Original Article

Oncogene (2011) 30, 3599–3611; doi:10.1038/onc.2011.71; published online 21 March 2011

Camptothecin-induced downregulation of MLL5 contributes to the activation of tumor suppressor p53

F Cheng1, J Liu1, C Teh2, S-W Chong3, V Korzh2, Y-J Jiang3,4 and L-W Deng1

  1. 1Department of Biochemistry, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore
  2. 2Cancer and Developmental Cell Biology Division, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore
  3. 3Genes and Development Division, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore
  4. 4Division of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli County, Taiwan

Correspondence: Dr L-W Deng, Department of Biochemistry, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, 8 Medical Drive, Singapore 117597, Singapore. E-mail: lih_wen_deng@nuhs.edu.sg

Received 29 August 2010; Revised 9 January 2011; Accepted 4 February 2011; Published online 21 March 2011.

Top

Abstract

Mixed lineage leukemia 5 (MLL5) has been implicated in multiple aspects of cell physiology, such as hematopoiesis, cell cycle control and chromatin regulatory network. In this study, we present evidence that MLL5 is involved in the camptothecin (CPT)-induced p53 activation. CPT promoted the degradation of MLL5 protein in a time- and dose-dependent manner in actively replicating cells. The downregulation of MLL5 led to phosphorylation of p53 at Ser392, which was abrogated by exogenous overexpression of MLL5. In MLL5-knockdown cells, p53 protein was stabilized and bound to DNA with higher affinity, leading to activation of downstream genes. Co-immunoprecipitation showed that MLL5 preferentially interacted with the tetramerized form of p53, and knockdown of MLL5 promoted chromatin accumulation of p53 tetramers, suggesting that the association of MLL5 with p53 may prevent the p53 tetramers from binding to the chromatin target sites. The role of MLL5 in CPT-induced p53 activation was conserved in developing zebrafish, where CPT downregulated zebrafish Mll5 protein, and the microinjection of zebrafish mll5 mRNA substantially blocked the CPT-induced apoptosis. In summary, our study proposed MLL5 as a novel component in the regulation of p53 homeostasis and a new cellular determinant of CPT.

Keywords:

MLL5; camptothecin; p53; p53 tetramer; Serine 392

Top

Introduction

Mixed lineage leukemia 5 (MLL5) is the latest addition to the mammalian MLL protein family, which share characteristic domain structures, including a variable number of PHD (plant homeodomain) zinc fingers and a single SET (Su(var)3-9, enhancer-of-zeste and trithorax) domain (Emerling et al., 2002). The epigenetic regulatory functions of MLL family proteins have gained interests in recent years (Dambacher et al., 2010). MLL1 and MLL2 were shown to methylate histone H3 Lys4 (H3K4) by forming histone methyltransferase complexes consisting of WDR5, RbBP5 and ASH2L (Hughes et al., 2004; Yokoyama et al., 2004; Wysocka et al., 2005). In contrast, MLL5 lacks the cysteine-rich post-SET domain found in the rest of family members, which is believed to be required for histone methyltransferase activity (Kouzarides, 2002). No intrinsic histone methyltransferase activity was found on MLL5, although it physically associates with cyclin A2 promoter and indirectly regulates H3K4 methylation through histone-modifying enzymes LSD1 and SET7/9 (Sebastian et al., 2009). However, a short N-terminal isoform of MLL5, containing both PHD and SET domain, once glycosylated was discovered to act as a mono- and di-methyltransferase of H3K4 in promyelocytic leukemia cells (Fujiki et al., 2009). Although the histone-modifying capability of MLL5 has not been clearly defined, the regulatory function of MLL5 in the cell cycle was highlighted in a number of studies. Ectopically overexpressed MLL5 caused cell cycle arrest at G1/S phase (Deng et al., 2004), whereas siRNA-mediated knockdown of MLL5 arrested cell cycle at both G1/S and G2/M phases (Cheng et al., 2008). Upon MLL5 knockdown, the entry of quiescent myoblasts into S-phase was delayed, but the completion of S-phase progression was hastened (Sebastian et al., 2009). Recently, we showed that phosphorylation of MLL5 by mitotic kinase Cdc2 is required for mitotic entry (Liu et al., 2010b). Taken together, these data imply that MLL5 have different regulatory roles throughout cell cycle.

Camptothecin (CPT) is a cytotoxic plant alkaloid isolated from Camptotheca acuminata of the Nyssaceae family. It interferes with the breakage-reunion reaction of DNA topoisomerase I (Top1) by reversibly generating the Top1–CPT–DNA intermediate, known as the cleavable complex (Liu, 1989). In the presence of CPT, the cleavable complex is stabilized, causing inhibition of both DNA and RNA synthesis; prolonged exposure to CPT leads to DNA-strand breaks and cell cycle arrest (Ryan et al., 1991; Staker et al., 2002). CPT selectively kills S-phase cells, possibly due to the association of Top1 with DNA replication complexes (Pommier, 2006). The antitumor activity of CPT and its derivatives have been well documented, although the molecular mechanism of their activity has not been fully understood. For now, Top1 has been shown to be the only cellular target of CPT by Top1-knockout yeast studies and single point mutagenesis (Eng et al., 1988; Nitiss and Wang 1988; Pommier et al., 1999); therefore, it is the covalently cleavable complex, rather than CPT alone, that leads to many downstream signaling cascades. CPT induces downregulation of Cdc25A, which potentiates the S and G2/M cell cycle checkpoints and prevents propagation of damaged DNA (Xiao et al., 2003). Top1 is also found effectively degraded by CPT in non-transformed, but not in transformed cells (Desai et al., 2001).

The activation of p53 by CPT was often attributable to the activation of ataxia telangiectasia mutated (ATM), which was induced by the DNA-strand breaks arising from prolonged exposure to CPT (Zhao et al., 2008). Here, we documented a new mechanism of p53 activation by CPT. We showed that CPT promoted proteasome-mediated degradation of MLL5, leading to activation of p53 through Ser392-phosphorylation. MLL5 was found to localize at the chromatin region, interact with p53 and prevent p53 tetramers from being recruited to their target sites. Consistently, a similar mechanism of p53 activation by CPT was observed in zebrafish. Our data provided more insights into the cellular determinants of CPT and shed light on a novel mechanism of the negative regulation of p53.

Top

Results

Camptothecin promotes degradation of MLL5

Ectopic overexpression of MLL5 arrests cells at G1/S phase, whereas MLL5 knockdown causes dual-phase cell cycle arrest at both G1/S and G2/M phases (Deng et al., 2004; Cheng et al., 2008). Phosphorylation of MLL5 by Cdc2 is required for mitotic entry, at least partly explained the G2/M phase arrest caused by knockdown of MLL5 (Liu et al., 2010b); however, the mechanism underlying the G1/S arrest remains obscure. The G1/S arrest is a typical response to the activation of G1/S checkpoint, which serves as an important defense mechanism against genomic instability, before the full commitment of cell division (Elledge, 1996; Bartek and Lukas, 2001). Therefore, we decided to investigate the response of MLL5 to the activation of G1/S checkpoint by stressing the cells with various DNA-damaging agents, including cisplatin, etoposide, CPT, aphidicolin (Aph) and methyl methanesulfonate (Nyberg et al., 2002). Surprisingly, the expression of MLL5 protein was almost completely abolished by CPT, but only slightly downregulated or unaffected by other DNA-damaging agents, in colorectal carcinoma HCT116 cells (Figure 1a) and osteosarcoma U2OS cells (Supplementary Figure S1). In addition, the downregulation of MLL5 by CPT analogs, topotecan and SN-38, further confirmed the specific effect of CPT-class of Top1 inhibitors on MLL5 (Figure 1b). The CPT-induced downregulation of MLL5 was then analyzed by time- and dose-dependency experiments, in which a reduction of MLL5 level was detected as early as 2h after treatment, with 2μM CPT (Figure 1c). In order to find out if such downregulation takes place at a transcriptional or post-translational level, we examined whether the proteasome pathway of protein degradation was involved. Downregulation of MLL5 by CPT was rescued by proteasome inhibitor MG132, suggesting that MLL5 was degraded through the 26S-proteasome pathway (Figure 1d). In addition, the stability of MLL5 protein in unstressed and CPT-treated cells was compared. Cycloheximide (100μg/ml) was added to HCT116 cells prior treated or untreated with 5μM CPT for 1h. Cell lysates were obtained at indicated time after addition of cycloheximide. Result showed that the stability of MLL5 protein was compromised by CPT treatment (Figure 1e). In contrast, no reduction in the level of MLL5 mRNA was observed in CPT-treated HCT116 cells by semi-quantitative reverse-transcription PCR (Figure 1f).

