CDK12 controls G1/S progression by regulating RNAPII processivity at core DNA replication genes
2019; Springer Nature; Volume: 20; Issue: 9 Linguagem: Inglês
10.15252/embr.201847592
ISSN1469-3178
AutoresAnil Paul Chirackal Manavalan, Květa Pilařová, Michael Kluge, Koen Bartholomeeusen, Michal Rájecký, Jan Oppelt, Prashant Khirsariya, Kamil Paruch, Lumír Krejčí, Caroline C. Friedel, Dalibor Blažek,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoArticle25 July 2019Open Access Source DataTransparent process CDK12 controls G1/S progression by regulating RNAPII processivity at core DNA replication genes Anil Paul Chirackal Manavalan Anil Paul Chirackal Manavalan orcid.org/0000-0002-2113-0715 Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic Search for more papers by this author Kveta Pilarova Kveta Pilarova Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic Search for more papers by this author Michael Kluge Michael Kluge Institut für Informatik, Ludwig-Maximilians-Universität München, München, Germany Search for more papers by this author Koen Bartholomeeusen Koen Bartholomeeusen Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic Search for more papers by this author Michal Rajecky Michal Rajecky Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic Search for more papers by this author Jan Oppelt Jan Oppelt orcid.org/0000-0002-3076-4840 Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic Search for more papers by this author Prashant Khirsariya Prashant Khirsariya Department of Chemistry, CZ Openscreen, Faculty of Science, Masaryk University, Brno, Czech Republic Center of Biomolecular and Cellular Engineering, International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic Search for more papers by this author Kamil Paruch Kamil Paruch Department of Chemistry, CZ Openscreen, Faculty of Science, Masaryk University, Brno, Czech Republic Center of Biomolecular and Cellular Engineering, International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic Search for more papers by this author Lumir Krejci Lumir Krejci Center of Biomolecular and Cellular Engineering, International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic Department of Biology, Masaryk University, Brno, Czech Republic National Centre for Biomolecular Research, Masaryk University, Brno, Czech Republic Search for more papers by this author Caroline C Friedel Caroline C Friedel Institut für Informatik, Ludwig-Maximilians-Universität München, München, Germany Search for more papers by this author Dalibor Blazek Corresponding Author Dalibor Blazek [email protected] orcid.org/0000-0003-4662-9982 Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic Search for more papers by this author Anil Paul Chirackal Manavalan Anil Paul Chirackal Manavalan orcid.org/0000-0002-2113-0715 Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic Search for more papers by this author Kveta Pilarova Kveta Pilarova Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic Search for more papers by this author Michael Kluge Michael Kluge Institut für Informatik, Ludwig-Maximilians-Universität München, München, Germany Search for more papers by this author Koen Bartholomeeusen Koen Bartholomeeusen Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic Search for more papers by this author Michal Rajecky Michal Rajecky Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic Search for more papers by this author Jan Oppelt Jan Oppelt orcid.org/0000-0002-3076-4840 Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic Search for more papers by this author Prashant Khirsariya Prashant Khirsariya Department of Chemistry, CZ Openscreen, Faculty of Science, Masaryk University, Brno, Czech Republic Center of Biomolecular and Cellular Engineering, International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic Search for more papers by this author Kamil Paruch Kamil Paruch Department of Chemistry, CZ Openscreen, Faculty of Science, Masaryk University, Brno, Czech Republic Center of Biomolecular and Cellular Engineering, International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic Search for more papers by this author Lumir Krejci Lumir Krejci Center of Biomolecular and Cellular Engineering, International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic Department of Biology, Masaryk University, Brno, Czech Republic National Centre for Biomolecular Research, Masaryk University, Brno, Czech Republic Search for more papers by this author Caroline C Friedel Caroline C Friedel Institut für Informatik, Ludwig-Maximilians-Universität München, München, Germany Search for more papers by this author Dalibor Blazek Corresponding