Article in press - uncorrected proof Horm Mol Biol Clin Invest 2011;5(1):11–15 2011 by Walter de Gruyter • Berlin • New York. DOI 10.1515/HMBCI.2010.074
The corepressor Alien as a novel tumor suppressor?
Aria Baniahmad* Institute of Human Genetics, Jena University Hospital, Jena, Germany
Abstract Alien has been characterized as a corepressor for nuclear hormone receptors that harbor a silencing domain such as the thyroid hormone receptor (TR), the vitamin D3 receptor (VDR) and DAX-1. In addition, the androgen receptor (AR), a steroid hormone receptor, interacts with Alien. Alien enhances gene silencing mediated by TR, VDR and DAX-1, whereas Alien inhibits AR-mediated transactivation. The inhibition of AR by Alien seems to be restricted to cases where AR is bound to AR antagonists. In line with this, Alien inhibits AR target gene expression and human prostate cancer cell proliferation in an antagonist-specific manner indicating that Alien has an inhibitory role for cell cycle progression. Alien mediates gene silencing by recruitment of histone deacetylase activity and interestingly through nucleosome assembly activity. Hereby, Alien enhances nucleosome positioning mediated by nucleosome assembly protein 1, which suggests a novel molecular mechanism of corepressor function. Using a proteomic approach to identify Alien interacting partners, we detected the cell cycle factor E2F1 to bind to Alien in vivo. The E2F1-mediated transactivation and E2F target gene expression is inhibited by Alien, and in line with this Alien is observed to repress cell cycle progression. Keywords: cell cycle; corepressor; E2F-transcription factors; interactom; retinoblastoma; signalosome subunit CSN2.
Introduction Originally, corepressors were defined as proteins that interact with DNA-bound transcriptional silencers to mediate and/or enhance their transrepression. It emerges, however, that corepressors also bind to many transcription factors to modulate and to reduce their transactivation. Moreover, corepressors are associated with histone deacetylase (HDAC) and histone methyltransferase (HMT) activity not only deacetylate or methylate histones but also other regulatory factors, for which modifications such as by acetylation or methylation, control their functionality (1–3). Thus, it seems *Corresponding author: Aria Baniahmad, Institute of Human Genetics, Jena University Hospital, D-07743 Jena, Germany Phone: q49-3641-935501, Fax: q49-3641-935502, E-mail: [email protected] Received November 15, 2010; accepted November 16, 2010; previously published online March 4, 2011
that corepressors play an important role not only directly at transcriptional and chromatin levels. The regulation of corepressor interaction with transcription factors is essential for proper gene regulation (4). Here, six mechanisms have been shown to modulate the interaction such as acetylation, phosphorylation, nuclear localization, competition, the specificity of the DNA sequence and in the case of nuclear hormone receptors the type of ligand (4). Receptor agonists switch nuclear receptors often into transcriptional activators, whereas antagonists inhibit the receptormediated transactivation. For the estrogen and androgen receptors (ER, AR), the model is that agonists of these receptors induce the binding of coactivators, whereas receptor antagonists promote the binding of corepressors. Both agonist-bound estrogen or androgen receptors promote the proliferation of breast and prostate, respectively, in a hormonally controlled manner. In addition, the tumors of these tissues respond initially mostly well to these hormones. Therefore, both receptors are important drug targets for which specific antagonists have been developed that are clinically in use and inhibit cancer proliferation. Thus, the agonist-bound AR and ER activate the cell cycle in a hormone-dependent manner, which can be inhibited by antagonists. Eventually, resistance to hormone therapy by the treatment with antagonists occurs, representing a major drawback for patients. Resistance to prostate cancer (PCa) therapy can occur within the first 1–2 years of treatment (5). Several mechanisms have been proposed, of which corepressors malfunctioning seems to be a major cause (6, 7). AR amplifications in refractory and castration-resistant PCa lead to less corepressor recruitment to chromatin indicating a weaker repression of AR target genes. Furthermore, activation of signal transduction by membrane-bound receptors such as tyrosine kinases or protein kinase A leads to dissociation of corepressors from the AR (8, 9). Similarly, the ER dissociates from corepressors by activated signaling pathways such as activated Her/neu and EGF receptors in the presence of ER antagonists (tamoxifen) (10). Thus, the efficacy of cancer treatment with nuclear receptor antagonists seems to depend on functional corepressors. The molecular mechanism of how corepressors inhibit cancer growth is not yet fully understood. Both the androgen-bound AR and the estrogen-bound ER not only activate gene expression but also repress important genes. The catalytic subunit of human telomerase, for example, being an essential factor for immortalization of tumor cells is repressed by the agonist-bound AR. The AR is recruited to the hTERT promoter sequences in vivo and represses hTERT expression and telomerase activity in an androgen-dependent manner (11). However, it is not known how a coactivator loaded agonist-bound receptor lead to gene repression. More-
2010/054
Article in press - uncorrected proof 12 Baniahmad: Alien as a tumor suppressor?
over, the role of corepressors for this type of gene regulation is yet unclear. Moreover, the specificity of corepressors needs to be addressed. The AR interaction data suggest that the corepressor Alien binds in the presence of AR antagonist to the AR, whereas for the SMRT corepressor binding was also observed in the presence of agonist (8, 12). However, SMRT binding does not necessarily lead to repression of AR, because SMRT inhibited the AR-mediated transactivation only in the presence of AR antagonists. This indicates that corepressor function can be regulated by the type of the ligand and that binding to transcription factors itself is not sufficient for corepressor functionality (8). It also suggests that different corepressors act in a distinct manner. This is in line with observations that several corepressors bind specifically to agonist-bound nuclear receptors (13). The corepressor Alien was named based on its high sequence conservation to the Drosophila Alien protein (14). Mammalian Alien was characterized as a factor with corepressor function for the thyroid hormone receptor (TR) and vitamin D3 receptor (VDR). Both receptors are known to regulate cell proliferation and differentiation. Later it was shown that Alien is an isoform of the CSN2 subunit that can be complexed in the COP9 signalosome (15), a highly conserved multifunctional protein complex. Mostly the COP9 function to regulate protein stability was investigated and more recently it was suggested that COP9 signalosome is involved in checkpoint control, signal transduction, development and the cell cycle. Recent evidence suggests that the COP9 complex is also associated with chromatin (16, 17). Interestingly, the expression of Alien is upregulated by thyroid hormone in brain in vivo (15). Taken together, it suggests that Alien is associated with factors that regulate cell cycle proliferation. Here, an overview is provided about the identified mechanisms of how Alien mediates gene repression and its role in cell proliferation.
