Immunofluorescence staining of formaldehyde-fixed, polytene chromosomes with the antibody to cyclin T provides a global view of its distribution on chromosomes. Cyclin T is present at 60 sites at moderate or high levels and another ~140 sites at lower levels. The most prominent sites include chromosomal puffs and contain genes that are transcriptionally active during this stage of development. Even the highly diffuse early, ecdysone-induced puffs show prominent labeling, although the signal is spread over a large area. This analysis was carried out at the third instar larval stage -- specifically at a time when early ecdysone puffs at 74E and 75B are near the end of their maximally active phase [puff stage 7]. These large puffs show high levels of P-TEFb as do many of the other major early ecdysone-inducible puffs including 2B5-6, 2B13-17, 62E, 71E, 72D, 88D, 89B, and 93D. At an earlier developmental stage (puff stage 1-2) the 68C puff is near maximal activity and is a major site of P-TEFb localization, as are other intermolt puffs at 3C, 71E, and 90BC, which all encode one or more abundantly expressed salivary gland secretion proteins. These changes in the distribution of P-TEFb during development indicate that P-TEFb is directed to a large number of genes when they become active. Although these experiments do not address whether or not cyclin T is providing a critical function at every site it occupies, this pattern of labeling is consistent with its participation in the transcriptional regulation of numerous, but not necessarily all, active Drosophila genes (Lis, 2000).
Phosphorylation of the large RNA Polymerase II subunit C-terminal domain (CTD) is believed to be important in promoter clearance and for recruiting protein factors that function in messenger RNA synthesis and processing. P-TEFb
is a protein kinase that targets the (CTD). The goal of this study was to identify chromatin modifications and associations that require P-TEFb activity in vivo. The catalytic subunit of P-TEFb, Cdk9, was knocked down in Drosophila using RNA interference. Cdk9 knockdown flies die during metamorphosis. Phosphorylation at serine 2 and serine 5 of the CTD heptad repeat were both dramatically reduced in knockdown larvae. Hsp 70 mRNA induction by heat shock was attenuated in Cdk9 knockdown larvae. Both mono- and trimethylation of histone H3 at lysine 4 were dramatically reduced, suggesting a link between CTD phosphorylation and histone methylation in transcribed chromatin in vivo. Levels of the chromo helicase protein CHD1 were reduced in Cdk9 knockdown chromosomes, suggesting that CHD1 is targeted to chromosomes through P-TEFb-dependent histone methylation. Dimethylation of histone H3 at lysine 36 was significantly reduced in knockdown larvae, implicating CTD phosphorylation in the regulation of this chromatin modification. Binding of the RNA Polymerase II elongation factor ELL was reduced in knockdown chromosomes, suggesting that ELL is recruited to active polymerase via CTD phosphorylation (Eissenberg, 2007).
Cdk9, the catalytic subunit of P-TEFb, is highly conserved among eukaryotes. The yeast kinases Ctk1 and Bur1 are both homologs of Cdk9, and both are CTD kinases in Drosophila, although loss of Bur1 has no effect on CTD phosphorylation yeast. Bur1 is essential but Ctk1 is not (Eissenberg, 2007).
RNAi knockdown of Cdk9 in transgenic flies results in lethality at the pupal stage. This is considerably later than the embryonic lethality reported for C. elegans RNAi knockdown of Cdk9. While this difference could reflect differences in the requirements for Cdk9 in these organisms, it is more likely that differences in timing or efficiency of RNAi, Cdk9 protein turnover and/or maternal Cdk9 loading accounts for the much later lethality in knockdown flies. Nevertheless, these results confirm and extend the finding that P-TEFb is essential in metazoan development (Eissenberg, 2007).
In contrast, Cdk9 homologs in fission yeast and Neurospora are not essential. Since CTD phosphorylation has been linked to promoter clearance, pre-mRNA processing and chromatin modification, it is not possible to say what aspect of P-TEFb activity is essential in metazoa. RNAi knockdown of the Drosophila Cdk9 in cultured cells causes arrest of the cell cycle at the G1-S transition, implicating this kinase in cell cycle control. It is unlikely that cell cycle arrest is causing the lethality in the knockdown flies, since cell cycle mutations in Drosophila generally are associated with reduced or missing imaginal discs, and the discs in Cdk9 knockdown larvae appear overtly normal. The finding that Hsp70 transcripts are reduced in Cdk9 knockdown larvae is consistent with the reduced Hsp70 transcription previously reported in Cdk9 RNAi cultured cells. Hsp 70 is not essential in Drosophila, but the effects on Hsp70 suggest that defects in gene expression could underlie the essential requirement for Cdk9 in Drosophila development (Eissenberg, 2007).
