Anti-apoptotic function of the E2F transcription factor 4 ...

YJMCC-07414; No. of pages: 9; 4C: Journal of Molecular and Cellular Cardiology xxx (2012) xxx–xxx

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Original article

Anti-apoptotic function of the E2F transcription factor 4 (E2F4)/p130, a member of retinoblastoma gene family in cardiac myocytes☆ Dharmendra Dingar ⁎, Filip Konecny, Jian Zou, Xuetao Sun, Rüdiger von Harsdorf University Health Network, Toronto, Canada

a r t i c l e

i n f o

Article history: Received 23 April 2012 Received in revised form 5 September 2012 Accepted 10 September 2012 Available online xxxx Keywords: E2F4 Apoptosis Cardiomyocytes Hypoxia

a b s t r a c t The E2F4–p130 transcriptional repressor complex is a cell-cycle inhibitor in mitotic cells. However, the role of E2F4/p130 in differentiated cells is largely unknown. We investigated the role of E2F4/p130 in the regulation of apoptosis in postmitotic cardiomyocytes. Here we demonstrate that E2F4 can inhibit hypoxia-induced cell death in isolated ventricular cardiomyocytes. As analyzed by chromatin immunoprecipitation, the E2F4–p130-repressor directly blocks transcription of essential apoptosis-related genes, E2F1, Apaf-1, and p73α through recruitment of histone deacetylase 1 (HDAC1). In contrast, diminution of the E2F4–p130–HDAC1-repressor and recruitment of E2F1 and histone acetylase activity to these E2F-regulated promoters is required for the execution of cell death. Expression of kinase-dead HDAC1.H141A or HDAC-binding deficient p130ΔHDAC1 abolishes the antiapoptotic effect of E2F4. Moreover, histological examination of E2F4−/− hearts revealed a markedly enhanced degree of cardiomyocyte apoptosis. Taken together, our genetic and biochemical data delineate an essential negative function of E2F4 in cardiac myocyte apoptosis. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction The E2f family of transcription factors plays an important role in the regulation of several intracellular processes such as proliferation, apoptosis, and differentiation. E2f family is composed of five family members (E2fs 1–5). Based on their physiological activity, E2fs 1–3 are transcription activators while E2fs 4–5 act primarily as gene repressors [1]. E2F4 is a major E2F-family member in cardiomyocytes and other differentiated and quiescent cells (collectively referred as G0 cells). E2F4 is regulated by the retinoblastoma (pRb)-related pocket protein p130 [2]. In contrast, the “activating” E2Fs (E2F1, E2F2, E2F3a) are regulated by pRb in proliferating cells [1]. The activating E2Fs induce S phase in serum starved primary fibroblasts whereas E2F4 does not [3]. Moreover, inhibitory E2F4/p130 complexes repress E2F-regulated promoters [4]. Since the activator E2Fs are not expressed in G0 they cannot account for the negative control of E2F-regulated promoters. Therefore, the E2F4/p130 transcriptional repressor complex clearly distinguishes G0 cells from G1 cells. This view is important, since the heart consists of G0 cardiac myocytes, which are differentiated, post-mitotic, and non-dividing [5–8]. In cells which has the proliferative capacity during transition from G0 to G1, E2F4 protein levels remain constant whereas p130 protein

☆ This work was supported by a grant from the Canadian Institutes of Health Research [MOP-89959 to R.v.H.]. ⁎ Corresponding author at: University Health Network, MaRS 3‐908, 101 College Street, Toronto, Ontario, Canada M5G 1L7. Tel.: +1 416 581 7530; fax: +1 416 581 7451. E-mail address: [email protected] (D. Dingar).

becomes phosphorylated and subsequently degraded in a proteosomal dependent manner in early G1 [2]. Furthermore, phosphorylated p130 releases E2F4 and this inactivation of the E2F4-repressor is associated with the loss of E2F4–DNA binding activity and relief from E2F4mediated gene repression [2]. In this simplified model, promoter repression in G0 depends on E2F4/p130 but by late G1 these proteins are replaced by activating E2Fs. Notably, in G0 cells pRb does not bind to E2F-sensitive promoters (e.g. p107, E2F1, cdc25A, cdc6, B-myb, cyclin A, cdc2). Thus, pRb is not involved in the repression of E2F-dependent promoters in cardiomyocytes. E2F4 is held in the nucleus of G0 cells through physical association with p130 and is translocated to the cytoplasm by the nuclear export receptor CRM1 due to its nuclear export signal (NES). E2F4 lacks nuclear localization signal (NLS). Thus, the nuclear import of E2F4 is mediated by association with p130 and is not an intrinsic function of E2F4 [9]. In contrast, the activating E2Fs are constitutively localized in the nucleus independently of their association with pRb [10]. Taken together, the divergent physiological effects of E2F4 and the activating E2Fs are simply explained by their different intracellular localization and association with pocket proteins [11]. Moreover, p130 also recruits histone deacetylase 1 (HDAC1) to promoters and HDAC1 can repress gene expression by altering chromatin structure, since decreased acetylation of histone H3 and H4 residues is associated with transcriptionally inactive chromatin [12,13]. p130, but not pRb is strictly required for HDAC1-binding to E2F-responsive promoters [14]. HDAC1activity is responsible for the underacetylated state of histones at E2F-regulated promoters and thus transcriptional repression during G0 [13]. An attractive model suggests that activating E2Fs displace E2F4/p130/HDAC1 complex from promoters. In turn, these activator

