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Funkce CDK12 a CDK13 V Regulaci Transkripce Hana Paculová

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Funkce CDK12 a CDK13 V Regulaci Transkripce Hana Paculová MASARYKOVA UNIVERZITA PŘÍRODOVĚDECKÁ FAKULTA ÚSTAV BIOCHEMIE Funkce CDK12 a CDK13 v regulaci transkripce Disertační práce Hana Paculová Školitel: Mgr. Jiří Kohoutek, Ph.D Brno 2018 Bibliogra cký záznam Autorka: Mgr. Hana Paculová Prrodovedecáá aaául,a鏈 Maaarkáova unvverv,a Úa,av bvochemve Název práce: Funáce CDK12 a CDK13 v regulacv ,ranaárvpce Studijní program: Bvochemve Studijní obor: Bvochemve Školitel: Mgr. Jvr Kohou,eá鏈 Ph.D Akademický rok: 2017/2018 Po et stran: 89 Klí ová slova: Ckálvn-dependen,n ávnaaa鏈 CDK12鏈 ,ranaárvpce鏈 RNA polkmeraaa II鏈 raáovvna vaječnáů鏈 CHK1 Bibliographic entry Author: Mgr. Hana Paculová Facul,k oa acvence鏈 Maaarká unvverav,k Department of Biochemistry Title oF dissertation: CDK12 and CDK13 aunc,von vn ,ranacrvp,von regula,von Degree programme: Bvochemva,rk Field oF study: Bvochemva,rk Supervisor: Mgr. Jvr Kohou,eá鏈 Ph.D Academic year: 2017/2018 Number oF pages: 89 Keywords: Ckclvn-dependen ávnaae鏈 CDK12鏈 ,ranacrvp,von鏈 RNA polkmeraae II鏈 ovarvan cancer鏈 CHK1 Abstrakt Ckálvn-dependen,n ávnaaa 12 (CDK12) je ,ranaárvpčn ávnaaa鏈 á,erá rd expreav avých clových genů ,m鏈 že aoaaorkluje RNA polkmeraau II v průbehu elongačn aáe ,ranaárvpce. CDK12 je apojena do neáolváa bunečných preceaů鏈 což ahrnuje odpoveď na pošáoen DNA鏈 vývoj a bunečnou dvaerencvacv a aea,rvh mRNA. CDK12 bkla popaána jaáo jeden genů鏈 á,eré jaou čaa,o mu,ovánk v hvgh-grade aerónm ovarválnm áarcvnomu鏈 nvcméne vlvv ,ech,o mu,ac na aunácv CDK12 a jejvch role v áarcvnogenev dopoaud nebkla a,anovena. Zjva,vlv jame鏈 že ve,švna mu,ac CDK12鏈 á,eré bklk naleenk v nádorech鏈 brán vk,voren áomplexu CDK12 a Ckálvnem K a vnhvbuj ávnaaovou aá,vvv,u CDK12. Pomoc analýk expreae mRNA e voráů nádorů neaoucch CDK12 mu,ace ae nám podarvlo vden,vfáova, aadu genů鏈 jejvchž expreae je ávvalá na CDK12. Dále uáaujeme鏈 že CDK12 ae prmo podl na na ,ranaárvpcv ,ech,o genů ,aá鏈 že aoaaorkluje RNA polkmeraau II na Servnu 2. Naáonec jame uááalv鏈 že mu,ace CDK12 anvžuj achopnoa, buneá opravova, dvoure,ecové lomk v DNA pomoc homologn reáombvnace. Z ,oho vkvoujeme鏈 že mu,ace鏈 á,eré deá,vvuj aunácv CDK12鏈 mohou véa, áe anžené achopnoa,v buneá opravova, pošáoenou DNA鏈 což může véa, áe genomové nea,abvlv,e a prvapva, á rovojv nádoru. U veláého množa,v nádorů ae vkvvne reava,ence pro,v exva,ujcm půaobům léčbk鏈 pro,o je po,reba hleda, nové možnoa,v ,erapve nádorů. Inhvbv,ork CHK1 vkšuj ,erapeu,vcáé účvnák lá,eá鏈 á,eré půaobuj pošáoen DNA. Je námo鏈 že deregulace BRCA1 v CDK12 půaobuje genomovou nea,abvlv,u鏈 rohodlv jame ae pro,o ,ea,ova, hkpo,éu鏈 že anžen BRCA1 a CDK12 povede á ve,šmu ck,oa,a,vcáému eaeá,u vnhvbv,oru CHK1. Zjva,vlv jame鏈 že anžen expreae BRCA1 v CDK12 vkšuje účvneá CHK1 vnhvbv,oru in vitro. Snžen expreae BRCA1 výšvlo aenv,vvv,u buneá á CHK1 vnhvbv,oru v mkšm xenograa,ovém modelu. Snžen BRCA1 v áombvnacv a CHK1 vnhvbvc vedlo áe výšené hladvne pošáoen DNA a á neachopnoa,v prej, S-aáv bunečného ckálu. Navrhujeme鏈 že vnhvbvce CHK1 bk mohla bý, vhodnou a,ra,egv pro léčbu nádorů a defcven,n BRCA1 nebo CDK12. V nádorové ,erapvv bk mohla bý, vkužv,a v áombvnace