Detecting'endogenous'SUMO'targets'in'mammalian'cells'and'tissues.'Nat' Struct'Mol'Biol'20:'525-531' Beg'AA'and'Scheiffele'P'(2006)'Neuroscience.'SUMO'wrestles'the'synapse.'Science'311:'962- 963' Bellini'E,'Pavesi'G,'Barbiero'I,'Bergo'A,'Chandola'C,'Nawaz'MS,'Rusconi'L,'Stefanelli'G,'Strollo' M,' Valente' MM,' Kilstrup-Nielsen' C' and' Landsberger' N' (2014)' MeCP2' post-translational' modifications:' a' mechanism' to' control' its' involvement' in' synaptic' plasticity' and' homeostasis?'Front'Cell'Neurosci'8:'236' Bilbiography' ' 151' Berdnik'D,'Torok'T,'Gonzalez-Gaitan'M'and'Knoblich'JA'(2002)'The'endocytic'protein'alpha- Adaptin'is'required'for' numb-mediated' asymmetric'cell'division' in' Drosophila.'Dev'Cell'3:' 221-231' Bernier-Villamor' V,' Sampson' DA,' Matunis' MJ' and' Lima' CD' (2002)' Structural' basis' for' E2- mediated'SUMO'conjugation'revealed'by'a'complex'between'ubiquitin-conjugating'enzyme' Ubc9'and'RanGAP1.'Cell'108:'345-356' Bertrand' N,' Castro' DS' and' Guillemot' F' (2002)' Proneural' genes' and' the' specification' of' neural'cell'types.'Nat'Rev'Neurosci'3:'517-530' Blader' P,' Fischer' N,' Gradwohl' G,' Guillemot' F' and' Strahle' U' (1997)' The' activity' of' neurogenin1'is' controlled'by' local' cues'in' the'zebrafish' embryo.' Development'124:' 4557- 4569' Bohren'KM,'Nadkarni'V,'Song'JH,'Gabbay'KH'and'Owerbach'D'(2004)'A'M55V'polymorphism' in'a'novel'SUMO'gene'(SUMO-4)'differentially'activates'heat'shock'transcription'factors'and' is'associated'with'susceptibility'to'type'I'diabetes'mellitus.'J'Biol'Chem'279:'27233-27238' Bray'SJ'(2006)'Notch'signalling:'a'simple'pathway'becomes'complex.'Nat'Rev'Mol'Cell'Biol'7:' 678-689' Castoralova' M,' Brezinova' D,' Sveda' M,' Lipov' J,' Ruml' T' and' Knejzlik' Z' (2012)' SUMO-2/3' conjugates' accumulating' under' heat' shock' or' MG132' treatment' result' largely' from' new' protein'synthesis.'Biochim'Biophys'Acta'1823:'911-919' Ceballos-Chavez' M,' Rivero' S,' Garcia-Gutierrez' P,' Rodriguez-Paredes' M,' Garcia-Dominguez' M,'Bhattacharya'S'and'Reyes'JC'(2012)'Control'of'neuronal'differentiation'by'sumoylation'of' BRAF35,'a'subunit'of'the'LSD1-CoREST'histone'demethylase'complex.'Proc'Natl'Acad'Sci'U'S' A'109:'8085-8090' Cepko' CL' (1999)' The' roles' of' intrinsic' and' extrinsic' cues' and' bHLH' genes' in' the' determination'of'retinal'cell'fates.'Curr'Opin'Neurobiol'9:'37-46' Bibliography' ' ' ' 152' Chang'CC,'Naik'MT,'Huang'YS,'Jeng'JC,'Liao'PH,'Kuo'HY,'Ho'CC,'Hsieh'YL,'Lin'CH,'Huang'NJ,' Naik'NM,'Kung'CC,'Lin'SY,'Chen'RH,'Chang'KS,'Huang'TH'and'Shih'HM'(2011)'Structural'and' functional' roles' of' Daxx' SIM' phosphorylation' in' SUMO' paralog-selective' binding' and' apoptosis'modulation.'Mol'Cell'42:'62-74' Chavrier'P,'Zerial'M,'Lemaire'P,'Almendral'J,'Bravo'R'and'Charnay'P'(1988)'A'gene'encoding' a'protein'with'zinc'fingers'is'activated'during'G0/G1'transition'in'cultured'cells.'EMBO'J'7:' 