ELL Inhibits E2F1 Transcriptional Activity by Enhancing E2F1 Deacetylation via Recruitment of Histone Deacetylase 1 Wei Zhang, Wei Ji, Xing Liu, Gang Ouyang and Wuhan Xiao Mol. Cell. Biol. 2014, 34(4):765. DOI: 10.1128/MCB.00878-13. Published Ahead of Print 16 December 2013.
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ELL Inhibits E2F1 Transcriptional Activity by Enhancing E2F1 Deacetylation via Recruitment of Histone Deacetylase 1 Wei Zhang, Wei Ji, Xing Liu, Gang Ouyang, Wuhan Xiao The Key Laboratory of Aquatic Biodiversity and Conservation, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, People’s Republic of China
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he ELL (eleven-nineteen lysine-rich leukemia) protein was first identified as a translocation partner of MLL in acute myeloid leukemia (AML) (1). Subsequent studies showed that ELL can serve as a transcription elongation factor that increases the rate of RNA polymerase II transcriptional elongation by preventing transient pausing (2, 3) or by facilitating RNA polymerase II pause site entry and release (4). Increasing evidence strongly supports the idea that ELL belongs to a super elongation complex that modulates gene expression in development and disease (5–10). In addition, the targeted knockout of ELL in mice causes embryonic lethality, suggesting that ELL has a crucial role in early embryogenesis (11). ELL has been also identified as a partner of steroid receptors p53, hypoxia-inducible factor 1␣ (HIF-1␣), and EAF1/2, modulating their binding partner activity in either a positive or negative manner (12–15). While multiple lines of evidence point to the importance of ELL in normal and cancerous cells, the exact mechanism of its activity remains elusive. The E2F family of transcription factors play important roles in regulating cell cycle progression by transactivating genes required for entry into the S phase (16). As a member of the family, E2F1 also has crucial roles in regulating apoptosis via stimulating the transcription of several genes in the apoptotic pathway, particularly upon DNA damage (17). DNA damage stabilizes and activates E2F1 through phosphorylation by ATM (18) or CHK2 (19), as well as acetylation by PCAF (20). To understand the underlying mechanism of E2F1 activity, multiple investigators have explored the binding partners of E2F1. Studies have shown that the classic tumor suppressor pRb serves as a major regulator of E2F1 (21) by directly recruiting histone deacetylase 1 (HDAC1) (22). pRb is often mutated and inactive in tumors, leading to deregulation of E2F1 activity (23). TopBP1 also interacts with E2F1 and subsequently inhibits E2F1 activity through a Brg1/Brm-dependent, pRb-independent mechanism (24). MCPH1/BRIT1 cooperates with E2F1 to regulate the expression of E2F target genes (25). One target gene is the NAD-dependent histone deacetylase SirT1, which in a negative feedback loop inhibits E2F1’s activity in regulating the apoptotic response to DNA damage (26). Overall, the acetylation status of E2F1 represents a major mechanism for regulating E2F1 function. In this study, we identified ELL as a downstream target of E2F1
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in response to DNA damage. Furthermore, we found that ELL interacted with E2F1, inhibiting E2F1’s function through the recruitment of HDAC1, but the MLL-ELL fusion protein had no inhibitory role on E2F1 transcriptional activity. Our findings not only connect ELL to the E2F1 signaling pathway and reveal a novel role of ELL in response to DNA damage but also provide an insight into the mechanism for MLL-ELL-associated leukemogenesis. MATERIALS AND METHODS Cell lines. HEK293T, H1299, Saos2, and HCT116 cells were originally obtained from ATCC. HEK293T, H1299, and HCT116 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; HyClone) with 10% fetal bovine serum (FBS; HyClone) at 37°C in a humidified atmosphere containing 5% CO2, while Saos2 cells were maintained in McCoy’s 5A medium with 15% FBS. Plasmid constructs. The original ELL and MLL-ELL constructs were kindly provided by Ali Shilatifard. The p73 promoter construct was kindly provided by Toshiyuki Sakai. The E2F4B luciferase reporter and pRb expression vector were kindly provided by Fred Dick. The ELL promoter (position ⫺1768 to ⫹404) for its luciferase reporter construct was amplified from total DNA extracted from 293 cells by PCR using primers 5=-ATATCGGTACCGGGCGTGGTGGTGGGCATC TGTAA-3= and 5=-ATATCAAGCTTCGGGCCACCCGCCTGTATTGCC TC-3=. The p27/Kip1 (position ⫺2292 to ⫹471) promoter for its luciferase reporter construct was amplified by PCR using primers 5=-TATCGG TACCTCAAGTGATCCTCCCACCTCTGCT-3= and 5=-TATCAAGCTT AATGCAGCTGTCTTGAGCCTGTG-3=. The Apaf1 promoter for its luciferase reporter construct was amplified by PCR using primers 5=-TA TCGGTACCAACAAGGCTGGGCTGTTTCCTTCC-3= and 5=-TATCAA GCTTTACTGGACACAAAGGGAGGAGGTCTT-3=. These three promoters were then subcloned into pGL3-Basic vector (Promega). ChIP assays. The experimental protocol for chromatin immunoprecipitation (ChIP) and the primers for -actin were described previously
Received 9 July 2013 Returned for modification 1 August 2013 Accepted 5 December 2013 Published ahead of print 16 December 2013 Address correspondence to Wuhan Xiao, [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/MCB.00878-13
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ELL (eleven-nineteen lysine-rich leukemia protein) was first identified as a translocation partner of MLL in acute myeloid leukemia; however, the exact mechanism of its action has remained elusive. In this study, we identified ELL as a direct downstream target gene of E2F1. Coimmunoprecipitation assays showed that ELL interacted with E2F1 in vitro and in vivo, leading to inhibition of E2F1 transcriptional activity. In addition, ELL enhanced E2F1 deacetylation via recruitment of histone deacetylase 1 (HDAC1). Notably, the MLL-ELL fusion protein lost the inhibitory role of ELL in E2F1 transcriptional activity. Furthermore, DNA damage induced ELL in an E2F1-dependent manner and ELL protected cells against E2F1-dependent apoptosis. Our findings not only connect ELL to E2F1 function and uncover a novel role of ELL in response to DNA damage but also provide an insight into the mechanism for MLL-ELL-associated leukemogenesis.
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tifying the protein levels based on the band density obtained in Western blot assays. Lentivirus-mediated gene knockdown. Each short hairpin RNA (shRNA) sequence was cloned into the pLKO.1 vector and verified by sequencing. The following shRNA sequences were used: scrambled shRNA, 5=-GCCTTGATCGCTCGCTAAACA-3=; ELL shRNA#1, 5=-CAA CACCAACTACAGCCAGGA-3=; ELL shRNA#2, 5=-GCGAGTACCTGC ACAGCAA-3=; HDAC1 shRNA, 5=-CTAATGAGCTTCCATACAA-3= E2F1 shRNA, 5=-GTCACGCTATGAGACCTCA-3=; and Rb shRNA, 5=-G ATACCAGATCATGTCAGA-3=. For lentiviral shRNA production, HEK293T cells in 10-cm dishes were transfected with 6 g psPAX2, 6 g pMD2.G, and 12 g pLKO.1 with shRNA inserts. After 8 h of transfection, the medium was replaced with 12 ml of fresh DMEM with 10% FBS. After 40 h, the medium containing the lentivirus particles was collected, centrifuged, and passed through a 0.45-m filter, and then added to HEK293 or H1299 cells. An amount of 8 g/ml Polybrene was added to the medium to improve infection efficiency. Apoptosis assay. For annexin V staining, H1299 cells were infected with lentivirus expressing scrambled control shRNA, ELL shRNA, Rb shRNA, E2F1 shRNA, or combinations of these shRNAs. The cells were then treated with etoposide (70 M) or dimethyl sulfoxide (DMSO) control for 40 h. Subsequently, the cells were collected and washed in cold phosphate-buffered saline (PBS), resuspended in 1⫻ annexin binding buffer, and incubated with annexin V-fluorescein isothiocyanate (FITC) and phosphatidylinositol (PI) at room temperature for 10 min in the dark using an annexin V-PI apoptosis kit (MultiSciences Biotech). Flow cytometry analysis was performed to detect apoptotic cells. The data were evaluated with Cell Quest software. For Hoechst staining, the cells were treated with etoposide (70 M) or the DMSO control for 36 h. Hoechst 33342 (1 g/ml, Molecular Probes) was used to visualize breakage of nuclei. The cells were counted under a fluorescence microscope. Each experiment was repeated at least three times.