Author Dalibor Blazek [email protected] orcid.org/0000-0003-4662-9982 Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic Search for more papers by this author Author Information Anil Paul Chirackal Manavalan1, Kveta Pilarova1, Michael Kluge2, Koen Bartholomeeusen1,7, Michal Rajecky1, Jan Oppelt1, Prashant Khirsariya3,4, Kamil Paruch3,4, Lumir Krejci4,5,6, Caroline C Friedel2 and Dalibor Blazek *,1 1Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic 2Institut für Informatik, Ludwig-Maximilians-Universität München, München, Germany 3Department of Chemistry, CZ Openscreen, Faculty of Science, Masaryk University, Brno, Czech Republic 4Center of Biomolecular and Cellular Engineering, International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic 5Department of Biology, Masaryk University, Brno, Czech Republic 6National Centre for Biomolecular Research, Masaryk University, Brno, Czech Republic 7Present address: Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium *Corresponding author. Tel: +420 730 588 450; E-mail: [email protected] EMBO Reports (2019)20:e47592https://doi.org/10.15252/embr.201847592 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract 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. Synopsis CDK12-dependent RNAPII processivity on core DNA replication genes is a rate limiting factor for G1/S progression. CDK12 inhibition leads to premature termination and transcript shortening of a subset of genes. CDK12 kinase does not globally control RNAPII Ser2 phosphorylation on transcription units. CDK12 kinase is crucial for RNAPII processivity on a subset of long and poly(A)-signal-rich genes, particularly those involved in DNA replication and DNA damage response. DK12 inhibition results in loss of RNAPII occupancy and transcription from the gene 3′ends, which coincides with a shift of RNAPII phosphorylated at Ser2 into the gene body. Introduction Transcription of protein-coding genes is mediated by RNA polymerase II (RNAPII) and represents an important regulatory step of many cellular processes. RNAPII directs gene transcription in several phases, including initiation, elongation, and termination 1-3. The C-terminal domain (CTD) of RNAPII contains repeats of the heptapeptide YSPTSPS, and phosphorylation of the individual serines within these repeats is necessary for individual steps of the transcription cycle 4, 5. Phosphorylation of RNAPII Ser2 is a hallmark of transcription elongation, whereas phosphorylation of Ser5 correlates with initiating RNAPII 1, 6. Various kinases have been implicated in CTD phosphorylation 7-10, and the kinase CDK12 is thought to phosphorylate predominantly Ser2 11-18. These findings were based on the use of phospho-CTD specific antibodies combined with various experimental approaches including in vitro kinase assays, long-term siRNA-mediated depletion of CDK12 from cells or application of the CDK12 inhibitor THZ531. However, each of these experiments has caveats with respect to the physiological relevance. The specific impact of a short-term CDK12-selective inhibition on CTD phosphorylation and genome-wide transcription in cells remains an important question to be addressed. CDK12 and cyclin K (CCNK) are RNAPII- and transcription elongation-associated proteins 11, 12, 19. CDK12 and its homolog CDK13 (containing a virtually identical kinase domain) associate with CCNK to form two functionally distinct complexes CCNK/CDK12 and CCNK/CDK13 11, 12, 16, 20. Transcription of several core homologous recombination (HR) DNA repair genes, including BRCA1, FANCD2, FANCI, and ATR, is CDK12-dependent 11, 16, 21-23. In agreement, treatment with low concentrations of THZ531 resulted in down-regulation of a subset of DNA repair pathway genes. Higher concentrations led to a much wider transcriptional defect 17. Mechanistically, it has been suggested that CCNK is recruited to the promoters of DNA damage response genes such as FANCD2 24. Other studies using siRNA-mediated CDK12 depletion showed diminished 3′end processing of C-MYC and C-FOS genes 18, 25. Roles for CDK12 in other co-transcriptionally regulated processes such as alternative or last exon splicing have also been reported 26-28. Nevertheless, comprehensive insights into CDK12 target genes and how CDK12 kinase activity regulates their transcription are lacking. CDK12 is frequently mutated in cancer. Inactivation of CDK12 kinase activity was recently associated with unique genome instability phenotypes in ovarian, breast, and prostate cancers 29-31. They consist of large (up to 2–10 Mb in size) tandem duplications, which are completely different from other genome alteration patterns, including those observed