Cyclin-Dependent Kinase 7 Controls mRNA Synthesis by Affecting Stability of Preinitiation Complexes, Leading to Altered Gene Expression, Cell Cycle Progression, and Survival of Tumor Cells

Abstract

b Cyclin-dependent kinase 7 (CDK7) activates cell cycle CDKs and is a member of the general transcription factor TFIIH. Although there is substantial evidence for an active role of CDK7 in mRNA synthesis and associated processes, the degree of its influence on global and gene-specific transcription in mammalian species is unclear. In the current study, we utilize two novel inhibitors with high specificity for CDK7 to demonstrate a restricted but robust impact of CDK7 on gene transcription in vivo and in in vitro-reconstituted reactions. We distinguish between relative low-and high-dose responses and relate them to distinct molecular mechanisms and altered physiological responses. Low inhibitor doses cause rapid clearance of paused RNA polymerase II (RNAPII) molecules and sufficed to cause genome-wide alterations in gene expression, delays in cell cycle progression at both the G 1 /S and G 2 /M checkpoints, and diminished survival of human tumor cells. Higher doses and prolonged inhibition led to strong reductions in RNAPII carboxyl-terminal domain (CTD) phosphorylation, eventual activation of the p53 program, and increased cell death. Together, our data reason for a quantitative contribution of CDK7 to mRNA synthesis, which is critical for cellular homeostasis. C yclin-dependent kinases (CDKs) form the enzymatic components of a group of heterodimeric serine/threonine kinases that have important roles in multiple cellular processes (1). CDK7/KIN28 was originally identified as a critical regulator of mRNA transcription in Saccharomyces cerevisiae (2-5). In vertebrates CDK7 has a dual function, influencing cell cycle progression and RNA polymerase II (RNAPII) transcription (6). Specifically, CDK7 forms the CDK-activating kinase (CAK) with two other TFIIH subunits, cyclin H and MAT1. The CAK activates downstream cell cycle CDKs, including cdc-2/CDK1, CDK2, CDK4, and CDK6, by phosphorylating key threonine residues in a process known as T-loop activation (7,8). In transcription, as RNAPII begins to lose contact with many of the general transcription factors (GTFs) during promoter escape, CDK7, functioning as part of the TFIIH complex, phosphorylates RNAPII and allows the elongation complex to move downstream away from the transcription start site (TSS) (reviewed in reference 9). Specifically, CDK7 directly targets the carboxyl-terminal domain (CTD) of the Rpb1 subunit of RNAPII, which is comprised of 52 heptad repeats (Y 1 S 2 P 3 T 4 S 5 P 6 S 7 ) in humans. While the serine 2 (Ser2), serine 5 (Ser5), and serine 7 (Ser7) residues are all subject to phosphorylation, CDK7 preferentially targets Ser5 and Ser7 (10-17). Phosphorylation patterning of the CTD is important as it influences the association of numerous nuclear factors with RNAPII (18,19), as was recently demonstrated in yeast, where KIN28-driven phosphorylation of Ser5 residues was shown to trigger dissociation of the coactivator Mediator (20). In mammals, the exact mechanisms linking CTD phosphorylation (CTD-P) with transcription are yet to be fully elucidate...

New insight into the phosphorylation-regulated intranuclear localization of human cytomegalovirus pUL69 mediated by cyclin-dependent kinases (CDKs) and viral CDK orthologue pUL97

“…As shown in Figs 3(b) and S2, LDC4297 induced the typical pattern of intranuclear fine-speckled accumulation of pUL69. The impact of CDK7 on pUL69 localization was further substantiated by a second CDK7-specific compound, LDC3140 (Kelso et al, 2014), inducing an identical pattern of speckled pUL69 aggregation (Fig. S2 panels q-t).…”

A Novel CDK7 Inhibitor of the Pyrazolotriazine Class Exerts Broad-Spectrum Antiviral Activity at Nanomolar Concentrations

“…It should be stressed that LDC4297 showed an extreme selectivity for CDK7 compared to CDK2 (Ͼ280-fold) under physiologically relevant concentrations of ATP (i.e., 3.5 mM, also representing levels of ATP contained in our cell culture systems). This CDK7 selectivity was much more pronounced for LDC4297 than for other compounds of this chemical class (e.g., LDC3140, ϳ38-fold) (33).…”

“…The assay for all enzymes contained 70 mM HEPES-NaOH (pH 7.5), 3 mM MgCl, 3 M sodium orthovanadate, 1.2 mM dithiothreitol, ATP-[␥- 33 P]ATP (variable amounts, corresponding to the apparent ATP K m of the respective kinase; approximately 8 ϫ 10 5 cpm per well), and purified protein kinase and substrate (both variable amounts). The chemicals that were used in specific assays were as follows: all protein kinase C (PKC) assays (except the PKC-and PKC-assays) also contained 1 mM CaCl 2 , 4 mM EDTA, 5 g/ml phosphatidylserine, and 1 g/ml 1,2-dioleyl-glycerol; the CAMK1D, CAMK2A, CAMK2B, CAMK2D, CAMK4, CAMKK1, CAMKK2, DAPK2, EEF2K, MYLK, MYLK2, and MYLK3 assays included 1 g/ml calmodulin and 0.5 mM CaCl; the PRKG1 and PRKG2 assays contained 1 M cGMP; and the DNA pharmacokinetic (PK) assay contained 2.5 g/ml DNA.…”

“…1B) and by competition with an ATP-directed chemical probe. Thereby, the affinities of hit compounds for binding to the ATP-binding pocket of CDKs were determined and binding affinities to CDK7 or CDK2 were quantitated as the ability to compete or displace the CDK binding of a labeled tracer (33). The affinity of LDC4297 for CDK7 proved to be extremely high, so that the exact CDK7 affinities could not be resolved by tracer competition approaches under conventional assay conditions.…”

“…After medicinal chemistry optimization of the hit series, distinct lead compounds were identified possessing IC 50 values for CDK7 below 5 nM, among them the primary hit LDC4297 ( Fig. 1A; for further compound information, see Kelso et al [33]; patent WO2013128028A1). A kinome-wide analysis with 333 individual kinases was performed to assess compound selectivity on the basis of the inhibition of in vitro standard kinase activity (PanQinase activity assay).…”