948 Vol. 1, 948–958, November 2003 Molecular Cancer Research Interaction and Functional Cooperation of the Cancer-Amplified Transcriptional Coactivator Activating Signal Cointegrator-2 and E2F-1 in Cell Proliferation Hee Jeong Kong,1 Hyun Jung Yu,2 SunHwa Hong,2 Min Jung Park,2 Young Hyun Choi,3 Won Gun An,4 Jae Woon Lee,5 and JaeHun Cheong2 1Laboratory of Molecular Growth Regulation, National Institute of Health, Bethesda, MD; 2Department of Molecular Biology, Pusan National University, Busan, Republic of Korea; 3Department of Biochemistry, College of Oriental Medicine, Dong-Eui University, Busan, Republic of Korea; 4Department of Biology, Kyungpook National University, DaeGu, Republic of Korea; and 5Department of Medicine/Endocrinology, Baylor College of Medicine Houston, TX. Abstract structure and recruitment of a transcription initiation complex Activating signal cointegrator-2 (ASC-2), a novel coac- containing RNA polymerase II to the promoter (1). Recent tivator, is amplified in several cancer cells and known to studies have shown that DNA-bound transcriptional activator interact with mitogenic transcription factors, including proteins accomplish these two tasks with the assistance of a serum response factor, activating protein-1, and nuclear class of proteins called transcriptional coactivators (2, 3). They factor-KB, suggesting the physiological role of ASC-2 in may be thought of as adaptors or components in a signaling the promotion of cell proliferation. Here, we show that pathway that transmits transcriptional activation signals from the expression pattern of ASC-2 was correlated with that DNA-bound activator proteins to the chromatin and transcrip- of E2F-1 for protein increases at G1 and S phase. tion machinery. Furthermore, cells stably overexpressing ASC-2 had an Activating signal cointegrator-2 (ASC-2) was originally increased cell proliferation profile. These results isolated as a novel nuclear coactivator, which interacts and prompted us to examine the functional interaction of activates numerous nuclear hormone receptors including ASC-2 and E2F-1. Biochemical evidence of protein retinoid X receptor, peroxisome proliferator-activated receptor, interaction indicated that the transactivation domain of and thyroid hormone receptor (4) subsequently isolated from E2F-1 interacted with the COOH-terminal region of ASC- several groups named RAP250, peroxisome proliferator- 2. The importance of the E2F-1-ASC-2 interaction was activated receptor interacting protein, and transactivation supported by the demonstration that the coexpression response RNA binding protein (5–7). ASC-2, a typical of ASC-2 and E2F-1 synergistically transactivated E2F- ligand- and activating function-2-dependent interactor of 1-driven gene transcription and the acetylation of E2F-1 nuclear receptors, enhances the receptor transactivation either protein was necessary for ASC-2-mediated transcrip- alone or in conjunction with steroid receptor coactivator-1 tional coactivation. Interestingly, overexpression of (SRC-1) and cAMP-responsive element-binding protein (CBP)/ ASC-2 increased the endogenous protein level of E2F- p300. Sequence analysis results showed that ASC-2 was 1 in cells, resulting from the prolonged protein stability identical to amplified in breast cancer 3, which was amplified of E2F-1. Taken together, these results suggest that the in breast and other human cancers and had potential biological cancer-amplified transcriptional coactivator ASC-2 may relevance to cancer progression. ASC-2 was also amplified in promote cell proliferation through enhancement of E2F- gene copy number and overexpressed in some cancer cell lines 1-dependent transactivation of the expression of genes such as BT-474, ZR-75-1, and MCF7. ASC-2 was recently associated with cell cycle progression that may be found to mediate transactivation by a series of mitogenic available to favor tumor growth in vivo. transcription factors, including activating protein-1, nuclear factor-nB, and serum response factor (8). Thus, ASC-2 may Introduction directly participate in tumorigenesis when overexpressed. Transcription regulation is a control mechanism that is The E2F family of transcription factors plays an important critical for fundamental biological processes, such as cell role in organizing early cell cycle progression by coordinating growth and differentiation. The subsequent transcriptional early cell cycle events with the transcription of genes required activation of the gene involves local modifications in chromatin for entry into S phase (9, 10). Overexpression of E2F promotes cell cycle progression and is sufficient to drive quiescent cells into S phase and can lead to the transformation of cells (11, 12). There are five E2F family members