Genome-wide functions of PML–RARα in acute promyelocytic leukaemia

PML—RAR (retinoic acid receptor)α is the hallmark protein of acute promyelocytic leukaemia, a highly malignant subtype of acute myeloid leukaemia that accounts for approximately 10% of all AML cases. Recently, several studies have been set out to obtain a comprehensive genome-wide view of the molecular actions of this chimeric protein. In this review, we highlight the new insights that arose from these studies, in particular focussing on newly identified PML–RARα target genes, its interplay with RXR and deregulation of epigenetic modifications.

Acute promyelocytic leukaemia (APL) is a distinctive subtype of acute myeloid leukaemia (AML) that accounts for approximately 10% of all AML cases (Jing, 2004). The disease represents a highly malignant form of leukaemia with high bleeding tendency and a fatal course of only few weeks (Wang and Chen, 2008). The main diagnostic feature of APL is an aberrant chromosomal translocation that juxtaposes the PML gene on chromosome 15 and the retinoic acid receptor (RAR)a gene on chromosome 17 (Kakizuka et al, 1991). The resultant chimeric protein, which is found in over 95% of human APLs (Di Croce, 2005), retains the DNA-binding and ligand-binding domains of RARa and the multimerisation domain of PML. In normal cells PML is a main constituent of nuclear bodies, which are matrix-associated multiprotein-containing domains involved in various biological functions like DNA-damage response and microorganism resistance through regulation of a wide range of proteins, among which are various transcription factors . In contrast, in APL, the expression of PML -RARa disrupts the localisation of the wild-type PML from nuclear bodies to numerous micro speckles (Brown et al, 2009) and induces a maturation block at the promyelocytic level (Wang and Chen, 2008). All-trans retinoic acid (ATRA) and arsenic trioxide (ATO) are the two most important drugs in clinical use for the treatment of earlydiagnosed APL. Both ATRA and ATO degrade the PML -RARa fusion protein by acting on the RARa and PML moieties, respectively. ATRA mainly degrades the protein through proteosomemediated pathways (Zhu et al, 1999) and caspases , while ATO-induced degradation is initiated through sumoylation of the PML moiety. Both treatments ultimately lead to restoration of PML nuclear bodies (Lallemand-Breitenbach et al, 2008;Zhang et al, 2010), but whether this is important for curing the disease is an open question.
Various mechanisms have been proposed for PML -RARa functioning. It has been suggested that PML -RARa can form homodimers without RXR (Minucci et al, 2000) or that it forms PML -RARa oligomers that heterodimerise with RXR (Perez et al, 1993;Jansen et al, 1995). In addition, it has been suggested that during transformation PML -RARa induces a multitude of alterations in the chromatin architecture. These alterations are achieved through the recruitment of various epigenetic-modifying factors, like histone deacetylase complexes such as SMRT (Lin et al, 1998) and N-CoR , and DNA methyltransferases (Di Croce et al, 2002). In addition, recent evidence suggests co-recruitment of the histone methyltransferases SUV39H1 and polycomb repressor complexes, which dictate the epigenetic state of H3K9 (Carbone et al, 2006) and H3K27 (Villa et al, 2007), respectively. Unfortunately, most of these studies showed epigenetic alterations only around a limited set of binding regions, in most cases the RARb promoter. This was largely due to unavailability of the genome-wide PML -RARa target site repertoire. However, the recent advances in high-throughput tools have, for the first time, made it possible to look at the genome-wide actions of PML -RARa and different epigenetic marks associated with its binding. Indeed, two recent studies have provided a more global picture of PML -RARa functioning by identifying binding regions of PML -RARa, using state-of-the-art ChIP-seq and ChIPon-chip technologies. Importantly, the new technologies even allowed extension from the commonly used model cell lines to primary APL blasts, highlighting the new opportunities that have now become available.
In this review we will focus on the various aspects of PML -RARa functioning with respect to its genome-wide binding spectrum, its interplay with RXR and its regulation of various epigenetic modifications. In addition, we will discuss some of the newly identified target genes and target pathways of PML -RARa.

