Regulation of CDX2 and intestinal differentiation in homeostasis and carcinogenesis: emphasis on the role of MEX3A

Facul y o Medicine o he Uni e si y o Po o Doc o al P og am in Biomedicine Regula ion o CDX2 and in es inal di e en ia ion in homeos asis and ca cinogenesis: emphasis on he ole o MEX3A Regulação do CDX2 e di e enciação in es inal em homeos asia e ca cinogénese: ele ância uncional do MEX3A B uno Miguel Co eia Pe ei a IPATIMUP – Di e en ia ion and Cance g oup Po o, Po ugal 2013 O ien ação P o esso a Dou o a Raquel Ma ia da Sil a G aça Almeida, In es igado a e líde do g upo Di e enciação e Canc o do IPATIMUP P o esso a A iliada da Faculdade de Medicina da Uni e sidade do Po o Co-o ien ação P o esso a Dou o a Ma ia Leono Ma ins Soa es Da id, In es igado a do g upo Di e enciação e Canc o do IPATIMUP P o esso a Ca ed á ica da Faculdade de Medicina da Uni e sidade do Po o DISSERTAÇÃO DE CANDIDATURA AO GRAU DE DOUTOR APRESENTADA À FACULDADE DE MEDICINA DA UNIVERSIDADE DO PORTO iii A igo 48º, § 3º – A Faculdade não esponde pelas dou inas expendidas na Disse ação (Regulamen o da Faculdade de Medicina da Uni e sidade do Po o - Dec e o-Lei nº 19337, de 29 de Janei o de 1931). JÚRI DAS PROVAS DE DOUTORAMENTO DISSERTATION DEFENSE COMMITTEE P esiden e: Dou o José Agos inho Ma ques Lopes, Di ec o da Faculdade de Medicina da Uni e sidade do Po o (po delegação ei o al) Vogais: Dou o Ma c Billaud, In es igado do Ins i u Albe Bonnio , CRI INSERM/UJF U823, G enoble, F ança Dou o Pe e Jo dan, In es igado do Ins i u o Nacional de Saúde D . Rica do Jo ge de Lisboa Dou o Manuel Albe o Coimb a Sob inho Simões, P o esso Ca ed á ico da Faculdade de Medicina da Uni e sidade do Po o Dou o a Ca la Isabel Gonçal es de Oli ei a, P o esso a A iliada da Faculdade de Medicina da Uni e sidade do Po o Dou o João An ónio Pin o de Sousa, P o esso Associado da Faculdade de Medicina da Uni e sidade do Po o Dou o a Raquel Ma ia da Sil a G aça Almeida, P o esso a A iliada da Faculdade de Medicina da Uni e sidade do Po o, e o ien ado a da ese ii LISTA DE PUBLICAÇÕES LIST OF PUBLICATIONS Ao ab igo do dispos o no nº 2 do A . 8º do Dec e o-Lei nº 388/70, cons i uem pa e in eg an e des a Disse ação os seguin es abalhos já publicados: Pe ei a B, Oli ei a C, Da id L, Almeida R. (2009) CDX2 p omo e me hyla ion is no associa ed wi h mRNA exp ession. In e na ional Jou nal o Cance , 125(7):1739-1742 Pe ei a B, Sousa S, Ba os R, Ca e o L, Oli ei a P, Oli ei a C, Cha ie NT, Pla e o i M, Rouaul JP, F eund JN, Billaud M, Almeida R. (2013) CDX2 egula ion by he RNA-binding p o ein MEX3A: impac on in es inal di e en ia ion and s emness. Nucleic Acids Resea ch, 41(7):3986-3999 Pe ei a B, Le Bo gne M, Cha ie NT, Billaud M, Almeida R. (2013) MEX-3 p o eins: ecen insigh s on no el pos - ansc ip ional egula o s. T ends in Biochemical Sciences, 38(10):477-479 Em cump imen o com o dispos o no Dec e o-Lei sup amencionado, o au o decla a que pa icipou ac i amen e na concepção e execução do abalho expe imen al, na in e p e ação e discussão dos esul ados, e na edacção dos espec i os a igos cien í icos. ix TABLE OF CONTENTS LIST OF PUBLICATIONS FUNDING ACKNOWLEDGEMENTS TABLE OF CONTENTS ABSTRACT RESUMO ABBREVIATIONS CHAPTER 1. GENERAL INTRODUCTION AND AIMS 1.1 Gas oin es inal ca cinogenesis 1.1.1 An o e iew on gas ic cance 1.1.2 In es inal me aplasia 1.1.3 Colo ec al ca cinogenesis 1.1.4 In es inal homeos asis 1.2 The in es inal ansc ip ion ac o CDX2 1.2.1 CDX2 homeobox gene and i s a ge s 1.2.2 Role in homeos asis 1.2.3 In ol emen in ca cinogenesis 1.2.4 CDX2 egula o y ne wo k 1.3 Pos - ansc ip ional egula ion o gene exp ession 1.3.1 F om ansc ip s o p o eins 1.3.2 RNA-binding p o eins and hei biological unc ions 1.4 The MEX-3 amily o RNA-binding p o eins 1.4.1 Caeno habdi is elegans MEX-3 1.4.2 The mammalian MEX3 p o eins 2 4 7 8 11 13 15 17 24 25 27 29 xx ix x xi x ii xxiii xxi 1 x ii 1.5 Long- e m goal and speci ic aims CHAPTER 2. CDX2 PROMOTER METHYLATION Pape I – CDX2 p omo e me hyla ion is no associa ed wi h mRNA exp ession. In e na ional Jou nal o Cance , 2009 CHAPTER 3. CDX2 POST-TRANSCRIPTIONAL REGULATION BY THE RNA-BINDING PROTEIN MEX3A Pape II – CDX2 egula ion by he RNA-binding p o ein MEX3A: impac on in es inal di e en ia ion and s emness. Nucleic Acids Resea ch, 2013 Pape II – Supplemen a y Da a CHAPTER 4. THE MAMMALIAN MEX3 PROTEIN FAMILY Pape III – MEX-3 p o eins: ecen insigh s on no el pos - ansc ip ional egula o s, T ends in Biochemical Sciences, 2013 CHAPTER 5. DISCUSSION AND CONCLUDING REMARKS 5.1 CDX2 egula ion by p omo e me hyla ion 5.2 3D AGS cell model and loss o CDX2 exp ession 5.3 Mechanis ic aspec s o MEX3A unc ion 5.4 MEX3A and ca cinogenesis 5.5 A conse ed unc ion o MEX3A in mammalian emb yogenesis? 5.6 Concluding ema ks 37 45 59 67 75 77 78 82 84 87 35 43 65 73 30 REFERENCES APPENDIX Appendix A. Lis o an ibodies and p ime s used in his s udy Appendiz B. Lis o di e en ially exp essed genes be ween 3D and 2D AGS cell cul u es 119 91 xix ABSTRACT The homeodomain ansc ip ion ac o CDX2 is a key playe in in es inal di e en ia ion. The e o e, i is no su p ising o ind i s exp ession signi ican ly al e ed in ca cinogenic p ocesses o he gas oin es inal ac . In he s omach, de no o CDX2 exp ession d i es a p eneoplas ic lesion known as in es inal me aplasia ha inc eases he isk o gas ic cance de elopmen . Qui e he opposi e, in he con ex o colo ec al cance , CDX2 has been classically desc ibed as a umou -supp esso , al hough his iew has been g adually challenged in he las ew yea s. No wi hs anding, i is known ha umou s e aining CDX2 exp ession a e mo e di e en ia ed and show be e ou come in e ms o su i al. Thus, a comp ehensi e unde s anding o CDX2 egula ion s ands ou as a esea ch p io i y, o in he long- un, he acqui ed knowledge could be in eg a ed in o al e na i e he apeu ic s a egies ha migh bene i he pa ien . Taking in o conside a ion ha he ex acellula mic oen i onmen is also impo an o he cellula malignan beha io as gene ic cons ains, we decided o ackle i s in luence o e CDX2 exp ession in wo on s:, analyze he possible ole o DNA epigene ic changes, hypo hesizing ha in ec ion by Helicobac e pylo i and he gene a ion o ch onic in lamma ion could in some way a ec he CDX2 gene me hyla ion pa e n and induce i s ansc ip ion; and in a mo e explo a o y app oach, es ablish 3D cul u e models ha be e mimic he in i o milieu, hypo hesizing ha hey would p o ide new insigh s ega ding signaling pa hways a ec ing CDX2 le els. We show he e ha he e is a lack o co ela ion be ween CDX2 me hyla ion s a us and i s exp ession in in es inal me aplasia and gas ic cance cell lines, excluding his mechanism as a CDX2 egula o y ea u e du ing gas ic malignancy. On he o he hand, h ough genome- wide sc eening o he 3D cul u e sys em, comp ising a gas ic cance cell line wi h CDX2 exp ession and he basemen ma ix ma igel, we we e able o iden i y he RNA-binding p o ein MEX3A as pu a i ely in ol ed in CDX2 egula ion. Fu he suppo ed by an e olu iona y unc ional link o i s Caeno habdi is elegans o hologue MEX-3, we demons a e ha MEX3A main ains a conse ed ep essi e unc ion o e CDX2 in gas ic and in es inal cellula models. This is dependen on he in e ac ion wi h a speci ic binding de e minan p esen in CDX2 mRNA 3`un ansla ed egion. Mo eo e , MEX3A o e exp ession ca ies pheno ypic consequences, impai ing in es inal di e en ia ion and cellula pola iza ion, while p omo ing gain o s emness p ope ies. As a esul , we desc ibe o he i s ime a xxi mechanism o CDX2 pos - ansc ip ional con ol, likely con ibu ing o in es inal homeos asis and ca cinogenesis, and add a new laye o he al eady in ica e CDX2 egula o y ne wo k. RESUMO O ac o de ansc ição homeobox CDX2 é um elemen o c í ico na di e enciação in es inal. Assim, não é inespe ado o ac o de ap esen a ní eis de exp essão signi ica i amen e al e ados em p ocessos ca cinogénicos do ac o gas oin es inal. No es ômago, a exp essão de no o de CDX2 induz uma lesão p é-neoplásica denominada me aplasia in es inal que con e e um isco ac escido de desen ol imen o de ca cinoma gás ico. Pelo con á io, no con ex o colo ec al, es e gene em sido ge almen e desc i o como um sup esso umo al, embo a es a classi icação enha indo a se pos a em causa nos úl imos anos. Mesmo assim, econhece-se que umo es com exp essão de CDX2 são mais di e enciados e de êm melho p ognós ico em e mos de sob e ida. To na-se, en ão, undamen al ob e uma comp eensão ap o undada dos p ocessos de egulação de CDX2, pois a longo p azo o conhecimen o adqui ido pode se in eg ado em es a égias e apêu icas al e na i as que pode ão bene icia o pacien e. Tendo em conside ação que o mic oambien e ex acelula é, al como as al e ações gené icas, impo an e pa a o compo amen o celula maligno, decidimos con on a a sua in luência sob e a exp essão de CDX2 em duas pa es: analisa o possí el papel de al e ações epigené icas ao ní el do p omo o do gene, colocando a hipó ese de que o p ocesso de in ecção po Helicobac e pylo i e a subsequen e espos a in lama ó ia pode ão c ia condições que a ec am o pe il de me ilação de CDX2 induzindo a sua ansc ição; e numa abo dagem de ca ác e mais explo a ó io, es abelece modelos de cul u a 3D que mime izem o ambien e in i o, colocando a hipó ese de que es es pe mi i iam ob e no os dados no que espei a às ias de sinalização que a ec am os ní eis de CDX2. Demons ámos que não exis e uma co elação en e o es ado de me ilação do p omo o de CDX2 e a sua exp essão na me aplasia e em linhas celula es de canc o gás ico, excluindo es e mecanismo de egulação no p ocesso de ca cinogénese gás ica. Po ou o lado, a a és de um a imen o ansc icional do sis ema de cul u a 3D, cons i uído po uma linha celula gás ica e a ma iz de memb ana basal ma igel, iden i icámos uma p o eína de ligação ao ARN designada MEX3A como es ando po encialmen e en ol ida na egulação de CDX2. Apoiados numa base uncional e olu i a em elação ao papel do o ólogo MEX-3 em Caeno habdi is elegans, p o ámos que o MEX3A man ém uma unção conse ada enquan o ep esso da exp essão de CDX2 em modelos celula es gás icos e in es inais. Es e e ei o é dependen e da xxiii in e acção com um mo i o de ligação especí ico p esen e na egião 3` não- aduzida do ARN mensagei o de CDX2. Adicionalmen e, a sob exp esão de MEX3A aca e a consequências eno ípicas, a ec ando a di e enciação in es inal e pola idade celula , p omo endo simul aneamen e o ganho de p op iedades es aminais. Em conclusão, desc e emos pela p imei a ez um mecanismo pós- ansc icional de con olo de CDX2, com ele ância pa a a homeos asia in es inal e p o a elmen e ca cinogénese, adicionando um no o elemen o à complexa ede de egulação do gene CDX2. ABBREVIATIONS The ollowing lis desc ibes ele an abb e ia ions and ac onyms used. Bcd Bicoid BMP Bone mo phogene ic p o ein Cad Caudal CDX Caudal ype homeobox CIN Ch omosomal ins abili y CpG Cy osine guanine dinucleo ide CRC Colo ec al cance eIF Euka yo ic ini ia ion ac o GI Gas oin es inal GLD-1 De ec i e in ge mline de elopmen hnRNP K He e ogeneous nuclea ibonucleop o ein K HOX Homeobox IM In es inal me aplasia KH K-homology LGR5 Leucine ich epea -con aining G-p o ein coupled ecep o 5 LRC Label- e aining cell mex Muscle excess MEX3 Mex-3 homologue miR mic oRNA MRE MEX-3 ecogni ion elemen mRNP Messenge ibonucleop o ein MSI Mic osa elli e ins abili y MSI1 Musashi-1 OLFM4 Ol ac omedin-4 P body P ocessing body QKI Quaking RBP RNA-binding p o ein RING Really in e es ing new gene RKHD RING inge and KH domain-con aining TE T ophec ode m UTR Un ansla ed egion xx p opose ha bone ma ow-de i ed cells migh be he ini ia o cells in he gas ic ca cinogenic p ocess, including IM and dysplasia, by homing in and eng a ing o he inju y si es (Hough on e al. 2004; Va on e al. 2012). Al hough en icing as a heo y, he e has been limi ed ansla ion o his model in o he human se ing. Whiche e he case, IM is mos p obably he ou come o an adap i e esponse ha goes amiss, in which he issue ies o cope wi h se e al agg essi e inpu s, bu ends up wi h he c ea ion o suscep ible g ound o neoplas ic ans o ma ion (Mesqui a e al. 2006). In e ms o classi ica ion, wo majo gas ic IM ypes can be ecognized, aking in o accoun no only mo phological, bu also molecula al e a ions, like loss and gain o mucinous di e en ia ion ma ke s and mucin-associa ed ca bohyd a e an igens (Reis e al. 1999; Sil a e al. 2002). The comple e IM o in es inal ype (p e iously denomina ed ype I) e lec s a comple e swi ch in he di e en ia ion p og am, wi h loss o gas ic mucins MUC1, MUC5AC, and MUC6, and de no o exp ession o he in es inal mucin MUC2. I is cha ac e ized by he p esence o abso p i e cells, Pane h cells and goble cells sec e ing sialomucins, simila o he small in es inal pheno ype. The incomple e IM o GI-mixed ype e lec s a mix u e o gas ic and in es inal componen s, bo h a he glandula and cellula le el (Niwa e al. 2005). I is cha ac e ized by he p esence o columna and goble cells sec e ing sialomucins and/o sulphomucins (p e iously denomina ed ypes II and III, espec i ely), simila o he colonic pheno ype. Rema kably, he ec opic in es inal glands s ill p ese e a no mal cell mig a ion pa e n, which is achie ed by a edeploymen o he p oli e a i e niche om he middle o he base o he gland (Inada e al. 2001; Sakamo o e al. 2011). Recip ocal in e ac ions be ween he epi helium and he mesenchyme migh be behind his a chi ec u al emodelling (Mu oh e al. 2005a), as he induc ion o a mesenchymal in es inal pheno ype du ing he ea ly s ages o IM migh lead o he es ablishmen o a posi i e egula o y loop in ol ing pa ac ine signals (Sakagami e al. 1984). I is no ye clea i he wo IM ypes can be conside ed sequen ial s eps in a sha ed p ocess o g adual in es inaliza ion o i hey a ise independen ly. The possibili y exis s ha he incomple e ype is biologically mo e uns able as i e lec s an abe an di e en ia ion p og am wi hou pheno ypic pa allel in he adul o ganism. In ag eemen , epidemiological da a, hough sca ce, shows ha i con e s inc eased isk o gas ic cance de elopmen compa ed o he comple e ype (Filipe and Jass 1986; Rokkas CHAPTER 1. GENERAL INTRODUCTION AND AIMS e al. 1991; Filipe e al. 1994), highligh ing he impo ance o s