Regulation of E2F4 Mitogenic Activity During Terminal Differentiation by Its Heterodimerization Partners for Nuclear Translocation1

[CANCER RESEARCH 58. 1325-1331, April I. 1998] Advances in Brief

Regulation of E2F4 Mitogenic Activity during Terminal Differentiation by Its Heterodimerization Partners for Nuclear Translocation1

Pier Lorenzo Puri,2 Letizia Cimino, Marcella Fulco, Christian Zimmerman, Nicholas B. La Thangue, Antonio Giordano, Adolf Graessmann, and Massimo Levrero3

Laboratory of Gene Expression. Fondazione Andrea Cesalpino. Policlinico Umberto I, University of Rome "La Sapienza." 00161 Rome. Italy Â¡P.L P.. L. C., M. F., M.L.Â¡; Institut fÃ¼rMolekularbiologie und Biochemie der Freien UniversitÃ¤t Berlin. Berlin 33, Germany /C Z.. A. Gr./; Istituto di Medicina Interna. UniversitÃ di Cagliari. ÃœVÃ•24 Cagliari, Italy Â¡M.L]: Division of Biochemistry and Molecular Biology, University of Glasgow GI2K QQ. Scotland, United Kingdom Â¡N.B. L TJ; Sbarro Institute for Cancer Research and Molecular Medicine, Pathology, Anatomy, and Cell Biology, Jefferson Medicai College, Philadelphia. Pennsvlvania IV107 Â¡A.Gi.J

Abstract and pRb2/pl30) and cyclin/cdks4 (7-16). In general, binding of free E2F/DP heterodimers to E2F sites activates transcription, whereas com 121/1)1* heterodimers play a pivotal role in the regulation of cell growth plex formation with pRb or other pocket proteins silences the transcrip and differentiation. A decrease in E2F/DP activity occurs during cell cycle tion of downstream target genes. arrest and differentiation. However, very little is known about the specific Both growth-inhibitory and E2F-suppressive properties of pRb. p 107. role of the various E2F/DP members along the transition from proliferation and pRb2/pl30 are regulated by cyclin/cdk-dependent phosphorylation to terminal differentiation. We have previously shown that E2F4 accounts for during the G,-S phase progression (17-22). During in vitro myogenic the vast majority of the endogenous E2F in differentiating muscle cells. Here, we show that E2F4, which lacks a nuclear localization signal (ills), is distrib differentiation, muscle cells irreversibly exit from the cell cycle (23). uted in both the nucleus and the cytoplasm, in either asynchronously growing Accordingly, in differentiating myocytes, the activity of cyclin/cdks is myoblasts or differentiated myotubes. E2F4 nuclear accumulation is induced suppressed, and both pRb and pRb2/pl30 accumulate in their unphos- by the binding in the cytoplasm with specific partners p 107. pRb2/pl30, and phorylated form to repress E2F-dependent transcription and inhibit cell DP3S, an nls-containing spliced form of DP3, which provide the nls. Although cycle progression (3, 4, 24-27). We have previously shown that E2F4- overexpression of E2F4/DP3S reactivates the cell cycle in quiescent cells, the pRb2/pl30 represents the most abundant E2F-DNA bound complex in E2F4 nuclear accumulation induced by pRb2/pl30 and p 107 correlates with differentiating myocytes and that E2F4 accounts for the vast majority of cell growth arrest Moreover, E2F4/DP3S-induced cell cycle reactivation is the endogenous E2F in either myoblasts or myotubes (6). It has been efficiently counteracted by either pl07 or pRb2/pl30 overexpression. Rein- reported recently that E2F4 lacks a nls and its nuclear localization duction in quiescent cells of DNA synthesis by E2F1/DP1 overexpression is depends on the binding in the cytoplasms with some proteins which abrogated by coexpression of pRb and is hampered by MyoD overexpression. provide the nls (28, 29). Here, we have studied the subcellular distribution Both pRb2/pl30 and pRb, as well as MyoD, are up-regulated in myotubes. of E2F4 and the mechanisms responsible for its nuclear translocation in Accordingly, multinucleated myotubes, which are induced to reenter the differentiating muscle cells. We also show that the nuclear accumulation S-phase by oncoviral proteins, are refractory to cell cycle reactivation by forced expression of E2F4/DP3S or I 21 I/DIM. Thus, I 21 /DI' repression of E2F4 induced by distinct cytoplasmic partners determines different consequences on the cell cycle, which are also dependent on the nuclear represents only one of multiple redundant circuits that control the postmitotic amount of antiproliferative proteins that are up-regulated in differentiat state in terminally differentiated cells and that are targeted by adenovirus EIA and SV40 large T antigen. ing muscle cells. Materials and Methods Introduction Cell Lines and Culture Conditions. C2C12 mouse cells were grown in E2F transcription factor regulates cell cycle progression by influencing DMEM supplemented with 20% FBS (cycling myoblasts) until they reached the expression of proteins required for the G,-S phase transition and 80-90% confluence, when differentiation was induced by switching cell DNA synthesis (1). A decrease in E2F activity is a prerequisite for cultures to DMEM containing 2% FBS (DM). C2CI2 myoblasts were made induction of quiescence and terminal differentiation in many cell types quiescent by culture in 0.1% FBS for 48 h. (2-5), and the reactivation of DNA synthesis in quiescent cells by Microinjection. For microinjection experiments, cells were grown on mitogens is accompanied by an increase in E2F-dependent transcription small glass slides subdivided into numbered squares of 2 mm X 2 mm. Cells were microinjected with 100 molecules of DNA per nucleus, as described (2, 6). Accordingly, overexpression of E2F1 prevents muscle differenti previously (30). When indicated, plasmid pCHllO, which encodes for ÃŸ- ation (7). E2F activity is generally believed to be regulated at the nuclear galactosidase under the control of the SV40 early enhancer-promoter, was used level by DNA binding of E2F/DP heterodimers and complex formation as a marker for microinjected cells, and its cytoplasmic expression was with the retinoblastoma "pocket" protein family (including pRb, p 107, visualized by a specific anti-ÃŸ-galactosidase antibody, followed by a secondary rhodamine-conjugated antibody. The expression of the plasmid of interest (Ha-tagged) and the localization of its product were visualized by using an Received 11/26/97; accepted 2/6/98. The costs of publication of this article were defrayed in part by the payment of page anti-Ha antibody (12CA5), kindly provided by O. Segallo (Laboratory of charges. This article must therefore be hereby marked advertisement in accordance with Immunology. Regina Elena Cancer Center Institute. Rome, Italy). 18 U.S.C. Section 1734 solely lo indicate this fact. Immunofluorescence. Cells were washed in PBS, fixed in a methanol- ' This work was supported by grants from Fondazione Andrea Cesalpino. Associazi acetone (1:2, v/v) solution, dried, and incubated with the different primary one Italiana Ricerca sul Cancro, Progetto Finalizzato. Applicazioni Cliniche della Ricerca antibodies at 37Â°Cfor 30 min. Specifically bound antibody was visualized by Oncologica. Consiglio Nazionale delle Ricerche. European Community. Biomed 2 and Telethon (to M. L.), Deutsche Forschungsgemeinschaft (Grant Gr 384/13-3; to A. Gr.). incubation with rhodaminate second-step antibody against immunoglobulin of and NIH (Grant ROI CA60999-01A1; to A. Gi.). 2 Present address: Department of Biology and Center for Molecular Genetics. Univer sity of California at San Diego. La Jolla, CA 92093-0347. 4 The abbreviations used are: cdk. cyclin-dependenl kina.se; nls. nuclear Itx'alization 3 To whom requests for reprints should be addressed, at Laboratory of Gene Expres signal; FBS, fetal bovine serum; DM. differentiation medium: DAP1, 4'.6-diamidino-2- phenylindole; BrdUrd. 5-bromo-2'-deoxyuridine; DMR. DM restimulated with scrum; sion, Fondazione Andrea Cesalpino. Policlinico Umberto I, Viale del Policlinico 155, 00161 Rome, Italy. Phone: 39-6-4468529; Fax: 39-6-4940594; E-mail: [email protected]. TAg. T antigen. 1325

