The CCAAT Enhancer-binding Protein α (C/EBPα) Requires a SWI/SNF Complex for Proliferation Arrest
2004; Elsevier BV; Volume: 279; Issue: 8 Linguagem: Inglês
10.1074/jbc.m312709200
ISSN1083-351X
AutoresChristine Müller, Cornelis F. Calkhoven, Xiaojing Sha, Achim Leutz,
Tópico(s)interferon and immune responses
ResumoThe transcription factor CCAAT enhancer-binding protein α (C/EBPα) is a tumor suppressor in myeloid cells and inhibits proliferation in all cell types examined. C/EBPα interacts with the SWI/SNF chromatin-remodeling complex during the regulation of differentiation-specific genes. Here we show that C/EBPα fails to suppress proliferation in SWI/SNF defective cell lines after knock-down of SWI/SNF core components or after deletion of the SWI/SNF interaction domain in C/EBPα, respectively. Reconstitution of SWI/SNF function restores C/EBPα-dependent proliferation arrest. Our results show that the anti-proliferation activity of C/EBPα critically depends on components of the SWI/SNF core complex and suggest that the functional interaction between SWI/SNF and C/EBPα is a prerequisite for proliferation arrest. The transcription factor CCAAT enhancer-binding protein α (C/EBPα) is a tumor suppressor in myeloid cells and inhibits proliferation in all cell types examined. C/EBPα interacts with the SWI/SNF chromatin-remodeling complex during the regulation of differentiation-specific genes. Here we show that C/EBPα fails to suppress proliferation in SWI/SNF defective cell lines after knock-down of SWI/SNF core components or after deletion of the SWI/SNF interaction domain in C/EBPα, respectively. Reconstitution of SWI/SNF function restores C/EBPα-dependent proliferation arrest. Our results show that the anti-proliferation activity of C/EBPα critically depends on components of the SWI/SNF core complex and suggest that the functional interaction between SWI/SNF and C/EBPα is a prerequisite for proliferation arrest. Transcription factors involved in terminal differentiation are frequently associated with cell cycle arrest. A prototype transcription factor that induces differentiation and proliferation arrest is the CCAAT enhancer-binding protein α (C/EBPα). 1The abbreviations used are: C/EBP, CCAAT/enhancer binding protein; BCER, BALB/c mouse fibroblasts expressing the C/EBPαER fusion protein; Brm, Brahma; Brg1, Brahma related gene 1; cdk, cyclin-dependent kinase; C/EBPαER, C/EBPα estrogen receptor C/EBPαER, C/EBPα estrogen receptor fusion protein; CR1, conserved region 1; HA, hemagglutinin A; hBrm, human Brm; Rb, retinoblastoma; siRNA, small interfering RNA. 1The abbreviations used are: C/EBP, CCAAT/enhancer binding protein; BCER, BALB/c mouse fibroblasts expressing the C/EBPαER fusion protein; Brm, Brahma; Brg1, Brahma related gene 1; cdk, cyclin-dependent kinase; C/EBPαER, C/EBPα estrogen receptor C/EBPαER, C/EBPα estrogen receptor fusion protein; CR1, conserved region 1; HA, hemagglutinin A; hBrm, human Brm; Rb, retinoblastoma; siRNA, small interfering RNA. 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Genes Dev. 2001; 15: 3208-3216Crossref PubMed Scopus (158) Google Scholar, 41Calkhoven C.F. Müller C. Leutz A. Genes Dev. 2000; 14: 1920-1932PubMed Google Scholar). An EcoRI fragment containing the rat C/EBPαER (21Umek R.M. Friedman A.D. McKnight S.L. Science. 1991; 251: 288-292Crossref PubMed Scopus (571) Google Scholar) was cloned into the pBABEpuro vector (42Morgenstern J.P. Land H. Nucleic Acids Res. 1990; 18: 3587-3596Crossref PubMed Scopus (1895) Google Scholar). EcoRI fragments of wild type or the ATP-binding site mutant of h-Brm (HA-tagged) (43Muchardt C. Yaniv M. EMBO J. 1993; 12: 4279-4290Crossref PubMed Scopus (522) Google Scholar) were cloned into the pCDNA3 and pBABEpuro vectors. The Brahma and Ini1-specific small interfering RNA (siRNA) oligonucleotides were designed as described in (44Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3949) Google