a i ica ion conce ning p ognos ic signi icance (González e al. 2013). Cu iously, bac e ial coloniza ion is ypically absen in oci o comple e IM (B a o and Co ea 1999), a ou ing a p esumably p o ec i e unc ion. 1.1.3 Colo ec al ca cinogenesis Colo ec al cance (CRC) is cu en ly he hi d mos common cance wo ldwide, as 10% o he newly diagnosed cases a e malignancies o he colon o ec um. Almos 60% o he cases occu in de eloped egions and he a eas wi h highes incidence a e No h Ame ica, Aus alia/New Zealand and Eu ope (Fe lay e al. 2010). Mo e han 600.000 people die each yea , making i he ou h leading cause o cance - ela ed dea h. In Po ugal, conside ing he o al numbe o cases, i places i s in e ms o incidence and mo ali y (IARC 2010). As o he small in es ine, i has a ema kably low incidence o p ima y ca cinomas, especially conside ing i s leng h and su ace a ea, and hose ha do occu a e o en ela ed o he edi a y synd omes. Abou 90% o spo adic umou s occu in indi iduals o e he age o i y. O he isk ac o s include amily his o y, a die low in fibe s and high in ed mea , alcohol, smoking, and seden a y occupa ion (Johnson e al. 2013). A p esen es ima e is ha 15-30% o CRCs ha e a amilial componen . Less han 5% o hese happen in a ecognizable se ing o highly pene an cance synd omes due o ge mline mu a ions, being he mos common he he edi a y nonpolyposis CRC and amilial adenoma ous polyposis (Fea on 2011). Despi e ad ances in su gical echniques and adju an he apy, he e has been only a modes imp o emen in su i al o pa ien s wi h ad anced neoplasms (Edwa ds e al. 2012). Hence, e ec i e p ima y and seconda y p e en i e app oaches mus be de eloped o educe mo bidi y and mo ali y. Since he desc ip ion o he classic adenoma-ca cinoma pa hway (Fea on and Vogels ein 1990), de ining CRC as he esul o a g adual accumula ion o changes ha ans o m no mal glandula epi helial cells in o adenoma, ollowed by in asi e ca cinoma and e en ually me as a ic cance , ou unde s anding o i s molecula pa hogenesis has ad anced and led o nume ous e isions o his linea umou p og ession model. I is now ecognized ha loss o genomic s abili y is a hallma k CHAPTER 1. GENERAL INTRODUCTION AND AIMS 7 ea u e o colo ec al ca cinogenesis (G ady and Ca e he s 2008), and his p ope y led o he es ablishmen o a classi ica ion sys em: (i) he ch omosomal ins abili y (CIN) pheno ype, ound in as many as 85% o umou s and de ined by he p esence o aneuploidy o s uc u al abe a ions; (ii) he MSI pheno ype, de ined by he p esence o uns able loci due o inac i a ion o genes in he DNA misma ch epai amily; and (iii) he CpG island me hyla o pheno ype, exhibi ing bo h global DNA hypome hyla ion and hype me hyla ion o gene p omo e s ha con ain CpG islands (P i cha d and G ady 2011). I is he accumula ion o mu a ions combined wi h mul iple cycles o clonal selec ion ha esul s in he de egula ion o signalling pa hways con olling cell p oli e a ion, di e en ia ion, apop osis, angiogenesis and in asion, ul ima ely culmina ing in cance de elopmen . 1.1.4 In es inal homeos asis The in es inal ac is ana omically di ided in o wo well-de ined segmen s, he small in es ine and la ge in es ine (o colon), lined by a specialized single laye o cells o ganized in o wo mo phologically and unc ionally dis inc compa men s: lask-shaped submucosal in agina ions known as c yp s o Liebe kühn, and inge - shaped luminal p o usions e med illi, which d ama ically inc ease he abso p i e su ace a ea o he small in es ine. The c yp cons i u es he p oli e a i e compa men o he in es inal epi helium; i is monoclonal and main ained by ou o six mul ipo en s em cells loca ed in he lowe hi d (Bje knes and Cheng 1999) ha gi e ise o in e media e descendan s e e ed o as ansi -ampli ying cells. The illus ep esen s he di e en ia ed compa men and is polyclonal as i s cells de i e om se e al c yp s (Po en and Loe le 1990). Abso p i e en e ocy es, mucous-p oducing goble cells, and ho mone-sec e ing en e oendoc ine cells mig a e upwa ds along he basemen memb ane o he epi helium apex, whe e hey unde go apop osis, being subsequen ly ex olia ed in o he in es inal lumen (Hall e al. 1994). Pane h cells a e unusual in ha hey se le a he c yp base and a e he only cell ype mig a ing downwa ds (B y e al. 1994). The modula o ganiza ion o he small in es ine and colon is globally compa able. His ologically, he e a e, howe e , wo impo an di e ences be ween hem. The colon ca ies no illi; ins ead, i has a la su ace epi helium and la ge colonic c yp s ex ending deep in o he submucosa. The ela i e abundance o each o he main cell ypes also a ies ma kedly wi hin he CHAPTER 1. GENERAL INTRODUCTION AND AIMS di e en in es inal segmen s. En e ocy es a e highly pola ized cells esponsible o abso bing and anspo ing nu ien s ac oss he epi helium, sec e ing a cock ail o hyd oly ic enzymes in o he gu . They make up mo e han 80% o all in es inal epi helial cells. Goble cells sec e e p o ec i e mucins and e oil p o eins equi ed o he mo emen and e ec i e emo al o luminal con en s, while p o iding p o ec ion agains shea s ess and chemical damage. Acco dingly, hei numbe s inc ease om he p oximal (4%) o dis al (16%) in es ine (Ka am 1999). En e oendoc ine cells coo dina e gu physiology h ough speci ic ho mone p oduc ion (Höcke and Wiedenmann 1998). They a e sca e ed as indi idual cells h oughou he mucosa, ep esen ing a small p opo ion (<1%) o he cells lining he epi helium (Schonho e al. 2004). Finally, Pane h cells ha e a unc ion in inna e immuni y, syn hesizing bac e icidal agen s such as de ensins and lysozyme (Po e e al. 2002). They a e absen om he colon and ha e a li e expec ancy o six o eigh weeks ( an de Flie and Cle e s 2009), much longe han ha o hei e minally di e en ia ed illus coun e pa s, wi h a u no e a e o oughly h ee o i e days (W igh and I win 1982). Cu en ly, some con o e sy exis s as o he p esence o dis inc ypes o in es inal s em cells. The “+4 posi ion” model assumes he c yp base is exclusi ely popula ed by e minally di e en ia ed Pane h cells and ha s em cells a e loca ed jus abo e hem, on a e age a he +4 posi ion (Po en e al. 1974). These cells we e shown o di ide once e e y day and o be unusually sensi i e o adia ion, possibly p e en ing he accumula ion o dele e ious genomic changes (Po en 1977). They we e desc ibed o e ain DNA labelling, and a e also called label- e aining cells (LRCs), a ea u e sugges ed as being he esul o asymme ic s and seg ega ion (Po en e al. 2002). The “s em cell zone” model s a es ha small, undi e en ia ed, cycling cells called c yp base columna (CBC) cells, esiding in a s em cell-pe missi e en i onmen and wedged be ween he Pane h cells a he base o he c yp s, a e likely o be he ue s em cells (Cheng and Leblond 1974; Bje knes and Cheng 1999). Ye , de ini i e p oo o s emness equi es pu a i e s em cells o be expe imen ally linked o hei p ogeny, and his has p o en elusi e due o lack o speci ic ma ke s (Ba ke e al. 2012). The musashi-1 (MSI1) gene encodes a RNA- binding p o ein ini ially desc ibed as a egula o o asymme ic di ision in neu al s em cells (Glaze e al. 2012). La e s udies showed ha i deno es mul ipo en s em cells in o he se ings, being also highly exp essed a he c yp base, wi h exp ession CHAPTER 1. GENERAL INTRODUCTION AND AIMS 9 e iden on he CBC cells, as well as in some LRCs (Po en e al. 2003). Howe e , i s b oad exp ession domain sugges s ha i is a ma ke o ea ly commi ed p ogeni o s alike. Lineage acing echniques in inducible mouse models led o he iden i ica ion o he leucine ich epea -con aining G-p o ein coupled ecep o 5 (LGR5), he i s speci ic CBC cell ma ke (Ba ke e al. 2007). The LGR5 p o ein ac s as he ecep o o a small amily o Wn agonis s called R-spondins. Lg 5+ cells a e highly uni o m in mo phology, in a iably ouch Pane h cells, and di ide each day. Ano he obus ma ke o Lg 5+ s em cells called ol ac omedin-4 (OLFM4) has ecen ly eme ged. I is a membe o he ol ac omedin domain-con aining amily and encodes a sec e ed glycop o ein ha appea s o ha e an i-apop o ic and cell cycle egula o y cha ac e is ics (G o e e al. 2010). Molecula in si u hyb idiza ion e ealed ha OLFM4 is highly exp essed in no mal CBC cells in human small in es ine and colon ( an de Flie e al. 2009). The mos eliable candida e as an LRC ma ke o da e is BMI1, which encodes a componen o he polycomb ep essing complex 1 ha ac s as a ch oma in modi ie , being implica ed in he s able main enance o gene ep ession (Valk-Lingbeek e al. 2004). I has a ac ed a en ion due o i s ole in egula ing sel - enewal o neu al and hema opoie ic p ogeni o s. Bmi1 was ound o ma k a e cells a he +4 cell posi ion uniquely in abou 10% o he p oximal small in es ine (Sangio gi and Capecchi 2008). Modelling o he in es inal epi helium is based on a delica e balance be ween sel - enewal and di e en ia ion, which mus be main ained h oughou li e. No ewo hy, in all species s udied, he c yp - illus axis junc ion ep esen s he physical h eshold om which in es inal cells acqui e hei inal unc ional cha ac e is ics, a guing o conse ed molecula mechanisms in ol ed in his p ocess. In ac , in es inal homeos asis is dependen on au oc ine and pa ac ine in e ac ions be ween he mucosa and he unde lying mesenchyme, and many o he in e ening signalling pa hways, such as Wn , Bone Mo phogene ic P o ein (BMP), and No ch, ha e been iden i ied (C osnie e al. 2006). These molecula signals p o ide some o he basic p inciples h ough which in es inal a chi ec u e is o ganized, bu i is s ill no clea a wha poin s em cell p ogeny loses i s po ency and becomes i e e sibly commi ed o di e en ia ion. I espec i ely o how his occu s, cell a e decisions need o be closely imed in ela ion o he pa e n o cell di isions. While he mechanisms con olling in es inal cell ansi ions a e a om comple ely CHAPTER 1. GENERAL INTRODUCTION AND AIMS unde s ood, i is ob ious ha hey in ol e ansc ip ion ac o s con e ing compa men -speci ic gene exp ession. When conside ing he molecula basis o in es inal di e en ia ion, a common poin o con e gence exis s, independen ly o he biological se ing – he caudal (cad) ype homeobox 2 gene (CDX2). This ansc ip ion ac o is exclusi ely p esen in he in es ine and de no o exp essed in e e y oci o ec opic in es inal di e en ia ion in he body, associa ed wi h ca cinogenic p ocesses. I s emb yonic equi emen and ansc ip ional ac i i y o e in es ine-speci ic genes inco po a e he p ope ies o a “mas e egula o ”. 1.2.1 CDX2 homeobox gene and i s a ge s One o he ea lies isola ed homeobox genes in D osophila showed o he i s ime ma e nal as well as zygo ic exp ession, accumula ing in a concen a ion g adien spanning he an e o-pos e io axis o he emb yo. Du ing la e emb yogenesis, i was exp essed in mo e pos e io s uc u es, he e o e i was named cad (Mlodzik e al. 1985). Soon a e , h ee mu ine o hologues designa ed Cdx1 (Dup ey e al. 1988), Cdx2 (James and Kazenwadel 1991) and Cdx4 (Game and W igh 1993) we e cha ac e ized. Wi h he excep ion o Cdx4, hey a e con ined o he pos e io gu endode m du ing la e de elopmen and he ma u e in es ine a e bi h. The human CDX2 gene was independen ly cloned om an adul jejunal cDNA lib a y (D ummond e al. 1997) and by di e en ial sc eening o mRNA om CRC (Mallo e al. 1997). I maps o he Pa aHox gene clus e in ch omosome 13q12.3, has h ee exons and encodes a 313 amino acid p o ein con aining a nuclea ansloca ion and ac i a ion domain in he amino e minus (T inh e al. 1999), and a highly conse ed helix- u n-helix DNA-binding mo i called he homeodomain owa ds he ca boxyl e minus. The CDX2 homeodomain sha es 100% homology wi h CHAPTER 1. GENERAL INTRODUCTION AND AIMS 11 1.2 THE INTESTINAL TRANSCRIPTION FACTOR CDX2 he hams e CDX3 homeodomain, 96% wi h he mouse CDX2, and e en 88% wi h he homeodomain om D osophila Cad, implying ecogni ion o simila DNA a ge s. CDX p o eins ha e been demons a ed o bind as a monome o dime o one o mo e CDX- esponsi e elemen s, ypically consis ing o he consensus sequence (A/C)TTTAT(A/G), in di ec o e e se o ien a ion bo h in p omo e and gene enhance egions (Ma gali e al. 1993; Taylo e al. 1997; Ve zi e al. 2010). These mo i s a e equen ly jux aposing (T oelsen e al. 1997) o e en in e sec ing (Lambe e al. 1996) a TATA-box sequence, usually pa o he RNA polyme ase II binding si e. Al hough some epo s demons a e ha CDX2 can ac as an indi ec ep esso , coun e ac ing CDX1 o o he ansc ip ion ac o -media ed ac i a ion, by compe ing o he same binding si es (Gau ie -S ein e al. 2003; Fu umiya e al. 2013) o h ough in e ac ion wi h basal componen s o he ansc ip ional machine y (Chun e al. 2007; Mu oh e al. 2010), mos s udies p o ide s ong e idence o a ole in ac i a ing gene ansc ip ion (Ve zi e al. 2011). Indeed, CDX2 is de e minan o he exp ession o nume ous in es inal di e en ia ion ma ke s, and i has been shown o egula e abso p i e lineage-speci ic ac o s such as suc ase-isomal ase (Suh e al. 1994; Boud eau e al. 2002), lac ase phlo izin hyd olase (T oelsen e al. 1997; Fang e al. 2000), ca bonic anhyd ase 1 (D ummond e al. 1996), guanylyl cyclase C (Pa k e al. 2000), calbindin-D9K (Lambe e al. 1996; Wang e al. 2004a), li e -in es ine cadhe in (Hinoi e al. 2002), and illin (Yamamichi e al. 2009); goble cell-speci ic ac o s such as MUC2 (Mesqui a e al. 2003; Yamamo o e al. 2003) and e oil ac o 3 (Shimada e al. 2007); and en e oendoc ine cell-speci ic p oglucagon (Jin and D ucke 1996). On he con a y, CDX2 le els consis en ly seem lowes in Pane h cells, a lineage ecen ly shown o be supp essed by CDX2 o e exp ession-media ed loss o nuclea β-ca enin (C issey e al. 2011). CDX2 is also in ol ed in con olling he exp ession o addi ional molecules ha con ibu e o cellula dynamics, including p ocesses o p oli e a ion (Uesaka e al. 2002), g ow h a es (Bai e al. 2003; Aoki e al. 2011), mig a ion (Coskun e al. 2010), adhesion (Lo en z e al. 1997; Sakaguchi e al. 2002; Hinkel e al. 2012), me abolism and anspo (Modica e al. 2009; Kakizaki e al. 2010), in lamma ion (Wang e al. 2005), glycop o eome modula ion (Isshiki e al. 2003), and apop osis (Mallo e al. 1998). In any case, egula o y ou come ul ima ely esul s om he coope a i e balance wi h o he impo an ansc ip ion ac o s, being mos ly ele an he hepa ocy e nuclea ac o (HNF1 and HNF4) and GATA-binding ac o (GATA4/5/6) amilies (Boud eau e al. 2002; Ve zi e al. 2010; CHAPTER 1. GENERAL INTRODUCTION AND AIMS Ve zi e al. 2013). On he o he hand, hese and o he ansc ip ion ac o s, such as KLF4, can be di ec ly egula ed by CDX2 (Maha an e al. 1999; Boyd e al. 2010). Hence, he complex hie a chies o con ol ha go e n gene exp ession du ing in es inal di e en ia ion and de elopmen a e de e mined by he s oichiome y o di e en ansc ip ion ac o s and co ac o s wi hin in es inal cells a any gi en ime, as well as by he ype, numbe , and a angemen o cis-ac ing elemen s in he egula o y egions o in es inal genes. 