RESULTS
ELL is a direct downstream target of E2F1. During our study of the regulation of thrombospondin-1 (TSP-1) by E2F1 (30), we noticed that ELL, a TSP-1 regulator (31), was also regulated by E2F1. To understand whether ELL was a direct downstream target of E2F1, we first did promoter assays. A 2.1-kb promoter region of ELL was cloned into the pGL3-Basic vector (Fig. 1A). In HEK293T cells, overexpression of E2F1 caused the ELL promoter activity to increase by about 20-fold but did not increase the activity of the pGL3-Basic vector control (Fig. 1B). Furthermore, overexpression of E2F1 (E132), a DNA binding-deficient mutant, failed to activate the ELL promoter luciferase reporter (Fig. 1C), indicating that the DNA binding domain must be kept intact for E2F1 to upregulate ELL expression. To map the E2F1 response region in the ELL promoter, we generated three truncated ELL promoter constructs (Fig. 1A). When E2F1 was overexpressed, all three truncated ELL promoter reporters showed activity (Fig. 1B), suggesting that the E2F1 response element is located between nucleotides ⫺32 and ⫹404. We identified a putative E2F1 binding site (GCGCCAGA) located at the ⫹198 position (Fig. 1D). To confirm that this site indeed acts as an E2F1 binding site, we mutated CG to AT (GATCCAGA) and found that E2F1 could not induce transcription from this mutant promoter (Fig. 1D and E). Semiquantitative RT-PCR revealed that overexpression of E2F1 in H1299 cells caused an increase in E2F1 mRNA (Fig. 1F) but that knockdown of E2F1 by E2F1 shRNA reduced the E2F1 mRNA level (Fig. 1G). To determine whether E2F1 bound directly to the ELL pro-
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(27). The primers specific for the ELL promoter region from position ⫹118 to ⫹324 were 5=-CGGGTTAGCGACGGCAGCAAGGTG-3= (forward) and 5=-CCGCGAGCAGCACCACAGACCTAG-3= (reverse). The primers (control) for the ELL promoter region from position ⫺1389 to ⫺1133, far from E2F1 binding sites, were 5=-CATGGTGAAATCCCGTC TCTAG-3= (forward) and 5=-CCTCCCAAAGTGCTAGGATTAC-3= (reverse). The primers for the p73 promoter region from ⫺1257 to ⫺1049 were 5=-TGAGCCATGAAGATGTGCGAG-3= (forward) and 5=-GCTGC TTATGGTCTGATGCTTATG-3=(reverse) (28). Antibodies and reagents. Human ELL polyclonal antibody was generated against synthesized peptides of ELL (Abmart, Shanghai, China). Other antibodies used were as follows: antihemagglutinin (antiHA) antibody (Covance), anti-Myc antibody (9E10; Santa Cruz), antiFlag antibody (M2; Sigma), anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) antibody (SC-47724; Santa Cruz), anti-E2F1 antibody (KH95; Santa Cruz), anti-HDAC1 antibody (3426-1; Epitomics), anti-p73 antibody (ab40658; Abcam), anti-p27/KIP1 antibody (ab32304; Abcam), anti-Apaf1 antibody (ab32372; Abcam), and antipoly(ADP-ribose) polymerase 1 (anti-PARP1) antibody (no. 9542; Cell Signaling Technology). Trichostatin A (TSA) (5 mM) was purchased from Sigma. Semiquantitative reverse transcription (RT)-PCR. Total RNA was extracted from cells by using TRIzol reagent (Invitrogen), and cDNA was synthesized using a first-strand cDNA synthesis kit (Fermentas). The following primers were used: for human ELL mRNA, forward, 5=-ACTGCA TCCAGCAGTATGTCTCCA-3=, and reverse, 5=-CTTGATGACAATGG CACTTCGGCT-3=; for human p73 mRNA, forward, 5=-CATGGTCTCG GGGTCCCACT-3=, and reverse, 5=-CGTGAACTCCTCCTTGATGG-3=; for human Apaf1 mRNA, forward, 5=-AATGGACACCTTCTTGGACG3=, and reverse, 5=-GCACTTCATCCTCATGAGCC-3=; for human p27 mRNA, forward, 5=-CCGGTGGACCACGAAGAGT-3=, and reverse, 5=GCTCGCCTCTTCCATGTCTC-3=; for human Rb mRNA, forward, 5=GCAGTATGCTTCCACCAGGC-3=, and reverse, 5=-AAGGGCTTCGAG GAATGTGAG-3=; and for 18S RNA, used as an internal control, forward, 5=-TCAACTTCGATGGTAGTCGCCGT-3=, and reverse, 5=-TCCTTGG ATGTGGTAGCCGTTCT-3=. Data are reported as the means ⫾ standard errors of the means (SEM) of three independent experiments performed in triplicate. The statistical analysis was performed using GraphPad Prism 5 (unpaired t test) (GraphPad Software Inc.). Luciferase reporter assays. HEK293T, H1299, or Saos2 cells were seeded in 24-well plates and transfected with the luciferase reporters indicated above in “Plasmid constructs” using Lipofectamine 2000 (Invitrogen) or VigoFect (Vigorous Biotech, Beijing, China). pTK-Renilla was used as an internal control. Luciferase activity was measured after transfection for 20 to 24 h using the dual-luciferase reporter assay system (Promega). Data are reported as the means ⫾ SEM of three independent experiments performed in triplicate. The statistical analysis was performed using GraphPad Prism 5 (unpaired t test) (GraphPad Software Inc.). Western blot assay and coimmunoprecipitation. For Western blot assay and coimmunoprecipitation of overexpressed proteins, the experimental procedures have been described previously (29). For endogenous coimmunoprecipitation, the cells indicated below were lysed and centrifuged at 13,000 rpm for 15 min. The supernatants were immunoprecipitated with agarose beads conjugated with the antibodies indicated below at 4°C overnight. The beads were washed three times using radioimmunoprecipitation assay (RIPA) buffer with 500 mM NaCl. The proteins were detected by Western blot assay using the antibodies indicated below. For detection of E2F1 acetylation, cell lysates containing Flag-tagged E2F1 were immunoprecipitated with anti-Flag beads at 4°C overnight. The beads were washed five times with ice-cold RIPA buffer, eluted with SDS loading buffer, and then subjected to SDS-PAGE and immunoblotted with an antiacetyllysine antibody (Millipore). The Fujifilm LAS4000 mini-luminescent image analyzer system was used to photograph the blots. Multi Gauge version 3.0 was used for quan-
ELL Inhibits E2F1 Activity
ELL promoter reporters. HEK293T cells were transfected with different truncated ELL promoter reporters together with either Flag-E2F1 expression vector or Flag empty vector (control). (C) Overexpression of the E2F1 (E132) mutant failed to activate the ELL promoter. The full-length ELL promoter (⫺1768 to ⫹404) reporter was transfected together with either the wild-type E2F1 construct or the E132 mutant construct. (D) Partial sequence of the ELL promoter (⫺32 to ⫹404); one potential E2F1 binding site is marked by the box, and substitution mutations are underlined. (E) E2F1 trans activation of the ELL promoter through the potential E2F1 binding site GCGCCAGA around the ⫹198 position. Transfection of the E2F1 expression vector failed to activate the ELL mutant promoter (⫹198 mut). (F) Overexpression of E2F1 in H1299 cells caused an increase in the ELL mRNA level as revealed by semiquantitative RT-PCR. (G) Knockdown of E2F1 by E2F1 shRNA in H1299 cells caused a reduction in the ELL mRNA level as revealed by semiquantitative RT-PCR. (H) Schematic of locations of fragments amplified in ELL promoter or -actin promoter. E2F1 binding region in ELL promoter is amplified between ⫹118 and ⫹324, and the control region in the ELL promoter within which E2F1 cannot bind is amplified between ⫺1389 and ⫺1133; the fragment amplified from -actin is used as the control. (I) Chromatin immunoprecipitation analysis indicated that E2F1 interacted directly with the ELL promoter region harboring the putative E2F1 binding site. (J) Semiquantitative RT-PCR confirmed that E2F1 binds to the ⫹118 to ⫹324 region of the ELL promoter. (K) Semiquantitative RT-PCR confirmed that E2F1 cannot bind to the ⫺1389 to ⫺1133 region of the ELL promoter. (L) Upregulation of ELL protein by overexpression of E2F1 was confirmed by Western blotting. (M) Knockdown of E2F1 shRNA decreased the ELL protein level; pSuper-GFP-shRNA was used as the control. (N) E2F1 induced by 4-OHT (500 nm; dissolved in ethyl alcohol) in H1299 cells stably expressing ER-E2F1 caused upregulation of ELL protein. If not otherwise specified, data are reported as the means ⫾ SEM of three independent experiments performed in triplicate.
moter harboring the E2F1 binding site, we performed chromatin immunoprecipitation (ChIP) assays. One pair of primers encompassing the E2F1 binding site was designed to amplify a 208-bp fragment, and another pair of primers was designed to amplify a 257-bp fragment far from the E2F1 binding site (control) (Fig. 1H). As shown in Fig. 1I, anti-E2F1 antibody could easily precipitate the 208-bp fragment of the ELL promoter encompassing the E2F1 binding site. Semiquantitative RT-PCR confirmed that antiE2F1 antibody pulled down the 208-bp fragment (position ⫹118 to ⫹324) (Fig. 1J) but not the 257-bp fragment (position ⫺1389 to ⫺1133) (Fig. 1K), which is far from the E2F1 binding region.
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Subsequently, we performed Western blot assays to determine whether the protein level of ELL was regulated by E2F1. In HEK293T cells, overexpression of E2F1 upregulated ELL (1.4-fold versus 1.0-fold) (Fig. 1L), but knockdown of E2F1 by E2F1 shRNA reduced ELL expression (0.6-fold versus 1.0-fold) (Fig. 1M). Similarly, we found that activation of ER-E2F1 expression by treating an H1299 cell line stably expressing an ER-E2F1 fusion protein with 4-hydroxytamoxifen (4-OHT; 500 nm) resulted in the upregulation of ELL (1.9-fold versus 1.0-fold) (Fig. 1N). Taken together, these results suggest that the ELL gene is a potential direct target of E2F1.
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FIG 1 The ELL gene is a direct target of E2F1. (A) Schematic of ELL promoter luciferase reporter constructs. (B) Overexpression of E2F1 induced the activity of
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RFP-tagged E2F1. (B and C) Immunoprecipitation assays showed that E2F1 interacted with ELL when they were overexpressed in HEK293T cells. (D and E) ELL interacted with E2F1 endogenously in both H1299 cells (p53 deficient) and Saos2 cells (Rb deficient). Anti-E2F1 monoclonal antibody was used for immunoprecipitation (IP), and mouse IgG was used as the control; ELL was detected by an anti-ELL polyclonal antibody. (F) E2F1 bound to ELL directly, as revealed by Ni-NTA assays. His-tagged ELL and GST-tagged E2F1 were expressed in Escherichia coli; Ni-NTA agarose beads were used for immunoprecipitation, and anti-E2F1 antibody was used for Western blotting. (G) Coomassie blue staining for GST and GST-E2F1 purified from E. coli. (H) Domain mapping for ELL interacting with E2F1. ⫺, no interaction; ⫹, interaction; IB, immunoblotting. ELL (aa 1 to 379) was essential for E2F1 binding. (I) Domain mapping for E2F1 interacting with ELL. E2F1 (aa 201 to 368) was essential for ELL binding.
ELL interacts with E2F1 in vitro and in vivo. As a nuclear protein, ELL binds to and inhibits p53 (13). Given that E2F1 is a well-defined transcription factor, we speculated that the interaction of ELL with E2F1 may affect E2F1 function. To test this, we initially did colocalization assays. As shown in Fig. 2A, after transfection into HeLa cells, green fluorescent protein (GFP)-tagged ELL colocalized with red fluorescent protein (RFP)-tagged E2F1, indicating that ELL might interact with E2F1 in the nucleus. To confirm the interaction, we conducted coimmunoprecipitation assays. In HEK293T cells transfected with HA-E2F1 and Myc-ELL or Myc-empty vector control, anti-Myc antibody-conjugated agarose beads pulled down HA-E2F1 (Fig. 2B). Similarly, HA-E2F1 also pulled down Flag-ELL in immunoprecipitation experiments using anti-HA antibody-conjugated agarose beads (Fig. 2C). To determine whether ELL interacted with E2F1 endogenously, we used anti-E2F1 antibody to perform endogenous immunoprecipitation in H1299 (p53-null) and Saos2 (Rb-null) cells. As shown in Fig. 2D and E, ELL interacted with E2F1 endogenously. This experiment also suggests that ELL can interact with E2F1 independently of both p53 and Rb. To test whether ELL bound to E2F1 directly, we conducted Ni-nitrilotriacetic acid (NTA) pulldown assays. His-tagged ELL expressed in Escherichia
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coli could pull down glutathione S-transferase (GST)-tagged E2F1 expressed in E. coli (Fig. 2F and G). Collectively, these experiments suggest that the two proteins can interact directly in a p53- and Rb-independent manner. We then mapped the domains of E2F1 and ELL responsible for interaction between the two proteins by using deletion mutants (Fig. 2H and I). The N terminus of ELL (amino acids [aa] 1 to 379) was crucial for interacting with E2F1 (Fig. 2H), and the domain from aa 201 to 368 of E2F1 was required for interacting with ELL (Fig. 2I). Surprisingly, the longer construct (aa 1 to 508) binds to E2F1 more weakly than the shorter one (aa 1 to 379), which might be due to the conformational difference between these two peptides. ELL inhibits E2F1 transcriptional activity. To investigate the functional importance of the interaction between ELL and E2F1, we evaluated the effect of ELL on promoter reporters of E2F1’s downstream target genes. In HEK293T cells, overexpression of E2F1 resulted in a dramatic activation of the E2F4B luciferase reporter (Fig. 3A). However, cotransfection of the ELL expression vector caused this activation to decrease significantly (Fig. 3A). The expression levels of transfected HA-ELL and Flag-E2F1 were confirmed by Western blot assay (Fig. 3A, right). We saw similar
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FIG 2 ELL interacts with E2F1 in vivo and in vitro. (A) ELL colocalized with E2F1 in the nucleus. HeLa cells were transfected with GFP-tagged ELL and
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cells (A), Rb-deficient Saos2 cells (B), Rb-intact H1299 cells (C), and HCT116 cells (D). The expression levels of HA-ELL and Flag-E2F1 were confirmed by Western blotting (A, right). (E, F, G) The activity of E2F1 downstream target gene promoter reporters (Apaf1, p73, and p27) induced by E2F1 was suppressed by cotransfection of ELL in HEK293T cells. (H) Cotransfection of pRB and ELL showed a synergistic effect on the inhibition of E2F1-mediated E2F4B luciferase reporter activity. (Right) The expression levels of HA-ELL, HA-Rb, and Flag-E2F1 were confirmed by Western blotting. (I) ELL deletion mutants were cotransfected with E2F1 and E2F4B luciferase reporter. The ELL mutant with aa 46 to 379 was essential for the inhibitory effect on E2F1 transcriptional activity. (J) Knockdown of ELL by ELL shRNA#1 and shRNA#2 in H1299 cells was confirmed by semiquantitative RT-PCR. (K) Knockdown of ELL by ELL shRNA in HEK293T cells enhanced the activity of the E2F4B luciferase reporter. (L, M, N) Knockdown of ELL by ELL shRNA in H1299 cells increased the mRNA levels of Apaf1, p73, and p27. Data are reported as the means ⫾ SEM of three independent experiments performed in triplicate. (O) Western blot assays for Apfa1, p73, and p27 in H1299 cells with ELL knockdown.
