The DNA Binding Property of PML/RARA but Not the Integrity of PML Nuclear Bodies Is Indispensable for Leukemic Transformation

PML/RARA is the oncoprotein driving acute promyelocytic leukemia (APL). It suppresses genes expression by recruitment of a number of transcriptional repressors, resulting in differentiation block and malignant transformation of hematopoietic cells. Here, we found that mice primary hematopoietic progenitor cells (HPCs), transduced by DNA-binding-defective PML/RARA mutants, were deficient in colony formation. Further experiments showed that DNA-binding-defective PML/RARA mutants could not repress the transcription of retinoic acid regulated genes. Intriguingly, there were no significant differences of the micro-speckled intracellular distribution between the mutants and wild-type PML/RARA. Some retinoic acid target genes regulated by PML/RARA are involved in not only differentiation block but also hematopoietic cell self-renewal. Altogether, our data demonstrate that direct DNA-binding is essential for PML/RARA to immortalize hematopoietic cells, while disruption of PML-nuclear body does not seem to be a prerequisite for hematopoietic cell transformation.


Introduction
Acute promyelocytic leukemia (APL) accounts for about 12-15% of the incidents of acute myeloid leukemia. In over 98% APL patients a specific chromosomal translocation t (15;17) fuses the PML gene on chromosome 15 to the RARA gene on chromosome 17. The resulting PML/RARA fusion protein is the molecular determinant of the disease [1]. The presence of a strong dimerization/oligomerization domain in the PML moiety makes PML/RARA as a potent repressor at least in part through the ability of PML/RARA homodimers to recruit nuclear receptor corepressors [2][3][4][5][6][7][8][9]. The K160 sumoylation site of PML/RARA which allows the recruitment of a potent repressor DAXX is decisive for ex vivo cell immortalization/differentiation arrest and in vivo APL pathogenesis [10]. The presence of RXR, the RARA heterodimeric partner, extends the binding of PML/RARA to more widely spaced direct repeats than the normal RARA/RXR heterodimers and is pivotal to transformation ex vivo [11][12][13][14]. These distinct properties of the fusion protein cooperate to enforce transcriptional repression. Intriguingly, the treatment with all trans retinoic acid (ATRA) induces complete remissions [15], at least in part through reactivation of PML/RARA-dependent transcription and ubiquitin-proteasome dependent degradation of PML/RARA fusion protein [16].
Recent studies have demonstrated that PML/RARA regulates a novel class of genes through interaction with other transcription factors, such as Sp1 and NF-Y, without directly binding to the DNA, defining a gain-of-function for the oncoprotein [17]. PML/ RARA was also shown to transactivate the TF promoter through an indirect interaction with an element composed of a GAGC motif and the flanking nucleotides [18]. It is also reported that PML/RARA predominantly targets PU.1-regulated promoters through both protein-protein interaction and DNA binding via retinoic acid response element (RARE) half sites forming complex binding motifs [19]. Yet, any contribution of this indirect and complex repression of target genes by PML/RARA to differentiation arrest and immortalization of primary hematopoietic progenitors remains unknown.
PML is a nuclear matrix protein that organizes PML nuclear bodies (NBs) [20]. NBs accumulate a wide variety of cellular or viral proteins, including key regulators of cell growth or transformation. They have been proposed to be important for senescence induction or growth control [20,21]. In APL, PML/ RARA alters the intranuclear distribution of PML and other NBassociated proteins. The phenomenon has been theoretically attributed to the hypothesis that PML by forming heterodimer with PML/RARA has been tethered to chromatin through DNA binding domain of the fusion protein. Therefore disruption of NBs has been suggested to contribute to APL pathogenesis.
In this study we have investigated the role of direct DNA binding of PML/RARA in cell transformation and demonstrated that the DNA binding property is critical to cell differentiation arrest and immortalization. Meanwhile, we show that the DNA binding domain is not the molecular determinant for PML/RARA intra-cellular distribution and the disruption of PML NBs integrity is not a prerequisite for full cell transformation.