0022-2828/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.yjmcc.2012.09.004

Please cite this article as: Dingar D., et al., Anti-apoptotic function of the E2F transcription factor 4 (E2F4)/p130, a member of retinoblastoma gene family in cardiac myocytes, J Mol Cell Cardiol (2012), http://dx.doi.org/10.1016/j.yjmcc.2012.09.004

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D. Dingar et al. / Journal of Molecular and Cellular Cardiology xxx (2012) xxx–xxx

E2Fs might recruit histone acetyltransferase (HAT) activity to promoters, enabling gene expression through relaxation of nucleosomal chromatin structure. E2F4 was shown to be abundantly expressed in embryonic mouse cardiac ventricle and its expression decline during development and shown to be involved in mitosis [15]. Previous report shows control of pro-apoptotic gene expression in cardiomyocytes by E2F4 [16]. In neurons and other cells E2F4 plays an important role in the regulation of apoptosis [17,18]. In the heart E2F4's role is not well characterized. Therefore, to further our understanding about E2F4, we have investigated its role in the regulation of apoptosis in isolated cardiomyocytes and E2F4−/− heart. Here we show that E2F4/p130 suppresses pro-apoptotic gene expression in cardiomyocyte and removal of this inhibition is required for cardiomyocyte apoptosis. 2. Material and methods 2.1. Cardiomyocyte culture and E2F4 knockouts Fisher (F344) inbred rats weighing 180–200 g were purchased from Charles River Laboratories (St. Laurent, Que.) and E2F4 knockout mice were obtained from J. Nevins (Duke University Medical Center, Durham, NC, USA) and bred as described [19]. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85‐23, revised 1996). The local Institutional Animal Care Committee at UHN, Toronto, Ontario, ensured compliance under protocols (2011 and 1415) with the abovementioned guidelines. Rat neonatal pups and E2F4 knockout mice were anesthetized by inhalation of Isoflurane (5%) in oxygen by draw over circulation system and then their spine was cervically dislocated. Adequacy of anesthesia was ensured by inspection of respiratory rate and pattern, color of mucous membranes, corneal reflex and reactivity on toe pinch. For isolation of ventricular cardiomyocytes from 3-day postnatal Wistar rats, the hearts were dissected, minced, enzymatically digested (collagenase II, 0.5 mg/ ml, Invitrogen; pancreatin 1 mg/ml, Sigma) to selectively enrich for cardiomyocytes. The resultant cell suspension (4 × 10 6 cells) was plated onto 10 cm-collagen I (Gibco) coated dishes in culture medium DMEM/F12 containing 3 mM Na-pyruvate, 2 mM glutamine, standard antibiotics (GIBCO), 0.2% (v/v) BSA, 0.1 mM ascorbic acid, and 0.5% (v/v) insulin–transferrin–selenium (Sigma). After preplating (to eliminate adherent non-cardiomyocyte cells), myocytes were held for 36 h in the presence of 25 mM araC (inhibits proliferating noncardiomyocyte cells) and 5% horse serum (Sigma). Cardiomyocytes were subjected to hypoxia in serum-free culture medium without BSA (hypoxia chamber; 5% CO2, 95% N2 in a humidified atmosphere; Labotect, Model 3015). Recombinant adenoviruses were generated with the pAdeasy vector system as per the manufacturer's instructions (Stratagene). Cardiomyocytes were transduced (100 plaqueforming units [pfu]/cell or 50 pfu/cell for double-transfections) after preplating and 36 h later exposed to hypoxic culture conditions for 24 h or other time points as mentioned. 2.2. Cellular extracts and cell fractionation NP40-buffer was used for whole cellular extracts: 50 mM Tris–HCl pH 7.5, 250 mM NaCl, 0.5% NP-40, 5 mM EDTA pH 8.0, 1 mM DTT, protease inhibitor cocktail (Roche), and phosphatase inhibitors (1 mM Na3VO4, 20 mM NaF, 10 mM β-glycerophosphate, and 1 mM NaP2O7). For subcellular cell fractions, 2.5 × 10 6 cardiomyocytes in 10 cm-dish were trypsinized and lysed in 500 μl harvest buffer (10 mM HEPES pH 7.9, 50 mM NaCl, 500 mM sucrose, 0.1 mM EDTA, 1 mM DTT, and protease inhibitors). After centrifugation, the supernatant was designated as cytoplasmic extract. The remaining pellet was washed in buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, protease inhibitors) and nuclei were lysed using 500 μl

