May 20, 1996 - The EMBO Journal vol.15 no.18 pp.4949-4958, 1996. Effects on differentiation by the promyelocytic leukemia PML/RARa protein depend on ...
The EMBO Journal vol.15 no.18 pp.4949-4958, 1996
Effects on differentiation by the promyelocytic leukemia PML/RARa protein depend on the fusion of the PML protein dimerization and RARa DNA binding domains Francesco Grignanil, Ugo Testa2, Daniela Rogaia1, Pier Francesco Ferrucci1, Paola Samoggia2, Antonello Pinto3, Donatella Aldinucci3, Vania Gelmettil, Marta Fagiolil, Myriam Alcalayl4, Jacob Seeler5, Fausto Grignani1, lido Nicoletti1, Cesare Peschle2'6 and Pier Giuseppe Pelicci 417 Ilstituto di Clinica Medica I, Policlinico Monteluce, Perugia University, 06100 Perugia, 2Department of Haematology and Oncology, Istituto Superiore di Sanita, 00161 Rome, 3Centro di Riferimento Oncologico, 33081 Aviano, 4European Institute of Oncology, Department of Experimental Oncology, 20141 Milan, Italy, 5Unite de Recombinaison et Expression Genetique, INSERM U 163, CNRS URA 271, Institute Pasteur, 75724 Paris Cedex 15, France and 6Thomas Jefferson University, Philadelphia, PA 19107-5541, USA 7Corresponding author The block of terminal differentiation is a prominent feature of acute promyelocytic leukemia (APL) and its release by retinoic acid correlates with disease remission. Expression of the APL-specific PML/RARa fusion protein in hematopoietic precursor cell lines blocks terminal differentiation, suggesting that PML/ RARa may have the same activity in APL blasts. We expressed different PML/RARa mutants in U937 and TF-1 cells and demonstrated that the integrity of the PML protein dimerization and RARa DNA binding domains is crucial for the differentiation block induced by PMLJRARc, and that these domains exert their functions only within the context of the fusion protein. Analysis of the in vivo dimerization and cell localization properties of the PML/RARa mutants revealed that PML/RARa-PML and PML/RARa-RXR heterodimers are not necssary for PML/RARa activity on differentiation. We propose that a crucial mechanism underlying PML/RARa oncogenic activity is the deregulation of a transcription factor, RARa, through its fusion with the dimerization interface of another nuclear protein, PML. Keywords: APL/differentiation block/PML/RARa/RXR
Introduction Chromosomal translocations are a frequent feature of leukemias and sarcomas. Chromosome breakpoints often involve nuclear proteins, mainly transcription factors, and lead to the formation of fusion proteins. As most of these translocations are primary karyotype alterations, the corresponding fusion proteins are considered crucial to the process of neoplastic transformation (reviewed in Rabbitts, 1994). However, we know little about the mechanisms through which these fusion proteins exert their K Oxford University Press
biological effects, particularly the relative contribution of each fusion protein component and the effects of the fusion itself. Acute promyelocytic leukemia (APL) is characterized by the 15;17 translocation which generates the PML/ RARcx chimeric gene (Warrel et al., 1993; Grignani et al., 1994). The resulting PML/RARax fusion protein is of particular interest because its expression correlates both with the promyelocytic phenotype and with disease remission and blast differentiation in response to retinoic acid (RA) treatment (LoCoco et al., 1991; Warrel et al., 1991). Thus, PML/RARx might be implicated in both the pathogenesis of the disease and the response to RA. This concept is supported by the finding that the ectopic expression of PML/RARx into human hematopoietic cell lines blocks differentiation by physiological stimuli, such as vitamin D3 (D3), D3 + transforming growth factorP1 (TGF) or hemin, but increases susceptibility to the differentiative stimulus of pharmacological concentrations of RA (Grignani et al., 1993, 1995). The mechanisms through which PML/RARa exerts its biological activities are poorly understood; available data are limited to results of in vitro functional activities of specific PML and RARa regions retained in the fusion protein. RARa binds RA target gene promoters in physical association with RXRs (reviewed in Kastner et al., 1994), a family of retinoid receptors which act as co-factors for other nuclear receptors, such as D3 (VDR) and thyroid hormone receptors (Yu et al., 1991; Kliewer et al., 1992; Zhang et al., 1992). As the PML/RARac fusion protein retains the RARa DNA and RA binding domains and the interface of dimerization with RXR, it is able to transactivate RA target genes in vitro and bind RXRs and RA (de The et al., 1991; Kakizuka et al., 1991; Pandolfi et al., 1991; Kastner et al., 1992; Nervi et al., 1992; Perez et al., 1993). The capacity of PML/RARoc to bind RXR exerts an indirect negative effect on VDR transactivation functions in vitro, probably by competing for RXRs (Perez et al., 1993). Sequestering of RXR has been proposed as a mechanism of PML/RARax oncogenic activity (Weis et al., 1994). The physiological function of PML is unknown. Its N-terminal portion includes three cysteine/histidine-rich regions. The first region includes the RING motif that defines a large family of proteins whose functions involve regulation of gene expression and/or DNA binding (Reddy et al., 1992; Lovering et al., 1993). C-terminal to the cysteine/histidine clusters is a coiled-coil region which mediates the formation of PML homodimers in vitro (Kastner et al., 1992; Perez et al., 1993). Both the PML cysteine-rich and coiled-coil regions are retained in the PML/RARcx fusion protein. The coiled-coil region directs the formation of PML/RARox homodimers and PML/ RARac-PML heterodimers in vitro (Kastner et al., 1992; Perez et al., 1993).