Results
DNA binding defective mutants of PML/RARA failed to bind the RARE and activate the RARE-reporter gene The mutation of C88 to G of RARA abrogates its DNA binding and transactivation ability [22]. To demonstrate that the same mutation in PML/RARA (PML/RARA C580G) recaptures this property ( Figure 1A), luciferase assay of the RA-sensitive reporter gene (RARE-3-Tk-Luc) [23] and ChIP analysis have been conducted. The PML/RARA C580G mutant was much less potent than the parental fusion in ligand (ATRA) triggered activation ( Figure 1B). Coexpression of RXR can dramatically increase the binding efficiency of PML/RARA. We had previously demonstrated that M883 and T886 of PML/RARA are essential sites for RXR binding [24]. To completely abrogate the DNA binding through heterodimer formation with RXR, M883R, T886R mutations were introduced in PML/RARA C580G mutant ( Figure 1A). Like PML/RARA C580G, the PML/RARA C580G, M883R, T886R triple mutant displayed very weak transactivation upon ATRA treatment ( Figure 1B). The transactivation property of PML/RARA M883R, T886R is fully consistent with our previous observations ( Figure 1B). Note that cells transduced by different PML/RARA mutants expressed similar levels of fusion proteins and had comparable PML/RARA modification adducts ( Figure 1C).
In ChIP analysis, as expected, the abundance of enriched genomic DNA fragments containing the RARB promoter region precipitated by anti-PML antibody was significantly reduced in transfected 293T cells expressing DNA binding defective mutants, which was consistent with the luciferase results ( Figure 1D). No obvious difference was observed in a neighbouring negative control of ChIP assay (data not shown). Similar results were obtained in stable NIH3T3 cells ( Figure S1). The same experiment was also performed with a PML/RARA DDBD mutant lacking entire DNA-binding domain. Unsurprisingly, this mutant displayed similar feature as the DNA binding defective point mutation mutants. ( Figure 1D).

DNA binding property of PML/RARA is required for differentiation arrest and transformation of primary mouse hematopoietic progenitors
Previous studies suggested that the RARA DNA binding region of the fusion protein contributed only partially to the differentiation block [25]. We then questioned whether DNA binding properties would be dispensable for PML/RARA-induced transformation of primary hematopoietic progenitors ex vivo [26]. The colony formation assay of mouse hematopoietic progenitors was carried out. The cells transduced by PML/RARA could be replated indefinitely in methylcellulose with immature myeloid progenitor morphology. In sharp contrast, those cells transduced by DNA binding defective mutants, PML/RARA C580G and PML/RARA C580G, M883R, T886R could not trigger a sharp differentiation arrest and failed to allow indefinite colony formation in the replating assay (Figure 2A and 2B). PML/RARA C580G expressing cells were differentiated into both granulocytes and monocytes, as assessed by both MGG staining and myeloid antigens Gr-1 and Mac1 expression analysis ( Figure 2C and 2D). PML/RARA C580G, M883R, T886R transduced cells behaved similarly to the PML/RARA C580G ones. Nevertheless, PML/ RARA M883R, T886R-transformed cells underwent a sharp differentiation arrest and became immortal as previously reported [14] (Figure 2A to 2D). Again, cells transduced by different PML/ RARA mutants expressed similar level of fusion protein and comparable PML/RARA sumoylation patterns were present for each mutant (data not shown).
The disruption of PML nuclear bodies is insufficient for full leukemic transformation of primary mouse hematopoietic progenitors PML nuclear bodies play an important role in the control of apoptosis or senescence and are disrupted upon PML/RARA expression [27,28]. To investigate whether the integrity of PML NBs is tightly linked to DNA binding and cell transformation, an immuno-fluorescence assay has been performed in the cells expressing different PML/RARA mutants. Surprisingly, PML/ RARA DNA binding defective mutants disrupted PML NBs both in stably transfected NIH 3T3 cells or transduced mouse hematopoietic progenitors ( Figure 3A and 3B). Note the expression of each mutant was comparable as examined by western blot ( Figure 3C and 3D). Since rabbit anti-PML antibodies used did not recognize endogenous murine PML, immunofluorescence experiments were re-performed in Hela cells. As expected, while the typical NBs were revealed by double staining of anti-PML and anti-SP100 in control cells, they were destroyed and replaced by PML/RARA specific micro-speckles in PML/RARA mutants transfected cells ( Figure S2). These observations therefore imply that disruption of PML NBs is not sufficient for PML/RARA to fully immortalize primary hematopoietic progenitor cells and the DNA binding is not the mechanism through which PML NBs are disrupted by PML/RARA.

