CDK8/19 Mediator kinases potentiate induction of transcription by NFκB

Abstract

The nuclear factor-κB (NFκB) family of transcription factors has been implicated in inflammatory disorders, viral infections, and cancer. Most of the drugs that inhibit NFκB show significant side effects, possibly due to sustained NFκB suppression. Drugs affecting induced, but not basal, NFκB activity may have the potential to provide therapeutic benefit without associated toxicity. NFκB activation by stress-inducible cell cycle inhibitor p21 was shown to be mediated by a p21-stimulated transcription-regulating kinase CDK8. CDK8 and its paralog CDK19, associated with the transcriptional Mediator complex, act as coregulators of several transcription factors implicated in cancer; CDK8/19 inhibitors are entering clinical development. Here we show that CDK8/19 inhibition by different small-molecule kinase inhibitors or shRNAs suppresses the elongation of NFκB-induced transcription when such transcription is activated by p21-independent canonical inducers, such as TNFα. On NFκB activation, CDK8/19 are corecruited with NFκB to the promoters of the responsive genes. Inhibition of CDK8/19 kinase activity suppresses the RNA polymerase II C-terminal domain phosphorylation required for transcriptional elongation, in a gene-specific manner. Genes coregulated by CDK8/19 and NFκB include IL8, CXCL1, and CXCL2, which encode tumor-promoting proinflammatory cytokines. Although it suppressed newly induced NFκB-driven transcription, CDK8/19 inhibition in most cases had no effect on the basal expression of NFκB-regulated genes or promoters; the same selective regulation of newly induced transcription was observed with other transcription signals potentiated by CDK8/19. This selective role of CDK8/19 identifies these kinases as mediators of transcriptional reprogramming, a key aspect of development and differentiation as well as pathological processes.

Keywords: CDK19; CDK8; NFκB; RNA polymerase II; regulation of transcription.

Fig. 1.

Effects of Senexin A (SnxA) on NFκB-dependent promoter activity and NFκB translocation. (A) Effect of Senexin A on GFP expression from NFκB-dependent consensus promoter in HT1080 cells, untreated or treated with TNFα (20 ng/mL, 18 h). The effects of Senexin A on TNFα-induced GFP were statistically significant (P < 0.05) at all concentrations. (B) Effects of Senexin A on luciferase expression from NFκB-dependent E-selectin promoter in HEK293 cells untreated or treated with IL1 (10 ng/mL, 4 h) or TNFα (10 ng/mL, 4 h). Asterisks indicate significant effects of Senexin A (P < 0.05). (C) Effects of TNFα (20 ng/mL, 30 min), on the appearance of p50 and p65 in the nuclear fractions of the HT1080 derivative used in Fig. 1A and in HEK293 cells treated or untreated with Senexin A (5 μM), TPCK (50 μM), or MG115 (20 μM) for 2 h.

Fig. S1.

Effects of Senexin A on TNFα-induced activation and translocation of NFκB proteins. (A) Effects of TNFα (10 ng/mL), in the absence or presence of 5 µM Senexin A, 50 µM TPCK, and 20 µM MG115, on NFκB site DNA binding of p50 and p65 in the nuclei of HT1080-p21-9-NFκB (Left) and HEK293 (Right) cells (TransAM NFκB assay; Active Motif). (B) Immunofluorescence analysis of the effects of 10 ng/mL TNFα treatment of different durations on the cytoplasm-to-nucleus translocation of p65 in HEK293 cells, in the absence (Left) or presence (Right) of 5 µM Senexin A.

Fig. 2.

qPCR analysis of the effects of TNFα and Senexin A on 15 NFκB-regulated genes following treatment with 10 ng/mL TNFα for indicated periods, with or without 5 μM Senexin A (added 2 h before TNFα). Asterisks indicate significant effects of Senexin A (P < 0.05).

Fig. S2.

Effects of Senexin A on NFκB-mediated transcription by microarray analysis. (A) Agilent microarray analysis of the effects of 10 ng/mL TNFα and 5 µM Senexin A at different time points (2, 6, 12, and 24 h) on the expression of TNFα-regulated genes in HEK293 cells. (B) Agilent microarray analysis of the effects of 10 ng/mL TNFα and 5 µM Senexin A at 30 min after TNFα stimulation on the expression of the top TNFα-regulated genes in HEK293 cells. (C) Illumina microarray analysis of the effects of 20 ng/mL IL1 and 5 µM Senexin A at 2 h after IL1 stimulation on expression of the top IL1-regulated genes in HEK293-IL1R-pELAM-Luc cells.

Fig. S3.

