Topoisomerase I inhibition induced a downregulation of Aurora-A expression
We first wanted to confirm in colorectal cell lines that Aurora-A was mainly expressed during the G2 phase of the cell cycle. To this end, HCT116 cells were synchronized in G1/S with hydroxyurea, washed and then grown again in serum for 5 to 13 hr. Under these conditions, FACS analysis showed that cells were synchronized after 8-9 hr in the G2 phase of the cell cycle and that they enter the next G1 phase after 12-13 hr of serum release (Figure 1A). As expected, we observed that Aurora-A was expressed in G2, both at the protein (Figure 1B, lanes 1-7) and mRNA levels (Figure 1B, lanes 8-9). The same results were obtained in a second colorectal cell line, the HT29 cells and with different kinds of synchronization such as double thymidine block and serum starvation (data not shown).
To determine whether topoisomerase I inhibition has any influence on Aurora-A expression, HCT116 cells were treated with sn38, the active metabolite of irinotecan [15]. Under these conditions, control cells were synchronized in the G2 phase of the cell cycle after 48-72 hr (Figure 1C, note that serum 9hr means G1/S synchronization followed by serum stimulation for 9 hr). Although this was the cell cycle stage when Aurora-A expression was supposed to be maximal, results indicated that the expression of the kinase was downregulated in response to sn38, both at the protein (Figure 1D, compare lane 1 with lanes 2-4) and mRNA levels (Figure 1E, normal mRNAs expression in G2 was normalized to 1). As a control, the p21waf1 mRNA increased as expected following genotoxic treatment. Finally, these experiments have also been repeated in a different colorectal cell line and sn38 also downregulated Aurora-A in HT29 cells (data not shown and see below Figure 2B).
Altogether, these results indicate that topoisomerase I inhibitors such as sn38 induced a downregulation of Aurora-A expression.
Myc binds to the promoter of the Aurora gene and is involved in its regulation
Following sn38 treatment, we observed as expected in HCT116 cells that p53 was stabilized and phosphorylated on its serine 15 residue. Consequently, p21waf1 level was also enhanced in response to drug treatment (Figure 2A, lanes 1-4). To check whether Aurora-A downregulation was dependent on the p53-p21 pathway [20, 21], we used the HCT116 p21-/- derivative cell line in which both p21waf1 alleles have been deleted by homologous recombination [22]. Results showed that sn38 reduced Aurora-A expression in HCT116 p21-/- cells (Figure 2B, lanes 1-4). The same effect was observed in the HT29 cell line that contains a mutated form of p53 (Figure 2B, lanes 5-6). These results indicate that Aurora-A downregulation is not cell-type specific and is independent of the p53-p21 pathway.
During the course of this study, we noticed that the expression of the c-Myc transcription factor was significantly reduced following topoisomerase I inhibition (Figure 2C, lanes 1-4). This suggested that c-Myc was involved in the regulation of the Aurora-A gene. To verify this hypothesis, we used doxycyclin-inducible expression vectors that stably drives the expression of two different Myc siRNAs in two different clones of the LS174T colorectal cell line. As previously shown [17], western blot analysis showed that doxycyclin induced a significant downregulation of c-Myc levels in the two clones (Figure 2D, lanes 4 and 6, top panel). Interestingly, we observed under these conditions that Aurora-A expression was inhibited upon c-Myc knockdown (Figure 2D, compare lanes 4 and 6 with lanes 3 and 5, middle panel). Note that c-Myc downregulation did not modify cell cycle distribution in the G2 phase of the cell cycle (data not shown) so that Aurora-A inhibition can not be explained by G0/G1 arrest.
Using the UCSC genome browser http://genome.ucsc.edu, we noticed that ChIP-ChIP experiments have already suggested that Myc can potentially bind to the Aurora-A promoter in Hela cells. Moreover, Ouyang and collaborators have shown by ChIP-seq that both c-Myc and N-Myc can be found associated with this gene in embryonic stem cells [23]. Effectively, transcription factor recognition site analysis of the Aurora-A promoter revealed the presence of non canonical E-boxes that could represent potential Myc binding sites (Figure 3A). To determine if Myc binds to the Aurora A promoter, its recruitment was analyzed by chromatin immunoprecipitation experiments (ChIP) in the LS174T cell line described above. Results presented Figure 3B, lanes 1-4, showed that Myc was effectively recruited to the -668/-400 region of the Aurora-A promoter and that this was associated with histone 3 acetylation (K9), which is indicative of gene transcription. Following siRNA induction and Myc downregulation, the binding of the transcription factor was downregulated and this inhibition was associated with histone H3 deacetylation (Figure 3B, lanes 2 and 4). As a control, no binding of a control IgG (Figure 3B, lanes 5-6), and Myc did not bind to the 5' part of the Aurora-A promoter (data not shown).