Expression of endogenous ATRA response genes in cells transduced by PML/RARA and DNA binding defective mutants
Transcriptional repression of ATRA target genes induced by PML/RARA was proposed to underlie the APL pathogenesis [6,29]. We thus examined whether the DNA binding defective mutants retain the ability to repress endogenous ATRA target genes and impair ATRA response in stably transduced NIH 3T3 cells and primary mouse hematopoietic progenitors by quantitative RT-PCR. We first focused on two types of genes: a well characterized RARA target Rarb [23] and one primary PML/ RARA target, transglutaminase type II (Tg II) [30]. Only PML/ RARA reproducibly activated the expressions of these two genes in NIH 3T3 cells, with PML/RARA C580G, PML/RARA M883R,T886R and PML/RARA C580G, M883R,T886R being completely inactive ( Figure 4A and 4B). In transduced primary hematopoietic progenitors a clear induction of Rarb and Tg II expression was observed in response to ATRA administration for both PML/RARA and PML/RARA M883R, T886R ( Figure 4C and 4D). The epigenetic chromatin environment of the PML/ RARA binding site(s) within the Tg II promoter may significantly differ between fibroblasts and transformed primary hematopoietic progenitors, which could explain why the activation was observed in one system, but not the other. Contrarily to the prevailing model, PML/RARA expression did not induce significant changes in the baseline levels of the two target genes (data not shown).
It is reported that PML/RARA predominantly targets PU.1regulated promoters through both protein-protein interaction and DNA binding via RARE half sites forming complex binding motifs [19]. We thus examined the transcriptional activation of PU.1 and HCK in the presence of different PML/RARA mutants. The activation of PU.1 and HCK were not detected in transduced NIH 3T3 cells, perhaps due to the absence of hematopoietic related genes expression in fibroblasts (data not shown). In contrast, the expression of PU.1 and HCK were only detected in the hematopoietic progenitors transduced by PML/RARA or PML/RARA M883R, T886R, but not in those expressing PML/ RARA C580G and PML/RARA C580G, M883R, T886R, again demonstrating that the inability of DNA binding affects ATRA response of endogenous genes ( Figure 4E and 4F).
Finally the expression of ID1 was tested. To our surprise, PML/ RARA and PML/RARA M883R, T886R were still capable of activating the endogenous ID1 gene in transduced cells, suggesting that a consensus RARE may exist within the promoter of ID1 ( Figure 4G).  ATRA treatment induced degradation of PML/RARA may remove corepressors and allow induction of target genes expression. No obvious difference of protein degradation was observed between PML/RARA and mutants after ATRA or arsenic trioxide (ATO) treatment ( Figure S3), suggesting the protein degradation and corepressors release may not play critical roles on the expression of genes tested.

Discussion
A combination of PML-mediated RARA homodimerization, enhanced corepressor binding, and repression of ATRA target genes was proposed to trigger the APL-specific differentiation block [6,29]. Three recently published reports demonstrated that PML/RARA regulates a novel class of target genes such as ID1 and TF through interaction with other transcription factors, without directly binding to the DNA, defining a gain-of-function for the oncoprotein [17,18]. The contribution of this gain of function of PML/RARA in the transformation of primary hematopoietic progenitor cells was never tested. This work, probing PML/RARA function in primary mouse hematopoietic progenitor cells, demonstrates that the direct DNA binding property is indispensible for the fusion protein to impair differentiation and immortalization of the primary cells ex vivo. Hence, reduced DNA-binding affinity likely plays a critical role in the weaker phenotype of the DNA binding defective PML/RARA C580G mutant. Our experiments in primary cells also confirm the critical importance of DNA binding previously shown in the U937-differentiation assay [25]. Note that, in the context of RARA-triggered ex vivo transformation, mutation in the DNAbinding domain abolished ex vivo transformation as well [26], likely reflecting the absence of any dimer formation.
The greater biological effects of PML/RARA, when compared to its defective mutants, do not reflect a higher level of protein expression, as the mutants were usually expressed at higher levels than the parental fusion. Moreover, the pattern of posttranslational modification was identical between the parental and the mutant proteins ( Figure 1B, Figure 3C and Figure 3D). We favor the idea that the greater expression of the mutant fusion could result from either its reduced toxicity and/or its greater stability. Both of these features may reflect its lower affinity for DNA, as DNA binding was shown to be a critical determinant of RARA degradation [16].
PML NBs are extrachromosomal nuclear matrix associated structures that contain the PML and numerous partner proteins and are disrupted in APL cells in a treatment-reversible manner. Several pathological states are associated with abnormalities of NBs structures, including transformation, viral infection and neurodegenerative disorders. In the transduced primary cells, the expression of DNA binding defective mutants were able to disrupt NBs while these cells could not be immortalized (Figure 2 and 3), suggesting that the disruption of PML NBs is not sufficient for leukemic transformation. Moreover, the K160 sumoylation site in the PML moiety of PML/RARA was required for efficient immortalization/differentiation arrest ex vivo as previously shown [10]. In supporting our observation, the PML NBs were also disrupted in PML/RARA K160R transduced cells.
Our findings provide evidence that direct DNA-binding is indispensible for PML/RARA to transform hematopoietic cells and disruption of PML-nuclear bodies is not sufficient for full cell leukemic transformation.

