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Cell Growth & Differentiation Vol. 12, 201-210, April 2001
© 2001 American Association for Cancer Research

Human RERE Is Localized to Nuclear Promyelocytic Leukemia Oncogenic Domains and Enhances Apoptosis

Thomas Waerner, Paola Gardellin, Klaus Pfizenmaier, Andreas Weith1 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
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
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
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
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 human 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 consistently 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, however, has been identified thus far. The neuroblastoma cell line NGP contains a reciprocal chromosomal translocation/duplication 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 chromosome 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 protein, the NH2-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 protein (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/polyglutamine 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 polyglutamine tract, which induces apoptosis in cultured cells (16 , 17) . DRPLA protein is a shuttle plying across the nuclear membrane; 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 overexpressed RERE correlates with the appearance of nuclear aggregates 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 subdomains. One such type of discrete nuclear matrix-associated structures is variably named as PML nuclear bodies, ND10, Kremer bodies, or PODs (20, 21, 22, 23, 24) . Cells typically contain 10–30 PODs per nucleus with diameters between 0.2 and 1 µm, although their number and size changes during the cell cycle (25) . PODs are rearranged in their intranuclear distribution in several pathogenic conditions including adenovirus infection (26) and acute promyelocytic leukemia (27, 28, 29, 30) in which PODs are dissociated to a diffuse distribution pattern in the nucleus. To date, several components of PODs have been identified in addition to the tumor suppressor PML, including the transcriptional regulators CBP, SP100, pRB, and p53, as well as the proapoptotic proteins BAX and DAXX (reviewed in Refs. 31 and 32 ). Another protein that is localized to PODs is the small ubiquitin-like modifier SUMO-1 (also known as PIC1 or sentrin). SUMO-1 is required for proper formation of PODs by covalent modification of PML through the binding of SUMO-1 at three lysine residues (33) . Unmodified PML is associated with the soluble nucleoplasmic fraction, whereas the SUMOylated PML fraction is tightly associated with the nuclear body (34) . Only the correct formation of PODs allows the transient localization of other proteins, including those described above, to nuclear bodies (34) . Several of these proteins that are transiently located to PODs are known to be involved in pathways important for tumorigenesis, such as maintaining genomic stability (35) , regulating cell growth (36) , and controlling cell survival (37, 38, 39, 40) . Functional connections and common signaling pathways between the POD components are currently being elucidated. Recent studies suggest a role of PML in p53-dependent apoptosis or senescence, depending on the cell type, that appears to involve PML-mediated acetylation of p53 by the acetyltransferase CBP in PODs (41, 42, 43) .

In this article, we report the colocalization of overexpressed nuclear RERE with PML and BAX in PODs, as well as colocalization of cytoplasmic RERE with the remaining fraction of BAX located in the cytoplasm. We further showed that cells overexpressing RERE can undergo caspase-dependent cell death.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Primary Sequence and Genomic Organization.
DNA sequence analysis of the NGP breakpoint gene has revealed significant homology to the DRPLA gene. However, the breakpoint gene is clearly distinct from the DRPLA gene because its product contains an additional 1104 amino acids at the NH2 terminus compared with the DRPLA protein. The reading frame of the breakpoint gene was nearly identical to the recently described protein, RERE (1). The predicted protein sequence described here revealed the following differences: position 65, transition of G to S; position 68, transition T to A; position 114, V to E; position 115, C to S; position 921, G to A; position 940, A to P; position 977, T to K; position 984, H to N; position 1489, I to M; and position 1490, V to L.

The exon-intron boundaries of the gene for RERE were determined by alignment of the cDNA sequence against genomic sequences. We found that the open reading frame of RERE was split into 22 exons (Fig. 1)Citation , and that it was flanked by multiple stop codons. We designated the exon containing the start codon and additional 147 bp of the 5' untranslated region as exon 1. In exon 22, we identified a stop codon and additional 572 bp of the 3' untranslated region identical to the cDNA that we isolated.



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Fig. 1. A, exon-intron boundaries within the RERE gene. Exon size was determined by BLASTN-alignment of the RERE cDNA with genomic sequences of EMBL and GenBank databases. Three genomic sequences (AC021953, AL096855, and AC024508) contained the complete open reading frame of RERE. These and representative examples of genomic sequences of chromosome 1 are listed in the accession no. of corresponding clone column. The sequence identity of each exon to the RERE cDNA was >99%. B, schematic illustration of the exon-intron boundaries. The position of the start codon is marked by an arrow in the direction of the reading frame. The stop codon is indicated by a dotted vertical line in exon 22.

