Transcription through early-elongation checkpoints requires phosphorylation of negative transcription elongation factors (NTEFs) by the cyclin-dependent kinase (CDK)9. Using CDK9 inhibitors and global run-on sequencing (GRO-seq), we have mapped CDK9 inhibitor-sensitive checkpoints genome-wide in human (Homo sapiens) cells. Our data indicate that early-elongation checkpoints are a general feature of RNA polymerase (pol) II-transcribed human genes and occur independently of polymerase stalling. Pol II that has negotiated the early-elongation checkpoint can elongate in the presence of inhibitors but, remarkably, terminates transcription prematurely close to the terminal polyadenylation (poly(A)) site. Our analysis has revealed a hitherto-unsuspected poly(A)-associated elongation checkpoint, which has major implications for the regulation of gene expression. Interestingly, the pattern of modification of the carboxyl-terminal domain (CTD) of pol II terminated at this novel checkpoint largely mirrors the pattern normally found downstream of the poly(A) site, suggesting common mechanisms of termination.
Regions of hypoxia occur in most solid tumors, and they are associated with a poor prognostic outcome. Despite the absence of detectable DNA damage, severe hypoxia (<0.1% O2) induces a DNA damage response, including the activation of p53 and subsequent induction of p53-dependent apoptosis. Factors affecting hypoxia-induced p53-dependent apoptosis are unclear. Here we asked whether H3K9me3, through mediating gene repression, could regulate hypoxia-induced p53-dependent apoptosis. Under hypoxic conditions, increases in H3K9me3 occur in an oxygen-dependent but HIF-1-independent manner. We demonstrate that under hypoxic conditions, which induce p53 activity, the negative regulator of p53, APAK, is repressed by increases in H3K9me3 along the APAK loci. APAK repression in hypoxia is mediated by the methyltransferase SETDB1 but not Suv39h1 or G9a. Interestingly, increasing hypoxia-induced H3K9me3 through pharmacological inhibition of JMJD2 family members leads to an increase in apoptosis and decreased clonogenic survival and again correlates with APAK expression. The relevance of understanding the mechanisms of APAK expression regulation to human disease was suggested by analysis of patients with colorectal cancer, which demonstrates that high APAK expression correlates with poor prognosis. Together, these data demonstrate the functional importance of H3K9me3 in hypoxia, and they provide a novel mechanistic link between H3K9me3, p53 and apoptosis in physiologically relevant conditions of hypoxia.
Positive transcription elongation factor b (P-TEFb), which comprises cyclin-dependent kinase 9 (CDK9) kinase and cyclin T subunits, is an essential kinase complex in human cells. Phosphorylation of the negative elongation factors by P-TEFb is required for productive elongation of transcription of protein-coding genes by RNA polymerase II (pol II). In addition, P-TEFbmediated phosphorylation of the carboxyl-terminal domain (CTD) of the largest subunit of pol II mediates the recruitment of transcription and RNA processing factors during the transcription cycle. CDK9 also phosphorylates p53, a tumor suppressor that plays a central role in cellular responses to a range of stress factors. Many viral factors affect transcription by recruiting or modulating the activity of CDK9. In this review, we will focus on how the function of CDK9 is regulated by viral gene products. The central role of CDK9 in viral life cycles suggests that drugs targeting the interaction between viral products and P-TEFb could be effective anti-viral agents.
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