Inherited Diseases - Transtricptional Regulation - Dalibor Blažek

Research areas

• Regulation of eukaryotic transcription
• Role of Cdk9, Cdk12 and Cdk13 in regulation of gene expression
• Role of transcription cycle-related Cdks in maintenance of genome stability
• Transcription cycle-related Cdks in human disease

Main objectives

• Role of Cdks in phosphorylation of the C-terminal domain of RNA polymerase II and in the regulation of gene expression
• Control of DNA damage response and genome stability via regulation of expression of DNA damage response genes

Content of research

RNA polymerase II (RNAPII) directs transcription of protein coding genes and this process consists of several stages including preinitiation complex formation, productive elongation and termination. This transcription cycle is tightly linked to co-transcriptional maturation of nascent transcripts including pre-mRNA splicing and polyadenylation. RNAPII contains an unstructured C-terminal domain (CTD) with repeats of evolutionarily conserved heptapeptide YSPTSPS, where individual serines get phosphorylated. Several cyclin-dependent kinases (Cdks) regulate the phosphorylation status of the CTD and subsequent binding of transcription and pre-mRNA processing factors. Thus, the patterns of phosphorylation of the CTD direct actions of RNAPII during transcriptional cycle and co-transcriptional processing of nascent transcripts. Moreover, CTD was also functionally linked to DNA damage response and maintenance of genome stability via regulation of transcription, mRNA processing and recombination. Thus, CTD and its posttranslational modifications, associated proteins and modifying enzymes are emerging as new players in cellular response to DNA damage. Our recent work led to the identification of the Cyclin K/Cdk12 complex that phosphorylates serine 2 in the CTD of RNAPII and directs expression of several crucial DNA damage response genes including BRCA1, ATR or FANCI. In my lab we apply a combination of biochemical, proteomics and genome-wide techniques to determine the molecular mechanism that regulates the expression of Cdk12-dependent genes with a focus on DNA damage response genes. The ultimate goal of our research is to uncover how the CycK/Cdk12 complex and the CTD of RNAPII contribute to the maintenance of genome stability, and how disruption of their functions lead to the onset of a malignant state.

list / cards

Shared laboratory: A35/111, phone: 549 49 6649

SELECTED PUBLICATIONS

2017

• GRODECKA, L; KOVACOVA, T; KRAMAREK, M; SENECA, S; STOUFFS, K; DE LAET, C; MAJER, F; KRSJAKOVA, T; HUJOVA, P; HRNCIROVA, K; SOUCEK, P; LISSENS, W; BURATTI, E; FREIBERGER, T, 2017:Detailed molecular characterization of a novel IDS exonic mutation associated with multiple pseudoexon activation. JOURNAL OF MOLECULAR MEDICINE-JMM 95 (3), p. 299 - 309.
• GROCHALOVA, M; KONECNA, H; STEJSKAL, K; POTESIL, D; FRIDRICHOVA, D; SRBOVA, E; ORNEROVA, K; ZDRAHAL, Z, 2017:Deep coverage of the beer proteome. JOURNAL OF PROTEOMICS 162 , p. 119 - 124.

2016

• BLAZEK, D, 2016:TRANSCRIPTIONAL KINASES Caught by a sticky drug. NATURE CHEMICAL BIOLOGY 12 (10), p. 765 - 766.

2015

• EKUMI, KM; PACULOVA, H; LENASI, T; POSPICHALOVA, V; BOSKEN, CA; RYBARIKOVA, J; BRYJA, V; GEYER, M; BLAZEK, D; BARBORIC, M, 2015:Ovarian carcinoma CDK12 mutations misregulate expression of DNA repair genes via deficient formation and function of the Cdk12/CycK complex. NUCLEIC ACIDS RESEARCH 43 (5), p. 2575 - 2589.
• EKUMI, KM; PACULOVA, H; POSPICHALOVA, V; BOSKEN, CA; BRYJA, V; GEYER, M; BLAZEK, D; BARBORIC, M, 2015:Ovarian carcinoma CDK12 mutations misregulate DNA repair genes via deficient formation and function of the Cdk12/CycK complex. CLINICAL CANCER RESEARCH 21
• JANOUSKOVA, E; NECASOVA, I; PAVLOUSKOVA, J; ZIMMERMANN, M; HLUCHY, M; MARINI, V; NOVAKOVA, M; HOFR, C, 2015:Human Rap1 modulates TRF2 attraction to telomeric DNA. NUCLEIC ACIDS RESEARCH 43 (5), p. 2691 - 2700.