Materials and Methods
Constructs and reagents P/R DDBD and P/R M883R, T886R mutants were described previously [24,25]. The P/R C580G and P/R C580G, M883R, T886R mutants were constructed by site-directed mutagenesis using QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene). Wild type and mutant P/R cDNAs were cloned into the pSG5m eukaryotic expression vector, pMSCV-hygromycin and pMSCV-neomycin retroviral vectors. All mutants were confirmed by DNA sequencing. All Trans-Retinoic Acid (ATRA) (Sigma) was dissolved in ethanol as a stock solution at 1 mM and added to cells at a final concentration of 10 26 M. Arsenic trioxide (ATO) (Sigma) was kept as a 10 23 M stock solution and added to a 10 26 M final concentration.

Cell lines and mice
293T, NIH-3T3 and Plat-E cells were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Front). For Plat-E cells, blasticidin (10 mg/ml) and puromycin (1 mg/ml) were added to the culture medium [31]. Cultures were placed in a 37uC humidified incubator with 5% CO2. Wild type C57BL/6 mice were bought bred and maintained under specific pathogen-free conditions in the facility at the Shanghai Jiao Tong University School of Medicine. All experimental manipulations were undertaken in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of Shanghai Jiao Tong University School of Medicine.

Virus production
For virus production, the indicated retroviral constructs were transfected into Plat-E packaging cells using Effectene Transfection Reagent (QIAGEN) according to the manufacturer's recommendations. 48 hours later, virus-containing supernatants were harvested, and stored at 280uC or immediately used for infection of cells.

Morphological staining
Mouse bone marrow cells harvested from colonies were cytospun onto slides and stained with Wright-Giemsa staining solution following manufacture's manual. The samples were observed under a light microscope (Olympus).

Immunoblotting
Cells were lysed with laemmli sample buffer (Sigma), and then cell lysates were resolved by 7% SDS-PAGE, and proteins were transferred onto nitrocellulose membrane using the iBlot Dry Blotting System (Invitrogen) according to the user's manual. Western blotting was performed with 1:1000 diluted rabbit anti-RARA (a gift from Dr Chambon's lab), and rabbit anti-Actin as an internal control. Protein signals were detected with the chemiluminescent substrate SuperSignal West Pico (Pierce Biotechnology).

Luciferase reporter assay
Cells were transiently cotransfected with indicated plasmids (120 ng expression vector, 40 ng RARE3-tk-Luc reporter [23], and 20 ng Renilla-tk-Luc vector) using Effectene Transfection Reagent (QIAGEN). Cells were treated with ATRA (10 26 M) or ethanol vehicle overnight. Luciferase activities were measured on a luminometer using the Dual Luciferase Reporter Assay Kit (Promega) according to the manufacturer's protocol. Firefly luciferase activities were normalized to the Renilla luciferase activities to correct for differences in transfection efficiency. Assays were performed in duplicate and standard deviations calculated.
Immunoprecipitated DNA as well as inputs were analyzed by quantitative real-time PCR for genomic sequences from the RARB promoter region containing RARE and from the RARB exon as negative control region. Quantitative real-time PCR was performed using SYBR Premix Ex Taq (Takara) according to the manufacturer's instructions. Primers used were described previously [17], sense 59-TTGGGTCATTTGAAGGTTAGCA-39 and antisense 59-CACACAGAATGAAAGATTGAATTGC-39. Figure S1 ChIP analysis of wild type or mutant PML/RARA binding to the endogenous RARB promoter (A) and the neighbouring negative control region (B) in stable NIH-3T3 cells. Means 6 standard deviation (SD). (TIF) Figure S2 Immunofluorescence analysis was carried out with a rabbit anti-PML (green) and a chicken anti-SP100 (red) to show distribution of NBs, and with DAPI to visualize nuclei in Hela cells. While the typical NBs were observed in control cells, they were destroyed and replaced by PML/RARA specific microspeckles in PML/RARA mutants transfected cells. Note that the endogenous SP100 only co-localized with PML but not PML/ RARA. (TIF) Figure S3 293T cells transfected with indicated PML/RARA mutants were treated or not with ATRA (A) or arsenic trioxide (ATO) (B) overnight, respectively. Western blots were performed with anti-RARA antibody. (TIF)