vnhvbv,orů CDK12 a CHK1. Abstract Ckclvn-dependen, ávnaae 12 (CDK12) va a ,ranacrvp,von-aaaocva,ed ávnaae whvch promo,ea expreaavon oa v,a ,arge, genea bk phoaphorkla,vng RNA Polkmeraae II durvng ,he ,ranacrvp,von elonga,von. CDK12 va vnvolved vn mul,vple bvologvcal proceaaea鏈 vncludvng DNA damage reaponae鏈 developmen,鏈 dvaaeren,va,von and mRNA aplvcvng. CDK12 waa vden,vfed aa one oa ,he recurren,lk mu,a,ed genea vn hvgh-grade aeroua ovarvan cancer. Never,heleaa鏈 ,he vmpac, oa ,heae mu,a,vona on CDK12 aunc,von and ,hevr role vn carcvngeneava have no, been de,ermvned. We ahow ,ha, moa, oa ,he CDK12 ovarvan cancer-aaaocva,ed mu,a,vona preven, aorma,von oa ,he CDK12/Ckclvn K complex and dvarup, v,a ávnaae ac,vvv,k. Emplokvng mRNA expreaavon analkava oa ,umor aamplea bearvng CDK12 mu,a,vona鏈 we vden,vfed a ae, oa CDK12-dependen, DDR genea. We ahow ,ha, CDK12 dvrec,lk par,vcvpa,ea vn ,ranacrvp,von oa ,hoae genea bk phoaphorkla,vng Servne 2 oa RNA polkmeraae II. Fvnallk鏈 we ahow ,ha, CDK12 mu,a,vona dvarup, ,he abvlv,k oa cella ,o repavr oa double a,rand breaáa bk HR. Taáen ,oge,her鏈 CDK12 loaa-oa-aunc,von mu,a,vona cauae defcvenck vn DNA repavr鏈 whvch can lead ,o genomvc vna,abvlv,k and carcvnogeneava. Our reaul,a auppor, ,he hkpo,heava ,ha, CDK12 va a ,umor auppreaaor vn hvgh-grade aeroua ovarvan cancer. Svnce large number oa ,umora develop reava,ance ,o exva,vng ,herapvea鏈 ,here va a need ,o develop new ,herapeu,vc op,vona. CHK1 vnhvbv,ora po,en,va,e ,he ck,oa,a,vc eaaec, oa varvoua DNA-damagvng agen,a. Svnce loaa oa BRCA1 or CDK12 reaul, vn genomvc vna,abvlv,k鏈 we ,ea,ed a hkpo,heava ,ha, BRCA1 or CDK12 deple,von wvll enhance ,he ck,oa,a,vc eaaec, oa CHK1 vnhvbv,ora. The avlencvng oa BRCA1 or CDK12 aenav,ved ,umor cella ,o CHK1 vnhvbv,ora in vitro. BRCA1 deple,von enhanced ,he CHK1 vnhvbv,or aenav,vvv,k vn a mouae xenograa, model. Fvnallk鏈 BRCA1 downregula,von combvned wv,h CHK1 vnhvbv,von vnduced exceaavve amoun,a oa DNA damage鏈 reaul,vng vn an vnabvlv,k ,o comple,e ,he S-phaae. We auggea, CHK1 vnhvbv,von aa a a,ra,egk aor ,arge,vng BRCA1- or CDK12-defcven, ,umora. Moreover鏈 ,he combvna,von oa CHK1 and CDK12 vnhvbv,von mak exhvbv, a ,herapeu,vc eaaec,. Poděkování Na ,om,o ma,e bkch ch,ela podeáova, predevšm avému šáolv,elv Jvrmu Kohou,áovv a veden a cenné radk po celou dobu a,udva. Dále bkch ch,ela podeáova, Dalvborovv Blažáovv a jeho aáupvne a možnoa, apolupráce a celému áoleá,vvu na Oddelen chemve a ,oxváologve na VÚVel a podporu v průbehu a,udva. Prohlášení Prohlašujv鏈 že jaem avojv dvaer,ačn prácv vkpracovala aamoa,a,ne a vkužv,m vnaormačnch drojů鏈 á,eré jaou v prácv cv,ovánk Brno 23.4.2018 Hana Paculová 0 Table of contents 1. Theoretical part.........................................................................................................................................2 1.1 Tranacrvp,vonal regula,von......................................................................................................................2 1.2 Ckclvn dependen, ávnaaea.......................................................................................................................2 1.3 CDK12 a,ruc,ure.......................................................................................................................................4 1.4 CDK12 aaaocva,vng