results in Sao2 cells, H1299 cells, and HCT116 cells (Fig. 3B, C, and D). In addition, overexpression of ELL in HEK293T cells also suppressed the activation of three other promoter reporters induced by E2F1, including Apaf1-luc, p73-luc, and p27-luc (Fig. 3E, F, and G). Rb, a well-defined E2F1 partner, suppresses E2F1 transactivity (21). To gain a more complete picture of the inhibitory role of ELL on E2F1, we compared ELL to Rb. As shown in Fig. 3H, in HEK293T cells, Rb and ELL suppressed E2F1 transactivity to similar degrees in E2F4B luciferase reporter assays. Interestingly, ELL and Rb suppressed E2F1 transactivity synergistically, as revealed by cotransfection experiments (Fig. 3H). The expression levels of transfected HA-ELL, HA-Rb, and Flag-E2F1 were confirmed by Western blot assay (Fig. 3H, right). To identify the ELL domain responsible for E2F1 suppression, we did domain mapping experiments in which we created ELL deletion mutants. We found that the aa 46 to 379 region of ELL
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was essential for inhibiting E2F1 activity (Fig. 3I). The HA-ELL mutants containing this region, for example, those with aa 1 to 379, aa 1 to 508, and aa 46 to 621, significantly inhibited E2F1 transactivity (P ⬍ 0.05). However, the HA-ELL mutants lacking this region, i.e., those with aa 447 to 621 and aa 509 to 621, did not do so (Fig. 3I). Not surprisingly, the ELL domains that suppressed activity were the same as those involved in E2F1 binding (Fig. 2H). To confirm the inhibitory role of ELL on E2F1, we knocked down ELL by using ELL shRNAs and then checked E2F4B luciferase reporter activity. Knockdown of ELL mediated by ELL shRNA#1 and shRNA#2 was confirmed by semiquantitative RTPCR (Fig. 3J). After ELL knockdown, the activity of the E2F4B luciferase reporter increased significantly (Fig. 3K), in contrast to the results of ELL overexpression. Moreover, knockdown of ELL in H1299 cells caused the mRNA levels of Apaf1, p73, and p27 to increase dramatically (Fig. 3L, M, and N). Consistent with these
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FIG 3 ELL inhibits E2F1 transcriptional activity. Cotransfection of ELL suppressed E2F1-mediated transactivity on the E2F4B luciferase reporter in HEK293T
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results, knockdown of ELL in H1299 cells caused the protein levels of Apaf1, p73, and p27, three E2F1 downstream targets, to increase (Fig. 3O). Thus, it appears that ELL binding suppresses E2F1 transactivity. ELL enhanced the deacetylation of E2F1 by recruiting HDAC1. We next investigated the underlying mechanism of ELL inhibition on E2F1. Rb inhibits E2F1 by recruiting histone
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deacetylase 1 (HDAC1) to deacetylate E2F1 (22). To test whether ELL also inhibits E2F1 activity though HDAC1 recruitment, we first generated an E2F1 K/R mutant with the set of lysines at positions 117, 120, and 125 mutated to arginine, which therefore lacks the sites required for acetylation (20). When the E2F1 K/R mutant was cotransfected with ELL, the ability of ELL to inhibit transcription from the E2F1 reporter disappeared (Fig. 4A) (P ⫽ 0.0646
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FIG 4 ELL enhances deacetylation of E2F1 by recruiting HDAC1. (A) Overexpression of ELL failed to inhibit E2F4B luciferase reporter activity mediated by the E2F1 K/R mutant (K117R/K120R/K125R). (B) Overexpression of ELL reduced exogenous E2F1 acetylation. The Flag-ELL expression vector was cotransfected together with either the Myc-ELL expression vector or the Myc empty vector into HEK293T cells; agarose beads conjugated with anti-Flag antibody were used for immunoprecipitation, and antiacetyllysine antibody (anti-AcLys) was used for evaluating E2F1 acetylation. (C) Knockdown of ELL enhanced exogenous E2F1 acetylation. (D) ELL interacted with HDAC1 when they were overexpressed, and the N terminus of ELL (aa 1 to 379) was essential for HDAC1 binding. (E) ELL interacted with HDAC1 endogenously in HCT116 cells. (F) Overexpression of ELL enhanced the interaction between HDAC1 and E2F1. (G) Knockdown of ELL reduced the interaction between endogenous HDAC1 and endogenous E2F1 in H1299 cells. (H) Knockdown of ELL reduced the interaction between endogenous HDAC1 and endogenous E2F1 in Saos2 cells. (I) Knockdown of ELL in Saos2 cells reduced endogenous HDAC1 binding to the E2F1 binding region in the p73 promoter (⫺1257 to ⫺1049). (J) Knockdown of HDAC1 partially reversed the inhibitory effect of ELL on E2F1-mediated E2F4B luciferase reporter activity (P ⬍ 0.0001). (K) shRNA-mediated knockdown of HDAC1 was confirmed by Western blot assays. (L) The HDAC inhibitor TSA reversed the suppressive role of ELL in E2F1-mediated E2F4B luciferase reporter activity. H1299 cells were treated with TSA (dissolved in DMSO) at a final concentration of 500 nM; DMSO was used as the control.
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versus P ⫽ 0.0008). Moreover, the E2F1 K/R mutant had lower transcriptional activity than wild-type E2F1 (Fig. 4A) (P ⫽ 0.0282). These results not only support the importance of acetylation for E2F1 activity but also suggest that ELL may suppress E2F1 through enhancing its deacetylation. Subsequently, we performed immunoprecipitation assays to analyze the acetylation status of E2F1 after ELL overexpression. As expected, overexpression of ELL indeed enhanced E2F1 deacetylation (Fig. 4B). In contrast, knockdown of ELL resulted in an increase in E2F1 acetylation (Fig. 4C). Studies have indicated that the acetylation of E2F1 is positively regulated by CBP/p300 and negatively regulated by HDAC1 (22, 32). To test whether ELL could recruit HDAC1 to E2F1, we first did coimmunoprecipitation assays to determine if ELL and HDAC could interact. As expected from our domain mapping studies, the N terminus of ELL (aa 1 to 379) bound efficiently to HDAC1 (Fig. 4D). In addition, the anti-ELL antibody could pull down endogenous HDAC1 in HCT116 cells (Fig. 4E), suggesting that ELL interacts with HDAC1 endogenously. Because E2F1 and HDAC1 bind to the same domain of ELL, we next examined whether ELL could promote an interaction between E2F1 and HDAC1. HEK293T cells were transfected with Flag-E2F1 and HAHDAC1 in the presence or absence of Myc-ELL. We found that E2F1 and HDAC coimmunoprecipitated and that ELL expression enhanced this interaction (Fig. 4F). To further confirm the recruitment of HDAC1 by ELL, we performed endogenous coimmunoprecipitation in H1299 cells that have a stable knockdown of ELL. As shown in Fig. 4G, ELL knockdown diminished the endogenous interaction between E2F1 and HDAC1. Because H1299 cells contain intact Rb, endogenous Rb might recruit E2F1 to interact with ELL. To rule out this possibility, we performed endogenous coimmunoprecipitation in Rb-deficient Saos2 cells. As
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shown in Fig. 4H, ELL knockdown still diminished the endogenous interaction between E2F1 and HDAC1, supporting the idea that ELL recruits HDAC1 to E2F1 independently of Rb. In addition, knockdown of ELL in Saos2 cells also diminished HDAC1 binding to the specific region of the p73 promoter, which was reported to be bound by E2F1 (28). Moreover, knockdown of HDAC1 by shRNA could partially reverse the suppressive role of ELL on E2F1 transactivity, as revealed by promoter assays (Fig. 4J). The efficiency of HDAC1 shRNA-mediated HDAC1 knockdown was confirmed by Western blot assay (Fig. 4K). The addition of the HDAC inhibitor TSA reversed the suppressive role of ELL on E2F1 transactivity (Fig. 4L), further supporting the notion that ELL recruits HDAC1 to suppress E2F1 activity. The MLL-ELL fusion protein has no inhibitory role on E2F1 transcriptional activity. To determine the physiological relevance of ELL in the inhibition of E2F1 transcriptional activity, we examined the effect of an MLL-ELL fusion protein on E2F1 transactivity. In H1299 cells, overexpression of ELL inhibited the E2F1induced activities of p27, Apaf1, and p73 promoter reporters, as well as that of the E2F4B luciferase reporter (Fig. 5A, B, C, and D), similar to the results in HEK293T cells and Saos2 cells, indicating that the inhibitory role of ELL in E2F1 activity is cell line independent. In contrast, overexpression of MLL-ELL had no obvious effect on these reporters (Fig. 5A, B, C, and D). The expression levels of transfected Flag-E2F1, HA-ELL, and HA-MLL-ELL were confirmed by Western blot assay (Fig. 5C, right). Consistent with these results, the expression of p73 and p27 induced by E2F1 activation via adding 4-OHT (500 nM) to H1299 cells stably transfected with ER-E2F1 was also inhibited by overexpression of ELL but not by overexpression of MLL-ELL, as revealed by semiquantitative RT-PCR (Fig. 5E and F). Moreover, the acetylation of E2F1 was not affected by overexpression of MLL-ELL (Fig. 5G).
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FIG 5 The MLL-ELL fusion protein has no inhibitory role in E2F1 transactivity. (A, B, C, and D) The activity of E2F1 downstream target gene promoter reporters (p27, Apaf1, and p73) and E2F4B luciferase reporter induced by E2F1 was suppressed by cotransfection of ELL but not by cotransfection of MLL-ELL in H1299 cells. The expression levels of HA-ELL, HA-MLL-ELL, and Flag-E2F1 were confirmed by Western blotting (C, right). (E and F) The expression of p73 and p27 induced by activation of E2F1 (by adding 4-OHT at a final concentration of 500 nM) in H1299 cells stably transfected with ER-E2F1 was suppressed by overexpression of ELL but not by overexpression of MLL-ELL. Data are reported as the means ⫾ SEM of three independent experiments performed in triplicate. (G) Overexpression of ELL reduced exogenous E2F1 acetylation, but overexpression of MLL-ELL did not do so.
