CDK12 is a kinase associated with elongating RNA polymerase II (RNAPII) and is frequently mutated in cancer. CDK12 depletion reduces the expression of homologous recombination (HR) DNA repair genes, but comprehensive insight into its target genes and cellular processes is lacking. We use a chemical genetic approach to inhibit analog-sensitive CDK12, and find that CDK12 kinase activity is required for transcription of core DNA replication genes and thus for G1/S progression. RNA-seq and ChIP-seq reveal that CDK12 inhibition triggers an RNAPII processivity defect characterized by a loss of mapped reads from 3'ends of predominantly long, poly(A)-signal-rich genes. CDK12 inhibition does not globally reduce levels of RNAPII-Ser2 phosphorylation. However, individual CDK12-dependent genes show a shift of P-Ser2 peaks into the gene body approximately to the positions where RNAPII occupancy and transcription were lost. Thus, CDK12 catalytic activity represents a novel link between regulation of transcription and cell cycle progression. We propose that DNA replication and HR DNA repair defects as a consequence of CDK12 inactivation underlie the genome instability phenotype observed in many cancers.

Center of Biomolecular and Cellular Engineering International Clinical Research Center St Anne's University Hospital Brno Czech Republic

Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Biology Masaryk University Brno Czech Republic

Department of Chemistry CZ Openscreen Faculty of Science Masaryk University Brno Czech Republic

Institut für Informatik Ludwig Maximilians Universität München München Germany

National Centre for Biomolecular Research Masaryk University Brno Czech Republic

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Fuda NJ, Ardehali MB, Lis JT (2009) Defining mechanisms that regulate RNA polymerase II transcription in vivo . Nature 461: 186–192 PubMed PMC

Harlen KM, Churchman LS (2017) The code and beyond: transcription regulation by the RNA polymerase II carboxy‐terminal domain. Nat Rev Mol Cell Biol 18: 263–273 PubMed

Adelman K, Lis JT (2012) Promoter‐proximal pausing of RNA polymerase II: emerging roles in metazoans. Nat Rev Genet 13: 720–731 PubMed PMC

Buratowski S (2003) The CTD code. Nat Struct Biol 10: 679–680 PubMed

Egloff S, Murphy S (2008) Cracking the RNA polymerase II CTD code. Trends Genet 24: 280–288 PubMed

Eick D, Geyer M (2013) The RNA polymerase II carboxy‐terminal domain (CTD) code. Chem Rev 113: 8456–8490 PubMed

Zaborowska J, Egloff S, Murphy S (2016) The pol II CTD: new twists in the tail. Nat Struct Mol Biol 23: 771–777 PubMed

Peterlin BM, Price DH (2006) Controlling the elongation phase of transcription with P‐TEFb. Mol Cell 23: 297–305 PubMed

Larochelle S, Amat R, Glover‐Cutter K, Sanso M, Zhang C, Allen JJ, Shokat KM, Bentley DL, Fisher RP (2012) Cyclin‐dependent kinase control of the initiation‐to‐elongation switch of RNA polymerase II. Nat Struct Mol Biol 19: 1108–1115 PubMed PMC

Drogat J, Hermand D (2012) Gene‐specific requirement of RNA polymerase II CTD phosphorylation. Mol Microbiol 84: 995–1004 PubMed

Blazek D, Kohoutek J, Bartholomeeusen K, Johansen E, Hulinkova P, Luo Z, 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 Dev 25: 2158–2172 PubMed PMC

Bartkowiak B, Liu P, Phatnani HP, Fuda NJ, Cooper JJ, Price DH, Adelman K, Lis JT, Greenleaf AL (2010) CDK12 is a transcription elongation‐associated CTD kinase, the metazoan ortholog of yeast Ctk1. Genes Dev 24: 2303–2316 PubMed PMC

Bosken CA, Farnung L, Hintermair C, Merzel Schachter M, Vogel‐Bachmayr K, Blazek D, Anand K, Fisher RP, Eick D, Geyer M (2014) The structure and substrate specificity of human Cdk12/Cyclin K. Nat Commun 5: 3505 PubMed PMC

Cheng SW, Kuzyk MA, Moradian A, Ichu TA, Chang VC, Tien JF, Vollett SE, Griffith M, Marra MA, Morin GB (2012) Interaction of cyclin‐dependent kinase 12/CrkRS with cyclin K1 is required for the phosphorylation of the C‐terminal domain of RNA polymerase II. Mol Cell Biol 32: 4691–4704 PubMed PMC