 
Tissue Distribution of RERE RNA.
Northern blots with poly(A)+ RNA from normal tissues, neuroblastoma, and other tumor cell lines, as well as a dot blot of normal tissue and tumor cell lines, were probed with a 5' radiolabeled RERE cDNA probe (see "Materials and Methods"). We detected two dominant transcripts of 7.4 and 9.4 kb in all investigated human tissues (Fig. 2A)Citation . Similar results were obtained with a cDNA probe that encodes the 3' region of the RERE reading frame, with the exception that the signal intensity of the 7.4-kb transcript was slightly enhanced, relative to the 9.4-kb transcript that showed equal intensity (data not shown). These transcript sizes were consistent with the transcript sizes of the RERE homologue in rat, the atrophin-related protein (44) , but showed subtle discrepancies concerning relative abundance and size of the larger and smaller transcripts (1) . These differences might be caused by the use of hybridization probes from different regions in the RERE cDNA, detecting different RNA transcripts. Some of the tissues analyzed revealed additional bands when probed with the 5' probe. In testis, transcripts were detected at 6.8 kb and, with low abundance, at 5.2 and 3 kb. A weakly expressed transcript of 5.2 kb was also detected in thyroid, adrenal cortex, adrenal medulla, pancreas, skeletal muscle, and heart. Because the hybridization pattern of the 5' probe on Northern blots was highly specific, we also used a dot blot procedure to determine the expression of the RERE mRNA in a large variety of tissues and tumor cell lines (see "Materials and Methods"). This analysis confirmed the presence of RERE mRNA transcripts in all investigated tissues and tumor cell lines. Expression was particularly abundant in cerebellum, testis, uterus, and prostate, and in kidney, both in fetal and adult kidney (Fig. 2B)Citation .



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Fig. 2. Broad expression of RERE mRNA in human tissues and detection of RERE splice variants. The same RERE cDNA was used to probe agarose gel blots and a dot blot (Clontech, PaloAlto, CA). A, two major RNA species of 9.4 and 7.4 kb, respectively, were detected on human tissue blots. In testis, three additional mRNA species with a molecular weight of approximately 6.8, 5.2, and 3 kb were marked with *. The 5.2-kb transcript was also detected in thyroid, adrenal cortex adrenal medulla, pancreas, skeletal muscle, and heart. Right, sizes (in kb). GAPDH RNA expression (lower bands) was used as quantity control. B, in dot blots strongest signals were obtained in uterus, prostate, and testis (D8, E8, and F8), cerebellum (A2 + B2), and kidney (A7 + C11). As control for uniform RNA loading, the same blot was probed with a cDNA probe of ubiquitin (lower panel). Description of the RNA spotted on the MTE array is shown in the lower right diagram. C, a radiolabeled PCR product containing the cDNA sequence of RERE exons 4 and 5 was used to probe human tissue mRNA blots. One predominant 9.4-kb RNA species was detected in all investigated human tissues with strongest expression in testis. An additional signal at 6.8 kb appeared in testis. Right, sizes (in kb). GAPDH mRNA expression (bands in the lower panel) was used as control for equal loading.

 
Identification of Alternatively Spliced RERE Transcripts.
The detection of different mRNA transcripts of 3, 5.2, 6.8, 7.4, and 9.4 kb in size, respectively, with the 5' probe of RERE suggested that they might correspond to different RNA splice variants. To address this possibility, we first carried out sequence alignments to identify public database entries that may correspond to alternatively spliced RERE RNAs. We identified three sequences that began or ended exactly at the transcriptional start point of an exon. This indicated that the cDNA sequences were alternatively spliced. The accession number (and exons of those sequences) is AB036737 (spliced at exon 1–22), which is the previously published RERE cDNA sequence (1) . AB036737 contains a 5' sequence that is not included in our cDNA sequence and that is localized on the genomic clone with the accession number AC25240, where it is encoded as two exons. These exons contain the sequence of nucleotide 3 to nucleotides 306 and 307 to 492 of AB036737. AB007927 (spliced at exon 7–22) contains a 5' alternatively spliced sequence of 183 nucleotides that is encoded as one contig on the genomic clone AL 096855. The cDNA sequence with the accession number AF016005 (spliced at exon 11–22) also contains a 5' alternatively spliced sequence of 107 nucleotides that is encoded as one exon at the genomic sequence AL 096855. As a second step, we hybridized a human multiple tissue Northern blot with a radiolabeled probe that contained a cDNA sequence that was not present in two of the three cDNA sequences that were identified as splice products of RERE RNA. With this probe that contained cDNA sequence of exon four and exon five, we detected the 9.4-kb RERE RNA species in all investigated tissues, the 6.8-kb mRNA species in testis, but not the 7.4-kb transcript. This indicated the absence of at least the exons four and five in the 7.4-kb mRNA transcript (Fig. 2C)Citation .

