Acute promyelocytic leukemia (APL), a subset of acute myeloid leukemias, is morphologically characterized by the accumulation of malignant myeloid cells blocked at the promyelocytic stage of maturation. Genetically, it is characterized by a translocation that fuses the retinoic acid (RA) receptor α (RARα) gene on chromosome 17 with either the promyelocytic leukemia (PML) gene on chromosome 15 or the PML zinc finger (PLZF) gene on chromosome 11 (11
). The resulting RAR chimeric genes encode fusion proteins PML-RAR and PLZF-RAR. Ectopic expression of RAR fusion proteins in hemopoietic precursor cells blocks their ability to undergo terminal differentiation, while transgenic mice overexpressing PML-RAR or PLZF-RAR develop an APL-like phenotype, indicating the causal role of these fusion proteins in APL (4
). Structure-function analysis of PML-RAR revealed that the RAR component of the fusion protein is indispensable for its ability to impair terminal differentiation (12
RARα is a member of the superfamily of nuclear hormone receptors that bind to specific RA response elements as heterodimers with retinoid X receptor (RXR) (21
). The activity of RAR/RXR is influenced by its physiological ligand, RA. In the absence of RA, the heterodimer recruits the nuclear corepressor N-CoR/histone deacetylase complex (HDAC), suggesting that it silences its target genes by altering chromatin structure (23
). Upon hormone addition, the corepressor complex is released and the receptor associates with transcriptional coactivators. A rapidly expanding repertoire of coactivators has recently been isolated, including proteins possessing histone acetyltransferase (HAT) activity (18
). The observation that transcriptional activation is associated with recruitment of HAT has reinforced the model in which modification of chromatin structure targeted by RAR/RXR contributes to transcriptional control. However, the precise molecular mechanisms that bring the RA signal from the nuclear receptors to the transcriptional machinery have yet to be elucidated.
It has recently been demonstrated that transcriptional activation by RAR/RXR requires both ATP-driven chromatin remodeling and coactivator HAT activities and that these multiprotein complexes act in a temporally ordered manner (7
). A simplistic model for the coactivator recruitment by nuclear receptors involves different steps. First, proteins that remodel nucleosomal structures are recruited to the liganded receptors. Subsequently, TRAP/DRIP complexes can replace the previous ones and bind the receptors. The recruitment of RNA polymerase II (PolII) to these coactivators completes this second step in transactivation (10
In divergence from this sequential model, it has recently been proposed that the cofactors essential for hormone-dependent activation are not all recruited through their direct interactions with the nuclear receptors but can be recruited through a combinatorial effect of direct or indirect interactions (15
). However, these two models are not considered mutually exclusive and might be receptor specific.
PML-RAR retains both DNA binding domains and ligand binding domains of RARα. It shows an affinity for RA comparable to that of wild-type RARα (2
) and binds to RA response elements as either homodimers or multimeric complexes containing or not containing RXR (20
). Since its ability to block hematopoietic differentiation depends on an intact DNA binding domain, RA target genes are thought to represent downstream effectors of PML-RAR (11
). Mechanistically, the fusion protein is thought to block differentiation by constitutively silencing RA-responsive genes involved in the control of differentiation of hematopoietic precursor cells. In accordance, it has been demonstrated that the fusion protein forms stable complexes with N-CoR/HDAC in the absence of RA or in the presence of physiological concentrations of RA, while pharmacological doses of ligand provoke the release of HDAC (13
). This abnormal recruitment of N-CoR by PML-RAR derives from the coiled-coil region of PML, which permits the formation of PML-RAR oligomers in vivo (20
). It is generally accepted that APL transformation correlates with PML-RAR repressive functions on the basis of the following observations: (i) mutations of the PML-RAR N-CoR binding site(s) impair the biological activity of the fusion protein, (ii) oligomerization per se is sufficient to activate the oncogenic potential of the transcription factor RAR, (iii) large doses of RA induce disease remission in PML-RAR APL patients (11
), and (iv) HDAC inhibitors combined with RA enhance terminal differentiation of APL cells. However, studies of the mechanisms by which PML-RAR regulates transcription are limited by the fact that repression has not been analyzed in a native chromatin context. Xenopus
oocytes are an attractive in vivo system with which to perform these studies since microinjection of double-stranded DNA leads to progressive chromatinization of the template (17
). Moreover, this system, unlike mammalian cells, contains undetectable levels of endogenous receptors, thus allowing unambiguous evaluation of the properties of the main regulators of APL.
In this work, we used Xenopus
oocytes to perform a comparative analysis of the wild-type transcription factor RARα and PML-RAR as both transcriptional regulators and modifiers of chromatin structure. As a target promoter, we used the RA receptor β2 (RARβ2) promoter, a well-defined in vivo target of PML-RAR (6
). Our results demonstrate that unliganded PML-RAR is a different repressor compared to the wild-type protein. In fact, whereas a heterodimer of RAR/RXR is required to silence RARβ2, the oncoprotein is a powerful repressor in the absence of its partner. Surprisingly, only PML-RAR owns a repressive pathway independent of nucleosome deposition and histone deacetylation. Also, in the presence of RA, the two proteins behave differently; in fact, RAR/RXR is a significantly more powerful activator than PML-RAR. We discuss possible molecular mechanisms justifying the observed phenomena.
Chromatin assays confirmed the new properties of PML-RAR. In fact, in the absence of RA, RAR/RXR binding to DNA does not produce topological changes, whereas unliganded PML-RAR closes the chromatin configuration. Since trichostatin A (TSA) can overcome this effect, we can state that the aberrant recruitment of HDAC activities is responsible for this closing effect. Analysis of DNase I-hypersensitive sites revealed that wild-type heterodimer and PML-RAR bind to minichromosomes, but only liganded RAR/RXR modifies the nucleoprotein structure organized on DNA. The absence of chromatin reconfiguration in the presence of RA-bound PML-RAR confirms that the fusion protein has lost some activating function(s).