1.2.2 Role in homeos asis Du ing emb yogenesis, CDX homologues pa icipa e in pa e ning o he e eb al column (Sub amanian e al. 1995; Chawengsaksophak e al. 1997; an Nes e al. 2006), as well as in haema opoiesis (Wang e al. 2008), ia HOX gene egula ion and wi h a ce ain deg ee o unc ional edundancy ( an den Akke e al. 2002; Sa o y e al. 2009). Bo h Cdx1 and Cdx2 a e exp essed du ing endode m de elopmen and in adul in es ine; howe e , he speci ic ole o each membe and he ex en o hei unc ional equi alence is s ill no comple ely unde s ood. In he la e emb yo, CDX1 and CDX2 le els a y quan i a i ely along he os ocaudal axis, wi h highes exp ession o CDX1 in he dis al po ion o he colon and highes CDX2 exp ession in he p oximal colon, diminishing in ei he di ec ion (James e al. 1994; Silbe g e al. 2000). A ai ly complemen a y g adien o exp ession has also been desc ibed along he e ical c yp - illus axis, wi h he o me p ima ily localized o he c yp , and he la e p ima ily along he illus, al hough wi h less s aining owa ds he ip (Silbe g e al. 1997; Rings e al. 2001; Kim e al. 2002; Silbe g e al. 2002). These pa e ns endu e h oughou he li espan o he animal and a e also obse ed in human issue (Wal e s e al. 1997; Boulange e al. 2005). They a e hough o e lec in insically di e en unc ionali ies, wi h CDX1 associa ed o a mo e p oli e a i e pheno ype and CDX2 o a mo e di e en ia ed one, conceding a pa ially in e changeable ac i i y in ce ain con ex s (Ve zi e al. 2011). Cdx1−/− mu an s a e iable and e ile, and like ansgenics o e exp essing Cdx1, display no o e in es inal pheno ype (Sub amanian e al. 1995; Bonhomme e al. 2008; C issey e al. 2008). Cdx2 p ecedes and is needed o Cdx1 onse du ing in es inal de elopmen (Eda e al. 2002; Silbe g e al. 2002; Mu oh e al. 2009), and Cdx1 equi emen is only unmasked upon Cdx2 loss, as hei combined absence in he adul s age CHAPTER 1. GENERAL INTRODUCTION AND AIMS 13 signi ican ly enhances le hali y e ec s o isola ed Cdx2 de iciency (Ve zi e al. 2010; Ve zi e al. 2011; S inge e al. 2012). I he e o e ollows ha CDX2 unc ion in he adul in es ine is mo e i al han ha o CDX1. The ole o CDX2 s a ed o be es ablished in i o, when i was obse ed ha o ced exp ession in he undi e en ia ed in es inal cell line IEC-6 a es s p oli e a ion and ini ializes an epi helial pola i y p og am (Suh and T abe 1996). Cdx2-/- mice emb yos display p e-implan a ion le hali y and die in u e o be ween emb yonic day 3.5 and 5.5, whe eas he Cdx2+/- mu an s a e iable, bu exhibi skele al anomalies o s un ed g ow h (Chawengsaksophak e al. 1997; Tamai e al. 1999). C ucial e idence ha CDX2 unc ions in speci ying in es inal cell a e was ini ially ob ained by obse ing ha 90% o he he e ozygous mice de elop mul iple polyps wi hin he i s h ee mon hs o li e, pa icula ly in he p oximal colon. These polyps p esen he emaining Cdx2 allele inac i a ed and e eal a homeo ic e e sion owa ds an e io di e en ia ion (Beck e al. 1999). This p ocess o in e cala y egene a ion means ha local spo adic Cdx2 haploinsu iciency con eys a signal o a pa hway o os al pheno ype; hence, a g adien o posi ional in o ma ion is obse ed, wi h a eas o s a i ied squamous epi helium simila o ha seen in he oesophagus, a eas esembling he gas ic mucosa, and e en a eas eminiscen o he small in es ine. Wild- ype male hos s wi h Cdx2–/– cells om mu an emale dono s also de elop chimae ic in es inal pa ches o o gano ypically no mal s omach epi helium (Beck e al. 2003). O in e es , he unde lying hos s oma concu en ly assumes a gas ic pheno ype, p o ing ha endode mal exp ession o CDX2 ini ia es endode mal/mesode mal c oss- alk and is he p ima y signal o gu di e en ia ion, subsequen ly in ol ing app op ia e eedback loops (S inge e al. 2008). A complemen a y app oach, elying in ansgenic exp ession o Cdx2 in he s omach, demons a ed ha CDX2 is su icien o induce in es inal en e ocy es wi h enzyma ic and abso p i e unc ions in i o (Mu oh e al. 2005b). This is ein o ced by hese mice being able o su i e o e one mon h a e ex ensi e small bowel esec ion, when compa ed o he sho se en day li espan o su ge y-con ols. O he animal models ha e been independen ly epo ed, in which emb yonic le hali y o Cdx2 abla ion was ci cum en ed by condi ionally a ge ing i s knockou a pos e io de elopmen al s ages. Pending on empo al es ic ions, his inac i a ion di e en ially impac s global in es inal mo phology. Ea ly loss esul s in an e io ans o ma ion o he small in es ine o an oesophageal pheno ype (Gao e al. 2009), CHAPTER 1. GENERAL INTRODUCTION AND AIMS CHAPTER 1. GENERAL INTRODUCTION AND AIMS whe eas in la e induc ion he in es ine shows a mo e gas ic-like iden i y, wi h impai ed endo-lysosomal a icking and ine icien seg ega ion o apico-basal memb ane domains (Gao and Kaes ne 2010; G ainge e al. 2010). Condi ional inac i a ion in he al eady adul mice comp omises en e ocy e unc ion, causing se e e malnu i ion and dea h in abou h ee weeks (Ve zi e al. 2010; Ve zi e al. 2011). A limi ed abla ion s a egy compa ible wi h long- e m su i al has been applied in his se ing e ealing he p esence o pa ial gas ic-na u e me aplasias associa ed wi h loss o all di e en ia ed in es inal cell ypes (H yniuk e al. 2012; S inge e al. 2012). Hence, CDX2 s ands ou as being he mos c i ical elemen in he de elopmen , di e en ia ion, and main enance o he in es inal pheno ype. 1.2.3 In ol emen in ca cinogenesis Beyond i s homeo ic unc ion, CDX2 is also in ol ed in p ocesses o leukemogenesis (Lenge ke and Daley 2012) and GI ca cinogenesis. Con lic ing da a ega ding he na u e o CDX2 unc ion in umou ini ia ion and de elopmen exis , and mos p obably CDX2 assumes di e en oles in dissimila con ex s. IM and gas ic cance An associa ion be ween IM and in es inal- ype gas ic ca cinoma has been p e iously es ablished (Co ea 1992). I is now widely ecognized ha he main molecula d i e o his p eneoplas ic condi ion is de no o exp ession o CDX2. Se e al s udies ha e desc ibed he p esence o bo h CDX1 and CDX2 in nea ly all gas ic IM oci (Bai e al. 2002; Almeida e al. 2003; Kim e al. 2006; Ba os e al. 2008). Bu p oo o concep suppo ing CDX2 in ol emen in his p ocess was p o ided by wo ansgenic mouse models achie ing inapp op ia e Cdx2 exp ession in he gas ic epi helium. These mice de elop IM, cha ac e ized by he p esence o abso p i e, goble , and en e oendoc ine cell- ypes (Mu oh e al. 2002; Silbe g e al. 2002). One model e en p og essed o gas ic cance a e long- e m induc ion (Mu oh e al. 2004), ein o cing he implica ion o IM in he genesis o gas ic ca cinoma. Besides being ec opically exp essed in IM, i has been epea edly demons a ed ha CDX2 exp ession is down egula ed in he p og ession om IM o gas ic cance (abou 50% a e posi i e), and ha CDX2 posi i i y signi ican ly co ela es wi h a mo e di e en ia ed (in es inal) his ology and be e p ognosis (Kaimak chie e al. 2004; 15 CHAPTER 1. GENERAL INTRODUCTION AND AIMS As o he in luence o s omal elemen s, i was obse ed ea ly on ha epi helial- mesenchymal cellula in e ac ions can di e en ially a ec he exp ession o CDX1 and CDX2 homeobox genes (Duluc e al. 1997). Fo example, di e en componen s o he basemen memb ane ma ix, which lie a he in e ace o and a e syn hesized by epi helial and mesenchymal cells, such as Laminin-α1, can posi i ely modula e CDX2 exp ession (Lo en z e al. 1997). A s imula ion o CDX2 exp ession by subepi helial colonic myo ib oblas s was also epo ed, media ed by he non- canonical Wn amily membe WNT5A and he epi helial ecep o ROR2, con ibu ing o inhibi ion o he canonical Wn signalling and in es inal di e en ia ion (Pacheco and Macleod 2008). On he o he hand, collagen ype I induces pheno ypic changes in CRC cells h ough he β1-in eg in/FAK signalling pa hway ha in ol e educed Cdx2 p omo e ac i i y and mRNA exp ession (B able z e al. 2004). In ag eemen wi h his, a model o o ho opic and he e o opic xenog a s in nude mice demons a ed ha CDX2 exp ession is adap able and s ongly dependen on he mic oen i onmen (Benahmed e al. 2007). Pos - ansc ip ional egula ion by mic oRNAs Due o he absence o CDX2 exp ession in abou hal o he gas ic cance cases and he e ogeneous loss in CRCs, i was hypo hesized ha mic oRNAs (miRs) could also be associa ed wi h CDX2 silencing in hese con ex s. Compu a ional p edic ion o miR-binding si es, using independen da abases, led o he selec ion o miR-9 and miR-204 as pu a i e candida es o CDX2 egula ion (Ro k ua e al. 2011). A compa a i e analysis o CDX2 and miR-9 exp ession in a panel o gas ic cance issues e ealed an o e all in e se co ela ion be ween bo h, and in i o expe imen al da a, con i med ha miR-9 in e ac s di ec ly and speci ically wi h he CDX2 3`un ansla ed egion (UTR), leading o down egula ion o CDX2 a ge genes and p omo ion o cell g ow h. Mo e ecen ly, i was demons a ed ha exogenous CDX1-induced exp ession o miR-9, miR-16 and miR-22 supp esses CDX2 mRNA by a ge ing i s 3`UTR in a CRC cell line (Tagawa e al. 2012). Pos - ansla ional egula ion Phospho yla ed o ms o p38 MAPKs we e shown o be mos ly e ained in he nuclei o illus cells, wi h di e en ial p38α kinase ac i i y cons i u ing an ea ly and necessa y e en o he ini ia ion o he in es inal di e en ia ion p og am (Houde e CHAPTER 1. GENERAL INTRODUCTION AND AIMS al. 2001). This s imula o y e ec is accomplished by CDX2 phospho yla ion, enhancing i s ansc ip ional abili y wi hou in ol emen in he loss o p oli e a i e po en ial o cell su i al. On he o he hand, phospho yla ion by ERK1/2 MAPKs o se ine 60 wi hin he amino- e minal ac i a ion domain o CDX2, educes i s ansc ip ional capabili y and impai s ce ain in es inal di e en ia ion p ope ies (Rings e al. 2001; Lemieux e al. 2011). This modi ica ion was mainly ound in he p oli e a ing compa men o he in es ine, while he nonphospho yla ed and mo e ac i e CDX2 was p edominan ly ound in he di e en ia ed egion, in ag eemen wi h he localiza ion o pERK1/2 (Aliaga e al. 1999). In addi ion, ac i a ion o he ERK1/2 MAPK cascade was shown o p omo e ubiqui in-p o easome-dependen u no e o he CDX2 p o ein (K uege e al. 2009). Two independen s udies ha e elucida ed how CDX2 egula ion can be coo dina ed wi h he cell cycle machine y. I was obse ed ha CDX2 unde goes CRM1-dependen nuclea expo and subsequen p o eoly ic deg ada ion by in e ac ion wi h CDK2 in p oli e a i e in es inal cells (Boulange e al. 2005). The CDK2-media ed phospho yla ion o CDX2 was ound o occu downs eam o he homeodomain a se ine 283, iden i ied as being pa o a conse ed mo i o ou e enly spaced se ines called he 4S mo i , simila o he one con olling β-ca enin deg ada ion by he p o easome (G oss e al. 2005). P e en ing phospho yla ion h ough his si e blocked polyubiqui ina ion and s abilized CDX2, wi h impac on o e all cell beha iou . In conclusion, CDX2 con ains mul iple phospho yla ion si es ha ei he posi i ely o nega i ely balance i s ac i i y and/o s abili y in esponse o di e en signalling pa hways, being concei able ha hei combined ac ion is equi ed o he onse o he comple e di e en ia ion p ocess. P esen ly, i is well es ablished ha gene exp ession is egula ed a mul iple le els, as p e iously demons a ed o CDX2, and ha he di e se p ocesses in ol ed a e in eg a ed wi hin each o he . T ansc ip ional con ol is one o he mos impo an s eps wi hin he gene egula ion cascade. Ne e heless, he signi icance o pos - 23 1.3 POST-TRANSCRIPTIONAL REGULATION OF GENE EXPRESSION CHAPTER 1. GENERAL INTRODUCTION AND AIMS ansc ip ional con ol is e ol ing, as he abili y o ep og am p o ein syn hesis is a common heme in emb yonic de elopmen , wound healing, in lamma ion, me abolic s ess and aging. An o e iew o he main concep s o pos - ansc ip ional egula ion is p esen ed, ocusing on RNA-binding p o eins (RBPs) and hei oles in de elopmen and disease. 1.3.1 F om ansc ip s o p o eins An inc easing numbe o publica ions show ha a poo co ela ion be ween s eady-s a e ansc ip abundances and co esponding p o ein pools gene ally exis s in almos e e y o ganism (de Sousa Ab eu e al. 2009; Maie e al. 2009; Vogel e al. 2010). In acco dance, a ecen s udy comp ehensi ely analysed mRNA and p o ein le els, hal -li es, ansc ip ion and ansla ion a e cons an s o housands o genes in NIH3T3 mouse ib oblas s, and ound ha mRNA le els only explain a ound 40% o he a iabili y in p o ein le els (Schwanhäusse e al. 2011). Whe he his exac pe cen age is alid o o he cell ypes is unknown. Thus, i is clea ha a signi ican ac ion o Euka yo ic gene egula ion is pos - ansc ip ional in na u e. Pos - ansc ip ional egula ion encompasses RNA p ocessing, localiza ion, ansla ion and decay, as well as RNA s abili y h oughou . These in e connec ed mechanisms p o ide complemen a y quali y-con ol laye s ha collec i ely de ine he a e o e e y ansc ip (Moo e 2005). Adding o he complexi y, RNAs do no dwell alone in he cell, as hey a e e e accompanied by ans-ac ing ac o s, namely RBPs and non-coding RNAs, such as miRs, ha bind cis-elemen s usually p esen in he 3´UTR (Kue s en and Goodwin 2003; Hun zinge and Izau alde 2011). These a e capable o adjus ing he amoun o gene p oduc mo e apidly, p ecisely and wi h a ac ical e e sibili y op ion ha ansc ip ional egula ion alone canno o e . I is his unique esco , hei ela i e posi ions, and in e ac ions ha c ea e a highly dynamic web o messenge ibonucleop o ein (mRNP) complexes, uling RNA li e. Indeed, Euka yo ic mRNPs ha e been unc ionally conside ed “pos - ansc ip ional ope ons” ha ma kedly expand he egula o y elas ici y o ou su p isingly small genomes (Keene and Tenenbaum 2002; Keene 2007). E idence in a ou o his en icing RNA ope on model on a genome-wide scale is moun ing, as s udies show ha disc e e subse s o mRNAs wi h sha ed sequence elemen s and CHAPTER 1. GENERAL INTRODUCTION AND AIMS encoding p o eins wi h common unc ions and loca ions a e coo dina ely egula ed by speci ic mRNPs (Ge be e al. 2004; Hogan e al. 2008). RNA p ocessing s a s in he nucleus, whe e p ecu so mRNAs acqui e a 7- me hylguanosine cap s uc u e a he 5' e minus (Lewis and Izau alde 1997), a specialized poly(A) ail a he 3` end (Mangus e al. 2003), and in ons a e emo ed by splicing (Ma lin e al. 2005). A majo emodelling in mRNP composi ion occu s as ma u e mRNAs pass h ough he nuclea -po e complex (Köhle and Hu 2007), wi h some ac o s emaining s ably associa ed, whe eas o he s a e dynamically eplaced by cy oplasmic coun e pa s. Upon expo , se e al mRNAs a e in eg a ed in he ansla ionally ac i e pool, a p ocess ha emodels he p o ein coa and assembles polysomes. The canonical mechanism o ansla ion is in ica e and in ol es h ee coo dina ed e en s: ini ia ion, elonga ion and e mina ion (Jackson e al. 2010). On he o he hand, some mRNAs a e p og ammed o delayed ansla ion, which allows ansc ip s o be anspo ed o a speci ic subcellula localiza ion o e en s o ed un il de elopmen al o en i onmen al cues call o hei p o ein syn hesis. These mRNAs a e packaged in o cy oplasmic mRNP g anules ha lack a limi ing memb ane and a e isible unde ligh mic oscopy, called p ocessing bodies (P bodies) and s ess g anules (Ande son and Kede sha 2006). 