CDK9: a signaling hub for transcriptional control

Abstract

Cyclin-dependent kinase 9 (CDK9) is critical for RNA Polymerase II (Pol II) transcription initiation, elongation, and termination in several key biological processes including development, differentiation, and cell fate responses. A broad range of diseases are characterized by CDK9 malfunction, illustrating its importance in maintaining transcriptional homeostasis in basal- and signal-regulated conditions. Here we provide a historical recount of CDK9 discovery and the current models suggesting CDK9 is a central hub necessary for proper execution of different steps in the transcription cycle. Finally, we discuss the current therapeutic strategies to treat CDK9 malfunction in several disease states. Abbreviations: CDK: Cyclin-dependent kinase; Pol II: RNA Polymerase II; PIC: Pre-initiation Complex; TFIIH: Transcription Factor-II H; snoRNA: small nucleolar RNA; CycT: CyclinT1/T2; P-TEFb: Positive Transcription Elongation Factor Complex; snRNP: small nuclear ribonucleo-protein; HEXIM: Hexamethylene Bis-acetamide-inducible Protein 1/2; LARP7: La-related Protein 7; MePCE: Methylphosphate Capping Enzyme; HIV: human immunodeficiency virus; TAT: trans-activator of transcription; TAR: Trans-activation response element; Hsp70: Heat Shock Protein 70; Hsp90/Cdc37: Hsp90- Hsp90 co-chaperone Cdc37; DSIF: DRB Sensitivity Inducing Factor; NELF: Negative Elongation Factor; CPSF: cleavage and polyadenylation-specific factor; CSTF: cleavage-stimulatory factor; eRNA: enhancer RNA; BRD4: Bromodomain-containing protein 4; JMJD6: Jumonji C-domain-containing protein 6; SEC: Super Elongation Complex; ELL: eleven-nineteen Lys-rich leukemia; ENL: eleven-nineteen leukemia; MLL: mixed lineage leukemia; BEC: BRD4-containing Elongation Complex; SEC-L2/L3: SEC-like complexes; KAP1: Kruppel-associated box-protein 1; KEC: KAP1-7SK Elongation Complex; DRB: Dichloro-1-ß-D-Ribofuranosylbenzimidazole; H2Bub1: H2B mono-ubiquitination; KM: KM05382; PP1: Protein Phosphatase 1; CDK9i: CDK9 inhibitor; SHAPE: Selective 2'-hydroxyl acylation analyzed by primer extension; TE: Typical enhancer; SE : Super enhancer.

Keywords: 7SK; CDK9; Cancer; Disease; Elongation; Enhancer; HIV; P-TEFb; Pausing; RNA polymerase II; Transcription.

Figure 1.

CDK9 activity is tightly regulated through incorporation into several regulatory complexes. (a) CDK9 Western Blot. CDK9 exists in two isoforms, a 55-kDa long form and the best characterized, 42-kDa short form. CDK9 probed in HCT-116 cells with anti-CDK9 (D-7, sc-13130). (b) CDK9 Crystal Structure. Structure of CDK9 showing the ATP binding site (green) and T186 in the T-loop (red). Note that the structure is of CDK9 complexed with CycT [27] but CycT was omitted for simplicity. CDK9 has not been crystallized in the apo form. (c) CDK9 kinase activity is regulated through incorporation into the 7SK snRNP complex. Phosphorylation of CDK9’s T-loop (T186) promotes its interaction with HEXIM and incorporation into the 7SK snRNP. The 7SK RNA/snRNP model depicted is based on Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) data from the Price lab [35]. (d) ES cells utilize the 7SK snRNP for CDK9 kinase regulation. In ES cells, CDK9 is inactivated through incorporation into the 7SK snRNP (left). Upon ES stimulation (e.g., retinoic acid), CDK9 release factors (CDK9 RF) promote the release of CDK9, resulting in kinase activation for target gene transcription and cell differentiation (right). (e) Resting CD4 T cells prevent CDK9/CycT assembly. In resting T cells, CDK9 is sequentially incorporated into the Hsp70 and the co-chaperone Hsp90/CDC37 complex in the cytoplasm, where it awaits assembly with CycT (left). Upon T cell activation (e.g., antigen, Toll-like receptor signaling), CDK9 is released from the co-chaperone complex, assembled with newly-synthesized CycT and delivered to T cell responsive genes for transcription activation (right).