2014

• BOSKEN, CA; FARNUNG, L; HINTERMAIR, C; SCHACHTER, MM; VOGEL-BACHMAYR, K; BLAZEK, D; ANAND, K; FISHER, RP; EICK, D; GEYER, M, 2014:The structure and substrate specificity of human Cdk12/Cyclin K. NATURE COMMUNICATIONS 5
• SMERDOVA, L; SVOBODOVA, J; KABATKOVA, M; KOHOUTEK, J; BLAZEK, D; MACHALA, M; VONDRACEK, J, 2014:Upregulation of CYP1B1 expression by inflammatory cytokines is mediated by the p38 MAP kinase signal transduction pathway. CARCINOGENESIS 35 (11), p. 2534 - 2543.

2011

• BLAZEK, D; KOHOUTEK, J; BARTHOLOMEEUSEN, K; JOHANSEN, E; HULINKOVA, P; LUO, ZP; CIMERMANCIC, P; ULE, J; PETERLIN, BM, 2011:The Cyclin K/Cdk12 complex maintains genomic stability via regulation of expression of DNA damage response genes. GENES & DEVELOPMENT 25 (20), p. 2158 - 2172.

GRANTY

• The roles of Cdk12 in ovarian tumorigenesis (MFR2013), Foundations - International - Marsha Rivkin Center, 2013 - 2014
• The novel isoform of cyclin K is a partner of cdk12 kinase regulating eukaryotic transcription and alternative splicing (GAP305/11/1564), Czech Science Foundation - Standard Grants, 2011 - 2013
• Novel methylation of Hexim1 and cyclin composition of positive trancription elongation factor b (P-TEFb) regulate kinase activity of cdk9 (ME09047), MEYS - KONTAKT, 2009 - 2013
• Regulation and function of P-TEFb complexes (SRGA 454), South Moravian Region - SoMoPro, 2010 - 2013

CURRENT RESEARCH INFRASTRUCTURE

Laboratories equipped with technologies used in molecular biological analysis: RealTime PCR cyclers, PCR cyclers, laminar boxes, CO2 boxes.

1. Characterization of Cdk12 and Cdk13 kinase substrates and their role in development of cancer

Supervisor: Mgr. Dalibor Blažek, Ph.D.
Consultant: doc. RNDr. Zbyněk Zdráhal, Dr.

Annotation

Cdk12 and Cdk13 are transcriptional cyclin-dependent kinases (Cdk) found mutated in various cancers. In previous studies we found that Cdk12 maintains genome stability via optimal transcription of key homologous recombination repair pathway genes including BRCA1. Cellular function of Cdk13 remains unclear. Apart from the C-terminal domain of RNA Polymerase II other cellular substrates for both kinases are not known. In this research we propose using a screen in cells carrying an analog sensitive mutants of both kinases to discover their novel cellular substrates. The substrates and their roles in normal and cancerous cells will be characterized by various techniques of molecular biology and biochemistry.

Keywords: cyclin-dependent kinases, Cdk12, Cdk13, recombination repair pathway genes, BRCA1, cancer

2. Role of Cdk12 in regulation of gene expression/DNA damage response in ovarian cancer

Supervisor: Mgr. Dalibor Blažek, Ph.D.
Consultant: doc. RNDr. Zbyněk Zdráhal, Dr.

Annotation

Gene expression of protein-coding genes is regulated at many levels. This project focuses on transcription cycle-related cyclin-dependent kinase 12 (Cdk12). We showed Cdk12 to be involved in the regulation of gene expression of key DNA damage response genes (BRCA1, FANCI, ATR) via phosphorylation of the C-terminal domain of RNA Polymerase II (see our recent papers: Blazek et al., Genes and Development 2011; 25(20):2158-72; Bosken et al. Nature Comm. 2014;24;5:3505; Ekumi at al., Nucleic Acids Research 2015;11;43(5):2575-89). Also, Cdk12 was found among the most often mutated genes in ovarian carcinoma (Ovarian Carcinoma Cancer Genome Atlas, Nature, 2011). In accordance with the proposed broad role of the Cdk12 in the regulation of gene expression/DNA damage response is its deregulation in many other cancers. However, mechanism how Cdk12 contributes to the regulation of gene expression is unknown. The main aim of the project will be deciphering of molecular mechanism that mediates expression of Cdk12-dependent genes. To address this question many techniques of molecular biology and biochemistry will be applied including state of the art mass spectrometry and next generation sequencing (ChIP-seq etc.).Ovarian carcinoma cell lines will be used as a model for the studies.

Keywords: cyclin-dependent kinase 12, Cdk12, regulation of gene expression, ovarian carcinoma, DNA damage response, mass spektrometry, next generation sequencing