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Structural and Functional Analysis of the Cdk13/Cyclin K Complex

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Structural and Functional Analysis of the Cdk13/Cyclin K Complex MASARYK UNIVERSITY FACULTY OF SCIENCE The role of the C-terminal domain of RNA polymerase II in transcriptionally regulated process of genomic instability Ph.D. Dissertation Květa Pilařová Supervisor: Mgr. Dalibor Blažek, Ph.D. Department of biochemistry Brno 2020 Bibliografický záznam Autor: Mgr. Květa Pilařová Přírodovědecká fakulta, Masarykova univerzita Ústav biochemie Název práce: Úloha C-terminální domény RNA polymerasy II v transkripčně regulovaném procesu genomové nestability Studijní program: Biochemie Vedoucí práce: Mgr. Dalibor Blažek, Ph.D. Akademický rok: 2019/2020 Počet stran: 132 Klíčová slova: CDK12, CDK13, kinázová aktivita, analog-senzitivní kináza, C- terminální doména RNAPII, transkripce, genová exprese, přechod mezi G1/S, genomová nestabilita, nádorový biomarker Bibliographic entry Author: Mgr. Květa Pilařová Faculty of Science, Masaryk University Department of biochemistry Title of thesis: The role of the C-terminal domain of RNA polymerase II in transcriptionally regulated process of genomic instability Degree programme: Biochemistry Supervisor: Mgr. Dalibor Blažek, Ph.D. Academic year: 2019/2020 Number of pages: 132 Keywords: CDK12, CDK13, kinase activity, analogue-sensitive kinase, C- terminal domain of RNAPII, transcription, gene expression, G1/S progression, genome instability, tumour biomarker Abstrakt Transkripce protein-kódujících genů je řízena v eukaryotických buňkách RNA polymerázou II (RNAPII). Cyklin-dependentní kinázy 12 a 13 (CDK12 a CDK13) se řadí do skupiny transkripčních CDKs, které asociují s RNAPII při elongaci a fosforylují její C-terminální doménu (CTD). Obě kinázy působí také na expresi genů. Abnormální funkce těchto proteinů jsou u lidských buněk spojovány s různými typy onemocnění a v posledním desetiletí se proto staly předmětem studia výzkumů v oblasti medicíny. Jakým způsobem se CDK12 a CDK13 konkrétně podílí na regulaci transkripce a fosforylačním statusu CTD však není příliš známo. Substrátová specifita a struktura lidského komplexu CDK12 s cyklinem K (CycK) byla definována in vitro (Bösken et al., 2014). V první studii této dizertační práce jsme provedli strukturní a funkční analýzu proteinů, které tvoří lidský komplex CDK13/CycK. V rámci této studie jsme také charakterizovali společné a specifické funkce obou komplexů vázajících CycK. Zjistili jsme, že in vitro kinázová aktivita na CTD RNAPII je u obou z nich stejná. Velmi odlišné jsou však skupiny genů a související procesy, které jednotlivé kinázy regulují. Výsledky této studie ukázaly, že proteiny CDK12 a CDK13 mají specifické funkce a mohly by pomoci objasnit mechanismus, jakým jejich aberace přispívají ke vzniku konkrétních onemocnění. U CDK12 bylo prokázáno, že deplece tohoto proteinu ovlivňuje fosforylační status CTD a reguluje expresi specifických skupin genů, které zahrnují mimo jiné DNA-reparační geny (Bartkowiak et al., 2010; Blazek et al., 2011; Cheng et al., 2012; Liang et al., 2015; Tien et al., 2017; Dubbury et al, 2018). Nadále však chybí komplexnější analýza funkce CDK12 v buňkách včetně určení mechanismu, kterým tato kináza reguluje své cílové geny. Za použití buněčné linie exprimující analog-senzitivní formu CDK12 jsme v přiložené studii #2 provedli celogenomové analýzy po krátkodobé inhibici buněk kompetitivním analogem ATP. Zjistili jsme, že kinázová aktivita CDK12 je potřebná pro optimální transkripci genů kódujících základní DNA-replikační proteiny, a tudíž pro G1/S přechod v rámci buněčného cyklu. Inhibice CDK12 vyvolává snížení procesivity RNAPII, což způsobuje předčasnou terminaci některých genů, které se vyznačují především dlouhou délkou a vysokým podílem polyadenylačních signálů, včetně DNA reparačních a replikačních genů. Podařilo se nám tak ukázat nový způsob, jakým CDK12 propojuje regulaci transkripce a průběh buněčného cyklu. Ukazuje se, že CDK12 může být účinným prostředkem a prediktivním biomarkem protinádorové léčby. U pacientů s nádory vaječníků a prostaty s genetickou inaktivací CDK12 byl detekován specifický typ genomové nestability charakteristický výskytem fokálních tandemových duplikací (Popova et al., 2016; Wu et al., 2018). V přiloženém článku #3 jsme shrnuli současné poznatky o regulaci genové exprese proteinem CDK12. Posuzovali jsme také, jakým způsobem změna exprese CDK12-regulovaných genů ovlivňuje proces buněčného cyklu a možné příčiny vedoucí ke genomové nestabilitě u nádorů s mutací CDK12. Abstract In eukaryotic cells, transcription of protein-coding genes is directed by RNA polymerase II (RNAPII). Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13, respectively) belong to a group of transcriptional CDKs that associate with elongating RNAPII, phosphorylate its C-terminal domain (CTD) and affect gene expression. Their aberrations in human cells are associated with various diseases and the proteins have become attractive targets for medical research in the last decade. However, specific contribution of these kinases to regulation of transcription and CTD phosphorylation is still poorly understood. The structure and substrate specificity of human CDK12/Cyclin K (CycK) complex have been determined in vitro (Bösken et al., 2014). In the first study included in this work, we performed functional analyses of the human CDK13/CycK complex and we assessed the common and specific functions of both CycK-bound complexes. We demonstrated that their CTD kinase activities in vitro are the same. However, they regulate expression of a markedly different set of genes involved in dissimilar biological processes. This points to different functions of the proteins and may help to understand the mechanism by which their aberrations contribute to the onset of specific diseases. CDK12 depletion was shown to affect bulk CTD phosphorylation and regulate expression of a specific subset of genes, including DNA repair genes (Bartkowiak et al., 2010; Blazek et al., 2011; Cheng et al., 2012; Liang et al., 2015; Tien et al., 2017; Dubbury et al, 2018). The comprehensive insight into CDK12´s cellular functions and the mechanism by which CDK12 regulates expression of its target genes remain to be determined. In our study (publication #2), we performed genome-wide analyses using short-term inhibition with a competitive ATP analogue in CDK12 analogue-sensitive cell line. We found that CDK12 kinase activity is required for optimal transcription of core DNA replication genes and thus for G1/S progression. CDK12 inhibition causes an RNAPII processivity defect that leads to premature termination of predominantly long, polyadenylation-site-rich genes, including DNA replication and repair genes. Thus, we provide evidence that CDK12 represents a novel link between regulation of transcription and cell cycle progression. CDK12 has emerged as a promising anti-cancer target and CDK12 aberrations found in different types of cancers have the potential to be used as biomarkers for therapeutic intervention. CDK12 inactivation in prostate and ovarian tumours is associated with unique genome instability phenotype characterized by formation of focal tandem duplications (Popova et al., 2016; Wu et al., 2018). In a review attached at the end of this work we summarized mechanisms that CDK12 utilizes for the regulation of gene expression and discussed how the perturbation of CDK12-sensitive genes contributes to the disruption of cell cycle progression and the onset of genome instability that is observed in CDK12-mutated tumours. Acknowledgements My entire PhD studies including the work on this thesis were full of enriching experiences. There were many successes, as well as difficult challenges. I could never reach the end of this journey without the guidance from many teachers and scientists at the Masaryk University and help of other people. They did not hesitate to share their valuable experience, gave me a lot of their free time and provided important support. First and foremost, I would like to thank my supervisor, Dalibor, for giving me the opportunity to work on very interesting projects in a friendly, inspiring and professional environment of his lab. He showed me how much enthusiasm scientific research can involve and how to stay persistent and focused on the right scientific tracks. I am also very grateful that he was always trying to foster my critical and scientific thinking. I am deeply thankful to everyone in our lab. For countless meetings, collective lunches and coffee breaks during which they shared ideas and constructive criticism as well as for the good times outside of the lab. For their constant willingness to listen to my problems of any kind and helping me to overcome them. My special thanks go to Pavla who was always available for giving me advice in the lab and has also become a very good friend of mine. Thank you, Pavla, for all your help with my experiments and for all the life advice. I am grateful to all the people I met during my work, especially at CEITEC, and to all my collaborators from the labs of Matthias Geyer, Caroline Friedel, Lumir Krejci and Kamil Paruch. None of this would have been possible without the unconditional love and support of my family. I would like to thank my parents who have always supported me in whatever I decided to do. Thanks to them I have learnt to believe in myself. To my sisters, for the long calls and many visits in Brno full of empowering talks. I thank all of them for always showing interest in my work. Finally, I would like to thank my boyfriend Vojta for believing that my studies are going to finish one day. For showing me how to stay positive in any situation
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