RERE RNA Levels in Tumor Cell Lines.
To detect the integrity and abundance of RERE transcripts in tumors, Northern blot analysis and a real-time quantitative RT-PCR approach using TaqMan technology (Perkin-Elmer) were performed. For this purpose, we analyzed poly(A)+ RNA of 18 neuroblastoma cell lines, one promyeloblast cell line (HL-60), and one neuroectodermal cell line (SK-N-MC). Poly(A)+ RNAs of these cell lines were analyzed with the radiolabeled 5' probe of RERE cDNA. We observed that both RNA species are present at equal levels in SK-N-MC and HL-60 cells. In contrast, both transcripts are of significantly lower abundance in the neuroblastoma cell lines NGP, SK-N-AS, GI-CA-N, GI-ME-N, SHEP, Vi856, and IMR32. A reduced expression specifically of the 7.4-kb transcript was also detected in the other 11 neuroblastoma cell lines studied (Fig. 3)Citation . An additional quantitative approach using probes and primers targeted against the 3' region of RERE in TaqMan assays correlated with the observations described above (Fig. 3Citation , lower panel). Moreover, weak expression of RERE mRNA, which may indicate residual gene expression, was detected with the RT-PCR approach in all but one cell line, where no bands had been seen in Northern blots. In the neuroblastoma cell line Vi856, no RERE RNA was detected, either by Northern blot analysis or by the more sensitive TaqMan assay.



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Fig. 3. Northern blot and RT-PCR analysis of RERE mRNA expression in tumor cell lines with the 5' radiolabeled cDNA probe. Bands were weak or absent in 7 of the 18 RNA samples. Note the low abundance or absence of the 7.4-kb band in all other tumor cell lines. Right, sizes (in kb). The only exceptions are the neuroectodermal tumor cell line SK-N-MC and the leukemia cell line HL-60. GAPDH expression (lower bands) was used as quantity control. Lower panel, RT-PCR quantification of the hARP 3' region. Very weak expression is seen in cell lines that were negative in Northern analyses. Expression level is indicated as ratio to SK-N-MC, the expression level of which has been set to 1.0.

 
Cellular Localization of Full-Length and Truncated RERE Proteins.
Because the RERE protein contained putative NLSs (1) and three putative protein binding sites of amino acid repeats of either R and D (RD repeat) or R and E (RE repeat), we predicted that RERE would localize to the nucleus and that RERE might interact with other proteins in the nucleus. To investigate this, we generated a series of GFP-tagged expression constructs (see "Materials and Methods"), which allowed the expression of either full-length RERE or of the truncated forms N381, N581, and C1087 (Fig. 4A)Citation . When SK-N-AS cells were transfected with full-length GFP-RERE, expression of RERE was detected in distinct dot-like nuclear structures of variable sizes (Fig. 4, C and D)Citation . This applied to 82% of all observed GFP-positive cells (Fig. 4B)Citation . Such structures had been described previously as "nuclear bodies" (reviewed in Ref. 24 ). The remaining 18% of cells expressed GFP-RERE at very low quantity. Here, the label was preferentially detected as being homogeneously distributed in the nucleus. When monitoring the subcellular distribution, 49% of the GFP-RERE-transfected cells showed exclusive nuclear staining, whereas the other 51% displayed staining both in the nucleus and in the cytoplasm (Fig. 4B)Citation .



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Fig. 4. Cellular distribution of GFP-RERE products in transfected SK-N-AS cells. A, schematic illustration of constructs to produce a full-length or truncated RERE protein fused to GFP. The GFP portion (green), RD repeats (yellow), putative NLSs (blue), acidic sequences (gray), RERE repeats (red), and deleted sequences (gray hatched) are shown. B, the fraction of cells showing a localized pattern of fluorescence is based on three experiments; in each experiment, 100 cells were analyzed 18 h after transfection with the respective RERE constructs. Left columns, the percentage of cells showing exclusively nuclear staining and the SD (N) or staining in both the nucleus and the cytoplasm (N+C). Right columns, the percentage of transfected cells that show localization of RERE to nuclear aggregates and the SD (N. aggr.) and the size range of the observed RERE aggregates. C–F, different distribution patterns of GFP-RERE products (green). DNA is counterstained with DAPI (blue). C, note the pattern of nuclear aggregates containing full-length RERE in the nucleus and D, its localization to both nucleus and cytoplasm. E, typical distribution pattern of GFP-C1087 showing localization to conspicuous aggregates in the nucleus and in the cytoplasm. F, diffuse distribution of the GFP-N581 product with additional localization to very small nuclear and cytoplasmic aggregates. Bar, 5µm.

 
Full-length and all artificially truncated RERE products contained at least one putative NLS and localized to the nucleus as well as to the cytoplasm. The nuclear distribution of NH2- and COOH-terminally truncated GFP-RERE proteins differed from that of the full-length RERE. The C1087 deletion product, which lacks the NH2-terminal portion of RERE, showed also diffuse nuclear distribution and formed large nuclear aggregates in 32% of the GFP-positive cells (Fig. 4E)Citation . Products of the constructs N581 (Fig. 4F)Citation and N381 lacking various portions of the COOH terminus showed diffuse nuclear distribution with additional nuclear structures of small size in a fraction of transfected cells (14 and 21%, respectively). The cytoplasmic fraction of GFP-N381 and GFP-N581 was distributed homogeneously in most transfected cells, whereas GFP-C1087 localization in the cytoplasm occurred in big aggregated structures. A quantification of RERE localization to different cellular structures as well as the size range of nuclear aggregates are shown in Fig. 4BCitation . Thus, it appeared that the stringent localization of RERE to nuclear bodies requires NH2-terminal amino acid sequences, supported by one or more COOH-terminal domains.

