MASARYKOVA UNIVERZITA Pøírodovìdecká fakulta Ústav experimentální biologie Diplomová práce Brno 2019 Marie Krátká Pøírodovìdecká fakulta Ústav experimentální biologie Adaptivní evoluce genu CenH3 u rostlin s holocentrickými chromozómy Diplomová práce Marie Krátká Vedoucí práce: Mgr. František Zedek, Ph.D. Brno 2019 Bibliograficky´za´znam Autor: Bc. Marie Kra´tka´ Prˇı´rodoveˇdecka´fakulta, Masarykova univerzita U´ stav experimenta´lnı´biologie Na´zev pra´ce: Adaptivnı´evoluce genu CenH3 u rostlin s holocentricky´mi chro- mozo´my Studijnı´program: Experimenta´lnı´biologie Studijnı´obor: Molekula´rnı´biologie a genetika Vedoucı´pra´ce: Mgr. Frantisˇek Zedek, Ph.D. Akademicky´rok: 2018/2019 Pocˇet stran: 54 Klı´cˇova´slova: CenH3; CENP-A; holocentricke´ chromozomy; adaptivnı´ evo- luce, centromericky´tah; asymetricka´meio´za; CENP-C; Cypera- ceae; kodon-substitucˇnı´modely; MEME; aBSREL Bibliographic Entry Author: Bc. Marie Kra´tka´ Faculty of Science, Masaryk University Department of Experimental Biology Title of Thesis: Adaptive evolution of the CenH3 gene in plants with holocent- ric chromosomes Degree Programme: Experimental biology Field of Study: Molecular biology and genetics Supervisor: Mgr. Frantisˇek Zedek, Ph.D. Academic Year: 2018/2019 Number of Pages: 54 Keywords: CenH3; CENP-A; holocentric chromosomes; centromeric drive; asymmetric meiosis; CENP-C; adaptive evolution Cyperaceae; codon-substitution models; MEME; aBSREL Abstrakt U organismu˚ s monocentricky´m usporˇa´da´nı´m chromozomu˚ probı´ha´ centromericky´ tah, evolucˇnı´proces iniciovany´expanzı´centromericky´ch repetic, ktere´jsou tak na sebe schopny nava´zat veˇtsˇı´mnozˇstvı´kinetochorovy´ch proteinu˚a pote´by´t dı´ky asymetrii deˇ- licı´ho vrˇete´nka prˇisamicˇı´meio´ze preferencˇneˇsegregova´ny do vajı´cˇka. V reakci na tyto sobecke´centromery docha´zı´k adaptivnı´evoluci kinetochorove´ho proteinu CenH3, ktery´ doka´zˇe zabra´nit tahu sobecky´ch centromer obnovenı´m jejich rovnocennosti. Alternativnı´m zpu˚sobem obrany mohou by´t holocentricke´chromozomy, ktere´majı´v meio´ze kinetochor sestaven pode´l cele´de´lky chromozomu, cozˇmu˚zˇe znemozˇnit rozsˇı´rˇeny´m repetitivnı´m sek- vencı´m zı´skat selekcˇnı´vy´hodu a iniciovat centromericky´tah. V takove´m prˇı´padeˇproto nedocha´zı´k adaptivnı´evoluci CenH3. Tato pra´ce popisuje evoluci genu CenH3 v 34 dru- zı´ch rostlin z holocentricke´cˇeledi Cyperaceae. Selekcˇnı´analy´zou byla nalezena pozitivnı´ selekce CenH3 srovnatelna´s monocentricky´mi organismy s asymetrickou samicˇı´meio´zou, z cˇehozˇvyply´va´, zˇe usporˇa´da´nı´kinetochoru˚beˇhem meio´zy v te´to cˇeledi nebra´nı´centrome- ricke´mu tahu. To by se dalo vysveˇtlit mozˇny´m polycentricky´m usporˇa´da´nı´m kinetochoru beˇhem meio´zy, ktere´vsˇak zatı´m bylo pozorova´no pouze v druhu Rhynchospora pubera. Abstract In organisms with monocentric chromosome morphology, an evolutionary process called centromere drive takes place. This process is initiated by centromeric repeats ex- pansion, which leads to the recruitment of excess kinetochore proteins. This allows the expanded centromere to be preferentially segregated to the egg during asymmetric female meiosis. In response