SMRT corepressor interacts with PLZF and with the PML-retinoic acid receptor a (RARa) and PLZF-RARa oncoproteins associated with acute promyelocytic leukemia

Retinoic acid receptors (RARs) are hormone-regulated transcription factors that control key aspects of normal differentiation. Aberrant RAR activity may be a causal factor in neoplasia. Human acute promyelocytic leukemia, for example, is tightly linked to chromosomal translocations that fuse novel amino acid sequences (denoted PML, PLZF, and NPM) to the DNA-binding and hormone-binding domains of RARa. The resulting chimeric receptors have unique transcriptional properties that may contribute to leukemogenesis. Normal RARs repress gene transcription by associating with ancillary factors denoted corepressors (also referred to as SMRT, N-CoR, TRAC, or RIP13). We report here that the PML-RARa and PLZF-RARa oncoproteins retain the ability of RARa to associate with corepressors, and that this corepressor association correlates with certain aspects of the leukemic phenotype. Unexpectedly, the PLZF moiety itself can interact with SMRT corepressor. This interaction with corepressor is mediated, in part, by a POZ motif within PLZF. Given the presence of POZ motifs in a number of known transcriptional repressors, similar interactions with SMRT may play a role in transcriptional silencing by a variety of both receptor and nonreceptor transcription factors. Retinoids regulate many aspects of vertebrate cell proliferation and differentiation through receptors that function as hormone-regulated transcription factors (1–6). Two major classes of retinoid receptors have been identified: retinoic acid receptors (RARs) and retinoid X receptors (RXRs) (3–6). Both RARs and RXRs bind to specific sites on the DNA, and they can activate or repress expression of adjacent target genes (1–6). Aberrant RARs appear to play a crucial role in human acute promyelocytic leukemia (APL) (7–15). In over 95% of all APL patients, specific chromosomal translocations create abnormal RARs by replacing the N terminus of RARa with novel ORFs. The breakpoint in RARa is identical in all cases, whereas the nature of the novel N-terminal sequences can vary. In the common t(15;17) translocation, the RARa N terminus is replaced with an ORF denoted PML (for promyelocytic leukemia) (7–12). Alternatively, t(11;17) and t(5;17) translocations result in chimeric proteins denoted PLZF (promyelocytic leukemia zinc finger)-RARa and NPM (nucleophosmin)-RARa, respectively (13–15). Although possessing a number of recognizable structural motifs, including several prevalent in transcription factors, the PML, PLZF, and NPMderived sequences exhibit little structural interrelatedness, and the functions of these proteins in the normal cell are not fully understood (reviewed in refs. 16–18). The near-invariant association of acute promyelocytic leukemia with the expression of chimeric RARa proteins, and the ability of retinoids to drive leukemias bearing the PML-RARa translocation into differentiation and clinical remission (16– 18), suggest an active role for these chimeras in conferring the leukemogenic phenotype. Furthermore, ectopic expression of PML-RARa in murine or avian systems can lead to leukemia and hepatocellular carcinoma (e.g., see refs. 19–22). However, the molecular basis by which these chimeric RARa proteins contribute to neoplasia is unclear. PML-RARa and PLZFRARa bind to many of the same target DNA sites recognized by RARa (14, 23–25). Therefore, the chimeric RARa proteins may function in leukemias by leading to aberrant regulation of genes that are targets for RARa in normal cells. Alternatively, if PML and PLZF are themselves involved in transcriptional regulation, the chimeras may aberrantly regulate genes normally under PML or PLZF control. In the absence of hormone, RARs (and the closely related thyroid hormone receptors, T3Rs) act as transcriptional repressors, a process mediated, in part, by the physical association of these receptors with ancillary proteins termed corepressors (26–33). Addition of hormone converts these receptors into transcriptional activators, a process that correlates with dissociation of the corepressors from the receptor and a corresponding recruitment of a novel set of coactivator proteins (26–34). In vertebrates, corepressors are encoded by two known, interrelated loci, denoted SMRT (or TRAC) and N-CoR (or RIP13), with each locus expressed as a variety of alternatively spliced mRNAs (e.g., TRAC-1, TRAC-2, RIP13A; refs. 26–33). Mutant T3Rs exhibiting aberrant interactions with corepressors display aberrant transcriptional regulatory properties (26–29, 33). Therefore we asked if the PML-RARa and PLZF-RARa chimeras exhibited similar aberrant interactions with corepressors that might contribute to their oncogenic proclivities. The results of these studies are reported below. MATERIALS AND METHODS In Vitro ReceptoryCorepressor Binding Assays. Glutathione S-transferase (GST), and GST-SMRT fusion constructions representing codons 71–769 of TRAC-1 were previously described (29, 33). Similar GST fusion constructs restricted to SMRT(TRAC-1) codons 71–394, 406–769, or 611–769 were created by subcloning of appropriate restriction fragments into pGEX-KG (33). GST-PLZF fusion proteins representing The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y949028-6$2.00y0 PNAS is available online at http:yywww.pnas.org. This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: RAR, retinoic acid receptor; PML, promyelocytic leukemia; PLZF, PML zinc finger; AD, activation domain; DBD, DNA-binding domain; T3R, thyroid hormone receptor; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase. †S.-H.H. and G.D. contributed equally to this work. §To whom reprint requests should be addressed. e-mail: mlprivalsky@ ucdavis.edu.