Yu M, Yang W, Ni T, Tang Z, Nakadai T, Zhu J, Roeder RG (2015) RNA polymerase II‐associated factor 1 regulates the release and phosphorylation of paused RNA polymerase II. Science 350: 1383–1386 PubMed PMC

Greifenberg AK, Honig D, Pilarova K, Duster R, Bartholomeeusen K, Bosken CA, Anand K, Blazek D, Geyer M (2016) Structural and functional analysis of the Cdk13/Cyclin K complex. Cell Rep 14: 320–331 PubMed

Zhang T, Kwiatkowski N, Olson CM, Dixon‐Clarke SE, Abraham BJ, Greifenberg AK, Ficarro SB, Elkins JM, Liang Y, Hannett NM et al (2016) Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors. Nat Chem Biol 12: 876–884 PubMed PMC

Davidson L, Muniz L, West S (2014) 3′ end formation of pre‐mRNA and phosphorylation of Ser2 on the RNA polymerase II CTD are reciprocally coupled in human cells. Genes Dev 28: 342–356 PubMed PMC

Edwards MC, Wong C, Elledge SJ (1998) Human cyclin K, a novel RNA polymerase II‐associated cyclin possessing both carboxy‐terminal domain kinase and Cdk‐activating kinase activity. Mol Cell Biol 18: 4291–4300 PubMed PMC

Kohoutek J, Blazek D (2012) Cyclin K goes with Cdk12 and Cdk13. Cell Div 7: 12 PubMed PMC

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 Res 43: 2575–2589 PubMed PMC

Juan HC, Lin Y, Chen HR, Fann MJ (2016) Cdk12 is essential for embryonic development and the maintenance of genomic stability. Cell Death Differ 23: 1038–1048 PubMed PMC

Liang K, Gao X, Gilmore JM, Florens L, Washburn MP, Smith E, Shilatifard A (2015) Characterization of human cyclin‐dependent kinase 12 (CDK12) and CDK13 complexes in C‐terminal domain phosphorylation, gene transcription, and RNA processing. Mol Cell Biol 35: 928–938 PubMed PMC

Hoshii T, Cifani P, Feng Z, Huang CH, Koche R, Chen CW, Delaney CD, Lowe SW, Kentsis A, Armstrong SA (2018) A non‐catalytic function of SETD1A regulates cyclin K and the DNA damage response. Cell 172: 1007–1021 e17 PubMed PMC

Eifler TT, Shao W, Bartholomeeusen K, Fujinaga K, Jager S, Johnson J, Luo Z, Krogan N, Peterlin BM (2014) CDK12 increases 3′ end processing of growth factor‐induced c‐FOS transcripts. Mol Cell Biol 35: 468–478 PubMed PMC

Ko TK, Kelly E, Pines J (2001) CrkRS: a novel conserved Cdc2‐related protein kinase that colocalises with SC35 speckles. J Cell Sci 114: 2591–2603 PubMed

Chen HH, Wang YC, Fann MJ (2006) Identification and characterization of the CDK12/cyclin L1 complex involved in alternative splicing regulation. Mol Cell Biol 26: 2736–2745 PubMed PMC

Tien JF, Mazloomian A, Cheng SG, Hughes CS, Chow CCT, Canapi LT, Oloumi A, Trigo‐Gonzalez G, Bashashati A, Xu J et al (2017) CDK12 regulates alternative last exon mRNA splicing and promotes breast cancer cell invasion. Nucleic Acids Res 45: 6698–6716 PubMed PMC

Popova T, Manie E, Boeva V, Battistella A, Goundiam O, Smith NK, Mueller CR, Raynal V, Mariani O, Sastre‐Garau X et al (2016) Ovarian cancers harboring inactivating mutations in CDK12 display a distinct genomic instability pattern characterized by large tandem duplications. Can Res 76: 1882–1891 PubMed

Wu YM, Cieslik M, Lonigro RJ, Vats P, Reimers MA, Cao X, Ning Y, Wang L, Kunju LP, de Sarkar N et al (2018) Inactivation of CDK12 delineates a distinct immunogenic class of advanced prostate cancer. Cell 173: 1770–1782 e14 PubMed PMC

