4. CyclinT versus CyclinK
4.2. Targeting of P-TEFb containing CyclinT to promoters stimulates transcription …
The absence of redundancy of the cyclin activities suggests that P-TEFb complexes containing CyclinT or CyclinK regulate transcription elongation in different ways. This may imply alternative mechanisms allowing P-TEFb to interact with the transcription machinery and/or to modify its substrates such as the CTD repeats of RNA polymerase II. To determine whether P-TEFb containing either cyclin is able to stimulate transcription, we performed tethering assays in tissue culture cells. In the following experiments, targeting of P-TEFb to the reporter was achieved using chimeric proteins in which the DNA-binding domain of GAL4 (GBD) was fused to the N-termini of full length CyclinT and CyclinK (Fig.33A). The ability of these fusion proteins to stimulate transcription was monitored on a firefly luciferase reporter gene which contains fourteen GAL4 binding sites (UAS sites) downstream of a minimal hsp70 promoter (Fig.33A).
4.2.1. P-TEFb tethering assays using GBD:CyclinT and GBD:CyclinK fusion proteins.
The assays were performed on a stable S2 cell line encoding the reporter construct, which were transiently transfected with inducible pMT plasmids encoding either GBD-fusion
Table 5 : Absence of rescue of cyclinT mutation by a transgenic expression of cyclinK.
Table 6 : Absence of rescue of cyclinK mutation by a transgenic expression of HA:cyclinT.
Genotype of Parents
protein, alone or together with wild-type or mutant Cdk9. As control experiments, cells were transfected with empty pMT plasmids or plasmids expressing only the GBD. Each transfections were repeated three times, and were normalized on the levels of a constitutively expressed renilla luciferase reporter (see Materials & Methods).
The graph in figure 33B indicates the mean levels of firefly luciferase activity obtained for each transfection (lanes 2-10) relative to the basal luciferase activity monitored in cells transfected with empty plasmids (lane 1). The expression of GBD:CyclinT (lane 3), GBD:CyclinK (lane 4) or Cdk9 (lane 5) alone has little effect on the reporter activity, ranging from 1,1 to 3,3 fold above the basal level. By contrast, co-expression of GBD:CyclinT with Cdk9 induced high level of luciferase activity (lane 6, 40-fold increase from transfection with empty plasmid and 13-fold compared to Cdk9 and or GBD:CyclinT alone). Surprisingly, no obvious activation of the reporter was obtained by co-expressing GBD:CyclinK with Cdk9 (lane 7). These data suggest that recruitment of P-TEFb containing CyclinT, but not CyclinK, to the promoter is sufficient to activate transcription. Similar results have been reported in previous studies, which showed that targeting of human CyclinT1 or CyclinT2 to reporters induced their expression while CyclinK had no effects (Peng et al., 1998b ; Napolitano et al., 2000 ; Taube et al., 2002). We next tested the effect of dominant-negative Cdk9DN on the reporter activity. Consistent with a loss of kinase activity, expression of Cdk9DN alone or together with GBD:CyclinK did not activate the luciferase reporter (lanes 8,10), which remained close to the basal level. Surprisingly, however, co-expression of Cdk9DN with GBD:CyclinT still activated transcription of the reporter to intermediate levels (compare lanes 6 and 9). This unexpected result may be explained if the induced GBD:CyclinT and Cdk9DN proteins could make complexes with endogenous CyclinT and Cdk9. In this hypothetical context, a certain proportion of P-TEFb made of GBD:CyclinT and wild-type Cdk9 could activate the reporter while some P-TEFb comprising the endogenous CyclinT and Cdk9DN might reduce the expression of the renilla luciferase reporter. Alternatively, the many UAS sites of the firefly construct might allow binding of both active and inactive P-TEFb and the recruitment of a single active complex could be sufficient for activation. Although our data did not suggest that S2 cells contained a lot of endogenous Cdk9 available (lane 3), it would be enough to produce some GBD:CyclinT/Cdk9 heterodimers. Another possibility comes from the observations that several intron-less genes such as the human histone and the Drosophila hsp70 genes can be transcribed in the absence of P-TEFb activity (Medlin et al., 2005 ; Ni et al, 2004). Despite the fact that P-TEFb function is necessary for the proper processing of mRNAs and that non-polyadenylated RNAs are rapidly degraded, it appears
that some mRNAs can still be produced in the absence of P-TEFb kinase activity (Ni et al, 2004). This suggests that other signals than Ser2-phosphorylation participate in the recruitment of the 3’ end processing machinery and could partially compensate the loss of P-TEFb function (reviewed by Egloff et al., 2008). This may also indicates that P-P-TEFb can stimulate mRNA processing independently of its kinase activity, by facilitating the recruitment of processing factors to the elongation complex. In our experiments, it is possible that similar events occurred as the luciferase constructs did not possess any intron, contributing to the significant level of luciferase expression monitored in lane89. This latter hypothesis will be further discussed in the last part of the manuscript. Taken together, our results have nevertheless demonstrated that Drosophila CyclinT, but not CyclinK, is able to trigger transcription elongation when targeted, along with Cdk9, to the promoter.
