It has been proposed that retinoic acid receptors (RARs) and thyroid hormone receptors (TRs) both bind to AGGTCA “half-site” sequences, but distinguish their different target genes by recognizing different half-site spacings. We report here that artificial DNA binding sites based on these AGGTCA half-sites confer high affinity, but poor specificity, and that spacing alone does not account for the divergent DNA recognition properties of TRs and RARs. Instead, we have determined that the non-consensus half-sites that are present in naturally occurring RAR and TR target genes play a crucial role in defining receptor DNA recognition specificity, and work together with flanking sequences and half-site spacing to produce receptor-specific DNA binding in vitro. We also provide evidence that auxiliary proteins in cells generate an additional layer of receptor-specific target gene recognition, in part by destabilizing the binding of nuclear receptors to the “wrong” response elements.

Human acute promyelocytic leukemia is causally linked to chromosomal translocations that generate chimeric retinoic acid receptor-␣ proteins (x-RAR␣ fusions). Wild-type RAR␣ is a transcription factor that binds to the SMRT/NCoR family of corepressors in the absence of hormone but releases from corepressor and binds coactivators in response to retinoic acid. In contrast, the x-RAR␣ fusions are impaired for corepressor release and operate in acute promyelocytic leukemia as dominant-negative inhibitors of wild-type RAR␣. We report that the two most common x-RAR␣ fusions, PML-RAR␣ and PLZF-RAR␣, have gained the ability to recognize specific splice variants of SMRT and NCoR that are poorly recognized by RAR␣. These differences in corepressor specificity between the normal and oncogenic receptors are further magnified in the presence of a retinoid X receptor heteromeric partner. The ability of retinoids to fully release corepressor from PML-RAR␣ differs for the different splice variants, a phenomenon relevant to the requirement for supraphysiological levels of this hormone in differentiation therapy of leukemic cells. We propose that this shift in the specificity of the x-RAR␣ fusions to a novel repertoire of corepressors contributes to the dominant-negative and oncogenic properties of these oncoproteins and helps explain previously paradoxical aspects of their behavior. Retinoic acid receptors (RARs)3 are members of the nuclear receptor family of ligand-regulated transcription factors. RARs bind to specific target genes and activate transcription in response to cognate agonists, such as all-trans-retinoic acid (ATRA) (1, 2). Conversely, in the absence of ATRA, RARs can repress transcription of their target genes below basal levels (1, 2). This bimodal transcriptional regulation is possible through the differential recruitment of coactivator and corepressor proteins to the RAR, which in turn creates the biochemical milieu to support or repress transcription (1, 2). Two corepressor paralogs, SMRT and NCoR, play important roles in nuclear receptor-mediated repression. Both SMRT and NCoR contain CoRNR box motifs ((I/L)XX(I/V)I) near their C termini that bind a hydrophobic groove on the surface of the unliganded nuclear receptors. The SMRT and NCoR N-terminal domains, in turn, recruit additional proteins that help confer repression, including histone deacetylases, TBL-1, TBLR-1, and GPS-2 (3-5). RARs can bind to their DNA-binding sites (retinoic acid response elements, or RAREs) as homodimers or as heterodimers with retinoic X receptors (RXRs) (1, 2).The RAR␣ locus, on chromosome 17, undergoes reciprocal chromosomal translocations at high frequency in human acute promyelocytic leukemia (APL), generating x-RAR␣ fusion proteins that play a causal role in this malignancy (6, 7). In Ͼ98% of APL, the x-sequences originate from the PML (promyelocytic leukemia) open reading frame on chromosome 15. Although more rare, yet other x-RAR␣ fusions have been identified, including PLZF (promyelocytic leukemia zinc finger)-RAR␣, STAT5b-R...