Research ArticleMOLECULAR BIOLOGY

CDK13 cooperates with CDK12 to control global RNA polymerase II processivity

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Science Advances  29 Apr 2020:
Vol. 6, no. 18, eaaz5041
DOI: 10.1126/sciadv.aaz5041
  • Fig. 1 CDK12 and CDK13 are functionally redundant.

    (A) Heat map of dependency scores for 55 CDKs and cyclins in 62 hematological cancer cell lines from the Avana CRISPR public 19Q1 dataset. (B) Box plot comparisons of dependency scores for cyclin K and CDK7, CDK9, CDK12, and CDK13 in 62 hematological and 496 nonhematological cancer cell lines. (C) Overview of CDK12 and CDK13 ATP AS mutant kinase generation through mutation of the gatekeeper residue. (D) Crystal structures of WT and AS CDK12 with 1-NM-PP1 interaction. (E) Propidium iodide (PI) incorporation assay of MV4;11 WT and AS clones treated with 1-NM-PP1 as indicated for 48 hours. (F) Representative CellTrace Violet dye (CTV) profiles and mean division number of PI-negative MV4;11 WT and AS clones treated with 1-NM-PP1 as indicated for 48 hours. (B) represents 62 hematological and 496 nonhematological cell lines. (E) and (F) represent the mean ± SEM of three independent experiments, and Student’s t tests were performed for (B), (E), and (F) (*P < 0.05, **P < 0.001, and ***P < 0.0001).

  • Fig. 2 Dual CDK12 and CDK13 inhibition drives global disruption of transcriptional landscapes.

    (A) Log2 fold change (FC) gene expression volcano plots for MV4;11 AS clones treated with 10 μM 1-NM-PP1 for 4 hours [relative to dimethyl sulfoxide (DMSO) treatment]. The top 15 differentially expressed genes [ranked by adjusted P value] are labeled. Genes exhibiting a log2FC of >2 (red) or <−2 (blue) with an adjusted P value of <0.01 are highlighted. (B) Venn diagram of the number of differentially expressed genes for MV4;11 AS clones treated with 10 μM 1-NM-PP1 for 4 hours (logFC < −1 and logFC > 1 and adjusted P < 0.05 relative to DMSO treatment). (C) Heat map of differentially expressed genes for MV4;11 AS clones treated with 10 μM 1-NM-PP1 for 4 hours (log2FC < −1 and log2FC > 1 and adjusted P < 0.001 relative to DMSO treatment). (D) Schematic of proximal versus distal UTR/polyadenylation sites. (E) MA plots representing proximal and distal 3′UTR peak shifts for MV4;11 AS clones treated with 10 μM 1-NM-PP1 for 4 hours. (F) Venn diagram of significant proximal and distal 3′UTR peak shift events for MV4;11 AS clones treated with 10 μM 1-NM-PP1 for 4 hours (adjusted P < 0.05 relative to DMSO treatment). (G) Proportion of genes with a significant change in proximal or distal 3′UTR peak signal for MV4;11 AS clones treated with 10 μM 1-NM-PP1 for 4 hours (relative to DMSO treatment). (H) Representative IGV profiles of differential 3′UTR peak usage for MV4;11 AS clones treated with 10 μM 1-NM-PP1 for 4 hours. Scale bars, 5 kb (SETDB1) and 10 kb (ZNF561). Data are representative of two biologically independent replicates.

  • Fig. 3 CDK12 and CDK13 redundantly control POLII CTD phosphorylation to control processivity.

    (A) Log2FC volcano plots of differentially phosphorylated peptides in MV4;11 AS clones treated with 10 μM 1-NM-PP1 for 2 hours (relative to DMSO treatment). Highlighted points represent proteins with an adjusted P value of <0.3 relative to DMSO treatment, and the number of increased (UP) and decreased (DOWN) phosphopeptides is highlighted. (B) Heat map of differentially phosphorylated peptides for MV4;11 AS clones treated with 10 μM 1-NM-PP1 for 2 hours (adjusted P < 0.3 relative to DMSO treatment). (C) Venn diagram, GO analysis, and (D) STRING interaction network for proteins with significantly decreased phosphopeptides in MV4;11 AS clones treated with 10 μM 1-NM-PP1 for 2 hours (adjusted P < 0.3 relative to DMSO condition). (E) Western blot analysis of MV4;11 parental cells and MV4;11 WT and AS clones treated as indicated with 200 nM THZ531 (6 hours) or 10 μM 1-NM-PP1 (4 hours). (F) Representative IGV images and (G) average gene profiles of 9552 expressed genes for total and phospho-Ser2 POLII ChIP-seq analysis of MV4;11 AS clones treated with 10 μM 1-NM-PP1 for 4 hours. Scale bars, 10 kb. (H) POLII processivity index (FC ratio of the 5′/3′ POLII signal ratio in the gene body) for MV4;11 WT and AS clones treated with 10 μM 1-NM-PP1 for 4 hours. Western blots are representative of three independent experiments. (H) is representative of 9552 expressed genes, and Student t test was performed (****P < 0.001).

  • Fig. 4 Perturbation of POLII transcription cycle at the elongation checkpoint by dual CDK12 and CDK13 inhibition.

    (A) Schematic overview of DRB-release PRO-seq assay for (B) to (G). (B) Representative IGV images for DRB-release PRO-seq of MV4;11 WT and AS clones treated with 10 μM 1-NM-PP1. Scale bars, 50 kb. (C) Average gene profiles for DRB-release PRO-seq analysis of 368 genes in MV4;11 AS clones treated with 10 μM 1-NM-PP1. Heat map analysis of (D) PRO-seq signal and (E) log2FC PRO-seq signal (DMSO relative to 1-NM-PP1) in MV4;11 WT and AS clones treated with 10 μM 1-NM-PP1. (F) POLII elongation rate and (G) log2FC in POLII elongation rate (relative to DMSO) in MV4;11 WT and AS clones treated with 10 μM 1-NM-PP1. (H) Average gene profiles for TT-seq analysis of 368 genes normalized to Drosophila S2 RNA spike-in in MV4;11 WT and AS clones treated as indicated for 25 min. (I) Schematic overview of POLII elongation rate and processivity computational modeling parameters. Computational modeling of simulated defects in POLII (J) elongation rates, (K) processivity half-life (T1/2), and (L) combined elongation and processivity. (F) and (G) are representative of 368 genes, and Student’s t test was performed for (F) (***P < 0.001).

Supplementary Materials

  • Supplementary Materials

    CDK13 cooperates with CDK12 to control global RNA polymerase II processivity

    Zheng Fan, Jennifer R. Devlin, Simon J. Hogg, Maria A. Doyle, Paul F. Harrison, Izabela Todorovski, Leonie A. Cluse, Deborah A. Knight, Jarrod J. Sandow, Gareth Gregory, Andrew Fox, Traude H. Beilharz, Nicholas Kwiatkowski, Nichollas E. Scott, Ana Tufegdzic Vidakovic, Gavin P. Kelly, Jesper Q. Svejstrup, Matthias Geyer, Nathanael S. Gray, Stephin J. Vervoort, Ricky W. Johnstone

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