ckclvn - Ckclvn K.....................................................................................................5 1.5 CDK12 aunc,von vn ,ranacrvp,von regula,von .......................................................................................6 1.6 CDK12 homologa.....................................................................................................................................7 1.7 CDK12 aunc,von vn aplvcvng regula,von.................................................................................................8 1.8 CDK12 aunc,von vn developmen, and dvaaeren,va,von.........................................................................9 1.9 CDK12 va mu,a,ed vn Hvgh-grade aeroua ovarvan cancer .................................................................9 1.10 CDK12 loaa conaera aenav,vvv,k ,o PARP1/2 vnhvbv,ora..................................................................10 1.11 CDK12 va amplvfed vn Her2 poav,vve breaa, cancer ........................................................................11 1.12 CDK12 vnhvbv,ora.................................................................................................................................12 1.13 CHK1 vnhvbv,ora aa an,v-cancer druga...............................................................................................13 2. Aims of the dissertation thesis.............................................................................................................15 3. Results ......................................................................................................................................................16 3.1 Ovarian carcinoma CDK12 mutations misregulate expression of DNA repair genes via de cient formation and function of the CDK12/Cyclin K complex .................16 3.1.1 Genera,von oa cell lvnea expreaavng mu,a,ed CDK12 aorma..........................................................16 3.1.2 Deaec,vve vn,erac,von be,ween CkcK and Cdá12 va ,he predomvnan, conaequence oa CDK12 mu,a,vona vn HGS-OvCa .................................................18 3.1.3 CDK12 mu,a,vona vn HGS-OvCa abroga,e ,he ávnaae ac,vvv,k oa Cdá12 ...................................20 3.1.4 CDK12 mu,a,vona vn HGS-OvCa decreaae ,ranacrvp,vonal ac,vva,von bk Cdá12 .....................20 3.1.5 HGS-OvCa pa,ven, aamplea wv,h CDK12 mu,a,vona exhvbv, downregula,von oa genea oa ,he HR repavr pa,hwak.................................................................23 3.1.6 CDK12 deple,von decreaaea expreaavon oa HR genea....................................................................25 3.1.7 The Cdá12/CkcK complex occupvea HR genea and promo,ea Ser2 phoaphorkla,von oa RNAPII................................................................................... 27 3.1.8 CDK12 mu,a,vona vn HGS-OvCa dvaable ,he a,vmula,ork role oa ,he Cdá12/CkcK complex vn ,he repavr oa DSBa bk HR...................................................29 3.1.9 Dvacuaavon ...........................................................................................................................................31 3.2 BRCA1 or CDK12 loss sensitizes cells to CHK1 inhibitors .......................................................33
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