The positive transcription elongation factor P-TEFb, originally identified in Drosophila cells by Marshall and Price (1992, 1995), has been shown to be an essential kinase for overcoming the early block to transcription elongation in vitro through phosphorylating the RNAPII CTD (Marshall et al., 1996 ; Peng et al., 1998a). Subsequently, the use of specific antibodies recognizing the Drosophila CyclinT subunit of P-TEFb and either forms of phosphorylated CTD led to the hypothesis that P-TEFb function is actually required for expression of many, if not all, protein-coding genes in the genome (Lis et al., 2000 ; Andrulis et al., 2000 ; Kaplan et al., 2000). While all these studies have concentrated
their efforts on the characterization of the role of Cdk9/CyclinT in regulating transcription, no data are available on the role of Drosophila CyclinK. In contrast to mammalian systems where several T-type cyclins regulate Cdk9 activity, a single cyclinT gene has been identified in Drosophila (Peng et al., 1998a). In addition, it is unlikely that alternative Cdk9 isoforms exist in this species to modulate P-TEFb function.
As we started our work, little was known about the physiological role of P-TEFb in Drosophila. This was notably due to the fact that no mutations of the Cdk9 kinase or of the cyclin subunits had been reported. To address this question, we opted for a transgenic approach allowing the conditional expression of a dominant-negative form of Cdk9. One aspect of our project was dedicated to the characterization of phenotypes associated with expression of dominant-negative Cdk9 in living Drosophila. By altering specifically the function of the kinase in a given tissue and at a precise period of development, we expected to identify cellular processes that are more sensitive to cdk9 mutation.
We also investigated the functional significance of the two cyclin subunits on P-TEFb activity. On the one hand, we wanted to compare the transcriptional activities of the two P-TEFb complexes containing CyclinT or CyclinK in reporter-transcription assays performed in tissue culture cells. On the other hand, we wanted to test the redundancy of the two cyclin activities in vivo. To this end, we examined the ability of CyclinK to rescue cyclinT mutation in developing Drosophila and reciprocally. Finally, we also expected that a different requirement of one or the other cyclin on P-TEFb function would be detectable through modification of the Cdk9 dominant-negative phenotypes when CyclinT or CyclinK is co-expressed.
Results
1. Cloning and fly transformation
To study the function of P-TEFb in living Drosophila we used the transgenic approach. This technique allowed us to direct the expression of the different complexes in various tissues by the UAS/GAL4 system (Brand and Perrimon, 1993). The two genes cdk9 and cyclinT were cloned from genomic DNA while a cyclinK cDNA was used (Mariot, 2002).
Because the cyclinK coding sequence does not contain any introns, all three constructs correspond to the genomic sequences of the protein-coding genes. In all three constructs a specific tag was introduced at the N-terminus allowing their detection by immunochemistry (Fig.12). The genes were then cloned into the UASp transformation vector (P. Rorth, 1998) and injected into flies.
1.1. Generating a dominant-negative form of Cdk9 kinase.
In order to analyse the function of the Cdk9 kinase in vivo, we introduced in the cdk9 genomic sequence a substitution of the aspartate residue in position 199 by an asparagine (D199N). A similar mutation of this amino acid conserved in the active site of all known protein kinases is known to produce a dominant-negative effect in other members of the cdc2 family of cyclin-dependant kinases (Van den Heuvel and Harlow, 1993). Such a mutation has been shown in vitro and in tissue culture cells to abolish the kinase activity of human (Garriga et al., 1996a ; Gold et al., 1998) or Drosophila Cdk9 (Peng et al, 1998a ; Lis et al, 2000). The location of this point mutation within the Cdk9 protein sequences is shown in annexe 1. The mutant gene, referred to below as cdk9DN, as well as a control wild-type cdk9 gene were modified by inserting a triple Myc epitope-tag at the N-termini. The expression of the resulting transgenes after generation of Drosophila transgenic lines was then tested by crossing with the heat-inducible hsp70-GAL4 driver, and the protein levels were analysed by western blotting. As shown in figure 12, the expression of the tagged transgenes was readily detectable in larval extracts (Fig.12B) as well as in adult flies few hours following heat shock induction (Fig. 12C). In absence of heat shock, we noticed that certain levels of proteins were produced in both larvae and adult flies, which is due to the leakiness of the hsp70 promoter at normal temperature (25°C).
Figure 12 : Transgenic expression of Cdk9, CyclinK and CyclinT. (A) Schematic representation of the tagged cdk9, cyclinK and cyclinT constructs. The length in amino acids and estimated molecular weight of the corresponding protein are indicated. (B) Time course expression of Myc:cdk9 (1) and Myc:cdk9DN (2) transgenes under control of the hsp70-GAL4 driver. Third-instar larvae (UASp-Myc:cdk9 or UASp-Myc:cdk9DN/+ ; hsp70-GAL4/+) were heat shocked 30 minutes at 37°C. The protein levels were analysed by western blotting from crude extract prepared after 1, 3 and 5 hours of recovery at 25°C using anti-Myc antibody. Anti-tubulin was used as a loading control. Due to leakiness of the hsp70 promoter, some Myc:Cdk9 and Myc:Cdk9DN are detectable in non-heat shocked larvae (no HS) but not in control larvae (w1118).
(C) Western blotting on crude extracts from non-heat shocked (HS-) and heat shocked (HS+ : 30 min at 37°C followed by 5 hours of recovery) flies expressing the tagged proteins under control of the hps70-GAL4 driver (UASp-Myc:cdk9 or UASp-Myc:cdk9DN/+ ; hsp70-GAL4/+, UASp-Flag:cyclinK/+ ; hsp70-GAL4/+ and UASp-HA:cyclinT/+ ; hsp70-GAL4/+ respectively).
Membranes were probed with either anti-Myc, anti-Flag or anti-HA antibodies, anti-Tubulin being used as a loading control.
1.2. cyclinT and cyclinK transgenes.
Several transgenes encoding the two known cyclin partners for Cdk9 in Drosophila, cyclinT and cyclinK, were also introduced into flies. A wild-type cyclinK gene as well as a tagged version of the gene containing a triple Flag epitope-tag at the 5’ end of the coding sequence were generated (Mariot, 2002). Similarly, a tagged version of cyclinT was produced by inserting a triple HA tag at its N-terminus. The expression of the two tagged transgenes was then tested in adult flies using the inducible hsp70-GAL4 driver. As observed for Cdk9, both cyclins were abundantly produced upon heat shock treatment and were detected close to their expected molecular weights (Fig.12C).