to the selfish centromeres, kinetochore protein CenH3 adaptively evol- ves. The evolution of CenH3 can avert the centromere drive by restoring the centromere parity. Alternative protection from the centromere drive can be provided by holocentric chromosomes, which have their kinetochore assembled along an entire chromosome du- ring meiosis. This can prevent expanded centromeres from acquiring selective advantage and initiating the centromere drive. In such a case, no adaptive evolution of CenH3 occurs. This thesis describes the evolution of the CenH3 gene in 34 species from the Cyperaceae family. Positive selection of CenH3 comparable with monocentric organisms with asym- metric female meiosis was found using selection analysis, which suggests that the structure of kinetochores in the Cyperaceae family doesn’t prevent the centromere drive. This could be explained by the possible polycentric structure of the kinetochore in this family during meiosis which has so far been observed only in Rhynchospora pubera species. Podeˇkova´nı´ Na tomto mı´steˇbych chteˇla podeˇkovat vedoucı´mu sve´diplomove´pra´ce Mgr. Frantisˇku Zedkovi, Ph.D. za odborne´vedenı´a rady, ktere´jsem od neˇj obdrzˇela beˇhem vypracova´va´nı´ sve´pra´ce, doc. RNDr. Petrovi Buresˇovi, Ph.D. a Mgr. Pavlovi Vesele´mu, Ph.D. za urcˇenı´ a sbeˇr vzorku˚a Ing. Jakubovi Sˇmerdovi, Ph.D. a KaterˇineˇLojdove´za pomoc v laboratorˇi. Da´le bych chteˇla podeˇkovat organizaci MetaCentrum za poskytnutı´vy´pocˇetnı´ch zdroju˚. Prohla´sˇenı´ Prohlasˇuji, zˇe jsem svoji diplomovou pra´ci vypracovala samostatneˇs vyuzˇitı´m infor- macˇnı´ch zdroju˚, ktere´jsou v pra´ci citova´ny. Brno 10. kveˇtna 2019 . Marie Kra´tka´ Obsah Kapitola 1. U´ vod........................................................... 10 1.1 CenH3............................................... 10 1.2 Centromericky´tah....................................... 10 1.3 Adaptivnı´evoluce CenH3.................................. 12 1.3.1 Zˇ ivocˇichove´....................................... 12 1.3.2 Rostliny.......................................... 12 1.3.3 Evoluce dalsˇı´ch kinetochorovy´ch proteinu˚................... 13 1.3.4 Evoluce CenH3 v organizmech se symetrickou meio´zou......... 13 1.3.5 Vliv usporˇa´da´nı´centromery na evoluci CenH3................ 13 1.4 Holocentricke´chromozomy................................. 14 1.4.1 Monocentricke´, metapolycentricke´, holocentricke´.............. 14 1.4.2 Centromericke´sekvence............................... 16 1.4.3 Centromericky´tah................................... 17 1.5 Metody studia adaptivnı´evoluce............................. 17 Cı´l diplomove´pra´ce......................................................... 19 Kapitola 2. Materia´l a metody.............................................. 20 2.1 Izolace RNA a DNA, prˇı´prava cDNA.......................... 20 2.2 Amplifikace genu CenH3 .................................. 20 2.2.1 Na´vrh primeru˚a PCR................................. 20 2.2.2 Agaro´zova´elektrofore´za............................... 22 2.2.3 Optimalizace PCR................................... 22 2.3 Sekvenace a u´prava sekvencı´................................ 