Colocalization of the RERE and PML Proteins.
The nuclear aggregates in which full-length RERE was found to be localized are reminiscent of nuclear PODs. We therefore tested antibodies against proteins either localized to PODs or to other distinct nuclear subdomains 18 h after RERE transfection in SK-N-AS cells. We found almost complete coincidence of RERE localization with that of PML in the larger sized bodies (95–98%; data not shown), indicating that RERE is localized largely in PODs (Fig. 5, A–C)Citation . This colocalization with PML is not influenced by the GFP portion fused to RERE, because we observed a similar extent of colocalization using native RERE (Fig. 5, M–O)Citation . Upon decreased size of the structures, the PML-RERE association was less tight; indeed, in the smallest sized aggregates, significant colocalization could not be detected, but rather a localization of RERE in close proximity to PML (Fig. 5, D–F)Citation . The fraction of transfected cells that showed both nuclear aggregates and complete colocalization of endogenous PML and full-length RERE was 79% (Fig. 5P)Citation .



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Fig. 5. Colocalization of RERE (green) and PML (red) proteins in nuclear PODs. SK-N-AS cells were transfected with GFP-RERE (A–F), native RERE (M–O), or truncated forms of RERE fused to GFP (G–I, GFP-N581 and J–L, GFP-C1087). Eighteen h after transfection, endogenous PML protein was immunolabeled, and DNA was counterstained with DAPI (blue). RERE and PML are associated in aggregates either containing full-length GFP-RERE (A–C), native RERE (M–O), or the NH2-terminal GFP-N581 protein (G–I). D–F, note that both proteins do not colocalize in a fraction of small RERE aggregates or in a fraction of nuclear structures expressing the COOH-terminal GFP-C1087 product (J–L). Bar, 5µm. P shows a statistical analysis of the results displayed in A–O. The third column lists the percentage of cells displaying nuclear-dot staining of RERE and its colocalization with PML in relation to the total number of transfected cells. In the fourth column, the number of cells displaying RERE/PML colocalization in relation to the number of cells showing nuclear aggregates is provided. Cells were counted positive if >95% of their detectable PODs showed complete overlay with GFP-RERE.

 
In cells that transiently expressed the truncated RERE products N581, N381 and also showed localization of RERE to nuclear aggregates, PODs matched RERE aggregates in a fraction of transfected cells (54 and 52%, respectively; Fig. 5, G–I and PCitation ). Nuclear aggregates of the C1087 product were not congruent with PODs in 71% of the transfected cells carrying nuclear RERE aggregates (Fig. 5, J–L and P)Citation . Thus, although the nuclear staining of RERE NH2-terminal products as well as RERE COOH-terminal products was diffuse in a large percentage of transfected cells, the fraction of transfected cells that showed both nuclear aggregates and colocalization with PML was 8% (N581), 11% (N381), and 10% (C1087), as shown in Fig. 5PCitation . We could further demonstrate that all transfected RERE constructs showed a mutually exclusive staining pattern to proteins that are associated with other types of nuclear structures. Examples include the cell cycle regulator Pin1 and the RNA-splicing protein SC35 (Ref. 45 ; data not shown). These results suggested that a significant subset of RERE containing nuclear aggregates correspond to the PODs described previously.

RERE Colocalizes with BAX and Enhances Caspase-dependent Cell Death.
After 4 days, we detected morphological changes of the nucleus, such as chromatin condensation and nuclear fragmentation in roughly 85% of GFP-RERE-positive cells (data not shown). After 6–7 days, essentially all GFP-RERE-transfected cells were eliminated by apoptosis. This compares well with observations of others (1) , that overexpression of RERE is "toxic" to cells. We observed apoptotic features earlier, i.e., after 2 days, when the medium was supplemented with IFN-{gamma}. As control, IFN-{gamma} alone was not sufficient to trigger cell death in SK-N-AS neuroblastoma cells (data not shown). The role of IFN-{gamma} for this enhancement of the "toxic" RERE function is not further investigated, but it is known that IFN-{gamma} cooperates in apoptosis of neuroblastoma cells (46) and that IFNs induce the transcription of the PML protein (47) . To further investigate the mechanism of cell death induced by overexpression of RERE, we performed immunostaining with antibodies against the proapoptotic protein BAX, which has been localized previously to PODs (38) . We discovered that 20 h after transfection of SK-N-AS cells with GFP-RERE, there was a significant colocalization of RERE and BAX, irrespective of the presence or absence of IFN-{gamma} in the medium. Cytoplasmic RERE was always found to colocalize with BAX. The observed overlay between cytoplasmic staining of RERE and BAX ranged from partial to nearly complete congruence in roughly 70% of all transfected cells (Fig. 6, A–C)Citation . Furthermore, nuclear RERE was colocalized with BAX in nuclear aggregates (most likely PODs) in roughly 40% of transfected cells, independent of RERE and BAX colocalization in the cytoplasm (Fig. 6, D–F)Citation . The fact that we never observed any BAX localization in nuclear aggregates in the absence of RERE overexpression (data not shown) indicates that RERE can induce a partial recruitment of BAX to these structures. A similar cellular distribution of RERE, with respect to its colocalization with BAX (as described above), was observed in cells transfected with untagged (native) RERE (data not shown). This was achieved by using an affinity-purified antibody that recognizes moderate concentrations of exogenous RERE but unfortunately fails to detect endogenous RERE.