Menghi F, Barthel FP, Yadav V, Tang M, Ji B, Tang Z, Carter GW, Ruan Y, Scully R, Verhaak RGW et al (2018) The tandem duplicator phenotype is a prevalent genome‐wide cancer configuration driven by distinct gene mutations. Cancer Cell 34: 197–210 e5 PubMed PMC

Rao M, Powers S (2018) Tandem duplications may supply the missing genetic alterations in many triple‐negative breast and gynecological cancers. Cancer Cell 34: 179–180 PubMed

Menghi F, Inaki K, Woo X, Kumar PA, Grzeda KR, Malhotra A, Yadav V, Kim H, Marquez EJ, Ucar D et al (2016) The tandem duplicator phenotype as a distinct genomic configuration in cancer. Proc Natl Acad Sci USA 113: E2373–E2382 PubMed PMC

Johnson SF, Cruz C, Greifenberg AK, Dust S, Stover DG, Chi D, Primack B, Cao S, Bernhardy AJ, Coulson R et al (2016) CDK12 inhibition reverses de novo and acquired PARP inhibitor resistance in BRCA wild‐type and mutated models of triple‐negative breast cancer. Cell Rep 17: 2367–2381 PubMed PMC

Joshi PM, Sutor SL, Huntoon CJ, Karnitz LM (2014) Ovarian cancer‐associated mutations disable catalytic activity of CDK12, a kinase that promotes homologous recombination repair and resistance to cisplatin and poly(ADP‐ribose) polymerase inhibitors. J Biol Chem 289: 9247–9253 PubMed PMC

Bajrami I, Frankum JR, Konde A, Miller RE, Rehman FL, Brough R, Campbell J, Sims D, Rafiq R, Hooper S et al (2014) Genome‐wide profiling of genetic synthetic lethality identifies CDK12 as a novel determinant of PARP1/2 inhibitor sensitivity. Can Res 74: 287–297 PubMed PMC

Iniguez AB, Stolte B, Wang EJ, Conway AS, Alexe G, Dharia NV, Kwiatkowski N, Zhang T, Abraham BJ, Mora J et al (2018) EWS/FLI confers tumor cell synthetic lethality to CDK12 inhibition in ewing sarcoma. Cancer Cell 33: 202–216 e6 PubMed PMC

Malumbres M, Barbacid M (2009) Cell cycle, CDKs and cancer: a changing paradigm. Nat Rev Cancer 9: 153–166 PubMed

Bertoli C, Skotheim JM, de Bruin RA (2013) Control of cell cycle transcription during G1 and S phases. Nat Rev Mol Cell Biol 14: 518–528 PubMed PMC

Bracken AP, Ciro M, Cocito A, Helin K (2004) E2F target genes: unraveling the biology. Trends Biochem Sci 29: 409–417 PubMed

Tatsumi Y, Sugimoto N, Yugawa T, Narisawa‐Saito M, Kiyono T, Fujita M (2006) Deregulation of Cdt1 induces chromosomal damage without rereplication and leads to chromosomal instability. J Cell Sci 119: 3128–3140 PubMed

Liontos M, Koutsami M, Sideridou M, Evangelou K, Kletsas D, Levy B, Kotsinas A, Nahum O, Zoumpourlis V, Kouloukoussa M et al (2007) Deregulated overexpression of hCdt1 and hCdc6 promotes malignant behavior. Can Res 67: 10899–10909 PubMed

Tsantoulis PK, Gorgoulis VG (2005) Involvement of E2F transcription factor family in cancer. Eur J Cancer 41: 2403–2414 PubMed

Baxley RM, Bielinsky AK (2017) Mcm10: a dynamic scaffold at eukaryotic replication forks. Genes (Basel) 8: E73 PubMed PMC

Lopez MS, Kliegman JI, Shokat KM (2014) The logic and design of analog‐sensitive kinases and their small molecule inhibitors. Methods Enzymol 548: 189–213 PubMed

Larochelle S, Batliner J, Gamble MJ, Barboza NM, Kraybill BC, Blethrow JD, Shokat KM, Fisher RP (2006) Dichotomous but stringent substrate selection by the dual‐function Cdk7 complex revealed by chemical genetics. Nat Struct Mol Biol 13: 55–62 PubMed

