Positive transcription elongation factor b (P-TEFb) is a kinase that phosphorylates the carboxyl-terminal domain (CTD) of RNA Polymerase II (Pol II). Here, we show that flavopiridol, a highly specific P-TEFb kinase inhibitor, dramatically reduces the global levels of Ser2--but not Ser5--phosphorylated CTD at actively transcribed loci on Drosophila polytene chromosomes under both normal and heat shocked conditions. Brief treatment of Drosophila cells with flavopiridol leads to a reduction in the accumulation of induced hsp70 and hsp26 RNAs. Surprisingly, the density of transcribing Pol II and Pol II progression through hsp70 in vivo are nearly normal in flavopiridol-treated cells. The major defect in expression is at the level of 3' end processing. A similar but more modest 3' processing defect was also observed for hsp26. We propose that P-TEFb phosphorylation of Pol II CTD coordinates transcription elongation with 3' end processing, and failure to do so leads to rapid RNA degradation.

Summary Changes of epigenetic status and chromatin structure have been shown to associate with aging in many organisms. Here, we report an RNAi screen of putative histone methyltransferases and demethylases in wild type C. elegans using reproduction inhibitor. We identified six genes that, when inactivated by RNAi, consistently extend lifespan. Five of these genes do not require germline proliferation to affect lifespan. We further characterized two of these genes, the highly homologous SET-domain containing genes, set-9 and set-26. They share redundant functions in maintaining normal lifespan, while exhibiting differential tissue expression patterns. Furthermore, we found that set-9 and set-26 partially act through the FOXO transcription factor, DAF-16, to modulate lifespan. Interestingly, inactivation of somatic SET-26 alone results in a robust lifespan extension, and alters the levels of histone H3 protein and the repressive histone marks, H3K9me3 and H3K27me3, in an age-dependent manner. We hypothesize that inactivation of SET-26 triggers compensation mechanisms to restore repressive chromatin structure, and hence affects chromatin stability to promote longevity.

Positive transcription elongation factor b (P-TEFb) is the major metazoan RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) Ser2 kinase, and its activity is believed to promote productive elongation and coupled RNA processing. Here, we demonstrate that P-TEFb is critical for the transition of Pol II into a mature transcription elongation complex in vivo. Within 3 min following P-TEFb inhibition, most polymerases were restricted to within 150 bp of the transcription initiation site of the active Drosophila melanogaster Hsp70 gene, and live-cell imaging demonstrated that these polymerases were stably associated. Polymerases already productively elongating at the time of P-TEFb inhibition, however, proceeded with elongation in the absence of active P-TEFb and cleared from the Hsp70 gene. Strikingly, all transcription factors tested (P-TEFb, Spt5, Spt6, and TFIIS) and RNA-processing factor CstF50 exited the body of the gene with kinetics indistinguishable from that of Pol II. An analysis of the phosphorylation state of Pol II upon the inhibition of P-TEFb also revealed no detectable CTD Ser2 phosphatase activity upstream of the Hsp70 polyadenylation site. In the continued presence of P-TEFb inhibitor, Pol II levels across the gene eventually recovered.RNA polymerase II (Pol II) undergoes a series of critical modifications as it progresses through a cycle of transcription. The carboxyl-terminal domain (CTD) of the largest subunit of Pol II, which consists of tandem heptapeptide repeats with the consensus sequence Y 1 S 2 P 3 T 4 S 5 P 6 S 7 , is a major target for these modifications, which include phosphorylation (primarily of serines 2 and 5 of the consensus repeat), proline cis/trans isomerization, and glycosylation (36). Distinct phosphorylation marks are associated with different transcription stages: in metazoans, Pol II enters the preinitiation complex in a hypophosphorylated state; it progresses from the initiation site to promoter-proximal regions, where it is phosphorylated primarily at Ser5; it matures into a doubly (Ser2 and Ser5) phosphorylated, productively elongating Pol II; and upon passing the polyadenylation site, it displays reduced phosphorylation (7,20,30). These phosphorylation states allow Pol II to interact with distinct repertoires of proteins at different stages of the transcription cycle and are critical in the coordination of transcription and coupled pre-mRNA processing (44,46). The Cdk7 and Cdk9 kinases are responsible for most CTD serine phosphorylation in metazoans (39). Cdk7, a subunit of the general transcription factor TFIIH, phosphorylates the CTD preferentially at Ser5 (47,53,60). By contrast, Cdk9, which together with cyclin T composes the P-TEFb kinase complex (42), phosphorylates the CTD predominantly at Ser2 (39, 47, 57, 71). P-TEFb and Cdk7 both have positive roles in transcription and function at postinitiation steps (3,22,33,56). P-TEFb recruitment to an activated gene is concomitant with the efficient release of Pol II from a promoter-proximally paused to a product...

Uninduced heat shock genes are poised for rapid activation, with RNA polymerase II (Pol II) transcriptionally engaged, but paused or stalled, within the promoter-proximal region. Upon heat shock, this Pol II is promptly released from the promoter region and additional Pol II and transcription factors are robustly recruited to the gene. Regulation of the heat shock response relies upon factors that modify the efficiency of elongation through the initially transcribed sequence. Here, we report that Pol II is susceptible to transcription arrest within the promoter-proximal region of Drosophila hsp70 and that transcript cleavage factor TFIIS is essential for rapid induction of hsp70 RNA. Moreover, using a tandem RNAi-ChIP assay, we discovered that TFIIS is not required to establish the stalled Pol II, but that TFIIS is critical for efficient release of Pol II from the hsp70 promoter region and the subsequent recruitment of additional Pol II upon heat induction.

Functional data indicate that specific histone modification enzymes can be key to longevity in Caenorhabditis elegans, but the molecular basis of how chromatin structure modulates longevity is not well understood. In this study, we profiled the genome-wide pattern of trimethylation of Lys36 on histone 3 (H3K36me3) in the somatic cells of young and old Caenorhabditis elegans. We revealed a new role of H3K36me3 in maintaining gene expression stability through aging with important consequences on longevity. We found that genes with dramatic expression change during aging are marked with low or even undetectable levels of H3K36me3 in their gene bodies irrespective of their corresponding mRNA abundance. Interestingly, 3′ untranslated region (UTR) length strongly correlates with H3K36me3 levels and age-dependent mRNA expression stability. A similar negative correlation between H3K36me3 marking and mRNA expression change during aging was also observed in Drosophila melanogaster, suggesting a conserved mechanism for H3K36me3 in suppressing age-dependent mRNA expression change. Importantly, inactivation of the methyltransferase met-1 resulted in a decrease in global H3K36me3 marks, an increase in mRNA expression change with age, and a shortened life span, suggesting a causative role of the H3K36me3 marking in modulating age-dependent gene expression stability and longevity.