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Fig. 6. Colocalization of GFP-RERE with the proapoptotic protein BAX and increased apoptotic rate in RERE-overexpressing SK-N-AS cells. Cells were transfected with GFP-RERE (green). After 20 h, endogenous BAX was immunolabeled (red), and DNA was DAPI counterstained (blue). A–C, note the conspicuous portion of costained BAX and cytoplasmic RERE, observed in roughly 70% of transfected cells that exhibit cytoplasmic RERE staining. D–F, colocalization of RERE and BAX to PODs, detected in roughly 40% of all transfected cells, which occurred irrespective of additional cytoplasmic colocalization of these proteins. Cells (n = 300) were analyzed for their portion of overlapping signals. G, SK-N-AS cells, transiently transfected with GFP or GFP-RERE cDNA were analyzed for the frequency of apoptotic cells, determined by TUNEL assay. Forty-eight h after transfection, GFP-RERE-transfected cells showed an increased amount of apoptotic cells compared with GFP-transfected control cells. This effect was abolished by adding caspase-3 inhibitor to the medium. The number of all transfected cells was determined by counting the corresponding GFP-positive cells 16 h after transfection. One of three comparable experiments is shown; bars, SD of TUNEL-positive cells with 10 x 1000 cells counted under a microscope.

 
To quantify the amount of apoptotic cells, we next performed TUNEL assays. We found that after transient transfection of GFP-RERE, the fraction of TUNEL-positive cells was 42% of all transfected cells after 48 h; compared with 12% TUNEL-positive GFP-only transfected cells, this corresponds to a 3.5-fold increase in their frequency to undergo apoptosis. No enhancement of apoptosis was detected at earlier time points (Fig. 6G)Citation . To ensure that the proapoptotic function of RERE is independent of the GFP-portion, we transfected SK-N-AS cells with different amounts of native RERE cDNA. We observed a dose-dependent increase of TUNEL-positive cells, reaching levels comparable with effects observed upon GFP-RERE transfection. Compared with cells transfected with an empty vector, a strong increase in the fraction of apoptotic cells was detected. The overall observed transfection efficiency of SK-N-AS cells that we observed was between 12 and 20% (data not shown).

Because RERE-induced cell death was associated with typical nuclear morphological features of apoptosis, such as chromatin condensation, we examined whether activated caspase-3, the major caspase executioner of apoptosis, was required for this process. We therefore treated GFP-RERE-transfected cells with a nonreversible, caspase-3-specific inhibitor (48) . It became apparent that application of the inhibitor efficiently prevented the increased appearance of TUNEL-positive cells (Fig. 6G)Citation . When treating the GFP-RERE-transfected cells with this caspase inhibitor, DNA fragmentation was prevented, but the formation of nuclear aggregates was not suppressed (data not shown).


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
In this study, we provide information on the human RERE gene, which is located across the translocation breakpoint in the neuroblastoma cell line NGP and which is homologous to the DRPLA gene. During the course of our experiments, Yanagisawa et al. (1) published the observation that RERE protein largely localizes to "nuclear speckles," without any further description of the nature of these domains. Here, our results demonstrate that in cells transiently expressing a high level of RERE, a large fraction of this protein is located to PODs (20 , 21 , 24) . It is known that the formation of PODs is important for proper cell division and for cell growth control (25 , 36 , 37) . The POD-associated proteins PML, pRB, p53, and BAX are characterized as tumor suppressors (31 , 38 , 41) . PML antagonizes the initiation and progression of tumors of various histological origins acting as a cell growth suppressor (49) . Because PML-/- mice and cells are resistant to different apoptotic signals, PML is also required for activation of caspase-1 and caspase-3 (39) . We have shown that a high cellular concentration of RERE induces apoptotic cell death, correlating with proapoptotic activity of PML and BAX. However, although strong expression of PML also induces caspase-independent cell death (38) , the expression of RERE in contrast appears to preferentially induce activation of caspases and concomitant apoptosis. Thus, the function of RERE may rather be comparable with the overexpression of proapoptotic BAX. Interestingly, we could show that upon transfection of RERE, almost all transfected cells showed cytoplasmic colocalization of RERE and BAX, and in addition, a fraction of cells showed recruitment of BAX from the cytosol to nuclear RERE aggregates. Because we detected a specific cytoplasmic colocalization of RERE protein with BAX but not with other proteins that are at least partially localized to the cytoplasm (BCL-2, ß-actin, and p58 Golgi protein),4 it might be possible that RERE plays a direct role in recruiting BAX to the nucleus and particularly to PODs. Thus, recruitment of proapoptotic proteins such as BAX to PODs may be the mechanism of how RERE promotes apoptosis. Future studies will address the question of whether RERE physically interacts with BAX and/or PML. Recruitment of cytoplasmic BAX to the nucleus has been described previously in apoptotic processes that are induced by anti-epidermal growth factor receptor antibodies in colorectal carcinoma cells (50) . Localization of BAX specifically to nuclear PODs has been described in HeLa cells expressing high levels of PML protein (38) , indicating a functional association of the two proteins.