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

CPT promotes proteasome-mediated degradation of MLL5. (ae) Total cellular protein of HCT116 cells under indicated treatments were extracted and analyzed by western blot with specified antibodies. Actin was used as a loading control. (a) Among the five DNA-damaging agents, CPT downregulated MLL5 protein most effectively. HCT116 cells were treated with 10μM cisplatin, 10μM etoposide, 10μM CPT, 5μM Aph or 0.1% methyl methanesulfonate for 6h before lysis. (b) MLL5 was downregulated by CPT analogs, topotecan and SN-38. HCT116 cells were treated with 5μM topotecan, 5μM SN-38 or 5μM CPT for 6h before lysis. (c) CPT downregulated MLL5 protein in a time- and dose-dependent manner. HCT116 cells were treated with increasing doses of CPT for 6h, or treated for increasing duration with 2μM CPT before lysis. (d) MG132 blocked the downregulation of MLL5 by CPT. HCT116 cells were treated with 5μM CPT, 20μM MG132, or both for 6h before lysis. (e) The stability of MLL5 protein was compromised in CPT-treated cells. HCT116 cells were treated with 5μM CPT for 1h before addition of 100μg/ml cycloheximide. Cell lysates were obtained at the indicatedh after cycloheximide treatment. (f) No downregulation of MLL5 mRNA was observed in CPT-treated cells. Total RNA was extracted from CPT-treated HCT116 cells and MLL5 was amplified by reverse-transcription PCR. GAPDH was used as a loading control. ‘NT’ stands for no treatment.

Full figure and legend (125K)

CPT-induced degradation of MLL5 independent of ATM and requires active DNA replication

CPT-induced degradation of proteins, such as Top1, exhibited a high degree of heterogeneity among different cell types (Desai et al., 2001). Therefore, we tested if downregulation of MLL5 by CPT was cell type-dependent. Human lung fibroblast WI-38, cervical carcinoma HeLa, U2OS, HCT116 p53+/+ and HCT116 p53/ were treated with CPT for 6h before harvest. Degradation of MLL5 was observed in all cell lines tested, regardless of cell type or the status of p53 (Figure 2a). Prolonged exposure of CPT is known to cause persistent DNA double-strand breaks, leading to the activation of DNA-dependent protein kinases including ATM and ATR (ataxia telangiectasia and Rad3 related) (Liu, 1989). To investigate if ATM/ATR acts upstream of MLL5 degradation, HCT116 cells were pre-treated with 5mM caffeine for 1h, a potent inhibitor to DNA-dependent protein kinases, before CPT treatment (Sarkaria et al., 1999). Caffeine remained in the culture medium until cell lysis at 6h after CPT addition. CPT-induced phosphorylation of Chk2-Thr68 was blocked in caffeine-treated cells, indicating an effective inhibition of ATM/ATR by caffeine. However, MLL5 underwent degradation even in the presence of caffeine (Figure 2b). S-phase cells are particularly sensitive to CPT due to the collision between the replication machinery and the cleavable complex (Liu, 1989). To study the role of DNA replication in the downregulation of MLL5, we examined whether cell proliferation was required for the CPT-induced MLL5-degradation. WI-38 cells, known to quit cell cycle and enter a quiescent G0/G1 state upon serum starvation (Dimri et al., 1994), were cultured in 10% or 0.2% serum for 72h before CPT treatment. In cells cultured in normal 10% serum, MLL5 was effectively degraded by CPT treatment. In contrast, the CPT-induced degradation of MLL5 was severely compromised in the cells cultured in 0.2% serum (Figure 2c). To study whether the active DNA replication was required for the degradation of MLL5, we pre-treated HCT116 cells for 2h with 2μM Aph, an inhibitor of DNA replication, before CPT treatment. Although MLL5 was efficiently degraded by CPT treatment alone, pre-treatment with Aph partially rescued MLL5 downregulation (Figure 2d, Supplementary Figure S2), supporting the idea that the degradation of MLL5 was caused by the interference of the cleavable complex with the DNA replication machinery. Aph alone did not cause degradation of MLL5 (Figures 1a and 2d), suggesting that the CPT-induced MLL5-degradation resulted from the cleavable complex rather than the stalled replication. In order to further confirm the DNA replication-dependent downregulation of MLL5, HCT116 cells were synchronized to G1/S boundary (0h) using double-thymidine block and then treated with CPT in the presence or absence of 2mM thymidine for 6h. Results showed that MLL5 was readily degraded by CPT when cells were released into S phase, whereas the blockade of S-phase progression abrogated the CPT-induced downregulation of MLL5 (Figure 2e). Cell cycle profiles were analyzed to ensure the success of G1/S synchronization and release (Supplementary Figure S3).

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

CPT-induced downregulation of MLL5 is independent of ATM, but requires active DNA replication. (ae) Total cellular protein from cells under indicated treatments were extracted and the protein expression of MLL5 was measured by western blot. Actin was used as a loading control. (a) CPT-induced downregulation of MLL5 was not cell type-specific. Cells were treated with 5μM CPT for 6h before lysis. (b) Caffeine could not block the CPT-induced downregulation of MLL5. HCT116 cells were pre-treated with 5mM caffeine for 1h before 5μM CPT treatment. Phosphor-Chk2 Thr68 was used as a marker for ATM activation. (c) CPT-induced downregulation of MLL5 was compromised in serum-starved cells. WI-38 cells were cultured in medium containing 10 or 0.2% serum for 72h before treatment with CPT for up to 6h. (d) Aph partially blocked the CPT-induced downregulation of MLL5. HCT116 cells were pre-treated with 2μM Aph for 2h, before 5μM CPT treatment. (e) MLL5 was degraded by CPT during the S phase progression, which was blocked by thymidine. HCT116 cells were synchronized to G1/S boundary, and treated with 5μM CPT in the presence or absence of 2mM thymidine for 6h before lysis.

Full figure and legend (92K)

CPT-induced MLL5-degradation induces Ser392 phosphorylation

CPT increases p53 stability and induces phosphorylation of p53 at both Ser15 and Ser392 residues (Houser et al., 2001; Zhao et al., 2008). Interestingly, knockdown of MLL5 induced hyper-phosphorylation of p53 at Ser392, but not at Ser15, Ser20 or Ser46, in multiple cell lines tested (Cheng et al., 2008). Therefore, we asked whether the downregulation of MLL5 contributed to the CPT-induced p53 activation via Ser392 phosphorylation. First, the protein expression and phosphorylation of p53 in response to CPT or MLL5-siRNA treatment was compared in WI-38 and HCT116 cells. Two different MLL5 siRNAs were selected to minimize off-target effects. Hyper-phosphorylation of p53 at Ser392 and up-regulation of p21 was observed in both CPT-treated and MLL5-knockdown cells (Figure 3a). However, Ser15 phosphorylation, known to result from CPT-induced activation of ATM (Zhao et al., 2008), was only increased in the CPT-treated cells. The expression of p53 protein was remarkably upregulated by both CPT treatment and MLL5 knockdown in WI-38 cells, but only slightly enhanced in HCT116 cells, probably due to the higher-than-normal basal level of p53 in cancer cells (Figure 3a and Supplementary Figure S4).

Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

CPT-induced downregulation of MLL5 leads to phosphorylation of p53 at Ser392. (a, b) Total cell lysates were obtained from cells under indicated treatments and subjected to western blot, using specified antibodies. (a) Both CPT and MLL5 siRNAs enhanced the Ser392 phosphorylation of p53 and expression of p21, but the Ser15 phosphorylation was only induced by CPT treatment rather than knockdown of MLL5. For CPT treatment, HCT116 and WI-38 cells were treated with 5μM CPT for 6h before lysis; for siRNA transfection, cells were transfected with either scrambled (Sc) siRNA or MLL5 (M5) siRNAs and harvested at 48h post transfection. (b) Exogenous overexpression of MLL5 blocked the CPT-induced Ser392 phosphorylation of p53, but not the Ser15 phosphorylation. WI-38 cells were transfected with GFP-MLL5 and subjected to a fluorescence-activated cell sorter at 48h post transfection. Both GFP-positive and negative cells were immediately treated with 5μM CPT after sorting and lysed after 6h. Because of the large size of MLL5 protein, endogenous MLL5 and exogenous overexpressed-GFP-MLL5 proteins could not be well separated by SDS–PAGE, so the total MLL5 detected by anti-MLL5 antibody comprised both endogenous MLL5 and GFP-MLL5.

Full figure and legend (72K)

Next, we tested whether overexpression of MLL5 could abrogate the CPT-induced Ser392 phosphorylation. WI-38 cells overexpressing GFP-MLL5 were isolated by a fluorescence-activated cell sorter at 48h post transfection. Both GFP-positive and negative cells were then treated with CPT for 6h before harvest. The ectopically-expressed GFP-MLL5 and the endogenous MLL5 were indistinguishable when probed with antibody against endogenous MLL5, because of its large protein size. In the GFP-negative cells, total MLL5 was effectively degraded by CPT with p53 being upregulated. In contrast, in the GFP-positive cells, the total MLL5 including both the endogenous MLL5 and GFP-MLL5 proteins was not as effectively degraded by CPT, and the CPT-induced p53 activation was also compromised (Figure 3b). Moreover, overexpression of GFP-MLL5 abrogated the CPT-induced Ser392 phosphorylation of p53, but not the ATM-dependent Ser15 phosphorylation, further confirming that the CPT-induced downregulation of MLL5 acted independently of the ATM pathway. Taken together, our data suggested that CPT-induced degradation of MLL5 induced hyper-phosphorylation of p53 at Ser392, which contributed to the activation of p53.

MLL5 negatively regulates p53 at a post-translational level

To investigate how such negative regulation of p53 mediated by MLL5 knockdown took place, we first examined the stability of p53 protein in MLL5-knockdown cells, as phosphorylation modification usually couples with its stabilization (Bode and Dong, 2004). HCT116 cells were transfected with either scrambled siRNA or MLL5-siRNA#1. Cycloheximide was added 48h post transfection to inhibit the de novo protein synthesis, and cells were harvested at indicated time points. The decay of p53 protein was delayed in MLL5-knockdown cells (Figures 4a and b), indicating that the downregulation of MLL5 rendered p53 protein more stable. In contrast, p53 mRNA level was unaffected upon MLL5 knockdown (Figure 4c). In unperturbed cells, p53 level is kept at minimum by constant ubiquitination-dependent degradation. Therefore, the enhanced stability of p53 upon knockdown of MLL5 might be due to decreased p53 ubiquitination. To test this, at 48h post transfection with either scrambled or MLL5-siRNAs, proteasome inhibitors MG132 or N-acetyl-leucyl-leucyl-norleucinal were added to the culture medium for 14h before harvest for western blot analysis. Ubiquitination of p53 was reduced when MLL5 was knocked down in both HCT116 and U2OS cells (Figure 4d and Supplementary Figure S5).

Figure 4.
Figure 4 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

MLL5 negatively regulates p53 at a post-translational level. (ah) HCT116 cells transfected with scrambled (Sc) siRNA or MLL5 (M5) siRNA #1. (a, b) The p53 from MLL5-knockdown cells showed an increased half-life compared with the Sc siRNA-transfected cells. Cycloheximide (100μg/ml) was added to cells at 48h post transfection and the lysates were collected at indicated time points after cycloheximide addition. Quantification of western blot was done by ImageJ (NIH, Bethesda, MD, USA) and plotted using Excel (Microsoft, Redmond, WA, USA). (c) No changes in p53 mRNA expression in MLL5-knockdown cells were observed. Total RNA was extracted from Sc or MLL5 siRNA-transfected HCT116 cells at 48h post transfection and the expression of p53 mRNA was measured by reverse-transcription PCR. (d) The poly ubiquitination of p53 in MLL5-knockdown cells was reduced compared with the Sc siRNA-transfected cells. MG132 (20μM) or N-acetyl-leucyl-leucyl-norleucinal (ALLN, 20μM) was added to cells at 48h post transfection and allowed for incubation of 14h before lysis. (e) Knockdown of MLL5 caused a translocation of cellular p53 from the nucleoplasmic fraction to chromatin-associated fraction. The cellular protein of HCT116 was fractionated into cytoplasmic (Cyt), nucleoplasmic (Nuc) and chromatin-associated (Chr) fractions at 48h post transfection. Chromatin-bound proteins were further illustrated by their dissociation from chromatin after treatment with micrococcal nuclease. Tubulin, MCM6 and histone H1 were used as markers for Cyt, Nuc and Chr fractions, respectively. ‘WCL’ stands for whole cell lysate. (f) More p53 accumulated at chromatin after knockdown of MLL5. Both total cellular p53 and chromatin-bound p53 were stained at 48h post transfection. (g) More p53 associated with the promoter sequences of p21 and Gadd45 in MLL5-knockdown cells compared with Sc siRNA-transfected cells. At 48h post transfection, cells were crosslinked and chromatin immunoprecipitation was performed using anti-p53 antibody. The eluates were used as templates and amplified with primers specific for p21 and Gadd45 promoters. (h) The mRNA of both p21 and Gadd45 were upregulated by knockdown of MLL5. The total RNA was extracted from Sc or MLL5 siRNA-transfected HCT116 cells. The p21 and Gadd45 mRNA expression was measured by semi-quantitative reverse-transcription PCR.

Full figure and legend (161K)

It has been suggested that Ser392 phosphorylation enhances the binding of p53 to the DNA (Sakaguchi et al., 1997). To study if the affinity of p53 to DNA was affected by downregulation of MLL5, cellular fractionation was performed in scrambled or MLL5-siRNA-transfected HCT116 cells. Consistent with the previous reports (Deng et al., 2004; Liu et al., 2010b), MLL5 was primarily detected in the chromatin-associated fraction (Figure 4e). Interestingly, a detectable translocation of p53 from the nucleoplasmic to the chromatin-associated fraction was observed upon MLL5 knockdown, an indication of an enhanced recruitment of p53 to its target site (Figure 4e). When subjected to micrococcal nuclease, a remarkable portion of the chromatin-bound p53 was released into the nucleoplasmic fraction, suggesting that the chromatin-accumulated p53 upon MLL5 knockdown was indeed associated with DNA. To confirm this observation, chromatin-bound p53 was visualized by immunofluorescent staining, with cytoplasmic and nucleoplasmic proteins washed away before fixation. Consistently, knockdown of MLL5 caused a remarkable increase in the level of chromatin-bound p53, but only a slight enhancement in the whole cellular p53 (Figure 4f). The percentage of cells with enhanced chromatin-bound p53 was calculated from three independent experiments, revealing a 4.1-fold increase after the knockdown of MLL5 (Supplementary Figure S6). To verify whether such chromatin accumulation of p53 was functional, chromatin immunoprecipitation was carried out in scrambled or MLL5-siRNA-transfected HCT 116 cells, using anti-p53 antibody. The chromatin immunoprecipitation eluates were tested for the abundance of the promoter regions of p53 downstream targets, p21 and Gadd45. Results showed that the chromatin accumulation of p53 did translate to an enhanced binding to the promoters of both genes (Figure 4g). Semi-quantitative RT–PCR of p21 and Gadd45 further confirmed that both genes were upregulated (Figure 4h).

