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CDK11 is required for transcription of replication-dependent histone genes

Abstract

Replication-dependent histones (RDH) are required for packaging of newly synthetized DNA into nucleosomes during the S phase when their expression is highly upregulated. However, the mechanisms of this upregulation in metazoan cells remain poorly understood. Using iCLIP and ChIP–seq, we found that human cyclin-dependent kinase 11 (CDK11) associates with RNA and chromatin of RDH genes primarily in the S phase. Moreover, its amino-terminal region binds FLASH, an RDH-specific 3′-end processing factor, which keeps the kinase on the chromatin. CDK11 phosphorylates serine 2 (Ser2) of the carboxy-terminal domain of RNA polymerase II (RNAPII), which is initiated when RNAPII reaches the middle of RDH genes and is required for further RNAPII elongation and 3′-end processing. CDK11 depletion leads to decreased number of cells in S phase, likely owing to the function of CDK11 in RDH gene expression. Thus, the reliance of RDH expression on CDK11 could explain why CDK11 is essential for the growth of many cancers.

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Fig. 1: CDK11 binds chromatin of RDH genes and promotes their transcription.
Fig. 2: FLASH recruits CDK11 to the RDH genes.
Fig. 3: CDK11 is recruited to RDH genes predominantly in S phase.
Fig. 4: RNA promotes CDK11 recruitment to the RDH chromatin.
Fig. 5: CDK11 promotes transcriptional elongation of RDH genes.
Fig. 6: Recruitment of 3′-end processing factor CPSF100 to the RDH genes depends on CDK11-mediated phosphorylation of Ser2.
Fig. 7: Summary of iCLIP and ChIP–seq data and working model.

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Data Availability

All next-generation-sequencing source and processed data are available at NCBI GEO (accession number GSE118051). Source data for Figs. 26 and Extended Data Figs. 1–8 are available with the paper online.

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Acknowledgements

We thank all members of the Blazek and Ule laboratories for discussions throughout the project and helpful comments on the manuscript. We also wish to thank O. Jasnovidova (Stefl lab) for the GST-CTD, B. G. Wouters for the HCT116 Flp-in cell line, Z. Slaba for preparation of His-FLASH constructs and proteins, T. Curk for iCount support and K. Bartholomeeusen, Life Science Editors and M. Dettenhofer for comments on the manuscript. The work was supported by grants from: the Czech Science Foundation (16-10930S), Masaryk University (MUNI/E/0080/2017), the CEITEC (Project CEITEC-Central-European Institute of Technology (CZ.1.05/1.1.00/02.0068) to D.B. and the European Research Council (617837-Translate) to J.U. The Francis Crick Institute receives its core funding from Cancer Research UK (FC001002), the UK Medical Research Council (FC001002) and the Wellcome Trust (FC001002).

Author information

Authors and Affiliations

Authors

Contributions

P.G. performed most experiments except for radioactive IVKA, some experiments in cell cycle synchronized cells (M.H.), nuclear run-on, RNase protection assay, GST-pulldown, FLASH ChIP–qPCR and IVKA with P-specific antibodies (M.R.) and CDK11 and P-Ser5 ChIP–qPCR (D.B). I.R.dl.M. performed all bioinformatics analyses under the supervision of J.U. and with some input from P.G. and D.B. D.B. conceived the study, acquired funding and wrote the manuscript with the support of P.G, I.R.dl.M. and J.U. All authors discussed the design of experiments, analyzed the data and commented on the manuscript.

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Correspondence to Dalibor Blazek.