Scholar) and are as follows: Brahma upper strand, 5′-GAT CCC CAA AGG ACC TTG CCT GGC ATT TCA AGA GAA TGC CAG GCA AGG TCC TTT TTT TTG AAA-3′, and Brahma lower strand, 5′-AGC TTT TCC AAA AAA AAG GAC CTT GCC TGG CAT TCT CTT GAA ATG CCA GGC AAG GTC CTT TGG G-3′); Ini1 upper strand, 5′-GAT CCC CCA CGG CCC CGG CCT GGT AAT TCA AGA GAT TAC CAG GCC GGG GCC GTG TTT TTG GAA A-3′, and Ini1 lower strand, 5′-AGC TTT TCC AAA AAC ACG GCC CCG GCC TGG TAA TCT CTT GAA TTA CCA GGC CGG GGC CGT GGG G-3′. The double-stranded oligonucleotides were cloned into BglII- and HindIII-digested pSUPER vector (44Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3949) Google Scholar). Cell Culture—Cells were incubated in 5% CO2 at 37 °C. NIH3T3 (American Type Culture Collection), C33A, SW13, and Phoenix A cells in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Invitrogen) and BCER (BALB/c mouse fibroblasts expressing the C/EBPαER fusion protein) as described earlier (16Müller C. Alunni-Fabbroni M. Kowenz-Leutz E. Mo X. Tommasino M. Leutz A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7276-7281Crossref PubMed Scopus (53) Google Scholar). Transfected or retrovirally infected cells were cultivated with puromycin (2 μg/ml) or G-418 (0.8 mg/ml). Transfection Methods and Colony Staining—For stable expression, cells (5 × 104) were transfected with 5 μg of DNA using calcium phosphate. Antibiotic-resistant colonies were pooled for protein expression analysis or fixed with 4% para-formaldehyde for 1 h and stained with Diff-Quick (Merz und Dade AG) to determine colony numbers. Gene Porter transfection reagent (Gene Therapy Systems) was used with 1 × 105 BCER cells and 10 μg of pSUPER-based vector plus 1 μg pBABEpuro in siRNA experiments. After 1-2 weeks of puromycin selection, single clones were isolated and analyzed for Brahma or Ini1 expression by Western blotting. For transient expression, 1 × 106 cells were transfected using calcium phosphate. 34-38 h later, cells were harvested. Reporter Assay—1 × 106 cells from C33A clones retrovirally infected with C/EBPαER-pBABEpuro or with the empty pBABEpuro were transfected with 1 μg of the C/EBP-responsive M82-luciferase construct as described earlier (45Sterneck E. Müller C. Katz S. Leutz A. EMBO J. 1992; 11: 115-126Crossref PubMed Scopus (72) Google Scholar). Transfection of each clone was done in parallel with four plates, two of which received β-estradiol (1 μm), whereas the other two received the solvent only. 36 h after transfection, cells were harvested for luciferase assay (45Sterneck E. Müller C. Katz S. Leutz A. EMBO J. 1992; 11: 115-126Crossref PubMed Scopus (72) Google Scholar). Retroviral Infection—Phoenix A cells were transiently transfected using calcium phosphate. 48 h after transfection, 5 × 105 target cells were infected and selected as described (46Pear W.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Crossref PubMed Scopus (2291) Google Scholar). Growth Curves—Cells (1 × 104 cells per well) were seeded in phenol red-free Dulbecco's modified Eagle's medium, and either β-estradiol (1 μm) or solvent was added. Every 24 h cells were harvested, and the number of living cells was determined by trypan blue exclusion. Western Blotting—Proteins were separated on 12, 10, or 8% SDS-polyacrylamide gels and blotted on polyvinylidene difluoride membrane (Immobilon-P, Millipore). Proteins were detected using antibodies against C/EBPα (14AA), Brm (N-19 and C-20), Ini1 (H-300), and α-Tubulin (TU-02) (0.5 μg/ml each, all from Santa Cruz Biotechnology Inc.), anti-HA (1:1000, Babco), and appropriate horseradish peroxidase-conjugated secondary antibodies (anti-rabbit, 1:5000 and anti-mouse, 1:5000, both from Amersham Biosciences, and anti-goat, 1:2000, from Santa Cruz Biotechnology Inc.) and detected by chemiluminescence (ECL, Amersham Biosciences). Alternatively, fluorochrome-conjugated secondary