MLL5 interacts with p53 tetramers and prevents them from binding to chromatin

Next, we asked whether MLL5 physically interacts with p53. We performed co-immunoprecipitation of endogenous MLL5 and p53 in HCT116 cells, where p53 was detected in the immune complex pulled down, using anti-MLL5 antibody (Figure 5a). To map the interacting domains of MLL5 and p53, we overexpressed FLAG-MLL5 and its deletion fragments, comprising of the PHDSET domain (MLL5-PS, 1-560aa), the central domain (MLL5-CD, 562-1112aa) and the C-terminal domain (MLL5-CT, 1113-1858aa) in 293T cells and performed immunoprecipitation using anti-FLAG antibody. As shown in Figure 5b, the central domain of MLL5 was required for the interaction with p53, which was confirmed by the absence of interaction between the CD-deletion mutant (MLL5-ΔCD) and p53. To find out the region of p53 that interacted with MLL5, hemagglutinin (HA)-tagged fragments of p53 were co-transfected into 293T cells with MLL5-CD fragment, followed by immunoprecipitation using anti-FLAG antibody. Results showed that the DNA-binding domain of p53 (p53-a) was required for the interaction with MLL5 (Figure 5c). Interestingly, the p53-e fragment, which included the tetramerization domain (TD), but lacked the C-terminal regulatory domain (363-393aa), showed a much stronger affinity to MLL5, as compared with the full-length p53 or fragment p53-d (Figure 5c). Therefore, MLL5 may preferentially bind to p53 tetramer, but the presence of C-terminal regulatory domain may inhibit the interaction or the tetramer formation. To test this hypothesis, HA-tagged TD-deletion mutant of p53 (p53ΔTD) was tested for its interaction with MLL5-CD. Results showed that p53ΔTD had similar affinity to MLL5-CD as compared with full-length p53 or p53-e, suggesting that the C-terminal regulatory domain may have an inhibitory effect on tetramerization (Supplementary Figure S7). However, TD and C-terminal regulatory domain alone (p53-b) was unable to interact with MLL5, implying that the DNA-binding domain was the key region responsible for the interaction.

Figure 5.
Figure 5 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Mapping of the interacting domains of MLL5 and p53. (a) Endogenous MLL5 and p53 interact. HCT116 cells were lysed and immunoprecipitated by rabbit IgG or anti-MLL5 antibody (Cheng et al., 2008). The eluates were subject to western blot, using anti-MLL5 or anti-p53 antibody. (b) The central domain of MLL5 interacted with p53. Full-length or fragments of MLL5 (MLL5-PS, CD, CT, ΔCD) were tagged with a FLAG epitope and transfected into 293T cells. Cells were lysed 48h post transfection and immunoprecipitated by mouse IgG or anti-FLAG antibodies. The eluates were subjected to western blot, using anti-FLAG or anti-p53 antibody. (c) The DNA-binding domain of p53 was required for interaction with MLL5. The interaction was enhanced by TD, whereas it was inhibited by the C-terminal regulatory domain. FLAG-tagged MLL5-CD and HA-tagged p53 fragments were co-transfected into 293T cells, lysed 48h post transfection and immunoprecipitated by anti-FLAG antibody. The eluates were subjected to western blot, using anti-FLAG or anti-HA antibody. ‘TAD’ stands for transactivation domain.

Full figure and legend (155K)

To further test whether the formation of p53-tetramers would enhance the interaction, a tetramerization assay was conducted to assess the tetramerization status of MLL5-interacting p53 fragment (p53-e). FLAG-MLL5-CD and HA-p53-e were co-transfected into 293T cells and immunoprecipitated using anti-FLAG or anti-HA antibodies. Before elution, crosslinking agent glutaraldehyde was added to the agarose beads to preserve the formation of p53 oligomer. After elution, equal amount of p53-e fragment, either pulled down by anti-FLAG or anti-HA antibodies, was analyzed by western blot, using anti-HA antibody. Results showed that the moiety of MLL5-CD-interacting p53-e (pulled down by anti-FLAG antibody) contained more tetramers compared with the total p53-e (pulled down by anti-HA antibody) (Figure 6a). The preference of MLL5 to interact with p53 tetramers led us to investigate whether the p53 tetramer formation was affected by knockdown of MLL5. HCT116 cells were transfected with either scrambled or MLL5-siRNAs, and a tetramerization assay was performed 48h post transfection. Interestingly, more p53 tetramers were formed in MLL5-knockdown HCT116 cells (Figure 6b). Taken into consideration that MLL5 primarily localized at chromatin and the chromatin-bound p53 increased upon MLL5 knockdown (Figures 4e and f), we then hypothesized that MLL5 may interact with the p53 tetramers and prevent them from being recruited to their target sites on chromatin. Additionally, when MLL5 was depleted by siRNA, p53 tetramers would be able to accumulate at chromatin region, become phosphorylated at Ser392 and activate its downstream targets.

Figure 6.
Figure 6 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

MLL5 preferentially interacts with p53 tetramers and knockdown of MLL5 causes the accumulation of p53 tetramers. (a) More MLL5-CD-interacting p53-e (pulled down by anti-FLAG antibody) existed as tetramers compared with the whole cellular p53-e (pulled down by anti-HA antibody). FLAG-tagged MLL5-CD and HA-tagged p53-e were co-transfected into 293T cells. Cells were lysed at 48h post transfection and immunoprecipitated, using either anti-FLAG or anti-HA antibody. The immune complexes were subject to a tetramerization assay by incubation with increasing doses of glutaraldehyde before elution. The eluates were analyzed by western blot, using anti-HA antibody. (b) Knockdown of MLL5 enhanced the formation of p53 tetramers. HCT116 cells were transfected with either scrambled (Sc) or MLL5 (M5) siRNA #1. Cells were lysed at 48h post transfection and the lysates were subjected to a tetramerization assay before western blot analysis. Actin was used as a loading control.

Full figure and legend (76K)

CPT-induced downregulation of MLL5 is conserved in developing zebrafish

CPT-induced cleavable complex formation is conserved from yeast to mammals (Liu, 1989). In zebrafish, CPT induces p53 activation and causes apoptosis (Langheinrich et al., 2002; Le et al., 2009). Therefore, we tested whether MLL5-mediated p53 activation by CPT was conserved in developing zebrafish. Embryos at 24h post fertilization were treated with CPT for 6h at indicated doses before collection. Results showed that zebrafish Mll5 was effectively degraded at a dose-dependent manner (Figure 7A). Next, we examined whether overexpression of zebrafish Mll5 protein could reverse the CPT-induced p53 activation. Embryos were microinjected with zebrafish mll5 mRNA at one-cell or two-cell stage, and treated with CPT at 24h post fertilization for 6h. Results showed that overexpression of zebrafish mll5 partially blocked the induction of p53 by CPT (Figure 7B), suggesting that zebrafish Mll5 may mediate the CPT-induced p53 activation. The effect of Mll5 overexpression on the p53 activation was confirmed by the restoration of p21 mRNA expression measured by RT–PCR (Figure 7B). To confirm that the p53 induction was functional, CPT-induced cellular apoptosis was measured using a terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay. Results showed that the CPT treatment indeed led to massive cell death, and such CPT-induced apoptosis was effectively rescued by overexpression of zebrafish mll5 (Figure 7C, a′–d′). The number of TUNEL-positive cells in the anterior hindbrain (Figure 7C, a′–d′) was counted from three independent experiments and plotted in a histogram (Figure 7D), showing there was about 4.6-fold decrease in CPT-induced apoptotic signal when zebrafish mll5 mRNA was microinjected. In order to confirm the participation of Mll5 in the regulation of p53-dependent apoptosis, similar TUNEL experiment was done in p53-null zebrafish. Results showed that the overexpression of mll5 could not rescue the p53-independent CPT-induced apoptosis (Supplementary Figure S8).

Figure 7.
Figure 7 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

CPT downregulates Mll5 and activates p53 in developing zebrafish. (A) Zebrafish Mll5 was downregulated by CPT in a dose-dependent manner. Zebrafish embryos at 24h post fertilization were treated with increasing doses of CPT for 6h before total protein extraction. (bd) Zebrafish mll5 mRNA was microinjected into wild-type embryos at one-cell or two-cell stage at 0.2ng per embryo. The embryos were allowed to grow for 24h until treatment, with 2μM CPT for 6h. Embryos were then harvested for western blot, reverse-transcription PCR or TUNEL assay. (B) Overexpression of zebrafish mll5 mRNA partially abrogated the CPT-induced upregulation of p53 protein and p21 mRNA. Tubulin and gapdh were used as loading controls for western blot and PCR, respectively. (C, D) Overexpression of zebrafish mll5 mRNA blocked the CPT-induced apoptosis in wild-type embryos. TUNEL-positive cells at the anterior hindbrain were counted (C, a′–d′), and data from three independent experiments were plotted in (D) using Excel.