29-35' Cheng' J,' Huang' M,' Zhu' Y,' Xin' YJ,' Zhao' YK,' Huang' J,' Yu' JX,' Zhou' WH' and' Qiu' Z' (2014)' SUMOylation'of'MeCP2'is'essential'for'transcriptional'repression'and'hippocampal'synapse' development.'J'Neurochem'128:'798-806' Chenn'A'and'McConnell'SK'(1995)'Cleavage'orientation'and'the'asymmetric'inheritance'of' Notch1'immunoreactivity'in'mammalian'neurogenesis.'Cell'82:'631-641' Chenn' A,' Zhang' YA,' Chang' BT' and' McConnell' SK' (1998)' Intrinsic' polarity' of' mammalian' neuroepithelial'cells.'Mol'Cell'Neurosci'11:'183-193' Childs' KS' and' Goodbourn' S' (2003)' Identification' of' novel' co-repressor' molecules' for' Interferon'Regulatory'Factor-2.'Nucleic'Acids'Res'31:'3016-3026' Chomette' D,' Frain' M,' Cereghini' S,' Charnay' P' and' Ghislain' J' (2006)' Krox20' hindbrain' cisregulatory' landscape:' interplay' between' multiple' long-range' initiation' and' autoregulatory' elements.'Development'133:'1253-1262' Chung'CD,'Liao'J,'Liu'B,'Rao'X,'Jay'P,'Berta'P'and'Shuai'K'(1997)'Specific'inhibition'of'Stat3' signal'transduction'by'PIAS3.'Science'278:'1803-1805' Davis' RL' and' Turner' DL' (2001)' Vertebrate' hairy' and' Enhancer' of' split' related' proteins:' transcriptional' repressors' regulating' cellular' differentiation' and' embryonic' patterning.' Oncogene'20:'8342-8357' Bilbiography' ' 153' Desmazieres'A,'Charnay'P'and'Gilardi-Hebenstreit'P'(2009)'Krox20'controls'the'transcription' of'its'various'targets'in'the'developing'hindbrain'according'to'multiple'modes.'J'Biol'Chem' 284:'10831-10840' Desmazieres'A,'Decker'L,'Vallat'JM,'Charnay'P'and'Gilardi-Hebenstreit'P'(2008)'Disruption'of' Krox20-Nab'interaction'in'the'mouse'leads'to'peripheral'neuropathy'with'biphasic'evolution.' J'Neurosci'28:'5891-5900' Desterro'JM,'Rodriguez'MS'and'Hay'RT'(1998)'SUMO-1'modification'of'IkappaBalpha'inhibits' NF-kappaB'activation.'Mol'Cell'2:'233-239' Desterro' JM,' Rodriguez' MS,' Kemp' GD' and' Hay' RT' (1999)' Identification' of' the' enzyme' required'for'activation'of'the'small'ubiquitin-like'protein'SUMO-1.'J'Biol'Chem'274:'10618- 10624' Desterro'JM,'Thomson'J'and'Hay'RT'(1997)'Ubc9'conjugates'SUMO'but'not'ubiquitin.'FEBS' Lett'417:'297-300' Di'Bacco'A,'Ouyang'J,'Lee'HY,'Catic'A,'Ploegh'H'and'Gill'G'(2006)'The'SUMO-specific'protease' SENP5'is'required'for'cell'division.'Mol'Cell'Biol'26:'4489-4498' Dohmen'RJ'(2004)'SUMO'protein'modification.'Biochim'Biophys'Acta'1695:'113-131' Dohmen'RJ,'Stappen'R,'McGrath'JP,'Forrova'H,'Kolarov'J,'Goffeau'A'and'Varshavsky'A'(1995)' An'essential'yeast'gene'encoding'a'homolog'of'ubiquitin-activating'enzyme.'J'Biol'Chem'270:' 18099-18109' Eaton'EM'and'Sealy'L'(2003)'Modification'of'CCAAT/enhancer-binding'protein-beta'by'the' small' ubiquitin-like' modifier' (SUMO)' family' members,' SUMO-2' and' SUMO-3.' J' Biol' Chem' 278:'33416-33421' Eckermann'K'(2013)'SUMO'and'Parkinson's'disease.'Neuromolecular'Med'15:'737-759'