CDK12 loss in cancer cells affects DNA damage response genes through premature cleavage and polyadenylation

Abstract

Cyclin-dependent kinase 12 (CDK12) modulates transcription elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and selectively affects the expression of genes involved in the DNA damage response (DDR) and mRNA processing. Yet, the mechanisms underlying such selectivity remain unclear. Here we show that CDK12 inhibition in cancer cells lacking CDK12 mutations results in gene length-dependent elongation defects, inducing premature cleavage and polyadenylation (PCPA) and loss of expression of long (>45 kb) genes, a substantial proportion of which participate in the DDR. This early termination phenotype correlates with an increased number of intronic polyadenylation sites, a feature especially prominent among DDR genes. Phosphoproteomic analysis indicated that CDK12 directly phosphorylates pre-mRNA processing factors, including those regulating PCPA. These results support a model in which DDR genes are uniquely susceptible to CDK12 inhibition primarily due to their relatively longer lengths and lower ratios of U1 snRNP binding to intronic polyadenylation sites.

Fig. 1

CDK12/13 inhibition results in selective cytotoxicity in NB cells and affects transcription elongation. a Dose-response curves for human NB cells treated with increasing concentrations of THZ531 for 72 h. Kelly E9R cells, which express a homozygous mutation at the Cys1039 THZ531-binding site in CDK12 (see Methods) were also included. Fibroblast cells (NIH-3T3, IMR-90, BJ) were used as controls. Cytotoxicity is reported as percent cell viability relative to DMSO-treated cells. Data represent mean ± SD; n = 3. b Western blot analysis of Pol II phosphorylation in NB cells treated with THZ531 or DMSO at the indicated concentrations for the indicated times. c Waterfall plot of fold-change in gene expression in IMR-32 NB cells treated with THZ531, 400 nM for 6 h; selected DDR genes are highlighted. d qRT-PCR analysis of the indicated DDR gene expression in Kelly WT (left) cells and Kelly E9R (right), treated with THZ531 or DMSO at the indicated concentrations for 6 h. Data are normalized to GAPDH and compared to the DMSO control. e Flow cytometry analysis of γ-H2AX staining in Kelly NB cells treated with 400 nM THZ531 for the indicated time points (left). Gating was performed as shown in the left panel. Numbers indicate the percentages of living cells that stained positive for γ-H2AX. Quantification of staining (right). f Immunofluorescence staining of RAD51 focus formation in Kelly NB cells treated with THZ531 (400 nM) or DMSO for 24 h prior to exposure to gamma radiation (IR, 8 Gy). Nuclei are stained with DAPI (scale bar, 10 µM). Quantification of staining (right) of RAD51⁺ cells (>5 RAD51 foci per cell). Throughout the figure, error bars indicate mean values ± SD of three independent experiments, **p < 0.01, ***p < 0.001; two-tailed Student’s t-test

Fig. 2

CDK12/13 inhibition leads to an elongation defect that is gene-length dependent. a Average metagene profiles of normalized TT-seq reads over gene bodies and extending −2 to +2 kb of all detected genes in cells treated with THZ531 400 nM for 2 h. Sense and antisense reads are depicted by solid and dashed lines respectively. b Average metagene profile depicting the change (red) and rate of change (blue) in TT-seq read densities in regions flanking the TSS (−0.5 to +1 kb) in cells treated with THZ531. c Scatter plot showing log2 fold-changes in gene expression vs. gene length in log2 scale for each protein coding gene in cells treated as in panel a (R² = 0.12, p = 2e−277, F-test, Spearman correlation coefficient = −0.42). Differentially expressed genes are indicated (FDR < 0.1 and log2 FC > 1). d Average metagene profiles for protein-coding genes (as in a) stratified according to quartiles of gene length distribution and for very short genes. Sense and antisense reads are depicted by solid and dashed lines respectively. e Gene ontology (GO) enrichment analysis of long genes (>64.5 kb) (top); TT-seq tracks of nascent RNA expression at the PCF11 locus in NB cells treated with DMSO or THZ531 as in panel a (bottom). f GO enrichment analysis of very short genes (<3.4 kb) (top); TT-seq tracks at the HIST1H3A/HIST1H4A loci (bottom)

