Relationship between E1A binding to cellular proteins, c-myc ... - Nature

Oncogene (2007) 26, 781–787

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Relationship between E1A binding to cellular proteins, c-myc activation and S-phase induction S Baluchamy1,3,4, N Sankar1,3, A Navaraj1,5, E Moran2 and B Thimmapaya1 1 Department of Microbiology–Immunology Feinberg School of Medicine, Northwestern University, Chicago, IL, USA and 2Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA, USA

We recently showed that p300/CREB-binding protein (CBP) plays an important role in maintaining cells in G0/ G1 phase by keeping c-myc in a repressed state. Consistent with this, adenovirus E1A oncoprotein induces c-myc in a p300-dependent manner, and the c-myc induction is linked to S-phase induction. The induction of S phase by E1A is dependent on its binding to and inactivating several host proteins including p300/CBP. To determine whether there is a correlation between the host proteins binding to the N-terminal region of E1A, activation of c-myc and induction of S phase, we assayed the c-myc and S-phase induction in quiescent human cells by infecting them with Ad N-terminal E1A mutants with mutations that specifically affect binding to different chromatin-associated proteins including pRb, p300, p400 and p300/CBP-associated factor (PCAF). We show that the mutants that failed to bind to p300 or pRb were severely defective for c-myc and S-phase induction. The induction of c-myc and S phase was only moderately affected when E1A failed to bind to p400. Furthermore, analysis of the E1A mutants that fail to bind to p300, and both p300 and PCAF suggests that PCAF may also play a role in c-myc repression, and that the two chromatinassociated proteins may repress c-myc independently. In summary, these results suggest that c-myc deregulation by E1A through its interaction with these chromatin-associated proteins is an important step in the E1A-mediated cell cycle deregulation and possibly in cell transformation. Oncogene (2007) 26, 781–787. doi:10.1038/sj.onc.1209825; published online 24 July 2006 Keywords: E1A; p300; c-myc repression; cell cycle

Correspondence: Dr B Thimmapaya, Department of Microbiology— Immunology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Olson 8452, Chicago, IL 60611, USA. E-mail: [email protected] 3 These two authors contributed equally to this work. 4 Current address: Department of Genetics and Biochemistry, University of Illinois at Chicago, Chicago, IL, USA. 5 Current address: Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA. Received 28 September 2005; revised 9 June 2006; accepted 11 June 2006; published online 24 July 2006

Cell transformation by the adenoviral E1A oncoprotein is dependent on its binding to and inactivating several host proteins including p400, p300, pRb, and the two Rb family proteins p130 and p107, PCAF, TRRAP and GCN5 (reviewed in Moran, 1993; Barbeau et al., 1994; Frisch and Mymryk, 2002); for an extensive list of references on this topic please refer to the web site http://www.geocities.com/jmymryk.geo/. These host proteins are found in distinct chromatin remodeling complexes that modulate gene expression either by activating or repressing transcription. In most cases, specific transcriptional targets of these complexes in the context of cell cycle and the mechanism by which E1A interferes in their function are not known. pRb, p130 and p107 proteins in association with histone deacetylases repress E2F1 function that is critical for the induction of S phase (Harbour, 2001). p400 is a component of the human TIP60/NuA4 complex containing histone acetyl transferase (HAT) activity that modulates both transcriptional repression and activation (Fuchs et al., 2001; Doyon et al., 2004). p300 and CREB-binding protein (CBP) are two large highly homologous nuclear phosphoproteins containing HAT activity that function as transcriptional coactivators and link chromatin remodeling with transcription (Goodman and Smolik, 2000). TRRAP is found in three distinct human HAT complexes including the TIP60 HAT complex and two other complexes that are similar to yeast SAGA (Spt-Ada-Gcn5-acetyl transferase complex) containing either GCN5 or PCAF (reviewed in Sterner and Berger, 2000). Until recently, the significance of E1A binding to p300 in the E1A-mediated cell cycle deregulation and cell transformation was not known. We recently showed that in quiescent human cells, p300/CBP plays an important role in keeping c-myc in a repressed state. Depletion and induction of p300/CBP in serum-starved cells led to induction and repression, respectively, of c-myc and DNA synthesis (Kolli et al., 2001; Baluchamy et al., 2003; Rajabi et al., 2005). Consistent with this, we showed that in quiescent cells, E1A induced c-myc in a p300-dependent manner indicating that E1A interferes with p300/CBP repression of c-myc (Kolli et al., 2001). To determine whether or not there is a correlation between E1A binding to host proteins, and induction of c-myc and S phase, we have studied a series of previously characterized E1A mutants for their capacity to induce