Full figure and legend (128K)

Top

Discussion

In this study, using Top1 poison CPT, we identified a new function of MLL5 in the homeostasis of p53 during S-phase progression. The antitumor activity of CPT mainly results from its specific interference with the breakage-reunion reaction of Top1, which causes the reversible formation of covalently cleavable complexes consisting of Top1, CPT and DNA (Desai et al., 2001). Most of the downstream effects caused by cleavable complex are viewed as DNA damage responses, including p53 stabilization, G2-phase arrest, nuclear factor-κB activation and replication protein A phosphorylation (Shao et al., 1999; Huang et al., 2000; Desai et al., 2001; Li and Liu, 2001). CPT has been previously reported to induce p53 phosphorylation at Ser15 through the ATM/Chk2 activation (Zhao et al., 2008). Although the phosphorylation at Ser392 by CPT was also reported, the mechanism is unclear (Houser et al., 2001). Here, we documented a novel function of MLL5 in CPT-induced p53 activation in actively replicating cells, CPT induced p53 phosphorylation at Ser392 through the downregulation of MLL5. MLL5 degradation was likely to be independent of ATM or ATR, as caffeine was unable to block the CPT-induced degradation of MLL5 (Figure 2b). Overexpression of MLL5 abolished phosphorylation of p53 at Ser392, but not at Ser15 (Figure 3b), further proving that CPT-MLL5-p53 pathway worked in parallel with the ATM/ATR. MLL5 was only effectively degraded in replicating cells (Figure 2c), and the replication inhibitor Aph partially abrogated the downregulation of MLL5 by CPT treatment (Figure 2d). Moreover, MLL5 in cells released from G1/S boundary was degraded by CPT during S-phase progression (Figure 2e). Therefore, it is reasonable to speculate that the degradation of MLL5 required the collision event between DNA replication machinery and the cleavable complex. However, such downregulation might not be directly related to the inhibition of Top1, as β-lapachone, another class of Top1 inhibitor with a mode of action distinct from CPT, was unable to degrade MLL5 or activate p53 through Ser392 phosphorylation (Supplementary Figure S9). RNAi-mediated knockdown of MLL5 was previously shown to delay or arrest the G1/S transition (Cheng et al., 2008; Sebastian et al., 2009). Therefore, it is plausible that the elimination of MLL5 is required for delaying the cell cycle progression, allowing deployment of damage repair mechanism.

Previously, CPT was shown to trigger proteasome degradation of cytoplasmic IκBα, which facilitates the entering of nuclear factor-κB into nucleus and activation of its target genes (Huang et al., 2000). Another protein targeted for degradation by CPT is Cdc25A, an essential cell cycle regulator implicated in both G1/S and G2/M phases. CPT induces Chk1-dependent degradation of Cdc25A, which leads to the activation of both S phase and G2/M checkpoints (Xiao et al., 2003). In view of our data, we hypothesize that the cleavable complex may selectively target for degrading a defined group of molecules, including IκBα, Cdc25A and MLL5. This in turn activates a multitude of signaling cascades resulting in cell cycle arrest, followed by DNA damage repair or apoptosis. Further investigation into the ubiquitin/26S proteasomal degradation of MLL5 may reveal the detailed mechanism of CPT-induced MLL5 downregulation.

Function of p53 is tightly regulated by a diverse array of post-translational modifications, including phosphorylation, acetylation, ubiquitination and sumoylation (Haupt et al., 1997; Gostissa et al., 1999; Liu et al., 1999; Tibbetts et al., 1999; Bode and Dong, 2004). Phosphorylation of p53 at Ser392 was known to be responsive to UV but not γ-irradiation (Lu et al., 1998; Keller et al., 2001). Moreover, Ser392 phosphorylation enhances sequence-specific DNA binding in vitro, possibly by stabilizing the tetramer formation of p53 (Sakaguchi et al., 1997). In support of this view, our results also demonstrated that the phosphorylation of p53 at Ser392 caused by downregulation of MLL5 resulted in the enhanced accumulation of p53 tetramers at their target chromatin region. So far, four kinases were known to directly or indirectly associate with the Ser392-phosphorylation of p53, including CDK9, CK2, p38 and PKR (Cuddihy et al., 1999; Huang et al., 1999; Keller et al., 2001; Claudio et al., 2006). Possible involvement of these kinases in the CPT-induced Ser392 phosphorylation was evaluated by treating kinase-knockdown cells with CPT, followed by detecting Ser392 phosphorylation signals. None of the kinases, when knocked down alone or together, could block the CPT-induced Ser392 phosphorylation in a substantial level (Supplementary Figure S10). This suggested that additional novel kinase(s) might mediate the CPT-induced Ser392 phosphorylation. We, hence, hypothesized that the absence of MLL5 would lead to more p53 tetramers accumulate at chromatin, which in turn recruit the kinases to phosphorylate p53 at Ser392. In support of our hypothesis, knockdown of MLL5 led to Ser392 phosphorylation and stabilization of p53 (Figures 3b and 4a), but not the S392A mutant or the tetramerization domain-deletion mutant (Supplementary Figure S11). Overexpression of MLL5 abrogated the CPT-induced p53 phosphorylation at Ser392 (Figure 3b), suggesting that the cellular sensitivity to CPT may depend on the intracellular level of MLL5. This has a great promise for future studies, including (a) an analysis of cellular mechanisms of MLL5 regulation, (b) the role of MLL5 in an acquisition of cellular resistance to CPT and its analogs (example, topotecan and SN-38). Further investigation should be done to explore the possibility of targeting MLL5 in Top1 poison-mediated chemotherapy.

We showed that MLL5 preferentially interacted with p53 tetramer and knockdown of MLL5 caused chromatin accumulation of p53 tetramers (Figures 5 and 6), which may serve as a novel mechanism of keeping in check the p53 target genes in unperturbed cells. The TD of p53 was not required for its interaction with MLL5, but rather enhanced the interaction. The DNA-binding domain alone (p53-a fragment) was sufficient for p53 to interact with MLL5. Interestingly, in the p53 mutants with mutations potentially affecting its DNA binding capability, including R175H (conformational mutant), R248W (DNA-binding mutant), and S392A, all three mutants were still able to interact with MLL5 (Supplementary Figure S12). In the nucleus, equilibrium exists between monomers, dimers and tetramers of p53 (Friedman et al., 1993; Chene, 2001). We speculate that in the presence of chromatin-bound MLL5, p53 tetramers are excluded from the chromatin and the equilibrium is shifted towards dimers or monomers that can shuttle to cytoplasm for degradation (Figure 8a). Depletion of MLL5 by CPT or siRNA would lead to accumulation of p53 tetramers at the chromatin, which subsequently would be stabilized through Ser392 phosphorylation (Figure 8b). Recently, several histone-modifying enzymes have emerged as potential regulators of p53 (Grossman et al., 2003; Chuikov et al., 2004). However, it is still unclear whether MLL5 can modify p53 upon interaction, or MLL5 merely serves as a chromatin-anchored adapter for a larger protein complex that negatively regulates p53. Better characterization of the function of conserved domains on MLL5, such as the PHD zinc finger (Scheel and Hofmann, 2003; Dul and Walworth, 2007; Kim and Buratowski, 2009) and SET domain (Berger, 2002), will be useful to understand the nature of the negative regulation of p53 by MLL5 in more details.

Figure 8.
Figure 8 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Proposed models for the role of MLL5 in CPT-induced p53 activation. (a) In unperturbed cells, chromatin-bound MLL5 interacts with p53 tetramers and prevents them from binding to chromatin, rendering the transcription of p53 target genes inactive. Presence of MLL5 shifts the equilibrium of nuclear p53 towards monomers and dimers. (b) When cells are treated with CPT, MLL5 is readily degraded and thereby p53 tetramers are able to bind to chromatin and activate its downstream target genes. The p53 tetramers are stabilized by Ser392 phosphorylation, contributing to shift the equilibrium of nuclear p53 towards tetramers.