1.3.2 RNA-binding p o eins and hei biological unc ions An ex ensi e compu a ional analysis es ablished ha RBPs comp ise 8 o 15% o he p o ein coding epe oi e in euka yo ic genomes, highligh ing bo h hei ancien o igin and he impo ance o RNA egula ion in cell unc ion (Anan ha aman e al. 2002). One possible explana ion is ha as highly speci ic p ocesses o ine- une gene exp ession e ol ed, a concomi an expansion o he numbe o RBPs needed has occu ed. Fo example, a leas 74% o human genes exp ess mul iple iso o ms by using di e en exonic combina ions h ough al e na i e splicing (Johnson e al. 2003). I s eme gence du ing e olu ion d o e he need o a co esponding inc ease in he numbe o RBPs, and i is also, in i sel , a mechanism by which cells can expand hei RBP epe oi e. A subse o hese p o eins ecognizes common ea u es o almos e e y message, such as he 5`cap o he 3`poly (A) ail. Howe e , he majo i y ha e a equi emen o a p ima y sequence o ype o seconda y s uc u e in which he 25 CHAPTER 1. GENERAL INTRODUCTION AND AIMS o me is embedded, ha a e p esen in some mRNAs bu no o he s. Thei speci ici y is media ed by unique s uc u al a angemen s o indi idual RNA-binding domains whose p ope ies a e u he mode a ed by auxilia y domains. This p ope y, coined as “coope a i e modula i y”, accommoda es di e en unc ionali ies and allows o eno mous combina o ial po en ial, u he inc easing a ini y owa ds RNA (Lunde e al. 2007). To da e, mo e han 40 di e en binding domains ha e been p oposed, being he mos equen he RNA ecogni ion mo i . O he common classes include he K-homology (KH) domain, zinc- inge s o he CCCH and CCHC ype, and he double-s anded RNA-binding domain. RBPs capaci y o i ually egula e e e y aspec o RNA biogenesis and unc ion is ema kable. In ac , hey play pi o al oles du ing emb yonic de elopmen (Kue s en and Goodwin 2003), and in a b ea h o se e al homeos a ic p ocesses such as synap ic plas ici y (Luo e al. 2010), immune esponses (Ande son 2008), epi helial cell p oli e a ion (Yang e al. 2011), di e en ia ion (Yang e al. 2010) and pola i y (Nagaoka e al. 2012). Wi h inc easing knowledge on hei impo ance, he mo e appa en i becomes ha ampe ing wi h RBPs exp ession o unc ion unde lies he onse o se e al pa hological condi ions, including cance (Lukong e al. 2008). Membe s o he signal ansduc ion and ac i a ion o RNA (STAR) amily o RBPs play i al oles in cell p oli e a ion and di e en ia ion. SAM68, o ins ance, is o e exp essed in b eas (Lukong e al. 2005) and p os a e cance cells (Busà e al. 2007). On he con a y, Quaking (QKI) seems o unc ion as a umou supp esso in CRCs (Yang e al. 2010) and glioblas omas (Chen e al. 2012). Membe s o an e olu iona ily-conse ed amily o RBPs, he MEX-3 amily, a e eme ging as impo an pos - ansc ip ional egula o s o se e al cellula p ocesses in di e se physiological se ings. Thei unc ions and unde lying molecula mechanisms, pa icula ly well-known o he ances al mex-3 in he model o ganism Caeno habdi is elegans, a e now desc ibed. 1.4 THE MEX-3 FAMILY OF RNA-BINDING PROTEINS CHAPTER 1. GENERAL INTRODUCTION AND AIMS 1.4.1 Caeno habdi is elegans MEX-3 In he wo m emb yo, no mal de elopmen equi es p ecise spa ial and empo al exp ession pa e ns o ma e nal mRNAs, such as he an e io ly localized memb ane ecep o glp-1 and he pos e io ly localized ansc ip ion ac o pal-1, he D osophila no ch and cad o hologues, espec i ely (E ans and Hun e 2005). The co ec dis ibu ion o hese messages ha ac o di ec lineage-speci ic commi men pa e ns om indi idual blas ome es, is de e mined by pa i ioning- de ec i e (pa ) genes, which a e equi ed o es ablish pola i y and whose dis up ion cause he ea lies and mos ex ensi e emb yonic abno mali ies (Kemphues e al. 1988; Golds ein and Maca a 2007). Molecula e idence places C. elegans MEX-3 amid bo h classes, an in e media e egula o o cell a e de e minan s unde pa - dependen con ol, hus ac ing as a c i ical componen in he link be ween cell pola i y and asymme ic gene exp ession (Huang e al. 2002). mex-3 gene encodes a p o ein wi h wo se en y-amino acid egions ha a e 40% iden ical o each o he . The epea ed sequences co espond o pu a i e RNA- binding mo i s, ini ially iden i ied in he p e-mRNA-binding he e ogeneous nuclea ibonucleop o ein (hnRNP) K and named KH domains (Siomi e al. 1993). MEX-3 in e ac s wi h a minimal MEX-3 ecogni ion elemen (MRE), de ined as he degene a e consensus sequence (A/G/U)(G/U)AGN(0-8)U(U/A/C)UA (Pagano e al. 2009). Mu a ions dis up ing he mex-3 locus a e ully pene an , ecessi e, s ic ma e nal-e ec and emb yonically le hal, esul ing in emb yos ha abno mally gene a e body-wall muscle om he an e io blas ome e (AB), hence he name mex o “muscle excess” (D ape e al. 1996). This homeo ic ans o ma ion is due, in pa , o he unc ion MEX-3 exe s o e he ansc ip ion ac o pal-1, he o hologue o CDX genes in mammals, and he soma ic de e minan o pos e io iden i y (Hun e and Kenyon 1996). mex-3 mRNA and p o ein accumula e cy oplasmically and a e une enly dis ibu ed in ea ly emb yos (D ape e al. 1996). A e e iliza ion and be ween he wo and ou -cell s age, MEX-3 is p e e en ially localized o he AB lineage, disappea ing a e wa ds in a pa e n simila o ha desc ibed o o he ma e nal mRNAs (Seydoux and Fi e 1994). On he o he hand, while pal-1 mRNA is p esen h oughou he ea ly emb yo, PAL-1 p o ein is asymme ically localized and only de ec ed om he ou -cell s age onwa ds in descendan s o he pos e io (P1) blas ome e lineage, hus co ela ing wi h low MEX-3 le els (Figu e 3A). In acco dance, PAL-1 is ec opically exp essed in all cells o mex-3 mu an emb yos and 27 CHAPTER 1. GENERAL INTRODUCTION AND AIMS MEX-3 has been shown o ep ess ansla ion o a lacZ RNA epo e cons uc con aining he pal-1 3`UTR in an e io blas ome es (Hun e and Kenyon 1996). The e o e, MEX-3 plays a c ucial ole as a ansla ional ep esso o pal-1, speci ying p ope blas ome e iden i y du ing ea ly emb yogenesis. Pos - ansc ip ional egula ion o ma e nal RNAs is also a p ima y mechanism o con ol gene exp ession in C. elegans ge mline, and MEX-3 con ibu es o he main enance o ge m cell o ipo ency in he adul wo m (Ciosk e al. 2006). The he maph odi e ge mline is a highly dynamic o gan p esen ing a s e eo ypical o ganiza ion o game es commi men : he mos dis al end o he gonad is a p oli e a i e compa men con aining mi o ic cells ha p og essi ely en e meiosis in a cen al syncy ial egion. Spon aneous ge m cell dea h by apop osis o di e en ia ion in o spe m cells (la e L4 la al s age) and oocy es (adul hood) occu s in he p oximal gonad. Swi ches be ween hese di e en s ages in ol e spa ially non- o e lapping ansla ional egula ion o a ge mRNAs by he RBPs de ec i e in ge mline de elopmen (GLD-1) and MEX-3 (Lee and Schedl 2001; Moo z e al. 2004) (Figu e 3B). In ma u ing oocy es, MEX-3 also media es pal-1 inhibi ion; combined wi h GLD-1 ac ion, his ensu es ha emb yos do no inhe i ma e nal PAL-1 p o ein and de elop p ope ly (D ape e al. 1996; Moo z e al. 2004). In acco dance, double mu an s ains o mex-3 and gld-1 de elop a ge mline umou con aining nume ous cells o muscula , neu onal and in es inal na u e, eminiscen o human CHAPTER 1. GENERAL INTRODUCTION AND AIMS e a omas (Ciosk e al. 2006). This ge m cell ansdi e en ia ion seems o be he end esul o an abno mal exp ession o soma ic de e minan s such as PAL-1. 1.4.2 The mammalian MEX3 p o eins C. elegans mex-3 was iden i ied and cha ac e ized in humans as a amily o ou homologous genes called MEX3A o MEX3D (Buche -Poyau e al. 2007). These a e loca ed a ch omosomic posi ions 1q22, 15q25.2, 18q21.2 and 19p13.3, espec i ely, and a e composed o wo exons and one in on. The closely ela ed encoded p o eins con ain wo andemly epea ed KH domains ha pu a i ely p o ide RNA-binding p ope ies. Addi ionally, hey possess a ca boxy- e minal eally in e es ing new gene (RING) inge module, no p esen in he nema ode MEX-3 and belie ed o media e p o ein-p o ein in e ac ions. Fou mouse o hologues we e also iden i ied displaying s ong simila i y a he amino acid le el o hei human coun e pa s. Fo hese cha ac e is ics hey a e also known as RING inge and KH domain-con aining (RKHD) p o eins. Exp ession analysis o he MEX3 ansc ip s in di e en human issues showed ha MEX3D is ubiqui ous, while he o he s ha e a a ied exp ession pa e n, wi h he highes le el ound in e al b ain and es is (Buche -Poyau e al. 2007). O e exp ession expe imen s demons a ed ha MEX3 a e phosphop o eins ha shu le be ween he nucleus and he cy oplasm by he CRM1-dependen expo pa hway and ia an N- e minally loca ed expo signal. Con ocal mic oscopy analysis e ealed co-localiza ion o MEX3A and MEX3B wi h DCP1A and AGO amily membe s, being he la e ca aly ic componen s o he RNA-induced silencing complex (RISC), he key e ec o o miR and RNA in e e ence (RNAi) pa hways. This associa ion was es ic ed o P bodies, which a e known si es o mRNA u no e (Eulalio e al. 2007). These esul s oge he wi h he abili y o MEX3 p o eins o in e ac in i o wi h poly(A) ibonucleo ide homopolyme s and ce ain cellula mRNAs, indica e ha MEX3 p o eins cons i u e no el human RBPs po en ially in ol ed in pos - ansc ip ional egula o y ne wo ks (Donnini e al. 2004; Buche -Poyau e al. 2007; Cou che e al. 2008). Ne e heless, he speci ic biological ole o each membe was basically unknown du ing he cou se o his wo k, and only ecen ly s a ed o be explo ed in mo e de ail. 29 CHAPTER 1. GENERAL INTRODUCTION AND AIMS The homeobox ansc ip ion ac o CDX2 is de e minan o p ope emb yonic de elopmen , as well as o endode mal induc ion and adul main enance o he in es inal epi helium. Besides his homeos a ic unc ion, in i o expe imen s, animal models and human lesions show ha de no o exp ession o CDX2 is associa ed wi h ec opic oci o in es inal di e en ia ion, which in u n con eys an inc eased isk o cance de elopmen . A pa adigma ic example o his link is p o ided by gas ic IM, a lesion ha encompasses a issue adap i e esponse o H. pylo i in ec ion and subsequen ch onic in lamma o y eac ion. The ole o bac e ial e adica ion as a common p e en i e s a egy agains gas ic cance de elopmen emains deba able and la gely dependen on he ex en o p eneoplas ic changes a he ime o ea men , gi en he appa en s abili y o he CDX2-dependen IM pheno ype. Mo eo e , he na u e o CDX2 in ol emen in he es ablishmen and/o p og ession o CRC is a om being consensual. Ea ly da a sugges ed ha loss o CDX2 was a common e en ; howe e , ensuing s udies ha e shown ha i s exp ession is e ained and in some cases e en inc eased. The e o e, de ining a ho ough po ayal o he CDX2 egula o y ne wo k s ands ou as a leading in es iga ion p io i y, no only o ad ance i s speci ic con ibu ion o hese ca cinogenic p ocesses, bu mos impo an ly o apply he acqui ed knowledge in he design o al e na i e and pe haps mo e a ge -o ien ed he apeu ic s a egies. The p esen wo k, amed wi hin he abo emen ioned concep ions, in ends o unco e new molecula mechanisms o CDX2 egula ion ocusing on he ole o ye poo ly-desc ibed mic oen i onmen al in luences as a esea ch guiding line. In his ega d, he ollowing speci ic objec i es a e p oposed: 1. E alua e i me hyla ion a he p omo e le el is a mechanism in ol ed in he egula ion o CDX2 gene exp ession DNA me hyla ion is an epigene ic mechanism o ansc ip ional egula ion and a di ec measu e o he en i onmen al e ec o e he genome, wi h an in ol emen in cance a ibu ed o he inapp op ia e silencing o umou supp esso genes (hype me hyla ion), o loss o oncogene ep ession (hypome hyla ion). P e ious s udies epo ed ha he me hyla ion s a us o CDX2 migh ha e a ele an egula o y ole in di e en con ex s. Bu hey p esen disc epancies, bo h conce ning he egion o que y and he me hods employed o analyse i . The lack o 1.5 LONG-TERM GOAL AND SPECIFIC AIMS CHAPTER 1. GENERAL INTRODUCTION AND AIMS a comp ehensi e analysis led us o e alua e i me hyla ion a he p omo e le el could be a mechanism in ol ed in he egula ion o CDX2 exp ession, and he ob ained esul s a e p esen ed in Chap e 2. We hypo hesized ha in in es ine, whe e CDX2 is exp essed, he gene would be unme hyla ed, while in he no mal gas ic mucosa, whe e CDX2 is no de ec ed, he gene would be me hyla ed. The p ocess o H. pylo i in ec ion and accompanying in lamma o y esponse would in some way be esponsible o p omo e deme hyla ion, cons i u ing he igge ing e en leading o he de elopmen o IM. The al e a ion in CDX2 me hyla ion p o ile along he gas ic ca cinogenic p ocess would cons i u e a p oo o concep , undoub edly es ablishing he ele ance o his mechanism and con ibu ing o se le a con en ious ques ion in he ield. 2. Assess he ole o cell-ma ix in e ac ions in he egula ion o CDX2 exp ession Fo many yea s now, adi ional me hods o cell cul u e ha e p oduced impo an concep ual ad ances in cance esea ch. Ne e heless, cells g own on la subs a es can di e conside ably in hei mo phology, di e en ia ion, cell-cell and cell-ma ix in e ac ions om hose g owing in mo e physiological condi ions. A he o he end o he spec um we ha e whole-animal models, which equen ly co obo a e he impo ance o pa icula p ocesses. Bu hese a e complex, expensi e and no easily manipula ed. By mimicking ea u es o he in i o mic oen i onmen and aking ad an age o he same ools used o s udy cells in s anda d cul u e, 3D cell/ issue models ha e been b idging he gap be ween bo h, p o iding unique pe spec i es on he beha iou o s em cells, epi helial issues and umou s. Ea lie publica ions desc ibed he ole o epi helial-mesenchymal in e ac ions as de e minan o he exp ession o CDX2, no only o p ope spa io- empo al induc ion o in es inal di e en ia ion, bu also in modula ing i s le els du ing ca cinogenesis. Howe e , he di ec molecula media o s esponsible o his adap able esponse emained o he mos pa elusi e. In Chap e 3, we p esen esul s ega ding he es ablishmen o in i o 3D cell cul u e models comp ising gas ic cance cell lines and an ex acellula ma ix. We hypo hesized ha his app oach, explo a o y in na u e, would allow pinpoin ing egula o y ac o s and signalling pa hways in ol ed in he con ol o CDX2 exp ession. 