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Peer review information Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 CDK11 is recruited to chromatin of RDH genes to regulate their transcription.

a,b, GO analyses of enriched cellular processes (a) and components (b) in genes down-regulated in the RNA-seq experiment. Total of 401 genes (log2FoldChange<−1.5; p-adj<0.01) were analysed by the Gorilla program. c, Depiction of histone mRNA and position of RT-qPCR primers. The stem shape and the black triangle depict SL and mRNA cleavage, respectively. Arrows display positions of total RT-qPCR primers. Blue and orange rectangle represents histone open reading frame (ORF) and histone downstream element (HDE), respectively. d, Graph shows relative levels of total mRNAs of described genes in HCT116 cells treated with control (siCTRL) or CDK11 (siCDK11) siRNA. Total RNA was reverse transcribed using random hexamer primers. mRNA levels were normalized to PPIA mRNA expression. n=3 biologically independent experiments, error bars=SEM, *P<0.05, Student´s two-sided t-test. e, HCT116 cells were transfected with control or CDK11 siRNA and nascent mRNA from nuclear run-ons were measured by RT-qPCR. Graph shows fold change of RDH nascent mRNAs normalized to control knockdown and PPIA housekeeping gene (2-ΔΔCq). Results show mean ± standard deviation of four biological replicates. n=4 biologically independent experiments, error bars=SD. f, GO analyses of enriched cellular functions of genes occupied by CDK11 in HCT116 cells. 393 genes (log2FoldChange<−1.5; p<0.01) were chosen for the analyses by the Gorilla program. g, CDK11 ChIP–seq occupancy on HIST2H2BE, HIST2H2AC, HIST2H2AB (upper panel) and HIST1H1E, HIST1H2BD RDH cluster genes (lower panel). Black lines below the RDH schema and the gene tracks show positions of the SL and CDK11 ChIP-seq peaks identified by the MACS2 program (p<0.05), respectively. h, CDK11 ChIP-qPCR analyses on the indicated RDH genes in HCT116 cells transfected with mock or CDK11 siRNA. ChIP-qPCR was performed with either CDK11 or no antibody (no Ab) control. Ir1 represents intergenic region. n=4, error bars=SEM.

Source data

Extended Data Fig. 2 CDK11 and FLASH interact directly and are present on the chromatin of RDH genes.

a,b, Western blot analyses of immunoprecipitates of endogenous CDK11 (a) and flag-tagged FLASH (F-FLASH) (b) from HCT116 cells expressing F-FLASH. The blots were probed against proteins indicated on the side. c, Correlation analyses of CDK11 and FLASH co-occupancy genome-wide (10 kb bins) (top panel) and on RDH genes (bottom panel). Spearman correlation coefficient between indicated ChIP-seq samples is shown and correlation strength is indicated by colour code. FLASH ChIP-seq from hTERT and U2OS cells. d, Western blot analyses of FLASH and CDK11 protein levels in cell lysates used for the CDK11 and FLASH ChIP-qPCR experiments in Fig. 2e and Extended Data Fig. 2e, respectively. FUS is a loading control. Representative replicate is shown. e, Endogenous FLASH ChIP-qPCR on indicated RDH genes or control intergenic region (Ir) in HCT116 cells treated either with control (CTRL) or FLASH siRNA for 24 h. n=3 biologically independent experiments, error bars=SEM, *P<0.05, Student´s two-sided t-test.

Source data

Extended Data Fig. 3 CDK11 is recruited to the RDH genes in S phase and maintains protein levels of FLASH.

a, Western blots show levels of indicated proteins in depicted cell cycle phases. FUS is a loading control and CCNE1 a G1/S phase marker. Endo and flag mark endogenous and F-CDK11, respectively. b, Graph presents mRNA levels of described histone genes in G1/S, S and G2/M phases. RT-qPCR is normalized relative to MAZ mRNA. n=3 biologically independent experiments, error bars=SEM. c, Histogram presents cell cycle profiles of either asynchronous or synchronized cells released in the indicated times after double thymidine synchronization. Cells in G1/S, S and G2/M phases were harvested at 0, 2 and 8 h after the release, respectively. d, Western blot analyses of FLASH protein levels in cells treated with control (CTRL) or CDK11 siRNA for indicated times. e, Western blot analyses of CDK11 protein levels in lysates from HCT116 cells treated with control (CTRL) or FLASH siRNA for 72 h. f, Gene tracks of HIST1H4E and HIST1H1C showing CDK11 occupancy during the cell cycle. CDK11 ChIP-seq in asynchronous cells, synchronized G2/M and S phase and ChIP-seq with no antibody (no Ab) control are depicted.