RERE-overexpressing cells showed typical apoptotic features, including chromatin condensation, nuclear fragmentation,4 and internucleosomal DNA cleavage as detected by TUNEL assay. We also detected the cleavage of poly(ADP-ribose) polymerase,4 which is a prior step to DNA fragmentation in the apoptotic process. Poly(ADP-ribose) polymerase cleavage is caused by activated caspase-3, a member of the interleukin-1ß converting enzyme/CED-3 family. We have shown that upon blocking caspase-3 activity with a caspase inhibitor, the induction of apoptosis is abolished. This indicates that RERE overexpression induces caspase-dependent apoptotic cell death. Another proapoptotic protein, DAXX, is also localized to nuclear PODs and interacts directly with PML (35 , 40) . The proapoptotic activity of DAXX is abolished in the PML-/- cells, where it is aberrantly localized in the nucleus. It remains to be seen whether RERE localization is similarly altered in PML mutant mice. DAXX can function as a transcriptional repressor, and also PML itself appears to be involved in transcriptional regulation of target genes such as p53 (41, 42, 43) , possibly as a cofactor via interaction with CREB-binding protein (31) . Furthermore, PODs represent a site of active gene transcription within nuclei (31) . In this context, it is interesting to note that atrophin-1, which shows sequence homology with RERE but which is apparently not localized to PODs (51) , associates with nuclear receptor corepressor complexes and thus may be involved in transcriptional regulation (51) . Further studies using a heterologous transcriptional reporter system are required to address a potential role of RERE in transcriptional regulation, possibly as a cofactor, because a DNA-binding domain is not evident.

Our investigations on the cellular localization of various RERE protein products (full-length RERE and NH2- or COOH-terminal deletion mutants of RERE) add further information on which regions of RERE enhance localization to PODs. Furthermore, our studies provide data on the morphology, number, and size of RERE-containing nuclear aggregates. In addition to the published uniform staining of the NH2-terminal RERE products (1) , we identified a significant fraction of nuclear aggregates containing NH2-terminal portions of RERE that colocalized with PODs in neuroblastoma cells. In contrast, the nuclear staining of the COOH-terminal RERE portion indicated the localization to nuclear aggregates of which only a small percentage coincided with PODs (see Fig. 5KCitation ). Thus, we propose that the NH2-terminal part of RERE is mainly responsible for the localization to small PML-containing nuclear aggregates. Our preliminary data suggest that localization to PODs correlates with the ability of RERE to induce apoptosis. Mutants lacking various portions of the COOH terminus that show less stringent colocalization to PODs also cause less apoptosis.4 Strikingly, this NH2-terminal RERE portion is not present in the RERE homologous protein atrophin-1 (and its mutated form DRPLA) and in two RERE splice variants that we identified in public databases; this suggests a lack of colocalization of these putative translation products with PODs. Indeed, this suggestion is supported by previous findings that overexpressed DRPLA, which interacts with RERE, localizes predominantly in the nucleus, either with almost uniform distribution or in discrete nuclear structures, but does not colocalize with PODs (51) . Localization of RERE to nuclear aggregates was also absent in cells with low exogenous RERE protein level4 (1) or in cells overexpressing both RERE and its interacting partner DRPLA (1) . The results presented here do not only indicate that RERE localizes to PODs when it is highly expressed but also defines the importance of the NH2-terminal protein portion in this process. Different splice variants and loss of function mutants as well as their involvement in tumorigenesis are described for both the BAX gene (52) and the PML gene (29 , 53) . It remains to be further tested whether the different RERE splice variants have different functional properties, such as interaction with PML and/or BAX as well as differences in their capability to induce apoptosis. Thus far, in the absence of a suitable antibody a study of endogenous RERE in neuroblastoma cells is presently not possible. Our Northern blot analyses indicate subtle differences between normal tissues and tumor cells, in which either both major transcripts or specifically the smaller, 7.4-kb transcript are expressed at reduced levels in neuroblastoma tumor cell lines. The RERE gene has been identified as a the breakpoint gene in the neuroblastoma cell line NGP3 and is located in a genomic region that is frequently mutated in neuroblastomas (4) . The functional relevance of the reduction of RERE transcript level for the development of neuroblastoma tumors remains to be seen.