Full figure and legend (81K)

In developing zebrafish, CPT treatment caused the degradation of zebrafish Mll5 and activation of p53, whereas ectopic overexpression of zebrafish mll5 mRNA abrogated the induction of p53 expression (Figure 7B). The Ser392 of human p53 is conserved in zebrafish as Ser372, so we speculated that CPT-induced downregulation of zebrafish Mll5 could lead to phosphorylation of zebrafish p53 at Ser372. However, we were unable to validate the hypothesis due to the lack of phosphor-specific antibodies for zebrafish p53. Ectopic overexpression of zebrafish mll5 abrogated the induction of p53 expression, and significantly blocked the CPT-induced apoptosis in p53 wild-type embryos (Figures 7C and D). However, the overexpressed mll5 had little effect on the CPT-induced apoptosis in p53-null embryos (Supplementary Figure S8), further confirming that the protective role of mll5 against apoptosis relied upon p53. CPT treatment caused significant apoptosis in developing zebrafish, but not in mammalian cells, possibly because the intensive DNA replication coupled with the rapid cell division made zebrafish embryos extremely vulnerable to CPT treatment. Our observations in developing zebrafish suggested an evolutionarily conserved role of MLL5 in the CPT-induced p53 activation.

In summary, our results provide evidence for a novel role of MLL5 in the CPT-induced p53 activation. The replication-dependent degradation of MLL5 by CPT treatment caused the Ser392 phosphorylation and stabilization of p53, further leading to the activation of its target genes. Function of MLL5 in preventing p53 tetramers from binding to chromatin has added one more layer of complexity to the already intricate regulation of p53. More studies are required to completely understand the nature of the negative regulation of p53 by MLL5.

Top

Materials and methods

Cell culture and plasmid construction

Human lung fibroblast WI-38, cervical carcinoma HeLa, osteosarcoma U2OS and embryonic kidney HEK 293T cells were obtained from American Type Culture Collection (Manassas, VA, USA). Colorectal carcinoma cell lines HCT116 p53+/+ and HCT116 p53/ were kind gifts from Dr Bert Vogelstein (Bunz et al., 1998). Cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, L-glutamine (2mM), penicillin (100 units/ml) and streptomycin (100μg/ml). Generation of FLAG-tagged MLL5 deletion mutants and GFP-tagged MLL5 were described before (Deng et al., 2004; Liu et al., 2010b). The HA-tagged p53 deletion mutants were cloned into pXJ40 in frame with BamHI and NotI sites.

Cell synchronization and cell cycle analysis

HCT116 cells were synchronized to G1/S boundary by incubation with 2mM thymidine for 18h, followed by releasing in drug-free medium for 9h, and a second incubation with 2mM thymidine for 18h. To analyze the cell cycle profile, cells were fixed with 70% ethanol on ice for at least 2h and stained in propidium iodide solution (20μg/ml propidium iodide, 100μg/ml RNase A and 0.1% Triton X-100) for 30min at 37°C. The cellular DNA content was analyzed by a flow cytomter (Dako CyAn ADP, Glostrup, Denmark) and the data were processed using Summit (Beckman Coulter, Fullerton, CA, USA).

Cell lysate preparation, cellular protein fractionation and western blot

For total cell lysate preparation, cells were lysed in Laemmli sample buffer (62.5mM Tris–HCl pH 6.8, 2.5% SDS, 10% glycerol, 0.01% bromophenol blue), boiled at 100°C for 5min and sonicated for 20s at 30% output power (Sonics VCX130, Newtown, CT, USA). Cellular protein fractionation was performed as previously described (Mendez and Stillman, 2000). In brief, HCT116 cells were lysed (40 million cells/ml) for 5min at 4°C in buffer A (10mM (4-(2-hydroxyethyl-1-piperazineethane sulfonic acid) pH7.9, 10mM KCl, 1.5mM MgCl2, 0.34M sucrose, 10% glycerol, 1mM dithiothreitol, 0.1% Triton X-100) supplemented with protease and phosphatase inhibitors (2mM phenylmethanesulfonylfluoride, 2μg/ml leupeptin, 2μg/ml aprotinin, 1μg/ml pepstatin A, 1mM Na3VO4 and 5mM NaF). After centrifugation at 1300g for 5min, the nuclei were collected in the pellet, whereas the supernatant was further clarified by centrifugation at 21000g for 10min and kept as the cytoplasmic fraction. Nuclei were then washed twice with buffer A, and lysed for 20min in buffer B (3mM ethylenediamine tetraacetic acid, 0.2mM ethylene glycol tetraacetic acid, 1mM dithiothreitol) supplemented with protease and phosphatase inhibitors. After centrifugation at 1700g for 5min, the supernatant was kept as nucleoplasmic fraction, whereas the remaining pellet was dissolved in Laemmli sample buffer and kept as chromatin-associated fraction. To release chromatin-bound protein, the nuclei were treated with 100 units of MNase (Fermentas #EN0181, Glen Burnie, MD, USA) at 37°C for 3min before adding buffer B. A flowchart of the fractionation protocol was illustrated in Supplementary Figure S13. Information on antibodies can be found in Supplementary Table 1.

Transfection and RT–PCR

Cells were seeded one day before transfection in antibiotic-free medium. Plasmids and siRNAs were transfected using TransIT (Mirus, Madison, WI, USA) and Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA), respectively. For RT–PCR, total RNA was extracted using TRIzol (Invitrogen) and the cDNA was synthesized using iScript kit (Bio-Rad, Hercules, CA, USA). Sequences of siRNAs and primers were included in Supplementary Table 1.

Chromatin immunoprecipitation

The chromatin immunoprecipitation was performed as previously described, with minor modifications (Lee et al., 2006). In brief, cells were crosslinked, using formaldehyde at a final concentration of 1% for 10min before the addition of glycine (final concentration 125mM). Cells were then scraped off, lysed and sonicated before overnight incubation with 2μg mouse IgG or p53 antibody at 4°C. The lysate-antibody mixture was then incubated with anti-mouse beads for 2h before reverse crosslinking and elution. DNA were purified and amplified by PCR, using primers included in Supplementary Table 1.

Immunofluorescence

To stain chromatin-bound proteins, HCT116 cells grown on cover-slips were incubated with buffer (150mM NaCl, 20mM Tris–HCl pH 8.0, 1% Triton X-100) supplemented with protease and phosphatase inhibitors for 30min on ice, before fixation with 100% methanol for 10min at −20°C. Cells were then re-hydrated with phosphate-buffered saline (PBS) before blocking in 5% bovine serum albumin for 1h. Incubation with p53 antibody was done overnight at 4°C, followed by washing with 0.05% Tween-20/PBS (PBST) for three times. Cells were then incubated with fluorescein isothiocyanate-conjugated anti-rabbit antibody for 1h. After washing with PBST, DNA was stained with 4′, 6-diamidino-2-phenylindole, dihydrochloride (Invitrogen, #D1306), and the coverslips were mounted to glass slides with FluorSave (Merck #345789, Cincinnati, OH, USA). Staining was visualized using inverted fluorescence microscopy (Olympus IX71, Tokyo, Japan) equipped with QICAM camera (QImaging, Surrey, BC, Canada), and analyzed by ImagePro software (Media Cybernetics, Bethesda, MD, USA).

Immunoprecipitation and tetramerization assay

The immunoprecipitation (Liu et al., 2010a) and tetramerization assay (Foo et al., 2007) were performed essentially, as previously described. For both assays, cells were first lysed in buffer (150mM NaCl, 20mM Tris–HCl pH 8.0, 1% Triton X-100) supplemented with protease and phosphatase inhibitors for 10min on ice. For immunoprecipitation, the lysates were pre-cleared with protein G beads before incubation with 15μg MLL5 antibody or 2μg FLAG antibody for 2h at 4°C, followed by 1h incubation with protein G beads. The immune complexes were then washed with PBST and eluted by Laemmli sample buffer. For tetramerization assay, glutaraldehyde (Sigma-Aldrich #G7776, St Louis, MO, USA) was added to the lysates at a final concentration of 0.01 or 0.1% to allow crosslinking for 5min on ice. The crosslinked-lysates were then mixed with equal volume of Laemmli sample buffer, boiled and analyzed by western blot.