buffer C (10 mM HEPES pH 7.9, 500 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% NP-40, 1 mM DTT, protease inhibitors). After centrifugation (14,000 ×g; 1 h; 4 °C) the supernatant was designated as nuclear fraction. 2.3. Reporter gene assays and chromatin immunoprecipitations E2F1-Luc (wt) and E2F1-Luc (mt) were from J. Nevins (Duke University Medical Center, Durham, NC, USA). Luciferase activity was determined as described [20]. At 12 h after induction of hypoxia, chromatin immunoprecipitations (4×107 cells/reaction) were done as described with minor modifications [21]. To amplify E2F-responsive promoter regions, the following primer sets were used: Rat E2F1 (GenBank XM_230765), 5′-GCCTTCGCCAGACCCCGCCACCCA-3′; 5′-CGCCGCGGCC TGCCGTCATGG-3′; and β-MHC, 5′-CAAGGAGCTACCTACCAGACAG-3′, 5′-GGCTCCAGGTCTCAGGGC-3′. PCR products were separated on 8% polyacrylamide gels. Radioactive label was analyzed in a PhosphorImager using TINA software. 2.4. Blotting applications, immunofluorescence, RT-PCR, and apoptosis assays Immunoprecipitation, western- and northern blotting, immunofluorescence microscopy, RT-PCR, and apoptosis-detection were done as described in our previous publications [20,22]. p130 (sc-317), actin (sc-7210), lamin (sc-20681), p16INK4 (sc-156), HDAC1 (sc-7872), HDAC5 (sc-11419), and HDAC6 (H-300 also recognizing HDAC9) antibodies were from Santa Cruz. Acetylated histone H3 (06–599) and acetylated histone H4 (06–598) antibodies were from Upstate. E2F3 (Ab-4), E2F4 (Ab-4), cdk4 (Ab-5), and cdk6 (Ab-3) antibodies were from Thermo Scientific. E2F1 (KH129) antibody was from Neomarkers, cdk2 (610145) antibody was from Pharmingen. 2.5. Functional two-dimensional M-mode echocardiography Experimental animals were randomly allotted into study groups, anesthetized and assessed by echocardiography as follows: Left Ventricle End Diastolic Distance (LVEDD), Left Ventricle End Systolic Diastolic Distance (LVESD), and Fractional Shortening (FS). All animals were pre-anesthetized in the induction chamber with mixture of oxygen and 3% of Isoflurane, and then transferred on the heating pad, secured to the pad in a left recumbent position and rectal temperature was maintained at 37.0 +/− 0.5 °C. Ultrasound system Vivid 7 system (GE Mississauga, ON) equipped with an il3L linear probe operated at 14 MHz was used, and animals imaged with 2% Isoflurane anesthesia at a room temperature. The heart was imaged in the 2-D mode in the parasternal long- and short-axis views with a depth setting of 1.0 cm and at a frame rate of 275 frames/s. M-mode echocardiography was performed by using a parasternal short-axis view at the level of the papillary muscles; images were obtained at a sweep speed of 200 mm/s. Measurements provided were done from leading edge to leading edge according to the American Society of Echocardiography guidelines (1). Wall thickness and LV dimensions were obtained from a short-axis view at the level of the papillary muscles at a frame rate of 260 Hz. FS was calculated according to the formulas FS = [(LVEDD − LVEDS) / LVEDD] × 100. 2.6. Statistical analyses Factorial design analysis of variance (ANOVA) or τ-tests were used to analyze data as appropriate. Significant ANOVA values were followed by simple main effect analyses or post hoc comparisons of individual mean using the Tukey's method were appropriate. The level of significance was less than 0.05.



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Fig. 1. E2F4 translocates from the nucleus to the cytoplasm during hypoxia-triggered cell death in neonatal ventricular cardiomyocytes. A, Subcellular localization of E2F4 and E2F1 (green) in normoxic and hypoxic (8 h) cardiomyocytes (sarcomeric myosin heavy chain, red; nuclear DNA, blue). CRM-1-mediated nuclear exclusion of E2F4 is inhibited by leptomycin B (LMB). B, Western blot analysis of nuclear and cytoplasmic protein extracts. Aliquot of 3 × 105 cells corresponding to 50 μg total protein was resolved per lane. To correct for equal loading, membranes were probed with antibodies to actin and laminin. C, Nuclear import of E2F4 inhibits cardiomyocyte apoptosis. Nuclear import of E2F4 requires binding to the retinoblastoma (pRb)-related pocket protein p130. Derivatives of the indicated E2F4 and p130 proteins were overexpressed in cardiomyocytes 72 h before 24 h of hypoxia. p130Δ21 lacks the E2F4-interaction site and E2FΔN is a DNA-binding deficient mutant. D, Immunocytochemical detection of apoptotic nuclei after 24 h of hypoxia was done by TUNEL assay. For quantitative analysis 200 nuclei were counted in random fields. Colorimetric assay for determination of caspase 3 activity in apoptotic cardiomyocytes (mean ± SD, n= 3). p value was less than 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


