Oncogene (2011) 30, 967–977 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE The reduced and altered activities of PAX5 are linked to the protein–protein interaction motif (coiled-coil domain) of the PAX5–PML fusion protein in t(9;15)-associated acute lymphocytic leukemia

JJ Qiu, H Chu1, X Lu, X Jiang1 and S Dong

Department of Medicine and Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX, USA

The paired box domain of PAX5 was reported to fuse Introduction with the sequence of promyelocytic leukemia (PML) to produce PAX5–PML chimeric protein in two patients Acute lymphoblastic leukemia (ALL) is characterized by with B-cell acute lymphoblastic leukemia. In the present the clonal proliferation and accumulation of malignant studies, we found, by gel shift assays, that PAX5–PML blast cells in the bone marrow (BM) and peripheral bound to a panel of PAX5-consensus sequence acts as a blood. It has been frequently reported that acquired homodimer with reduction of its DNA-binding affinities in chromosomal translocation in the leukemic blasts of comparison with wild-type PAX5. In transient transfec- ALL patients are involved in leukemogenesis. The most tion assays using 293T and HeLa cells, and retrovirus significant chromosomal translocations in ALL include: transduction of murine hematopoietic stem/progenitor (1) the poor-risk translocations; BCR–ABL fusion gene cells together with quantitative real-time polymerase from t(9;22)(q34;q11), MLL fusion genes from the chain reaction analysis, PAX5–PML inhibited wild-type 11q23 chromosomal rearrangements; E2A–PBX1 from PAX5 target gene transcriptional activity. Studies com- t(1;19)(q23;p13), and rearrangements of MYC with the paring PAX5–PML with PAX5–PML(DCC) demon- immunoglobulin genes; and (2) the good risk transloca- strated that the coiled-coil (CC) protein interaction tion; ETV6(TEL)–AML1 fusion gene from t(12;21) domain located within the PML moiety was required for (p13;q22). These abnormalities occur most frequently in PAX5–PML homodimer complex formation and partial B-lineage leukemias, although rearrangements of the transcriptional repression of genes controlled by PAX5. T-cell receptor genes are associated with T-lineage ALL Fluorescent microscopic examination of transiently (Look, 1997). expressed YFP-tagged proteins in HeLa and 293T cells PAX5 (also known as BSAP for B cell-specific demonstrated that YFP–PAX5–PML and YFP–PAX5– activating protein) is one of the conserved paired-box PML(DCC) exhibited a diffuse granular pattern within (PAX) domain-containing genes, which encodes the the nucleus, similar to PAX5 but not PML. By fluorescent transcription factor whose expression persists exclu- recovery after photobleach (FRAP), we have shown that sively in the B-lymphocyte lineage, indicating that it has PAX5–PML fusion protein has reduced intranuclear a pivotal role in B-cell development (Urbanek et al., mobility compared with wild-type PAX5. Furthermore, 1994; Nutt et al., 1999; Mikkola et al., 2002; Cobaleda the dimerization domain (CC) of PML was responsible et al., 2007). One of the target genes for PAX5 is the for the reduced intranuclear mobility of PAX5–PML. CD19 gene, which has two PAX5 binding sites in its These results indicate that the CC domain of PAX5–PML 50-upstream region. PAX5 is considered as the most is important for each of the known activities of PAX5– important transcription factor for the expression of PML fusion proteins. CD19 gene. PAX5-binding sites have also been identi- Oncogene (2011) 30, 967–977; doi:10.1038/onc.2010.473; fied in the regulatory sequences of a number of other published online 25 October 2010 genes, including BLK, BLNK and RAG2 (Lauring and Schlissel, 1999; Schebesta et al., 2002, 2007; Delogu Keywords: PAX5; PAX5–PML; dimerization; FRAP; et al., 2006). PAX5 can act as an activator or a repressor acute lymphocytic leukemia (ALL) of transcription in a promoter-dependent way. The proliferation and differentiation of B-lymphocyte is controlled by PAX5, and in PAX5-deficient mice there are no mature B cells and B-lymphocyte lineage progression is arrested at the early pro-B stage without Correspondence: Dr S Dong or Dr JJ Qiu, Department of Medicine, expressing CD19. Although PAX5-deficient pro-B cells Baylor College of Medicine, One Baylor Plaza, BCM 286, Room can be propagated in vitro under appropriate culture N1317.03, Houston, TX 77030, USA. conditions, further progression down the B-lymphocyte E-mail: [email protected] or [email protected] lineage is prevented unless PAX5 is reintroduced into 1Current address: The Central Hospital of Wuhan, Wuhan Second Hospital, Wuhan, China the cells, reinforcing the importance of PAX5 in Received 7 December 2009; revised 30 August 2010; accepted 3 the development of B lymphocyte (Nutt et al., 1999; September 2010; published online 25 October 2010 Mikkola et al., 2002). PAX5–PML in ALL JJ Qiu et al 968 The chromosomal translocations involving the PAX PAX5–C20orf112 (Kawamata et al., 2008; Nebral et al., gene locus have been reported in human cancer patients 2009). In addition, the decreased PAX5 gene expression (Robson et al., 2006). The PAX3–FKHR and PAX7– was reported in one third of B-cell ALL patients FKHR fusion genes were identified in alveolar rhabdo- (Mullighan et al., 2007). Recently, the new PAX5-fusion myosarcoma patients, which are most frequent in protein PAX5–PML resulting from the recombination adolescent and young adult rhabdomyosarcoma patie- between PAX5 gene on chromosome 9 and promyelo- nts (Barr, 2001). The PAX8–PPARgamma chimeric gene cytic leukemia (PML) gene on chromosome 15, was is a molecular event associated with follicular thyroid identified in two cases of childhood ALL, which leads to tumorigenesis and is generated by a chromosomal an in-frame fusion of PAX5 to the PML gene (Nebral rearrangement between PAX8 and PPARgamma genes et al., 2007). The paired box region is a DNA-binding (Robson et al., 2006). The t(9;14)(pl3;q32) chromosomal domain that is highly conserved during evolution in all translocation involving PAX5 and the potent Emu PAX genes, which was retained in all fusion genes enhancer of the IgH gene was recently reported in involving a PAX gene (Robson et al., 2006). The cases of B-cell lymphoma leading to elevated PAX5 putative PAX5–PML chimeric protein also retains the expression. Moreover, the rearrangements of PAX5 paired-box domain from PAX5 and most functional gene were also found in patients with B-cell acute lympho- domains from PML protein. blastic leukemia such as PAX5–ETV6 (TEL), PAX5– In this study, we examined the effect of the PAX5– EVI3, PAX5–ELN, PAX5–FOXP1, PAX5–ZNF521 and PML versus PAX5–PML(DCC) in which the potential