Source data

Extended Data Fig. 4 CDK11 is an evolutionary conserved RNA-binding protein.

a, Alignment of CDK11 protein sequences from different species by the MultAlin program. CDK11 protein sequences from human (Homo-hCDK11B (NP_001778.2) and hCDK11A (NP_001300825.1)), mouse (Mus-mCDK11 (NP_031687.2)), cow (Bos-bCDK11 (NP_001007812.2)), chicken (Gallus-gCDK11 (NP_001026042.2)), frog (Xenopus-xCDK11(NP_001086696.1), and zebra fish (Danio-dCDK11 (NP_001008646.1)) were obtained from the NCBI database. Numbers above the alignment indicate amino acid numbering. Red, blue and black colored letters indicate amino acid consensus, similarity, and difference, respectively. b, Schema of iCLIP workflow. Big and small grey ovals correspond to a full length F-CDK11 protein and a CDK11 peptide, respectively crosslinked to RNA (black line). RE=restriction enzyme. c, 293 cell lines stably expressing flag-tag CDK11 (F-CDK11) or endogenous hnRNPC (used as iCLIP procedure positive control with the canonical RNA-binding protein) were treated with 4SU for 6 hr and irradiated with 200 mJ/cm2 at 365 nm. Lysates were either treated with decreasing concentrations of RNase I or left untreated. The RNA-protein complexes were resolved on SDS-PAGE and visualized by autoradiography. Clamps on the side of the panels and asterisk indicate RNA-protein complexes and collapsed RNA-protein band after high RNase I treatment, respectively. Band corresponding to autophosphorylated CDK11 is shown by the arrow. d, Significant iCLIP crosslinks (FDR > 0.05) were matched with their transcripts and correlated between the indicated biological replicates of depicted F-CDK11 and no Ab iCLIP libraries. Numbers inside blue balls correspond to R2 (Pearson correlation coefficient). e, Percentage of significant bound genomic regions (FDR > 0.05) normalized by region length. Biological replicates of F-CDK11 iCLIP libraries are labelled on the left, individual genomic regions are differentiated by a color code.

Source data

Extended Data Fig. 5 CDK11 binds predominantly RDH RNAs in cells.

a, GO analyses of enriched cellular processes in CDK11-bound mRNAs. 371 mRNAs (with CDK11 CLIP cDNA density > 0.01) were analysed by the Gorilla program. b–d, Biodalliance genome browser view of F-CDK11, F-CDK11(226–783) and uncrosslinked control (no UV) iCLIP binding at canonical RDH HIST1H1E (b), non-canonical histones H3F3A (upper panel) and H1F0 (lower panel) (c) and protein coding FOS (upper panel) and PPIA (lower panel) transcripts (d). For HIST1H1E, the expanded view of its 3´end sequence (stop codon and stem loop are indicated with black lines) and CDK11 binding there is shown (b). e, Highly expressed transcripts bind to CDK11 with much lower intensity when compared to RDH transcripts. Comparison of expression (upper panel) and binding to CDK11 (lower panel) of RDH (black line) and highly expressed genes (grey line). Data are based on RNA-seq normalized to reads per kilobase per million (RPKM) and CDK11 iCLIP experiments in 293 cells. f, g, Graph represents RIP analyses of endogenous CDK11 binding to indicated RNAs in HCT116 cells. Immunoprecipitations were performed with either CDK11 or no antibody (Ab) in cell lysates treated with mock or CDK11 siRNA (siCDK11). n=4 biologically independent experiments, error bars=SEM. (f). Western blot analyses of efficiency of CDK11 depletion with indicated siRNAs (lanes 1, 2) and immunoprecipitations with shown antibodies (lanes 3, 4, 5) in cell lysates used for the RIP experiment. Antibodies used for western blotting are displayed on the right of the panel (g). h,i, Graph displays RIP analyses carried out in cell lysates from HCT116 cells stably expressing indicated F-CDK11 proteins. Immunoprecipitations were done with flag or no antibody (no Ab); qPCR was performed in triplicate for each biological replicate. n=4 biologically independent experiments, error bars=SEM (h). Western blot analyses of levels of doxycycline induced F-CDK11 proteins in cell lysates input into the RIP (left panel, lanes 1–3); efficiency of immunoprecipitation of the proteins with flag antibody in RIP (lane 4–6, right panel). Anti-flag antibody was used for western blotting (i). j, Myc-tagged CDK11 (M-CDK11) protein was either expressed (lanes 1, 3, and 4) or not (lane 2) in 293 cells carrying indicated stably integrated flag-tagged proteins. Lysates were immunoprecipitated with anti-flag agarose and immunoprecipitates of M-CDK11 and flag-tagged proteins were identified as presented on the lower and middle western blots, respectively. The upper western blot shows amounts of M-CDK11 proteins in 5% input into the immunoprecipitations (IP). F-EV corresponds to cell line stably expressing flag tag alone.