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together, these results suggest that ELL protects cells from E2F1dependent cell death upon DNA damage. The above-described observations indicate that upon DNA damage, E2F1 upregulates ELL effectively. However, ELL can subsequently suppress E2F1 activity, which might result in turning off the DNA damage response. To understand how these two processes were operated, we examined the expression of p73, a welldefined downstream target of E2F1 in its response to DNA damage (28), as well as the expression of ELL, by kinetic/temporal analysis. As shown in Fig. 7, upon DNA damage, p73 expression was steadily upregulated and reached its highest level at 24 h, after which it went down sharply (Fig. 7A). In contrast, ELL expression was upregulated at a relatively lower rate and reached its highest level at 36 h, after which it went down slowly (Fig. 7B). These results indicate that, in response to DNA damage, E2F1 upregulates ELL at a relatively lower rate at the later stage, which turns off the E2F1-dependent DNA damage response to protect cells from DNA damage-induced cell death and also shuts down its own expression. These two processes appeared to be temporally regulated. Based on these data, we propose a model for the role of ELL in E2F1 activity (Fig. 8). Upon DNA damage, E2F1 is acetylated by PCAF or other acetylases, resulting in E2F1 activating its downstream targets, including ELL (Fig. 8A). Once ELL is induced, it recruits HDAC1 to E2F1, leading to deacetylation of E2F1 and inhibition of its transactivity; ELL and Rb have a synergistic role in the suppression of E2F1 activity (Fig. 8B). DISCUSSION
Although studies have demonstrated that ELL serves as an important translocation partner of MLL in acute myeloid leukemia, the role of ELL in leukemogenesis remains largely unknown. Here, we provide evidence to show that ELL behaves in a manner similar to Rb, a classic tumor suppressor. Both Rb and ELL appear to inhibit E2F1 activity by recruiting HDAC1. Furthermore, we provide evidences to show that the MLL-ELL fusion protein resulting from chromosome translocation t(11;19)(q23;p13.1) in acute myeloid leukemia has no effect on the suppression of E2F1 transcriptional activity. These data suggest that disruption of ELL function may represent one of the mechanisms for leukemogenesis in MLLrearranged leukemia. Of note, MLL-ELL retains the greater part of ELL (aa 46 to 621). This region of ELL in the fusion protein can still interact well with E2F1, although its interaction with E2F1 is weaker than that of the N terminus (aa 1 to 379). Moreover, the region of ELL (aa 46 to 621) that is included in the fusion protein can still inhibit the transactivity of E2F1 as well as full-length ELL. However, after becoming fused with MLL, it loses the ability to suppress E2F1 transactivity. This phenomenon might be due to the conformational change of this part of the ELL protein (aa 46 to 621) after its fusion with the N terminus of MLL (aa 1 to 1406), as was also suggested by Wiederschain et al. (13). To date, more than 60 different MLL translocation partners have been identified (33). Among these partners, some of the most common MLL translocation partners, including AF4, AF9, ENL, and ELL, are physically associated in complexes involved in transcriptional elongation. It is evident that the deregulation of transcriptional elongation represents a major mechanism for MLLassociated leukemogenesis (6, 34–39). Here, we showed that the MLL-ELL fusion protein also disrupts ELL’s function in suppress-
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These data suggest that the MLL-ELL fusion protein resulting from the chromosome translocation t(11;19)(q23;p13.1) in acute myeloid leukemia loses the inhibitory role of ELL in E2F1 transcriptional activity, indicating a possible mechanism for MLLELL-based AML. ELL protects cells from E2F1-dependent cell death upon DNA damage. DNA damage stabilizes E2F1, thereby inducing the expression of proapoptotic genes (16). Thus, we examined whether ELL modulated this biological function of E2F1 by using the DNA damage reagent etoposide. Treatment of H1299 cells with etoposide (70 M) significantly induced ELL promoter activity (Fig. 6A). However, E2F1 knockdown by E2F1 shRNA abolished the etoposide-mediated induction of ELL promoter activity (Fig. 6A). Similar results were obtained by semiquantitative RTPCR (Fig. 6B). As such, in H1299 cells stably transfected with a scrambled shRNA as a control, treatment with etoposide (70 M) resulted in an increase in ELL protein level which was not evident in the absence of E2F1 (Fig. 6C). These results suggest that E2F1 induces ELL upon DNA damage. Subsequently, we examined whether ELL could affect the E2F1-dependent apoptosis upon DNA damage. We treated control H1299 cells (scrambled shRNA) and stable ELL knockdown cells, as well as stable Rb knockdown cells, with etoposide (70 M) for 40 h and then stained the cells using an annexin V-PI apoptosis kit. The flow cytometry analysis showed that ELL knockdown resulted in a greater number of dead cells (22.8% ⫹ 10.3% [later apoptotic cell ratio plus early apoptotic cell ratio] and 25.2% ⫹ 13.5% compared to 7.61% ⫹ 7.82%) (Fig. 6D). As expected, Rb knockdown also resulted a greater number of dead cells (28.7% ⫹ 9.69% [later apoptotic cell ratio ⫹ early apoptotic cell ratio] compared to 7.61% ⫹ 7.82%). Interestingly, simultaneous knockdown of ELL and Rb caused the dead cell number to increase further (38.7% ⫹ 14.1% and 33.1% ⫹ 15.6%, respectively) (Fig. 6D), suggesting a synergistic effect of Rb and ELL on protecting cells from DNA damage-induced death, consistent with the observation that Rb and ELL have a synergistic inhibitory role in E2F4B luciferase reporter activity (Fig. 3H). However, knockdown of E2F1 diminished the effect of ELL knockdown, as well as that of Rb knockdown, on the enhancement of DNA damage-induced cell death (Fig. 6E). To reinforce this conclusion, we employed Hoechst staining to do further assays. We treated control H1299 cells (scrambled shRNA) and stable ELL knockdown cells with etoposide (70 M) for 36 h and then stained the cells using Hoechst 33342. Using fluorescence microscopy, we found that ELL knockdown resulted in a greater number of dead cells (Fig. 6F, top). However, this difference was not evident when E2F1 was knocked down (Fig. 6F, bottom). Quantitative analysis of cell survival validated this tendency (Fig. 6G). As a key downstream factor of E2F1 in response to DNA damage (28), we examined p73 expression after knockdown of ELL, Rb, or both. As shown in Fig. 6H, knockdown of either Rb or ELL enhanced p73 expression, and simultaneous knockdown of Rb and ELL enhanced p73 expression further, again suggesting a synergistic role of Rb and ELL in the suppression of E2F1 transactivity. Moreover, knockdown of ELL caused a stronger induction of p73 and more PARP1 cleavage after treatment with etoposide (Fig. 6I and J) (30). However, when E2F1 was knocked down, the induction of p73 by knockdown of ELL became weaker (Fig. 6I and J). In addition, the PARP1 cleavage was also diminished after E2F1 knockdown (Fig. 6J). Taken
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ing E2F1 transactivity, which may represent another mechanism for MLL-ELL-based leukemogenesis. Probably due, in part, to the embryonic lethality of ELL knockout mice, the function of ELL in vivo and the exact mechanism of
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its action remain unclear (11). Here, we show that ELL is induced in response to DNA damage and that it protects cells from E2F1dependent cell death upon DNA damage, revealing a novel function of ELL. As an important member of the E2F family, the func-
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FIG 6 ELL inhibits E2F1 apoptotic function in response to DNA damage. (A) ELL promoter reporter activity was induced by etoposide treatment (70 m), but knockdown of E2F1 abolished this induction in H1299 cells. (B and C) Etoposide treatment (70 m) of H1299 cells also induced higher ELL mRNA (B) and ELL protein (C) levels, increases that were abolished by the knockdown of E2F1. (D) Knockdown of ELL, as well as knockdown of Rb in H1299 cells, increased DNA damage-induced cell death (1st to 5th panels from left to right); Knockdown of both Rb and ELL enhanced DNA damage-induced cell death synergistically (compare 6th and 7th panels to 3rd, 4th, and 5th panels from left to right). (E) Knockdown of E2F1 counteracted the effect of ELL, as well as that of Rb, in DNA damage-induced cell death. H1299 cells were treated with 70 M etoposide for 40 h; DMSO was used as the control. (F) Knockdown of ELL in H1299 cells increased DNA damage-induced, E2F1-dependent cell death. (G) Quantitative analysis for cell survival ratio (%) after treatment with 70 M etoposide for 36 h. (H) Knockdown of ELL in H1299 cells, as well as that of Rb, enhanced DNA damage-induced p73 expression, as revealed by semiquantitative RT-PCR; knockdown of both Rb and ELL enhanced DNA damage-induced p73 expression synergistically. (I) Semiquantitative RT-PCR for p73 expression in H1299 cells with knockdown of E2F1, ELL, or both after treatment with 50 M etoposide for 16 h; DMSO was used as the control. Data are reported as the means ⫾ SEM of three independent experiments performed in triplicate. (J) Western blot analysis for p73 expression and PARP1 cleavage in H1299 cells with knockdown of E2F1, ELL, or both after treatment with 50 M etoposide for 16 h; DMSO was used as the control.
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FIG 7 Kinetic/temporal expression patterns of p73 and ELL in response to DNA damage. (A) Kinetic/temporal analysis of p73 expression in response to DNA
tion of E2F1 in response to the stress of DNA damage has drawn much attention. Upon DNA damage, E2F1 directly upregulates SirT1, which then, in a negative feedback loop, directly deacetylates E2F1 to inhibit its activity (26). Similarly, in this study, we found that E2F1 also upregulates ELL, leading to the enhancement of E2F1 deacetylation and inhibition of its activity. However, we also showed that SirT1 suppressed the transactivity of the E2F1 K/R mutant, while ELL did not, ruling out the possibility that
SirT1 serves as a mediator of ELL function. It has been demonstrated that E2F1 lysines K117, K120, and K125, which are mainly acetylated by PCAF, can be deacetylated by multiple deacetylases, including HDAC1 (20). In this study, we found that ELL can recruit HDAC1 to E2F1 and that the addition of the HDAC1 inhibitor TSA can restore the ability of ELL to suppress E2F1 activity, indicating that HDAC1 may serve as the key mediator for ELL function. However, we still cannot rule out the possibility that other deacetylases can also mediate the ability of ELL to affect E2F1, and this requires further investigation. ACKNOWLEDGMENTS We are grateful to Ali Shilatifard, Fred Dick, and Toshiyuki Sakai for the generous gifts of reagents. W.X. is supported by 973 grant 2010CB126306, CAS Major Scientific and Technological Project grant XDA08010208, and NSFC grants 31071212 and 91019008.
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FIG 8 Schematic model of the role of ELL in the suppression of E2F1 transcriptional activity. (A) Upon DNA damage, E2F1 is acetylated by PCAF or other acetylases, resulting in E2F1 activating its downstream targets, including ELL. (B) After ELL is induced upon DNA damage, it recruits HDAC1 to E2F1, resulting in deacetylation of E2F1 and inhibition of its transactivity; ELL and Rb have a synergistic role in the suppression of E2F1 activity.