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3. Results 3.1. E2F4 exits the nucleus during cardiomyocyte apoptosis We have previously reported that hypoxic cell death in cardiomyocytes requires E2F1 [20]. Therefore, to study the role of E2F4 we analyzed the subcellular compartmentalization of endogenous E2F4 in cardiomyocytes at an early time point (0–8 h) after induction of hypoxia. At this stage, cells did not exhibit any morphologic signs of death. Under normoxia, E2F4 protein localization was nuclear, whereas E2F1 was detected in the cytoplasm (Figs. 1A, B). In contrast, E2F4 was sequestered in the cytoplasm and E2F1 to the nucleus in hypoxic cells. The nuclear export inhibitor leptomycin B (LMB) prevented nuclear export of E2F4 in hypoxic cells, suggesting that E2F4 is actively exported from the nucleus. It has been shown that different members of the DP family form heterodimers with E2F family members and this heterodimerization increases E2Fs DNA binding, transactivation, and pocket protein binding activities [23]. Similar to E2F4, DP2 and p130, which interact with E2F4, localized in nucleus under normoxia and DP2 was exported to the cytoplasm and p130 was degraded in hypoxic cells (Fig. 1B). Thus, our results show that E2F4 is specifically sequestered in the cytoplasm of cardiomyocytes during hypoxia. 3.2. p130 is required for the nuclear localization of E2F4 Our findings may suggest that when E2F4 was retained in the nucleus, it can inhibit cell death. Therefore, we thought that forced expression of nuclear E2F4 would inhibit cardiomyocyte apoptosis. For this purpose, we used p130 that binds to and transfers E2F4 in the nucleus [24]. When E2F4 was transduced alone, it was localized in cytoplasm and cardiomyocyte apoptosis was not suppressed (Figs. 1C, D). In contrast, when p130 was co-expressed, E2F4 was nuclear and cell death was inhibited (Figs. 1C, D). Moreover, cell death inhibition was not observed for E2F4-binding deficient mutant, p130Δ21 and E2F4ΔN that are transactivating deficient (Figs. 1C, D). Derivatives of the indicated E2F4 and p130 proteins were overexpressed in cardiomyocytes 72 h before 24 h of hypoxia. Cell death assay and immunofluorescence detection were done at similar time point. This shows that cell death inhibition occurred when overexpressed E2F4 was present in the nuclei. Therefore, our results show that p130 can direct E2F4 to the nucleus, which is associated with the inhibition of cardiomyocyte apoptosis. 3.3. E2F4/p130 is recruited to the E2F1 promoter in normoxic but not in hypoxic cardiomyocytes Our previous data have shown that E2F1 is required for hypoxic cardiomyocyte death [20]. To test whether E2F4 can repress the E2F1 promoter, we employed chromatin immunoprecipitations (ChIP) to analyze E2F1 gene promoter occupancy by E2F4 and p130. Endogenous E2F4 and p130 bound to the E2F1 promoter in normoxic cells (Fig. 2A). However, E2F4- and p130-binding diminished dramatically in hypoxic cardiomyocytes and that correlated with the recruitment of E2F1 to the E2F1 promoter. We did not detect significant binding of pRb to the E2F1 promoter. The specificity of our ChIP assay was confirmed with primers annealing to the β-MHC promoter because transcription of this gene is not thought to be under control of either E2Fs. Thus, significant levels of β-MHC were never amplified. In conclusion, our results indicate that E2F4/p130 transcriptional repressor complex is specifically recruited to the E2F1 promoter in normoxic but not hypoxic cardiomyocytes. 3.4. The E2F4/p130 repressor complex inhibits E2F1 promoter activation in cardiomyocytes When the E2F4/p130-transcriptional repressor is recruited to the E2F1 promoter then the gene transcription from this promoter should