Zebrafish, microinjection and in situ TUNEL assay

The wild-type zebrafish were maintained by standard protocols (Westerfield, 2000) and IACUC regulations (Biopolis IACUC application #050096). The zebrafish mll5 was cloned into pCS2 vector, and mll5 mRNA was synthesized using mMessenger (Ambion, Austin, TX, USA). The mll5 mRNA was microinjected into embryos at one-cell to two-cell stage using PLI-100 pico-injector (Harvard Apparatus, Holliston, MA, USA) at 0.2ng per embryo. For TUNEL assay, embryos were fixed with 4% paraformaldehyde for overnight at 4°C. Fixed embryos were then rinsed with PBS, dehydrated in 100% methanol for 2h at −20°C and treated with 100% acetone for 10min at −20°C. Embryos were rehydrated in a successive dilution of methanol in PBS (75, 50, 25% methanol) and rinsed with PBS twice. Embryos were subsequently permeabilized with 10μg/ml protease K (Roche, Mannheim, Germany) for 10min and re-fixed in paraformaldehyde for 20min. The remaining steps were then performed using in situ cell death detection kit (Roche), according to manufacturer's protocol.

Top

Conflict of interest

The authors declare no conflicts of interest.

Top

References

  1. Bartek J, Lukas J. (2001). Mammalian G1- and S-phase checkpoints in response to DNA damage. Curr Opin Cell Biol 13: 738–747. | Article | PubMed | ISI | ChemPort |
  2. Berger SL. (2002). Histone modifications in transcriptional regulation. Curr Opin Genet Dev 12: 142–148. | Article | PubMed | ISI | ChemPort |
  3. Bode AM, Dong Z. (2004). Post-translational modification of p53 in tumorigenesis. Nat Rev Cancer 4: 793–805. | Article | PubMed | ISI | ChemPort |
  4. Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, Brown JP et al. (1998). Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 282: 1497–1501. | Article | PubMed | ISI | ChemPort |
  5. Chene P. (2001). The role of tetramerization in p53 function. Oncogene 20: 2611–2617. | Article | PubMed | ISI | ChemPort |
  6. Cheng F, Liu J, Zhou SH, Wang XN, Chew JF, Deng LW. (2008). RNA interference against mixed lineage leukemia 5 resulted in cell cycle arrest. Int J Biochem Cell Biol 40: 2472–2481. | Article | PubMed |
  7. Chuikov S, Kurash JK, Wilson JR, Xiao B, Justin N, Ivanov GS et al. (2004). Regulation of p53 activity through lysine methylation. Nature 432: 353–360. | Article | PubMed | ISI | ChemPort |
  8. Claudio PP, Cui J, Ghafouri M, Mariano C, White MK, Safak M et al. (2006). Cdk9 phosphorylates p53 on serine 392 independently of CKII. J Cell Physiol 208: 602–612. | Article | PubMed | ISI | ChemPort |
  9. Cuddihy AR, Wong AH, Tam NW, Li S, Koromilas AE. (1999). The double-stranded RNA activated protein kinase PKR physically associates with the tumor suppressor p53 protein and phosphorylates human p53 on serine 392 in vitro. Oncogene 18: 2690–2702. | Article | PubMed | ISI | ChemPort |
  10. Dambacher S, Hahn M, Schotta G. (2010). Epigenetic regulation of development by histone lysine methylation. Heredity 105: 24–37. | Article | PubMed | ISI |
  11. Deng LW, Chiu I, Strominger JL. (2004). MLL 5 protein forms intranuclear foci, and overexpression inhibits cell cycle progression. Proc Natl Acad Sci USA 101: 757–762. | Article | PubMed |
  12. Desai SD, Li TK, Rodriguez-Bauman A, Rubin EH, Liu LF. (2001). Ubiquitin/26S proteasome-mediated degradation of topoisomerase I as a resistance mechanism to camptothecin in tumor cells. Cancer Res 61: 5926–5932. | PubMed | ISI | ChemPort |
  13. Dimri GP, Hara E, Campisi J. (1994). Regulation of two E2F-related genes in presenescent and senescent human fibroblasts. J Biol Chem 269: 16180–16186. | PubMed | ISI | ChemPort |
  14. Dul BE, Walworth NC. (2007). The plant homeodomain fingers of fission yeast Msc1 exhibit E3 ubiquitin ligase activity. J Biol Chem 282: 18397–18406. | Article | PubMed | ISI |
  15. Elledge SJ. (1996). Cell cycle checkpoints: preventing an identity crisis. Science 274: 1664–1672. | Article | PubMed | ISI | ChemPort |
  16. Emerling BM, Bonifas J, Kratz CP, Donovan S, Taylor BR, Green ED et al. (2002). MLL5, a homolog of Drosophila trithorax located within a segment of chromosome band 7q22 implicated in myeloid leukemia. Oncogene 21: 4849–4854. | Article | PubMed | ISI | ChemPort |
  17. Eng WK, Faucette L, Johnson RK, Sternglanz R. (1988). Evidence that DNA topoisomerase I is necessary for the cytotoxic effects of camptothecin. Mol Pharmacol 34: 755–760. | PubMed | ISI | ChemPort |
  18. Foo RS, Nam YJ, Ostreicher MJ, Metzl MD, Whelan RS, Peng CF et al. (2007). Regulation of p53 tetramerization and nuclear export by ARC. Proc Natl Acad Sci USA 104: 20826–20831. | Article | PubMed |
  19. Friedman PN, Chen X, Bargonetti J, Prives C. (1993). The p53 protein is an unusually shaped tetramer that binds directly to DNA. Proc Natl Acad Sci USA 90: 3319–3323. | Article | PubMed | ChemPort |
  20. Fujiki R, Chikanishi T, Hashiba W, Ito H, Takada I, Roeder RG et al. (2009). GlcNAcylation of a histone methyltransferase in retinoic-acid-induced granulopoiesis. Nature 459: 455–459. | Article | PubMed | ISI | ChemPort |
  21. Gostissa M, Hengstermann A, Fogal V, Sandy P, Schwarz SE, Scheffner M et al. (1999). Activation of p53 by conjugation to the ubiquitin-like protein SUMO-1. EMBO J 18: 6462–6471. | Article | PubMed | ISI | ChemPort |
  22. Grossman SR, Deato ME, Brignone C, Chan HM, Kung AL, Tagami H et al. (2003). Polyubiquitination of p53 by a ubiquitin ligase activity of p300. Science 300: 342–344. | Article | PubMed | ISI | ChemPort |
  23. Haupt Y, Maya R, Kazaz A, Oren M. (1997). Mdm2 promotes the rapid degradation of p53. Nature 387: 296–299. | Article | PubMed | ISI | ChemPort |
  24. Houser S, Koshlatyi S, Lu T, Gopen T, Bargonetti J. (2001). Camptothecin and Zeocin can increase p53 levels during all cell cycle stages. Biochem Biophys Res Commun 289: 998–1009. | Article | PubMed | ISI | ChemPort |
  25. Huang C, Ma WY, Maxiner A, Sun Y, Dong Z. (1999). p38 kinase mediates UV-induced phosphorylation of p53 protein at serine 389. J Biol Chem 274: 12229–12235. | Article | PubMed | ISI | ChemPort |
  26. Huang TT, Wuerzberger-Davis SM, Seufzer BJ, Shumway SD, Kurama T, Boothman DA et al. (2000). NF-kappaB activation by camptothecin. A linkage between nuclear DNA damage and cytoplasmic signaling events. J Biol Chem 275: 9501–9509. | Article | PubMed | ISI | ChemPort |
  27. Hughes CM, Rozenblatt-Rosen O, Milne TA, Copeland TD, Levine SS, Lee JC et al. (2004). Menin associates with a trithorax family histone methyltransferase complex and with the hoxc8 locus. Mol Cell 13: 587–597. | Article | PubMed | ISI | ChemPort |