Galbraith MD, Andrysik Z, Pandey A, Hoh M, Bonner EA, Hill AA, Sullivan KD, Espinosa JM (2017) CDK8 kinase activity promotes glycolysis. Cell Rep 21: 1495–1506 PubMed PMC

Bartkowiak B, Yan C, Greenleaf AL (2015) Engineering an analog‐sensitive CDK12 cell line using CRISPR/Cas. Biochem Biophys Acta 1849: 1179–1187 PubMed PMC

Blazek D (2012) The cyclin K/Cdk12 complex: an emerging new player in the maintenance of genome stability. Cell Cycle 11: 1049–1050 PubMed PMC

Shiloh Y, Ziv Y (2013) The ATM protein kinase: regulating the cellular response to genotoxic stress, and more. Nat Rev Mol Cell Biol 14: 197–210 PubMed

Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES et al (2005) Gene set enrichment analysis: a knowledge‐based approach for interpreting genome‐wide expression profiles. Proc Natl Acad Sci USA 102: 15545–15550 PubMed PMC

Fragkos M, Ganier O, Coulombe P, Mechali M (2015) DNA replication origin activation in space and time. Nat Rev Mol Cell Biol 16: 360–374 PubMed

Beltran M, Yates CM, Skalska L, Dawson M, Reis FP, Viiri K, Fisher CL, Sibley CR, Foster BM, Bartke T et al (2016) The interaction of PRC2 with RNA or chromatin is mutually antagonistic. Genome Res 26: 896–907 PubMed PMC

Jackson SP, Bartek J (2009) The DNA‐damage response in human biology and disease. Nature 461: 1071–1078 PubMed PMC

Rahl PB, Lin CY, Seila AC, Flynn RA, McCuine S, Burge CB, Sharp PA, Young RA (2010) c‐Myc regulates transcriptional pause release. Cell 141: 432–445 PubMed PMC

Sanso M, Fisher RP (2013) Pause, play, repeat: CDKs push RNAP II's buttons. Transcription 4: 146–152 PubMed PMC

Ardehali MB, Yao J, Adelman K, Fuda NJ, Petesch SJ, Webb WW, Lis JT (2009) Spt6 enhances the elongation rate of RNA polymerase II in vivo . EMBO J 28: 1067–1077 PubMed PMC

Vos SM, Farnung L, Boehning M, Wigge C, Linden A, Urlaub H, Cramer P (2018) Structure of activated transcription complex Pol II‐DSIF‐PAF‐SPT6. Nature 560: 607–612 PubMed

Yoh SM, Cho H, Pickle L, Evans RM, Jones KA (2007) The Spt6 SH2 domain binds Ser2‐P RNAPII to direct Iws1‐dependent mRNA splicing and export. Genes Dev 21: 160–174 PubMed PMC

Anders S, Reyes A, Huber W (2012) Detecting differential usage of exons from RNA‐seq data. Genome Res 22: 2008–2017 PubMed PMC

Gu B, Eick D, Bensaude O (2013) CTD serine‐2 plays a critical role in splicing and termination factor recruitment to RNA polymerase II in vivo . Nucleic Acids Res 41: 1591–1603 PubMed PMC

Herzel L, Ottoz DSM, Alpert T, Neugebauer KM (2017) Splicing and transcription touch base: co‐transcriptional spliceosome assembly and function. Nat Rev Mol Cell Biol 18: 637–650 PubMed PMC

Dubbury SJ, Boutz PL, Sharp PA (2018) CDK12 regulates DNA repair genes by suppressing intronic polyadenylation. Nature 564: 141–145 PubMed PMC

Singh J, Padgett RA (2009) Rates of in situ transcription and splicing in large human genes. Nat Struct Mol Biol 16: 1128–1133 PubMed PMC

Fitz J, Neumann T, Pavri R (2018) Regulation of RNA polymerase II processivity by Spt5 is restricted to a narrow window during elongation. EMBO J 37: e97965 PubMed PMC

Jonkers I, Kwak H, Lis JT (2014) Genome‐wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons. Elife 3: e02407 PubMed PMC

Branzei D, Szakal B (2017) Building up and breaking down: mechanisms controlling recombination during replication. Crit Rev Biochem Mol Biol 52: 381–394 PubMed