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1998 American Association for Cancer Research. REGULATION OF E2F4 MITOOENIC ACTIVITY the relevant species. Nuclei were counterstained with DAPI. Immunofluores- body (kindly provided by K. Heiin, European Institute of Oncology, cence for the detection of BrdUrd, as an indicator of DNA synthesis, was Milan, Italy), we were able to detect endogenous E2F4 localized in both performed using the BrdUrd labeling and detection Kit (Boehringer). For the nuclei and the cytoplasm of mononucleatedmyoblastscultured in the costaining with mouse monoclonal antibodies, an anti-BrdUrd antibody that presence of serum, whereas in myotubes E2F4, mainly accumulates into was directly conjugated with fluorescein was used. the nuclei (data not shown). Finally, we observed that the cytoplasmic Western Blot. Total cell lysates were loaded and then resolved on SDS- fraction of E2F4 increases following serum stimulation of GO arrested PAGE. After overnight electrophoresis, gels were equilibrated for 30 min in cells, either myoblasts (data not shown) or myotubes (Fig. 2). transfer buffer (25 mw Tris-200 mM glycine) and transferred to polyvinylidene difluoride membranes (Millipore) at 0.25 mA for 5 h at 4Â°C.Membranes were first On the basis of these results, we speculated that the nuclear import incubated in 1X TBS [20 mM Tris (pH 6.5)-0.5 M NaCl] with 5% BSA for 1 h, of E2F/DP members lacking the nls might be regulated by the avail with the primary antibody overnight, and, finally, with a secondary antibody ability of cytoplasmic carrier(s), likely in a serum-dependent manner. conjugated to horseradish peroxidase for 1 h. The antigen-antibody interaction was We, therefore, performed a set of experiments aimed at identifying the visualized by incubation for 30 s in a chemiluminescent reagent (ECL Western cellular factors responsible for nuclear accumulation of E2F/DP mem blotting detection; Amersham) and exposure to an X-ray film. bers lacking the nls. According to results previously reported in other Subcellular Localization. Nuclear and cytoplasmic fractions were pre cell types (29), coexpression of E2F1 is sufficient for exogenous DPI pared by swelling the cells for 10 min in 300 jal of hypotonie buffer A [10 mM to localize into the nuclei of injected myoblasts (Fig. 3, A and C). Tris (pH 7.5), 10 mM NaCl, 3 mM MgCl2, 1 mM EGTA, 10 mM sodium Among all of the E2F4 interacting proteins (which include all of the phosphate, 20 mg/ml aprotinin, 20 mg/ml leupeptin, 10 mM NaF, 1 mM pocket proteins, DPI, and the DP3 alternative splicing variants, DP3ÃŸ phenylmethylsulfonyl fluoride, and 1 mM Na3VO4], homogenizing them with and DP3S), only pl07, pRb2/pl30, and DP3Ã”,the version encom 10 slow strokes with a Dounce homogenizer, and spinning them for 5 min at 375 X g at 4Â°C.The supernatant was mixed with an equal volume of 2X passing a nls (29), induce, upon coinjection in quiescent myoblasts, Laemmli sample buffer to give the cytoplasmic fraction. The nuclei were the nuclear localization of exogenously expressed E2F4 (Fig. 3, B and washed three times in buffer A plus 0.1% Nonidet P-40 and lysed in IX C). The same results were obtained in C2C12 myotubes, in which the Laemmli sample buffer. Total lysates were prepared by direct lysis of cells in differences in subcellular distribution of exogenous proteins are more 1x Laemmli sample buffer. easily detected, due to the larger size of the cells and the presence of Northern Blot Total cellular RNA was extracted from either proliferating multiple nuclei (Fig. 3, D and E). or quiescent myoblasts and from myotubes cultured either in DM or DMR, as We then evaluated whether the nuclear import of exogenous E2F4, described previously (6). Thirty /Â¿gof RNA were size-fractionated on 1.2% as induced by coexpression of its cytoplasmic carriers, results in the agarose-0.6% formaldehyde gels and transferred to nylon filters (Duralon; reactivation of the cell cycle in quiescent myocytes. Although forced Stratagene). The E2F1, DPI, E2F4, DP3, and GAPDH probes were restriction DNA fragments derived from the corresponding cDNAs and 32P-labeled by expression of E2F4 in conjunction with DP3S reactivates DNA syn thesis in serum-starved myocytes (Fig. 4A), induction of E2F4 nuclear random priming. Hybridization was carried out as described previously (6). accumulation