In summary, we have shown that RERE is localized to PODs and colocalizes with the proapoptotic proteins PML and BAX. Furthermore, overexpression of RERE triggers caspase-3 activation, leading to cell death. These findings shed considerable light on the function of RERE, which may provide an important link between the role of PODs in the control of apoptosis and tumorigenesis.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Reagents and Cells.
All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO), and restriction enzymes were obtained from Roche (Basel, Switzerland), unless specified otherwise. Cells were cultured in DMEM (Life Technologies, Inc., Rockville, MD) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 50 units/ml penicillin, and 0.1 mg/ml streptomycin at 37°C under a humidified atmosphere of 5% CO2. Cells were grown in either flasks, 100-mm dishes, or chamber slides (Nalgene Nunc International, Naperville, IL). The human neuroblastoma cell lines STA-NB-3, STA-NB-1.1, and Vi856 were kindly provided by P. Ambros, (Childrens Cancer Research Institute, Vienna, Austria), and SJ-NB-5, 6,7,8,10,12 were kindly provided by V. Kidd (St. Jude’s Hospital, Philadelphia, PA). Other cell lines were obtained from general providers. The medium of all cells that were cultured for RNA isolation was supplemented with nonessential amino acids (Life Technologies, Inc.). For transient cell transfection, FUGENE 6 transfection reagent (Roche) was used as described in the manufacturer’s protocol.

RNA Detection.
For TaqMan analysis (ABI PRISM 7700 Sequence Detector; Perkin-Elmer Corp., Norwalk, CT), all probe signals were quantified relatively by comparison to the GAPDH signal (GAPDH control reagents; Perkin-Elmer Corp.). All primers were synthesized by MWG-Biotech AG (Ebersberg, Germany). For detection of the RERE 3' RNA sequence, the following primers were used: sense primer, 5'-GATGCTTCGCCACCCAGTT-3', antisense primer 5'-TCGACTGTAATAATCTTCCTGACTTGG-3', and probe 5'-CTCCATGGCCAGTCTCTGCAGCTC-3'.

Northern blots were prepared using a standard protocol (54) . Poly(A)+ RNA of 20 tumor cell lines was purified using a direct mRNA purification kit (Qiagen, Hilden, Germany), separated by gel electrophoresis, and subjected to nylon membrane (DuPont, Boston, MA). The following blots were purchased from Clontech: MTE blot; Human Multiple Tissue Northern blot panel I, IV; and Human Endocrine blot. Two different probes were generated for Northern blot analysis, i.e., a RERE 5' probe, encoding the cDNA sequence nucleotides 364-1411, and the RERE 3' probe containing the cDNA sequence nucleotides 4165–5095. The probes were gel purified, eluted with a gel purification kit (Qiagen), and labeled with [33P]dCTP (Amersham) using the Megaprime labeling system (Amersham). For demonstration of RERE RNA splice variants, a [33P]dATP (Amersham)-labeled PCR product (cDNA sequence nucleotides 724–877) of the RERE cDNA was amplified (sense primer, 5'-AGTAAGAGGGACCATTC-3'; antisense primer, 5'-AAGGGCAGCATGGT-3'). All probes were purified using a nucleotide removal kit (Qiagen), and hybridization was performed according to the manufacturer’s instructions (Clontech). For control of equal RNA loading, a GAPDH probe was generated using the antisense primer 5'-TTCTACCACTACCCTAAAG-3 (MWG-Biotech) and the ECL 3'-Oligolabeling and Detection System (Amersham), as it is described in the manufacturer’s protocol. Blots were placed on a BioMax MR film (Eastman Kodak, Rochester, NY) with BioMax TranScreen-LE intensifying screen (Kodak) for 2 days (GAPDH) and 4 days (RERE) at -70°C.

RERE cDNA and Deletion Constructs.
A partial RERE cDNA sequence was used to identify expressed sequence tags with overlapping sequences in public databases. For this and all further described sequence homology searches, we used the bioScout software (LION Bioscience AG, Heidelberg, Germany). The sequence of one clone (accession no. AA721677) was identified that completed the partial RERE cDNA sequence. This clone was sequenced and digested with the restriction enzymes NotI and SgrAI. The obtained 746-nucleotide insert was gel purified (QIAquick purification kit; Qiagen) and ligated to the previously NotI- and SgrAI-digested pSG5/partial RERE cDNA construct to receive the entire coding sequence of RERE (pSG5; Stratagene, La Jolla, CA; kindly provided by P. Gardellin, Institute of Molecular Pathology, Vienna, Austria). To obtain an NH2-terminal GFP fusion protein (pEGFP-RERE), the RERE cDNA was digested with the restriction enzymes PmlI (New England Biolabs, Beverly, MA) and SacII and ligated into the previously Ecl136 II (New England Biolabs)- and SacII-opened vector pEGFPC2 (Clontech, Palo Alto, CA). To obtain truncated NH2-terminal RERE, the full-length cDNA was either digested with the enzymes PstI (N381, amino acids 1–381) or SalI (N581, amino acids 1–581), purified, and religated. To receive the COOH-terminal RERE portion (C1087, amino acids 480-1567), the cDNA was digested with EcoRI and BamHI, purified, and cloned into EcoRI/BamHI-opened pEGFPC2. To obtain native RERE cDNA, RERE cDNA deletion constructs, and vector-only construct, the GFP part of the respective pEGFPC2 construct was excised using the enzymes NheI and XhoI. Subsequently, DNA overhangs were blunted using Klenow enzyme, and constructs were ligated after a gel purification step.