22 2.4 Kodonovy´alignment a konstrukce fylogeneticke´ho stromu........... 22 2.5 Selekcˇnı´analy´za........................................ 23 Kapitola 3. Vy´sledky....................................................... 24 Kapitola 4. Diskuze......................................................... 29 4.1 Zı´skane´sekvence a fylogeneticky´strom........................ 29 4.2 Adaptivnı´evoluce CenH3 .................................. 29 4.3 Centromericky´tah....................................... 30 4.4 Symetrie meio´zy........................................ 30 –– 4.5 Adaptivnı´role holocentricky´ch chromozomu˚..................... 31 Souhrn..................................................................... 33 Summary................................................................... 34 Seznam pouzˇite´literatury................................................... 35 Prˇı´lohy..................................................................... 41 Tabulka P1: Seznam zı´skany´ch sekvencı´........................... 41 Soubor P1: Skript pro maskova´nı´alignmentu........................ 42 Soubor P2: Maskovany´alignment zı´skany´ch sekvencı´.................. 43 Soubor P3: Fylogeneticky´strom zı´skany´ch sekvencı´................... 52 Kapitola 1 U´ vod 1.1 CenH3 Protein CenH3 (centromericka´varianta H3, u obratlovcu˚oznacˇovana´jako CENP-A) tvorˇı´ jaky´si ”za´kladnı´ka´men kinetochoru”. CenH3 je varianta histonu H3, ktera´se nacha´zı´ pouze v oblastech centromery (prˇedstavujejaky´si marker funkcˇnı´centromery) a narozdı´l od klasicke´ho H3 umozˇnˇuje vazbu dalsˇı´ch kinetochorovy´ch proteinu˚a sestavenı´kineto- choru v centromericke´oblasti (Van Hooser et al., 2001; Heun et al., 2006). Strukturneˇ je CenH3 velmi podobny´histonu H3 (obzvla´sˇt’v oblasti HFD - histone fold dome´ny na C-konci), lisˇı´se hlavneˇna N-konci, ktery´zajisˇt’uje interakci s DNA a dalsˇı´mi proteiny (naprˇ.kinetochorovy´mi) (Chen et al., 2000; Malik et al., 2002), a loop 1 oblastı´uvnitrˇ HFD, ktera´je typicky v CenH3 o neˇkolik nukleotidu˚delsˇı´nezˇv H3 a je zodpoveˇdna´za specifickou interakci s centromerickou DNA (Malik a Henikoff, 2003). CenH3 je take´specificky´tı´m, zˇe se v pru˚beˇhu evoluce oproti sve´mu kanonicke´mu proteˇjsˇku H3 velmi rychle vyvı´jı´, a to obzvla´sˇteˇv regionech zodpoveˇdny´ch za vazbu DNA. Takova´to rychla´evoluce proteinu naprosto nezbytne´ho pro spra´vny´pru˚beˇh buneˇcˇne´ho deˇlenı´je velmi nezvykla´, cozˇvede k u´vaze, zˇe adaptivnı´evoluce CenH3 je podmı´neˇna rychlou evolucı´ centromericky´ch sekvencı´ (Malik a Henikoff, 2003). Podle hypote´zy centromericke´ho tahu se CenH3 vyvı´jı´v reakci na negativnı´du˚sledky rozsˇı´rˇenı´sobecke´ centromery a tı´m se sta´va´u´cˇastnı´kem evolucˇnı´ch za´vodu˚ve zbrojenı´. 1.2 Centromericky´tah Podle Mendelovy´ch za´konu˚deˇdicˇnosti je soubor alel v gameta´ch du˚sledkem na´hodne´ho procesu segregace. Tato na´hodnost zajisˇt’uje, zˇe se konkre´tnı´alela vyskytuje v 1/2 vy- tvorˇeny´ch gamet. Meioticky´tah je proces, ktery´tuto rovnova´hu alel v gameta´ch porusˇuje (Sandler a Novitski, 1957). Prˇiasymetricke´samicˇı´meio´ze, ktera´je typicka´pro veˇtsˇinu zˇi- vocˇichu˚a vysˇsˇı´ch rostlin (Gorelick et