E2FBP1/DRIL1, an AT-Rich Interaction Domain-Family Transcription Factor, Is Regulated by p53 Kaiwen Ma, 1 Keigo Araki, 1 Solachuddin J.A. Ichwan, 1 Tamaki Suganuma, 1,2 Mimi Tamamori-Adachi, 2 and Masa-Aki Ikeda 2 1 Section of Molecular Embryology, Graduate School and 2 Department of Biochemical Genetics, Medical Research Institute, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan Abstract E2FBP1/DRIL1 is an AT-rich interaction domain DNA- binding protein and is ubiquitously expressed in various tissues. It has been shown that Bright, the mouse orthologue of E2FBP1/DRIL1, exhibits sequence-specific DNA binding and regulates immunoglobulin transcrip- tion. Here we show a novel connection between E2FBP1/ DRIL1 and the p53 tumor suppressor, a key regulator of growth arrest or apoptosis in response to cellular stress. We found a putative p53-binding site, which specifically responded to p53, in the second intron of the E2FBP1/ DRIL1 gene. E2FBP1/DRIL1was induced by p53 and up- regulated following DNA damage caused by UV radiation or doxorubicin treatment in a manner dependent on endogenous p53. The ectopic expression of E2FBP1/ DRIL1 induced growth arrest in U2OS cells expressing normal p53 , but not Saos-2 cells lacking p53 . These results suggest that E2FBP1/DRIL1 may play a role in growth suppression mediated by p53. Introduction E2FBP1 was originally isolated as a cDNA encoding a protein that interacts with E2F-1, a member of the E2F transcription factor family (1). It was also independently discovered as DRIL1 (2), a human orthologue of murine Bright [B-cell regulator of immunoglobulin heavy-chain (IgH) transcription] (3), and Drosophila dead ringer (DRI ) (4), both of which encode a conserved DNA-binding domain termed AT- rich interaction domain (ARID). In addition to the core ARID, these proteins share a broader homologous region that extends outside the core ARID, thus leading to the classification of these proteins as the extended ARID (eARID) subfamily (5). E2FBP1/DRIL1 exhibits overall 79% identity and 83% similarity to Bright (2). Bright has been shown to act as a B cell-specific, matrix associating region-binding protein that transactivates the IgH enhancer and regulates B cell-specific gene expression (3, 6, 7). Bright binds to specific AT-rich sequences that are important for nuclear matrix association within matrix associating regions located in the IgH gene (3). Although Bright has been reported to specifically express in differentiating and mature B cells (3, 8), it has been shown that E2FBP1/DRIL1 is ubiquitously expressed in all tissues examined (2), suggesting that E2FBP1/DRIL1 might participate in various biological processes. Here, we report a novel connection between E2FBP1/DRIL1 and the p53 tumor suppressor, a key regulator in a wide range of cellular processes, including cell cycle arrest and apoptosis (9–11). We provide evidence that E2FBP1/DRIL1 is a novel p53-target that may play a role in growth arrest mediated by p53. Results E2FBP1 Is Identical to DRIL1 E2FBP1 was originally isolated as a cDNA encoding an E2F-binding protein from an expression library of the human embryonal carcinoma cell line NEC14 using a GST-E2F-1 fusion protein as a probe (1). A search of the Genbank database revealed that the E2FBP1 matched DRIL1 (2), but the reported E2FBP1 cDNA lacked a 5V portion of DRIL1 . We later sequenced another E2FBP1 cDNA clone that was 700 bp longer than the previous clone. 1 This full-length cDNA contained the additional 5V -sequence, confirming that these two genes are identical (termed E2FBP1/DRIL1 in this report). E2FBP1/DRIL1 Is a Potential Target for p53 The ubiquitous expression of E2FBP1/DRIL1 raised the possibility that E2FBP1/DRIL1 might be involved in universal cellular processes. To examine the regulation of E2FBP1/ DRIL1 , we searched the genomic sequence of the E2FBP1/ DRIL1 gene (2) and found a putative p53-binding site in its second intron (Fig. 1A). We thus examined whether the putative p53-binding site represents a functional p53-binding site. Gel mobility shift assays using a probe containing the putative p53-binding sequence detected a shift band in extracts of p53-deficient Saos-2 cells infected with an adenovirus expressing wild-type p53 (Ad-p53) (Fig. 1B). The band was competed with an unlabeled competitor containing the putative Received 2/22/02; revised 1/27/03; accepted 2/27/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Grant support: Grants-in-aid for Cancer Research (08264105 to M.I.) and Scientific Research (10470400, 10877295 to M.I. and 10307043, 08557097 to K. Eto) from the Ministry of Education, Science, Sports and Culture of Japan, the Japan Society for the Promotion of Science, and Ground Research for Space Utilization promoted by NASDA and the Japan Space Forum (to K. Eto). Requests for reprints: Masa-Aki Ikeda, Section of Molecular Embryology, Graduate School, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. Phone: 81-3-5803-5579; Fax: 81-3-5803- 0213. E-mail: mikeda.emb@tmd.ac.jp Copyright D 2003 American Association for Cancer Research. 1 K. Oda, unpublished results. Vol. 1, 438 – 444, April 2003 Molecular Cancer Research 438 Research. on December 4, 2021. © 2003 American Association for Cancer mcr.aacrjournals.org Downloaded from