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ELL Inhibits E2F1 Transcriptional Activity by Enhancing E2F1 Deacetylation via Recruitment of Histone Deacetylase 1 Wei Zhang, Wei Ji, Xing Liu, Gang Ouyang, Wuhan Xiao The Key Laboratory of Aquatic Biodiversity and Conservation, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, People’s Republic of China
T
he ELL (eleven-nineteen lysine-rich leukemia) protein was first identified as a translocation partner of MLL in acute myeloid leukemia (AML) (1). Subsequent studies showed that ELL can serve as a transcription elongation factor that increases the rate of RNA polymerase II transcriptional elongation by preventing transient pausing (2, 3) or by facilitating RNA polymerase II pause site entry and release (4). Increasing evidence strongly supports the idea that ELL belongs to a super elongation complex that modulates gene expression in development and disease (5–10). In addition, the targeted knockout of ELL in mice causes embryonic lethality, suggesting that ELL has a crucial role in early embryogenesis (11). ELL has been also identified as a partner of steroid receptors p53, hypoxia-inducible factor 1␣ (HIF-1␣), and EAF1/2, modulating their binding partner activity in either a positive or negative manner (12–15). While multiple lines of evidence point to the importance of ELL in normal and cancerous cells, the exact mechanism of its activity remains elusive. The E2F family of transcription factors play important roles in regulating cell cycle progression by transactivating genes required for entry into the S phase (16). As a member of the family, E2F1 also has crucial roles in regulating apoptosis via stimulating the transcription of several genes in the apoptotic pathway, particularly upon DNA damage (17). DNA damage stabilizes and activates E2F1 through phosphorylation by ATM (18) or CHK2 (19), as well as acetylation by PCAF (20). To understand the underlying mechanism of E2F1 activity, multiple investigators have explored the binding partners of E2F1. Studies have shown that the classic tumor suppressor pRb serves as a major regulator of E2F1 (21) by directly recruiting histone deacetylase 1 (HDAC1) (22). pRb is often mutated and inactive in tumors, leading to deregulation of E2F1 activity (23). TopBP1 also interacts with E2F1 and subsequently inhibits E2F1 activity through a Brg1/Brm-dependent, pRb-independent mechanism (24). MCPH1/BRIT1 cooperates with E2F1 to regulate the expression of E2F target genes (25). One target gene is the NAD-dependent histone deacetylase SirT1, which in a negative feedback loop inhibits E2F1’s activity in regulating the apoptotic response to DNA damage (26). Overall, the acetylation status of E2F1 represents a major mechanism for regulating E2F1 function. In this study, we identified ELL as a downstream target of E2F1
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in response to DNA damage. Furthermore, we found that ELL interacted with E2F1, inhibiting E2F1’s function through the recruitment of HDAC1, but the MLL-ELL fusion protein had no inhibitory role on E2F1 transcriptional activity. Our findings not only connect ELL to the E2F1 signaling pathway and reveal a novel role of ELL in response to DNA damage but also provide an insight into the mechanism for MLL-ELL-associated leukemogenesis. MATERIALS AND METHODS Cell lines. HEK293T, H1299, Saos2, and HCT116 cells were originally obtained from ATCC. HEK293T, H1299, and HCT116 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; HyClone) with 10% fetal bovine serum (FBS; HyClone) at 37°C in a humidified atmosphere containing 5% CO2, while Saos2 cells were maintained in McCoy’s 5A medium with 15% FBS. Plasmid constructs. The original ELL and MLL-ELL constructs were kindly provided by Ali Shilatifard. The p73 promoter construct was kindly provided by Toshiyuki Sakai. The E2F4B luciferase reporter and pRb expression vector were kindly provided by Fred Dick. The ELL promoter (position ⫺1768 to ⫹404) for its luciferase reporter construct was amplified from total DNA extracted from 293 cells by PCR using primers 5=-ATATCGGTACCGGGCGTGGTGGTGGGCATC TGTAA-3= and 5=-ATATCAAGCTTCGGGCCACCCGCCTGTATTGCC TC-3=. The p27/Kip1 (position ⫺2292 to ⫹471) promoter for its luciferase reporter construct was amplified by PCR using primers 5=-TATCGG TACCTCAAGTGATCCTCCCACCTCTGCT-3= and 5=-TATCAAGCTT AATGCAGCTGTCTTGAGCCTGTG-3=. The Apaf1 promoter for its luciferase reporter construct was amplified by PCR using primers 5=-TA TCGGTACCAACAAGGCTGGGCTGTTTCCTTCC-3= and 5=-TATCAA GCTTTACTGGACACAAAGGGAGGAGGTCTT-3=. These three promoters were then subcloned into pGL3-Basic vector (Promega). ChIP assays. The experimental protocol for chromatin immunoprecipitation (ChIP) and the primers for -actin were described previously
Received 9 July 2013 Returned for modification 1 August 2013 Accepted 5 December 2013 Published ahead of print 16 December 2013 Address correspondence to Wuhan Xiao, [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/MCB.00878-13
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ELL (eleven-nineteen lysine-rich leukemia protein) was first identified as a translocation partner of MLL in acute myeloid leukemia; however, the exact mechanism of its action has remained elusive. In this study, we identified ELL as a direct downstream target gene of E2F1. Coimmunoprecipitation assays showed that ELL interacted with E2F1 in vitro and in vivo, leading to inhibition of E2F1 transcriptional activity. In addition, ELL enhanced E2F1 deacetylation via recruitment of histone deacetylase 1 (HDAC1). Notably, the MLL-ELL fusion protein lost the inhibitory role of ELL in E2F1 transcriptional activity. Furthermore, DNA damage induced ELL in an E2F1-dependent manner and ELL protected cells against E2F1-dependent apoptosis. Our findings not only connect ELL to E2F1 function and uncover a novel role of ELL in response to DNA damage but also provide an insight into the mechanism for MLL-ELL-associated leukemogenesis.
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tifying the protein levels based on the band density obtained in Western blot assays. Lentivirus-mediated gene knockdown. Each short hairpin RNA (shRNA) sequence was cloned into the pLKO.1 vector and verified by sequencing. The following shRNA sequences were used: scrambled shRNA, 5=-GCCTTGATCGCTCGCTAAACA-3=; ELL shRNA#1, 5=-CAA CACCAACTACAGCCAGGA-3=; ELL shRNA#2, 5=-GCGAGTACCTGC ACAGCAA-3=; HDAC1 shRNA, 5=-CTAATGAGCTTCCATACAA-3= E2F1 shRNA, 5=-GTCACGCTATGAGACCTCA-3=; and Rb shRNA, 5=-G ATACCAGATCATGTCAGA-3=. For lentiviral shRNA production, HEK293T cells in 10-cm dishes were transfected with 6 g psPAX2, 6 g pMD2.G, and 12 g pLKO.1 with shRNA inserts. After 8 h of transfection, the medium was replaced with 12 ml of fresh DMEM with 10% FBS. After 40 h, the medium containing the lentivirus particles was collected, centrifuged, and passed through a 0.45-m filter, and then added to HEK293 or H1299 cells. An amount of 8 g/ml Polybrene was added to the medium to improve infection efficiency. Apoptosis assay. For annexin V staining, H1299 cells were infected with lentivirus expressing scrambled control shRNA, ELL shRNA, Rb shRNA, E2F1 shRNA, or combinations of these shRNAs. The cells were then treated with etoposide (70 M) or dimethyl sulfoxide (DMSO) control for 40 h. Subsequently, the cells were collected and washed in cold phosphate-buffered saline (PBS), resuspended in 1⫻ annexin binding buffer, and incubated with annexin V-fluorescein isothiocyanate (FITC) and phosphatidylinositol (PI) at room temperature for 10 min in the dark using an annexin V-PI apoptosis kit (MultiSciences Biotech). Flow cytometry analysis was performed to detect apoptotic cells. The data were evaluated with Cell Quest software. For Hoechst staining, the cells were treated with etoposide (70 M) or the DMSO control for 36 h. Hoechst 33342 (1 g/ml, Molecular Probes) was used to visualize breakage of nuclei. The cells were counted under a fluorescence microscope. Each experiment was repeated at least three times.