ALTERED RAR SIGNALLING IN APL CELLS
All-trans retinoic acid belongs to the group of vitamin A-derived substances and binds three major RARs, RARa, b and g. The full execution of the ATRA signalling pathway operates by inducibly controlling the expression of the genes that have a direct repeat with spacing 2 or 5 (DR2 or DR5) in their promoter (de The et al, 1991). Retinoid signalling has a key role in various developmental and differentiation processes. According to the classical model, RAR and RXR, another nuclear receptor, heterodimerise under non-ATRA conditions, recruit co-repressors and silence target gene expression. In this model, RAR binding to ATRA results in a conformational change in the RXR -RAR heterodimer, allowing recruitment of gene-activating complexes and resultant gene expression. In APL, PML -RARa is thought to behave functionally as an altered RARa that has lost the potential to respond to fluctuations in physiological ATRA concentration, and as a result acts as a constitutive transcriptional repressor for DR2-or DR5containing genes. Strikingly, recent studies that analysed the genome-wide PML -RARa binding sites not only identified DR2 and DR5 elements as the primary PML -RARa response elements, but also regions containing DR1, DR3 and DR4 motifs and even more atypical DR motifs with various spacing and orientation were detected , thereby extending in vivo previous in vitro data (Kamashev et al, 2004). This rather diverse repertoire of response elements present at the PML -RARa binding sites underscores the idea of a gain of DNA-binding capacity as an essential feature of PML -RARa-mediated transformation. In addition to this extended binding potential, global binding data of PML -RARa using ChIP-seq revealed PML -RARa binding to the RARa, RARb and RARg genes itself (Table 1), suggesting that expression of these proteins is directly regulated by PML -RARa . All these data suggest that PML -RARa affects ATRA signalling at multiple levels: first by regulating expression of the genes involved in transmitting the ATRA signals, second through an altered regulation of classical DR2-and DR5containing RAR/RXR target genes and third through an extension of the (PML-)RAR binding potential towards more degenerate DR-containing regulatory sites.

PML-RARa HETERODIMERISES WITH RXR
Although oligomerisation of the RARa fusion proteins has been considered to be a crucial requirement to their oncogenic potential (Minucci et al, 2000), several studies support a role of RXR in the PML-RARa transformation process. RXR was already described as part of the PML -RARa oncogenic complex in in vitro settings (Kamashev et al, 2004). In addition, recent studies highlighted the importance of RXR in PML-RARa-mediated transcriptional repression (Zeisig et al, 2007;Zhu et al, 2007). These studies showed that impaired RXR binding by PML-RARa mutants impairs APL development in transgenic mice while still retaining the transforming potential in vitro. Furthermore, they showed that silencing of RXR by shRNA suppresses the RARa fusion-mediated transformation in vitro. These studies were corroborated and extended by the identification of genome-wide association of RXR with PML-RARa . For 99% of PML-RARa binding sites, association of RXR was detected. Together, these studies highlight the importance of RXR in the PML -RARa-mediated transformation process. It may therefore be interesting to also test RXR antagonists (Altucci et al, 2007) in this subtype of AML.

PML-RARa CROSS TALKS WITH OTHER TRANSCRIPTION FACTORS
Apart from interacting with RXR, PML -RARa has been suggested to interact with many other proteins. Amongst these are various key haematopoietic regulatory transcription factors such as GATA-2, PU.1 and AP-1 factors. PML -RARa is proposed to be involved in inhibition of AP-1 transcriptional activity in an ATRA-dependent manner. This is illustrated by the observation that PML -RARa interacts with c-Jun and c-Fos and that the repressive effect on AP1 target sites is reversed by ATRA treatment (Doucas et al, 1993). There is also evidence of a physical association of GATA2 with PML -RARa. This interaction is suggested to result in enhanced GATA-2-dependent transactivation capacity (Tsuzuki et al, 2000). In line with these two studies is the observation that PML -RARa binding was detected not only near several AP1 factors, such as JUNB and JUND, but also near GATA2 (Table 1)  , suggesting that these factors are affected both at the level of their expression and in their targeting capacities. In addition, we observed PML -RARa binding at several other key regulators of haematopoiesis such as PU.1 (see below) RUNX1, RUNX3 and GFI1 (Table 1). This extends the observation that PML -RARa regulates the classical targets of the retinoic acid signalling pathway to many other key players in haematopoiesis.