Cdk9 knock-down flies show dramatic reductions in both serine 2 and serine 5 phosphorylation. In contrast, flavopiridol treatment of cultured cells has been found to selectively reduce serine 2 phosphorylation. The significance of this difference is unclear, but could reflect differences in experimental protocol. For example, flavopiridol treatments were limited to 15-20 min, while RNAi knockdown third instar larvae are subject to knockdown conditions for several days before assay. Longer periods of Cdk9 inactivation may be required for reduction in serine 5 phosphorylation. Alternatively, it is possible that knockdown of Cdk9 protein levels results in inhibition of TFIIH, the other known CTD kinase. Regardless of the mechanism, the RNAi knockdown clearly results in reduced phosphorylation of the CTD, enabling a test of the consequences of loss of CTD phosphorylation on chromatin modification and recruitment of RNA Polymerase II-associated factors (Eissenberg, 2007).
Loss of CTD phosphorylation in Cdk9 knockdown larvae is associated with reduced binding of the RNA Polymerase II elongation factor ELL genome-wide. ELL is broadly co-localized with phosphorylated RNA Polymerase II on polytene chromosomes, and is rapidly recruited to heat shock loci after a brief heat shock. These results suggest that the efficient recruitment of ELL to transcribed loci requires CTD phosphorylation. Whether this reflects a direct interaction of ELL with the CTD is unknown (Eissenberg, 2007).
Despite the fact that Elongin A affects the same kinetic parameter in RNA Polymerase II catalysis as ELL, Elongin A binding is not reduced by loss of CTD phosphorylation. As with ELL, the nature of Elongin A binding to RNA Polymerase II is unknown, but these observations suggest their binding can be distinguished by sensitivity to the phosphorylation state of the CTD. Since no increase of Elongin A was observed under conditions of reduced ELL binding, it seems unlikely that ELL and Elongin A compete for RNA Polymerase II binding (Eissenberg, 2007).
Spt4 and Spt5 are subunits of DSIF, which is implicated in the regulation of RNA Polymerase II elongation. Previous work suggested that reduced serine 2 phosphorylation of the RNA Polymerase II CTD has no effect on Spt5 recruitment to a heat shock gene in cultured cells (Ni, 2004). In Cdk9 knockdown flies, in which both serine 2 and 5 phosphorylation are reduced, the chromosomal distribution of Spt5 is unchanged genome-wide. This is consistent with previous reports that Spt5 interacts with both phosphorylated and unphosphorylated RNA Polymerase II (Wen, 1999; Lindstrom, 2001; Lindstrom, 2003; Eissenberg, 2007 and references therein).
The chromo domain motif is a binding site for methylated histone tails. The role of the CHD1 chromo domain in methylated histone binding is controversial. However, recent structural data determined that the double chromo domain of mammalian CHD1 binds methylated H3K4 in vitro (Flanagan, 2005). This study shows that Cdk9 knockdown leads to a loss of chromosomal CHD1. This observation is most easily interpreted as the result of loss of H3K4 methylation that also occurs in Cdk9 knockdown chromosomes. Thus, the finding reported in this study lends support to the in vitro binding data and strongly suggests that the chromo domain-methylated histone interaction plays a dominant role in targeting CHD1 to active chromatin in vivo (Eissenberg, 2007).
The observation that both H3K4 and H3K36 methylation are significantly reduced in Cdk9 knockdown chromosomes suggests a linkage between phosphorylation of the CTD and histone methylation at transcribed genes. In this respect, Cdk9 subsumes activities found in yeast Bur1/Bur2 and yeast Ctk1. Since no significant difference was observed in ASH1 protein levels on Cdk9 knockdown chromosomes, a model is favored in which Cdk9-dependent RNA Polymerase II elongation plays a mechanistic role in H3 tail methylation. In this model, RNA Polymerase II passage destabilizes histone-DNA contacts, making the histones better substrates for efficient methylation. Reduced CTD phosphorylation would lead to reduced rates of RNA Polymerase II transcription genome-wide, resulting in reduced efficiency of histone tail methylation. While the mechanism connecting CTD phosphorylation to RNA Polymerase II elongation rate is likely to be complex in vivo, the observation that reduced CTD phosphorylation is associated with reduced dELL binding suggests that loss of dELL association could be a contributing factor (Eissenberg, 2007).
Mutation in Ash1 in Drosophila results in loss of all detectable H3K4 methylation, but has no effect on H3K36 methylation. This is consistent with independent mechanisms for these two chromatin modifications. A Polymerase II passage model provides a simple mechanism to account for similar effects on both modifications based on substrate availability (Eissenberg, 2007).
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