31 CHAPTER 2. CDX2 PROMOTER METHYLATION 1740 PEREIRA ET AL. F IGURE 1 – Me hyla ion s a us o CDX2 in a panel o human gas ic cance cell lines and unc ional ela ion wi h CDX2 mRNA exp ession. (a) Schema ic ep esen a ion o he 5`p oximal lanking egion o CDX2 gene. Thin e ical lines ma k he loca ion o CpG dinucleo ides. A box indica es he i s exon, including non-coding (whi e) and coding (black) sequences. Two pu a i e CpG islands we e iden i ied wi h he online bioin o ma ic ool CpGPlo (www.ebi.ac.uk/ ools/emboss/cpgplo ), one in he p omo e egion (–1603bp o –1197bp) and ano he in he i s exon, including he coding egion (–22bp o +660bp). A ow-heads indica e he posi ion o bisul i e speci ic p ime s. (b) RT-PCR analysis o CDX2 mRNA exp ession in gas ic cance cell lines. GAPDH mRNA exp ession was used as an in e nal loading con ol and NTC indica es no empla e con ol. (c) Me hyla ion p o ile o CDX2 in gas ic cance cell lines ob ained by di ec sequencing o bisul i e- ea ed genomic DNA. M, me hyla ed; U, unme hyla ed. (d) Real- ime PCR o CDX2 exp ession in human gas ic cance cell lines a e ea men wi h he deme hyla ing agen 5-aza-2`-deoxycy idine (5azaDc, Sigma) du ing di e en ime-poin s. Each expe imen was ca ied ou in iplica e a leas wice; he esul s a e exp essed as mean ± SD o biological eplicas. The alues ob ained wi h ehicle- ea ed cells we e e e ed o as 1. CDX2 mRNA le els we e no malized wi h he co esponding 18S RNA le els. CHAPTER 2. CDX2 PROMOTER METHYLATION CDX2 PROMOTER METHYLATION AND mRNA EXPRESSION 1741 F IGURE 2 – Bisul i e DNA sequencing o he i s and second agmen s o CDX2 CpG island 2 in wo specimens o he gas ic mucosa and ad- jacen IM oci (S1 and S2) and in wo specimens o no mal colonic mucosa (S3 and S4). Only clea ly iden i iable no mal gas ic glands and me aplas ic glands we e selec ed using a PALM Mic obeam Mic oscope (Zeiss). Wi h his me hodology we we e able o minimize s omal cell con amina ion in ou issue samples. PCR amplicons om he issues we e cloned in o pCR4-TOPO ec o using he TOPO TA Cloning ki (In i ogen), and a leas i e indi idual clones om each sample we e sequenced using he ABI P ism BigDye Te mina o 3.1 Cycle Sequenc- ing ki (Applied Biosys ems) wi h a e e se M13 p ime . Each ho izon al ow o ci cles ep esen s analysis in a single clone o bisul i e- ea ed DNA o he 42 CpG si es con ained in he men ioned egion. Solid and open ci cles ep esen me hyla ed and unme hyla ed CpG si es, espec- i ely. 39 CHAPTER 2. CDX2 PROMOTER METHYLATION 1742 PEREIRA ET AL. Re e ences 1. Yuasa Y, Nagasaki H, Akiyama Y, Hashimo o Y, Takizawa T, Kojima K, Kawano T, Sugiha a K, Imai K, Nakachi K. DNA me hyla- ion s a us is in e sely co ela ed wi h g een ea in ake and physical ac i i y in gas ic cance pa ien s. In J Cance 2009;124:2677–82. 2. Almeida R, Sil a E, San os-Sil a F, Silbe g DG, Wang J, De Bol'os C, Da id L. Exp ession o in es ine-speci ic ansc ip ion ac o s. CDX1 and CDX2, in in es inal me aplasia and gas ic ca cinomas. J Pa hol 2003;199:36–40. 3. Mesqui a P, Raquel A, Nuno L, Reis CA, Sil a LF, Se pa J, an Seu- ningen I, Ba os H, Da id L. Me aplasia—a ansdi e en ia ion p o- cess ha acili a es cance de elopmen : he model o gas ic in es i- nal me aplasia. C i Re Oncog 2006;12:3–26. 4. Guo M, House MG, Suzuki H, Ye Y, B ock MV, Lu F, Liu Z, Rus gi AK, He man JG. Epigene ic silencing o CDX2 is a ea u e o squa- mous esophageal cance . In J Cance 2007;121:1219–26. 5. Yuasa Y, Nagasaki H, Akiyama Y, Sakai H, Nakajima T, Ohku a Y, Takizawa T, Koike M, Tani M, Iwai T, Sugiha a K, Imai K, e al. Rela ionship be ween CDX2 gene me hyla ion and die a y ac o s in gas ic cance pa ien s. Ca cinogenesis 2005;26:193–200. 6. Kawai H, Tomii K, Toyooka S, Yano M, Mu akami M, Tsukuda K, Shimizu N. P omo e me hyla ion down egula es CDX2 exp ession in colo ec al ca cinomas. Oncol Rep 2005;13:547–51. 7. Liu T, Zhang X, So CK, Wang S, Wang P, Yan L, Mye s R, Chen Z, Pa e son AP, Yang CS, Chen X. Regula ion o Cdx2 exp ession by p omo e me hyla ion, and e ec s o Cdx2 ans ec ion on mo phol- ogy and gene exp ession o human esophageal epi helial cells. Ca ci- nogenesis 2007;28:488–96. 8. Fujii S, Ochiai A. Enhance o zes e homolog 2 down egula es E-cad- he in by media ing his one H3 me hyla ion in gas ic cance cells. Cance Sci 2008;99:738–46. 41 43 CDX2 pos - ansc ip ional egula ion by he RNA-binding p o ein MEX3A Chap e 3 CHAPTER 3. CDX2 POST-TRANSCRIPTIONAL REGULATION BY THE RNA-BINDING PROTEIN MEX3A The esul s conce ning his chap e a e published in: Pe ei a B, Sousa S, Ba os R, Ca e o L, Oli ei a P, Oli ei a C, Cha ie NT, Pla e o i M, Rouaul JP, F eund JN, Billaud M, Almeida R. (2013) CDX2 egula ion by he RNA- binding p o ein MEX3A: impac on in es inal di e en ia ion and s emness. Nucleic Acids Resea ch, 41(7):3986-3999 doi: 10.1093/na /gk 087 This wo k was suppo ed by Fundação pa a a Ciência e a Tecnologia (FCT) – P og ama Ope acional Ciência e Ino ação 2010 do Quad o Comuni á io de Apoio III and FEDER [PTDC/SAU-OBD/64490/2006]. __________________________________________________________________________________ The au ho decla es ha he pe o med and/o was in ol ed in all he expe imen al wo k, pa icipa ed in he s udy design and w o e he manusc ip . Mic oa ay expe imen s we e conduc ed a NFDM (Na ional Facili y o DNA Mic oa ays, Uni e sidade de A ei o, Po ugal) and 3D cell cys assays a Ins i u Albe Bonnio (INSERM-UJF U823, G enoble, F ance). CHAPTER 3. CDX2 POST-TRANSCRIPTIONAL REGULATION BY THE RNA-BINDING PROTEIN MEX3A 3986–3999 Nucleic Acids Resea ch, 2013, Vol. 41, No. 7 Published online 13 Feb ua y 2013 doi:10.1093/na /gk 087 CDX2 egula ion by he RNA-binding p o ein MEX3A: impac on in es inal di e en ia ion and s emness B uno Pe ei a 1 , So ia Sousa 1 , Ri a Ba os 1 , Lau a Ca e o 2 , Pa ícia Oli ei a 1 , Ca la Oli ei a 1,3 , Nicolas T. Cha ie 4 , Michelina Pla e o i 5 , Jean-Pie e Rouaul 6 , Jean-Noël F eund 7 , Ma c Billaud 4 and Raquel Almeida 1,3, * 1IPATIMUP – Ins i u e o Molecula Pa hology and Immunology o he Uni e si y o Po o, 4200-465 Po o, Po ugal, 2Depa men o Biology and CESAM, RNA Biology Labo a o y, Uni e si y o A ei o, 3810-193 A ei o, Po ugal, 3FMUP – Facul y o Medicine, Uni e si y o Po o, 4200-319 Po o, Po ugal, 4INSERM-UJF U823, Ins i u Albe Bonnio , BP 170, 38042 G enoble Cedex 9, F ance, 5Cen e de Géné ique e de Physiologie Moléculai e e Cellulai e, Uni e si é Claude Be na d Lyon 1, UMR5534, 69622 Villeu banne, F ance, 6Ins i u de Génomique Fonc ionnelle de Lyon, UMR5242 CNRS/INRA/UCBL/ENS Ecole No male Supé ieu e de Lyon, 69364 Lyon Cedex 07, F ance and 7INSERM UMR_S1113, Uni e si é de S asbou g, Fédé a ion de Médecine T ansla ionnelle, 67200 S asbou g, F ance Recei ed No embe 5, 2012; Re ised Janua y 22, 2013; Accep ed Janua y 23, 2013 ABSTRACT The homeobox ansc ip ion ac o CDX2 plays a c ucial ole in in es inal cell a e speci ica ion, bo h du ing no mal de elopmen and in umo igenic p ocesses in ol ing in es inal ep og amming. The CDX2 egula o y ne wo k is in ica e, bu i has no ye been ully unco e ed. Th ough genome-wide sc eening o a 3D cul u e sys em, he RNA-binding p o ein MEX3A was iden i ied as pu a i ely in ol ed in CDX2 egula ion; he e o e, i s biological ele- ance was add essed by se ing up cell-based assays oge he wi h exp ession s udies in mu ine in es ine. We demons a e he e ha MEX3A has a ep essi e unc ion by con olling CDX2 le els in gas ic and colo ec al cellula models. This is dependen on he in e ac ion wi h a speci ic binding de e minan p esen in CDX2 mRNA 3`un ansla ed egion. We ha e u he de e mined ha MEX3A impai s in es inal di e en ia ion and cellula pola iza ion, a ec s cell cycle p og ession and p omo es inc eased exp ession o in es inal s em cell ma ke s, namely LGR5, BMI1 and MSI1. Finally, we show ha MEX3A is exp essed in mouse in es ine, suppo ing an in i o con ex o in e ac ion wi h CDX2 and modula ion o s em cell p ope ies. The e o e, we desc ibe a no el CDX2 pos - ansc ip ional egula o y mechanism, h ough he RNA-binding p o ein MEX3A, wi h a majo impac in in es inal di e en ia ion, pola i y and s emness, likely con ibu ing o in es inal homeos asis and ca cinogenesis. INTRODUCTION The homeodomain ansc ip ion ac o CDX2 is a c i ical de e minan o in es inal homeos asis, bo h du ing de el- opmen and h oughou adul li e. CDX2 is in ol ed in he an e o-pos e io pa e ning o he mammalian emb yo and is he key molecula media o o in es inal di e en i- a ion (1–4). Fu he mo e, mul iple e idences subs an ia e CDX2 c ucial ole in ca cinogenesis o he diges i e ac . I was shown o inhibi cell g ow h and mig a ion in i o as well as dissemina ion o colon umou cells in i o (5). CDX2 he e ogeneous loss has also been obse ed in colo- ec al ca cinomas (CRCs), pa icula ly in in asi e cells a he umou edge (6). Mo eo e , CDX2 educ ion inc eases he p og ession o chemically induced CRCs (7). Con e sely, unde ce ain pa hological condi ions, CDX2 becomes abno mally exp essed in o he o gans o he diges i e ac besides in es ine, namely he esophagus *To whom co espondence should be add essed. Tel: +351 2255 70700; Fax: +351 2255 70799; Email: almeida@ipa imup.p P esen add ess: Sofia Sousa, School o Pha macy, Facul y o Heal h Sciences, Uni e si y o Eas e n Finland, Yliopis on an a 1 C, FIN-70211 Kuopio, Finland. © The Au ho (s) 2013. Published by Ox o d Uni e si y P ess. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion Non-Comme cial License (h p://c ea i ecommons.o g/licenses/ by-nc/3.0/), which pe mi s un es ic ed non-comme cial use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. 45 CHAPTER 3. CDX2 POST-TRANSCRIPTIONAL REGULATION BY THE RNA-BINDING PROTEIN MEX3A Nucleic Acids Resea ch, 2013, Vol. 41, No. 7 3987 (8) and s omach (9,10), d i ing a p ecance ous lesion known as in es inal me aplasia, a p ocess confi med in ansgenic mouse models (11,12). Owing o he essen ial unc ion in in es inal de elopmen , di e en ia ion and ca cinogenesis, CDX2 egula ion has been ex ensi ely s udied. We ha e p e iously iden ified di e en mechanisms in ol ed in he an- sc ip ional egula ion o his gene such as he Bone mo phogene ic p o ein (BMP) pa hway (13), SOX2 (14) and a CDX2 au o egula o y loop (15). Se e al ansc ip ion ac o s including HNF4a, GATA6, TCF4 and β-ca enin we e shown o in e ac wi h Cdx2 p omo e agmen s (16). Howe e , mu a ions a he CDX2 locus a e a a e e en in CRC (17), and i s exp ession does no depend on me hyla ion o he p oximal p omo e (18). On he o he hand, CDX2 p o ein phospho yla ion has also been shown o modi y i s ac i i y in in es inal cells (19,20). These and o he s udies suppo he no ion ha CDX2 egula ion is in ica e and s ic ly con olled. Du ing he pas wo decades, pos - ansc ip ional egu- la ion eme ged as a undamen al mechanism guiding gene exp ession in highe euka yo ic cells, being a he co e o no mal cellula p ocesses bu also cance ini ia ion and de elopmen . I is now inc easingly clea ha RNA ma u - a ion, localiza ion, ansla ion and s abili y p o ide mul iple laye s o spa io- empo al con ol de e mining a ansc ip ’s a e (21,22). These coupled e en s a e gene ally dependen on he coope a ion be ween cis- egula o y elemen s and ans-ac ing ac o s, such as non-coding RNAs and RNA-binding p o eins (RBPs). RBPs ha e been implica ed in i ually e e y aspec o RNA me abol- ism (22,23), pa icula ly, hei ep essi e ole is c i ical o es ablish p ecise ansla ional pa e ns ha define de elop- men al and di e en ia ion swi ches in many o ganisms. In Caeno habdi is elegans, MEX-3 is a ansla ional ep esso ha egula es blas ome e iden i y du ing ea ly emb yogenesis (24) and ge mline o ipo ency in he adul wo m (25). MEX-3 has wo K homology domains, which a e conse ed single-s anded RNA-binding mo i s (26). Mu a ions dis up ing mex-3 locus a e le hal, esul ing in emb yos ha inapp op ia ely gene a e body-wall muscle om he an e io blas ome e; hence, he name mex o ‘muscle excess’. This is specified, in pa , by he ep essi e unc ion ha MEX-3 exe s o e he ansc ip ion ac o pal-1, he C. elegans o hologue o caudal in D osophila and CDX in mammals (24,27). In humans, mex-3 was iden ified and cha ac e ized as ou homologous genes, MEX3A–D (28,29), whose biological ele ance is s a ing o be explo ed. Recen ly, he unc ional ole o MEX3C as a RNA-binding ubiqui in E3 ligase was es ablished, media ing he pos - ansc ip ional decay o HLA-A allo ypes (30). I was also shown o be necessa y o no mal pos na al g ow h in mu an mice by enhancing he local exp ession o insulin-like g ow h ac o 1 in bone (31) and appea s o be in ol ed in me abolic egula ion o ene gy balance (32), h ough ye unknown e ec o s. A a ian o m o MEX3D called TINO was shown o nega i ely egula e he an iapop o ic p o ein BCL-2 in HeLa cells (33). Finally, knockdown o MEX3A by siRNAs was shown o supp ess cell p oli e - a ion and mig a ion