Myc is a basic helix-loop-helix zipper transcription factor that heterodimerizes with Max to activate gene transcription. Its activity is inhibited by Mad which associates with Max to recruit repressor complexes to promoters [24]. To determine if Myc and Max are associated with the Aurora-A promoter and if this association is cell cycle dependent, HCT116 cells were synchronized in G1/S with hydroxyurea, washed and then grown again in serum for 5 hr (S/early G2), 9 hr (G2) and 13 hr (next G1). ChIP experiments were then performed as described above. Results presented Figure 3C, lanes 3 and 7, indicate that the two proteins are effectively recruited to this promoter in the G2 phase of the cell cycle. To determine if the two proteins are associated on DNA, a serial ChIP experiment (Re-ChIP) was then performed. For this, the soluble chromatin was immunoprecipitated with Myc antibodies, the immune complexes were released with DTT and the chromatin was further divided into two aliquots and reimmunoprecipitated with IgG or Max antibodies. Under these conditions, subsequent Re-IPs with Max antibodies were able to immunoprecipitate the Aurora-A promoter whereas this was not the case with the control antibody (Figure 3D). Importantly, the association of the two proteins was only detected during the G2 phase of the cell cycle. ChIP result have been obtained by semi-quantitative PCR (Figure 3D, lanes 1-4) and quantified by quantitative-PCR (Figure 3E). As a control, the PCR analysis did not detect any occupancy of a control DNA region (Figure 3D, lanes 5-8) or of the proxymal promoter during the G1 phase of the cell cycle and at the G1/S transition (Figure 3D, lane 4 and 1).
We concluded from these results that the Myc/Max complex binds to the promoter of the Aurora A gene during the G2 phase of the cell cycle and that Myc is involved in the regulation of this gene.
Topoisomerase I inhibition prevents the association of the Myc-Max complex with the Aurora-A promoter
To determine the links between the Myc/Max/Mad pathway and the regulation of the Aurora-A gene following topoisomerase I inhibition, Max/Mad expression was first evaluated following sn38 treatment. Whereas no significant effect was observed on Max expression, Mad levels increased at the protein and mRNA levels (Figure 4A, lanes 1-2 and Figure 4B). As a control, Myc and Aurora-A expressions were downregulated as expected. To determine if the binding of these proteins to the Aurora-A gene was affected by sn38, their recruitment was analyzed by ChIP following treatment. Results showed that the recruitment of Myc and Max was inhibited following topoisomerase I inhibition (Figure 4C, compare lanes 2 and 5, 7 and 9). Note that a weak association of Myc and Max was detected in growing conditions, probably due to the percentage of cells, which are in the G2 phase of the cell cycle (Figure 4C, lanes 1 and 6). Interestingly, these proteins were also found associated with the initiation site, suggesting that the upstream and initiation regions might associate in a transcriptional loop (data not shown). Myc and Max bindings were also inhibited on this initiation site following sn38 treatment. To extend these observations, ChIP experiments were then performed to analyze the recruitment of the Mad protein. In growing conditions or during the G2 phase of the cell cycle, Mad was not found associated with the Aurora-A promoter. Interestingly, when cells were treated with sn38, this protein was significantly recruited to this gene (Figure 4D, lanes 3-4).
Altogether, we concluded from these results that the Myc/Max complex binds to the promoter of the Aurora-A gene in the G2 phase of the cell cycle and that this binding is inhibited upon topoisomerase I inhibition.