4.2.2. P-TEFb tethering assays using GBD:Cdk9 fusion proteins.
To confirm the results presented above, we performed another set of P-TEFb-tethering experiments on the same reporter construct by using either GBD:Cdk9 or GBD:Cdk9DN fusion proteins (Fig.34A). As previously, the ability of each protein or P-TEFb complex to induce expression of the reporter was tested three times independently, and the levels of firefly luciferase monitored were normalized to the levels of a constitutively expressed renilla luciferase reporter. As shown in figure 34B, the expression of GBD:Cdk9 or either cyclin individually had only minor effects on the reporter activity (lanes 3-5).
Consistent with our previous results, the co-expression of GBD:Cdk9 with CyclinT strongly activated transcription of the reporter (lane 6) while CyclinK had no effect (lane 7). In contrast, however, the recruitment of GBD:Cdk9DN/CyclinT to the promoter did not activate the expression of the reporter as previously observed with GBD:CyclinT/Cdk9DN (compare lanes 8 and 9), even though a slight increase of the reporter activity was obtained compared to
Figure 33. P-TEFb tethering assays using GBD:CyclinT and GBD:CyclinK. (A) Schematic drawings of the UAS reporter and the GAL4 fusion proteins. The UAS-hsp-luciferase reporter contains 14 GAL4-binding sites (UAS) inserted downstream of a minimal hsp70 promoter followed by the firefly luciferase coding sequence. The fusion proteins were obtained by insertion of the GAL4-binding domain (GBD, positions 1-151) at the 5’ end of the tagged CyclinT and CyclinK constructs.
(B) Luciferase assays. Cells encoding the UAS reporter were transiently transfected with empty plasmid (pMT, lane 1) and/or plasmids encoding the different constructs as indicated below the graph (lanes 2-10). Transfections were repeated 3 times independently and normalized to the levels of a renilla luciferase reporter expressed under control of the tubulin promoter. The standard deviations are indicated by errors bars. The graph represents the fold activation of the UAS reporter monitored in each sample relative to cells transfected with empty plasmid.
the basal level (compare lanes 1 and 9). The transactivation of the reporter by the GBD:Cdk9 fusion protein did not appear less efficient than that obtained with the non-chimeric Cdk9 protein used in the previous experiment (lane 6/lane 3 : 21.7-fold increase), indicating that the GBD does not strongly interfere with the kinase activity of Cdk9 nor its ability to interact with the elongation complex. Altogether, these data led us to conclude that the direct recruitment of the Cdk9/CyclinT heterodimer to the promoter is sufficient to activate transcription while the recruitment of P-TEFb containing CyclinK is not.
The absence of transcriptional activation by Cdk9/CyclinK when tethered to sites upstream of the promoter might be due to the inability of the complex to interact with its substrate such as the CTD repeats of RNA polymerase II. This hypothesis is supported by the works of Lin et al. (2002c), who showed that human Cdk9/CyclinK can efficiently phosphorylate the polymerase CTD and trigger transcription only when the complex is artificially targeted to the transcription initiation site of a reporter. Therefore, in contrast to CyclinT, CyclinK has to be recruited in close proximity to the polymerase CTD to enable phosphorylation by Cdk9. This observation also suggests that CyclinK is unable to interact with the CTD by itself. This assumption is notably supported by the finding of two CTD-binding domains within the C-terminal halves of human CyclinT1 and CyclinT2, which are absent from the shortest CyclinK protein (Taube et al., 2002 ; Kurosu et al., 2004).