in human gas ic cance cells, bu he molecula mechanisms behind hese findings we e no ad- d essed (34). Pu suing he aim o unco e ing new CDX2 egula o y mechanisms, we explo ed a pu a i e ansla- ional ep ession by MEX3A, which was in e sely co ela ed wi h CDX2 in a 3D expe imen al model. By s udying MEX3A exp ession in i o and using a cell line-based app oach o modula e i s le els in i o, ou s udy desc ibes a no el pos - ansc ip ional p ocess by which CDX2 exp ession is impai ed in he gas oin es inal se ing and in es inal-like homeos asis comp omised, h ough al e a ions in di e en ia ion, pola i y and s emness ea u es. Ano he laye o con ol is hus added o he complex CDX2 egula o y ne wo k, in ol ing MEX3A as a key egula o o in es inal homeos asis, which migh ha e significan implica ions o gas oin es- inal ca cinogenesis. MATERIALS AND METHODS Cell cul u e and ea men s Human gas ic ca cinoma cell line AGS (ATCC, Ame ican Type Cul u e Collec ion) and CRC cell line Caco-2 (ATCC) we e cul u ed unde s anda d condi ions in RPMI-1640 medium and Dulbecco’s modified Eagle’s medium, espec i ely, con aining 10% e al bo ine se um, 100 U/ml o penicillin and 100 µg/ml o s ep omycin (Li e Technologies). Fo he AGS 3D cul u e, flasks we e coa ed wi h 50 µl/cm2 o ma igel basemen memb ane (BD Biosciences) a a 1.5:1 p opo ion o se um- ee medium. A 2 x 104 cells/cm2 suspension was seeded on op and main ained o 14 days wi h medium change e e y 2 days. Fo he Caco-2 3D cul u e, co e slips we e coa ed wi h 60 µl/cm2 o ma igel. A 6 x 103 cells/cm2 suspension plus 2% ma igel was seeded on op and main- ained o 8 days wi h medium change e e y 2 days. To quan i y lumen o ma ion, >100 cys s we e mic oscop- ically examined. Fo p o easome inhibi ion, cells we e ea ed wi h 25 µM MG132 (Calbiochem) o ehicle ea ed wi h DMSO. To inhibi ansc ip ion, cells we e exposed o 10 µg/ml o Ac inomycin D (Sigma). Cons uc s and si e-di ec ed mu agenesis The p e iously published pCMV-MEX3A exp ession ec o (28) was used oge he wi h a pCMV-Tag3B emp y ec o (Agilen Technologies) in ans ec ions. A pRLCon ol cons uc con aining a humanized Renilla luci e ase (Rluc) coding sequence (35) was used as a backbone o c ea e he pRLCDX2 ec o , encoding a luci e ase usion ansc ip o he pa en al CDX2 3`un ansla ed egion (UTR). The QuickChange si e- di ec ed mu agenesis ki (S a agene) was hen used o in oduce specific mu a ions in he p e ious plasmid, o gene a e he pRL∆CDX2 cons uc , wi h a mu a ed MEX-3 ecogni ion elemen (MRE). Oligonucleo ides con aining he desi ed mu a ions we e designed acco ding o he manu ac u e ’s ins uc ions (Supplemen a y Table S1). Taking in o accoun he degene a e consensus sequence desc ibed o MEX-3 binding in C. elegans, a mu a ional backg ound o Cy idine was used because p e- sumably his base is no ole a ed in he MRE (36). Nucleic Acids Resea ch, 2013, Vol. 41, No. 7 3987 3988 Nucleic Acids Resea ch, 2013, Vol. 41, No. 7 CHAPTER 3. CDX2 POST-TRANSCRIPTIONAL REGULATION BY THE RNA-BINDING PROTEIN MEX3A T ans ec ions, RNAi and luci e ase assays T ansien ans ec ions we e done using Lipo ec amine 2000 eagen acco ding o he manu ac u e ’s guidelines (Li e Technologies). A DNA (µg) o Lipo ec amine 2000 eagen (µl) a io o 1:1.5 in OPTI-MEM medium was used o ou ine ans ec ion expe imen s o exp ession ec o s. Fo s able ans ec ions, selec ion was ini ia ed 48h pos - ans ec ion in medium supplemen ed wi h 0.6 mg/ml o G418 (Sigma). Neomycin- esis an posi i e clones ob ained h ough limi ing dilu ion we e ou inely main ained wi h 0.2 mg/ml o G418. A comme cial se o h ee S eal h Selec siRNA duplexes (HSS150674, HSS150675 and HSS150676) di ec ed agains human MEX3A (Li e Technologies) and a cus om se o h ee siRNA duplexes di ec ed agains CDX2 we e used wi h sc ambled con ols. An siRNA duplexes (pmol) o Lipo ec amine 2000 eagen (µl) a io o 20:1 in OPTI- MEM medium was used o inhibi ion expe imen s. Luci e ase epo e assays we e pe o med wi h he Rluc Assay Sys em (P omega), and β-galac osidase ac i i y was used o no maliza ion o expe imen al a ia ions. RNA isola ion and quan i a i e eal- ime PCR To al RNA was ex ac ed using TRI Reagen (Sigma) and e e se ansc ibed using he Supe sc ip II Re e se T ansc ip ase ki (Li e Technologies). Analysis o BMI1, CDX2, GAPDH, LGR5, MEX3A and Rluc mRNA exp ession was pe o med in an ABI P ism 7500 sys em using SYBRg een eagen (Li e Technologies) and specific p ime pai s (Supplemen a y Table S1). Each sample was amplified in iplica e and specifici y confi med by dissoci- a ion analysis. The 18S RNA exp ession was measu ed o no maliza ion o a ge gene abundance. Mic oa ays and da a p ocessing Expe imen s we e pe o med a he Na ional Facili y o DNA Mic oa ays (A ei o, Po ugal). Th ee independen 2D and 3D AGS cell cul u es we e selec ed o o al RNA ex ac ion using TRI Reagen . RNA quan i y and quali y we e assessed using he Nanod op ND-1000 (The mo Scien ific) and 2100 Bioanalyze (Agilen Technologies) sys ems, and only samples wi h a RNA in eg i y numbe abo e nine we e conside ed o u he s udy. P epa a ion and labelling o he RNA was pe o med using he One-Colo Mic oa ay-Based Gene Exp ession Analysis Quick Amp Labelling ki (Agilen Technologies). B iefly, 600 ng o o al RNA om 2D and 3D biological eplicas was used as inpu oge he wi h spike-in con ols o gene a e Cyanine 3-labelled cRNA. The amplified cRNA samples we e pu ified wi h RNeasy Mini ki (Qiagen) and hyb idized o Human Gene exp ession 4 x 44K 2 Mic oa ay slides (Agilen Technologies) a 65 o C o 17h. A e washing p ocedu es, images o he hyb idiza- ions we e acqui ed using a G2505B Mic oa ay Scanne (Agilen Technologies). The Fea u e Ex ac ion so wa e was used o spo iden ifica ion, backg ound sub ac ion and quan ifica ion o he fluo escen signal. Raw exp es- sion da a we e p ocessed using BRB-A ayTools 3.8.1 so wa e (37). A e base 2 log ans o ma ion and a e age o p obe eplica es, no maliza ion was applied using he median in ensi y o e he en i e a ay o minimize sys ema ic a iance. Di e en ially exp essed ansc ip s be ween he wo cul u e condi ions we e fil e ed using he Class Compa ison ool, pe o ming an unpai ed sample - es wi h a P < 0.01 and conside ing a minimal 1.5- old change. Hie a chical clus e ing analysis o significan ly al e ed genes was conduc ed in TIGR Mul iExpe imen Viewe 4.8.1 so wa e (38) using Euclidean co ela ion and a e age linkage clus e ing, and exp ession alues indica ed colo ime ically. Func ional anno a ion o di e en ially exp essed genes, iden ified by ex ending he unpai ed - es pa ame e o P < 0.05 and main aining he minimal old change was pe o med wi h DAVID p og am (39). Mic oa ay da a ha e been submi ed o he A ayExp ess da abase (h p://www.ebi. ac.uk/a ayexp ess/) and assigned he iden ifie E-MTAB- 1234. The comple e lis o di e en ially exp essed genes is de ailed in Supplemen a y Table S2. P o ein ex ac ion and wes e n blo analysis Cells we e lysed o 30 min on ice in a lysis bu e con aining 20 mM T is–HCl (pH 7.5), 150 mM NaCl, 2 mM e hylenediamine e aace ic acid and 1% IGEPAL (Sigma), supplemen ed wi h Comple e p o ease inhibi o cock ail (Roche Applied Science), 1 mM PMSF and 1 mM Na 3 VO 4 . Lysa es we e cen i uged a 12 000 pm o 20 min a 4 o C and he supe na an eco e ed. P o ein con- cen a ion was de e mined using he BCA P o ein Assay Reagen (The mo Scien ific). P o ein ex ac s (30–50 µg) we e un on 10% sodium dodecyl sulpha e–polyac yl- amide gel elec opho esis, ans e ed o a ni ocellulose memb ane, blo ed o e nigh wi h app op ia e an ibodies (Supplemen a y Table S1), and signals e ealed wi h ECL de ec ion ki (GE Heal hca e Li e Sciences). Ac in le els we e used o no malize p o ein exp ession, and quan ifica ion o wes e n blo s was pe o med using Fiji so wa e (40). Flow cy ome y Caco-2 cells we e ha es ed 48 h a e ans ec ion a con- fluence and fixed wi h 1% pa a o maldehyde o 20 min a oom empe a u e ollowed by pe meabiliza ion wi h 0.1% T i on X-100 (Sigma) o 5 min on ice. S aining was pe o med wi h an i-MEX3A an ibody and isualized wi h goa an i- abbi FITC-conjuga ed seconda y an ibody. Fo DNA con en assessmen , cells we e incuba ed wi h a p opidium iodide (50 µg/ml) and RNAse A (200 µg/ml) solu ion (Sigma) o 30 min a oom empe a u e. Samples we e ead in a FACSCan o II (BD Biosciences) flow cy ome e , and analysis was pe o med using he FlowJo so wa e. RNA-immunop ecipi a ion assay Cells we e lysed 48 h a e ans ec ion as indica ed o p o ein ex ac ion bu wi h he addi ion o 20 U/ml o RNAseOUT Ribonuclease inhibi o (Li e Technologies). Be o e he lysis p ocedu e, cells we e washed wi h phos- pha e bu e ed saline (PBS) and subjec ed o ul a iole c osslink (254-nm wa eleng h) a an ene gy le el 47 Nucleic Acids Resea ch, 2013, Vol. 41, No. 7 3995 CHAPTER 3. CDX2 POST-TRANSCRIPTIONAL REGULATION BY THE RNA-BINDING PROTEIN MEX3A Figu e 7. Pola i y al e a ions induced by MEX3A in Caco-2 cells. (A) Immunofluo escence showing ZO-1 exp ession in Caco-2 mock and MEX3A s ably ans ec ed cell lines a day -2 o cul u e (o iginal magnifica ion, x400). (B) Mo phology o Caco-2 mock and MEX3A cys s in b igh field mic oscopy du ing 3D cul u e (o iginal magnifica ion, x100) and CDX2/MEX3A exp ession (o iginal magnifica ion, x630). (C) Quan ifica ion o cys s wi h lumen o no lumen a cul u e day 8. (D) Exp ession o E-cadhe in and Phalloidin s aining in Caco-2 cys s (o iginal magnifica ion, x630; all scale ba s 20 µm). 3D cell cul u e sys ems ha e been shown o enable physiological and unc ional di e en ia ion o se e al epi- helial cell ypes (47,48), cons i u ing a p omising al e na- i e o o e come s anda d cell cul u e limi a ions. Acco dingly, ou ansc ip omic analysis pe o med on a 3D model o AGS cells, which ha e a significan le el o endogenous CDX2 esponsi e o di e en molecula s imuli (13–15), allowed us o disclose MEX3A as a mo- lecula playe in ol ed in he egula ion o CDX2 ansla- ion. The a ay p esen ed an inc eased MEX3A exp ession in 3D cul u e and no al e a ion in he le els o he o he MEX3 amily membe s. We u he demons a ed ha MEX3A o e exp ession leads o ma ked CDX2 p o ein dec ease in wo cell lines. We ied o o e come he limi- a ion o using o e exp ession sys ems by using wo di - e en cell lines ha we e bo h ansien ly and s ably ans ec ed wi h a MEX3A plasmid, which ga e conco d- an esul s. In addi ion, CDX2 nega i e egula ion by MEX3A was confi med in a mo e physiological con ex using a siRNA app oach owa ds endogenous MEX3A in Caco-2 cells. We p o ed ha MEX3A is able o in e ac wi h CDX2 mRNA h ough a canonical MRE p esen CHAPTER 3. CDX2 POST-TRANSCRIPTIONAL REGULATION BY THE RNA-BINDING PROTEIN MEX3A 3996 Nucleic Acids Resea ch, 2013, Vol. 41, No. 7 Figu e 8. Exp ession pa e ns o MEX3A and CDX2 in mouse no mal in es ine. Rep esen a i e immunofluo escence da a o MEX3A and CDX2 in small in es ine and colon a e shown (o iginal magnifica ion, x200; scale ba s 50 µm; inse o iginal magnifica ion, x400). in he 3´UTR. This sequence, which we now show o be unc ionally ele an in humans, seems o be he single de e minan o MEX3A binding, independen ly o he ups eam 5`coding egion. In ac , bioin o ma ics analysis shows ha he degene a e MRE mo i has been e olu iona ily conse ed in di e en CDX2 homologues (Supplemen a y Figu e S3), namely in chimpanzee, mouse, a , zeb afish, ui fly and og, sugges ing ha MEX3A is c i ical o CDX2 egula ion. Endogenously, MEX3A was mainly localized in he nuclea compa men o in es inal epi helial cells, bo h in i o and in i o. This esul is c ucial o show ha a biological backg ound o he egula ion o CDX2 by MEX3A exis s. In his ega d, a a ian o m o MEX3D 55 Nucleic Acids Resea ch, 2013, Vol. 41, No. 7 3997 CHAPTER 3. CDX2 POST-TRANSCRIPTIONAL REGULATION BY THE RNA-BINDING PROTEIN MEX3A called TINO, which has been shown o nega i ely egula e BCL-2 exp ession by ansc ip des abiliza ion, is also p e- dominan ly localized in he nuclei o HeLa cells (33). On he o he hand, MEX3A ans ec an s showed p e- dominan cy oplasmic s aining. A po en ial p oblem could be an ibody specifici y; howe e , his was success- ully e alua ed by ans ec ion o siRNA duplexes di ec ed agains MEX3A (Supplemen a y Figu e S4). The e o e, he di e en ial localiza ion in dis inc exp ession backg ounds migh be because a ce ain h eshold in ex- p ession le els has o be achie ed o cy oplasmic ans- loca ion o occu , gi en ha MEX3 p o eins a e capable o pe o ming nucleocy oplasmic shu ling (28). On he o he hand, specific subcellula dis ibu ion migh be ela ed wi h p o ein phospho yla ion, in acco dance wi h published da a desc ibing MEX3 membe s as phospho- p o eins (28,29). Whiche e he case, his does no con a- dic pos - ansc ip ional egula ion, as i can be elici ed a mul iple poin s o he ansc ip li espan, including p e-mRNA p ocessing in he nucleus, expo om he nucleus o he cy oplasm and subsequen coo dina ed a ficking o he ma u e mRNA o he ansla ion ma- chine y. I also emains o be ully cla ified he conse- quence o pa ial accumula ion in P bodies, s uc u es in ol ed in p ocesses o mRNA deg ada ion, nonsense- media ed mRNA decay, ansla ional ep ession and RNA-media ed gene silencing (49), al hough he e a e se e al RBPs wi h es ablished oles in ansla ional egu- la ion known o colocalize wi h P bodies (50,51). We used he Caco-2 cell line model o modula e MEX3A exp ession and assess i s pheno ype. Su p isingly, MEX3A inhibi ion p oduced dis inc e ec s o e CDX2 le els de- pending on cellula confluence. MEX3A migh selec i ely egula e unique subse s o a ge s in di e en cul u e con- di ions. In ag eemen , i is known ha ansc ip omic and p o eomic changes occu du ing he p og ession om he p oli e a i e s a e o spon aneous di e en ia ion o Caco-2 cells. Con e sely, MEX3A migh ha e a dual ole, ac ing bo h as a ep esso o enhance con ingen on he cellula mic oen i onmen , as obse ed o o he RBPs, like HuR (52). I is possible ha in ica e associa ions o MEX3A wi h o he molecula e ec o s, namely o he p o eins o mic oRNAs, de e mine di e gen egula o y endings. MEX3A o e exp ession in Caco-2 cells esul ed in p onounced pheno ypic al e a ions. CDX2 and Villin down egula ion we e indica i e o a loss in in es inal di - e en ia ion, which was u he confi med