be inhibited. To test this, we introduced an E2F1 promoter luciferasereporter construct in cardiomyocytes. The E2F-Luc (wt) contains a 728-bp fragment of the human E2F1 promoter fused to a luciferase reporter gene. In E2F1-Luc (mt) the E2F1 sites in the same E2F1 promoter fragment have been mutated [20]. Hypoxia led to an increase in luciferase activity of E2F1-Luc (wt) (Fig. 2B). While ectopic p130 and E2F4 led to a marked reduction of luciferase activity in hypoxic myocytes. This inhibitory impact of E2F4/p130 on E2F1 promoter was not observed when DNA-binding deficient E2F4ΔN or E2F4 nonbinding p130Δ21 were expressed. Collectively, we infer from our results that the E2F4/p130 repressor functionally inhibits the E2F1 promoter. Loss of E2F4 dependent transcriptional repression was accompanied with substantial increase in mRNA levels of each pro-apoptotic factor in dying cardiomyocytes [25,26]. To verify that promoter occupancy by E2F4/p130 actually reflects alterations in transcript levels, we checked expression of pro-apoptotic factor Apaf-1, p73, and caspase 3 by northern blotting. p73 is p53 homologue [25] and Apaf-1 is apoptosis protease activating factor-1. Apaf-1 expression is under the control of p53 and E2F [26]. When E2F4 was directed into the nucleus by p130 during hypoxia, a strong inhibition of Apaf-1, p73, and caspase 3 transcription was observed (Fig. 2C). Interestingly this effect was reversed in cardiomyocytes coexpressing E2F4 together with p130ΔHDAC1, an HDAC1 binding deficient mutant or HDAC1.H141A, an HDAC1 deacetylase inactive mutant. This suggests that HDAC1 can act as a physiological effector contributing to E2F4/p130-mediated gene silencing in cardiomyocytes. 3.5. The E2F4/p130 transcriptional repressor occupies the promoters of key apoptotic genes in cardiomyocytes Having shown that E2F4/p130 negatively regulates the expression of key pro-apoptotic genes (Fig. 2C), we then asked whether or not E2F4/p130 directly regulates their promoter activity. Thus, we used ChIP analysis to determine whether E2F4/p130 binds to these target promoters. In normoxic cardiomyocytes, the E2F-sites of Apaf-1 and p73 were clearly occupied by E2F4 and p130 (Fig. 2D). However, we detected very weak signals for E2F- and pocket protein binding to the caspase 3 promoter, indicating that the caspase 3 promoter is not transrepressed in an E2F-dependent fashion under basal conditions. On the contrary, during hypoxia, E2F4- and p130-binding diminished on promoters of p73 and Apaf-1. Instead, E2F1 appeared on the promoters of caspase 3, p73, and Apaf-1. These results strongly suggest that the E2F4/p130 repressor directly inhibits transcription of pro-apoptotic genes (Apaf-1 and p73). 3.6. E2F4 recruits HDAC1 to target promoters in normoxic cardiomyocytes After seeing inhibition of E2F1 and pro-apoptotic gene expression by E2F4/p130, we then checked histone acetylation status of E2F1 and pro-apoptotic gene promoters. ChIP of acetylated histones (AcH3 and AcH4) showed increased acetylation of E2F1, Apaf-1, p73, and caspase 3 promoters during hypoxia (Fig. 3A) which is consistent with an increase in mRNA expression (Fig. 2C). Based on this we thought that E2F4/p130 may recruit HDAC for this gene repression in normoxic cardiomyocytes. Therefore, we performed co-immunoprecipitation with E2F4 or p130 specific antibody from normoxic cardiomyocyte lysate and looked for HDAC1 immunoreactivity. E2F4 and p130 antibody immunoprecipitated HDAC1 immunoreactivity (Fig. 3B). Consistently, ChIP with HDAC1 antibody revealed that HDAC1 is recruited to E2F1, Apaf-1, and p73 only during normoxia (Fig. 3C). We did not detect HDAC1 on the caspase 3 promoter, supporting our previous observation (Fig. 2D) that this promoter is not inhibited by E2F4. Moreover, we failed to observe any significant binding of HDAC5 and HDAC9 to E2F1 target promoters tested suggesting a certain degree of specificity at the level of corepressor recruitment. We constantly observed that HDAC1 dramatically disappeared from promoters of E2F1, Apaf-1, and p73 in dying cardiomyocytes. In



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Fig. 2. The E2F4-transcriptional repressor recruits p130 and HDAC1 to promoters of key apoptotic genes that are required for the suppression of apoptosis. A, The E2F4 repressor transcriptionally silences the E2F1 gene promoter. Chromatin immunoprecipitations (ChIP) were performed with antibodies specific for individual E2F- or pRb-family members as indicated (top). Immunoprecipitations were amplified with gene specific primer pairs corresponding to E2F1 and β-MHC (left). B, Relief from E2F4-dependent transrepression activates the E2F1-gene promoter. The luciferase reporter E2F1-Luc (wt) contains a 728-bp fragment of the human E2F1 promoter fused to a luciferase reporter cDNA. In E2F1-Luc (mt), the E2F sites in the same E2F1 promoter fragment have been mutated. Luciferase activity was determined from whole-cell extracts containing 80 μg total protein. Fold activation of luciferase activity was corrected for β-Gal expression (values mean ± SD, n = 4). C, Nuclear E2F4 blocks E2F1-dependent transcription of pro‐apoptotic genes. At 24 h after induction of hypoxia, RNA (30 μg per lane) was collected from cardiomyocytes ectopically expressing the indicated constructs and subjected to northern blotting. One result of at least two experiments is shown. D, The E2F4/p130-repressor complex is recruited to promoters of essential pro-apoptotic genes in cardiomyocytes. For ChIP analysis, immunoprecipitations with antibodies as indicated (top) were amplified with gene specific primer pairs (left). Representative blot of two experiments is shown. p value was less than 0.05.

summary, the transcriptional activation of pro-apoptotic genes (Apaf-1 and p73) and E2F1 coincides with loss of E2F4/p130 and HDAC1 from their promoters. To determine, whether the ectopic E2F4/p130 exhibits HDAC-activity we measured the p130-associated deacetylase activity. Fig. 3D shows that a significant deacetylase activity was detected in anti-p130 immunoprecipitates from hypoxic cardiomyocytes overexpressing E2F4/p130. We failed to detect p130-associated HDAC-activity when HDAC1-binding deficient p130ΔHDAC1 was substituted for p130 or in the presence of trichostatin (TSA), an inhibitor of HDAC. Finally to further confirm p130 associated HDAC activity on pro-apoptotic genes, we performed ChIP with E2F4, p130, E2F3, or pRB and looked for Apaf-1 promoter. Consistently, E2F4/p130 overexpression reestablished its association to the Apaf-1 promoter in hypoxic cardiomyocytes (Fig. 3E). This association was not observed in E2F4ΔN. Consistently similar association was observed with HDAC1, but not with E2F4ΔN or p130ΔHDAC1 mutant (Fig. 3F). Therefore, our results suggest that sustained gene silencing by E2F4/p130 is mediated through HDAC1. E2F4/p130 recruits HDAC1 activity to the E2F1-regulated promoters during normoxia and the ectopic reconstitution of this repressor complex can prevent hypoxic cell death. 3.7. Mice lacking E2F4 show significantly more apoptosis in the myocardium Our data indicate that nuclear E2F4 blocks cardiomyocyte apoptosis by recruitment of the E2F4/p130/HDAC1 transcriptional repressor complex. In this inhibitor complex, E2F4 determines the specificity to target promoters. Therefore, we questioned, whether the loss of E2F4 would also affect the viability of cardiomyocytes in vivo. To answer this question we examined apoptosis in E2F4 −/− mouse heart. E2F4 −/− mice