Novel mechanisms of Alien-mediated repression by nucleosome assembly Alien mediates gene silencing in part by recruiting HDAC activity (14). This is presumably mediated by the recruitment of mSIN3A to Alien (18). It was shown that Alien and Sin3A exhibit a dynamic corecruitment to chromatin sites of VDR responsive gene CYP24 in vivo. The CYP24 gene is repressed in the absence of vitamin D3 and activated by the hormone through two vitamin D3 response elements (VDREs) located in the promoter region. Accordingly, the histone H4 at these VDREs is acetylated within 1 h of hormone treatment. Interestingly, although hormone is still present the level of acetylation of histone H4 at the VDREs is decreased within a few hours suggesting that HDAC activity is recruited to these VDREs. In line with these observations, in the absence of vitamin D3 mSIN3A and Alien are corecruited specifically to the VDREs of the CYP24 promoter (18). SIN3A exists in a large protein complex that contains
HDAC activity. Addition of ligand leads to dissociation of both Alien and mSIN3A from the VDREs (18). However, within 60 min both Alien and SIN3A are corecruited again in a cycling manner despite the presence of hormone. This suggests that the observed deacetylation of histone H4 at the vicinity of the VDREs might be mediated by the recruitment of Alien and mSIN3A and the associated HDAC activity. This indicates that corepressors also play a gene regulatory role in the presence of hormones such as turning down gene expression after a limited time period of hormone-induced gene activation. Interestingly, a novel mechanism of corepressors was postulated by Alien-mediated gene repression. Nucleosome assembly on template DNA was shown to inhibit transcription in various assay systems (19). It is thought that nucleosome assembly inhibits the DNA access of general transcription factors, transcription initiation factors and thus the recruitment of the RNA polymerases leading to gene silencing. One factor known to assemble nucleosomes onto DNA is the nucleosome assembly protein 1 (NAP1) (20). NAP1 functions as a histone chaperone, binding to the histones H2A–H2B dimers and H3–H4 tetramers (21, 22). NAP1 also modulates binding of linker histone H1, functions as a histone exchanger and as an ATP-independent histone-sliding factor (23). In Drosophila, a knockout of NAP1 leads to embryonic lethality and in yeast knockout experiments to significant increase in atypical histone-DNA complexes, as well as in deregulated transcription activation and repression (24). Using a yeast two-hybrid screen with Alien as bait, we identified various independent cDNA clones encoding NAP1 spanning a central region of NAP1 (25). The interaction of Alien with NAP1 was verified in vitro and in vivo. Interestingly, Alien modulated the nucleosome assembly activity of NAP1 by enhancing nucleosome assembly on DNA templates. Furthermore, Alien reduces the accessibility of the histones H3 and H4 for NAP1-promoted assembly reaction. This indicates that Alien sustains and reinforces the formation of nucleosomes. Employing deletion mutants of Alien suggest that different regions of Alien are involved in enhancement of NAP1-mediated nucleosome assembly and in inhibiting the accessibility of the histones H3/H4 (25). Using the VDR for which Alien was shown to act as a corepressor (18), a hormone-induced transcription system was chosen. Chromatin immunoprecipitation experiments (ChIP and re-ChIP) suggest that NAP1 and Alien are complexed and are corecruited in a vitamin D3-induced manner to the endogenous VDR target gene CYP24 promoter in vivo (25). These data suggest a novel pathway with Alien to bind to NAP1 and to enhance/enforce nucleosome assembly, thus linking corepressor function with nucleosome assembly activity.
Inhibition of PCa cell growth by Alien Alien was shown to interact with the AR seemingly in an AR antagonist-specific manner (26). Interestingly, AR ago-
Article in press - uncorrected proof Baniahmad: Alien as a tumor suppressor? 13
nists such as dihydrotestosterone (DHT) or R1881 did not promote binding of Alien to the AR indicating a ligand-specific induced interaction with AR. This could be due to conformational changes of the AR. One possibility is that agonists induce an intramolecular N-/C-terminal interaction, whereas AR antagonists do not and thus the N-terminus of AR might be better accessible (27). Analyzing the interaction sites of Alien with AR suggests that Alien binds to the AR/ N-terminus in the presence of antagonists explaining the observed ligand-specific interaction. Interestingly, the SUMOylation sites located in the AR/N-terminus are required for Alien/AR interaction because mutation of the SUMO sites of AR abrogates binding of Alien to the AR (26). Using the androgen-dependent LNCaP cells as a model for androgen-promoted PCa growth, the role of Alien on endogenous AR target gene expression and cell proliferation was analyzed by stable Alien expression. In line with the ligand-specific interaction of Alien with the AR, it was observed that Alien inhibited the prostate specific antigen gene expression as well as the androgen-dependent LNCaP cell growth in an AR antagonist-dependent manner. By contrast, in the presence of AR agonists, Alien reduced cell growth only marginally (26). This indicates that Alien inhibits in an antagonist-specific manner AR and AR-controlled PCa cell proliferation.
Alien interacts with evolutionarily conserved factors regulating cell proliferation Alien/CSN2 is a highly conserved factor present in multicellular organisms from plants to Drosophila and humans. Alien mediates gene silencing not only for mammalian nuclear receptors possessing a silencing domain. Drosophila Alien interacts with nuclear hormone receptors such as the ecdysone receptor (14). Interestingly, nuclear hormone receptors such as COUP-TFI and -TFII, to which Alien binds, exhibit a considerable sequence homology between Drosophila (svp) and human with up to 90% identity in amino acid sequence. Similarly, the NAP1 sequence is conserved in evolution sharing high homology between human and Drosophila NAP1, indicating that Alien interacts with evolutionary conserved factors. Further insights of Alien-mediated corepressor function were gained by using an in vivo proteomic approach to identify novel Alien interacting partners (28–30). Interestingly, conserved cell cycle factors were among proteins that interact with Alien. Specifically, among those factors, the E2F1 transcriptional activator was identified. Alien inhibited E2F1-mediated transactivation, was corecruited to chromatic E2F1 sites in vivo and repressed E2F target gene expression. E2F1 is a promoter of cell cycle progression. In line with this, using a tetracycline inducible system cell proliferation was reduced in a doxycycline-dependent manner. We noted that cells did not accumulate in a specific-cell cycle phase. This could be due to the observation that the protein levels of Alien itself change within the cell cycle phases (28–30).
