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3. Characterization of Cdk9 mutation

3.3. Ubiquitous expression of Cdk9 DN affects larval endoreplicating tissues

3.3.2. Ectopic expression of dMyc suppresses the endoreplication defect of mutant Cdk9

Our data suggest that the larval growth defect, triggered by the overexpression of cdk9 dominant-negative, is primarily due to a decrease of DNA content within the larval endoreplicating tissues. Such a phenotype has notably been reported in diminutive (dm)

Figure 22. Cdk9DN expression affects larval endoreplicating tissues. (A) DAPI staining of second (upper panel) and third (lower panel) instar salivary glands from control (da-GAL4/+) and wild-type or mutant cdk9 expressing larvae under control of the da-GAL4 driver (UASp-cdk9 or UASp-cdk9DN/+

; da-GAL4/+). The salivary glands nuclei in the mutant failed to grow between the second and the third stage compare to control larvae. (B) Immunostaining of salivary glands expressing the tagged Cdk9 proteins under control of the AB1 driver. Salivary glands from control (AB1/+), Myc:Cdk9 (UASp-Myc:cdk9/+ ; AB1/+) and Myc:Cdk9DN (UASp-Myc:cdk9DN/+ ; AB1/+) expressing larvae were stained with DAPI (upper panel) and anti-Myc antibody (lower panel). The endoreplication defects induced by Cdk9DN expression in salivary glands was similar to phenotype obtained with da-Gal4.

Anti-Myc staining showed that both wild-type and mutant proteins are predominantly located in the nuclei.

mutant larvae, were both cell growth and DNA replication are affected (Gallant et al., 1996 ; Maines et al., 2004 ; Pierce et al., 2004). The dMyc transcription factor, encoded by dm, has been linked to a wide range of cellular processes including growth and proliferation, differentiation, apoptosis and oncogenesis (reviewed by Secombe et al, 2004). The dMyc protein and its antagonist dMnt, are thought to regulate these processes through transcriptional control of genes required for growth and proliferation. Both proteins bind DNA at so called E-box sequences within the promoter, and recruit various co-factors involved in transcription activation or repression. Recent studies have shown that c-Myc, the mammalian ortholog of dMyc, directly interacts with CyclinT1 in tissue-culture cells, allowing recruitment of P-TEFb to its target promoters (Eberhardy and Farnham, 2002 ; Kanazawa et al., 2003 ; Bouchard et al., 2004). Moreover, inhibition of P-TEFb activity results in specific inactivation of several Myc-responsive genes and impairs Myc-induced S-phase and apoptosis (Gargano et al., 2007). Therefore, P-TEFb appears to be an important co-activator for Myc-dependant transactivation in mammals.

To test whether the lack of DNA synthesis in Cdk9DN salivary glands is due to a decrease in expression of dMyc target genes and/or dm itself, we attempted to rescue at least partially the phenotype by overexpressing dMyc. To this end, we co-expressed our cdk9 mutant transgene with a UAS-dMyc transgene in salivary glands under control of the AB1 driver. Previous studies have shown that an ectopic expression of dMyc in endoreplicating tissues results in larger cells with a higher ploidy than normal (Pierce et al., 2004). We indeed found that a constitutive expression of dMyc in salivary glands throughout larval development leads to a dramatic increase of the size of the nuclei (Fig.224B). The excess of DNA content, visualized by DAPI staining, is particularly striking in the proximal region of the gland where the rate of endoreplication is normally limited compared to the distal region (compare a and b). As shown previously, the salivary gland nuclei expressing Cdk9DN failed to endoreplicate

Figure 23. Salivary glands nuclei expressing Cdk9DN have reduced DNA replication activity. (A) Immunostaining of salivary glands from control (AB1/+) and Cdk9 or Cdk9DN expressing larvae (UASp-cdk9/+ or UASp-cdk9DN/+ ; AB1/+). Third instar larvae were fed with BrdU-containing food for 2-3 days at 25°C, and the salivary glands were stained with anti-BrdU (red) and DAPI (green).

Most nuclei in control and wild-type Cdk9 salivary glands were highly labeled with anti-BrdU compared to mutant Cdk9 nuclei. In contrast, fat bodies surrounding the glands were similarly labeled in both wild-type and mutant Cdk9 expressing larvae (arrowhead) indicating that DNA replication operates normally in the absence of GAL4. (B) Percentage of BrdU-positive cells in control (1), wild-type Cdk9 (2) and mutant Cdk9 (3) expressing salivary glands determined for the experiment above.

Eleven to 16 glands were analyzed for each genotype. The average of BrdU incorporating cells, indicated for each bar of the graph, showed that only 20% of the nuclei endoreplicates in the mutant and more than 95% in control animals.

and the gland is filled with small nuclei of similar size (Fig. 24C). When co-expressed with dMyc, the cdk9 mutant phenotype was completely suppressed (Fig. 24D). Furthermore, we observed that the nuclei in the proximal region of the gland underwent excessive rounds of DNA replication, and reached a size comparable to nuclei overexpressing dMyc alone (compare b and d). This result suggests that overexpressed dMyc is likely to be epistatic to Cdk9DN, at least in our experimental conditions. The strong suppression of the cdk9 mutant phenotype by dMyc might however simply reflect a difference in terms of protein expression, as both transgenes are not regulated by same promoter. Additionally, a fast degradation of the overexpressed Cdk9 proteins, as demonstrated in human cells (Garriga et al., 2003), might also contribute to a low capacity of the mutant kinase to interfere with dMyc-mediated transcription. For technical reasons, we were not able to increase the levels of Cdk9DN expression in this experiment (i.e. expression of an additional copy of the cdk9DN transgene), and attempt to limit the rate of DNA replication caused by dMyc. Nevertheless, these data strongly suggest that the Cdk9DN endoreplication defect is due to a reduced expression and/or activity of dMyc in salivary glands.