show abnormalities in hematopoietic lineage development, and increased frequency of apoptotic cells. They also show growth deficiency and high rate of mortality after birth [19]. Consistent with our data on isolated cardiomyocytes, the hearts from E2F4−/− mice [19] displayed significantly more TUNEL-positive nuclei than those from wt-littermates (Figs. 4A, B). Additionally, we have detected cleaved caspase 3 in E2F4−/− heart lysate, which is indicative of mitochondrial apoptosis (Fig. 4C). We further analyzed the impact of the loss of E2F4 on E2F1, Apaf-1, and p73 gene expressions by RT-PCR and western blot. The E2F1, Apaf-1 and p73 mRNAs and protein levels were significantly increased in E2F4 −/− hearts compared to wt-littermates (Figs. 4D, E). This result is consistent with our previous conclusion that loss of E2F4 contributes to transcriptional activation of E2F-responsive genes in isolated cardiomyocytes. After detecting more apoptotic nuclei in E2F4 −/− heart we then measured heart function by echocardiography. E2F4 −/− heart fractional shortening (FS) was less as compared to E2F4 +/+ (Figs. 5A, B), suggesting that more apoptosis in E2F4 −/− heart deteriorates heart function. Furthermore, from hearts of E2F4 −/− and E2F4 +/+ mice collected, weighted and compared to their body weights, significant increase of HW/BW was noted in E2F4 −/− group as compared to E2F4 +/+ littermates (Fig. 5C). E2F4 expression in wild type and absence in knockout heart was confirmed by western blot (Fig. 5D). Given that E2F4 −/− mice show growth abnormalities, E2F4 −/− mice are smaller in size and weight compared to wild type littermates [19], this complicate HW/BW ratio interpretation. Taken together, our findings lead us to believe that E2F4 is required for the control of E2F-responsive genes, cardiomyocyte survival and physiological heart function.


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Fig. 3. The E2F4/p130-transcriptional repressor recruits HDAC1 to promoters of key apoptotic genes that are required for the inhibition of cardiomyocyte apoptosis. A, Induction of histone acetylation of E2F responsive genes during cardiomyocyte apoptosis. ChIP was performed with antibodies specific for acetylated histones (AcH3, AcH4) and PCR was performed with primer pairs for each of the indicated genes (top). B, The E2F4/p130-repressor is specifically associated with HDAC1. Immunoprecipitations of normoxic cardiomyocyte extracts (2.0 mg total protein) were performed with antibodies to E2F4 or p130 and western blot detection was done with specific antibodies (top panel). C, HDAC1 is recruited to promoters of apoptotic factors in normoxic cardiomyocytes. Promoter occupancy by HDAC was analyzed by ChIP using the indicated antibodies (right) and gene specific PCR primers (top). D, Ectopic E2F4/p130 recruit HDAC-activity to suppress cardiomyocyte apoptosis. Cells were transduced with the indicated adenoviruses (bottom). Immunoprecipitations were carried out with p130-specific antibody. p130-Associated HDAC-activity was measured against acetylated histone H4 peptide in the presence or absence of TSA (trichostatin), an inhibitor of HDAC. Results are representative of three independent experiments. E, Overexpressed E2F4 and p130 proteins are recruited to the Apaf-1 promoter in hypoxic cardiomyocytes. ChIP was done with antibodies to E2Fs and pRb-family members and PCR was performed with primers (right). F, The ability of E2F4/p130 to inhibit cell death depends on HDAC1 corepressor recruitment to E2F-responsive promoters in cardiomyocytes. For ChIP analysis, chromatin was immunoprecipitated with the indicated antibodies (left) and Apaf-1 specific PCR primers.

4. Discussion Here we presented evidence that E2F4 under basal conditions was localized in the cardiomyocyte's nucleus where it binds in a sequencespecific manner to promoters of key apoptotic factors including E2F1, p73, and Apaf-1. E2F4 serves as a molecular scaffold for p130 and HDAC1 thereby functioning as a transcriptional repressor. In apoptotic cardiomyocytes, this E2F4 repressor complex is inactivated through the nuclear exclusion of E2F4. Abrogation of the E2F4-dependent repression induces apoptosis in isolated cardiomyocytes and in the adult mice myocardium. In conclusion, our study revealed the role of E2F4 in the maintenance of cardiac integrity.