antibodies were used (goat anti-mouse IgG (H+L) and goat anti-rabbit IgG (H+L), both from Alexa Fluor, each in a dilution of 1:5000), and the blots were analyzed with the Odyssey-Imager (Li-COR). The anti-proliferative activity of C/EBPα and various C/EBPα mutants (Fig. 1A) was determined in a colony assay using mouse NIH3T3 fibroblasts and the SWI/SNF-defective human cell lines C33A and SW13 (43Muchardt C. Yaniv M. EMBO J. 1993; 12: 4279-4290Crossref PubMed Scopus (522) Google Scholar). As shown in Table I, full-length C/EBPα (C/EBPα FL) inhibits colony formation in NIH3T3 but not in SWI/SNF-defective cells. Removal of the SWI/SNF interaction domain in the center of C/EBPα (C/EBPα Δ126-200) (31Pedersen T.A. Kowenz-Leutz E. Leutz A. Nerlov C. Genes Dev. 2001; 15: 3208-3216Crossref PubMed Scopus (158) Google Scholar) largely abrogates the inhibitory effect on colony formation, suggesting a general contribution of SWI/SNF in C/EBPα-mediated proliferation arrest. Notably, when SWI/SNF recruitment of the internal deletion mutant was restored by adding the heterologous SWI/SNF recruiting domain of C/EBPβ (Δ126-200+CR1; CR1 was derived from the C/EBPβ N terminus; Refs. 31Pedersen T.A. Kowenz-Leutz E. Leutz A. Nerlov C. Genes Dev. 2001; 15: 3208-3216Crossref PubMed Scopus (158) Google Scholar and 47Kowenz-Leutz E. Leutz A. Mol. Cell. 1999; 4: 735-743Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar), the ability to suppress colony formation in NIH3T3 cells was also restored. In SWI/SNF-defective cells the same chimeric C/EBP protein, like the full-length C/EBPα, fails to suppress proliferation. The C/EBPα p30 isoform, which lacks the major N-terminal transactivation function, has been shown previously to be defective in the activation of genes and the suppression of proliferation (17Porse B.T. Pedersen T.A. Xu X. Lindberg B. Wewer U.M. Friis-Hansen L. Nerlov C. Cell. 2001; 107: 247-258Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 41Calkhoven C.F. Müller C. Leutz A. Genes Dev. 2000; 14: 1920-1932PubMed Google Scholar, 48Lin F.T. MacDougald O.A. Diehl A.M. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9606-9610Crossref PubMed Scopus (258) Google Scholar). In a control experiment, C/EBPα p30 was expressed and found to be unable to inhibit proliferation in both NIH3T3- and SWI/SNF-defective cells (Table I).Table ISuppression of colony formation by C/EBP α depends on SWI/SNFNIH3T3C33ASW13Empty vector71.0 ± 3.6143.3 ± 9.3131.3 ± 8.2C/EBP α FL24.3 ± 4.9124 ± 7112.7 ± 8Δ 126-20053.3 ± 5130.6 ± 15.9NDΔ 126-200 + CR124.7 ± 2.1133.3 ± 11.9NDC/EBP α p3075.3 ± 7.7127.3 ± 6.8105 ± 12.2 Open table in a new tab A comparison of C/EBPα protein expression levels after transient and stable transfection in NIH3T3 fibroblasts and SWI/SNF-defective cells further supports the idea that C/EBPα-induced proliferation arrest depends on interaction with SWI/SNF. As shown in Fig. 1B, C/EBPα proteins are expressed at equal levels in NIH3T3 cells shortly after transfection (2 days). After 2 weeks, however, only traces of full-length C/EBPα or C/EBPα Δ126-200 + CR1 were found in NIH3T3 cells, suggesting counter selection, whereas expression of C/EBPα Δ126-200 and p30 remained high. In contrast, expression from all constructs was maintained in the SWI/SNF-defective C33A cells (Fig. 1B) or SW13 cells (data not shown), indicating that, in the absence of SWI/SNF, C/EBPα expression does not restrain proliferation. These results show that the SWI/SNF interaction domain of C/EBPα and a functional SWI/SNF complex are required for efficient induction of proliferation arrest. Furthermore, SWI/SNF-defective cells are the first cells identified that tolerate high levels of C/EBPα expression during proliferation. In addition to the SWI/SNF defect, C33A and SW13 cells might have accumulated other mutations that may help to overcome C/EBPα-induced proliferation arrest. To examine this possibility, SWI/SNF-defective cells were reconstituted with intact hBrm and with a conditional version of C/EBPα (16Müller C. Alunni-Fabbroni M. Kowenz-Leutz E. Mo X. Tommasino M. Leutz A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7276-7281Crossref PubMed Scopus (53) Google Scholar, 21Umek R.M. Friedman A.D. McKnight S.L. Science. 