by p107 or pRb2/pl30 fails to induce S-phase entry in Results quiescent myoblasts (Fig. 4A). Accordingly, reinduction of DNA synthesis in quiescent myocytes (Fig. 4<4)and fibroblasts (data not To address the functional relevance of different E2F/DP complexes in shown), as obtained by overexpression of E2F4/DP38, is hampered by reinducing DNA synthesis in differentiating muscle cells, we microin- the comicroinjection of p107, pRb2/pl30, and, less efficiently, pRb jected various combinations of expression vectors encoding members of and MyoD (Fig. 4A), two other cell growth suppressors in muscle the E2F and DP families in undifferentiatedquiescent.Forced expression cells (37). Instead, E2F1/DP1-induced S-phase progression is effi of E2F1 is known to induce S-phase entry, followed by apoptosis, in ciently inhibited only by pRb and, to a lesser extent, by MyoD (Fig. quiescent fibroblasts (31-35) and DNA synthesis in cardiac myocytes 4ÃŸ).Theseresults indicate that the pocket proteins pRb, p107, and (36). Indeed, upon microinjectionof an E2F1 expression vector, reinduc- pRb2/pl30 and the myogenic factor MyoD, exert their cell growth- tion of DNA synthesis has been observed in C2C12 myoblasts (Fig. 1) inhibitory function during terminal differentiation of muscle cells, at and C3H10T1/2 fibroblasts (data not shown) rendered quiescent by least in part by counteracting the proliferative potential of E2F/DP. serum starvation,and the phenomenon is enhanced by DPI coexpression Notably, the levels of unphosphorylated pRb2/pl30 and pRb increase (Fig. 1).At variance with E2F1, forced expressionof either E2F4 or DPI during muscle differentiation and the antiproliferative function of alone fails to reactivate DNA synthesis in both quiescent undifferentiated these proteins is induced by their concomitant dephosphorylation (3, C2C12 myocytes (Fig. 1)and serum-starvedfibroblasts(data not shown). 4, 26, 38, 39). The different abilities of E2F1, E2F4, and DPI to reinduce cell cycle A quantitative analysis of the expression levels of the various activation were found to correlate with their different intracellulardistri E2F/DP members at the different stages of muscle differentiation (Fig. butions. Previous reports have shown that E2F4 and DPI lack a nls (28, 5) shows that, in proliferating myoblasts, DP3 transcripts are ex 29). Consistentwith this notion, although exogenous E2F1 protein local pressed at high levels and decrease during muscle differentiation, and izes into the nuclei of injected cells (Fig. IÃŸ),forcedexpression of E2F4 likewise, the synthesis of both E2F1 and DPI protein is specifically (Fig. IÃŸ)andDPI (see Fig. 3Q, either alone or in combination (data not repressed at the transcriptional level in differentiating myocytes (Fig. shown), results in their cytoplasmic accumulation. By immunoblottÃ¬ng 5). In contrast, E2F4 RNA levels are only slightly modified during the after subcellular fractionation, we found that a significant proportion of different stages of muscle differentiation, with a small increase in endogenous E2F4 (Fig. 2) and DPI (data not shown) is normally present myotubes (Fig. 5). Although the expression of all of the E2F/DP in the cytoplasm of both asynchronouslygrowing myoblasts and differ members can be reinducible by serum (Fig. 5), in quiescent myoblasts, entiated myotubes. Because both the nuclear and cytoplasmic fractions a rise of E2F/DP levels correlates with reinduction of DNA synthesis may result during cell fractionation, to some extent, the fractions are by serum (6). cross-contaminated, and it is difficult to determine with precision the We and others (3, 6) have previously shown that, in terminally actual subcellular distribution of these proteins. Furthermore, differenti differentiated myotubes, E2F-dependent transcription is inhibited by ating muscle cells include an asynchronous population of myocytes at pocket protein binding, and this contributes to the maintenance of different stages of differentiation, and this can further complicate a myotubes that are refractory to cell cycle stimulation by mitogens. definitive interpretation of the results obtained. However, in a parallel Accordingly, both adenovirus EIA and SV40 large TAg, two onco- approach by immunofluorescence,using an anti-E2F4 monoclonal anti viral proteins that disrupt the interaction between pocket proteins and 1326