Fig. 3

CDK12 inhibition leads to PCPA of long genes. a Average metagene profiles of normalized poly(A) 3′-seq reads at the transcription end sites (TES) (−1 to +4 kb) of all long genes (>64.5 kb) (left), and short genes (RD histone genes) (right). b Average metagene profiles of normalized poly(A) 3′-seq reads over gene bodies and extending −2 to +2 kb of all detected genes in cells treated with THZ531 400 nM for 2 and 6 h. Sense and antisense reads are depicted by solid and dashed lines, respectively. c Histograms showing the genomic distributions and rankings of the top 5000 poly(A) 3′-seq peaks in DMSO- and THZ531-treated cells (400 nM, 6 h). The poly(A) 3′ peaks were binned according to the depicted genomic regions and their intensities (x-axis). d Bar plot indicating the number of protein-coding genes that underwent premature cleavage and polyadenylation (PCPA) with THZ531. The expanded window on the right shows the genomic distribution of the identified intronic poly(A) sites. e Average metagene profiles of normalized poly(A) 3′-seq reads at the TSS (−1 to +10 kb) for all detected genes in Kelly WT (left) and Kelly E9R (right) cells. Changes (insets) in read density between DMSO- and THZ531 (200 nM, 6 h)-treated Kelly WT (p = 5.8e−41) and Kelly E9R (p = 0.11) cells; comparisons between groups by Wilcoxon rank-sum test. The center line indicates the median for each data set. f Density plot of odds-ratios of poly(A) site usage (intronic vs 3′ UTR) for genes in Kelly WT and E9R cells (p = 0, Kolmogorov-Smirnov test)

Fig. 4

CDK12/13 inhibition results in minimal splicing alterations. a Diagrammatic representation (left) and bar plot of splicing events (right) observed in TT-seq analysis of NB cells treated with THZ531 (400 nM) for 2 h. b Scatterplot of intron retention index (IR index) vs. the ratio of exon and intron lengths in log2 scale. Genes with an IR index >1 or ≤1 display intron retention and loss respectively (adjusted p < 0.05, Fisher’s exact test). c Box plot illustrating the length distributions of genes that display intron loss or retention. The center line indicates the median for each data set. d Density plots illustrating the contributions of the proximal (first intron/exon) and distal (last intron/exon) gene regions in calculation of the IR index. Comparison of IR index distribution between proximal and distal intron/exon pairs (p = 0, Kolmogorov-Smirnov test)

Fig. 5

Gene length and a lower U1/PAS ratio predispose DDR genes to PCPA. a GO enrichment analysis of the 809 genes that underwent PCPA (FDR < 0.01) based on TT-seq analysis of cells treated with THZ531 (400 nM for 2 h). b Box plots and bar plots showing the distribution and numbers of PCPA and DDR genes in the different gene-length categories established in Fig. 2d (****p < 0.0001, **p < 0.01, Fisher’s exact test). The center line indicates the median for each data set. c TT-seq and poly(A) 3′-seq tracks at the BLM DDR gene locus depicting the loss of annotated terminal polyadenylation signal and the presence of early termination due to PCPA in cells treated with THZ531 as in a. d Number of intronic poly(A) sites as a function of transcript length. A polynomial regression curve is plotted for all genes (black) and DDR genes only (red) (p = 1.7e−13, predicted vs. observed, Wilcoxon rank-sum test). e Box plots comparing the indicated determinants of PCPA in all genes vs. PCPA genes only and the proportion of DDR genes within the latter subset (see figure for p and d values; Wilcoxon rank-sum test & Cohen’s d effect-size, respectively). The black and red center lines indicate the median of all PCPA and DDR genes respectively. f Cumulative fraction plot showing the change in expression of PCPA (p = 2.2e−16, Kolmogorov-Smirnov test) and DDR (p = 1.9e−14, Kolmogorov-Smirnov test) transcripts relative to other transcripts following THZ531 treatment as in a

Fig. 6

CDK12/13 phosphorylates RNA processing proteins. a Volcano plot of proteome-wide changes in phosphorylation site occupancy identified through SILAC analysis of NB cells treated with THZ531, 400 nM for 2 h. Expanded box shows selected co-transcriptional RNA processing proteins. b GO terms for candidate CDK12/13 substrates. c In vitro kinase assays of CDK12/CycK (red)-mediated and CDK13/CycK (green)-mediated phosphorylation of CDC5L (aa 370-505) and GST-SF3B1 (aa 113-462) at the indicated time points. A negative control measurement without kinase is shown in blue. Radioactive kinase reactions were performed with 0.2 µM CDK12/CycK or CDK13/CycK and 50 µM substrate protein, respectively. Data are reported as mean ± SD, n = 3. d Dose-response curves of THZ531 incubated with recombinant CDK12 (left) and CDK13 (right) protein and CDC5L (aa 370-505) and GST-SF3B1 (aa 113-462). Radioactive kinase reactions were performed after 30 min preincubation with increasing concentrations of THZ531. For all incubation time series, the counts per minute of the kinase activity measurements were normalized to the relative ³²P transfer. Data are reported as mean ± SD; n = 3. IC₅₀ values shown in parentheses. e Model of CDK12 as a regulator of pre-mRNA processing. CDK12 phosphorylates and thus likely stimulates the orchestrated action of RNA Pol II CTD and RNA processing proteins. CDK12 inhibition leads to a gene-length-dependent productive elongation defect associated with early termination through premature cleavage and polyadenylation (PCPA). Especially vulnerable to PCPA are long genes with a lower ratio of U1 snRNP binding to poly(A) sites, which include many of those involved in the DDR. Among short genes, including genes that normally terminate through stem-loop binding, CDK12 inhibition increases intron retention and leads to longer polyadenylated transcripts