PML-RARA specific consensus sites

However, two studies of PML-RARA’s in vitro DNA binding specificity provided early evidence that PML-RARA was not merely a dominant negative RARA, but had its own unique binding properties. Perez et al investigated the binding of RARA and PML-RARA in in vitro gel shift assays to DR1-5 elements containing either

GGGTCA(n1-5)AGGTCA or GGTTCA(n1-5)AGTTCA half sites, termed DRnG and

DRnT sites respectively (91). While RARA/RXR bound equally well to DRnG and DRnT DR2 and DR5 probes, PML-RARA showed a marked preference for the DRnG probes. However, in light of the fact that the RARβ DR5 site used as the prototypical PML-RARA binding site is a DRnT site, it remains unclear what effect, if any, these preferences would have in the proper cellular context. Additionally, PML-RARA bound DR2 probes as well as DR3 probes, unlike RARA/RXR homodimers, which bind DR2 and DR5 sites most strongly. This study was the first evidence that PML-RARA has an extended binding repertoire compared to RARA. A subsequent study by Hauksdottir and Privalsky examined the binding of RARA and PML-RARA to a canonical

AGAGGTCAACGAGAGGTCA DR5 site when half site residues or preceeding residues were systematically mutated (92). PML-RARA proved less sensitive than RARA to changes in the base immediately preceeding a half site, and to changes in the third residue of a half site. The presence of RXR further enhanced PML-RARA binding to less favorable sites in vitro. However, the correlation of binding with activation was imperfect. While PML-RARA alone could activate mutated DR5 sites better than RARA/RXR in reporter assays, the presence of RXR reversed this trend. PML-

RARA/RXR transactivation was less than that seen with RARA/RXR, suggesting that repression by PML-RARA may be dependent upon RXR.

A 2004 study by Kamashev et al provided the first unbiased screen of PML- RARA consensus sites (93). PML-RARA protein was incubated with 25 base pair random DNA duplexes, and bound sequences were selected by gel shifts and amplified. After six rounds of selection and amplification, the duplexes were cloned and sequenced. The identified binding sites contained not only canonical DR2, DR3, DR4 and DR5 RAREs, but also widely spaced RAREs containing up to 13 base pair spacers between half sites. Inverted repeats, most commonly IR0 sites, and everted repeat sites (most often ER8 sites) were also identified. The addition of RXR to gel shift assays extended the binding of PML-RARA to RARES with up to 20 residues separating half sites (DR20 sites). Interestingly, transgenic mice expressing PML-RARA which cannot bind RXRA do not develop leukemia (94), suggesting that the role of RXR in DNA binding may also be relevant in vivo. Kamashev et al demonstrated that IR0, ER8 and DR sites with more than 5 base pair spacers are minimally activated by RARA in the presence of RA, but are strongly induced by PML-RARA. The relaxed binding specificity of PML-RARA is a clear gain-of-function above basal RARA functions; PML-RARA can no longer be considered to be solely a dominant negative RARA. Accordingly, in other studies, when APL blasts or NB4 cells are treated with ATRA, many of the induced or repressed genes do not contain known RAREs in their promoters (95). These relaxed binding sites provide a potential mechanism by which PML-RARA can alter expression of genes other than RARA targets.

However, the study by Kamashev et al did not examine whether PML-RARA binds altered RAREs in a chromosomal context, instead relying upon gel shift and luciferase reporter assays. In recent years, several chromatin immunoprecipitation studies have been published which have validated non-canonical RAREs as bona fide PML-RARA binding sites, as well as demonstrating new motifs. Hoemme et al reported a ChIP-chip study of PR9 cells, which contain a zinc inducible PML-RARA construct (96). Only 40% of the identified PML-RARA targets contained classical RAREs, and many of the remaining genomic regions contained altered RAREs. While this study was limited by the design of the arrays used, which contained only 12,000 known promoter regions and 12,000 CpG islands, it did demonstrate that PML-RARA had altered DNA properties in a chromosomal context. Two later reports expanded upon these

observations. Martens et al performed chromatin precipitation coupled to high

throughput next-generation DNA sequencing (ChIP-seq), using PR9 cells, NB4 cells and primary APL patient samples (97). Nearly all possible combinations of half sites were found within PML-RARA binding regions, including DR elements with up to 13

nucleotide spacers and half sites in everted or inverted orientation. Wang et al reported a separate study ChIP-chip study (98) at the same time as the Martens study. This study represented an improvement over the previous ChIP-chip study because of improved array design (probes covering over 25,500 promoters versus only 12,000 promoters) and advances in bioinformatic identification of binding regions and potential motif sequences. Consistent with previous reports, binding regions with various orientations of RARE half sites were identified. However, bioinformatic motif discovery approaches demonstrated that RARE half sites frequently appeared near PU.1 consensus sites. A separate ChIP-

chip experiment demonstrated that PU.1 protein was in fact occupying the consensus sites near RARE half sites. PML-RARA selectively binds RARE half sites in proximity to occupied PU.1 consensus sites, via both coiled-coiled domain dependent protein- protein interactions with PU.1 and DNA binding to the half site. Formation of this PML- RARA-PU.1 complex leads to repression of PU.1 transcriptional targets.

1.9. Protein-protein interactions of PML-RARA