a PAX5 PML

aa1 aa260 aa442aa578 aa858

Paired RING B1 B2 Coiled-Coil PAX5-PML

Paired RING B1 B2 PAX5-PML(ΔCC)

Paired 5’-PAX5

b 14 c 14 293T 293T

12 12

10 10

8 8

6 6

4 4

2 2 Relative luciferase activities with CD19-Luc Relative luciferase activities with hRAG2-Luc 0 0 1 2 3 4 5 1 2 3 4 5

CC) pSG5 PAX5 Δ pSG5 PAX5 ΔCC) 5’-PAX5 5’-PAX5 PAX5-PML PAX5-PML PAX5-PML( PAX5-PML( Figure 1 Structure and transactivating properties of PAX5–PML fusion protein. (a) Schematic illustration of the structure of PAX5– PML, PAX5–PML(DCC) and 50-PAX5. The PAX5–PML fusion protein contains the paired box-domain (DNA-binding domain) of PAX5 at its N-terminal and the RING domain, B1 and B2 domain, CC domain of PML at its C-terminal end. Transcriptional activation of a CD19-luc reporter (b) or a hRAG2-luc reporter (c) in 293T cells with the indicated PAX5-containing constructs. The results represent the mean±s.d. of four wells normalized for transfection efficiency using the b-Galactosidase assay. (d) The murine hematopoietic stem/progenitor cells were transduced with virus expressing MSCV, MSCV-PAX5–PML, MSCV-PAX5–PML(DCC) and MSCV-PAX5, respectively. The selected cells were harvested around 10 days after transduction for RNA preparation and CD19, Blnk, Rag2, Mb1/CD79a, Blk, Atp1b1 and Nedd9 mRNA levels were evaluated by real-time RT–PCR. The data were analyzed by using the formula 2ÀDDCt and b2-microglobulin gene was used as an endogenous control. The data are mean±s.d. for three independent reactions. An asterisk (*) indicates that the expression level is very low in the marked subgroup (see Supplementary Figure 4).