Another explanation could be that Alien also interacts with other E2F members including the inhibitory E2F family members, which could result in an overall mixed effect so that the Alien-mediated inhibition of cell proliferation is due to a general inhibition of the cell cycle phases. Taken together, this indicates that Alien inhibits cell cycle promoting factors and inhibits cell proliferation. The proteomic approach delivered further information. Other interesting cell cycle regulators were identified as interacting partners of Alien. Both tumor suppressors p33ING1 and p33ING2 interact with Alien in vivo (31). Both factors are associated with the phenomenon of cellular senescence. Cellular senescence arrests the cell growth in an irreversible manner. The cells, however, remain metabolically active. It is thought to be a major mechanism to prevent cancer. Notably, p33ING1 expression is enhanced in senescent cells and premature expression of p33ING1 induces premature senescence. Both ING factors are chromatin-associated and bind to the histone mark histone H3 trimethylated lysine 4, a molecular mark of activated chromatin. However, both ING factors are potent transcriptional repressors and are associated with the SIN3A complex. The association-dissociation of ING with the SIN3A complex is mediated by acetyltransferase and HDAC activity. Because Alien enhances the p33ING1 and p33ING2 mediated gene repression, it is suggested that Alien could link activating chromatin marks with repression through p33ING (31). It is important to note that ING factors are highly conserved from yeast to human. Interestingly, other factors such as two transcription factor IIH (TFIIH) subunits, XBP and XPD/p44, were identified by in vivo proteomic approaches (28–30). TFIIH is an eukaryotic multiprotein complex exhibiting exceptional evolutionary conservation of its structure. Although initially considered to be exclusively a basal transcription factor, it is now considered to be responsible for initiation of transcription and transition from initiation to elongation. TFIIH is also important in nucleotide excision repair for unwinding DNA by helicase activity at lesion suites (32). It was also suggested that intact holocomplex of TFIIH is required for cell cycle regulation. Interestingly, recent reports suggest that the TFIIH subunit XBP is required for transcription but not for DNA repair (33). The functional relevance of the Alien-XPD or -XBP interaction is not yet investigated and is open for speculation but indicates interaction of Alien with evolutionarily conserved factors with cell cycle regulatory function. Other conserved factors that interact with Alien were found to be the retinoblastoma pocket domain protein pRb. Alien interacts with pRb but not with a mutant Rb isolated from a patient with pRb with a mutation in the pocket domain that abrogates pRb-mediated gene silencing. pRb is a tumor suppressor and inhibits cell cycle progression. This is mediated by interaction and repression of E2F activity. pRB not only blocks the surface of the E2F transactivation domain but mediates gene repression via recruitment of HDAC and HMT activity to repress actively chromatin at E2F-DNA sites and thus E2F target genes. The functional role of Alien-pRb interaction remains unclear. However, E2F-mediated repression of Alien seems to be independent
Article in press - uncorrected proof 14 Baniahmad: Alien as a tumor suppressor?
of pRB because in cells lacking pRb Alien represses E2F1mediated transactivation. This could indicate that Alien can, in part, replace pRb function. It is important to note that pRb is also conserved between Drosophila and mammals.
Discussion The data suggest that the expression of Alien inhibits cell proliferation. Alien presumably possesses at least two underlying mechanisms. One is a receptor-antagonist mediated pathway and the other is a receptor-independent pathway. Antagonist-bound AR inhibits cell proliferation, which seems to strongly depend on corepressor presence and activity. By contrast, the inhibition of E2F target genes by Alien suggests another basal mechanism to inhibit cancer cell proliferation. Thereby, Alien can inhibit E2F1 directly in a pRbindependent manner and through interaction with pRb, which presumably enhances its role to inhibit E2F transactivation indirectly. Alien is a highly conserved protein and the proteomic data suggest an interaction pattern with cell cycle regulators and transcriptional repressors. It further suggests that Alien binds to factors that exhibit evolutionarily conserved sequence conservation. The knockout mice for the CSN2 gene, which revealed a preimplantational lethal phenotype, suggest that Alien plays an essential role very early in development. So far, the observations suggest that the corepressor activity of Alien can be regulated at two levels. On the one hand, the expression of Alien can be triggered by hormone and on the other hand, Alien interacts with the mixed-lineage kinase 2 and enhances Alien-mediated gene repression (34). Taken together, Alien interacts with many evolutionarily highly conserved factors that are involved in cell cycle regulation and inhibits cell proliferation. The detailed molecular mechanism how the Alien interacting factors are functionally involved in the putative tumor suppressor function needs to be further analyzed.
Acknowledgment I am grateful to W. Hessenkemper for critically reading the manuscript. This research was supported by the Deutsche Krebshilfe to A.B.
References 1. Spange S, Wagner T, Heinzel T, Kra¨mer OH. Acetylation of nonhistone proteins modulates cellular signalling at multiple levels. Int J Biochem Cell Biol 2009;41:185–98. 2. Grzenda A, Lomberk G, Zhang JS, Urrutia R. Sin3: master scaffold and transcriptional corepressor. Biochim Biophys Acta 2009;1789:443–50. 3. Stanisic´ V, Lonard DM, O’Malley BW. Modulation of steroid hormone receptor activity. Prog Brain Res 2010;181:153–76. 4. Burke LJ, Baniahmad A. Co-repressors 2000. FASEB J 2000;14: 1876–88.