Source data

Extended Data Fig. 6 RNA promotes CDK11 recruitment to the FLASH-containing RDH chromatin.

a, Workflow of nuclear pellet fractionation procedure. b,c, Western blot analysis of: immunoprecipitates of endogenous FLASH from cell lysates either treated or not with RNase A (b), association of indicated factors in soluble and insoluble fractions of chromatin either treated or not with RNase A/T1 (c) Antibodies are shown on the side of the panels. d, Western blot analysis of immunoprecipitates of endogenous FLASH from HCT116 cells carrying stably integrated flag-tagged CDK11 (F-CDK11), its indicated deletion mutants and control flag-tagged empty vector (F-EV) (right panel). Western blot analyses of inputs into FLASH immunoprecipitations are shown (left panel). Amounts of flag-tagged CDK11 proteins were monitored by anti-flag antibody. e, Western blot analysis of endogenous (endo) and flag-tagged CDK11 protein levels in the cells treated with amanitin or triptolide or untreated control (CTRL) used for F-CDK11 ChIP-qPCR (Fig. 4e). Antibodies are shown on the side of the panels.

Source data

Extended Data Fig. 7 CDK11 regulates Ser2 phosphorylation and RNAPII elongation specifically on the RDH genes.

a, ChIP-seq analyses of P-Ser2 occupancies on highly expressed (n=200) and other genes (except RDH, up- and down-regulated genes, n= 56751 genomic features) in HCT116 cells treated with either control (siCTRL) or CDK11 (siCDK11) siRNA. P-Ser2 noAb=control input into P-Ser2 ChIP-seq. b, HIST1H1C and HIST1H2AJ gene tracks as in Fig. 5e. c, ChIP-seq analyses of RNAPII occupancies as in Extended Data Fig. 7a d–f, ChIP-qPCR analyses of RNAPII (d) and phosphorylated Ser5 (e) occupancies on coding regions of the indicated RDH genes in HCT116 cells transfected with control (siCTRL) or CDK11 (siCDK11) siRNA. Ratio of P-Ser5 and total RNAPII ChIP-qPCR signals is displayed (f). n=3 biologically independent experiments, error bars=SEM, Ir=intergenic region. g, ChIP-qPCR analyses of P-Thr4 occupancies on coding regions of the indicated RDH and protein coding genes in HCT116 cells transfected with control (siCTRL) or CDK11(siCDK11) siRNA. n=5 biologically independent experiments, error bars=SEM, Ir=intergenic region.