Oncogene PAX5–PML in ALL JJ Qiu et al 969 d 6 4.5 CD19 4 Blnk 5 3.5 on

4 ti 3 2.5 3

induc 2 2 1.5 Fold Fold induction 1 1 0.5 0 * * 0 MSCV PAX5-PML PAX5-PML(ΔCC) PAX5 MSCV PAX5-PML PAX5-PML(ΔCC) PAX5 3 6 Rag2 Mb1/CD79a 2.5 5

2 on 4 cti

1.5 du 3 n 1 2 Fold i Fold induction 0.5 1 0 0 MSCV PAX5-PML PAX5-PML(ΔCC) PAX5 MSCV PAX5-PML PAX5-PML(ΔCC) PAX5 1.2 1.2 Blk Atp1b1 1 1 on

ti 0.8 0.8 ction 0.6 u 0.6 ind

dinduc 0.4

l 0.4 o Fold F 0.2 0.2 0 0 MSCV PAX5-PML PAX5-PML(ΔCC) MSCV PAX5-PML PAX5-PML(ΔCC)

1.2 Nedd9 1 n 0.8 ctio u

d 0.6 n i

d 0.4 ol

F 0.2 0 MSCV PAX5-PML PAX5-PML(ΔCC) Figure 1 Continued. dimerization domain was deleted, on PAX5-consensus chimeric oncogenic protein on transcriptional activities sequence binding, PAX5 targeting gene transcriptional using CD19-luc or human RAG2-luc as reporter function, its intracellular localization and mobility. We (Figure 1, Supplementary Figure 1). Transfection of demonstrated that PAX5–PML bound PAX5-consensus the 293T or HeLa cell line with both of PAX5-related sequence as a homodimer, inhibited PAX5 targeting reporter vectors resulted in its transactivation by gene transcriptional activity, and was shown to have the endogenous PAX5. The addition of a wild-type PAX5 reduction of intranucleus mobility. Thus, PAX5–PML expression vector resulted in dramatically increased appears to contribute to leukemic phenotype by binding transactivation, indicating that exogenous PAX5 could to PAX5-consensus sequence and interfering with transactivate these PAX5 consensus-containing ele- PAX5 functional activity, in which, the coiled-coil ments (Figure 1, Supplementary Figure 1). However, (CC) dimerization domain of PAX5–PML may have the expression of PAX5–PML fusion protein, which was an important role. identified in two B-cell acute lymphocytic leukemia (ALL) patients (Nebral et al., 2007), had no such an effect and instead inhibited the transcriptional luciferase activity by endogenous PAX5 (Figure 1, Supplementary Results Figure 1). In contrast, 50-PAX5 lacking the C-terminal transactivation domain of PAX5 gene has no (with PAX5–PML inhibits the transcriptional activity CD19-luc reporter) or a little (with hRAG2-luc reporter) of PAX5 target gene luciferase activity from PAX5-reporter. To evaluate It is well known that wild-type PAX5 has a transactiva- whether or not the potential dimerization of PAX5– tion effect on CD19 gene which is one of the PAX5 PML protein is important for its transcriptional target genes (Cobaleda et al., 2007). In the present inhibition activity, we have generated a PAX5– study, we determined the effects of PAX5–PML PML(DCC) construct, in which the CC domain from