Genomic DNA.
The genomic sequence of the RERE reading frame was determined with sequence alignment of the RERE cDNA sequence with genomic sequences using bioScout (LION Bioscience AG).

Nucleotide Sequencing.
Nucleotide sequences were determined with LONG READIR4200 (Licor, Lincoln, NE) using Thermo Sequenase cycle sequencing kit (Amersham Pharmacia Biotech, Uppsala, Sweden) and vector- or gene-specific primers synthesized at MWG-Biotech AG. Sequence data were assembled and manipulated using LI-COR AlignIR software (MWG-Biotech AG).

Immunofluorescence Microscopy.
Cells were fixed on glass chamber slides (Nalgene Nunc International) with 4% paraformaldehyde/PBS (pH 7.4) for 20 min at 4°C and permeated with Triton X-100/PBS (0,5%) for 10 min at room temperature. Slides were blocked with 4% BSA/PBS solution for 30 min and then incubated for 1 h with the respective primary antibody, diluted 1:200 in 4% BSA/PBS:anti-PML (PG-M3; Santa Cruz Biotechnology, Santa Cruz, CA), anti-SC 35 (Sigma Chemical Co.), anti-Pin1 (kindly provided by Dr. Schnapp, Boehringer Ingelheim Pharma KG, Germany), and anti-BAX (B-9; Santa Cruz Biotechnology). We also used a second BAX antibody (Ab-1; Calbiochem, Cambridge, MA), yielding identical results. Affinity-purified RERE antibody was generated against the following peptide (ESTKKNKKKPPKKKSRY; amino acids 70–86). After washing twice for 5 min in PBS, bound antibodies were detected with either Alexa Fluor 568 goat antimouse IgG F(ab')2 or Alexa Fluor 488 antimouse IgG F(ab')2 (Molecular Probes, Eugene, OR). Finally, slides were washed twice with PBS, mounted with Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL) including DAPI (1 ng/ml) for DNA counterstaining. Cells were viewed using a Leica DMRA microscope (Leica, Wetzlar, Germany) and analyzed for their portion of overlapping signals using dhs(M.I.S) software (Leica, Wetzlar, Germany). Colocalization was also demonstrated using a confocal microscope (DMIRBE; Leica).

TUNEL Assay.
Cells were either assayed immediately after transfection or were cultured for 16 or 48 h in DMEM as described above, supplemented with 2 x 103 units/ml IFN-{gamma} (Sigma Chemical Co.). The TUNEL assay was performed with formaldehyde-fixed cells, according to the manufacturer’s instructions (In Situ Cell Death Detection kit; Roche). For caspase inhibition, medium supplemented with 100 µM caspase-3 inhibitor III (Ac-DEVD-CMK; Calbiochem, La Jolla, CA) was given to the cells 1 h before the cDNA transfection.


    Acknowledgments
 
We are grateful to Dr. Wolfgang Rettig and our colleagues of the Department of Oncology Research, Boehringer Ingelheim Pharma KG, for discussion and technical support. We also thank Dr. Andreas Koehler for discussion and bio-informatic support, Dr. Peter Seither for discussion, and Dagmar Knebel and Werner Rust for excellent technical support.


    Footnotes
 
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.

1 To whom requests for reprints should be addressed, at Division of Research, Genomics Group, Boehringer Ingelheim Pharma KG, Birkendorfer Strasse 65, 88397 Biberach an der Riss, Germany. Phone: 49-7351-54-5354; Fax: 49-7351-54-5991; E-mail: andreas.weith{at}bc.boehringer-ingelheim.com Back

2 The abbreviations used are: RERE, RE repeats encoded; DRPLA, dentatorubral and pallidoluysian atrophy; SCA, spinocerebellar ataxia; DAPI, 4',6'-diamino-2-phenylindol; GFP, green fluorescent protein; PML, promyelocytic leukemia; POD, PML oncogenic domain; NLS, nuclear localization signal sequence; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-PCR, reverse transcription-PCR. Back

3 P. Gardellin, K. Paiha, R. Kurzbauer, and A. Weith, manuscript in preparation. Back

4 T. Waerner, unpublished data. Back

Received for publication 10/12/00. Revision received 2/ 1/01. Accepted for publication 3/ 6/01.


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 Discussion
 Materials and Methods
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