RESULTS
ELL is a direct downstream target of E2F1. During our study of the regulation of thrombospondin-1 (TSP-1) by E2F1 (30), we noticed that ELL, a TSP-1 regulator (31), was also regulated by E2F1. To understand whether ELL was a direct downstream target of E2F1, we first did promoter assays. A 2.1-kb promoter region of ELL was cloned into the pGL3-Basic vector (Fig. 1A). In HEK293T cells, overexpression of E2F1 caused the ELL promoter activity to increase by about 20-fold but did not increase the activity of the pGL3-Basic vector control (Fig. 1B). Furthermore, overexpression of E2F1 (E132), a DNA binding-deficient mutant, failed to activate the ELL promoter luciferase reporter (Fig. 1C), indicating that the DNA binding domain must be kept intact for E2F1 to upregulate ELL expression. To map the E2F1 response region in the ELL promoter, we generated three truncated ELL promoter constructs (Fig. 1A). When E2F1 was overexpressed, all three truncated ELL promoter reporters showed activity (Fig. 1B), suggesting that the E2F1 response element is located between nucleotides ⫺32 and ⫹404. We identified a putative E2F1 binding site (GCGCCAGA) located at the ⫹198 position (Fig. 1D). To confirm that this site indeed acts as an E2F1 binding site, we mutated CG to AT (GATCCAGA) and found that E2F1 could not induce transcription from this mutant promoter (Fig. 1D and E). Semiquantitative RT-PCR revealed that overexpression of E2F1 in H1299 cells caused an increase in E2F1 mRNA (Fig. 1F) but that knockdown of E2F1 by E2F1 shRNA reduced the E2F1 mRNA level (Fig. 1G). To determine whether E2F1 bound directly to the ELL pro-
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(27). The primers specific for the ELL promoter region from position ⫹118 to ⫹324 were 5=-CGGGTTAGCGACGGCAGCAAGGTG-3= (forward) and 5=-CCGCGAGCAGCACCACAGACCTAG-3= (reverse). The primers (control) for the ELL promoter region from position ⫺1389 to ⫺1133, far from E2F1 binding sites, were 5=-CATGGTGAAATCCCGTC TCTAG-3= (forward) and 5=-CCTCCCAAAGTGCTAGGATTAC-3= (reverse). The primers for the p73 promoter region from ⫺1257 to ⫺1049 were 5=-TGAGCCATGAAGATGTGCGAG-3= (forward) and 5=-GCTGC TTATGGTCTGATGCTTATG-3=(reverse) (28). Antibodies and reagents. Human ELL polyclonal antibody was generated against synthesized peptides of ELL (Abmart, Shanghai, China). Other antibodies used were as follows: antihemagglutinin (antiHA) antibody (Covance), anti-Myc antibody (9E10; Santa Cruz), antiFlag antibody (M2; Sigma), anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) antibody (SC-47724; Santa Cruz), anti-E2F1 antibody (KH95; Santa Cruz), anti-HDAC1 antibody (3426-1; Epitomics), anti-p73 antibody (ab40658; Abcam), anti-p27/KIP1 antibody (ab32304; Abcam), anti-Apaf1 antibody (ab32372; Abcam), and antipoly(ADP-ribose) polymerase 1 (anti-PARP1) antibody (no. 9542; Cell Signaling Technology). Trichostatin A (TSA) (5 mM) was purchased from Sigma. Semiquantitative reverse transcription (RT)-PCR. Total RNA was extracted from cells by using TRIzol reagent (Invitrogen), and cDNA was synthesized using a first-strand cDNA synthesis kit (Fermentas). The following primers were used: for human ELL mRNA, forward, 5=-ACTGCA TCCAGCAGTATGTCTCCA-3=, and reverse, 5=-CTTGATGACAATGG CACTTCGGCT-3=; for human p73 mRNA, forward, 5=-CATGGTCTCG GGGTCCCACT-3=, and reverse, 5=-CGTGAACTCCTCCTTGATGG-3=; for human Apaf1 mRNA, forward, 5=-AATGGACACCTTCTTGGACG3=, and reverse, 5=-GCACTTCATCCTCATGAGCC-3=; for human p27 mRNA, forward, 5=-CCGGTGGACCACGAAGAGT-3=, and reverse, 5=GCTCGCCTCTTCCATGTCTC-3=; for human Rb mRNA, forward, 5=GCAGTATGCTTCCACCAGGC-3=, and reverse, 5=-AAGGGCTTCGAG GAATGTGAG-3=; and for 18S RNA, used as an internal control, forward, 5=-TCAACTTCGATGGTAGTCGCCGT-3=, and reverse, 5=-TCCTTGG ATGTGGTAGCCGTTCT-3=. Data are reported as the means ⫾ standard errors of the means (SEM) of three independent experiments performed in triplicate. The statistical analysis was performed using GraphPad Prism 5 (unpaired t test) (GraphPad Software Inc.). Luciferase reporter assays. HEK293T, H1299, or Saos2 cells were seeded in 24-well plates and transfected with the luciferase reporters indicated above in “Plasmid constructs” using Lipofectamine 2000 (Invitrogen) or VigoFect (Vigorous Biotech, Beijing, China). pTK-Renilla was used as an internal control. Luciferase activity was measured after transfection for 20 to 24 h using the dual-luciferase reporter assay system (Promega). Data are reported as the means ⫾ SEM of three independent experiments performed in triplicate. The statistical analysis was performed using GraphPad Prism 5 (unpaired t test) (GraphPad Software Inc.). Western blot assay and coimmunoprecipitation. For Western blot assay and coimmunoprecipitation of overexpressed proteins, the experimental procedures have been described previously (29). For endogenous coimmunoprecipitation, the cells indicated below were lysed and centrifuged at 13,000 rpm for 15 min. The supernatants were immunoprecipitated with agarose beads conjugated with the antibodies indicated below at 4°C overnight. The beads were washed three times using radioimmunoprecipitation assay (RIPA) buffer with 500 mM NaCl. The proteins were detected by Western blot assay using the antibodies indicated below. For detection of E2F1 acetylation, cell lysates containing Flag-tagged E2F1 were immunoprecipitated with anti-Flag beads at 4°C overnight. The beads were washed five times with ice-cold RIPA buffer, eluted with SDS loading buffer, and then subjected to SDS-PAGE and immunoblotted with an antiacetyllysine antibody (Millipore). The Fujifilm LAS4000 mini-luminescent image analyzer system was used to photograph the blots. Multi Gauge version 3.0 was used for quan-
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ELL promoter reporters. HEK293T cells were transfected with different truncated ELL promoter reporters together with either Flag-E2F1 expression vector or Flag empty vector (control). (C) Overexpression of the E2F1 (E132) mutant failed to activate the ELL promoter. The full-length ELL promoter (⫺1768 to ⫹404) reporter was transfected together with either the wild-type E2F1 construct or the E132 mutant construct. (D) Partial sequence of the ELL promoter (⫺32 to ⫹404); one potential E2F1 binding site is marked by the box, and substitution mutations are underlined. (E) E2F1 trans activation of the ELL promoter through the potential E2F1 binding site GCGCCAGA around the ⫹198 position. Transfection of the E2F1 expression vector failed to activate the ELL mutant promoter (⫹198 mut). (F) Overexpression of E2F1 in H1299 cells caused an increase in the ELL mRNA level as revealed by semiquantitative RT-PCR. (G) Knockdown of E2F1 by E2F1 shRNA in H1299 cells caused a reduction in the ELL mRNA level as revealed by semiquantitative RT-PCR. (H) Schematic of locations of fragments amplified in ELL promoter or -actin promoter. E2F1 binding region in ELL promoter is amplified between ⫹118 and ⫹324, and the control region in the ELL promoter within which E2F1 cannot bind is amplified between ⫺1389 and ⫺1133; the fragment amplified from -actin is used as the control. (I) Chromatin immunoprecipitation analysis indicated that E2F1 interacted directly with the ELL promoter region harboring the putative E2F1 binding site. (J) Semiquantitative RT-PCR confirmed that E2F1 binds to the ⫹118 to ⫹324 region of the ELL promoter. (K) Semiquantitative RT-PCR confirmed that E2F1 cannot bind to the ⫺1389 to ⫺1133 region of the ELL promoter. (L) Upregulation of ELL protein by overexpression of E2F1 was confirmed by Western blotting. (M) Knockdown of E2F1 shRNA decreased the ELL protein level; pSuper-GFP-shRNA was used as the control. (N) E2F1 induced by 4-OHT (500 nm; dissolved in ethyl alcohol) in H1299 cells stably expressing ER-E2F1 caused upregulation of ELL protein. If not otherwise specified, data are reported as the means ⫾ SEM of three independent experiments performed in triplicate.
moter harboring the E2F1 binding site, we performed chromatin immunoprecipitation (ChIP) assays. One pair of primers encompassing the E2F1 binding site was designed to amplify a 208-bp fragment, and another pair of primers was designed to amplify a 257-bp fragment far from the E2F1 binding site (control) (Fig. 1H). As shown in Fig. 1I, anti-E2F1 antibody could easily precipitate the 208-bp fragment of the ELL promoter encompassing the E2F1 binding site. Semiquantitative RT-PCR confirmed that antiE2F1 antibody pulled down the 208-bp fragment (position ⫹118 to ⫹324) (Fig. 1J) but not the 257-bp fragment (position ⫺1389 to ⫺1133) (Fig. 1K), which is far from the E2F1 binding region.
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Subsequently, we performed Western blot assays to determine whether the protein level of ELL was regulated by E2F1. In HEK293T cells, overexpression of E2F1 upregulated ELL (1.4-fold versus 1.0-fold) (Fig. 1L), but knockdown of E2F1 by E2F1 shRNA reduced ELL expression (0.6-fold versus 1.0-fold) (Fig. 1M). Similarly, we found that activation of ER-E2F1 expression by treating an H1299 cell line stably expressing an ER-E2F1 fusion protein with 4-hydroxytamoxifen (4-OHT; 500 nm) resulted in the upregulation of ELL (1.9-fold versus 1.0-fold) (Fig. 1N). Taken together, these results suggest that the ELL gene is a potential direct target of E2F1.
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FIG 1 The ELL gene is a direct target of E2F1. (A) Schematic of ELL promoter luciferase reporter constructs. (B) Overexpression of E2F1 induced the activity of
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RFP-tagged E2F1. (B and C) Immunoprecipitation assays showed that E2F1 interacted with ELL when they were overexpressed in HEK293T cells. (D and E) ELL interacted with E2F1 endogenously in both H1299 cells (p53 deficient) and Saos2 cells (Rb deficient). Anti-E2F1 monoclonal antibody was used for immunoprecipitation (IP), and mouse IgG was used as the control; ELL was detected by an anti-ELL polyclonal antibody. (F) E2F1 bound to ELL directly, as revealed by Ni-NTA assays. His-tagged ELL and GST-tagged E2F1 were expressed in Escherichia coli; Ni-NTA agarose beads were used for immunoprecipitation, and anti-E2F1 antibody was used for Western blotting. (G) Coomassie blue staining for GST and GST-E2F1 purified from E. coli. (H) Domain mapping for ELL interacting with E2F1. ⫺, no interaction; ⫹, interaction; IB, immunoblotting. ELL (aa 1 to 379) was essential for E2F1 binding. (I) Domain mapping for E2F1 interacting with ELL. E2F1 (aa 201 to 368) was essential for ELL binding.
ELL interacts with E2F1 in vitro and in vivo. As a nuclear protein, ELL binds to and inhibits p53 (13). Given that E2F1 is a well-defined transcription factor, we speculated that the interaction of ELL with E2F1 may affect E2F1 function. To test this, we initially did colocalization assays. As shown in Fig. 2A, after transfection into HeLa cells, green fluorescent protein (GFP)-tagged ELL colocalized with red fluorescent protein (RFP)-tagged E2F1, indicating that ELL might interact with E2F1 in the nucleus. To confirm the interaction, we conducted coimmunoprecipitation assays. In HEK293T cells transfected with HA-E2F1 and Myc-ELL or Myc-empty vector control, anti-Myc antibody-conjugated agarose beads pulled down HA-E2F1 (Fig. 2B). Similarly, HA-E2F1 also pulled down Flag-ELL in immunoprecipitation experiments using anti-HA antibody-conjugated agarose beads (Fig. 2C). To determine whether ELL interacted with E2F1 endogenously, we used anti-E2F1 antibody to perform endogenous immunoprecipitation in H1299 (p53-null) and Saos2 (Rb-null) cells. As shown in Fig. 2D and E, ELL interacted with E2F1 endogenously. This experiment also suggests that ELL can interact with E2F1 independently of both p53 and Rb. To test whether ELL bound to E2F1 directly, we conducted Ni-nitrilotriacetic acid (NTA) pulldown assays. His-tagged ELL expressed in Escherichia
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coli could pull down glutathione S-transferase (GST)-tagged E2F1 expressed in E. coli (Fig. 2F and G). Collectively, these experiments suggest that the two proteins can interact directly in a p53- and Rb-independent manner. We then mapped the domains of E2F1 and ELL responsible for interaction between the two proteins by using deletion mutants (Fig. 2H and I). The N terminus of ELL (amino acids [aa] 1 to 379) was crucial for interacting with E2F1 (Fig. 2H), and the domain from aa 201 to 368 of E2F1 was required for interacting with ELL (Fig. 2I). Surprisingly, the longer construct (aa 1 to 508) binds to E2F1 more weakly than the shorter one (aa 1 to 379), which might be due to the conformational difference between these two peptides. ELL inhibits E2F1 transcriptional activity. To investigate the functional importance of the interaction between ELL and E2F1, we evaluated the effect of ELL on promoter reporters of E2F1’s downstream target genes. In HEK293T cells, overexpression of E2F1 resulted in a dramatic activation of the E2F4B luciferase reporter (Fig. 3A). However, cotransfection of the ELL expression vector caused this activation to decrease significantly (Fig. 3A). The expression levels of transfected HA-ELL and Flag-E2F1 were confirmed by Western blot assay (Fig. 3A, right). We saw similar
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FIG 2 ELL interacts with E2F1 in vivo and in vitro. (A) ELL colocalized with E2F1 in the nucleus. HeLa cells were transfected with GFP-tagged ELL and
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cells (A), Rb-deficient Saos2 cells (B), Rb-intact H1299 cells (C), and HCT116 cells (D). The expression levels of HA-ELL and Flag-E2F1 were confirmed by Western blotting (A, right). (E, F, G) The activity of E2F1 downstream target gene promoter reporters (Apaf1, p73, and p27) induced by E2F1 was suppressed by cotransfection of ELL in HEK293T cells. (H) Cotransfection of pRB and ELL showed a synergistic effect on the inhibition of E2F1-mediated E2F4B luciferase reporter activity. (Right) The expression levels of HA-ELL, HA-Rb, and Flag-E2F1 were confirmed by Western blotting. (I) ELL deletion mutants were cotransfected with E2F1 and E2F4B luciferase reporter. The ELL mutant with aa 46 to 379 was essential for the inhibitory effect on E2F1 transcriptional activity. (J) Knockdown of ELL by ELL shRNA#1 and shRNA#2 in H1299 cells was confirmed by semiquantitative RT-PCR. (K) Knockdown of ELL by ELL shRNA in HEK293T cells enhanced the activity of the E2F4B luciferase reporter. (L, M, N) Knockdown of ELL by ELL shRNA in H1299 cells increased the mRNA levels of Apaf1, p73, and p27. Data are reported as the means ⫾ SEM of three independent experiments performed in triplicate. (O) Western blot assays for Apfa1, p73, and p27 in H1299 cells with ELL knockdown.
