Human RERE Is Localized to Nuclear Promyelocytic Leukemia Oncogenic Domains and Enhances Apoptosis Thomas Waerner, Paola Gardellin, Klaus Pfizenmaier, Andreas Weith, 1 and Norbert Kraut Department of Research, Boehringer Ingelheim Pharma KG, 88397 Biberach, Germany [T. W., A. W., N. K.]; Institute of Cell Biology and Immunology, University of Stuttgart, 70596 Stuttgart, Germany [K. P.]; and Institut Curie, Centre Universitaire, 91405 Orsay, France [P. G.] Abstract RE repeats encoded (RERE) was identified recently as a protein with high homology to the atrophin-1 protein, which appears to be causal in the hereditary neurodegenerative disorder termed dentatorubral- pallidoluysian atrophy (DRPLA) caused by an abnormal glutamine expansion. We have independently identified RERE in a search for genes localized to the translocation breakpoint region at chromosome 1p36.2 in the neuroblastoma cell line NGP. Here we show that neuroblastoma tumor cell lines display reduced abundance of RERE transcripts. Furthermore, we detected RERE protein mainly in the nucleus, where it colocalizes with the promyelocytic leukemia protein in promyelocytic leukemia oncogenic domains (PODs). Overexpression of RERE recruits a fraction of the proapoptotic protein BAX to PODs. This observation correlates with RERE-induced apoptosis, which occurs in a caspase-dependent manner. These results identify RERE as a novel component of PODs and suggest an important role of RERE in the control of cell survival. Introduction RERE, 2 a novel gene described recently by Yanagisawa et al. (1), is located to the distal region of chromosome 1p. In this genomic region, molecular and cytogenetic studies have unveiled frequent structural rearrangements in several hu- man malignancies (2, 3). In neuroblastoma, comparison among extensive studies indicates a 10-cM region between genomic markers D1S47 and D1S244 at 1p36.2 that is con- sistently deleted (4, 5). These results, and the observation that introduction of a normal chromosome 1p region into neuroblastoma (6) or colorectal carcinoma (7) abolishes the tumor phenotype, suggest that this region contains one or more tumor suppressor genes. No strong candidate, how- ever, has been identified thus far. The neuroblastoma cell line NGP contains a reciprocal chromosomal translocation/du- plication t(1;15)(p36.2;q24), dup(1)(p36.2–p36.3) (Ref.8), which maps within the minimal deleted region (9). The genomic sequence of RERE was identified recently to span this reciprocal translocation/duplication breakpoint in the cell line NGP. 3 RERE is related to the DRPLA gene that resides on chromo- some 12p. RERE RNA as well as DRPLA RNA, in general, are widely expressed (1, 10). DRPLA protein contains an unstable expansion of a “CAG” repeat which, upon extension, leads to a polyglutamine stretch. In contrast, RERE does not contain such a polyglutamine repeat. The COOH-terminal portion of the RERE protein is homologous to the DRPLA protein with 67% identical residues at corresponding positions, including two RERE repeats. In addition to its homology to the DRPLA pro- tein, the NH 2 -terminal portion of the RERE protein contains an arginine-aspartic acid (RD) dipeptide repeat and two putative NLSs of monopartite and bipartite basic amino acid stretches (1, 11). The polyglutamine stretch in DRPLA protein appears to be causal for a progressive neurodegenerative disorder through interaction with specific effector molecules including RERE pro- tein (1, 12). Affected brain areas are predominantly located in the dentate nucleus of the cerebellum and in the globus pallidus (12, 13). The molecular mechanism underlying neuronal death seems to be common among seven different CAG/polyglu- tamine disorders including Huntington’s disease, spinobulbar muscular atrophy, and SCA (SCA1, SCA2, SCA3, SCA6, and SCA7). In affected brains of patients and model organisms (14, 15), nuclear inclusion bodies are detected. These inclusion bodies seem to be consistent with aggregates formed in cells by overexpression of small proteins carrying an extended poly- glutamine tract, which induces apoptosis in cultured cells (16, 17). DRPLA protein is a shuttle plying across the nuclear mem- brane; it functions in a signal transduction pathway coupled with insulin/insulin-like growth factor-I (18) and is a caspase substrate (19). Association of DRPLA protein with overex- pressed RERE correlates with the appearance of nuclear ag- gregates of different sizes and with the induction of cell death (1). In the nucleus, different kinds of small aggregates exist that delineate highly organized structural and functional subdo- mains. One such type of discrete nuclear matrix-associated structures is variably named as PML nuclear bodies, ND10, Kremer bodies, or PODs (20 –24). Cells typically contain 10 –30 Received 10/12/00; revised 2/1/01; accepted 3/6/01. 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 indi- cate this fact. 1 To whom requests for reprints should be addressed, at Division of Research, Genomics Group, Boehringer Ingelheim Pharma KG, Birken- dorfer Strasse 65, 88397 Biberach an der Riss, Germany. Phone: 49- 7351-54-5354; Fax: 49-7351-54-5991; E-mail: andreas.weith@bc. boehringer-ingelheim.com. 2 The abbreviations used are: RERE, RE repeats encoded; DRPLA, den- tatorubral and pallidoluysian atrophy; SCA, spinocerebellar ataxia; DAPI, 4',6'-diamino-2-phenylindol; GFP, green fluorescent protein; PML, pro- myelocytic leukemia; POD, PML oncogenic domain; NLS, nuclear local- ization signal sequence; TUNEL, terminal deoxynucleotidyl transferase- mediated nick end labeling; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-PCR, reverse transcription-PCR. 3 P. Gardellin, K. Paiha, R. Kurzbauer, and A. Weith, manuscript in preparation. 201 Vol. 12, 201–210, April 2001 Cell Growth & Differentiation