- MHC 100

- E2F4

NC N CMC 2 60 il i GM DM DMR Fig. 2. Subcellular localization of endogenous E2F4. Nuclear (Lanes N) and cytoplasmic (Lanes O fractions were prepared from myoblasts cultured in growth medium (CM) and myotubes cultured in either DM or DMR. as described in "Materials and Methods." The relative amount of E2F4 was determined by Western blot using a polyclonal anti-E2F4 antibody (C-20; Santa Cruz Biotechnology). A monoclonal antimyosin heavy chain (MHC] antibody, MF20, was used as a marker for cytoplasmic fraction from differentiated myotubes.

E2F, reactivate DNA synthesis when expressed in myotubes (40, 41). s g Further analysis of different EIA deletion mutants revealed that the r"M + !* !*+ Â« potential of EIA to reinduce S phase in myotubes is, indeed, depend II â€¢¿*â€¢*â€¢â€¢9 M Â»i Â»i M M 4 ent on the integrity of its pocket protein-binding domain (42, 43). M 0 0 M M H However, it remains unclear whether the reactivation of DNA syn thesis by EIA entirely relies on its ability to reinduce E2F-dependent B transcription. To evaluate the potential role of E2F/DP in reactivating anti-Ha anti-BrdU the cell cycle in myotubes, we overexpressed in these cells different E2F/DP combinations by microinjection. Overexpression and nuclear accumulation of either E2F1/DP1 or E2F4/DP38, two heterodimers that induced cell cycle progression in quiescent myoblasts (see Fig. 4), as well as in E2F4/pl07 and E2F4/pRb2/p 130, failed to reinduce DNA synthesis in myotubes (Fig. 6), as can be observed after forced HaE2Fl expression of EIA and TAg (Fig. 6). Forced expression by microin jection of E2F1, together with E2F4/DP36, was also unable to reac tivate the cell cycle in the same cells (data not shown). The failure to reinduce S-phase entry by E2F/DP overexpression was also confirmed by the absence of nuclear cyclin A expression in the injected myo tubes (data not shown). Thus, terminally differentiated cells, unlike quiescent undifferentiated fibroblasts and myoblasts, become resistant to the aberrant cell cycle reactivation occurring in response to the overexpression of transcription factors that stimulate G,-S phase progression. Oncoviral proteins overcome this refractory state by virtue of their ability to HaE2Fl affect multiple pathways. + Discussion DPI Here, we show that E2F4, which lacks a nls, has both a nuclear and extranuclear localization in C2C12 muscle cells during the different stages of differentiation. It has been reported recently that, in other cell types, E2F4 subcellular localization is regulated by the cell cycle

Fig. I. Effects on DNA synthesis of E2FI, E2F4. and DPI microinjection into C2C12 quiescent myoblasts. A, nonconfluent C2C12 cells were cultured for 36 h in 0.1% FBS-containing medium before direct nuclear microinjection with the indicated plasmids (pCMV-E2Fl. pCMV-DPI. pCMV-E2F4. and pSV-TAg) and the corresponding control empty vectors, together with the pCHl 10 ÃŸ-galactosidase expression plasmid. as a marker for productively injected cells. Immediately after injection, 0.1 % FBS containing medium with BrdUrd was added. After additional 18 h. cells were fixed and processed for indirect immunofluorescence to delect cytoplasmic ÃŸ-galactosidase expression with an anti-ÃŸ- galactosidase antibody and nuclear BrdUrd incorporation (see "Materials and Methods"). HaE2F4 A SV40 TAg-encoding plasmid was used as positive control. B, BrdUrd incorporation (right) of C2C12 quiescent myoblasts after microinjection of the indicated plasmids and subcellular localization of their products (left). Cells that are positive for the expression of the injected plasmid are visualized by anti-HA antibody (rhodamine). and nuclei are stained for BrdUrd incorporation (fluorescein). For each diagram shown in this figure, the mean of two independent microinjection experiments performed under identical condi tions is represented. For each experiment, at least 100 injected cells were considered. 1327

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HaE2F4 + pRb2/pl3

HaE2F4 ,'O.j'â€¢¿DP38

Fig. 3. A, subcellular localization of exogenous E2FI, E2F4, and DPI after microinjection in C2C12 myoblasts. Cells were microinjected directly into the nuclei with the indicated plasmids (pCMV-HAE2Fl, pCMV-HADPl, and pCMV-HAE2F4), alone or in combination the following plasmids: pCMV-E2Fl, pCMVE2F4, and pCMVDPl. After 18 h, cells were fixed and stained by immunofluorescence to detect the localization of the product of the injected plasmid. To discriminate between the exogenous and the endogenous proteins, an anti-HA antiserum was used as primary antibody. Columns, percentage of nuclei positively stained for the exogenous protein; Bars, SEM. fl, subcellular localization of exogenous E2F4

1328

tÂ» e P Â¡gomo g .< .N Â£ o, o. e. H ** E2F4 E2F4+DP3S E2F1+DP1 Fig. 4. A. Stimulation of DNA synthesis in C2C12 quiescent myoblasts by microinjection of E2F4. either alone or in combination with DP3S. and effect of coexpression of different growth-suppressive proteins (pCMVpRb, pCMVplO?, pCMVpRb2/pl30, and CMVMyoD). Nonconfluent quiescent C2C12 cells were microinjected directly into the nuclei with a pCMVHAE2F4-encoding plasmid. either alone or in combination with pG4DP38. The effect of the coinjection of the pocket proteins pRb. pl()7. and pRb2/pl3() or MyoD was also evaluated. Immediately after injection, 0.\% FBS containing medium with BrdUrd was added. After additional 18 h, cells were lived and processed for indirect immunofluorescence to detect cytoplasmic ÃŸ-galactosidase expression and nuclear BrdUrd incorporation (see "Materials and Methods." B. stimulation oi DNA synthesis in C2C12 quiescent myoblasts by microinjection of E2FI/DP1 and effect of coexpression of different growth-suppressive proteins (pCMVpRb. pCMVplOV, pC'MVpRbl/p IW. and CMVMyoD). Nonconfluent quiescent C2C12 cells were microinjected directly into the nuclei with DCMVHAE2F1 in combination with CMVDP1 in either the presence or absence of the indicated plasmids. Immediately after injection, 0.1% FBS containing medium with BrdUrd was added. After additional 18 h. cells were fixed and processed for iniliivct immunofluorescence to detect cytoplasmic ÃŸ-galactosidase expression and nuclear BrdUrd incorporation (see "Materials and Methods"). For each diagram shown in this IIJJIIK. the mean of two independent microinjection experiments performed under identical conditions is represented. For each experiment, at least 100 injected cells were considered.