results in Sao2 cells, H1299 cells, and HCT116 cells (Fig. 3B, C, and D). In addition, overexpression of ELL in HEK293T cells also suppressed the activation of three other promoter reporters induced by E2F1, including Apaf1-luc, p73-luc, and p27-luc (Fig. 3E, F, and G). Rb, a well-defined E2F1 partner, suppresses E2F1 transactivity (21). To gain a more complete picture of the inhibitory role of ELL on E2F1, we compared ELL to Rb. As shown in Fig. 3H, in HEK293T cells, Rb and ELL suppressed E2F1 transactivity to similar degrees in E2F4B luciferase reporter assays. Interestingly, ELL and Rb suppressed E2F1 transactivity synergistically, as revealed by cotransfection experiments (Fig. 3H). The expression levels of transfected HA-ELL, HA-Rb, and Flag-E2F1 were confirmed by Western blot assay (Fig. 3H, right). To identify the ELL domain responsible for E2F1 suppression, we did domain mapping experiments in which we created ELL deletion mutants. We found that the aa 46 to 379 region of ELL
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was essential for inhibiting E2F1 activity (Fig. 3I). The HA-ELL mutants containing this region, for example, those with aa 1 to 379, aa 1 to 508, and aa 46 to 621, significantly inhibited E2F1 transactivity (P ⬍ 0.05). However, the HA-ELL mutants lacking this region, i.e., those with aa 447 to 621 and aa 509 to 621, did not do so (Fig. 3I). Not surprisingly, the ELL domains that suppressed activity were the same as those involved in E2F1 binding (Fig. 2H). To confirm the inhibitory role of ELL on E2F1, we knocked down ELL by using ELL shRNAs and then checked E2F4B luciferase reporter activity. Knockdown of ELL mediated by ELL shRNA#1 and shRNA#2 was confirmed by semiquantitative RTPCR (Fig. 3J). After ELL knockdown, the activity of the E2F4B luciferase reporter increased significantly (Fig. 3K), in contrast to the results of ELL overexpression. Moreover, knockdown of ELL in H1299 cells caused the mRNA levels of Apaf1, p73, and p27 to increase dramatically (Fig. 3L, M, and N). Consistent with these
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FIG 3 ELL inhibits E2F1 transcriptional activity. Cotransfection of ELL suppressed E2F1-mediated transactivity on the E2F4B luciferase reporter in HEK293T
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results, knockdown of ELL in H1299 cells caused the protein levels of Apaf1, p73, and p27, three E2F1 downstream targets, to increase (Fig. 3O). Thus, it appears that ELL binding suppresses E2F1 transactivity. ELL enhanced the deacetylation of E2F1 by recruiting HDAC1. We next investigated the underlying mechanism of ELL inhibition on E2F1. Rb inhibits E2F1 by recruiting histone
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deacetylase 1 (HDAC1) to deacetylate E2F1 (22). To test whether ELL also inhibits E2F1 activity though HDAC1 recruitment, we first generated an E2F1 K/R mutant with the set of lysines at positions 117, 120, and 125 mutated to arginine, which therefore lacks the sites required for acetylation (20). When the E2F1 K/R mutant was cotransfected with ELL, the ability of ELL to inhibit transcription from the E2F1 reporter disappeared (Fig. 4A) (P ⫽ 0.0646
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FIG 4 ELL enhances deacetylation of E2F1 by recruiting HDAC1. (A) Overexpression of ELL failed to inhibit E2F4B luciferase reporter activity mediated by the E2F1 K/R mutant (K117R/K120R/K125R). (B) Overexpression of ELL reduced exogenous E2F1 acetylation. The Flag-ELL expression vector was cotransfected together with either the Myc-ELL expression vector or the Myc empty vector into HEK293T cells; agarose beads conjugated with anti-Flag antibody were used for immunoprecipitation, and antiacetyllysine antibody (anti-AcLys) was used for evaluating E2F1 acetylation. (C) Knockdown of ELL enhanced exogenous E2F1 acetylation. (D) ELL interacted with HDAC1 when they were overexpressed, and the N terminus of ELL (aa 1 to 379) was essential for HDAC1 binding. (E) ELL interacted with HDAC1 endogenously in HCT116 cells. (F) Overexpression of ELL enhanced the interaction between HDAC1 and E2F1. (G) Knockdown of ELL reduced the interaction between endogenous HDAC1 and endogenous E2F1 in H1299 cells. (H) Knockdown of ELL reduced the interaction between endogenous HDAC1 and endogenous E2F1 in Saos2 cells. (I) Knockdown of ELL in Saos2 cells reduced endogenous HDAC1 binding to the E2F1 binding region in the p73 promoter (⫺1257 to ⫺1049). (J) Knockdown of HDAC1 partially reversed the inhibitory effect of ELL on E2F1-mediated E2F4B luciferase reporter activity (P ⬍ 0.0001). (K) shRNA-mediated knockdown of HDAC1 was confirmed by Western blot assays. (L) The HDAC inhibitor TSA reversed the suppressive role of ELL in E2F1-mediated E2F4B luciferase reporter activity. H1299 cells were treated with TSA (dissolved in DMSO) at a final concentration of 500 nM; DMSO was used as the control.
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versus P ⫽ 0.0008). Moreover, the E2F1 K/R mutant had lower transcriptional activity than wild-type E2F1 (Fig. 4A) (P ⫽ 0.0282). These results not only support the importance of acetylation for E2F1 activity but also suggest that ELL may suppress E2F1 through enhancing its deacetylation. Subsequently, we performed immunoprecipitation assays to analyze the acetylation status of E2F1 after ELL overexpression. As expected, overexpression of ELL indeed enhanced E2F1 deacetylation (Fig. 4B). In contrast, knockdown of ELL resulted in an increase in E2F1 acetylation (Fig. 4C). Studies have indicated that the acetylation of E2F1 is positively regulated by CBP/p300 and negatively regulated by HDAC1 (22, 32). To test whether ELL could recruit HDAC1 to E2F1, we first did coimmunoprecipitation assays to determine if ELL and HDAC could interact. As expected from our domain mapping studies, the N terminus of ELL (aa 1 to 379) bound efficiently to HDAC1 (Fig. 4D). In addition, the anti-ELL antibody could pull down endogenous HDAC1 in HCT116 cells (Fig. 4E), suggesting that ELL interacts with HDAC1 endogenously. Because E2F1 and HDAC1 bind to the same domain of ELL, we next examined whether ELL could promote an interaction between E2F1 and HDAC1. HEK293T cells were transfected with Flag-E2F1 and HAHDAC1 in the presence or absence of Myc-ELL. We found that E2F1 and HDAC coimmunoprecipitated and that ELL expression enhanced this interaction (Fig. 4F). To further confirm the recruitment of HDAC1 by ELL, we performed endogenous coimmunoprecipitation in H1299 cells that have a stable knockdown of ELL. As shown in Fig. 4G, ELL knockdown diminished the endogenous interaction between E2F1 and HDAC1. Because H1299 cells contain intact Rb, endogenous Rb might recruit E2F1 to interact with ELL. To rule out this possibility, we performed endogenous coimmunoprecipitation in Rb-deficient Saos2 cells. As
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shown in Fig. 4H, ELL knockdown still diminished the endogenous interaction between E2F1 and HDAC1, supporting the idea that ELL recruits HDAC1 to E2F1 independently of Rb. In addition, knockdown of ELL in Saos2 cells also diminished HDAC1 binding to the specific region of the p73 promoter, which was reported to be bound by E2F1 (28). Moreover, knockdown of HDAC1 by shRNA could partially reverse the suppressive role of ELL on E2F1 transactivity, as revealed by promoter assays (Fig. 4J). The efficiency of HDAC1 shRNA-mediated HDAC1 knockdown was confirmed by Western blot assay (Fig. 4K). The addition of the HDAC inhibitor TSA reversed the suppressive role of ELL on E2F1 transactivity (Fig. 4L), further supporting the notion that ELL recruits HDAC1 to suppress E2F1 activity. The MLL-ELL fusion protein has no inhibitory role on E2F1 transcriptional activity. To determine the physiological relevance of ELL in the inhibition of E2F1 transcriptional activity, we examined the effect of an MLL-ELL fusion protein on E2F1 transactivity. In H1299 cells, overexpression of ELL inhibited the E2F1induced activities of p27, Apaf1, and p73 promoter reporters, as well as that of the E2F4B luciferase reporter (Fig. 5A, B, C, and D), similar to the results in HEK293T cells and Saos2 cells, indicating that the inhibitory role of ELL in E2F1 activity is cell line independent. In contrast, overexpression of MLL-ELL had no obvious effect on these reporters (Fig. 5A, B, C, and D). The expression levels of transfected Flag-E2F1, HA-ELL, and HA-MLL-ELL were confirmed by Western blot assay (Fig. 5C, right). Consistent with these results, the expression of p73 and p27 induced by E2F1 activation via adding 4-OHT (500 nM) to H1299 cells stably transfected with ER-E2F1 was also inhibited by overexpression of ELL but not by overexpression of MLL-ELL, as revealed by semiquantitative RT-PCR (Fig. 5E and F). Moreover, the acetylation of E2F1 was not affected by overexpression of MLL-ELL (Fig. 5G).
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FIG 5 The MLL-ELL fusion protein has no inhibitory role in E2F1 transactivity. (A, B, C, and D) The activity of E2F1 downstream target gene promoter reporters (p27, Apaf1, and p73) and E2F4B luciferase reporter induced by E2F1 was suppressed by cotransfection of ELL but not by cotransfection of MLL-ELL in H1299 cells. The expression levels of HA-ELL, HA-MLL-ELL, and Flag-E2F1 were confirmed by Western blotting (C, right). (E and F) The expression of p73 and p27 induced by activation of E2F1 (by adding 4-OHT at a final concentration of 500 nM) in H1299 cells stably transfected with ER-E2F1 was suppressed by overexpression of ELL but not by overexpression of MLL-ELL. Data are reported as the means ⫾ SEM of three independent experiments performed in triplicate. (G) Overexpression of ELL reduced exogenous E2F1 acetylation, but overexpression of MLL-ELL did not do so.