in BRCA1- and other HR-inactivated tumors. Furthermore, they are characterized by an increased sensitivity to cisplatin and thus represent potential biomarker for treatment response 29-33. Although inactivation of CDK12 kinase activity clearly leads to HR defects and sensitivity to PARP inhibitors in cells 21, 34-37, the discovery of the CDK12 inactivation-specific tandem duplication phenotype indicated a distinct function of CDK12 in maintenance of genome stability. The size and distribution of the tandem duplications suggested that DNA replication stress-mediated defect(s) are a possible driving force for their formation 30, 31. Proper transcriptional regulation is essential for all metabolic processes including cell cycle progression 38. Transition between G1 and S phase is essential for orderly DNA replication and cellular division, and its deregulation leads to tumorigenesis 39. G1/S progression is transcriptionally controlled by the well-characterized E2F/RB pathway. E2F factors activate transcription of several hundred genes involved in regulation of DNA replication, S phase progression, and also DNA repair by binding to their promoters 40. Expression of many DNA replication genes (including CDC6, CDT1, TOPBP1, MCM10, CDC45, ORC1, CDC7, CCNE1/2), like many other E2F-dependent genes, is highly deregulated in various cancers 41-44. However, it is not known whether or how their transcription is controlled downstream of the E2F pathway, for instance during elongation. To answer the above questions, we used a chemical genetic approach to specifically and acutely inhibit endogenous CDK12 kinase activity. CDK12 inhibition led to a G1/S cell cycle progression defect caused by a deficient RNAPII processivity on a subset of core DNA replication genes. Loss of RNAPII occupancy and transcription from gene 3′ends coincided with a shift of the broad peaks of RNAPII phosphorylated at Ser2 from gene 3′ends into the gene body. Our results show that CDK12-regulated RNAPII processivity of core DNA replication genes is a key rate-limiting step of DNA replication and cell cycle progression and shed light into the mechanism of genomic instability associated with frequent aberrations of CDK12 kinase activity reported in many cancers. Results Preparation and characterization of AS CDK12 HCT116 cell line The role of the CDK12 catalytic activity in the regulation of transcription and other cellular processes is poorly characterized. Most of the previous studies of CDK12 involved long-term depletion, which is prone to indirect and compensatory effects 11, 12, 14, 23. The recent discovery of the covalent CDK12 inhibitor THZ531 made it possible to study CDK12 kinase activity; however, THZ531 also inhibits its functionally specialized homolog CDK13 and transcriptionally related JNK kinases 17. To overcome these limitations and determine the consequences of specific inhibition of CDK12, we modified both endogenous alleles of CDK12 in the HCT116 cell line to express an analog-sensitive (AS) version that is rapidly and specifically inhibited by the ATP analog 3-MB-PP1 45 (Fig 1A). This chemical genetic approach has been used to study other kinases 9, 46, 47 and was also attempted for CDK12 by engineering HeLa cells carrying a single copy of AS CDK12 (with the other CDK12 allele deleted) 48. Figure 1. Preparation and characterization of AS CDK12 HCT116 cell line A. Scheme depicting preparation of AS CDK12 HCT116 cell line. Gate keeper phenylalanine (F) and glycine (G) are indicated in red, and adjacent amino acids in CDK12 active site are shown in black letters (left). ATP and ATP analog 3-MB-PP1 are shown as black objects in wild-type (WT) and AS CDK12 (blue ovals), respectively (right). B. Genotyping of AS and WT CDK12 clones. Ethidium bromide-stained agarose gel visualizing PCR products from genomic DNA of AS (AS-PCR) and WT (WT-PCR) CDK12 HCT116 cells and their digest with BslI enzyme (indicated as AS- BslI and WT- BslI). Primer positions and BslI restriction sites are depicted at Fig EV1A. Numbers on the left and right indicate DNA marker and DNA fragment sizes, respectively. C. Detailed insight into sequencing of genomic DNA from WT and AS CDK12 HCT116 cell lines. The genomic region in WT and AS CDK12 subjected to genome editing is shown in red rectangle; gate keeper amino acids F and G are in red. The full ˜ 500 kb sequence surrounding the edited genomic region is in the Appendix Fig S1A and B. D. Effect of CDK12 inhibition on phosphorylation of the CTD of RNAPII. Western blot analyses of protein levels by the indicated antibodies in AS CDK12 HCT116 cells treated with 5 μM 3-MB-PP1 for indicated times. Long and short