1991; 251: 288-292Crossref PubMed Scopus (571) Google Scholar). First, the hormone-inducible C/EBPα estrogen receptor fusion protein, C/EBPαER, was introduced by retroviral gene transfer into C33A cells. After confirming the expression and transcriptional function of the C/EBPαER fusion protein by Western blotting and reporter assay (see "Experimental Procedures" for details) (45Sterneck E. Müller C. Katz S. Leutz A. EMBO J. 1992; 11: 115-126Crossref PubMed Scopus (72) Google Scholar), proliferation was found to be insensitive to estrogen (data not shown). Stable expression of hBrm, however, rendered these cells sensitive to estrogen-induced proliferation arrest as shown in Fig. 2A (left panel). In contrast, expression of a dominant negative hBrm mutant (defective in its ATPase function; Refs. 43Muchardt C. Yaniv M. EMBO J. 1993; 12: 4279-4290Crossref PubMed Scopus (522) Google Scholar and 49Khavari P.A. Pedersen C.L. Tamkun J.W. Mendel D.B. Crabtree G.R. Nature. 1993; 366: 170-174Crossref PubMed Scopus (533) Google Scholar) instead of wild type hBrm was unable to restore the anti-proliferative C/EBPα activity (Fig. 2A, right panel). Fig. 2B shows that C33A cells expressing hBrm in the absence of conditional C/EBPα are also not estrogen-sensitive, ruling out the possibility that hBrm mediates estrogen sensitivity independently of the C/EBPαER construct. Thus, failure of C/EBPα to arrest proliferation of C33A cells is due to the lack of the functional SWI/SNF ATPase Brm. Interestingly, C33A cells are also defective in the tumor suppressor Rb (50Scheffner M. Munger K. Byrne J.C. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5523-5527Crossref PubMed Scopus (759) Google Scholar), supporting previous studies (13Hendricks-Taylor L.R. Darlington G.J. Nucleic Acids Res. 1995; 23: 4726-4733Crossref PubMed Scopus (113) Google Scholar, 14Johansen L.M. Iwama I. Lodie T.A. Sasaki K. Felsher D.W. Golub T.R. Tenen D.G. Mol. Cell. Biol. 2001; 21: 3789-3806Crossref PubMed Scopus (221) Google Scholar, 16Müller C. Alunni-Fabbroni M. Kowenz-Leutz E. Mo X. Tommasino M. Leutz A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7276-7281Crossref PubMed Scopus (53) Google Scholar, 17Porse B.T. Pedersen T.A. Xu X. Lindberg B. Wewer U.M. Friis-Hansen L. Nerlov C. Cell. 2001; 107: 247-258Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 19Slomiany B.A. D'Arigo K.L. Kelly M.M. Kurtz D.T. Mol. Cell. Biol. 2000; 20: 5986-5997Crossref PubMed Scopus (144) Google Scholar) that have shown that a functional Rb is not required for the proliferation-suppressive activity of C/EBPα. BALB/c mouse fibroblasts that stably express the C/EBPαER protein were previously shown to respond to estrogen with proliferation arrest (BCER cells; Ref. 16Müller C. Alunni-Fabbroni M. Kowenz-Leutz E. Mo X. Tommasino M. Leutz A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7276-7281Crossref PubMed Scopus (53) Google Scholar). These cells were employed to independently assess the dependence on Brm and SWI/SNF for proliferation arrest in a different cell type. As shown in Fig. 3, expression of the dominant negative hBrm mutant abrogates estrogen-induced proliferation arrest in BCER cells. Thus, functional Brm, in conjunction with C/EBPα, is also required in BALB/c fibroblasts to inhibit proliferation. Similar results were obtained when Brm protein expression was knocked down by RNA interference in BCER cells, as shown in Fig. 4. A Brm-specific siRNA expressed from the pSUPER vector (44Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3949) Google