BINDING OF PML-RARa WITH PU.1
PU.1 is a protein that is essential for the haematopoietic differentiation process. PU.1 mutants lead to embryonic lethality at a late gestational stage (Scott et al, 1994). Mice that have homozygous disruption of the DNA-binding domain of PU.1 have severe septicaemia and die within 48 h of their birth (McKercher et al, 1996). In addition, PU.1 has been shown to be essential for reprogramming B-cells into the myeloid lineage (Xie et al, 2004). Already in 2006 it was proposed that PML -RARa interacts with PU.1 and that the action of PU.1 is suppressed upon expression of PML -RARa, thereby resulting in a differentiation block (Mueller et al, 2006). In these studies, ATRA treatment and the ensuing PML -RARa degradation resulted in restoration of PU.1 expression and a release of the differentiation block. These studies already hinted at the molecular interplay of PU.1 with PML -RARa. A recent study based on genome-wide binding of PML -RARa using ChIP-sequencing in a PML -RARa inducible cell model further shed light on the PU.1 and PML -RARa interaction (Wang et al, 2010). In this study, more than 84% of the detected PU.1 motifs were found in the close vicinity of variably spaced direct repeats (DRs). Further functional analysis suggested that the binding of PML-RARa to the regions containing both PU.1 and DRs is a prerequisite for subsequent repression of chromatin at Genome-wide functions of PML -RARa S Saeed et al PU.1 targeted regions. In addition to these observations, the PU.1 gene was found to be a direct target of PML -RARa . Interestingly, binding of PML -RARa was not detected at the promoter regions but rather in the third intron of the PU.1 gene ( Table 1). As this intronic region has also been reported to contain the transcription start site of an antisense transcript that acts as a putative negative regulator of PU.1 expression (Ebralidze et al, 2008), these results identify for the first time PML -RARamediated regulation of a non-coding transcript.

PML-RARa AFFECTS THE EPIGENOME
Multiple studies have suggested that central to oncogenic transformation in APLs is the PML -RARa-induced mis-targeting of the epigenetic machinery, thereby causing a perturbation of the normal epigenetic landscape. Genome-wide binding analysis of PML -RARa  revealed that various enzymes that can set different chromatin modifications are targeted by PML -RARa, including JMJD3 (H3K27me3 demethylation), SETDB1, JMJD1A (H3K9 modifiers), deacetylases like HDAC4 and 9, and genes involved in DNA methylation, such as DNMT3A (Table 1). These findings suggested that PML -RARa expression has the potential to confer a genome-wide alteration in epigenetic make-up. In addition to the direct transcriptional regulation of epigenetic enzymes, different labs have explored the epigenetic marks that are recruited by PML -RARa itself. Histone marks such as H3K27me3 and H3K9me3, as well as DNA methylation, have been proposed to be positively correlated with PML -RARa binding, whereas H3 acetylation was associated with loss of PML -RAR binding. The dynamic changes of DNA methylation, H3K9me3 and H3K27me3 functioning are suggested to be regulated by DNA methyltransferases, histone methyltransferase (SUV39H1) and polycomb repressive complex 2 (PRC2), respectively, and all these proteins have been suggested to interact with the PML -RARa complex in several independent studies (Di Croce et al, 2002;Carbone et al, 2006;Villa et al, 2007). H3 acetylation was the first chromatin modification associated with PML -RARa binding and is a mark that is negatively correlated with PML-RARa binding. Mechanistically, this is thought to be achieved through PML -RARa-mediated recruitment of HDACs Lin et al, 1998). Indeed, in vivo studies could show that there is a significant increase of H3 acetylation upon ATRAmediated degradation of PML -RARa at the RARb promoter (Villa et al, 2007). Still, the limiting factor to this observation and also other studies that investigated epigenetic marks was the number of PML -RARa binding regions addressed and, therefore, the generality of the proposed mechanisms. The recent genome-wide interrogation of APL  allowed for the first time expansion to all binding regions of PML-RARa. This showed that there was a significant regulation of local H3 acetylation at more than 80% of the PML -RARa binding regions, illustrated by the observation that H3 acetylation levels at these sites were significantly elevated upon ATRA-induced PML -RARa degradation. In contrast, changes in other epigenetic marks such as H3K9me3, H3K27me3 and DNA methylation could not be generalised towards all PML -RARa binding sites, as the vast majority of sites did not show a significant change after ATRA treatment. These findings are of significant worth, as they point out the importance of the role of histone deacetylases in maintenance of repressed chromatin architecture at PML -RARa binding sites. This sanctions the idea of making HDACs direct targets for therapeutic treatment of APL by using specific inhibitors for these proteins.