Gaillard H, Garcia‐Muse T, Aguilera A (2015) Replication stress and cancer. Nat Rev Cancer 15: 276–289 PubMed

Zeman MK, Cimprich KA (2014) Causes and consequences of replication stress. Nat Cell Biol 16: 2–9 PubMed PMC

Choi SH, Martinez TF, Kim S, Donaldson C, Shokhirev MN, Saghatelian A, Jones KA (2019) CDK12 phosphorylates 4E‐BP1 to enable mTORC1‐dependent translation and mitotic genome stability. Genes Dev 33: 418–435 PubMed PMC

Paculova H, Kramara J, Simeckova S, Fedr R, Soucek K, Hylse O, Paruch K, Svoboda M, Mistrik M, Kohoutek J (2017) BRCA1 or CDK12 loss sensitizes cells to CHK1 inhibitors. Tumour Biol 39: 1010428317727479 PubMed

Lei T, Zhang P, Zhang X, Xiao X, Zhang J, Qiu T, Dai Q, Zhang Y, Min L, Li Q et al (2018) Cyclin K regulates prereplicative complex assembly to promote mammalian cell proliferation. Nat Commun 9: 1876 PubMed PMC

Qiu H, Hu C, Hinnebusch AG (2009) Phosphorylation of the Pol II CTD by KIN28 enhances BUR1/BUR2 recruitment and Ser2 CTD phosphorylation near promoters. Mol Cell 33: 752–762 PubMed PMC

Devaiah BN, Lewis BA, Cherman N, Hewitt MC, Albrecht BK, Robey PG, Ozato K, Sims RJ III, Singer DS (2012) BRD4 is an atypical kinase that phosphorylates serine2 of the RNA polymerase II carboxy‐terminal domain. Proc Natl Acad Sci USA 109: 6927–6932 PubMed PMC

Booth GT, Parua PK, Sanso M, Fisher RP, Lis JT (2018) Cdk9 regulates a promoter‐proximal checkpoint to modulate RNA polymerase II elongation rate in fission yeast. Nat Commun 9: 543 PubMed PMC

Egloff S, O'Reilly D, Chapman RD, Taylor A, Tanzhaus K, Pitts L, Eick D, Murphy S (2007) Serine‐7 of the RNA polymerase II CTD is specifically required for snRNA gene expression. Science 318: 1777–1779 PubMed PMC

Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR‐Cas9 system. Nat Protoc 8: 2281–2308 PubMed PMC

Gratzner HG (1982) Monoclonal antibody to 5‐bromo‐ and 5‐iododeoxyuridine: a new reagent for detection of DNA replication. Science 218: 474–475 PubMed

Schlacher K, Christ N, Siaud N, Egashira A, Wu H, Jasin M (2011) Double‐strand break repair‐independent role for BRCA2 in blocking stalled replication fork degradation by MRE11. Cell 145: 529–542 PubMed PMC

Bonfert T, Kirner E, Csaba G, Zimmer R, Friedel CC (2015) ContextMap 2: fast and accurate context‐based RNA‐seq mapping. BMC Bioinformatics 16: 122 PubMed PMC

Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows‐Wheeler transform. Bioinformatics 25: 1754–1760 PubMed PMC

Liao Y, Smyth GK, Shi W (2014) featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30: 923–930 PubMed

Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA‐seq data with DESeq2. Genome Biol 15: 550 PubMed PMC

Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate – a practical and powerful approach to multiple testing. J R Stat Soc Series B Stat Methodol 57: 289–300

Eden E, Navon R, Steinfeld I, Lipson D, Yakhini Z (2009) GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists. BMC Bioinformatics 10: 48 PubMed PMC

Kluge M, Friedel CC (2018) Watchdog – a workflow management system for the distributed analysis of large‐scale experimental data. BMC Bioinformatics 19: 97 PubMed PMC

Gruber AR, Martin G, Keller W, Zavolan M (2014) Means to an end: mechanisms of alternative polyadenylation of messenger RNA precursors. Wiley Interdiscip Rev RNA 5: 183–196 PubMed PMC

Quinlan AR, Hall IM (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26: 841–842 PubMed PMC

Hahne F, Ivanek R (2016) Visualizing genomic data using gviz and bioconductor. Methods Mol Biol 1418: 335–351 PubMed

R Core Team (2016) R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing;

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