Scholar) diminished the Brm protein level and rendered BCER cells unresponsive to estrogen (Fig. 4A). Interestingly, knock-down of Brm was transient, and the protein reappeared after 3-5 weeks, probably because the interfering RNA, expressed from a non-selectable plasmid, was lost upon prolonged cultivation. As shown in Fig. 4B, the recurrence of endogenous Brm expression coincided with the restoration of C/EBPα-mediated proliferation arrest after estrogen induction. These results show that the transient down-regulation of Brm expression resulted in the reversible abrogation of C/EBPα-induced proliferation arrest. Brm is an essential component of a subgroup of SWI/SNF complexes, but it might also display proliferation control activity independently of the complex. To determine whether the SWI/SNF complex is required for C/EBPα-induced proliferation arrest, we knocked down expression of Ini1/SNF5, another essential SWI/SNF component (51Geng F. Cao Y. Laurent B.C. Mol. Cell. Biol. 2001; 21: 4311-4320Crossref PubMed Scopus (55) Google Scholar, 52Phelan M.L. Sif S. Narlikar G.J. Kingston R.E. Mol. Cell. 1999; 3: 247-253Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar), by RNA interference. Initially, 12 BCER clones were isolated that expressed reduced Ini1 protein levels, as examined by Western blotting (data not shown, and Fig. 5). Notably, all Ini1/SNF5 knock-down clones displayed significantly prolonged doubling times that might be due to enhancement of apoptosis in the absence of Ini1 (53Roberts C.W. Leroux M.M. Fleming M.D. Orkin S.H. 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Importantly, we could restore the anti-proliferative C/EBPα function in C33A cells by ectopic expression of hBrm. This ruled out the possibility that other defects that might have accumulated in the tumor cell line contributed to the loss of the anti-proliferative activity of C/EBPα. The strict SWI/SNF dependence of C/EBPα-mediated proliferation arrest was also found in BALB/c mouse fibroblasts. Reducing functional Brm levels by RNA interference or through competitive ectopic expression of a dominant negative Brm mutant resulted in loss of C/EBPα-mediated proliferation arrest. Because the knock-down of Brm by siRNA in BALB/c cells was only transient, we also obtained a rigorous and conditional control that linked C/EBPα-mediated proliferation arrest to endogenous Brm expression. Furthermore, we did not detect any residual anti-proliferative activity of C/EBPα in the investigated cells in the absence of Brm/SWI/SNF. Hence, our data imply that Brm and/or SWI/SNF is involved in all the mechanisms proposed to mediate C/EBPα-induced proliferation arrest. In addition to its function in the SWI/SNF complex, Brm might also be involved in the regulation of the cell cycle through other mechanisms. Although we can not rule out the possibility that multiple Brm-based mechanisms are simultaneously involved in C/EBPα-mediated cell cycle arrest, it is intriguing that elimination of Ini1/SNF5, another essential component of the SWI/SNF complex (51Geng F. Cao Y. Laurent B.C. Mol. Cell. Biol. 2001; 21: 4311-4320Crossref PubMed Scopus (55) Google Scholar, 52Phelan M.L. Sif S. Narlikar G.J. Kingston R.E. Mol. Cell. 1999; 3: 247-253Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar), also abrogated the anti-proliferative C/EBPα activity. The data therefore suggest that a Brm-based SWI/SNF complex is involved in C/EBPα-induced proliferation arrest. In a subset of mammalian SWI/SNF complexes, Brg1 is the ATPase subunit instead of Brm (49Khavari P.A. Pedersen C.L. Tamkun J.W. Mendel D.B. Crabtree G.R. Nature. 