5. Feldman BJ, Feldman D. The development of androgen-independent prostate cancer. Nat Rev Cancer 2001;1:34–45. 6. Chen CD, Welsbie DS, Tran C, Baek SH, Chen R, Vessella R, Rosenfeld MG, Sawyers CL. Molecular determinants of resistance to antiandrogen therapy. Nat Med 2004;10:33–9. 7. Baniahmad A. Nuclear hormone receptor co-repressors. J Steroid Biochem Mol Biol 2005;93:89–97. 8. Dotzlaw H, Moehren U, Mink S, Cato AC, In˜iguez Lluhı´ JA, Baniahmad A. The amino terminus of the human AR is target for corepressor action and antihormone agonism. Mol Endocrinol 2002;16:661–73. 9. Eisold M, Asim M, Eskelinen H, Linke T, Baniahmad A. Inhibition of MAPK-signaling pathway promotes the interaction of the corepressor SMRT with the human androgen receptor and mediates repression of prostate cancer cell growth in the presence of antiandrogens. J Mol Endocrinol 2009;42:429–35. 10. Ko YJ, Balk SP. Targeting steroid hormone receptor pathways in the treatment of hormone dependent cancers. Curr Pharm Biotechnol 2004;5:459–70. 11. Moehren U, Papaioannou M, Reeb CA, Grasselli A, Nanni S, Asim M, Roell D, Prade I, Farsetti A, Baniahmad A. Wild-type but not mutant androgen receptor inhibits expression of the hTERT telomerase subunit: a novel role of AR mutation for prostate cancer development. FASEB J 2008;22:1258–67. 12. Dotzlaw H, Papaioannou M, Moehren U, Claessens F, Baniahmad A. Agonist-antagonist induced coactivator and corepressor interplay on the human androgen receptor. Mol Cell Endocrinol 2003;213:79–85. 13. Gurevich I, Flores AM, Aneskievich BJ. Corepressors of agonist-bound nuclear receptors. Toxicol Appl Pharmacol 2007; 223:288–98. 14. Dressel U, Thormeyer D, Altincicek B, Paululat A, Eggert M, Schneider S, Tenbaum SP, Renkawitz R, Baniahmad A. Alien, a highly conserved protein with characteristics of a corepressor for members of the nuclear hormone receptor superfamily. Mol Cell Biol 1999;19:3383–94. 15. Tenbaum SP, Juenemann S, Schlitt T, Bernal J, Renkawitz R, Mun˜oz A, Baniahmad A. Alien/CSN2 gene expression is regulated by thyroid hormone in rat brain. Dev Biol 2003;254: 149–60. 16. Kato JY, Yoneda-Kato N. Mammalian COP9 signalosome. Genes Cells 2009;14:1209–25. 17. Chamovitz DA. Revisiting the COP9 signalosome as a transcriptional regulator. EMBO Rep 2009;10:352–8. 18. Moehren U, Dressel U, Reeb CA, Va¨isa¨nen S, Dunlop TW, Carlberg C, Baniahmad A. The highly conserved region of the co-repressor Sin3A functionally interacts with the co-repressor Alien. Nucleic Acids Res 2004;32:2995–3004. 19. Svaren J, Ho¨rz W. Regulation of gene expression by nucleosomes. Curr Opin Genet Dev 1996;6:164–70. 20. Mellor J. Dynamic nucleosomes and gene transcription. Trends Genet 2006;22:320–9. 21. Park YJ, Chodaparambil JV, Bao Y, McBryant SJ, Luger K. Nucleosome assembly protein 1 exchanges histone H2A-H2B dimers and assists nucleosome sliding. J Biol Chem 2005;280: 1817–25. 22. McBryant SJ, Park YJ, Abernathy SM, Laybourn PJ, Nyborg JK, Luger K. Preferential binding of the histone (H3-H4)2 tetramer by NAP1 is mediated by the amino-terminal histone tails. J Biol Chem 2003;278:44574–83. 23. Shintomi K, Iwabuchi M, Saeki H, Ura K, Kishimoto T, Ohsumi K. Nucleosome assembly protein-1 is a linker histone chaperone in Xenopus eggs. Proc Natl Acad Sci USA 2005;102: 8210–5.
Article in press - uncorrected proof Baniahmad: Alien as a tumor suppressor? 15
24. Andrews AJ, Chen X, Zevin A, Stargell LA, Luger K. The histone chaperone Nap1 promotes nucleosome assembly by eliminating nonnucleosomal histone DNA interactions. Mol Cell 2010;37:834–42. 25. Eckey M, Hong W, Papaioannou M, Baniahmad A. The nucleosome assembly activity of NAP1 is enhanced by Alien. Mol Cell Biol 2007;27:3557–68. 26. Moehren U, Papaioannou M, Reeb CA, Hong W, Baniahmad A. Alien interacts with the human androgen receptor and inhibits prostate cancer cell growth. Mol Endocrinol 2007;21: 1039–48. 27. Schaufele F, Carbonell X, Guerbadot M, Borngraeber S, Chapman MS, Ma AA, Miner JN, Diamond MI. The structural basis of androgen receptor activation: intramolecular and intermolecular amino-carboxy interactions. Proc Natl Acad Sci USA 2005;102:9802–7. 28. Kob R, Baniahmad A, Escher N, von Eggeling F, Melle C. Detection and identification of transcription factors as interaction partners of Alien in vivo. Cell Cycle 2007;6:993–6. 29. Tenbaum SP, Papaioannou M, Reeb CA, Goeman F, Escher N, Kob R, von Eggeling F, Melle C, Baniahmad A. Alien inhibits
30.
31.
32.
33.
34.
E2F1 gene expression and cell proliferation. Biochim Biophys Acta 2007;1773:1447–54. Escher N, Kob R, Tenbaum SP, Eisold M, Baniahmad A, von Eggeling F, Melle C. Various members of the E2F transcription factor family interact in vivo with the corepressor Alien. J Proteome Res 2007;6:1158–64. Fegers I, Kob R, Eckey M, Schmidt O, Goeman F, Papaioannou M, Escher N, von Eggeling F, Melle C, Baniahmad A. The tumor suppressors p33ING1 and p33ING2 interact with Alien in vivo and enhance Alien-mediated gene silencing. J Proteome Res 2007;6:4182–8. Mydlikova Z, Gursky J, Pirsel M. Transcription factor IIH – the protein complex with multiple functions. Neoplasma 2010; 57:287–90. Savolainen L, Cassel T, Helleday T. The XPD subunit of TFIIH is required for transcription-associated but not DNA doublestrand break-induced recombination in mammalian cells. Mutagenesis 2010;25:623–9. Eckey M, Tenbaum SP, Mun˜oz A, Baniahmad A. Mixed lineage kinase 2 enhances trans-repression of Alien and nuclear receptors. Mol Cell Endocrinol 2003;213:71–8.