exp. = long (4–14 min) and short (10–60 s) exposures, respectively. FUS and tubulin are loading controls. A representative image from three replicates is shown. E, F. Inhibition of CDK12 in AS CDK12 HCT116 cells results in down-regulation of CDK12-dependent HR genes. Graph shows RT–qPCR analysis of relative levels of mRNAs of described genes in AS CDK12 HCT116 (E) and WT CDK12 HCT116 (F) cells treated for indicated times with 3-MB-PP1. mRNA levels were normalized to HPRT1 mRNA expression and the mRNA levels of untreated control (CTRL) cells were set to 1. n = 3 replicates, error bars indicate standard error of the mean (SEM). Source data are available online for this figure. Source Data for Figure 1 [embr201847592-sup-0006-SDataFig1.pdf] Download figure Download PowerPoint We applied CRISPR-Cas technology to mutate the gatekeeper phenylalanine (F) 813 to glycine (G) in both CDK12 alleles in HCT116 cells (Figs 1A and EV1A). The single-strand oligo donor used as a template for CRISPR-Cas editing introduced a silent GTA>GTT mutation to prevent alternative splicing 48, and a TTT>GGG mutation to implement the desired F813G amino acid change and created a novel BslI restriction site used for screening (Fig EV1A). We validated our intact homozygous AS CDK12 HCT116 cell line by several approaches, including allele-specific PCR (Fig EV1B), BslI screening (Fig 1B; for expected restriction patterns see Fig EV1A), and Sanger sequencing (Fig 1C and Appendix Fig S1A and B). Immunoprecipitation (IP) of CDK12 from the WT and AS CDK12 HCT116 cells followed by Western blotting showed that equal amounts of CCNK associated with CDK12, and that comparable levels of CDK12 were expressed in both cell lines, confirming the functionality of the AS variant (Fig EV1C). To investigate the putative role of CDK12 as a RNAPII CTD kinase, we treated AS CDK12 cells with 3-MB-PP1 or control vehicle for 1, 2, 3, and 6 h and monitored changes in CTD phosphorylation by probing Western blots with phospho-specific antibodies (Figs 1D and EV1D). However, we did not observe any substantial changes in the global levels of phosphorylated Ser2 or Ser5 compared to untreated cells. Only short exposures of Western blots revealed a subtle, but noticeable trend toward accumulation of P-Ser2 after 3 h and P-Ser5 at 6 h and a slight decrease of P-Ser5 at 1–3 h, respectively, consistent with previous observations in AS CDK12 HeLa cells 48. Surprisingly, P-Ser7 levels were noticeably diminished starting with 1-h treatment but started recovering at 6 h. To functionally characterize AS CDK12 HCT116 cells, we treated them with 3-MB-PP1 for 1, 3, 5, and 24 h and monitored the expression of DNA repair genes that were previously shown to be regulated by CDK12 (BRCA1, BRCA2, ATR, and FANCI). We observed rapid down-regulation of all four CDK12-dependent genes (Fig 1E). Importantly, similarly treated WT HCT116 cells showed no down-regulation of these genes (Fig 1F), and RNA-seq of WT HCT116 cells treated with 3-MB-PP1 showed differential expression of only six protein-coding genes compared to the control (data not shown), confirming the absence of off-target effects of the ATP analog on other transcription-related kinases. Click here to expand this figure. Figure EV1. Preparation and characterization of AS CDK12 HCT116 cell line Depiction of CDK12 locus, genome editing, and genotyping strategy. Schema of CDK12 locus, with exon numbers shown above the CDK12 gene depiction (top). Primers used for genotyping PCR surrounding exon 6 of CDK12 gene are shown as horizontal arrows, PCR product is depicted as full horizontal line, and BslI restriction sites are indicated by vertical arrows. BslI restriction site created by genome editing is shown in green. Size (bp) of genotyping PCR product and BslI restriction fragments are indicated (middle). DNA subjected to genome editing and corresponding protein sequences in exon 6 of CDK12 genes are shown; the underlined DNA sequence in WT CDK12 allele underwent genome editing to create silent mutation preventing alternative splicing (nucleotide in blue), BslI restriction site, and to convert F813 to G813 (nucleotides in red) in AS CDK12. Engineered G813 in AS CDK12 is indicated in red (bottom). Characterization of AS CDK12 clone by a AS primer-specific PCR. Exon 6 in CDK12 gene is shown as a black box. Edited DNA in the AS CDK12 is marked by a red vertical line in the exon 6. Genotyping primers specific for WT (black arrows) and AS CDK12 (red arrow) are shown, and genotyping PCR product is depicted by a dashed line with size (in bp) indicated above (top). Ethidium bromide-stained agarose gel visualizing 352 bp PCR product from PCR