1993; 366: 170-174Crossref PubMed Scopus (533) Google Scholar, 54Chiba H. Muramatsu M. Nomoto A. Kato H. Nucleic Acids Res. 1994; 22: 1815-1820Crossref PubMed Scopus (287) Google Scholar). Brm and Brg1 are highly homologous proteins and were found to be at least partially redundant in the regulation of the cell cycle in several studies in cell culture (32Dunaief J.L. Strober B.E. Guha S. Khavari P.A. Alin K. Luban J. Begemann M. Crabtree G.R. Goff S.P. Cell. 1994; 79: 119-130Abstract Full Text PDF PubMed Scopus (551) Google Scholar, 35Strober B.E. Dunaief J.L. Guha S. Goff S.P. Mol. Cell. Biol. 1996; 16: 1576-1583Crossref PubMed Scopus (225) Google Scholar, 36Trouche D. Le Chalony C. Muchardt C. Yaniv M. Kouzarides T. Proc. Natl. Acad. Sci. U. S. 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Cell. 2000; 6: 1287-1295Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). We and others have observed that C/EBPs may interact with both Brm- and Brg1-based SWI/SNF complexes to activate differentiation-specific genes (31Pedersen T.A. Kowenz-Leutz E. Leutz A. Nerlov C. Genes Dev. 2001; 15: 3208-3216Crossref PubMed Scopus (158) Google Scholar, 47Kowenz-Leutz E. Leutz A. Mol. Cell. 1999; 4: 735-743Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 58Kadam S. Emerson B.M. Mol. Cell. 2003; 11: 377-389Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). This raises the question of whether Brg1 is also able to cooperate with C/EBPα for its proliferation-suppressing function. In the present study, the down-regulation of Brm in BALB/c fibroblasts was sufficient to abrogate C/EBPα-mediated cell cycle arrest, although the expression of Brg1 was not affected by the Brm-specific siRNA (data not shown). This suggests that Brg1 can not substitute for Brm in the anti-proliferative C/EBPα-SWI/SNF complex, pointing at a Brm-specific role in proliferation arrest. Experiments designed to address a role of Brg1 directly, however, failed because C33A cells that stably express Brg1 could not be obtained, probably because of cell toxicity, as was also observed by others (32Dunaief J.L. Strober B.E. Guha S. Khavari P.A. Alin K. Luban J. Begemann M. Crabtree G.R. Goff S.P. Cell. 1994; 79: 119-130Abstract Full Text PDF PubMed Scopus (551) Google Scholar). The possible existence of SWI/SNF complexes with different compositions that interact with C/EBPα brings up the question of whether differentiation and proliferation are controlled by the same or by different C/EBPα-SWI/SNF complexes. Of note in this context are data from our previous study showing that C/EBPα-induced proliferation arrest and differentiation can be uncoupled from each other by human papilloma virus E7 oncoproteins from the high malignancy strains HPV16 and HPV18 (16Müller C. Alunni-Fabbroni M. Kowenz-Leutz E. Mo X. Tommasino M. Leutz A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7276-7281Crossref PubMed Scopus (53) Google Scholar). These oncogenes are also the only ones identified to date that overcome C/EBPα-mediated proliferation arrest, and they do so in a Rb/pocket protein-independent fashion. Furthermore, we observed that abrogation of C/EBPα-mediated proliferation arrest by HPV16E7 simultaneously enhanced C/EBPα-induced differentiation (16Müller C. Alunni-Fabbroni M. Kowenz-Leutz E. Mo X. Tommasino M. Leutz A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7276-7281Crossref PubMed Scopus (53) Google Scholar). A tempting hypothesis, therefore, is that E7 interferes with the function of a "proliferation control SWI/SNF complex" to the benefit of a "differentiation control SWI/SNF complex." Brm as well as Brg1 interact with the tumor suppressor Rb, and both are involved in Rb-mediated proliferation arrest (32Dunaief J.L. Strober B.E. Guha S. Khavari P.A. Alin K. Luban J. Begemann M. Crabtree G.R. Goff S.P. Cell. 1994; 79: 119-130Abstract Full Text PDF PubMed Scopus (551) Google Scholar, 34Strobeck M.W. Knudsen K.E. Fribourg A.F. De Cristofaro M.F. Weissman B.E. Imbalzamo A.N. Knudsen E.