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The corepressor Alien as a novel tumor suppressor?
Aria Baniahmad* Institute of Human Genetics, Jena University Hospital, Jena, Germany
Abstract Alien has been characterized as a corepressor for nuclear hormone receptors that harbor a silencing domain such as the thyroid hormone receptor (TR), the vitamin D3 receptor (VDR) and DAX-1. In addition, the androgen receptor (AR), a steroid hormone receptor, interacts with Alien. Alien enhances gene silencing mediated by TR, VDR and DAX-1, whereas Alien inhibits AR-mediated transactivation. The inhibition of AR by Alien seems to be restricted to cases where AR is bound to AR antagonists. In line with this, Alien inhibits AR target gene expression and human prostate cancer cell proliferation in an antagonist-specific manner indicating that Alien has an inhibitory role for cell cycle progression. Alien mediates gene silencing by recruitment of histone deacetylase activity and interestingly through nucleosome assembly activity. Hereby, Alien enhances nucleosome positioning mediated by nucleosome assembly protein 1, which suggests a novel molecular mechanism of corepressor function. Using a proteomic approach to identify Alien interacting partners, we detected the cell cycle factor E2F1 to bind to Alien in vivo. The E2F1-mediated transactivation and E2F target gene expression is inhibited by Alien, and in line with this Alien is observed to repress cell cycle progression. Keywords: cell cycle; corepressor; E2F-transcription factors; interactom; retinoblastoma; signalosome subunit CSN2.
Introduction Originally, corepressors were defined as proteins that interact with DNA-bound transcriptional silencers to mediate and/or enhance their transrepression. It emerges, however, that corepressors also bind to many transcription factors to modulate and to reduce their transactivation. Moreover, corepressors are associated with histone deacetylase (HDAC) and histone methyltransferase (HMT) activity not only deacetylate or methylate histones but also other regulatory factors, for which modifications such as by acetylation or methylation, control their functionality (1–3). Thus, it seems *Corresponding author: Aria Baniahmad, Institute of Human Genetics, Jena University Hospital, D-07743 Jena, Germany Phone: q49-3641-935501, Fax: q49-3641-935502, E-mail: [email protected] Received November 15, 2010; accepted November 16, 2010; previously published online March 4, 2011
that corepressors play an important role not only directly at transcriptional and chromatin levels. The regulation of corepressor interaction with transcription factors is essential for proper gene regulation (4). Here, six mechanisms have been shown to modulate the interaction such as acetylation, phosphorylation, nuclear localization, competition, the specificity of the DNA sequence and in the case of nuclear hormone receptors the type of ligand (4). Receptor agonists switch nuclear receptors often into transcriptional activators, whereas antagonists inhibit the receptormediated transactivation. For the estrogen and androgen receptors (ER, AR), the model is that agonists of these receptors induce the binding of coactivators, whereas receptor antagonists promote the binding of corepressors. Both agonist-bound estrogen or androgen receptors promote the proliferation of breast and prostate, respectively, in a hormonally controlled manner. In addition, the tumors of these tissues respond initially mostly well to these hormones. Therefore, both receptors are important drug targets for which specific antagonists have been developed that are clinically in use and inhibit cancer proliferation. Thus, the agonist-bound AR and ER activate the cell cycle in a hormone-dependent manner, which can be inhibited by antagonists. Eventually, resistance to hormone therapy by the treatment with antagonists occurs, representing a major drawback for patients. Resistance to prostate cancer (PCa) therapy can occur within the first 1–2 years of treatment (5). Several mechanisms have been proposed, of which corepressors malfunctioning seems to be a major cause (6, 7). AR amplifications in refractory and castration-resistant PCa lead to less corepressor recruitment to chromatin indicating a weaker repression of AR target genes. Furthermore, activation of signal transduction by membrane-bound receptors such as tyrosine kinases or protein kinase A leads to dissociation of corepressors from the AR (8, 9). Similarly, the ER dissociates from corepressors by activated signaling pathways such as activated Her/neu and EGF receptors in the presence of ER antagonists (tamoxifen) (10). Thus, the efficacy of cancer treatment with nuclear receptor antagonists seems to depend on functional corepressors. The molecular mechanism of how corepressors inhibit cancer growth is not yet fully understood. Both the androgen-bound AR and the estrogen-bound ER not only activate gene expression but also repress important genes. The catalytic subunit of human telomerase, for example, being an essential factor for immortalization of tumor cells is repressed by the agonist-bound AR. The AR is recruited to the hTERT promoter sequences in vivo and represses hTERT expression and telomerase activity in an androgen-dependent manner (11). However, it is not known how a coactivator loaded agonist-bound receptor lead to gene repression. More-
2010/054
Article in press - uncorrected proof 12 Baniahmad: Alien as a tumor suppressor?
over, the role of corepressors for this type of gene regulation is yet unclear. Moreover, the specificity of corepressors needs to be addressed. The AR interaction data suggest that the corepressor Alien binds in the presence of AR antagonist to the AR, whereas for the SMRT corepressor binding was also observed in the presence of agonist (8, 12). However, SMRT binding does not necessarily lead to repression of AR, because SMRT inhibited the AR-mediated transactivation only in the presence of AR antagonists. This indicates that corepressor function can be regulated by the type of the ligand and that binding to transcription factors itself is not sufficient for corepressor functionality (8). It also suggests that different corepressors act in a distinct manner. This is in line with observations that several corepressors bind specifically to agonist-bound nuclear receptors (13). The corepressor Alien was named based on its high sequence conservation to the Drosophila Alien protein (14). Mammalian Alien was characterized as a factor with corepressor function for the thyroid hormone receptor (TR) and vitamin D3 receptor (VDR). Both receptors are known to regulate cell proliferation and differentiation. Later it was shown that Alien is an isoform of the CSN2 subunit that can be complexed in the COP9 signalosome (15), a highly conserved multifunctional protein complex. Mostly the COP9 function to regulate protein stability was investigated and more recently it was suggested that COP9 signalosome is involved in checkpoint control, signal transduction, development and the cell cycle. Recent evidence suggests that the COP9 complex is also associated with chromatin (16, 17). Interestingly, the expression of Alien is upregulated by thyroid hormone in brain in vivo (15). Taken together, it suggests that Alien is associated with factors that regulate cell cycle proliferation. Here, an overview is provided about the identified mechanisms of how Alien mediates gene repression and its role in cell proliferation.