mixture using either WT- (left) or AS-specific (right) forward primer (bottom). CCNK/CDK12 complex shows comparable properties in the AS and WT CDK12 HCT116 cell lines. Western blot analysis of protein levels (input) and association [determined by immunoprecipitation (IP)] of CCNK and CDK12 in the indicated cell lines. No Ab corresponds to a control immunoprecipitation without antibody. A representative image of three replicates is shown. Quantification of individual P-Ser modifications in the CTD of RNAPII after CDK12 inhibition. Amounts of individual proteins and CTD modifications presented in Fig 1D and in another two biological replicates from short film exposures were quantified by ImageJ software. All protein levels were normalized to a corresponding tubulin loading control, and samples without treatment in each time point (CTRL) were considered as 1; n = 3 biological replicates and error bars are standard error of the mean (SEM). Source data are available online for this figure. Download figure Download PowerPoint In summary, these results demonstrated the generation of a fully functional, homozygous AS CDK12 HCT116 cell line. CDK12 kinase activity is essential for optimal G1/S progression independently of DNA damage cell cycle checkpoint In our previous work, we noted that long-term CDK12 depletion leads to an accumulation of cells in G2/M phase, consistent with diminished transcription of CDK12-dependent DNA repair genes and activation of a DNA damage cell cycle checkpoint 11, 49. To determine whether CDK12 kinase activity directly regulates cell cycle progression, we arrested AS CDK12 HCT116 cells at G0/G1 by serum withdrawal for 72 h, released them into serum-containing media in the presence or absence of 3-MB-PP1, and harvested cells for flow cytometry analyses every 6 h after the release (Fig 2A). Figure 2. CDK12 kinase activity is essential for optimal G1/S progression independently of DNA damage cell cycle checkpoint A. Experimental outline. AS CDK12 HCT116 cells were arrested by serum starvation for 72 h and released into the serum-containing medium with or without 3-MB-PP1. DNA content was analyzed by flow cytometry at indicated time points after the release. B. CDK12 kinase activity is needed for G1/S progression in cells arrested by serum starvation. Flow cytometry profiles of control (−3-MB-PP1) or inhibitor (+3-MB-PP1) treated cells from the experiment depicted in Fig 2A. The red arrow points to the onset of the G1/S progression defect in 3-MB-PP1-treated cells. To better visualize the G1/S delay in the presence of the inhibitor, the 24-h time point is also shown. n = 3 replicates; representative result is shown. C. Quantification of cells (%) in individual cell cycle phases based on flow cytometry profiles of the representative replicate in Fig 2B. D. CDK12 protein levels peak in the G0/G1 phase of the cell cycle. Western blots show levels of proteins at indicated time points after the release of serum-starved AS CDK12 HCT116 cells. Corresponding cell cycle phases are depicted above time points. A representative Western blot from three replicates is shown. E. Experimental outline. AS CDK12 HCT116 cells were arrested by serum starvation for 72 h and released into the serum-containing medium. 3-MB-PP1 was either added or not at indicated time points after the release. Propidium iodide- or BrdU-stained DNA content was measured by flow cytometry at 16 h after the release. Note, that for the BrdU staining the 3-MB-PP1 was added only at the time of the release (0 h) and 3, 4, 5, and 6 h after the release. F, G. Inhibition of CDK12 in early G1 perturbs normal cell cycle progression. Quantification of cells (%) in cell cycle phases from flow cytometry profiles of propidium iodide (F)- and BrdU (G)-labeled cells upon addition of 3-MB-PP1 at indicated time points after serum addition in the experiment depicted in Fig 2E. CTRL in Fig 2G = control sample without 3-MB-PP1. n = 3 replicates, representative result is shown. H. Short-term CDK12 inhibition does not activate DNA damage checkpoints. Western blot analyses of phosphorylation of depicted DNA damage response markers upon inhibition of CDK12 for indicated times. CPT corresponds to 5 μM camptothecin. A representative Western blot from three replicates is shown. FUS is a loading control. Source data are available online for this figure. Source Data for Figure 2 [embr201847592-sup-0007-SDataFig2.pdf] Download figure Download PowerPoint In the absence of the inhibitor, the cells entered S phase in ~ 12 h, reached G2/M phase in ~ 18 h, and completed the full cell cycle in ~ 20 h (Fig 2B and C). In contrast, in the presence of 3-MB-PP1, cells started