Oncogene PAX5–PML in ALL JJ Qiu et al 970 PML moiety was deleted. Indeed, coexpression of PAX5–PML(DCC) protein had modest inhibition effect 2a γ of transcriptional activity with CD19-luc reporter or γ s-1 ε CD19-2 CD19-2,Ains e5 BLNK CD19-1 α I RAG2 BLK slightly augmented this activity using hRAG2-luc 5'S reporter vector (Figure 1, Supplementary Figure 1), suggesting that the dimerization, mediated by CC PAX5 domain, may have a certain role in the ability of PAX5–PML fusion protein to inhibit the gene transac- tivation by wild-type PAX5. PAX5-PML To extend our luciferase results in the context of hematological lymphoid cells, we transduced PAX5– PML, PAX5–PML(DCC) or PAX5 into murine BM PAX5-PML hematopoietic stem/progenitor cells and plated in methylcellulose medium containing cytokines for lym- phoid cell development (Supplementary Figure 2). We PAX5-PML(ΔCC) found that there are no obvious differences regarding the colony formation among these constructs (Supple- Figure 2 Binding of PAX5–PML to a series of PAX5-response elements by gel shift assays. The equivalent amounts of in vivo mentary Figure 3). Next, we extracted RNA from these PAX5 (a) PAX5–PML (b) PAX5–PML(DCC) (c) proteins were transduced murine BM cells for quantitative real-time incubated with the following radio-labeled PAX5-response element PCR analysis. The seven mouse Pax5 target genes, probes: CD19-1, CD19-2, CD19-2(Ains), 50Sg2a, Ie, as-1 and e5 including CD19, Blnk, Rag2, Mb1/CD79a, Blk, Atp1b1 (Adams et al., 1992) as well as the PAX5-response element and Nedd9 (Kawamata et al., 2008; Pridans et al., 2008), sequence from the natural enhancer/promoter regions of the human RAG2, BLNK and BLK gene. The overexposure of were selected in our studies. We found that, compared retardation bands of PAX5–PML with probes, as-1, e5, RAG2 with vector transduction only, introduction of PAX5 and BLK were shown in the bottom (b). into murine BM cells remarkably enhanced the expres- sion of these Pax5 target genes (Figure 1d), which supported these genes are positively regulated by PAX5 transcriptional factor as reported (Cobaleda et al., which the retardation bands could be supershifted 2007). In contrast, the gene expression levels of all these by addition of the antibodies against PAX5 or PML PAX5 target genes, including CD19 and Rag2 used in (Figure 3b). However, following the deletion of CC our luciferase studies, were lower in PAX5–PML domain from PML, the DNA-binding pattern and transduced BM cells, which were consistent with our affinity of PAX5–PML were similar to the wild-type luciferase results that PAX5–PML chimeric protein PAX5 except BLK probe (Figures 2b and c). These inhibited the transcriptional activity of PAX5 target results suggested that the PAX5–PML protein had genes. However, the expression levels of these Pax5 altered its DNA-binding to a series of PAX5-consensus target genes in PAX5–PML(DCC) transduced BM cells sites in comparison with wild-type PAX5, in which the were also lower, although we have also observed that CC domain of PML might be responsible for this effect. the mRNA levels of PAX5–PML(DCC) transduced BM were higher than that of PAX5–PML transduced BM in some Pax5 target genes, including CD19, PAX5–PML forms homodimer to bind the PAX5 Rag2, Mb1/CD79a, Blk and Atp1b1 (Figure 1d, Supple- consensus sequence and the CC domain of PML mentary Figure 4). is responsible for this effect It was reported that PAX5 bound to its DNA-response element by forming monomer (Czerny et al., 1993). PAX5–PML binds the PAX5 consensus DNA-binding We have demonstrated that the dimerization domain sequence (CC-domain) of PML has an important role in the To determine whether or not PAX5–PML inhibition of ability of PAX5–PML inhibition of PAX5 transcrip- PAX5 transcriptional function is owing, in part, to its tional potential (Figure 1). To see if this domain is also ability to directly bind PAX5-response element se- responsible for the DNA-binding of PAX5–PML to quences, PAX5–PML protein was tested in gel shift PAX5 consensus sequence, we have performed the gel assay using a series of PAX5-consensus duplex oligo- shift assay using PAX5–PML(DCC) construct lacking nucleotides (Figure 2). As expected, the wild-type PAX5 CC-domain. As shown in Figure 3a, the deletion of this efficiently binds to these sequences as reported elsewhere dimerization domain from PAX5–PML did not effect its (Figure 2a) (Adams et al., 1992; Czerny et al., 1993). ability to form PAX5–PML/DNA complex, rather, In contrast, chimeric PAX5–PML protein could also bound as efficiently to PAX5 binding sites as wild-type bind the most of these sequences but with the reduction PAX5 protein, indicating that the CC domain had no of DNA-binding ability in comparison of wild-type effect for PAX5–PML binding to its DNA sequence but PAX5, especially when the as-1, e5 and RAG2 probes influenced its DNA-binding affinity (Figure 3a, upper were tested. In addition, we found that PAX5–PML panel). However, we could also see that the PAX5– fusion protein bound more efficiently to, CD19 and PML(DCC)/DNA complex moved much faster than its BLNK, than to other sequences tested (Figure 2b), in parent form (PAX5–PML), its mobility was more close