(44, 45) and its nuclear translocation is induced by two class of transcription of E2F-responsive genes. Therefore, the recruitment of proteins, i.e., retinoblastoma and DP family members (28, 44), which E2F4 by p 107 and pRb2/pl30 into the nuclei of differentiating cells provide the nls. Here, we demonstrate that, in differentiating muscle may be viewed as a necessary step for the pocket proteins to bind the cells, E2F4 nuclear localization can be determined by its association DNA at specific regulatory sequences and, hence, repress S-phase with only two pocket proteins, i.e., pl07, pRb2/pl30, and a specific gene transcription and cell proliferation. DP3 spliced form, DP3S, which contains the nls. The availability of As opposed to E2F4, inactivation of the mitogenic potential of these cytoplasmic partners changes along the transition from myo E2F1 during muscle differentiation is achieved through its transcrip- blasts to myotubes and might determine, in concert with nuclear tional down-regulation. This is of particular relevance because E2F1, proteins differently expressed during terminal differentiation (i.e., in addition to its own mitogenic effect, induces the nuclear import of cyclins, pRb, and MyoD), the biological function of E2F4. The E2F4 DPI. which lacks a nls. Thus, differentiating cells might reduce the nuclear import, as induced by DP3S, stimulates cell cycle progression, nuclear pool of two potential mitogenic E2F/DP members by the whereas E2F4 nuclear localization imposed by pocket proteins corre extinction of the one (E2F1) required for the nuclear localization of lates with cell growth arrest. Accordingly, overexpression of either the other (DPI). pl07 or pRb2/pl30 and, partially, of pRb and MyoD extinguishes the An important cytoplasmic partner of E2F4, capable of inducing its mitogenic potential of the E2F4/DP36 heterodimer. Both p 107 and nuclear localization, is pRb2/pl30. It is known to be a nuclear inhib pRb2/pl30 are implicated in the induction of the cell cycle arrest itor of the cell cycle progression through the repression of the E2F- occurring in differentiating myocytes, whereas pRb plays a more dependent transcription (14, 15, 47) and is specifically up-regulated critical role in the maintenance of the postmitotic state in myotubes (4, during muscle differentiation (26). The results presented here indicate 6, 43, 46). pRb, p 107, and pRb2/pl30 lack a DNA-binding domain that pRb2/pl30 might also have a function in the cytoplasm of and exert their antiproliferative activity mainly by repressing the myocytes induced to differentiate, where it interacts with and recruits

upon coinjection with different partners in C2C12 myoblasts. Cells were microinjected directly into the nuclei with CMV-HAE2F4 either alone or in conjunction with the indicated plasmids (pCMV-DPl, pG4-DP3ÃŸ, pG4-DP3S, pCMV-pRb. pCMV-p107. pCMV-pRb2/pl30. and pSV-TAg). After 18 h. cells were fixed and stained for expression of exogenous E2F4 using an anti-HA antiserum. TAg (which contains a nls similar to that of DP35) was used as a negative control. Columns, percentage of nuclei positively stained for the exogenous protein; bars, SEM. C, subcellular localization of the products of the indicated plasmids is shown by immunofluorescence. Lefi, cells that are positive for the expression of the injected plasmid are visualized by anti-HA antibody, (rhodamine); right, nuclei are counterstained with DAPI. D. subcellular localization of exogenous E2F4 upon coinjection with different partners in C2C12 myotubes. Cells were microinjected directly into the nuclei with CMV-HAE2F4. either alone or in conjunction with the indicated plasmids (pCMV-DPl, pG4-DP3ÃŸ, pG4-DP38, pCMV-pRb, pCMV-pl07. pCMV-pRb2/p 130. and pSV-TAg). After 18 h, cells were fixed and stained for expression of exogenous E2F4 using an anti-HA antiserum. Columns, percentage of nuclei positively stained for the exogenous protein; bars. SEM. E, subcellular localization of the products of the indicated plasmids is shown by immunofluorescence. Right, cells that are positive for the expression of the injected plasmid are visualized by anti-HA antibody (rhodamine); middle, nuclei are counterstained with DAPI; left, differentiated myotubes are visualized by the cytoplasmic staining of MHC (fluorescein). For each diagram shown in this figure, the mean of two independent microinjection experiments performed under identical conditions is represented. For each experiment, at least 100 injected cells were considered. 1329

DPI

E2F4

DP3

GAPDH

Fig. 5. Regulation of mRNA levels of E2F1, E2F4, and DP3 isoforms during C2C12 differentiation and in response to serum. Northern analysis of different E2F/DP messages at different stages of C2C12 differentiation and their response to serum. RNAs were extracted from myoblasts cultured in growth medium (CM}, and from myotubes cultured either in DM or DMR. as described in "Materials and Methods."