by he flow cy ome ic p ofile, showing educed G0/G1 popula ion, usually associa ed wi h less-di e en ia ed cells. Ano he hallma k ea u e o MEX3A o e exp ession was he al e ed cellula pola iza ion in s anda d cul u e, as well as he impai ed abili y o o m pola ized s uc u es in he p esence o ma igel. These e ec s may be media ed by CDX2, as his ansc ip ion ac o was p e iously shown o egula e in es inal Villin h ough ec ui men o he B m- ype SWI/SNF complex o i s p omo e (53), and MEX3A-exp essing cys s closely esemble he ones ob ained wi h Caco-2 cells in which CDX2 supp ession was achie ed by len i i al sho -hai pin RNA pa icles (4). I is, he e o e, impo an o asce ain he biological se ing whe e his egula ion migh be de e minan , which is sugges ed by he p edominan exp ession o MEX3A in he s em, ansi -ampli ying and mig a ing pos -mi o ic cells o he in es ine. Al hough CDX2 p o ein can be de ec ed in mos o hese cells, i s le el is lowe in he c yp s compa ed wi h he uppe mos di e en ia ed cells o he illi (2,19,20). A simila inc easing bo om-up g adien has been desc ibed along he colonic gland axis in he dis al colon epi helium. By con as , in si u hyb id- iza ion e ealed ha CDX2 mRNA was homogeneously dis ibu ed along he en i e c yp - illus axis (20). Gi en his CDX2 p o ein exp ession g adien , ha we also show, and lack o co ela ion wi h mRNA, i is likely ha MEX3A fine- unes CDX2 le els in i o as well, in a ansc ip ion-independen manne , p o iding swi a ail- abili y o he p o ein o mee he physiological equi e- men s o he con inuously enewed gu epi helium. Mo eo e , i has been shown ha in es inal s em cells canno di e en ia e in o any o he in es inal lineages in a backg ound o Cdx2 abla ion (54), e ealing he need o igh CDX2 egula ion as de e minan o p ope pheno ype swi ching. Mos in e es ingly, we obse ed highe exp es- sion o di e en in es inal s em cell ma ke s when we o e exp essed MEX3A in Caco-2, sugges ing ha his p o ein is associa ed wi h s em cell ea u es. Ou AGS 3D model also showed a significan up egula ion o OLFM4 (Supplemen a y Table S2), p e iously iden ified as a ma ke o LGR5+ s em cells in human in es ine (46), al hough his gene was no di ec ly up egula ed in Caco-2 cells, which migh be due o in insic p ope ies o each cell line. S eng hening he hypo hesis o a ole o MEX3A in s em cell po en ial, a ecen publica ion showed ha MEX3A is pa o he molecula signa u e o he LGR5+ in es inal s em cells, p esen ing a 1.64- old inc ease in ela ion o daugh e cells, along wi h MSI1, LGR5 and OLFM4 (55). Fu he mo e, ou s udy ein o ces he inc easing knowledge ha o he egula o y mechanisms in addi ion o ansc ip ional ones ha e impo an unc- ions in s em cells. Al hough we do no know ye how MEX3A ela es wi h he in es inal s em cell pheno ype, we hypo hesize ha MEX3A o e exp ession in i sel , oge he wi h he induc ion o a lessened di e en ia ed pheno ype and pola i y de ec s media ed by CDX2 down egula ion, migh be c i ical o allow a pe missi e en- i onmen o he appea ance o s emness ea u es. Fu he mo e, he MEX3A–CDX2 axis migh be impo an du ing ea ly emb yogenesis whe e CDX2 is equi ed o co ec ophec ode m di e en ia ion, while absen om he inne cell mass, in a p ocess ha equi es igh egula- ion (56). In e es ingly, he pa e n o exp ession o he C. elegans o hologues o bo h p o eins was ound o be mu ually exclusi e in ea ly s ages o emb yogenesis (24,27). S ill, i emains o be assessed he ele ance o MEX3A in mul iple pa hological con ex s o he gas o- in es inal ac whe e di e en ia ion abno mali ies di ec ed by CDX2 a e key e en s. In conclusion, we ha e iden ified a no el ole o MEX3A p o ein in he egula ion o in es inal di e en i- a ion, pola i y and s emness ea u es, pa ially media ed by he ep ession o CDX2. This is he fi s desc ip ion o a CDX2 egula o y mechanism based on i s mRNA con ol by an RBP, ha ing a significan impac in CHAPTER 3. CDX2 POST-TRANSCRIPTIONAL REGULATION BY THE RNA-BINDING PROTEIN MEX3A 3998 Nucleic Acids Resea ch, 2013, Vol. 41, No. 7 in es inal homeos asis and likely in gas oin es inal ca cinogenesis. ACCESSION NUMBERS The mic oa ay da a om his publica ion ha e been submi ed o he A ayExp ess da abase (h p://www.ebi. ac.uk/a ayexp ess/) and assigned he iden ifie E-MTAB-1234. SUPPLEMENTARY DATA Supplemen a y Da a a e a ailable a NAR Online: Supplemen a y Tables 1 and 2 and Supplemen a y Figu es 1–4. ACKNOWLEDGEMENTS The au ho s a e g a e ul o P o esso Leono Da id o ui ul discussion, aluable expe imen al ad ice h ough- ou he p ojec and c i ical eading o he manusc ip . They hank D Ca a ina Lei ão o echnical assis ance in flow cy ome y and D Pa ícia Cas o o echnical assis ance in con ocal mic oscopy. The o e all s udy was coo dina ed by R.A. wi h con ibu ions om M.B.; B.P., S.S., R.B. and L.C. pe o med he expe imen s; M.P., JP.R. and JN.F. p o ided essen ial eagen s; B.P. and R.A. concei ed and designed he expe imen s; B.P. and R.A. w o e he manusc ip ; all au ho s con ibu ed o di e en aspec s o da a analysis and c i ical e ision o he manusc ip . FUNDING Fundação pa a a Ciência e a Tecnologia (FCT)— P og ama Ope acional Ciência e Ino ação 2010 do Quad o Comuni á io de Apoio III and FEDER [PTDC/ SAU-OBD/64490/2006]. IPATIMUP is an Associa e Labo a o y o he Po uguese Minis y o Science, Technology and Highe Educa ion and is pa ially sup- po ed by FCT. 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(2009) Cdx2 and he B m- ype SWI/SNF complex coope a i ely egula e illin exp ession in gas oin es inal cells. Exp. Cell Res., 315, 1779–1789. 54. S inge ,E.J., Duluc,I., Saandi,T., Da idson,I., Bialecka,M., Sa o,T., Ba ke ,N., Cle e s,H., P i cha d,C.A., Win on,D.J. e al. (2012) Cdx2 de e mines he a e o pos na al in es inal endode m. De elopmen , 139, 465–474. 55. Muñoz,J., S ange,D.E., Schepe s,A.G., an de We e ing,M., Koo,B.K., I zko i z,S., Volckmann,R., Kung,K.S., Kos e ,J., Radulescu,S. e al. (2012) The Lg 5 in es inal s em cell signa u e: obus exp ession o p oposed quiescen ‘+4’ cell ma ke s. EMBO J., 31 , 3079–3091. 56. S ump ,D., Mao,C.A., Yamanaka,Y., Rals on,A., Chawengsaksophak,K., Beck,F. and Rossan ,J. (2005) Cdx2 is equi ed o co ec cell a e specifica ion and di e en ia ion o ophec ode m in he mouse blas ocys . De elopmen , 132 , 2093–2102. CHAPTER 3. CDX2 POST-TRANSCRIPTIONAL REGULATION BY THE RNA-BINDING PROTEIN MEX3A SUPPLEMENTARY DATA Supplemen a y Figu e S1. Subcellula localiza ion o MEX3A in P bodies (A) Immuno luo escence o MEX3A s ably ans ec ed AGS cells o myc- ag and o endogenous DCP1A and EDC4 p o eins. (B) Immuno luo escence o MEX3A s ably ans ec ed Caco-2 cells o myc- ag and endogenous DCP1A and EDC4 p o eins. Whi e a ow-heads poin o di e en si es o co-localiza ion (o iginal magni ica ion, x630; scale ba s 20 µm). 59 CHAPTER 3. CDX2 POST-TRANSCRIPTIONAL REGULATION BY THE RNA-BINDING PROTEIN MEX3A Supplemen a y Figu e S2. S udy o he exp ession p o ile o endogenous MEX3A in Caco-2 cells. (A) Wes e n blo o MEX3A and CDX2 exp ession du ing Caco-2 di e en ia ion. The ime-poin in which con luence is achie ed is e e ed o as day 0 (cul u e day 6). (B) qPCR o MEX3A and CDX2 mRNA exp ession du ing he same ime-poin s. Values o CDX2 and MEX3A a day -2 we e e e ed o as 1. CHAPTER 3. CDX2 POST-TRANSCRIPTIONAL REGULATION BY THE RNA-BINDING PROTEIN MEX3A Pan oglody es CDX2 - One p edic ed MRE in he 3`UTR bp Sequence 1991 AGAGTTTTTA Mus musculus Cdx2 - Two p edic ed MREs in he CDS bp Sequence 873 GGAGTTTCACTTTA 1171 GGAGGGGTTTTA Ra us no egicus Cdx2 - Th ee p edic ed MREs, one in he CDS and wo in he 3`UTR bp Sequence 1010 GGAGGGGTTTTA 1228 TTAGATTTTTTTTTTA 2027 GGAGCTTTA Danio e io cdx1a - One p edic ed MRE in he 3`UTR bp Sequence 1176 TGAGTTTA Xenopus lae is cdx2 - Se en p edic ed MREs in he 3`UTR bp Sequence 1269 ATAGACTTTTA 1640 TTAGATCCCTTCTA 1860 GTAGCATTTTA 2251 GTAGGCAGCCTTTA 2355 GGAGACGTTTA 2488 TGAGTGTTTTATA 2546 GGAGGATATATTTA D osophila melanogas e cad - Fou p edic ed MREs in he 3´UTR bp Sequence 2235 ATAGCCGCATATATA 2251 AGAGTTTTAACGTTTA 2298 GTAGTTAATATA 2469 ATAGCTATTTA Supplemen a y Figu e S3. Bioin o ma ics sea ch o MREs in CDX2 homologues o se e al species. Di e en MRE sequences loca ed in he coding sequence (CDS) o 3`UTR o he ansc ip s a e shown. 61 CHAPTER 3. CDX2 POST-TRANSCRIPTIONAL REGULATION BY THE RNA-BINDING PROTEIN MEX3A Supplemen a y Figu e S4. Inhibi ion o endogenous MEX3A in Caco-2 cells. (A) Immuno luo escence o endogenous MEX3A a di e en ime-poin s, upon inhibi ion wi h speci ic siRNAs o 24h (o iginal magni ica ion, x400; scale ba 20 µm). (B) Co esponding wes e n blo analysis o MEX3A down egula ion. 63 71 73 Discussion and concluding ema ks Chap e 5 CHAPTER 5. DISCUSSION AND CONCLUDING REMARKS GI malignancies emain a leading cause o mo bidi y wo ldwide, a ec ing almos ou million indi iduals pe yea . This heal h bu den has been in ensi ely ackled du ing he las decades in he sea ch o new app oaches o p e en ion and managemen , and hough slow, some p og ess has been achie ed. A majo ad ancemen in unde s anding cance ae iology is he ecogni ion ha al hough in insically gene ic in o igin, i s he e ogeneous na u e s ems om complex in e ac ions be ween en i onmen al and hos ac o s. A he cellula le el, his implies ha cance cells a e no isola ed en i ies solely de ined by he linea accumula ion o i e e sible gene ic al e a ions. Ca cinogenesis is a a he dynamic p ocess cha ac e ized by ongoing adap a ions o he su ounding mic oen i onmen . The homeobox ansc ip ion ac o CDX2 is a mas e egula o o in es inal di e en ia ion. No su p isingly, CDX2 de egula ion is associa ed wi h GI ca cinogenic p ocesses, including IM onse du ing gas ic cance de elopmen and CRC, hough i s ole in he la e is no consensual. Since s uc u al al e a ions in he CDX2 locus a e a e, i has become e iden ha egula ion mus accoun o he majo al e a ions epo ed in CDX2 le els in di e en pa hological condi ions and consequen ly, o main aining i s no mal homeos a ic balance. Hence o h, e o s ha e been concen a ed in de ining he egula o y mechanisms unde lying CDX2 exp ession. Indeed, se e al ansc ip ional, pos - ansc ip ional and pos - ansla ional mechanisms ha e been desc ibed o con ol CDX2, making his a highly complex and igh ly o ganized egula o y sys em. Wi h he objec i e o un eiling new molecula mechanisms o CDX2 egula ion, we ocused on he ole o mic oen i onmen al in luences as a guiding line. In his ega d, we ha e s udied he e ec o me hyla ion a he CDX2 p omo e le el and o MEX3A p o ein o e CDX2 exp ession, an RNA-binding ac o unco e ed in a cell- ma ix in e ac ion model. The di e en pa s o his wo k a e now deba ed in dis inc sec ions, being poin ed ou wi hin each one u u e esea ch a enues o in e es . A he end, a gene al conclusion is p o ided, in eg a ing all da a om a molecula and e olu iona y s andpoin . CHAPTER 5. DISCUSSION AND CONCLUDING REMARKS Ou analysis ocused on wo classically de ined CpG islands, iden i ied in he 5`p oximal lanking egion, and hus, mo e likely in ol ed in CDX2 egula ion gi en he ansc ip ion s a si e p oximi y. We ini ially chose a panel o gas ic cance cell lines displaying di e en ial exp ession o CDX2 mRNA o alida e a bisul i e-modi ied DNA sequencing p o ocol and es ablish possible co ela ions wi h he CDX2 gene me hyl-pa e n. The choice o s a egy was based on he ac ha me hyla ion- speci ic PCR uses di e en pai s o p ime s o speci ically ampli y me hyla ed o unme hyla ed sequences, p o iding a semi-quan i a i e ou pu ha migh no be ep esen a i e o he whole CpG island, while sequencing o indi idual PCR amplicons wi h an unbiased p ime pai gene a es ex ensi e me hyla ion maps a single-nucleo ide esolu ion. He ewi h, we we e able o de e mine ha p omo e me hyla ion s a us does no co ela e wi h CDX2 exp ession le els, which has been con i med in ollowing s udies (Va on e al. 2012; Zhang e al. 2013). This is in acco dance wi h he CDX2 me hyla ion equency no showing any ela ionship o di e en clinicopa hological cha ac e is ics, like umou his ological ype o in asion (Yuasa e al. 2005), a ac a odds wi h he knowledge ha gas ic cance s e aining CDX2 exp ession a e less agg essi e and mo e di e en ia ed (Liu e al. 2007a). Con adic o y esul s ha e also been published in oesophageal issues, wi h he p esence o me hyla ed and unme hyla ed alleles in cance , and unme hyla ed in ma ched no mal epi helia, despi e no de ec able mRNA o p o ein in he la e (Vanine i e al. 2009). We we e in e es ed in assessing me hyla ion in IM p e-malignan condi ion compa ed o he no mal gas ic mucosa. We no iced he uppe CpG island was hea ily me hyla ed in all cell lines es ed, consis en wi h a p e ious desc ip ion (Kawai e al. 2005), and hence could no be disc imina i e. As a esul , we limi ed he issue analysis o he lowe CpG island only. Analysis o no mal colonic mucosa, IM oci and ma ched adjacen no mal epi helium showed a me hyla ion pa e n deemed inconsis en wi h he CDX2 issue- ype exp ession. Hence, CDX2 exp ession in IM is no a ibu able o deme hyla ion, bu o ansc ip ional ac i a ion by o he mechanisms. This lack o co ela ion has also been obse ed in s omachs om no mal mice and IM cases om Cdx2- ansgenic mice (Mu oh e al. 2009). Conce ning he in es inal backg ound, a ecen epo s a ed ha CDX2 p omo e hype me hyla ion is a e in CRC cases and cell lines (Sala i e al. 2012). The e o e, i 75 5.1 CDX2 REGULATION BY PROMOTER METHYLATION CHAPTER 5. DISCUSSION AND CONCLUDING REMARKS seems CDX2 p omo e me hyla ion is no causa i e o i s own le els in in es ine ei he , and canno accoun o he subse o CRCs p esen ing he e ogeneous CDX2 exp ession. Mos s udies, including ou own, assumed ha unc ionally ele an DNA me hyla ion occu s in p oximal p omo e a eas, bu i is possible ha o he si es migh ha e been neglec ed. In ac , bioin o ma ic analysis e eals ha a leas h ee addi ional CpG islands exis in mo e dis al 5` in e genic loca ions, app oxima ely 6kb, 8kb and 11kb om he CDX2 ansc ip ional s a si e, he las wo o e lapping he neighbou ing gene PRHOXNB. CpG sho e me hyla ion is an eme ging concep ha migh also be wo hwhile explo ing in his con ex . These a e egions o lowe CpG densi y ha lie in close p oximi y, bu o en no wi hin, CpG islands. No ably, and