to enter S phase at 18 h, indicating a delay in G1/S progression by 6–9 h. (Fig 2B and C). WT HCT116 cells treated with 3-MB-PP1 showed no defect in cell cycle progression excluding unspecific inhibition of other kinases (Fig EV2A). Importantly, serum-synchronized WT HCT116 cells treated with the CDK12 inhibitor THZ531 (Fig EV2B), as well as AS CDK12 HeLa 48 or AS CDK12 HCT116 cells synchronized by thymidine–nocodazole and inhibited by 3-MB-PP1 also demonstrated the G1/S progression delay (Fig EV2C and data not shown). Thus, the function of CDK12 in optimal G1/S progression appears to be general, rather than cell type- or treatment-specific. Click here to expand this figure. Figure EV2. CDK12 kinase activity is essential for optimal G1/S progression 3-MB-PP1 does not affect cell cycle progression in WT HCT116 cells. The experiment was performed as shown in Fig 2A. n = 3; representative result is shown. THZ531 causes G1/S progression defect in WT HCT116 cells arrested by serum starvation. Flow cytometry profiles of control (−THZ531) or 350 nM THZ531(+THZ531)-treated cells from the experiment outlined in Fig 2A. Red arrow points to the onset of the G1/S progression defect in THZ531-treated cells. n = 3 replicates; representative result is shown. CDK12 inhibition delays G1/S progression in thymidine/nocodazole-arrested AS CDK12 HeLa cells. Flow cytometry profiles of control (−3-MB-PP1) or 3-MB-PP1 (+3-MB-PP1) treated cells from the experiment shown in Fig 2A. Red arrow points to the onset of the G1/S progression defect in 3-MB-PP1-treated cells. n = 3 replicates; representative result is shown. Experimental outline. AS CDK12 HCT116 cells were arrested by serum starvation for 72 h and released into the serum-containing medium with (+) or without (−) 3-MB-PP1. 3-MB-PP1 was washed away and replaced with fresh medium at indicated times after the release, and all samples were subjected to flow cytometry analyses at 15 h after the release. G1/S progression delay can be rescued by removal of CDK12 inhibitor at early G1 phase. Flow cytometry profiles of propidium iodide-labeled cells from the experiment depicted in Fig EV2D. CTRL = control samples without the 3-MB-PP1. n = 3 replicates; representative result is shown. Download figure Download PowerPoint The protein levels of numerous cell cycle regulators fluctuate during cell cycle progression according to their function in a specific phase 38. To examine whether CDK12 levels change during cell cycle progression, we arrested AS CDK12 HCT116 cells by serum starvation, released them, and analyzed CDK12 proteins by Western blotting (Fig 2D). Strikingly, CDK12 levels were highest during early G0/G1 phase, started to diminish in G1/S transition, reached lowest levels in late S phase, and started to slightly recover in G2/M (Fig 2D). Similar trends, however much less distinct, were observed for CDK13 and CCNK. We verified cell cycle synchronization and individual phases of the cell cycle by the expression of CCNE1 in G1/S and accumulation of CCNA2 in G2/M phases (Fig 2D) and by the flow cytometry DNA content profiles (Fig 2B). To define when CDK12 kinase activity is needed for early cell cycle progression, serum-synchronized AS CDK12 HCT116 cells were released into serum-containing medium and 3-MB-PP1 was added at various times post-release, ranging from 0 to 12 h. Cell cycle progression was measured by flow cytometry at 16 h post-release (Fig 2E). Whereas treatments at 9 and 12 h had a weak or no effect on the G1/S transition, treatments within 6 h post-release delayed the transition, suggesting that CDK12 kinase activity is needed at very early G1 phase (Fig 2F). Similar results were obtained by flow cytometry analyses of BrdU-labeled cells (Fig 2G). As an additional approach, we released cells in the presence and absence of 3-MB-PP1 and washed away 3-MB-PP1 after 2, 3, 4, and 5 h (Fig EV2D). When the inhibitor was washed away between 2 and 5 h, the cells were able to progress to S phase comparably to untreated cells (Fig EV2E), indicating the requirement of CDK12 kinase activity in very early G1 phase for optimal G1/S progression. As long-term CDK12 depletion causes down-regulation of DNA repair genes resulting in endogenous DNA damage 11, 23, we asked whether the observed G1/S delay upon CDK12 inhibition was due to secondary activation of DNA damage cell cycle checkpoints 50. However, the levels of phosphorylated P-ATM and P-P53, markers of an activated DNA damage pathway, increased in cells only after 48-h inhibition of CDK12 (Fig 2H), coincident with onset of endogenous DNA damage upon long-term CDK12 deple
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