B B R 2 WWMMWMH E2F4 into the nucleus, thereby providing sequence-specific DNA binding to the pocket proteins. Likewise, DP38 could also contribute B Anti-Ha Anti-BrdU to induce the E2F4 nuclear accumulation in myotubes. In fact, al DAPI though DP3 transcription declines in differentiating cells, how the ratio between the different spliced forms changes is unknown. Thus, HaE2Fl it could be possible that DP3S stimulates E2F4 nuclear translocation in both myoblasts and myotubes and that the nuclear concentration of mitogenic (i.e., cyclin/cdks) and antimitogenic (i.e., pRb, pRb2/pl30, and MyoD) proteins finally determines the effect on the cell cycle. Nevertheless, because most of the results presented in this paper derive from experiments performed through protein overexpression, we believe that a definitive picture of the mechanisms regulating E2F4 subcellular regulation during myogenesis requires further study. We are currently addressing the regulation of both endogenous E2F4 HAE2F4 subcellular distribution and activity during muscle differentiation in + more detail, and we are investigating the possibility that cytoplasmic DP35 E2F4 might also have a biological function. Anti-El A DAPI Anti-BrdU Finally, we show that myotubes become refractory to reentering the S phase following the overexpression of E2F1/DP1 and E2F4/DP3Ã”, indicating that, in terminally differentiated cells, the mitogenic poten EIA tial of E2F/DP can be efficiently repressed. In keeping with this notion, the potential of EIA and SV40 large TAg to reactivate the DNA synthesis in myotubes (40, 41) cannot be simply E2F/DP dependent but would be actually related to their ability to affect multiple circuits, which cooperate to maintain the postmitotic state. TAg Because the EIA ability to stimulate DNA synthesis in myotubes is dependent on the region required for binding to the pocket proteins (42, 43), it is likely that most of the pathways that are targeted by EIA Fig. 6. Stimulation of DNA synthesis in C2C12 myotubes by microinjection of to reverse terminal muscle differentiation are pRb and/or pRb2/pl30 CMVHAE2F1 and CMVHAE2F4, either alone or in combination with the indicated plasmids (CMVDP1, pG4DP3e, and pCMVplO?). A. myotubes were injected with the dependent. Moreover, genetic evidence indicates that, in the absence indicated plasmids. As positive controls. pElA12S and pSVTAg were microinjected an of functional pRb, pRb2/pl30 is not sufficient to prevent serum- their expression detected by specific antibodies. Immediately after injection, 20% FBS- induced S-phase reentry (46). Thus, in terminally differentiated myo containing medium with BrdUrd was added. After an additional 18 h, cells were fixed and processed for indirect immunofluorescence to detect the expression of the injected tubes, pRb might exert some antiproliferative activity that could not plasmid with anti-HA antibodies and nuclear BrdUrd incorporation (see "Materials and Methods"). B, BrdUrd incorporation (left) of C2C12 myotubes after microinjection of the be reverted by E2F/DP overexpression. An increase in free E2F DNA indicated plasmids and subcellular localization of their products (right). Cells that are binding has been observed in response to serum stimulation in the positive for the expression of the injected plasmid are visualized by anti-HA antibody absence of S-phase progression (43). It is tempting to speculate that (rhodamine), and nuclei are stained for BrdUrd incorporation (fluorescein). Nuclei are counterstained with DAPI (middle). For each diagram shown in this figure, the mean of this reinduction of E2F free activity in myotubes might be involved in two independent microinjection experiments performed under identical conditions is other than proliferative processes (i.e., hypertrophie growth; Ref. 48). represented. For each experiment, at least 100 injected cells were considered. 1330