4949
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PML proteins are normally localized in specific nuclear subdomains (PML nuclear bodies, PML-NB) which contain other proteins, including the SplOO autoantigen (Szostecki et al., 1990). In APL cells, the PML-NBs are disrupted and PML/RARa and PML co-localize in poorly characterized microspeckled nuclear structures (Dyck et al., 1994; Koken et al., 1994; Weis et al., 1994). Delocalization of PML by PML/RARx is thought to be the consequence of PML/RARoc-PML heterodimer formation in vivo (Dyck et al., 1994; Koken et al., 1994; Weis et al., 1994). The capacity of PML/RARax to regulate RA target genes, to bind RXR and PML in vitro and to disrupt PML-NBs in vivo has led to the hypothesis that PML/ RARa might interfere with the function of a variety of nuclear proteins that include RARa, PML, other RXRdependent nuclear receptors and other components of PML-NBs. The process through which RA releases the differentiation block of APL blasts remains unknown. Notably, RA induces the redistribution of PML/RARa and PML into re-aggregated PML-NBs (Dyck et al., 1994; Koken et al., 1994; Weis et al., 1994). We investigated the mechanisms through which PML/ RARa interferes with terminal differentiation by expressing a series of PML/RARx mutants with deletions of relevant PML and RARa domains in the U937 and TF-1 hemopoietic precursor cell lines. The results suggest a novel mechanism of oncogene activation, whereby the activity of a transcription factor is modified by its fusion with the dimerization interface of another nuclear protein.
4950
Results Production of PMLIRARa mutants deficient in PML/RARac and PML dimerization or RA-dependent
enhancer functions We generated PML/RARa mutants with deletions of the PML cysteine/histidine-rich region (AC/R), the PML coiled-coil region (AH/R) or the RARox zinc finger region (P/AR) (Figure 1). To verify whether the deletions selectively abolished individual PML/RARoc functions, the capacity of each mutant to dimerize with PML or PML/RARoc and to act as an RA-inducible transcription factor was tested in co-immunoprecipitation and transactivation experiments. For PML/RARa-PML co-immunoprecipitations, HeLa cells were transiently co-transfected with expression vectors for the wild-type PML isoform 3 (PML3) (Fagioli et al., 1992) and each of the PML/RARax deletion mutants. Cell lysates were immunoprecipitated with an antiserum (anti-PML3) specific for a PML3 epitope not retained within PML/RARa and blotted with the RPa(F) anti-RARa antiserum (Gaub et al., 1989) (Figure 2A). The capacity of each PML/RARoc mutant to dimerize with PML/RARx was tested in similar coimmunoprecipitations that employed a hemagglutinin (HA) epitope-tagged PML/RARa cDNA and the various deletion mutants. Anti-HA immunoprecipitates were analyzed with the anti-RARa antiserum (Figure 2B). Transactivation experiments were performed by co-transfecting HeLa cells with an RA-responsive reporter gene (Pandolfi et al., 1991) and expression vectors for PML/RARoc deletion mutants in the presence of 10- M RA (not
Mechanisms of PML/RARa effects on differentiation
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