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together, these results suggest that ELL protects cells from E2F1dependent cell death upon DNA damage. The above-described observations indicate that upon DNA damage, E2F1 upregulates ELL effectively. However, ELL can subsequently suppress E2F1 activity, which might result in turning off the DNA damage response. To understand how these two processes were operated, we examined the expression of p73, a welldefined downstream target of E2F1 in its response to DNA damage (28), as well as the expression of ELL, by kinetic/temporal analysis. As shown in Fig. 7, upon DNA damage, p73 expression was steadily upregulated and reached its highest level at 24 h, after which it went down sharply (Fig. 7A). In contrast, ELL expression was upregulated at a relatively lower rate and reached its highest level at 36 h, after which it went down slowly (Fig. 7B). These results indicate that, in response to DNA damage, E2F1 upregulates ELL at a relatively lower rate at the later stage, which turns off the E2F1-dependent DNA damage response to protect cells from DNA damage-induced cell death and also shuts down its own expression. These two processes appeared to be temporally regulated. Based on these data, we propose a model for the role of ELL in E2F1 activity (Fig. 8). Upon DNA damage, E2F1 is acetylated by PCAF or other acetylases, resulting in E2F1 activating its downstream targets, including ELL (Fig. 8A). Once ELL is induced, it recruits HDAC1 to E2F1, leading to deacetylation of E2F1 and inhibition of its transactivity; ELL and Rb have a synergistic role in the suppression of E2F1 activity (Fig. 8B). DISCUSSION
Although studies have demonstrated that ELL serves as an important translocation partner of MLL in acute myeloid leukemia, the role of ELL in leukemogenesis remains largely unknown. Here, we provide evidence to show that ELL behaves in a manner similar to Rb, a classic tumor suppressor. Both Rb and ELL appear to inhibit E2F1 activity by recruiting HDAC1. Furthermore, we provide evidences to show that the MLL-ELL fusion protein resulting from chromosome translocation t(11;19)(q23;p13.1) in acute myeloid leukemia has no effect on the suppression of E2F1 transcriptional activity. These data suggest that disruption of ELL function may represent one of the mechanisms for leukemogenesis in MLLrearranged leukemia. Of note, MLL-ELL retains the greater part of ELL (aa 46 to 621). This region of ELL in the fusion protein can still interact well with E2F1, although its interaction with E2F1 is weaker than that of the N terminus (aa 1 to 379). Moreover, the region of ELL (aa 46 to 621) that is included in the fusion protein can still inhibit the transactivity of E2F1 as well as full-length ELL. However, after becoming fused with MLL, it loses the ability to suppress E2F1 transactivity. This phenomenon might be due to the conformational change of this part of the ELL protein (aa 46 to 621) after its fusion with the N terminus of MLL (aa 1 to 1406), as was also suggested by Wiederschain et al. (13). To date, more than 60 different MLL translocation partners have been identified (33). Among these partners, some of the most common MLL translocation partners, including AF4, AF9, ENL, and ELL, are physically associated in complexes involved in transcriptional elongation. It is evident that the deregulation of transcriptional elongation represents a major mechanism for MLLassociated leukemogenesis (6, 34–39). Here, we showed that the MLL-ELL fusion protein also disrupts ELL’s function in suppress-
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These data suggest that the MLL-ELL fusion protein resulting from the chromosome translocation t(11;19)(q23;p13.1) in acute myeloid leukemia loses the inhibitory role of ELL in E2F1 transcriptional activity, indicating a possible mechanism for MLLELL-based AML. ELL protects cells from E2F1-dependent cell death upon DNA damage. DNA damage stabilizes E2F1, thereby inducing the expression of proapoptotic genes (16). Thus, we examined whether ELL modulated this biological function of E2F1 by using the DNA damage reagent etoposide. Treatment of H1299 cells with etoposide (70 M) significantly induced ELL promoter activity (Fig. 6A). However, E2F1 knockdown by E2F1 shRNA abolished the etoposide-mediated induction of ELL promoter activity (Fig. 6A). Similar results were obtained by semiquantitative RTPCR (Fig. 6B). As such, in H1299 cells stably transfected with a scrambled shRNA as a control, treatment with etoposide (70 M) resulted in an increase in ELL protein level which was not evident in the absence of E2F1 (Fig. 6C). These results suggest that E2F1 induces ELL upon DNA damage. Subsequently, we examined whether ELL could affect the E2F1-dependent apoptosis upon DNA damage. We treated control H1299 cells (scrambled shRNA) and stable ELL knockdown cells, as well as stable Rb knockdown cells, with etoposide (70 M) for 40 h and then stained the cells using an annexin V-PI apoptosis kit. The flow cytometry analysis showed that ELL knockdown resulted in a greater number of dead cells (22.8% ⫹ 10.3% [later apoptotic cell ratio plus early apoptotic cell ratio] and 25.2% ⫹ 13.5% compared to 7.61% ⫹ 7.82%) (Fig. 6D). As expected, Rb knockdown also resulted a greater number of dead cells (28.7% ⫹ 9.69% [later apoptotic cell ratio ⫹ early apoptotic cell ratio] compared to 7.61% ⫹ 7.82%). Interestingly, simultaneous knockdown of ELL and Rb caused the dead cell number to increase further (38.7% ⫹ 14.1% and 33.1% ⫹ 15.6%, respectively) (Fig. 6D), suggesting a synergistic effect of Rb and ELL on protecting cells from DNA damage-induced death, consistent with the observation that Rb and ELL have a synergistic inhibitory role in E2F4B luciferase reporter activity (Fig. 3H). However, knockdown of E2F1 diminished the effect of ELL knockdown, as well as that of Rb knockdown, on the enhancement of DNA damage-induced cell death (Fig. 6E). To reinforce this conclusion, we employed Hoechst staining to do further assays. We treated control H1299 cells (scrambled shRNA) and stable ELL knockdown cells with etoposide (70 M) for 36 h and then stained the cells using Hoechst 33342. Using fluorescence microscopy, we found that ELL knockdown resulted in a greater number of dead cells (Fig. 6F, top). However, this difference was not evident when E2F1 was knocked down (Fig. 6F, bottom). Quantitative analysis of cell survival validated this tendency (Fig. 6G). As a key downstream factor of E2F1 in response to DNA damage (28), we examined p73 expression after knockdown of ELL, Rb, or both. As shown in Fig. 6H, knockdown of either Rb or ELL enhanced p73 expression, and simultaneous knockdown of Rb and ELL enhanced p73 expression further, again suggesting a synergistic role of Rb and ELL in the suppression of E2F1 transactivity. Moreover, knockdown of ELL caused a stronger induction of p73 and more PARP1 cleavage after treatment with etoposide (Fig. 6I and J) (30). However, when E2F1 was knocked down, the induction of p73 by knockdown of ELL became weaker (Fig. 6I and J). In addition, the PARP1 cleavage was also diminished after E2F1 knockdown (Fig. 6J). Taken
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ing E2F1 transactivity, which may represent another mechanism for MLL-ELL-based leukemogenesis. Probably due, in part, to the embryonic lethality of ELL knockout mice, the function of ELL in vivo and the exact mechanism of
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its action remain unclear (11). Here, we show that ELL is induced in response to DNA damage and that it protects cells from E2F1dependent cell death upon DNA damage, revealing a novel function of ELL. As an important member of the E2F family, the func-
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FIG 6 ELL inhibits E2F1 apoptotic function in response to DNA damage. (A) ELL promoter reporter activity was induced by etoposide treatment (70 m), but knockdown of E2F1 abolished this induction in H1299 cells. (B and C) Etoposide treatment (70 m) of H1299 cells also induced higher ELL mRNA (B) and ELL protein (C) levels, increases that were abolished by the knockdown of E2F1. (D) Knockdown of ELL, as well as knockdown of Rb in H1299 cells, increased DNA damage-induced cell death (1st to 5th panels from left to right); Knockdown of both Rb and ELL enhanced DNA damage-induced cell death synergistically (compare 6th and 7th panels to 3rd, 4th, and 5th panels from left to right). (E) Knockdown of E2F1 counteracted the effect of ELL, as well as that of Rb, in DNA damage-induced cell death. H1299 cells were treated with 70 M etoposide for 40 h; DMSO was used as the control. (F) Knockdown of ELL in H1299 cells increased DNA damage-induced, E2F1-dependent cell death. (G) Quantitative analysis for cell survival ratio (%) after treatment with 70 M etoposide for 36 h. (H) Knockdown of ELL in H1299 cells, as well as that of Rb, enhanced DNA damage-induced p73 expression, as revealed by semiquantitative RT-PCR; knockdown of both Rb and ELL enhanced DNA damage-induced p73 expression synergistically. (I) Semiquantitative RT-PCR for p73 expression in H1299 cells with knockdown of E2F1, ELL, or both after treatment with 50 M etoposide for 16 h; DMSO was used as the control. Data are reported as the means ⫾ SEM of three independent experiments performed in triplicate. (J) Western blot analysis for p73 expression and PARP1 cleavage in H1299 cells with knockdown of E2F1, ELL, or both after treatment with 50 M etoposide for 16 h; DMSO was used as the control.
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FIG 7 Kinetic/temporal expression patterns of p73 and ELL in response to DNA damage. (A) Kinetic/temporal analysis of p73 expression in response to DNA
tion of E2F1 in response to the stress of DNA damage has drawn much attention. Upon DNA damage, E2F1 directly upregulates SirT1, which then, in a negative feedback loop, directly deacetylates E2F1 to inhibit its activity (26). Similarly, in this study, we found that E2F1 also upregulates ELL, leading to the enhancement of E2F1 deacetylation and inhibition of its activity. However, we also showed that SirT1 suppressed the transactivity of the E2F1 K/R mutant, while ELL did not, ruling out the possibility that
SirT1 serves as a mediator of ELL function. It has been demonstrated that E2F1 lysines K117, K120, and K125, which are mainly acetylated by PCAF, can be deacetylated by multiple deacetylases, including HDAC1 (20). In this study, we found that ELL can recruit HDAC1 to E2F1 and that the addition of the HDAC1 inhibitor TSA can restore the ability of ELL to suppress E2F1 activity, indicating that HDAC1 may serve as the key mediator for ELL function. However, we still cannot rule out the possibility that other deacetylases can also mediate the ability of ELL to affect E2F1, and this requires further investigation. ACKNOWLEDGMENTS We are grateful to Ali Shilatifard, Fred Dick, and Toshiyuki Sakai for the generous gifts of reagents. W.X. is supported by 973 grant 2010CB126306, CAS Major Scientific and Technological Project grant XDA08010208, and NSFC grants 31071212 and 91019008.
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FIG 8 Schematic model of the role of ELL in the suppression of E2F1 transcriptional activity. (A) Upon DNA damage, E2F1 is acetylated by PCAF or other acetylases, resulting in E2F1 activating its downstream targets, including ELL. (B) After ELL is induced upon DNA damage, it recruits HDAC1 to E2F1, resulting in deacetylation of E2F1 and inhibition of its transactivity; ELL and Rb have a synergistic role in the suppression of E2F1 activity.
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