c-myc activation by adenovirus E1A S Baluchamy et al

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c-myc and to enter S phase. Our results show that the E1A mutants that do not bind to p300 or Rb do not induce c-myc and S phase, whereas a mutant that does not bind to p400 induces both c-myc and cell cycle efficiently. In addition, our studies suggest that E1A binding to PCAF may also independently contribute to c-myc induction. Figure 1 shows a schematic of the E1A mutants analysed in this study that were characterized extensively by several laboratories with respect to their capacity to bind to different host proteins in vivo, induce DNA synthesis and transform rodent cells in culture in the presence of activated ras (references cited above; also see Egan et al., 1988; Fuchs et al., 2001; Lang and Hearing, 2003). To determine the capacity of these mutants to induce c-myc and DNA synthesis in human cells, we used MCF10A cells (an immortalized non-transformed human breast epithelial cell line; Soule et al., 1990) that can be growth arrested by serum starvation and show normal serum-stimulated induction of c-myc and cell cycle (Kolli et al., 2001). To determine the levels of c-myc induced by the E1A mutants, serum-starved MCF10A cells were infected with Ad mutants described above for 12 h, and the RNA samples were analysed using a quantitative real-time PCR assay as described in the legend to Figure 2. An Ad vector expressing beta-galactosidase (Adb-gal) was used as a control, and the fold-increase in c-myc induction by the E1A mutants was compared to that of Adb-galinfected cells. As shown in Figure 2a, there was about sixfold induction of c-myc in WT-infected cells. In contrast, in cells infected with RG2 (arginine-2 changed to glycine) that does not bind to p300, c-myc induction was only about 2.5-fold. Similarly, the mutant 928 (cysteine-128 changed to glycine) that does not bind to Rb also was severely defective for c-myc induction with a 1.7-fold increase of c-myc RNA levels, suggesting a role for pRb in c-myc repression. E1A mutant that does not bind to p400 (dl1102, deletion of aa 26–35; referred to here as dl26–35) induced near-normal levels of c-myc RNA (5.5-fold). When RG2 and 928 mutations are combined in one molecule (RG2.928; in baby rat kidney cells, this mutant is more defective in cell cycle than

either of the mutations alone; Wang et al., 1995), it was as defective as 928 in inducing c-myc, suggesting that the effects of RG2 mutation were further reduced by 928 mutation suggesting that WT E1A may recruit both p300 and Rb simultaneously as suggested earlier (Wang et al., 1995). In contrast, mutant dl2–36 (aa 2–36 deleted) that binds to neither p300 nor p400 showed a dramatic decrease in c-myc RNA levels that was comparable to that of control samples. All of the E1A mutants used in these studies expressed their respective proteins, at reasonable levels as shown by a Western blot in Figure 2d. Comparison of the c-myc induction and the protein binding properties of the mutant dl26–35, RG2 and dl2– 36 suggested that host proteins other than Rb and p300 might also repress c-myc. For example, mutant dl26–35 induces c-myc to near-normal levels (Figure 2a), and it does not bind to p400, and also TRRAP and GCN5; its p300 and PCAF binding capacity is not affected (Lang and Hearing, 2003). RG2, which retains about 40% of its Myc-inducing capacity, binds to p400 at normal levels, whereas it does not bind to p300 (TRRAP and GCN5 binding is not tested). In contrast, dl2–36 that is inactive in inducing c-myc does not bind to both p400 and p300. As dl2–36 also contains dl26–35 deletion, it is not expected to bind to TRRAP and GCN5 as well. Thus, it seemed possible that the total loss of c-myc induction by dl2–36 may be owing to its inability to bind to p300 and another protein such as PCAF as PCAF and p300 binding regions on E1A overlap (Lang and Hearing, 2003). To test this possibility, MCF10A cells were infected for 16 h with WT, RG2 and dl2–36 along with an Ad vector expressing FLAG epitope-tagged PCAF from the cytomegalovirus (CMV) promoter (AdPCAF). The cell lysates were immunoprecipitated with an anti-FLAG antibody followed by Western immunoblotting with an anti-E1A antibody. As shown in Figure 2e, both WT and RG2 E1A proteins bound to PCAF at comparable levels. Reverse experiment (Immunoprecipitation with anti-E1A and Western with anti-FLAG) also indicated that RG2 binds to PCAF at near-normal levels. A Western blot experiment shown in Figure 2e indicated comparable levels of expression of

