We have purified a human histone H4 lysine 20 methyltransferase and cloned the encoding gene, PR/SET07. A mutation in Drosophila pr-set7 is lethal: second instar larval death coincides with the loss of H4 lysine 20 methylation, indicating a fundamental role for PR-Set7 in development. Transcriptionally competent regions lack H4 lysine 20 methylation, but the modification coincided with condensed chromosomal regions on polytene chromosomes, including chromocenter and euchromatic arms. The Drosophila male X chromosome, which is hyperacetylated at H4 lysine 16, has significantly decreased levels of lysine 20 methylation compared to that of females. In vitro, methylation of lysine 20 and acetylation of lysine 16 on the H4 tail are competitive. Taken together, these results support the hypothesis that methylation of H4 lysine 20 maintains silent chromatin, in part, by precluding neighboring acetylation on the H4 tail.

Recent studies have demonstrated roles for Spt4, Spt5, and Spt6 in the regulation of transcriptional elongation in both yeast and humans. Here, we show that Drosophila Spt5 and Spt6 colocalize at a large number of transcriptionally active chromosomal sites on polytene chromosomes and are rapidly recruited to endogenous and transgenic heat shock loci upon heat shock. Costaining with antibodies to Spt6 and to either the largest subunit of RNA polymerase II or cyclin T, a subunit of the elongation factor P-TEFb, reveals that all three factors have a similar distribution at sites of active transcription. Crosslinking and immunoprecipitation experiments show that Spt5 is present at uninduced heat shock gene promoters, and that upon heat shock, Spt5 and Spt6 associate with the 5 and 3 ends of heat shock genes. Spt6 is recruited within 2 minutes of a heat shock, similar to heat shock factor (HSF); moreover, this recruitment is dependent on HSF. These findings provide support for the roles of Spt5 in promoter-associated pausing and of Spt5 and Spt6 in transcriptional elongation in vivo.

The uninduced Drosophila hsp70 gene is poised for rapid activation. Here we examine the rapid changes upon heat shock in levels and location of heat shock factor (HSF), RNA polymerase II (Pol II) and its phosphorylated forms, and the Pol II kinase P-TEFb on hsp70 in vivo by using both real-time PCR assays of chromatin immunoprecipitates and polytene chromosome immunofluorescence. These studies capture Pol II recruitment and progression along hsp70 and reveal distinct spatial and temporal patterns of serine 2 and serine 5 phosphorylation: in uninduced cells, the promoter-paused Pol II shows Ser5 but not Ser2 phosphorylation, and in induced cells the relative level of Ser2-P Pol II is lower at the promoter than at regions downstream. An early time point of heat shock activation captures unphosphorylated Pol II recruited to the promoter prior to P-TEFb, and during the first wave of transcription Pol II and the P-TEFb kinase can be seen tracking together across hsp70 with indistinguishable kinetics. Pol II distributions on several other genes with paused Pol II show a pattern of Ser5 and Ser2 phosphorylation similar to that of hsp70. These studies of factor choreography set important limits in modeling transcription regulatory mechanisms.Recent studies of posttranslational modifications of, and factor interactions with, the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) have implicated this region of Pol II in productive transcriptional elongation and the coupling of transcription and pre-mRNA processing (for reviews see references 32 and 37). The CTD is highly conserved among eukaryotes, containing multiple repeats of a consensus heptad YSPTSPS; mammalian Pol II CTD consists of 52 repeats, yeast consists of 25 to 26 repeats, while Drosophila has 42 repeats (1, 6). This large flexible arm of Pol II appears to serve as a docking site for, or to stimulate the recruitment of, an orchestrated assembly of factors involved in pre-mRNA capping, splicing, and 3Ј polyadenylation at different stages in production of the nascent transcript (5,9,13,14,20,26). One way this coordination has been proposed to occur is through the known phosphorylation of serines 2 and 5 (Ser2-P and Ser5-P, respectively) of the heptad repeat (52, 56). Cdk7, the kinase subunit of general transcription factor TFIIH, has been shown to phosphorylate the CTD at Ser5, an event proposed to occur early in the transcription cycle (12,24,57). This in turn appears to influence the association and activity of the capping machinery (5,15,18,27,28,42,45). Positive transcription elongation factor b (P-TEFb) is able to phosphorylate the CTD at Ser2 and, under certain conditions, Ser5 (25, 38, 57). P-TEFb is a kinase composed of the proteins cyclin T (CycT) and cdk9 and is known to be recruited upon gene activation, overcoming the negative effects of factors like Spt5 and negative elongation factor (50) and aiding in the transition from transcription initiation to elongation (36). Cdk8, a component of the coactivator complex Mediator, h...

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.

The RNA polymerase II elongation complex contains several factors that facilitate transcription elongation and catalyse the processing of precursor messenger RNAs (pre-mRNAs). The conserved elongation factor Spt6 is recruited rapidly and robustly to sites of active transcription. Here we show that Drosophila Spt6 (dSpt6) co-purifies with the exosome, a complex of 3' to 5' exoribonucleases that is implicated in the processing of structural RNA and in the degradation of improperly processed pre-mRNA. Immunoprecipitation assays of Drosophila nuclear extracts show that the exosome also associates with the elongation factor dSpt5 and RNA polymerase II. In vivo, exosome subunits colocalize with dSpt6 at transcriptionally active loci on polytene chromosomes during normal development and are strongly recruited to heat-shock loci on gene induction. At higher resolution, chromatin immunoprecipitation analysis shows that the exosome is recruited to transcriptionally active units of heat-shock genes. These data provide a physical basis for the hypothesis that exosome-mediated pre-mRNA surveillance accompanies transcription elongation.