Source data

Extended Data Fig. 8 Depletion of CDK11 leads to changes in transcriptional read-through and/or use of cryptic polyA sites in RDH genes.

a,b, Depiction of histone mRNA and position of RT-qPCR primers. Blue and orange rectangle represents histone open reading frame (ORF) and histone downstream element (HDE), respectively. The stem shape depicts SL RNA and the black triangle indicates position of mRNA cleavage. Solid and dotted arrows display positions of total and read-through RT-qPCR primers, respectively (a). Graph shows relative levels of read-through mRNA of described genes in HCT116 cells transfected with control (CTRL) or CDK11 siRNA. Total RNA was reverse transcribed using random hexamer primers. Read-through mRNA levels are normalized to the total mRNA levels of the corresponding gene. The read-though transcription in CTRL cells was set as 1. n=4 biologically independent experiments, error bars=SEM. *P<0.05, Student´s two-sided t-test (b). c, RNase protection assay shows levels of differentially 3’ end processed HIST1H1C transcripts in HCT116 cells treated with control (CTRL), CDK11 or SLBP siRNA. Total HCT116 or yeast RNA was incubated with an antisense RNA probe targeting canonically processed and polyadenylated HIST1H1C transcripts. The mix was treated with RNase T1, resolved on denaturing gel and visualized. The arrows on the left indicate positions of full-length probe (probe) and polyadenylated (polyA) or canonically processed (stem loop) HIST1H1C transcripts. No RNase sample shows full-length anti-sense probe without RNase T1 treatment. d–g, Schema of alternative 3´end processing pathways of histone mRNA and position of RT-qPCR primers. The stem shape depicts SL RNA and the black triangle indicates position of mRNA cleavage. Absence of proper cleavage results in polyadenylated histone mRNA, proper cleavage produces mature non-polyadenylated histone mRNA. Solid arrows display positions of total RT-qPCR primers (d). Graphs show relative levels of mRNA of described genes in HCT116 cells treated with either control (CTRL), CDK11, SLBP or ARS2 siRNA. Total RNA was reverse transcribed using oligo(dT) primers. mRNA levels were normalized to PPIA mRNA expression. n=3 biologically independent experiments, *P<0.05, Student´s two-sided t-test (e,f). Depletion of proteins was verified by western blotting with indicated antibodies. Tubulin is a loading control (g).

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Extended Data Fig. 9 Presentation of iCLIP and ChIP-seq data on example RDH genes.

a–f Biodalliance genome browser view of HIST1H1E, HIST1H2BD (a); HIST1H4AJ (b); HIST2H2BE, HIST2H2AC, HIST2H2AB (c); HIST1H2AM, HIST1H1BO (d); HIST1H3A, HIST1H4A, HIST1H4B, HIST1H3B, HIST1H2AB (e); HIST1H2BL, HIST1H2AI, HIST1H3H, HIST1H2AJ and HIST1H2BM (f) genes with the indicated iCLIP and ChIP-seq data in control and CDK11 depleted cells.

Supplementary information

Supplementary Information

Supplementary Note and Supplementary Note References.

Reporting Summary

Supplementary Table 1

List of protein-coding mRNAs identified in CDK11 knockdown RNA-seq (genes with significant differential expression, adjusted P < 0.01, are shown).

Supplementary Table 2

The list of the significant CDK11 ChIP–seq peaks in the genome identified by the MACS2 program.

Supplementary Table 3

Table shows experimental conditions and number of unique cDNAs for individual iCLIP libraries.

Supplementary Table 4

List of protein-coding mRNAs identified in CDK11 iCLIP (mRNAs with significant iCLIP (FDR < 0.05) cDNA densities > 0.01 are shown).

Supplementary Table 5

The list of differential P-Ser2 ChIP–seq peaks in the genome identified by the MACS2 program.

Supplemetnary Table 6

The list of differential RNAPII ChIP–seq peaks in the genome identified by the MACS2 program.

Supplementary Table 7

Positions of RT–qPCR primers, SL and cryptic polyA sites in RDH genes tested in Extended Data Fig. 8.

Supplementary Table 8

Plasmids and stable cell lines used in this study.

Supplementary Table 9

siRNAs used in this study.

Supplementary Table 10

Primers used in this study.

Supplementary Table 11

Antibodies used in this study.

Supplementary Table 12

Gating strategy for flow cytometry.

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Gajdušková, P., Ruiz de los Mozos, I., Rájecký, M. et al. CDK11 is required for transcription of replication-dependent histone genes. Nat Struct Mol Biol 27, 500–510 (2020). https://doi.org/10.1038/s41594-020-0406-8

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