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Figure 2 Quantification of c-myc and c-jun mRNA levels by real-time PCR (a and b) and luciferase activity in response to c-myc activation (c). (d) Western blot showing E1A protein levels in cell lysates prepared from virus-infected cells, (e and f) PCAF interaction with the WT and the E1A mutant proteins. MCF10A cell were seeded overnight, serum-starved for 36 h and then infected with Ad variants at 10 PFU per cell as described (Rajabi et al., 2005). At 12 h after infection, total RNA was isolated and 2 mg of RNA was reverse transcribed by Retroscript First strand synthesis kit (Ambion, Austin, USA), and 1/20th volume of the cDNA was used as template for the real-time PCR assay. Reactions were carried out in a final volume of 20 ml in SYBR Green Jump Start Taq Ready mix (Sigma) using ABI Prism 7700 Sequence Detection System. Fold increase was calculated by 2DDC t method (Applied Biosystems). The following primers were used for RT–PCR amplification. c-myc, forward: 50 -gccacgtctccacacatcag; c-myc, reverse: 50 -tggtgcattttcggttgttg; c-jun, forward: 50 -aggaggagcctcagacagtg; c-jun, reverse: 50 -agcttcctttttcggcactt. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH), forward: 50 -gtgaaggtcggagtcaacg; GAPDH, reverse: 50 -tgaggtcaatgaaggggtc. The experiment was carried out in triplicate and the average values with s.d.’s are shown. (c) Myc activity was assayed by measuring the luciferase activity in virusinfected cells by using a Myc responsive Ad vector (AdM4; see text for details). Assays were carried out in triplicate and the average values with s.d.’s are shown. (e and f) Proliferating MCF10A cells were infected with WT or E1A mutants along with AdPCAF for 24 h, and 1 mg of proteins from each cell lysate was immunoprecipitated with antibodies as shown, followed by Western immunoblotting. Anti-E1A Ab M73 (obtained from E Moran) and anti-FLAG Ab (Sigma, cat. no. M4) were used in these assays. For input in (e and f), cell lysates equivalent to 50 mg protein were loaded directly onto the gel and Western immunoblotted with antibodies as shown.

PCAF in WT- and the mutant-infected cells (input in Figure 2e). In summary, the significantly reduced c-myc induction in RG2-infected cells and the total lack of c-myc induction in dl2–36-infected cells suggested that PCAF might also contribute to c-myc repression. We also studied another E1A mutant in which aa 55–60 were changed to alanine (E55); it binds to PCAF at normal levels whereas it binds to p300 very weakly

(Lang and Hearing, 2003). As shown in Figure 2a, c-myc induction in E55-infected cells is reduced to about 50% of the WT, consistent with its weak interaction with p300. To ensure that E55 binding to PCAF in virusinfected MCF10A cells was unaffected, cells were coinfected with E55 and AdPCAF, lysed and immunoprecipitated with an anti-E1A antibody. The immunoprecipitated proteins were Western immunoblotted with Oncogene