Acknowledgments 22. Claudio, P. P., De Luca. A., Haward, C. M., Baldi, A., Firpo, E. J., Koff, A., Paggi. M. G., and Giordano, A. Functional analysis of pRb2/pl30 interaction with cyclins. We thank F. Tato and A. La Rocca for helpful discussions and critical Cancer Res., 56: 2003-2008, 1996. Nadal-Ginard. B. Commitment, fusion and biochemical differentiation of a myogenic comments during the preparation of this manuscript; O. Segallo for providing 23. cell line in the absence of DNA synthesis. Cell. /5: 855-864, 1978. the anti-Ha 12Ca5 antibody; and K. Helin for the E2F1, E2F4Ha. and DPIHa 24. Halevy, O., Novitch, B. G.. Spicer, D. B., Skapek. S. X., Rhee, J.. Hannon. G. J., expression vectors and the anti-E2F4 monoclonal antibody TFE44. P. L. P. Beach. D.. and Lassar A.B. Correlation of terminal cell cycle arrest of skeletal muscle expresses his personal gratitude to A. Gr., from whom he learned the manual with induction of p21 by MyoD. Science (Washington DC). 267: 1018-1021, 1995. 25. Guo, K., Wang, J.. Andres, V., Smith, R.. and Walsh. K. MyoD-induced expression microinjection technique. P. L. P. also thanks Eva Guhl for her invaluable of p21 inhibits cyclin-dependent kinase activity upon myocyte terminal differentia technical assistance during the microinjection and immunofluorescence exper tion. Mol. Cell. Biol.. /5: 3823-3829, 1995. iments. 26. Kiess. M.. Gill. M., and Hamel, P. Expression and activity of the retinoblastoma protein (pRb) family proteins. pl07 and pl30. during L6 myoblasls differentiation. Note Added in Proof Cell Growth Differ.. 6: 1287-1298. 1995. 27. De Luca, A.. MacLachlan. T. K., flagella, L.. Dean, C.. Howard, C. M., Claudio, During revision of this manuscript, we learned lhat two additional papers have reported P. P., Baldi. A.. Khalili, K.. and Giordano, A. A unique domain of pRb2/pl.30 acts as an inhibitor of cdk2 kinase activity. J. Biol. Chem., 272: 20971-20974, 1997. observations on the regulation of E2F4 (49) and E2F5 (50) subcellular localization and the 28. Magae. J.. Wu. C-L.. Illenye. S.. Harlow. E., and Heintz. N. H. Nuclear localization correlation with the cell cycle. of DP and E2F transcription factor* by heterodimeric partners and retinoblastoma protein family members. J. Cell Sci., 709: 1717-1726, 1996. References 29. de la Luna, S., Burden, M. !.. Lee, C-W., and La Thangue, N. B. Nuclear accumu lation of the E2F heterodimer regulated by subunit composition and alternative 1. La Thangue. N. B. DP and E2F proteins: components of a heterodimeric transcription splicing of a nuclear localization signal. J. Cell Sci., 109: 2443-2452. 1996. factor implicated in cell cycle control. Curr. Opin. Cell. Biol., 6: 443-450, 1994. 30. Graessmann. A., and Graessmann, M. Microinjection of tissue culture cells. Methods 2. Chittenden, T.. Livingston, D. M., and De Caprio, J. A. Cell cycle analysis of E2F in Enzymol., 101: 482-492, 1983. primary T cells reveals novel E2F complexes and biochemically distinct forms of free 3 ' â€¢¿Johnson, D. G., Schwarz, J. K., Cress, W. D., and Nevins J. R. Expression of E2F. Mol. Cell. Biol.. 13: 3975-3983, 1993. transcription factor E2FI induces quiescent cells to enter S phase. Nature (Lond.), 3. Shin, E. K., Shin. A., Paulding. C.. Schaffausen, B., and Yee, A. S. Multiple changes 365: 349-352. 1993. in E2F function occur upon muscle differentiation. Mol. Cell. Biol., /5: 2252-2262. 32. Qin. X-Q., Livingston. D. M.. Kaelin. W. G.. and Adams. P. Deregulated transcription 1995. factor E2F1 expression leads to S-phase entry and p53-mediated apoptosis. Proc. 4. Corbeil, H. B.. Whyte, P., and Branton, P. E. Characterization of transcription factor Nati. Acad. Sci. USA, 91: 10918-10922. 1994. E2F complexes during muscle and neuronal differentiation. Oncogene. //: 909-920. 33. Wu, X.. and Levine. A. J. p53 and E2F1 cooperate to mediate apoptosis. Proc. Nati. 1995. Acad. Sci. USA. 91: 3602-3606, 1994. 5. Jiang. H.. Lin. J.. Young, S-M., Goldestein, N. I., Waxman. S., Davila, V., Chellapan. 34. Shan. B., and Lee. W-H. Deregulated expression of E2F1 induces S-phase entry and S. P., and Fisher, P. B. Cell cycle gene expression and E2F transcription factor leads to apoptosis. Mol. Cell. Biol., 14: 8166-8173. 1994. complexes in human melanoma cells induced to terminally differentiate. Oncogene. 35. Lukas, J., Petersen. B. O., Holm, K., Bartek, J., and Helin, K. Deregulated expression //: 1179-1189, 1995. of E2F family members induces S-phase entry and overcomes p!6-mediated growth 6. Puri, P. L., Balsano. C.. Burgio, V. L., Chinilo. P.. Natoli. G., Ricci, L., Mattei, E.. suppression. Mol. Cell. Biol.. 16: 1047-1057, 1996. Graessmann. A., and Levrero, M. MyoD prevents cyclin A/cdk2 containing E2F 36. Kirshenbaum, L. A., Abdellatif, M., Chakraborty, S.. and Schneider, M. D. Human complexes formation in terminally differentiated myocytes. Oncogene. 14: 1171- E2F-I reactivates cell cycle progression in ventricular myocytes and represses cardiac 1184. 1997. gene transcription. Dev. Biol., 179: 402-411. 1996. 7. Wang, J., Helin. K., Jin, P., and Nadal-Ginard, B. Inhibition of in vitro myogenic 37. Gu. W., Schneider. J. W.. Condorelli, G., Kaushal, S., Mandavi, V., and Nadal- differentiation by cellular transcription factor E2F1. Cell Growth Differ.. 6: 1299- Ginard. B. Interaction of myogenic factors and the retinoblastoma protein mediates 1306, 1995. muscle cell commitment and differenliation. Cell, 72: 309-324. 1993. 8. Weintraub. S. J.. Prater, C. A., and Dean, D. C. Retinoblastoma protein switches the 38. Endo. T., and Goto, S. Retinoblastoma gene product Rb accumulates during myo E2F sites from positive to negative. Nature (Lond.). 358: 259-261. 1992. genic differentiation and is deinduced by expression of SV40 large T antigen. 9. Bandara. L.. Buck. V., Zamanian. M., Johnston. L., and La Thangue, N. B. Functional J. Biochem. (Tokyo), 112: 427-430. 