  28. Keller DM, Zeng X, Wang Y, Zhang QH, Kapoor M, Shu H et al. (2001). A DNA damage-induced p53 serine 392 kinase complex contains CK2, hSpt16, and SSRP1. Mol Cell 7: 283–292. | Article | PubMed | ISI | ChemPort |
  29. Kim T, Buratowski S. (2009). Dimethylation of H3K4 by Set1 recruits the Set3 histone deacetylase complex to 5’ transcribed regions. Cell 137: 259–272. | Article | PubMed | ISI | ChemPort |
  30. Kouzarides T. (2002). Histone methylation in transcriptional control. Curr Opin Genet Dev 12: 198–209. | Article | PubMed | ISI | ChemPort |
  31. Langheinrich U, Hennen E, Stott G, Vacun G. (2002). Zebrafish as a model organism for the identification and characterization of drugs and genes affecting p53 signaling. Curr Biol 12: 2023–2028. | Article | PubMed | ISI | ChemPort |
  32. Le MT, Teh C, Shyh-Chang N, Xie H, Zhou B, Korzh V et al. (2009). MicroRNA-125b is a novel negative regulator of p53. Genes Dev 23: 862–876. | Article | PubMed | ISI | ChemPort |
  33. Lee TI, Johnstone SE, Young RA. (2006). Chromatin immunoprecipitation and microarray-based analysis of protein location. Nat Protoc 1: 729–748. | Article | PubMed | ISI | ChemPort |
  34. Li TK, Liu LF. (2001). Tumor cell death induced by topoisomerase-targeting drugs. Annu Rev Pharmacol Toxicol 41: 53–77. | Article | PubMed | ISI | ChemPort |
  35. Liu H, Takeda S, Kumar R, Westergard TD, Brown EJ, Pandita TK et al. (2010a). Phosphorylation of MLL by ATR is required for execution of mammalian S-phase checkpoint. Nature 467: 343–346. | Article | ISI |
  36. Liu J, Wang XN, Cheng F, Liou YC, Deng LW. (2010b). Phosphorylation of mixed lineage leukemia 5 by CDC2 affects its cellular distribution and is required for mitotic entry. J Biol Chem 285: 20904–20914. | Article | ISI |
  37. Liu L, Scolnick DM, Trievel RC, Zhang HB, Marmorstein R, Halazonetis TD et al. (1999). p53 sites acetylated in vitro by PCAF and p300 are acetylated in vivo in response to DNA damage. Mol Cell Biol 19: 1202–1209. | PubMed | ISI | ChemPort |
  38. Liu LF. (1989). DNA topoisomerase poisons as antitumor drugs. Annu Rev Biochem 58: 351–375. | Article | PubMed | ISI | ChemPort |
  39. Lu H, Taya Y, Ikeda M, Levine AJ. (1998). Ultraviolet radiation, but not gamma radiation or etoposide-induced DNA damage, results in the phosphorylation of the murine p53 protein at serine-389. Proc Natl Acad Sci USA 95: 6399–6402. | Article | PubMed | ChemPort |
  40. Mendez J, Stillman B. (2000). Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis. Mol Cell Biol 20: 8602–8612. | Article | PubMed | ISI | ChemPort |
  41. Nitiss J, Wang JC. (1988). DNA topoisomerase-targeting antitumor drugs can be studied in yeast. Proc Natl Acad Sci USA 85: 7501–7505. | Article | PubMed | ChemPort |
  42. Nyberg KA, Michelson RJ, Putnam CW, Weinert TA. (2002). Toward maintaining the genome: DNA damage and replication checkpoints. Annu Rev Genet 36: 617–656. | Article | PubMed | ISI | ChemPort |
  43. Pommier Y, Pourquier P, Urasaki Y, Wu J, Laco GS. (1999). Topoisomerase I inhibitors: selectivity and cellular resistance. Drug Resist Updat 2: 307–318. | Article | PubMed | ChemPort |
  44. Pommier Y. (2006). Topoisomerase I inhibitors: camptothecins and beyond. Nat Rev Cancer 6: 789–802. | Article | PubMed | ISI | ChemPort |
  45. Ryan AJ, Squires S, Strutt HL, Johnson RT. (1991). Camptothecin cytotoxicity in mammalian cells is associated with the induction of persistent double strand breaks in replicating DNA. Nucleic Acids Res 19: 3295–3300. | Article | PubMed | ISI | ChemPort |
  46. Sakaguchi K, Sakamoto H, Lewis MS, Anderson CW, Erickson JW, Appella E et al. (1997). Phosphorylation of serine 392 stabilizes the tetramer formation of tumor suppressor protein p53. Biochemistry 36: 10117–10124. | Article | PubMed | ISI | ChemPort |
  47. Sarkaria JN, Busby EC, Tibbetts RS, Roos P, Taya Y, Karnitz LM et al. (1999). Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res 59: 4375–4382. | PubMed | ISI | ChemPort |
  48. Scheel H, Hofmann K. (2003). No evidence for PHD fingers as ubiquitin ligases. Trends Cell Biol 13: 285–287; author reply 287–288. | Article | PubMed | ISI | ChemPort |
  49. Sebastian S, Sreenivas P, Sambasivan R, Cheedipudi S, Kandalla P, Pavlath GK et al. (2009). MLL5, a trithorax homolog, indirectly regulates H3K4 methylation, represses cyclin A2 expression, and promotes myogenic differentiation. Proc Natl Acad Sci USA 106: 4719–4724. | Article | PubMed |
  50. Shao RG, Cao CX, Zhang H, Kohn KW, Wold MS, Pommier Y. (1999). Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes. EMBO J 18: 1397–1406. | Article | PubMed | ISI | ChemPort |
  51. Staker BL, Hjerrild K, Feese MD, Behnke CA, Burgin Jr AB, Stewart L. (2002). The mechanism of topoisomerase I poisoning by a camptothecin analog. Proc Natl Acad Sci USA 99: 15387–15392. | Article | PubMed | ChemPort |
  52. Tibbetts RS, Brumbaugh KM, Williams JM, Sarkaria JN, Cliby WA, Shieh SY et al. (1999). A role for ATR in the DNA damage-induced phosphorylation of p53. Genes Dev 13: 152–157. | Article | PubMed | ISI | ChemPort |
  53. Westerfield M. (2000). The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio), 4th edn. Univ. of Oregon Press: Eugene.
  54. Wysocka J, Swigut T, Milne TA, Dou Y, Zhang X, Burlingame AL et al. (2005). WDR5 associates with histone H3 methylated at K4 and is essential for H3 K4 methylation and vertebrate development. Cell 121: 859–872. | Article | PubMed | ISI | ChemPort |
  55. Xiao Z, Chen Z, Gunasekera AH, Sowin TJ, Rosenberg SH, Fesik S et al. (2003). Chk1 mediates S and G2 arrests through Cdc25A degradation in response to DNA-damaging agents. J Biol Chem 278: 21767–21773. | Article | PubMed | ISI | ChemPort |
  56. Yokoyama A, Wang Z, Wysocka J, Sanyal M, Aufiero DJ, Kitabayashi I et al. (2004). Leukemia proto-oncoprotein MLL forms a SET1-like histone methyltransferase complex with menin to regulate Hox gene expression. Mol Cell Biol 24: 5639–5649. | Article | PubMed | ISI | ChemPort |
  57. Zhao H, Traganos F, Darzynkiewicz Z. (2008). Phosphorylation of p53 on Ser15 during cell cycle caused by Topo I and Topo II inhibitors in relation to ATM and Chk2 activation. Cell Cycle 7: 3048–3055. | Article | PubMed | ISI |
Top

Acknowledgements

We are grateful to Dr Thilo Hagen and Dr Qiang Yu for their valuable suggestions. We thank Dr Bert Vogelstein for HCT116 p53+/+ and HCT116 p53/ cells and Dr Victor Yu for pXJ-HA-p53 plasmid. This work was supported in part by BMRC-A*STAR, R-183-000-164-305; NMRC-A*STAR, R-183-000-220-275; and Ministry of Education Academic Research Fund Tier2, R-183-000-195-112 to LWD, and A*STAR-IMCB funding to YJJ and VK Both FC and JL are recipients of research scholarships from Ministry of Education, Singapore.

Supplementary Information accompanies the paper on the Oncogene website