Effects of CDK8/19 inhibition on NFκB-dependent gene expression. Asterisks denote statistically significant effects of Senexin B (P < 0.05). (A) Effects of different concentrations of TNFα (2-h treatment) in the absence or presence of 1 μM Senexin B on the expression of CXCL1, CXCL2, and NFKBIA in HEK293 cells. (B) Effects of 10 ng/mL TNFα and 1 μM Senexin B on the expression of CXCL1, CXCL2, and IL8 in HEK293 cells in serum-free (SF) media or in the presence of 1% or 10% FBS. (C) Effects of 10 ng/mL TNFα and 1 μM Senexin B on CXCL1 protein expression in conditioned media of HEK293 cells (ELISA).

Fig. 3.

Effects of CDK8/19 inhibition by different kinase inhibitors or shRNAs on NFκB-induced transcription. (A) Effects of the CDK8/19 kinase inhibitors Senexin A (SnxA), Senexin B (SnxB), and an equipotent derivative of Cortistatin A (CA) on CXCL1 and IL8 expression in HEK293 cells, pretreated with CDK8/19 inhibitors for 1 h and then treated with 10 ng/mL TNFα for 2 h. (B) Immunoblotting analysis of CDK8 and CDK19 knockdown (KD) in HEK293 derivatives. (C) Effects of TNFα and Senexin A on the mRNA expression of indicated genes in HEK293 cells and their derivatives with single or double knockdown of CDK8 and CDK19, pretreated with or without 5 μM Senexin A for 2 h, followed by 30 min treatment with 10 ng/mL TNFα. Asterisks mark significant effects of Senexin A (P < 0.05). (D) Immunoblotting analysis of CDK8 knockdown in HT1080 subline used in Fig. 1A and its CDK8 knockdown derivative. (E) Effects of TNFα and Senexin A on the expression of the indicated genes in HT1080 cells with or without CDK8 knockdown (the same treatment as in Fig. 3C). Asterisks denote significant effects of Senexin A (P < 0.05).

Fig. 4.

Mechanism of NFκB coregulation by CDK8/19. HEK293 cells were pretreated with or without 5 μM Senexin A (SnxA) for 2 h, followed by a 30-min treatment with 10 ng/mL TNFα. (A–E) ChIP analysis of the effects of TNFα and Senexin A on binding of p65 (A), CDK8/19 (B), Pol II (C), Pol II phosphorylated at S5 of the CTD (D), and Pol II phosphorylated at S2 of the CTD (E), to three NFκB-regulated and two housekeeping genes. Gene maps are shown at the top. (F) Schematic illustration of the mechanism of NFκB coregulation by CDK8/19.

Fig. S4.

ChIP analysis of the effects of TNFα and Senexin A on binding of CDK9 (A) and H3K9/14Ac (B) to three NFκB-regulated genes and two housekeeping genes. Gene maps are shown at the top.

Fig. 5.

Effects of Senexin B (SnxB) on the induction of gene expression by NFκB in different cell lines and by different transcription factors in HEK293. (A) Comparison of the effects of TNFα and Senexin B on the expression of TNFα-regulated genes in HEK293 and HCT116 cells (RNA-Seq data). Cells were pretreated with or without 1 μM Senexin B for 1 h, followed by 2 h treatment with 10 ng/mL TNFα. Red dots indicate genes significantly affected by Senexin B. (B) Effects of TNFα and Senexin B on the expression of CXCL1, CXCL2, and IL8 in HCT116 derivatives control, p21^−/−, and p53 ^−/−, with the same treatment as in A. Asterisks indicate statistically significant effects of Senexin B (P < 0.05). (C) Comparison of the effects of TNFα and Senexin B on the expression of CXCL1, CXCL2, and IL8 in the indicated cell lines. Cells were treated as in A except for LNCAP cells, which were pretreated with 5 μM Senexin B for 1 h, followed by a 30-min treatment with TNFα. Asterisks denote significant effects of Senexin B (P < 0.05). (D) Effects of hypoxia (∼2–3% O₂, 24 h) and Senexin B (1 µM) on the expression of a hypoxia-inducible gene in HEK293 cells. Asterisks indicate statistically significant differences between hypoxia and hypoxia + SnxB groups (P < 0.05). (E) Effects of IFNγ (250 IU/mL, 5 h) and Senexin B (1 µM) on STAT1 expression in HEK293 cells. Asterisks denote statistically significant effects of Senexin B (P < 0.05).

Fig. S5.

Comparison of the effects of TNFα and Senexin B on the expression of CXCL1, CXCL2, and IL8 in the indicated cell lines. Asterisks denote statistically significant effects of Senexin B (P < 0.05).