1992. synergy between DPI and E2F1 in the cell cycle regulating transcription factor 39. Martelli. F.. Cenciarelli, C.. Santarelli, G., Polikar. B.. Felsani. A., and Caruso. M. DRTF1/E2F. EMBO J., 12: 4314-4324. 1993. MyoD induces retinoblastoma gene expression during myogenic differentiation. 10. Helin. K., Wu, C-L., Fattaey, A. R., Lees, J. A., Dynlacht, B. D.. Ngwu, C., and Oncogene. 9: 3579-3590, 1994. Harlow, E. Heterodimerizalion of the transcription factors E2F1 and DPI leads to 40. Cardoso. M. C.. Leonhardt. H.. and Nadal-Ginard. B. Reversal of terminal differen cooperative (rani-activation. Genes Dev.. 7: 1850-1861. 1992. tiation and control of DNA replication: cyclin A and cdk2 specifically localize at 11. Krek, W., Livingston, D. M., and Shirodkar, S. Binding to DNA and the retinoblas subnuclear sites of DNA replication. Cell. 74: 976-992. 1993. toma gene product promoted by complex formation of different E2F family members. 41. Crescenzi, M., Soddu. S., and Tato. F. Mitotic cycle reactivation in terminally Science (Washington DC), 262: 1557-1559, 1993. differentiated cells by adenovirus infection. J. Cell. Physiol., 762: 26-35. 1995. 12. Krek, W., Ewen, M., Shirodkar, S., Arany, Z., Kaelin. W., and Livingston, D. 42. Tiainen. M.. Spitkovsky. D., Jansen-Durr, P.. Sacchi, A., and Crescenzi. M. Expres Negative regulation of the growth promoting transcription factor E2F1 by a stably sion of EIA in terminally differentiated muscle cells reactivates the cell cycle and bound cyclin A-dependent protein kinase. Cell, 78: 161-172, 1994. suppresses tissue-specific genes by separable mechanisms. Mol. Cell. Biol., 16: 13. Lees, E., Faha, B.. Dulie, V., Reed, S., and Harlow. E. Cyclin E/cdk2 and Cyclin 5302-5312, 1996. A/cdk2 kinases associate with pl07 and E2F in a temporally distinct manner. Genes 43. Puri, P. L.. Avantaggiati. M. L., Balsano. C.. Sang. N.. Graessmann. A.. Giordano. Dev., 6: 1874-1885, 1994. A. A., and Levrero. M. p300 is required for MyoD-dependent cell cycle arrest and 14. Vairo, G., Livingston, D. M., and Ginsberg. D. Functional interaction between E2F4 muscle specific gene transcription. EMBO J., 16: 369-383, 1997. and pl30: evidence for distinct mechanisms underlying growth suppression by 44. Lindeman, G. J., Gaubatz, S., Livingston, D. M., and Ginsberg. D. The subcellular different retinoblastoma family members. Genes Dev.. 9: 869-881. 1995. localization of E2F4 is cell cycle-dependent. Proc. Nati. Acad. Sci. USA. 94: 15. Tommasi. S., and Pfeiffer. G. P. In vivo structure of the human cdc2 promoter: release 5095-5100. 1997. of a pl30-E2F4 complex from sequences immediately upstream of the transcription 45. Muller. H., Moroni, M. C., Vigo, E., Petersen. B. O., Bartek. J.. and Helin, K. initiation site coincides with induction of cdc2 expression. Mol. Cell. Biol.. 15: Induction of S-phase entry by E2F transcription factors depends on their nuclear 6901-6913, 1995. localization. Mol. Cell. Biol.. 17: 5508-5520, 1997. 16. Zwicker, Jâ€žLiu, N., Engeland, K., Lucibello, F. C., and Muller, R. Cell cycle 46. Schneider. J. W., Gu, W., Zhu. L.. Mandavi. V.. and Nadal-Ginard. B. Reversal of regulation of E2F site occupation in vivo. Science (Washington DC), 27/: 1595- terminal differentiation mediated by pl07 in Rb â€”¿/â€”muscle cells. Science (Wash 1597, 1996. ington DC). 264: 1467-1471. 1994. 17. Minara, K., Cao, X-R., Yen, A.. Chandler. S., Driscoll. B.. Murphree, A. L., Tang. A.. 47. Claudio, P. P., Howard. C. M., Baldi, A.. De Luca. A., Fu, Y., Condorelli. G., Sun, and Fung, Y-K. T. Cell cycle dependent regulation of phosphorylation of the human Y., Colbum. B., Calabretta, B., and Giordano. A. pl30/pRb2 has growth suppressive retinoblastoma gene product. Science (Washington DC), 246: 1300-1303, 1989. properties similar to yet distinctive from those of retinoblastoma family members pRb 18. Lin, B. T-Y., Gruenwald, S., Moria, A. O., Lee, W-H, and Wang, Y. J. Y. Retino and pl07. Cancer Res.. 54: 5556-5560, 1994. blastoma cancer suppressor gene product is a substrate of the cell cycle regulator cdc2 48. Puri, P. L., Avantaggiati, M. L., Burgio, V. L., Chinilo, P., Collepardo, D., Natoli kinase. EMBO J., 10: 857-864, 1992. G.. Balsano. C. A., and Levrero, M. Reactive oxygen intermediates mediate 19. Hinds. P. W., Mittnacht, S.. Dulie, V., Arnold, A.. Reed, S. I., and Weinberg. R. A. angiotensin Il-induced cJun/cFos heterodimer DNA binding activity and prolif- Regulation of retinoblastoma protein function by ectopie expression of human cyc- erative/hypertrophic response in myogenic cells. J. Biol. Chem.. 270: 22129- lins. Cell, 70: 993-1006, 1992. 22134, 1995. 20. Beijersbergen, R. L., Carlee, L., Kerkhoven, R. M., and Bernard. R. Regulation of the 49. Verona, R.. Moberg. K.. Estes, S.. Stare. M.. Vemon, J. P., Lees, J. A. EZF activity retinoblastoma protein-related pl07 by cyclin complexes. Genes Dev.. 9: 1340-1353, is regulated by cell cycle-dependent changes in subcellular localization. Mol. Cell 1995. Biol.. 17: 7268-7282, 1997. 21. Baldi, A. De Luca, A., Claudio, P. P., Baldi, F., Giordano, G. G., Tommasino, M., 50. Allen, K. E., de la Luna. S., Kerkhoven. Rm, Bernards. R., La Thangue, N. B. Distinct Paggi, M., and Giordano, A. The Rb2/pl30 gene product is a nuclear protein whose mechanisms of nuclear accumulation regulate the functional consequence of ELF phosphorylation is cell cycle regulated. J. Cell. Biochem.. 59: 402-408. 1995. iranscription factors. J. Cell Sci., 110: 2819-2831. 1997. 1331

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1998 American Association for Cancer Research. Regulation of E2F4 Mitogenic Activity during Terminal Differentiation by Its Heterodimerization Partners for Nuclear Translocation

Pier Lorenzo Puri, Letizia Cimino, Marcella Fulco, et al.

Cancer Res 1998;58:1325-1331.

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