4.1. Unique role for E2F4 in transcriptional control of apoptotic genes in cardiomyocytes Our study delivers substantial experimental evidence that E2F4 and p130 are transcriptional repressors on the promoters of crucial apoptosis-inducing genes (E2F1, p73, Apaf-1) [20,25,26]. ChIP analysis of promoter occupancy by E2F4 and p130 proteins in cardiomyocytes revealed that E2F4/p130 was specifically recruited to these promoters (Fig. 2). In contrast, a striking diminution of E2F4 and strong E2F1binding to promoters were observed in apoptotic cells which coincided with transcriptional activation [27]. Our results indicate that E2F4 in cardiomyocytes is required for cardiac viability. In cardiomyocytes, crucial factors of the apoptotic machinery are controlled mainly at the transcriptional level and E2F4 makes a major contribution to the transcriptional silencing of these genes. p73-Dependent apoptosis is negatively regulated by E2F4/p130 in human osteosarcoma cells [27] supporting our view that genetic disruption of E2F4 can evoke apoptosis in the mice myocardium (Fig. 4). Similarly, Rempel et al. reported increased frequency of apoptosis in hematopoietic cells in the E2F4-deficient mice [19]. Interestingly, the E2F4−/− mice display a high incidence of an early

postnatal mortality and are substantially smaller than their wild-type littermates. Taken together, our findings suggest that E2F4 is required for the control of E2F-responsive genes and thus cardiomyocyte survival.

4.2. Recruitment of the corepressor HDAC1 by E2F4/p130 to E2F-responsive promoters We present evidence that HDAC1 is the key player in the E2F4/p130 complex, which directly contributes to silencing of pro-apoptotic genes. To the best of our knowledge, our study is the first to demonstrate that the E2F4–p130–HDAC1 complex acts as a survival factor in postmitotic cells. HDAC1, and not other HDAC family members, was recruited by E2F4/p130 to target promoters in normoxic cardiomyocytes. Through binding of the HDAC1 corepressor, E2F4/p130 is able to block apoptosis. This conclusion is based on our finding that both HDAC1-binding deficient p130ΔHDAC and deacetylase inactive HDAC1.H141A mutant prevented E2F4 from inhibiting cell death. Furthermore, the HDAC1corepressor was specifically recruited by E2F4/p130 complexes to E2F-sensitive promoters as shown by the analysis of endogenous and overexpressed proteins in ChIP experiments (Fig. 3). Correspondingly, loss of HDAC7 leads to derepression of Nur77 and apoptosis in thymocytes which is in accordance with our findings [28]. Moreover, HDAC inhibitors evoked p73-dependent apoptosis in transformed human cell lines [29]. In addition to establishing the identity of the E2F4-complex bound in viable cardiomyocytes, our use of ChIP analysis allowed us to show that p130 and E2F4 but not pRb or other E2Fs were required to specifically recruit HDAC1. Thus, our results define HDAC1 as a major contributor of an essential antiapoptotic activity of the E2F4-repressor complexes. Our study shows an absolute dependence on HDAC1 recruitment by p130 to the E2F1, p73, and Apaf-1 promoters. We do not rule out the possibility that other HDACs are able to associate with certain promoters in an E2F- and p130-independent manner. Cardiac histone deacetylases are important stress-responsive regulators of gene



Fig. 4. E2F4 is essential for cardiomyocyte survival. A, Apoptosis was analyzed by TUNEL assay in the left ventricular heart myocardium of a 6-week-old E2F4−/− mice. B, Cleaved caspase 3 positive nuclei were counted in E2F4−/− left ventricular myocardium. At least 200 nuclei were counted in random fields. Data are mean±SEM. C, Western blot detection of proteolytic cleavage of caspase 3 in the E2F4−/− myocardium. D, Analysis of expression of E2F-responsive genes in wild-type versus E2F4−/− myocardium. Total RNA was subjected to RT-PCR analysis by using cDNA specific primers. GAPDH expression was used as an internal control. Data are expressed are mean±SEM. E. Protein expression of E2F-responsive genes in wild-type versus E2F4−/− myocardium. Representative images of two independent experiments are shown. p value was less than 0.05.

expression in the heart [30]. Previous work has shown that phosphorylation site mutants of class II HDACs HDAC5 and HDAC9 inhibit cardiomyocyte hypertrophy and that the HDAC9 deficient mice are sensitized to hypertrophic signals [31]. HDAC9 binds to the myocyte enhancer factor-2 (MEF2) and blocks transcription from MEF2responsive promoters of genes necessary for cardiac growth. Targeted disruption of HDAC1 in mice results in early embryonic lethality before E10.5 due to severe proliferation and differentiation defects [32]. HDAC1 loss leads to an overall reduced HDAC activity and hyperacetylation of histones H3 and H4. These findings corroborate our view, that the E2F4/p130-dependent functions of HDAC1 are essential for a cellular integrity. Taken together, HDAC1 and HDAC9 suppressors play negative roles in cardiomyocyte survival and growth, respectively. These findings raise an intriguing possibility that interacting with different sets of transcription factors class I