Oncogene PAX5–PML in ALL JJ Qiu et al 971 Ab- Ab- Ab- Ab- Ab- PAX5 PAX5 PML PAX5 PML ΔCC)

pSG5 PAX5-PMLPAX5-PML(PAX5

CD19-1 pSG5 PAX5-PML PAX5-PML(ΔCC) CD19-2,Ains 100Kd

75Kd PAX5-PML + + + + + Cold Probe 2× 5× 20× 50×

50Kd

Ab-PAX5 CD19-2

12 BD-PAX5-PML 10 8 6

activities 4

Relative luciferase 2 0 1 2 3 4 5

AD ΔCC) AD-PML AD-PAX5 AD-PAX5-PML AD-PAX5-PML( Figure 3 Binding of PAX5-response element by PAX5–PML fusion protein homodimer. (a) Binding of PAX5–PML homodimer to a CD19-1 probe by gel shift assays. The equivalent amounts of in vivo PAX5–PML, PAX5–PML(DCC), PAX5 proteins were incubated with the radio-labeled CD19-1 probes. The level of protein expression, determined by immunoblotting with antibody against the N-terminal of PAX5, was shown in the bottom panel. (b) Gel shift assays were performed using in vivo PAX5–PML or PAX5– PML(DCC) protein incubated with radio-labeled CD19-2(Ains). Antibody against PAX5 or PML serum was added to the reactions for supershift assays where indicated. (c) Gel shift assays were performed using in vivo expressed PAX5–PML protein incubated with radiolabeled CD19-2 (hot-probe) without or with unlabeled CD19-2 (cold-probe) in the amounts indicated. (d) The protein–protein interactions between PAX5–PML proteins, PAX5–PML and wide-type PML were analyzed using mammalian two-hybrid assay. The AD (activation-domain) alone or its fusion with PAX5–PML, PAX5–PML(DCC), PML or PAX5 was expressed in HeLa cells together with a luciferase reporter containing four copies of Gal4-binding sites (4 Â UAS-TK luc) as well as Gal4 DBD-fused PAX5–PML (BD-PAX5–PML). The relative luciferase activities are averages of three independent transfection experiments normalized to b-galactosidase activity.

with the one of wild-type PAX5. Giving that the PAX5–PML can form complex with PAX5–PML as molecular mass of PAX5–PML(DCC) is much bigger well as wild-type PML than wild-type PAX5 and is a little less than its parent To confirm that the PAX5–PML protein homodimer form (PAX5–PML) (Figure 1a, Figure 3a, bottom complex formation resulted from our gel shift assay panel), we can conclude that the full-length PAX5– (Figures 3a and b), we also performed the mammalian PML protein could bind to PAX5-consensus sequence two-hybrid system assay (Qiu et al., 2010b). Transient as a homodimer, in contrast, PAX5–PML(DCC), like expression of a Gal4 DBD–PAX5–PML fusion (BD– wild-type PAX5, forms monomer to bind the PAX5 PAX5–PML) and AD-fused PAX5–PML (AD–PAX5– consensus sequence owing to lacking the dimerization PML) (Figure 3d, lane 2) or AD-fused PML (AD–PML) motif (CC-domain) from PML moiety (de The et al., (Figure 3d, lane 4) into HeLa cells resulted in strong 1991). stimulation of gene expression from a luciferase reporter