Novel mechanisms of Alien-mediated repression by nucleosome assembly Alien mediates gene silencing in part by recruiting HDAC activity (14). This is presumably mediated by the recruitment of mSIN3A to Alien (18). It was shown that Alien and Sin3A exhibit a dynamic corecruitment to chromatin sites of VDR responsive gene CYP24 in vivo. The CYP24 gene is repressed in the absence of vitamin D3 and activated by the hormone through two vitamin D3 response elements (VDREs) located in the promoter region. Accordingly, the histone H4 at these VDREs is acetylated within 1 h of hormone treatment. Interestingly, although hormone is still present the level of acetylation of histone H4 at the VDREs is decreased within a few hours suggesting that HDAC activity is recruited to these VDREs. In line with these observations, in the absence of vitamin D3 mSIN3A and Alien are corecruited specifically to the VDREs of the CYP24 promoter (18). SIN3A exists in a large protein complex that contains
HDAC activity. Addition of ligand leads to dissociation of both Alien and mSIN3A from the VDREs (18). However, within 60 min both Alien and SIN3A are corecruited again in a cycling manner despite the presence of hormone. This suggests that the observed deacetylation of histone H4 at the vicinity of the VDREs might be mediated by the recruitment of Alien and mSIN3A and the associated HDAC activity. This indicates that corepressors also play a gene regulatory role in the presence of hormones such as turning down gene expression after a limited time period of hormone-induced gene activation. Interestingly, a novel mechanism of corepressors was postulated by Alien-mediated gene repression. Nucleosome assembly on template DNA was shown to inhibit transcription in various assay systems (19). It is thought that nucleosome assembly inhibits the DNA access of general transcription factors, transcription initiation factors and thus the recruitment of the RNA polymerases leading to gene silencing. One factor known to assemble nucleosomes onto DNA is the nucleosome assembly protein 1 (NAP1) (20). NAP1 functions as a histone chaperone, binding to the histones H2A–H2B dimers and H3–H4 tetramers (21, 22). NAP1 also modulates binding of linker histone H1, functions as a histone exchanger and as an ATP-independent histone-sliding factor (23). In Drosophila, a knockout of NAP1 leads to embryonic lethality and in yeast knockout experiments to significant increase in atypical histone-DNA complexes, as well as in deregulated transcription activation and repression (24). Using a yeast two-hybrid screen with Alien as bait, we identified various independent cDNA clones encoding NAP1 spanning a central region of NAP1 (25). The interaction of Alien with NAP1 was verified in vitro and in vivo. Interestingly, Alien modulated the nucleosome assembly activity of NAP1 by enhancing nucleosome assembly on DNA templates. Furthermore, Alien reduces the accessibility of the histones H3 and H4 for NAP1-promoted assembly reaction. This indicates that Alien sustains and reinforces the formation of nucleosomes. Employing deletion mutants of Alien suggest that different regions of Alien are involved in enhancement of NAP1-mediated nucleosome assembly and in inhibiting the accessibility of the histones H3/H4 (25). Using the VDR for which Alien was shown to act as a corepressor (18), a hormone-induced transcription system was chosen. Chromatin immunoprecipitation experiments (ChIP and re-ChIP) suggest that NAP1 and Alien are complexed and are corecruited in a vitamin D3-induced manner to the endogenous VDR target gene CYP24 promoter in vivo (25). These data suggest a novel pathway with Alien to bind to NAP1 and to enhance/enforce nucleosome assembly, thus linking corepressor function with nucleosome assembly activity.
Inhibition of PCa cell growth by Alien Alien was shown to interact with the AR seemingly in an AR antagonist-specific manner (26). Interestingly, AR ago-
Article in press - uncorrected proof Baniahmad: Alien as a tumor suppressor? 13
nists such as dihydrotestosterone (DHT) or R1881 did not promote binding of Alien to the AR indicating a ligand-specific induced interaction with AR. This could be due to conformational changes of the AR. One possibility is that agonists induce an intramolecular N-/C-terminal interaction, whereas AR antagonists do not and thus the N-terminus of AR might be better accessible (27). Analyzing the interaction sites of Alien with AR suggests that Alien binds to the AR/ N-terminus in the presence of antagonists explaining the observed ligand-specific interaction. Interestingly, the SUMOylation sites located in the AR/N-terminus are required for Alien/AR interaction because mutation of the SUMO sites of AR abrogates binding of Alien to the AR (26). Using the androgen-dependent LNCaP cells as a model for androgen-promoted PCa growth, the role of Alien on endogenous AR target gene expression and cell proliferation was analyzed by stable Alien expression. In line with the ligand-specific interaction of Alien with the AR, it was observed that Alien inhibited the prostate specific antigen gene expression as well as the androgen-dependent LNCaP cell growth in an AR antagonist-dependent manner. By contrast, in the presence of AR agonists, Alien reduced cell growth only marginally (26). This indicates that Alien inhibits in an antagonist-specific manner AR and AR-controlled PCa cell proliferation.
Alien interacts with evolutionarily conserved factors regulating cell proliferation Alien/CSN2 is a highly conserved factor present in multicellular organisms from plants to Drosophila and humans. Alien mediates gene silencing not only for mammalian nuclear receptors possessing a silencing domain. Drosophila Alien interacts with nuclear hormone receptors such as the ecdysone receptor (14). Interestingly, nuclear hormone receptors such as COUP-TFI and -TFII, to which Alien binds, exhibit a considerable sequence homology between Drosophila (svp) and human with up to 90% identity in amino acid sequence. Similarly, the NAP1 sequence is conserved in evolution sharing high homology between human and Drosophila NAP1, indicating that Alien interacts with evolutionary conserved factors. Further insights of Alien-mediated corepressor function were gained by using an in vivo proteomic approach to identify novel Alien interacting partners (28–30). Interestingly, conserved cell cycle factors were among proteins that interact with Alien. Specifically, among those factors, the E2F1 transcriptional activator was identified. Alien inhibited E2F1-mediated transactivation, was corecruited to chromatic E2F1 sites in vivo and repressed E2F target gene expression. E2F1 is a promoter of cell cycle progression. In line with this, using a tetracycline inducible system cell proliferation was reduced in a doxycycline-dependent manner. We noted that cells did not accumulate in a specific-cell cycle phase. This could be due to the observation that the protein levels of Alien itself change within the cell cycle phases (28–30).