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anti-FLAG antibody. As shown in Figure 2f, and in agreement with a previous report (Lang and Hearing, 2003), the amount of PCAF coimmunoprecipitated with anti-E1A antibody was comparable in WT- and E55infected cell lysates. A separate Western blot experiment confirmed that E55 binds to p300 very weakly (data not shown). Thus, the p300 and PCAF binding properties and the Myc induction capacity of RG2 and E55 mutants are very similar. Because RG2 and E55 retain almost normal capacity to bind to PCAF, and are defective for p300 binding, it suggests that E1A can bind to p300 and PCAF independently, which is in agreement with previous reports (Reid et al., 1998; Lang and Hearing, 2003). Further, these results also suggest that these two chromatin proteins repress c-myc independently. The repression effects may be additive as a mutant that does not bind either p300 or PCAF (dl2–36) lacks c-myc induction capacity completely.

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To determine whether E1A binding to different host proteins affects another early response gene, the RNA samples used in the c-myc experiments were analysed for c-jun expression using real-time PCR assay. The results presented in Figure 2b show that the induction levels of c-jun in Adb-gal-, WT- and the mutant-infected cells were all comparable (less than 1.5-fold difference). These results are consistent with our previous report (Baluchamy et al., 2003) and suggest that relief of repression of gene expression by E1A is not global. We also assayed Myc activity levels in virus-infected cells by infecting cells with WT and various E1A mutants along with a c-myc reporter Ad vector (AdM4; Baluchamy et al., 2003) and assaying luciferase activity at 14 h after infection. As shown in Figure 2c Myc activity in RG2- and E55-infected cells dropped to 38 and 42%, respectively. In cells infected with dl2–36 and 928, Myc activity levels dropped to control levels. The mutant dl26–35 that is defective for binding to

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0 al al al al yc Myc Myc Myc -g β-g β-g β-g β +M T+ + + T+ + l 2+ 6+ l a 6 W RG l2-3 -g ga W RG2 l2-3 β β d d Figure 4 Reversal of the S-phase induction defect in RG2- and dl2–36-infected cells by c-myc overexpression at 24 h post infection. Serum-starved cells were coinfected with Ad WT or the E1A mutants with Adc-myc that overexpress c-myc (Mitchell and El-Deiry, 1999). Experimental conditions were essentially as described earlier in the legend to Figure 2. Adb-gal was used where appropriate to keep the multiplicity of infection constant. The experiment was carried out in triplicate and the average values with s.d.’s are shown.

p400, TRRAP and GCN5 showed only a moderate decrease. Thus the Myc RNA levels detected in real-time PCR assay correlated with the Myc activity levels. To determine the capacity of the E1A mutants to induce S phase, serum-starved MCF10A cells were infected with different E1A mutants, and beginning with 20 h post-infection, cells in G1, S and G2/M fractions were quantified by flow cytometry. The number of cells in S phase at 20, 24 and 28 h time points is shown in Figure 2a and representative FACS profiles of these samples at 28 h are shown in Figure 3b. As evident, the WT- and dl26–35-infected cells showed a robust S-phase induction (about 38 and 34% at 28 h respectively, Figure 3a). In contrast, the S-phase induction by all other mutants was greatly reduced (5–10%). These results are in broad agreement with RNA and Myc activity levels (Figure 2a and c). Under these conditions, less than 10% of the virus-infected cells were in the sub G1 fraction (cell debris and apoptosing cells, Figure 3b), indicating that there was no significant apoptosis. As RG2 mutant was severely defective in inducing S phase in human cells, the additive effects, if any, by the second mutation that prevents binding to pRb (RG2.928) or PCAF (dl2–36) was not evident in these studies. We note here that the cell cycle defect displayed by the E1A mutants presented here is more severe than that reported earlier using primary BRK cells (Wang et al., 1993; Nees et al., 2000). For example, the RG2 or 928 mutations individually were moderately defective for DNA synthesis in BRK cells, whereas, the two mutations when combined together in one E1A molecule (RG2.928 mutant) led to total inactivation of DNA synthesis (Wang et al., 1993). In contrast, in

