Research

Transcription of genes is a fundamental property of the living. We employ biochemistry, genetics and genome wide approaches to understand how gene transcription by Pol II is controlled by the positive transcription elongation factor (P-TEFb; CDK9/CycT) and its inhibitory 7SK snRNP complex at the promoter-proximal Pol II pause release step. Moreover, we are exploring how another critical transcriptional elongation kinase, the Cdk12/CycK complex, impacts target gene expression, and how frequent CDK12 cancer mutations perturb gene transcription programs to enable cancerogenesis. Finally, through the design of novel methodologies, we are aiming to uncover mechanisms that facilitate integration of Pol II transcription with maturation of precursor RNA transcripts.

While transcribing protein-coding and non-coding genes, Pol II goes through a series of specific steps, including loading of Pol II to gene promoter, initiation of RNA synthesis from transcription start site (TSS), promoter-proximal pausing immediately downstream of TSS, elongation along the body of a gene, termination, and eventual re-initiation. Historically, it has been thought that Pol II initiation is the rate-limiting step in gene transcription.

However, pioneering work in late 1980’s and early 90’s on human, fruit fly genes and HIV-1 transcription began to challenge the prevailing paradigm by revealing that Pol II could fail to generate full-length RNA transcripts because of a block soon after the start of transcription. Supporting this early work, novel genome wide approaches have revealed recently that upon transcription initiation, the bulk of Pol II is found at promoter-proximal pause sites, 30-100 nucleotides downstream of TSS (Figure 1, top). Thus, controlling the release of Pol II from pausing into productive elongation has emerged as the critical step in gene expression in multicellular organisms.

Perhaps not surprisingly, a mounting body of evidence has linked deregulation of Pol II elongation control to the onset of many hypertrophic and hyperproliferative diseases. These include cardiac hypertrophy and a variety of cancers, such as breast, gastric, skin, ovarian cancer and childhood hematological malignancies. Because addiction to transcriptional programs under the control of P-TEFb (CDK9/CycT) and CDK12/CycK is likely driving the identity of the hypertrophic and cancer cells, there is an enormous potential for including the targeting of the Pol II elongation kinases in future therapeutic interventions against the diseases.

The critical factor that triggers the release of paused Pol II into productive elongation at almost all genes of multicellular organisms is P-TEFb, which is composed of the catalytic Cdk9 and a regulatory CycT1, T2a or T2b subunits (Figure 1, bottom). To accomplish the goal, P-TEFb phosphorylates the C-terminal domain of Pol II at Serine 2 residues (Ser2-P), which is the defining mark active of Pol II elongation, to tether additional elongation, RNA maturation and chromatin modifying factors to Pol II. At the same time, P-TEFb counteracts the actions of multi-subunit Pol II pause-inducing factors NELF and DSIF. Here, phosphorylation of the NELF-E subunit evicts NELF from Pol II, and phosphorylation of the Spt5 subunit of DSIF transforms it into a positive elongation factor, promoting the Pol II pause release.

Befitting the general role of P-TEFb in Pol II transcription, its kinase activity is kept under a tight control by the inhibitory 7SK snRNP complex (Figure 2). In fact, up to 90% of P-TEFb can be sequestered inside 7SK snRNP, in which kinase activity of CDK9 is repressed by dimeric P-TEFb inhibitors HEXIM1 and HEXIM2. In addition to these proteins, canonical 7SK snRNP is composed of the following core 7SK snRNP components: the non-coding 7SK snRNA, La-related protein family member LARP7 and the 7SK γ-methylphosphate capping enzyme MePCE. While 7SK snRNA functions as an RNA scaffold of the snRNP, LARP7 and MePCE promote 7SK stability by binding 3’ end of 7SK and capping 5’ end of 7SK, respectively.

Importantly, P-TEFb-dependent Pol II pause release is frequently dysregulated in cancers, particularly in those addicted to c-MYC and translocations of mixed-lineage leukemia (MLL) gene. Hence, understanding fundamental biology of P-TEFb will provide foundation for anti-cancer therapies of the future.

These are some of the questions that we are currently addressing:

  • What are molecular mechanisms that drive the release of P-TEFb from 7SK snRNP?
  • Does P-TEFb activation play a prominent role in response to genotoxic stress?
  • Could we exploit the knowledge of P-TEFb biology for therapeutic purposes? 

In addition to the well-established P-TEFb kinase, recent work in yeast, fruit fly and human systems indicates that the Cdk12/CycK complex catalyzes the active Ser2-P elongation mark as well. Illustrating the significance of this novel transcription elongation kinase, genetic inactivation of CYCK and CDK12 in mice is embryonic lethal. In contrast to the broad requirement for P-TEFb in transcription, depletion of CDK12/CycK in human cells leaves steady-state levels of most gene transcripts unaltered. However, a prominent group among down-regulated genes includes many core players that ensure genomic stability, such as BRCA1, ATR, FANCI and FANCD2. Consistent with this finding, cells without CDK12/CycK exhibit spontaneous DNA damage and are sensitive to a variety of DNA damage agents. Together, these findings suggest that CDK12/CycK plays a vital role in maintaining genomic stability.

Critically, studies of CDK12/CycK are important from the biomedical standpoint. Namely, The Cancer Genome Atlas (TCGA) work on the high-grade serous ovarian carcinoma (HGS-OvCa) identified CDK12 as one of only nine genes with statistically recurrent somatic mutations, raising the possibility that inactivation of CDK12 is important for the development of this common cause of death in women worldwide. Indeed, our recent work has examined the HGS-OvCa CDK12 mutations biochemically and functionally, providing fundamental insights into how inactivation of CDK12/CycK could contribute to cancerogenesis.

These are some of the questions that we are currently addressing:

  • What are molecular mechanisms that enable the role of CDK12/CycK in transcription?
  • How do the loss-of-function CDK12 patient mutations triggers genomic instability?