Another explanation could be that Alien also interacts with other E2F members including the inhibitory E2F family members, which could result in an overall mixed effect so that the Alien-mediated inhibition of cell proliferation is due to a general inhibition of the cell cycle phases. Taken together, this indicates that Alien inhibits cell cycle promoting factors and inhibits cell proliferation. The proteomic approach delivered further information. Other interesting cell cycle regulators were identified as interacting partners of Alien. Both tumor suppressors p33ING1 and p33ING2 interact with Alien in vivo (31). Both factors are associated with the phenomenon of cellular senescence. Cellular senescence arrests the cell growth in an irreversible manner. The cells, however, remain metabolically active. It is thought to be a major mechanism to prevent cancer. Notably, p33ING1 expression is enhanced in senescent cells and premature expression of p33ING1 induces premature senescence. Both ING factors are chromatin-associated and bind to the histone mark histone H3 trimethylated lysine 4, a molecular mark of activated chromatin. However, both ING factors are potent transcriptional repressors and are associated with the SIN3A complex. The association-dissociation of ING with the SIN3A complex is mediated by acetyltransferase and HDAC activity. Because Alien enhances the p33ING1 and p33ING2 mediated gene repression, it is suggested that Alien could link activating chromatin marks with repression through p33ING (31). It is important to note that ING factors are highly conserved from yeast to human. Interestingly, other factors such as two transcription factor IIH (TFIIH) subunits, XBP and XPD/p44, were identified by in vivo proteomic approaches (28–30). TFIIH is an eukaryotic multiprotein complex exhibiting exceptional evolutionary conservation of its structure. Although initially considered to be exclusively a basal transcription factor, it is now considered to be responsible for initiation of transcription and transition from initiation to elongation. TFIIH is also important in nucleotide excision repair for unwinding DNA by helicase activity at lesion suites (32). It was also suggested that intact holocomplex of TFIIH is required for cell cycle regulation. Interestingly, recent reports suggest that the TFIIH subunit XBP is required for transcription but not for DNA repair (33). The functional relevance of the Alien-XPD or -XBP interaction is not yet investigated and is open for speculation but indicates interaction of Alien with evolutionarily conserved factors with cell cycle regulatory function. Other conserved factors that interact with Alien were found to be the retinoblastoma pocket domain protein pRb. Alien interacts with pRb but not with a mutant Rb isolated from a patient with pRb with a mutation in the pocket domain that abrogates pRb-mediated gene silencing. pRb is a tumor suppressor and inhibits cell cycle progression. This is mediated by interaction and repression of E2F activity. pRB not only blocks the surface of the E2F transactivation domain but mediates gene repression via recruitment of HDAC and HMT activity to repress actively chromatin at E2F-DNA sites and thus E2F target genes. The functional role of Alien-pRb interaction remains unclear. However, E2F-mediated repression of Alien seems to be independent
Article in press - uncorrected proof 14 Baniahmad: Alien as a tumor suppressor?
of pRB because in cells lacking pRb Alien represses E2F1mediated transactivation. This could indicate that Alien can, in part, replace pRb function. It is important to note that pRb is also conserved between Drosophila and mammals.
Discussion The data suggest that the expression of Alien inhibits cell proliferation. Alien presumably possesses at least two underlying mechanisms. One is a receptor-antagonist mediated pathway and the other is a receptor-independent pathway. Antagonist-bound AR inhibits cell proliferation, which seems to strongly depend on corepressor presence and activity. By contrast, the inhibition of E2F target genes by Alien suggests another basal mechanism to inhibit cancer cell proliferation. Thereby, Alien can inhibit E2F1 directly in a pRbindependent manner and through interaction with pRb, which presumably enhances its role to inhibit E2F transactivation indirectly. Alien is a highly conserved protein and the proteomic data suggest an interaction pattern with cell cycle regulators and transcriptional repressors. It further suggests that Alien binds to factors that exhibit evolutionarily conserved sequence conservation. The knockout mice for the CSN2 gene, which revealed a preimplantational lethal phenotype, suggest that Alien plays an essential role very early in development. So far, the observations suggest that the corepressor activity of Alien can be regulated at two levels. On the one hand, the expression of Alien can be triggered by hormone and on the other hand, Alien interacts with the mixed-lineage kinase 2 and enhances Alien-mediated gene repression (34). Taken together, Alien interacts with many evolutionarily highly conserved factors that are involved in cell cycle regulation and inhibits cell proliferation. The detailed molecular mechanism how the Alien interacting factors are functionally involved in the putative tumor suppressor function needs to be further analyzed.
Acknowledgment I am grateful to W. Hessenkemper for critically reading the manuscript. This research was supported by the Deutsche Krebshilfe to A.B.
References 1. Spange S, Wagner T, Heinzel T, Kra¨mer OH. Acetylation of nonhistone proteins modulates cellular signalling at multiple levels. Int J Biochem Cell Biol 2009;41:185–98. 2. Grzenda A, Lomberk G, Zhang JS, Urrutia R. Sin3: master scaffold and transcriptional corepressor. Biochim Biophys Acta 2009;1789:443–50. 3. Stanisic´ V, Lonard DM, O’Malley BW. Modulation of steroid hormone receptor activity. Prog Brain Res 2010;181:153–76. 4. Burke LJ, Baniahmad A. Co-repressors 2000. FASEB J 2000;14: 1876–88.