Topoisomerase I inhibition promotes Miz-1 recruitment to the Aurora-A promoter
The Miz-1 transcription factor is a POZ-domain-containing zinc-finger protein that can form a transcriptional repressor complex with Myc to inhibit gene transcription [24]. In addition, it has also been proposed that Miz-1 functions as a transcriptional repressor in a Myc-independent manner through its association with cofactors such as BCL6 or Gfi-1 [25, 26]. To determine if this protein was involved in Aurora-A inhibition, its expression was evaluated in HCT116 cells treated or not with sn38 (Figure 5A and 5B). Under these conditions, a weak increase in Miz-1 protein level was observed whereas no significant effect was detected on its mRNA expression. ChIP experiments performed in the G2 phase of the cell cycle showed that Miz-1 was associated with the -668/-400 region (Figure 5C, lane 2). By contrast, this protein was not significantly recruited to this gene in growing cells. Interestingly, Miz-1 recruitment was significantly increased following sn38 treatment (Figure 5C, lanes 3-5 and data not shown). Importantly, this binding was associated with a decreased recruitment of the CBP transcriptional coactivator, of the RNA type II polymerase and with a downregulation of histone H3 acetylation (Figure 5D, lanes 2, 4 and 6). We did not observe any recruitment of the HDAC1 histone deacetylase to this promoter.
Altogether, we concluded from these results that topoisomerase I inhibition induces a recruitment of Miz-1 to the Aurora-A promoter and decreases the binding of transcriptional coactivators.
The Aurora-A promoter is located within SAHF foci following topoisomerase I inhibition
We have recently shown that sn38 treatment induced senescence in colorectal cell lines (see [19, 27] and text below). Senescence is an irreversible proliferation-arrest that is characterized by the formation of isolated heterochromatin foci called Senescence Associated Heterochromatin Foci (SAHF, [28]). SAHF foci contain marks of transcriptional silencing such as heterochromatin protein 1 (HP1) and tri-methylation of the lysine 9 of histone H3 (H3K9Me3). During senescence, proliferative genes such as E2F targets are compacted within these heterochromatin foci to prevent cell cycle progression, generally as a consequence of Rb-mediated silencing. To extend our results, we then determined if the Aurora-A promoter was included within these SAHFs foci. As a first approach, we used immunofluorescence and western blot experiments to shown that sn38 induced a global increase in H3K9 trimethylation in HCT116 cells. As expected, a significant phosphorylation of histone H2Ax was also detected, reflecting the induction of DNA double strand breaks following topoisomerase I inhibition (Figure 6A and 6B). Results were quantified by Facs analysis to show a significant increase of the two signals (Figure 6C). DAPI staining also showed an increase in the presence of punctuate heterochromatin foci in the nucleus of sn38-treated cells which were not detected in control conditions (Figure 6D). ChIP experiments were then used to determine if proteins involved in transcriptional silencing could be found associated with the proxymal promoter of the Aurora-A gene following treatment. Interestingly, results presented Figure 6E, lanes 4-9, showed that HP1γ was recruited to this gene in sn38-treated cells. In addition, we also noticed a significant increase in the amount of tri-methylated H3K9 on the proxymal Aurora-A promoter. By contrast, when ChIP experiments were repeated with an antibody directed against the phosphorylated form of histone H2AX, no signs of DNA double strand breaks were detected within this gene.
In light of these results, we concluded that the Aurora-A proxymal promoter is located within SAHF foci following genotoxic treatment and that its inhibition is probably related to the recruitment of cofactors involved in transcriptional silencing such as HP1γ and to the tri-methylation of H3K9.
Topoisomerase I inhibition prevents centrosome separation
It has been shown that Aurora-A is involved in the maturation and separation of centrosome during progression from S phase towards mitosis [29]. To determine if topoisomerase I inhibition prevents this maturation, centrosome formation was analyzed by immunofluorescence and γ-tubulin staining. When cells were synchronized in the G2/M phase of the cell cycle, the centrosomes were effectively stained as a doublet and Aurora-A was essentially localized on the centrosomes. As expected, when cells were treated with sn38, Aurora-A became undetectable by immunofluorescence (data not shown). Interestingly, genotoxic treatment dit not prevent centrosome duplication, however, no separation was observed under these conditions (Figure 7A). Probably as a consequence of the absence of centrosomal separation and of progression towards mitosis, we observed using clonogenic assays that sn38 induced a complete inhibition of cell proliferation (Figure 7B). Using beta-galactosidase staining, we also noticed an induction of senescence following genotoxic treatment (Figure 7C).
Thus, we concluded from these results that topoisomerase I inhibition prevents centrosome separation, probably as a consequence of Aurora-A inhibition, and that this leads to G2 arrest and senescence induction.