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7748-7753Crossref PubMed Scopus (209) Google Scholar, 35Strober B.E. Dunaief J.L. Guha S. Goff S.P. Mol. Cell. Biol. 1996; 16: 1576-1583Crossref PubMed Scopus (225) Google Scholar, 36Trouche D. Le Chalony C. Muchardt C. Yaniv M. Kouzarides T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11268-11273Crossref PubMed Scopus (261) Google Scholar, 37Zhang H.S. Gavin M. Dahiya A. Postigo A.A. Ma D. Luo R.X. Harbour J.W. Dean D.C. Cell. 2000; 101: 79-89Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar). C/EBPα can also interact with Rb (59Chen P.-L. Riley D.J. Chen Y. Lee W.-H. Genes Dev. 1996; 10: 2794-2804Crossref PubMed Scopus (390) Google Scholar), and a complex comprising C/EBPα, Rb, E2F4, and Brm was suggested to repress E2F target genes (30Iakova P. Awad S.S. Timchenko N.A. Cell. 2003; 113: 495-506Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). However, a C/EBPα mutant that lacks its Rb interaction domain is still proliferation inhibitory (17Porse B.T. Pedersen T.A. Xu X. Lindberg B. Wewer U.M. Friis-Hansen L. Nerlov C. Cell. 2001; 107: 247-258Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Furthermore, C/EBPα efficiently induces proliferation arrest also in Rb-deficient cells (13Hendricks-Taylor L.R. Darlington G.J. Nucleic Acids Res. 1995; 23: 4726-4733Crossref PubMed Scopus (113) Google Scholar, 14Johansen L.M. Iwama I. Lodie T.A. Sasaki K. Felsher D.W. Golub T.R. Tenen D.G. Mol. Cell. Biol. 2001; 21: 3789-3806Crossref PubMed Scopus (221) Google Scholar, 19Slomiany B.A. D'Arigo K.L. Kelly M.M. Kurtz D.T. Mol. Cell. Biol. 2000; 20: 5986-5997Crossref PubMed Scopus (144) Google Scholar). We could restore the anti-proliferative potential of C/EBPα in C33A cells that lack functional Rb solely through ectopic expression of Brm. Therefore, our data support the view that Rb is not an essential requirement for C/EBPα-mediated proliferation arrest. A particularity of C/EBPα is that full-length and truncated (p30) isoforms are expressed through differential translation initiation (41Calkhoven C.F. Müller C. Leutz A. Genes Dev. 2000; 14: 1920-1932PubMed Google Scholar, 60Ossipow V. Descombes P. Schibler U. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8219-8223Crossref PubMed Scopus (322) Google Scholar). Several studies have demonstrated that the C/EBPα p30 isoform, which lacks the major transactivation domain, fails to suppress proliferation (17Porse B.T. Pedersen T.A. Xu X. Lindberg B. Wewer U.M. Friis-Hansen L. Nerlov C. Cell. 2001; 107: 247-258Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 41Calkhoven C.F. Müller C. Leutz A. Genes Dev. 2000; 14: 1920-1932PubMed Google Scholar, 48Lin F.T. MacDougald O.A. Diehl A.M. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9606-9610Crossref PubMed Scopus (258) Google Scholar). Nevertheless, the C/EBPα p30 isoform can still interact with SWI/SNF (31Pedersen T.A. Kowenz-Leutz E. Leutz A. Nerlov C. Genes Dev. 2001; 15: 3208-3216Crossref PubMed Scopus (158) Google Scholar). Therefore, a supplementary transcriptional activity may be required for effective induction of proliferation arrest by the full-length C/EBPα. Alternatively, additional functions, such as inhibition of E2F-mediated transcription (17Porse B.T. Pedersen T.A. Xu X. Lindberg B. Wewer U.M. Friis-Hansen L. Nerlov C. Cell. 2001; 107: 247-258Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), may reside in the transactivation domain that still have to be explored in greater detail. In summary, data presented here and elsewhere suggest that both proliferation and differentiation control by C/EBPα are tightly associated with SWI/SNF functions. We thank Christian Muchardt and Moshe Yaniv for human Brm constructs, Claus Nerlov for the C/EBPα Δ126-200 mutant construct, Walter Birchmeier and Jürgen Behrens for C33A and SW13 cells, Reuven Agami, Rene Bernards, and Thijn R. Brummelkamp for providing the pSUPER vector, and Garry Nolan for the Phoenix A cells.
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