5. Feldman BJ, Feldman D. The development of androgen-independent prostate cancer. Nat Rev Cancer 2001;1:34–45. 6. Chen CD, Welsbie DS, Tran C, Baek SH, Chen R, Vessella R, Rosenfeld MG, Sawyers CL. Molecular determinants of resistance to antiandrogen therapy. Nat Med 2004;10:33–9. 7. Baniahmad A. Nuclear hormone receptor co-repressors. J Steroid Biochem Mol Biol 2005;93:89–97. 8. Dotzlaw H, Moehren U, Mink S, Cato AC, In˜iguez Lluhı´ JA, Baniahmad A. The amino terminus of the human AR is target for corepressor action and antihormone agonism. Mol Endocrinol 2002;16:661–73. 9. Eisold M, Asim M, Eskelinen H, Linke T, Baniahmad A. Inhibition of MAPK-signaling pathway promotes the interaction of the corepressor SMRT with the human androgen receptor and mediates repression of prostate cancer cell growth in the presence of antiandrogens. J Mol Endocrinol 2009;42:429–35. 10. Ko YJ, Balk SP. Targeting steroid hormone receptor pathways in the treatment of hormone dependent cancers. Curr Pharm Biotechnol 2004;5:459–70. 11. Moehren U, Papaioannou M, Reeb CA, Grasselli A, Nanni S, Asim M, Roell D, Prade I, Farsetti A, Baniahmad A. Wild-type but not mutant androgen receptor inhibits expression of the hTERT telomerase subunit: a novel role of AR mutation for prostate cancer development. FASEB J 2008;22:1258–67. 12. Dotzlaw H, Papaioannou M, Moehren U, Claessens F, Baniahmad A. Agonist-antagonist induced coactivator and corepressor interplay on the human androgen receptor. Mol Cell Endocrinol 2003;213:79–85. 13. Gurevich I, Flores AM, Aneskievich BJ. Corepressors of agonist-bound nuclear receptors. Toxicol Appl Pharmacol 2007; 223:288–98. 14. Dressel U, Thormeyer D, Altincicek B, Paululat A, Eggert M, Schneider S, Tenbaum SP, Renkawitz R, Baniahmad A. Alien, a highly conserved protein with characteristics of a corepressor for members of the nuclear hormone receptor superfamily. Mol Cell Biol 1999;19:3383–94. 15. Tenbaum SP, Juenemann S, Schlitt T, Bernal J, Renkawitz R, Mun˜oz A, Baniahmad A. Alien/CSN2 gene expression is regulated by thyroid hormone in rat brain. Dev Biol 2003;254: 149–60. 16. Kato JY, Yoneda-Kato N. Mammalian COP9 signalosome. Genes Cells 2009;14:1209–25. 17. Chamovitz DA. Revisiting the COP9 signalosome as a transcriptional regulator. EMBO Rep 2009;10:352–8. 18. Moehren U, Dressel U, Reeb CA, Va¨isa¨nen S, Dunlop TW, Carlberg C, Baniahmad A. The highly conserved region of the co-repressor Sin3A functionally interacts with the co-repressor Alien. Nucleic Acids Res 2004;32:2995–3004. 19. Svaren J, Ho¨rz W. Regulation of gene expression by nucleosomes. Curr Opin Genet Dev 1996;6:164–70. 20. Mellor J. Dynamic nucleosomes and gene transcription. Trends Genet 2006;22:320–9. 21. Park YJ, Chodaparambil JV, Bao Y, McBryant SJ, Luger K. Nucleosome assembly protein 1 exchanges histone H2A-H2B dimers and assists nucleosome sliding. J Biol Chem 2005;280: 1817–25. 22. McBryant SJ, Park YJ, Abernathy SM, Laybourn PJ, Nyborg JK, Luger K. Preferential binding of the histone (H3-H4)2 tetramer by NAP1 is mediated by the amino-terminal histone tails. J Biol Chem 2003;278:44574–83. 23. Shintomi K, Iwabuchi M, Saeki H, Ura K, Kishimoto T, Ohsumi K. Nucleosome assembly protein-1 is a linker histone chaperone in Xenopus eggs. Proc Natl Acad Sci USA 2005;102: 8210–5.
Article in press - uncorrected proof Baniahmad: Alien as a tumor suppressor? 15
24. Andrews AJ, Chen X, Zevin A, Stargell LA, Luger K. The histone chaperone Nap1 promotes nucleosome assembly by eliminating nonnucleosomal histone DNA interactions. Mol Cell 2010;37:834–42. 25. Eckey M, Hong W, Papaioannou M, Baniahmad A. The nucleosome assembly activity of NAP1 is enhanced by Alien. Mol Cell Biol 2007;27:3557–68. 26. Moehren U, Papaioannou M, Reeb CA, Hong W, Baniahmad A. Alien interacts with the human androgen receptor and inhibits prostate cancer cell growth. Mol Endocrinol 2007;21: 1039–48. 27. Schaufele F, Carbonell X, Guerbadot M, Borngraeber S, Chapman MS, Ma AA, Miner JN, Diamond MI. The structural basis of androgen receptor activation: intramolecular and intermolecular amino-carboxy interactions. Proc Natl Acad Sci USA 2005;102:9802–7. 28. Kob R, Baniahmad A, Escher N, von Eggeling F, Melle C. Detection and identification of transcription factors as interaction partners of Alien in vivo. Cell Cycle 2007;6:993–6. 29. Tenbaum SP, Papaioannou M, Reeb CA, Goeman F, Escher N, Kob R, von Eggeling F, Melle C, Baniahmad A. Alien inhibits
30.
31.
32.
33.
34.
E2F1 gene expression and cell proliferation. Biochim Biophys Acta 2007;1773:1447–54. Escher N, Kob R, Tenbaum SP, Eisold M, Baniahmad A, von Eggeling F, Melle C. Various members of the E2F transcription factor family interact in vivo with the corepressor Alien. J Proteome Res 2007;6:1158–64. Fegers I, Kob R, Eckey M, Schmidt O, Goeman F, Papaioannou M, Escher N, von Eggeling F, Melle C, Baniahmad A. The tumor suppressors p33ING1 and p33ING2 interact with Alien in vivo and enhance Alien-mediated gene silencing. J Proteome Res 2007;6:4182–8. Mydlikova Z, Gursky J, Pirsel M. Transcription factor IIH – the protein complex with multiple functions. Neoplasma 2010; 57:287–90. Savolainen L, Cassel T, Helleday T. The XPD subunit of TFIIH is required for transcription-associated but not DNA doublestrand break-induced recombination in mammalian cells. Mutagenesis 2010;25:623–9. Eckey M, Tenbaum SP, Mun˜oz A, Baniahmad A. Mixed lineage kinase 2 enhances trans-repression of Alien and nuclear receptors. Mol Cell Endocrinol 2003;213:71–8.
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