OUTLOOK
In this review, we focused on two recent studies that describe a global analysis of PML-RARa Wang et al, 2010). Both studies have shown nearly 3000 genome-wide direct targets of PML-RARa by using high-throughput sequencing and array-based technologies, and provided a significant step forward in understanding PML-RARa-mediated leukaemogenesis. Although these studies used independent platforms, several common conclusions were drawn (Figure 1). One of these is the extended binding repertoire of PML-RARa in comparison with the non-fused RAR. Indeed, direct, inverted and everted repeats with various spacing and orientation were detected in vivo for PML-RARa binding sites in comparison with the classical DR2 and DR5 motifs that are the hallmarks of heterodimerised RARa/RXR binding regions. In addition to the DR motifs, the discovery of DNA motifs for PU.1 resulted in the finding that PU.1 colocalises with PML-RAR.
The above studies highlighted the fact that PML-RARa not only binds to the promoter regions of target genes but also has a rather diverse repertoire of binding sites. This complex binding spectrum suggests a potential influence on long-range chromosomal interactions by PML -RARa. The wide-ranging molecular alterations induced by PML-RARa are further illustrated by the fact that PML -RARa regulates several key regulators of normal haematopoiesis, such as PU.1, GATA-2, RUNX1 and many others, as well as different pathways such as RAR signalling. In addition, exploration of the epigenetic environment of the APL genome before and after ATRA treatment gave significant insights into PML -RARa chromatin regulation. An inverse correlation of PML -RARa with H3 acetylation was revealed at the PML-RARa binding sites themselves, while the genome-wide epigenetic environment was also significantly remodelled. Still, more in-depth functional studies are needed to provide an answer as to whether histone H3 acetylation levels or other epigenetic markings are crucial in the PML-RARainduced transformation process. Nevertheless, current data already hint at potential drug targets such as acetyltransferases and deacetylases, as well as RXR for treatment of APL.
Despite the wealth of important insights on PML-RARa functioning provided by the above studies, the question still remains as to what the crucial determinant for PML-RARa binding to a particular region actually is. Is it the underlying motif, interaction with other transcription factors, the chromatin accessibility, or a combination of all three? In order to obtain a better comprehension of normal haematopoiesis and leukaemia, it will be important to address these crucial questions. At the same time, deeper insight is required into the molecular behaviour of other oncofusion proteins that harbour functional properties similar to PML-RARa's such as AML1-ETO, which is the result of the t(8;21) chromosomal translocation, or the inv (16) translocation that gives rise to the CBFb-MYH11 oncofusion protein . A comparative analysis of the molecular actions of several oncofusion proteins is expected to uncover some of the more general mechanisms that are used by these proteins to transform cells.