Figure 2.

CDK9 is a central hub for proper signaling of each step in the transcription cycle. (a) CDK9 phosphorylation signals for Pol II pause release and initiation. Pausing of Pol II is facilitated by the negative elongation factors, DSIF and NELF (top) and blocks initiation of new Pol II molecules. Upon transcription stimulation, CDK9 phosphorylates the negative elongation factors and Ser2 of the Pol II CTD at promoters resulting in the removal of elongation blocks, release of Pol II into elongation, and initiation of new Pol II molecules (bottom). Inhibition of CDK9 (CDK9i) prohibits release of Pol II into elongation thereby blocking new polymerases from initiating (right). b) CDK9 is critical for proper transcription termination. Pol II pauses at poly(A) sites allowing for the CDK9-dependent phosphorylation of the Pol II CTD and recruitment of termination factors. CTD phosphorylation then provides a platform for the recruitment of termination factors at poly(A) sites and pause-release for proper termination and 3ʹ-end formation. CDK9 also phosphorylates a regulatory subunit of the PP1 phosphatase, preventing dephosphorylation of DSIF and premature termination. Once released from its paused state, Pol II transcribes through the poly(A) site resulting in the activation of PP1, dephosphorylation of DSIF, and transcription termination. CDK9 inhibitor (CDK9i) studies found that inhibition of CDK9 decreases transcript levels downstream of the poly(A) site resulting in a termination defect (right). Note that the different Pol II forms (initiating, pausing, elongating, and terminating) are color-coded for ease visualization.

Figure 3.

CDK9-containing elongation complexes and their functional interplay. (a) CDK9 assembles into multiple elongation complexes. Active CDK9 is bound to the SEC and BEC. Inactive CDK9, as part of the 7SK snRNP, is bound to KAP1 forming the KEC “pre-elongation” complex. (b) CDK9-containing complexes deliver CDK9 to multiple genomic loci The BEC and SEC deliver CDK9 to enhancer and super-enhancer regions. Additionally, both the SEC and KEC have been shown to deliver CDK9 to gene promoters. Also, BRD4 has been shown to recruit CDK9 to gene-specific promoters through interactions with gene-specific transcription factors. One hypothetical model of cooperation among the CDK9 delivery complexes is that positioning of inactive CDK9 by the KEC could allow for timely release and incorporation of active CDK9 into the SEC and/or BEC at enhancers and/or promoters.

Figure 4.

Diseases driven by malfunctioning CDK9 and therapeutic strategies (a) MLL promotes differentiation of stem cells. In ES cells, MLL recruits SET methyltransferases to promoters of HOX genes, leading to deposition of H3K4me3, gene activation, and proper differentiation. (b) MLL malfunction and therapeutic strategies in disease. In leukemia cells that express MLL/SEC fusion protein, WT-MLL is targeted by the UBE2O ubiquitin ligase for degradation, leading to an increase in SEC occupancy at HOX gene promoters and aberrant HOX gene activation. A promising new therapeutic strategy for leukemia inhibits the degradation of WT-MLL leading to increased occupancy of WT-MLL at HOX gene promoters, which in turn displaces MLL/SEC fusion protein leading to HOX downregulation. (c) HIV hijacks CDK9 for transcription of its own genome. The HIV Tat protein tethers CDK9/CycT to the HIV promoter through its interaction with the HIV TAR RNA (left). Tat inhibitors suppress HIV and are crucial for the success of the “block and lock” curative strategy of HIV infection (right).