CDK9 keeps RNA polymerase II on track

Abstract

Cyclin-dependent kinase 9 (CDK9), the kinase component of positive transcription elongation factor b (P-TEFb), is essential for transcription of most protein-coding genes by RNA polymerase II (RNAPII). By releasing promoter-proximally paused RNAPII into gene bodies, CDK9 controls the entry of RNAPII into productive elongation and is, therefore, critical for efficient synthesis of full-length messenger (m)RNAs. In recent years, new players involved in P-TEFb-dependent processes have been identified and an important function of CDK9 in coordinating elongation with transcription initiation and termination has been unveiled. As the regulatory functions of CDK9 in gene expression continue to expand, a number of human pathologies, including cancers, have been associated with aberrant CDK9 activity, underscoring the need to properly regulate CDK9. Here, I provide an overview of CDK9 function and regulation, with an emphasis on CDK9 dysregulation in human diseases.

Keywords: 7SK RNA; Cyclin T1; HIV; Promoter-proximal pausing; RNA polymerase II CTD; Transcriptional checkpoint.

Fig. 1

P-TEFb promotes the release of RNAPII from pause sites. Shortly after initiation, progression of RNAPII along the gene is halted by the coordinated action of NELF and DSIF, while protein phosphatase 4 (PP4) helps maintain the Spt5 subunit of DSIF in an unphosphorylated state. Once P-TEFb is recruited, it phosphorylates DSIF, NELF and the RNAPII CTD. Phosphorylation of Spt5 converts DSIF in a positive elongation factor, and NELF is evicted from the RNAPII complex. Phosphorylation of Spt5 is further reinforced by inhibitory phosphorylation of PP4. Upon pause release, accessory proteins join the elongation complex to increase RNAPII processivity and to increase the elongation rate. Phosphorylation of the CTD facilitates the recruitment of CTD reader proteins required for pre-mRNA processing. PIC: pre-initiation complex. CTD: carboxyl-terminal domain. NELF: negative elongation factor. DSIF: DRB-sensitivity inhibitory factor. TSS: transcription start site. Pre-mRNA: pre-messenger RNA

Fig. 2

Functions of promoter-proximal pausing and regulation of pause duration. Promoter-proximal pausing limits the rate of new initiation by RNAPII, facilitates capping of the nascent pre-mRNA and provides a window of time for integration of external signals or stimuli. Pause duration is tightly regulated and can vary from one gene to another. The residence time of RNAPII at pause sites is driven by the balance between P-TEFb recruitment, phosphatase activities and recruitment of termination factors such as Integrator, which can promote premature termination. CTD: carboxyl-terminal domain. NELF: negative elongation factor. DSIF: DRB-sensitivity inhibitory factor. PP: protein phosphatase

Fig. 3

CDK9 regulates transcription across the polyA site. During elongation, CDK9 phosphorylation inactivates PP4 and PP1 phosphatase activities, thus maintaining high levels of Spt5 phosphorylation. Once RNAPII transcribes through the polyA (pA) site, Ser2P recruits the cleavage and polyadenylation machinery (CPA), which is critical for proper termination. Concomittantly, CDK9 activity drops and PP1 becomes active, leading to dephosphorylation of SPT5. Unphosphorylated Spt5 acts as a brake for RNAPII that becomes a preferential substrate for CDK9-activated Xrn2 exonuclease. PP: protein phosphatase. CPA: cleavage and polyadenylation factors

Fig. 4

Cyclin T1 promotes the formation of ‘elongation condensates’ through liquid–liquid phase separation. Intrinsically disordered regions (IDRs) of transcription factors trigger the formation of ‘initiation’ condensates at gene promoters and enhancers and favour recruitment of RNAPII and gene activity. The Cyclin T1 histidine-rich region (HRD) organizes a phase-separated environment that promotes incorporation of P-TEFb into another type of condensate associated with transcription elongation (or nuclear speckles). These condensates simultaneously concentrate pause release, elongation and processing factors. CTD phosphorylation by CDK7 relocates RNAPII from ‘initiation’ to ‘elongation’ condensates, providing RNAPII with critical activities required for the next stage of the transcription cycle. CTD: carboxy-terminal domain

Fig. 5

Regulation of CDK9 availability by the 7SK snRNP. In the nucleoplasm, a fraction of P-TEFb is sequestered into the 7SK/P-TEFb snRNP, where CDK9 activity is inhibited by HEXIM1 in a 7SK-dependent manner. After cellular stress, disassociation of the 7SK/P-TEFb snRNP releases active P-TEFb, which can be loaded onto target genes by BRD4, Super Elongation Complexes (SEC) or gene-specific transcription factors (TF). The 7SK/P-TEFb snRNP is also directly targeted to gene promoters by KAP1. Factors such as SRSF2, DDX21 or PPM1G can activate P-TEFb from chromatin-anchored 7SK/P-TEFb snRNP in the vicinity of the RNAPII complex for ‘on site’ transcriptional activation. Note that only the active form of P-TEFb can be incorporated into transcription-associated condensates

Fig. 6

Involvement of CDK9 in human disease. a CDK9 is a primary target of HIV-1. The virus-encoded protein Tat extracts P-TEFb from the 7SK/P-TEFb RNP and increases the level of active P-TEFb in infected cells. P-TEFb is then tethered to the viral genome through interaction with the TAR structure that forms at the 5′ end of the viral nascent RNA and serves as a landing pad for the P-TEFb/Tat/SEC complex. Once recruited, P-TEFb releases paused RNAPII in the same way as it does on host protein-coding genes. b In cardiomyocytes, hypertrophic signals shift the equilibrium towards the P-TEFb active form. The resulting elevated CDK9 activity leads to the establishment of a hypertrophic transcription program and a global release of paused RNAPII into gene bodies. Elevated transcription leads to an increased level of mRNA and proteins and enlargement of cardiomyocytes. c Misregulation of CDK9 activity can lead to development of cancers. P-TEFb drives the expression of key pro-survival genes such as C-MYC and MCL-1 (top). Mistargeting of P-TEFb by MYC or MLL-SEC fusion proteins promotes tumour-specific transcriptional amplification (middle). P-TEFb represses tumor-suppressor genes through inhibitory phosphorylation of the BRG1 subunit of the BAF chromatin-remodeling complex