a leas o colon, mos cance -associa ed al e a ions in me hyla ion we e p edominan ly obse ed in hese a eas, a he han es ic ed o CpG islands (I iza y e al. 2009). In p inciple, hese pu a i e me hyl-si es should be esponsi e o inhibi o s o DNA me hyl ans e ase ac i i y as well. Ne e heless, he b oad ange na u e o his ype o ea men s is expec ed o gene a e indisc imina e e ec s on mul iple a ge s, whose ne balance migh no e lec inc eased CDX2 ansc ip ion. Thus, me hyla ion p o ile analysis o hese al e na i e egions s ands as a u u e esea ch goal. Ano he key poin o be conside ed is he ole o ch oma in s a e. Cell ype- speci ic ch oma in o ganiza ion enables di e en ial access o and ac i i y o egula o y elemen s and he mani es a ion o unique cellula pheno ypes. Al e a ions in hese signa u es a e a common ea u e o cance ini ia ion and de elopmen (Su à e al. 2013). Ac i e p omo e egions include heigh ened nuclease sensi i i y implying nucleosome deple ion, and his one modi ica ions associa ed wi h ansc ip ional ac i a ion, such as me hyla ion o his one H3 a lysine 4 (H3K4me1 o H3K4me3) and his one H3 ace yla ed a lysine 27 (H3K27Ac). Qui e he opposi e, nucleosome compac ion, me hyla ion o his one H3 a lysine 9 (H3K9me2 o H3K9me3) and o his one H3 a lysine 27 (H3K27me3) a e essen ially ep essi e ai s (Zhou e al. 2011). In his ega d, ch oma in changes associa ed wi h CDX1 and CDX2 exp ession we e only desc ibed in one publica ion and in colon cance cell lines (Lu e al. 2008). The in i o pa e n o hese epigene ic ma ks, pa icula ly in IM de elopmen , emains o be examined. CHAPTER 5. DISCUSSION AND CONCLUDING REMARKS Gi en ha p e ious epo s desc ibed he ole o epi helial-mesenchymal c oss alk as ele an o CDX2 exp ession, we es ablished in i o 3D cul u e models in o de o s udy in e ac ions be ween gas ic epi helial cells and s omal elemen s ha migh egula e CDX2. These sys ems ha e now been applied wi h success o gene a e and main ain gas ic and in es inal a chi ec u e in he o m o o ganoids om single Lg 5+ s em cells (Sa o e al. 2009; Ba ke e al. 2010). Ou ansc ip omic analysis compa ing 3D wi h s anda d AGS cell cul u e e ealed a emodeling o he gene exp ession p og am. Such adap a ion was pa icula ly no iceable by al e a ions in ac in eo ganiza ion media ed by inc eased exp ession o GTPases o he Rho amily, like ARHGEF9, which ac s as a guanine nucleo ide exchange ac o o he pola i y- ela ed p o ein CDC42. An inc ease in COL4A5 and LAMB1 ansc ip s, o example, was also obse ed, which encode o ype IV collagen and laminin, espec i ely, componen s o he basal lamina ha has been shown o be p ima ily a p oduc o epi helial cells, poin ing owa ds an induc i e eedback loop be ween he ma ix and epi helial cells. In e es ingly, we no iced a signi ican inc ease in cell cycle ela ed elemen s, o ins ance in SGOL1, which encodes a p o ein ha shields he cohesin complex om clea age and is c ucial o ai h ul ch omosome seg ega ion du ing mi osis and meiosis (Kahyo e al. 2011); ERCC6L, a DNA helicase ha is essen ial o main enance o genome in eg i y (Baumann e al. 2007); and he polyme ase POLD1, which unc ions in DNA eplica ion/ epai and whose mu a ions a ec ing he p oo eading domains ha e ecen ly been shown o inc ease he isk o colo ec al ca cinoma (Palles e al. 2013). T ansc ip s encoding in lamma o y esponse ac o s, like chemokine ligands, in e e ons and in e leukins, we e all down egula ed. Fu he mo e, an inc ease in No ch1, DLL1, and FZD1 was de ec ed. Combina o ial con ol be ween hese No ch and Wn pa hway membe s seems necessa y o main ain in es inal s em cells (F e e al. 2009). In line, he a ay showed a 60- old inc ease in he exp ession o OLFM4, a speci ic ma ke o Lg 5+ in es inal s em cells ( an de Flie e al. 2009). The p e ious esul s, oge he wi h he dec eased CDX2 exp ession sugges ha ou AGS 3D cells esemble undi e en ia ed in es inal cells. Since AGS was i s es ablished om an in es inal- ype gas ic adenoca cinoma, we specula e ha a pa ial e e sion o he malignan owa ds an in es inal pheno ype migh occu unde a physiologically mo e ele an mic oen i onmen , eminiscen o wha has been 77 5.2 3D AGS CELL MODEL AND LOSS OF CDX2 EXPRESSION CHAPTER 5. DISCUSSION AND CONCLUDING REMARKS obse ed in a mamma y model (Wea e e al. 1997). The ull po en ial o his model hus emains o be explo ed, as i migh p o ide addi ional insigh s on ac o s in ol ed in he egula ion o in es inal di e en ia ion. Th ough mic oa ay analysis o dissec he ansc ip ional egula o y ci cui y o AGS cells in cul u e wi h an ex acellula ma ix, and u he suppo ed by an e olu iona y link, we iden i ied and alida ed MEX3A as a CDX2 ep esso . Conside ing he dis inc expe imen al se ings es ablished, ou in e p e a ion is ha MEX3A ac i ely con ols CDX2 exp ession du ing p oli e a i e s a es, when ine- uning o CDX2 a he ansc ip le el seems mo e ele an o couple cell cycle a es wi h he eme gence o a di e en ia ion p og am (Bai e al. 2003). In subsequen di e en ia ed s a es, CDX2 exp ession has al eady been demons a ed o ely mos ly in i s p o ein s abili y (Boulange e al. 2005). Hence, MEX3A migh exhibi a ge speci ici y, and his hypo hesis is a ou ed by he ac ha i is s ill de ec ed in he di e en ia ed sec ion o he c yp - illus uni , albei weake han in he lowe po ions, a guing o some ac i i y in his compa men ha migh no be CDX2-o ien ed. Rega ding he spa ial compa men aliza ion in P bodies, we did no add ess i hese a e unc ionally ele an o i MEX3A e ec on mRNA akes place as e icien ly in he cy oplasm. Ne e heless, e olu iona y compa a i e da a suppo s his speci ic localiza ion. C. elegans MEX-3 is in ol ed in he co ec seg ega ion o la ge oci known as P g anules o he inal ge mline p ecu so (D ape e al. 1996; Schisa e al. 2001; Jud e al. 2007). P g anules a e memb ane- ee ibonucleop o ein cy oplasmic s uc u es ha p ima ily con ain ma e nally exp essed mRNAs alongside componen s sha ed wi h bo h P bodies and s ess g anules (Updike and S ome 2010). These h ee disc e e mic odomains a e dynamically and e e sibly induced in esponse o en i onmen al signals. Thus, hei na u e seems o be in acco dance wi h a unc ion in main aining o modi ying ce ain RNA p ope ies, delaying o e en p e en ing he ansla ional p ocess. Inhibi ion o speci ic P body componen s h ough an RNAi s a egy will help o shed ligh o e MEX3A unc ional equi emen s. We showed ha MEX3A in e ac s wi h CDX2 mRNA h ough a canonical MRE p esen in he 3`UTR. This 5.3 MECHANISTIC ASPECTS OF MEX3A FUNCTION CHAPTER 5. DISCUSSION AND CONCLUDING REMARKS does no exclude he possibili y o MEX3A ecognizing ei he di e en a ia ions o he degene a e sequence desc ibed o MEX-3 binding in C. elegans (Pagano e al. 2009) o e en o he unknown sequences; howe e , i p o ides an ancho poin o sea ch o new a ge s. We ha e no assessed i he in e ac ion is di ec o media ed by ano he RBP ha complexes wi h MEX3A, ei he h ough i s RING domain o e en h ough he KH domains, as hese ha e also been shown o media e p o ein oligome iza ion in some ins ances (Chen e al. 1997). RNA elec opho e ic mobili y gel shi assays wi h a p obe con aining he CDX2 3`UTR MRE sequence oge he wi h a MEX3A ecombinan p o ein will p o ide a de ini i e answe conce ning his issue. Ano he aspec ha was no ad anced is MEX3A own sequence and/o s uc u al in e ac ion de e minan s. MEX-3 p o eins possess ype I KH domains, which consis o h ee-s anded an ipa allel β-shee s, o ien ed agains h ee α-helices. The s able β-α-α-β-β-α con o ma ion exposes a lexible loop be ween he i s wo helices, occupied by semi-conse ed posi i ely cha ged esidues in a Gly-X-X-Gly mo i (Val e de e al. 2008). E en hough he o al numbe o sol ed s uc u es wi h 79 CHAPTER 5. DISCUSSION AND CONCLUDING REMARKS eigh -cell s age, which p ecedes es ablishmen o dis inc cell popula ions (Rals on and Rossan 2008; Jed usik e al. 2010). Since hese cells will gi e ise o bo h he inne cell mass and TE, he co olla y o his delay is ha Cdx2 mus be ep essed du ing ini ial apola di isions, and upon induc ion i mus be silenced and main ained as cells a e alloca ed o inside o ou side posi ions, espec i ely. T ansc ip ional con ol in esponse o lineage seg ega ion is an al eady es ablished means o egula ing Cdx2 (Nishioka e al. 2009), bu i is no well unde s ood how his is imposed in he i s place. Asymme ic cell di isions p oduce daugh e cells wi h dis inc a es, and ely on he dis inc seg ega ion o key de e minan s, including localized mRNAs. Acco dingly, i was epo ed ha Cdx2 ansc ip s become p e e en ially localized apically a he la e eigh -cell s age and asymme ically inhe i ed du ing mouse de elopmen a he eigh - o six een-cell s age ansi ion (Jed usik e al. 2008). This was no seen o ansc ip s encoding o he cell commi men ansc ip ion ac o s, like Nanog. A compelling hypo hesis is ha MEX3A migh con ibu e o he ini ial Cdx2 mRNA ep ession and possibly o i s apical en ichmen , ecapi ula ing C. elegans MEX-3 e ec o e pal-1. This possibili y is s eng hened by a epo showing ha in mouse Cdx2 asymme ic localiza ion depends on a minimal cis-elemen comp ising he las 97 nucleo ides o he ansc ip open eading ame (Skamagki e al. 2013), whe e we had al eady iden i ied one MRE and ano he one close ups eam. Assuming his scena io, MEX3A migh be in ol ed in he egula ion o he Cdx2 ansc ip ansla ion in an emb yonic se ing o e en in i s anspo , as i was demons a ed ha in ac mic o ubules and ac in cy oskele on, oge he wi h he ac i i y o mo o p o eins o he kinesin supe amily a e a equi emen o his asymme ic localiza ion (Skamagki e al. 2013). Thus, MEX3 p o eins migh ac as conse ed componen s o he cell pola iza ion pa hway con ibu ing o CDX2 egula ion du ing mammalian emb yogenesis, he eby linking gene egula ion and posi ional in o ma ion. CHAPTER 5. DISCUSSION AND CONCLUDING REMARKS Wi h his wo k, we ha e ad anced he knowledge on he molecula mechanisms o CDX2 egula ion. On one hand, ou esul s suppo he lack o e idence in a o o a ole o me hyla ion on de no o CDX2 exp ession in he gas i is-me aplasia-ca cinoma sequence, con ibu ing o sol e an ambiguous opic in he ield. On he o he hand, we p o ide e idence o a new mechanism o CDX2 pos - ansc ip ional egula ion media ed by he RNA-binding p o ein MEX3A. The ele ance o MEX3A jus s a ed o be un a eled, bu ou da a poin s owa ds i s in ol emen in in es inal homeos asis and possibly gas oin es inal ca cinogenesis, wi h e ec s in cellula pola i y and s emness. In his ega d, we will now asce ain he ole o MEX3A by s udying i s exp ession in p eneoplas ic and neoplas ic lesions, as well as i s co ela ion wi h CDX2 and clinicopa hological pa ame e s. In pa allel, we will es ablish a ansgenic mouse model wi h condi ional and inducible MEX3A exp ession in he illi compa men o he in es ine, in o de o assess he e ec s o ec opic MEX3A o e he in es inal pheno ype. This will be complemen ed by a ansc ip ome-wide analysis o MEX3A egula o y in e ac ions. O e all, we expec o signi ican ly con ibu e o he unde s anding o he impac o MEX3A pos - ansc ip ional con ol on gene exp ession, bo h in homeos asis and disease. E en conside ing MEX-3 well-desc ibed ole in C. elegans emb yonic de elopmen and main enance o cell o ipo ency, i is ema kable o ind such a ma ked deg ee o unc ional pa allelism in highe o ganisms, a ea u e ha one can p edic o e lec he impo ance o his p o ein amily. Consequen ly, i seems we ha e disclosed a case o e olu iona y conse a ion ha migh ha e majo implica ions o basic and clinical esea ch. 87 5.6 CONCLUDING REMARKS 89 Re e ences 91 REFERENCES Aliaga JC, Deschênes C, Beaulieu JF, Cal o EL, Ri a d N. 1999. Requi emen o he MAP kinase cascade o cell cycle p og ession and di e en ia ion o human in es inal cells. Am J Physiol 277: G631-641. Almeida R, Sil a E, San os-Sil a F, Silbe g DG, Wang J, De Bolós C, Da id L. 2003. 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J Biol Chem 274: 6011-6019. 111 119 Appendix ANTIBODIES An igen Applica ion Desc ip ion Dilu ion Sou ce Comme cial e e ence Ac in WB Rabbi polyclonal 1:8000 San a C uz Bio echnology sc-1616-R CDX2 IF and WB Mouse monoclonal IgG1 (Clone CDX2-88) 1:50 and 1:1000 Biogenex MU392A-UC Cyclin D1 WB Rabbi monoclonal (Clone SP4) 1:500 The mo Scien i ic RM-9104 DCP1A IF Rabbi polyclonal 1:500 Sigma Ald ich D5444 E-cadhe in IF Mouse monoclonal IgG1 (Clone HECD-1) 1:300 Taka a Biochemicals M106 EDC4 IF Rabbi polyclonal 1:400 Cell Signaling Technology 2548 F-ac in IF Te ame hyl hodamine B iso hiocyana e (TRITC)-conjuga e o phalloidin 1:10000 Sigma Ald ich 77418 MEX3A IF and WB Rabbi polyclonal 1:600 and 1:1000 Sigma Ald ich PRS4869 MSI1 WB Rabbi polyclonal 1:1000 Millipo e AB5977 c-Myc IF and WB Mouse monoclonal IgG1 (Clone 9E10) 1:600 and 1:1000 Clon ech 631206 Villin WB Mouse monoclonal 1:3000 Kindly p o ided by D . Syl ie Robine, Ins i u e Cu ie, Pa is, F ance ― ZO-1 IF Rabbi polyclonal 1:50 In i ogen 61-7300 Seconda y Goa an i-Mouse IgG IF Alexa Fluo 488 o Alexa Fluo 594 conjuga ed 1:100 In i ogen A-11001 o A-11005 Goa an i-Mouse IgG WB Ho se adish pe oxidase (HRP) conjuga ed 1:2000 San a C uz Bio echnology sc-2005 Goa an i-Rabbi IgG IF Alexa Fluo 488 o Alexa Fluo 594 conjuga ed 1:100 In i ogen A-11008 o A-11012 Goa an i-Rabbi IgG WB Ho se adish pe oxidase (HRP) conjuga ed 1:2000 San a C uz Bio echnology sc-2004 PRIMER SEQUENCES (5`- 3`) Bisul i e sequencing CpG1 F: TTTTAGAAATGATAGGATGAAGG R: ACCAAAAAACCTAAAACTAAAAA CpG2.1 F: GTAGGTTAGAGGGAGGGAT R: TCCTTATCCAAAAAATAACTCAC CpG2.2 F: GAGTTATTTTTTGGATAAGGA R: TAACCATTCCAATCCTCCC CpG2.3 F: GGGAGGATTGGAATGGTTA qPCR R: TTTACAACAACCCAAAAAC 18S F: CGCCGCTAGAGGTGAAATTC R: CATTCTTGGCAAATGCTTTCG BMI1 F: GTCTACATTCCTTCTGTAAAACG R: CTTGGAGAGTTTTATCTGACC CDX2 F: TTCACTACAGTCGCTACATCACCAT R: TTGTTGATTTTCCTCTCCTTTGCT GAPDH F: TCAAGGCTGAGAACGGGAAG R: AGAGGGGGCAGAGATGATGA LGR5 F: CAGCGTCTTCACCTCCTACCTA R: CCTTGGGAATGTATGTCAGAGC MEX3A F: CAAGCTCTGCGCTCTCTACAAA R: GGCCTTAATCTTGCAGCCTTG Rluc F: TGCAAGCAAATGAACGTGCTG R: TCTAGCCACGGGCTCGATGT Si e-di ec ed mu agenesis pRL∆CDX2 ec o *S: GTAACATCCAAGCCAGCCTTTTCCAAGCCTTCTGGATCC AS:GGATCCAGAAGGCTTGGAAAAGGCTGGCTTGGATGTTAC * Mu a ed nucleo ides a e in bold and unde lined Appendix A. Lis o an ibodies and p ime s used in his s udy.