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HDACs (HDAC1) and class II HDACs (HDAC9) might repress a different subset of genes with opposite functions in cardiomyocytes. We noticed a striking diminution of the E2F4-transcriptional repressor complex to all promoters tested in hypoxic cardiomyocytes. Promoter binding by E2F1 then resulted in an increased histone acetylation and transcriptional activation, possibly via recruitment of HATs. Cardiogenic transcription factors as MEF2 and GATA4 require association with HATs to activate gene expression [33,34]. Thus, our study might provide a new connection among HATs and E2F4/p130-regulated HDACs. Since E2F1 recruits HATs to facilitate transcriptional activation, the regulation of apoptotic gene expression and cardiomyocytes survival seems to depend on what the chromatin-modifying factor is directed to E2F-responsive promoters. Although we have not yet identified the identity of specific HATs recruited to these E2F-regulated promoters, our experiments for the first time link the potential activities of HATs and HDACs to the transcriptional regulation of important E2F-responsive apoptotic genes in cardiomyocytes. All these observations provide compelling evidence that the E2F4–p130–HDAC1 transcriptional repressor complex is required for the suppression of cell death in cardiomyocytes [35]. Similarly in post-mitotic neurons, E2F4 inactivation leads to caspase activation and apoptosis through expression of pro-apoptotic protein Bim, a BH3-only protein belonging to Bcl-2 family protein [17]. On the contrary, E2F4 in the nucleus promotes cell death in human intestinal epithelial crypt cells. But in the same study E2F4 does not induce cell death in colon cancer cells [36]. This suggests E2F4 function could be cell type specific. In the previous study overexpression of E2F4 mitigated expression of pro-apoptotic genes in cardiomyocytes [16] and in Chinese hamster cell lines E2F4 induced growth arrest and caspase dependent apoptosis [18] which is consistent with our observation that E2F4 in the nucleus block apoptosis. In summary, our study provides crucial information for the importance of E2F4 in the maintenance of myocardial viability. Overexpression of E2F4 in neonatal cardiomyocytes can induce S-phase entry, but increased expression of E2F1 and E2F3 induced apoptosis along with cell cycle progression [16]. Based on our data in isolated ventricular cardiomyocytes we can only conclude that E2F4/p130 directly blocks transcription of essential apoptosis-related genes, E2F1, Apaf-1, and p73α through recruitment of histone deacetylase 1 (HDAC1) and apoptosis after hypoxia. However in vivo, E2F4−/− heart shows higher abundance of apoptotic cardiomyocytes but still heart functions although with some effect on LV functions, so this suggest that E2F4 is required to suppress pro-apoptotic genes and plays a major role during apoptosis but may not be a common survival pathway. In broad view, p130 expression hits the highest level in the neonatal period and is subsequently down-regulated and expressed at lower levels, but still detectable, in adult myocardium. Phosphorylation of p130 by Cdk2 and 4 results in the release of E2F complexes, enabling them to activate transcription and trigger the expression of genes required for DNA synthesis promoting cell cycle. p130 has been implicated as a promoter of quiescence in cardiomyocytes and may contribute to the post-mitotic state of cardiomyocytes. Thus, stimuli that are leading to dissolution of E2f4/p130 complexes in heart trigger transcription of essential apoptosis-related genes, E2F1, Apaf-1, and p73α. Similar to E2F4/p130 role in cardiomyocytes, in terminally differentiated neurons E2F4/p130 also regulates apoptosis and it has been suggested that activation of cdk4 leads to hyperphosphorylation of p130 which leads to dissociation of E2F4/p130/ HDAC1 complex [17]. However, in cardiomyocytes we have not determined whether pro-survival kinases played a role in this process and whether other kinases might have been involved in p130/E2F4 subcellular localization. Given the important role of E2F4 in cardiomyocyte apoptosis during hypoxia future research will be directed to study enzymes involved in nuclear export of E2F4/p130, which then can be targeted to therapeutically inhibit their activity in cardiovascular diseases to protect from cardiomyocyte loss by apoptosis and improve heart function.


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Fig. 5. Reduction of cardiac function in e2f4−/− at baseline. A, Representative echo tracing images at baseline as assessed by M-mode echocardiography; 6–10 weeks of age (n > 4). B, Heart function FS (%) E2F4+/+, E2F4−/− measured at baseline. C, Heart weight/body weight ratio of E2F4+/+, E2F4−/− (***p b 0.001, Student's t-test). D, Western blot was performed on heart lysates obtained from six weeks old E2F4+/+, E2F4−/− male mice. Β-tubulin was used as a loading control.

5. Conclusions In this study, we report that E2F4 is part of a transcriptional repressor complex comprising p130 and HDAC1 in cardiac myocytes. Relief from E2F4-dependent gene silencing induces the expression of pro-apoptotic genes. E2F4-deficient mice exhibit a markedly enhanced degree of cardiomyocyte apoptosis. Taken together, our genetic and biochemical data demonstrate that E2F4 exerts a negative regulatory role in cardiac myocyte apoptosis. Disclosure The authors declare no conflicts of interest. Acknowledgments We thank the Canadian Institutes of Health Research for the funding and RVH lab members for their help. References [1] Trimarchi JM, Lees JA. Sibling rivalry in the E2F family. Nat Rev Mol Cell Biol 2002;3:11-20.

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