that have highly conserved Received 4/24/03; revised 9/5/03; accepted 9/25/03. The costs of publication of this article were defrayed in part by the payment of DNA binding and activation domains (13, 14), whereas a sixth page charges. This article must therefore be hereby marked advertisement in protein, E2F binding site modulating activity protein, is only accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Grant support: Korea Health Ministry of Health & Welfare, Republic of Korea conserved in the DNA binding domain (DBD). Three of these (02-PJ1-PG3-20908-0009). (E2F-1, E2F-2, and E2F-3) have the ability to induce S phase Requests for reprints: JaeHun Cheong, Department of Molecular Biology, Pusan (11, 15, 16). However, all E2F members are able to bind to a National University, Busan 609-735, Republic of Korea. Phone: 82-51-510-2277; Fax: 82-51-513-9258. E-mail: [email protected] similar consensus sequence when heterodimerized with a Copyright D 2003 American Association for Cancer Research. member of the DP family (17). Downloaded from mcr.aacrjournals.org on September 26, 2021. © 2003 American Association for Cancer Research. Molecular Cancer Research 949 The transcriptional activity of E2F is controlled through the binding of several proteins that regulate its activity. Positive regulators include TATA box binding protein (TBP; 18, 19), the coactivator CBP/p300 (20, 21), and the oncoprotein murine double minute clone 2 (22). Negative regulators include the tumor suppressor protein p53 (23) and the most important regulator, the retinoblastoma protein (Rb; 24–28). During G1 phase, Rb is hyperphosphorylated, which blocks the binding of E2F-Rb and releases transcriptionally active E2F at the G1-S transition. Other levels of control that influence E2F activity include its own phosphorylation (29), acetylation (30, 31), protein stabilization (32–34), and regulation of its intracellular localization (35). The transcription activity of E2F is increased by CBP/p300 and P/CAF, which have histone acetyltransferase (HAT) activities. These cofactors may regulate transcription through acetylation (36–38). Although the major targets of CBP/p300 and P/CAF are histones, these HATs also modify certain transcription factors such as p53, TFIIF, HMGI(Y), and GATA-1 (39–42). FIGURE 1. Expression of ASC-2 during cell cycle transition. HeLa cells In this study, we show that ASC-2 is associated with highly were synchronized by double thymidine block method at the indicated times and analyzed by FACS analysis (top). G1; n S; y G2-M. The increased cell proliferation by mediating the physical and different protein levels dependent on cell cycle transition were measured functional interactions with E2F-1. We also found that ASC-2 by using Western blot analysis with anti-ASC-2 (A3C1), E2F-1, and TBP antibodies for 1 h at room temperature. The protein bands were visualized stabilizes E2F-1 protein in vivo, which may explain one of the by the enhanced chemiluminescence detection as described in ‘‘Materials mechanisms of E2F-1-dependent transactivation by ASC-2. and Methods’’ (bottom). Control cells indicate asynchronous cells. Furthermore, the acetylation of the E2F-1 protein is required for the stabilization and cooperation of E2F-1 by ASC-2. These results give a clue why ASC-2 is highly expressed in a synergistically activated mitogenic transcription factors activat- variety of cancer cells and how ASC-2 promotes cell ing protein-1, nuclear factor-nB, and serum response factor as proliferation. well as nuclear hormone receptors (8). These results led us to examine whether ASC-2 may directly regulate cellular prolif- eration and cell cycle progression by acting as a novel Results coactivator molecule for mitogenic transcription factors. To The ASC-2 Expression Is Associated with Cell identify the possibility, we investigated the effect of over- Cycle Transition expression of ASC-2 on cell cycle progression and cell ASC-2 protein is rapidly induced in resting cells trans- proliferation. NIH3T3 and HeLa cells were stably transfected forming to proliferating ones following mitogenic stimulation with an expression plasmid of ASC-2 and then subjected to cell (unpublished data), suggesting that it plays an important role in proliferation and cell cycle analysis by fluorescence-activated the transition from quiescence state to proliferation. In an effort cell sorting (FACS) analysis. Stable transfectant clones of to clarify the regulation of ASC-2 expression dependent on cell NIH3T3 cells (clones 1 and 2) that expressed ASC-2 at moderate cycle transition, the differential expression patterns of ASC-2 and high levels, respectively, were selected and confirmed by the and cell cycle regulating molecules including cyclins, cyclin- protein