human MCF10 cells, DNA synthesis was severely affected when either of the sites is inactive (Figure 3a and b). This could be owing to more stringently controlled growth regulation of human cells compared to that of BRK cells. In summary, our results show that the inability of the mutants RG2, 928, E55 and dl2–36 to induce S phase in quiescent cells correlates with their impaired capacity to induce c-myc. To determine whether the cell cycle defect associated with RG2 and dl2–36 mutants is the result of reduced c-myc synthesis, we performed a Myc reversal experiment in which we infected cells with WT or the RG2 mutant along with an Ad vector that overexpresses c-myc (detailed in Kolli et al., 2001; also see the legend to Figure 4). Cells were harvested at 24 h after infection, and the distribution of cells in different cell cycle fractions was determined by flow cytometry. Data presented in Figure 4 indicate that overexpression of cmyc in RG2- and dl2–36-infected cells abrogated the cell cycle defect associated with these mutations. For example, at 24 h after infection without c-myc overexpression, about 35% of the WT-infected cells were in S phase, whereas only about 5–10% of the RG2- and dl2–36-infected cells moved to S phase. In contrast, when c-myc was overexpressed in these cells, the number of cells in the S-phase fraction was comparable between WT- and the mutant-infected cells. Note that all the c-myc-overexpressing samples moved into S phase faster than the corresponding control samples (table in Figure 4) presumably because of mitogenic effects of the Myc protein. In summary, our studies show that the capacity of E1A to induce c-myc in quiescent cells is significantly Oncogene


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impaired when it fails to bind to p300, Rb and PCAF, and this in turn affects its capacity to induce S phase. These interactions are not redundant because inactivation of any one of the above mentioned host protein binding sites in E1A results in significant loss of both cmyc and S-phase induction, indicating that c-myc repression by each of the chromatin-associated proteins is independent. The pocket family proteins in association with histone deacetylases block the E2F1 activity. The promoter proximal region of the c-myc promoter contains an E2F site, and E2F4/5 in association with p107 has been shown to repress this promoter in response to TGF-beta treatment (Chen et al., 2002). The mutation 928 selectively inactivates Rb binding, whereas its p107 binding is unaffected and binds to p130 at significant levels (Wang et al., 1993). Rb appears to actively participate in c-myc repression, which is consistent with our published data in which we showed that overexpression of Rb in quiescent MCF10A cells represses c-myc (Buchmann et al., 1998). A role for PCAF (first isolated as p300/CBP-associated factor) in Myc repression is intriguing and appears to be independent of that of p300, as E1A mutants with mutations that abrogate p300 but not PCAF binding (RG2 and E55) are significantly impaired in their capacity to induce c-myc. Further, overexpression of a mutant p300 that cannot bind PCAF represses c-myc as efficiently as the WT p300 (our unpublished results). It has been shown that purified p300 can repress transcrip-

tion in vitro (Santoso and Kadonaga, 2006). There are also several published reports that show that these two proteins can independently regulate gene expression (see for example, Puri et al., 1997). Currently, we do not know the mechanism by which p300 or PCAF represses c-myc. It is conceivable that pRb, p300 and PCAF may be present in the cell as a single or in separate complexes that are also associated with histone deacetylases. In quiescent cells, one or more Myc promoter-specific transcription factors may recruit these complexes and repress the promoter. Finally, the observation that dl26– 35 mutation has no significant effect on c-myc induction or DNA synthesis is interesting. This mutation abrogates E1A binding to p400, TRRAP and GCN5, and studies have shown that these proteins exist as a complex that modifies gene expression in the cell (Taubert et al., 2004). These data combined with 928, RG2 and dl2–36 results suggest that specific chromatin complexes may be involved in c-myc repression.

Acknowledgements This work was supported by the PHS grant CA74403. We are grateful to S Bayely (U McMaster, Canada) for providing certain adenovirus mutants, and W El-Deiry (U Pennsylvania) for providing Adc-myc. We sincerely apologize that because of space limitations, we could not include all the relevant references in this report.

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