Oncogene PAX5–PML in ALL JJ Qiu et al 972

PAX5-PML(YFP) DAPI Merge

PAX5-PML (GFP-LacR) PML(ChFP) DAPI Merge

PAX5-PML(ΔCC) (YFP) DAPI Merge

PAX5-PML PAX5-PML (GFP-LacR) (ChFP) DAPI Merge

PAX5(YFP) DAPI Merge

PAX5-PML PAX5-PML(ΔCC) (GFP-LacR) (ChFP) DAPI Merge

5’-PAX5(YFP) DAPI Merge

PAX5-PML (GFP-LacR) PAX5(ChFP) DAPI Merge

3’-PML(YFP) DAPI Merge

Figure 4 Cellular localization of PAX5-containing proteins. (a) Effect of GFP–LacR–PAX5–PML expression on the localization and distribution of ChFP-tagged PML, PAX5–PML, PAX5–PML(DCC) or PAX5 within A03_1 cells. A03_1 cells coexpressed with ChFP-PAX5–PML plus GFP–LacR–PAX5–PML (first row); ChFP-PML plus GFP–LacR–PAX5–PML (second row), ChFP-PAX5– PML(DCC) plus GFP–LacR–PAX5–PML (third row), ChFP-PAX5 plus GFP–LacR–PAX5–PML (fourth row) were subjected to deconvolution microscopy analysis. DNA was visualized by 40, 6-diamidino-2-phenylindole-staining (blue-color). (b) The deconvolution fluorescent microscopy was applied to analyze the HeLa cells transient-expressed with the indicated YFP-tagged proteins, PAX5–PML, PAX5–PML(DCC),PAX5,50-PAX5 or 30-PML. Nuclei were visualized by DNA staining with 40, 6-diamidino-2-phenylindole (Sigma).

containing four copies of Gal4-binding sites, in compar- coexpression of GFP–LacR–PAX5–PML with ChFP– ison with the combination of one plus AD empty vector PAX5–PML in A03_1 cells also revealed that part of (Figure 3d, lane 1). However, this gene activation effect the cherry fluorescent signal was localized onto the lac was not observed from the combination of BD–PAX5– operator array by GFP–LacR–PAX5–PML (Figure 4a, PML plus AD–PAX5–PML(DCC) or AD–PAX5 in the second row), although coexpression of either GFP– same experimental setting (Figure 3d, lane 3, lane 5). LacR–PAX5–PML plus ChFP–PAX5–PML(DCC) or The results of these two-hybrid studies provide strong GFP–LacR–PAX5–PML plus ChFP-PAX5 in A03_1 evidence that the PAX5–PML fusion protein could form cells did not reveal the recruitment of ChFP–PAX5– a complex with itself or wild-type PML and that the CC PML(DCC) or ChFP-PAX5 onto the array (Figure 4a, domain of PAX5–PML is required to mediate the third row and fourth row), confirming the above studies formation of these complexes. by gel shift, and two-hybrid assays that the CC domain The next question we want to address is whether was required for the formation of PAX5–PML homo- or not wild-type PML, could be recruited into the dimer complex and PAX5–PML/PML complex. chromatin region occupied by PAX5–PML fusion protein. Toward this end, the potential interaction between PAX5–PML and PML in the context of Nuclear localization of PAX5–PML chimeric protein chromatin was studied using the lac operator/lac We next wanted to determine the distribution of PAX5– repressor tethering system (Qiu et al., 2007, 2010a). PML in cells, the contribution of the PML domain to its The coexpression of GFP–LacR–PAX5–PML with distribution and to compare its distribution with that of ChFP–PML in A03_1 cells showed that part of the each component of the fusion gene product, namely ChFP–PML colocalized with GFP–LacR–PAX5–PML wild-type PAX5 and wild-type PML. To accomplish on the lac operator array, although the plenty of this, we made the expression vectors for PAX5–PML, ChFP-signal still remained in the PML nuclear body PAX5–PML(DCC), PAX5, 50-PAX5 and 30-PML, (Koken et al., 1994) (Figure 4a, first row), suggested that which were YFP-tagged. Fluorescent microscopic ex- wild-type PML could form PML/PAX5–PML/DNA amination of HeLa and 293T cells was performed complex in the context of chromatin. In addition, the following transient expression of these YFP-tagged

Oncogene PAX5–PML in ALL JJ Qiu et al 973

PAX5

Pre-bleach Bleach 1s 5s

PAX5-PML

Pre-bleach Bleach 20s 40s

PAX5-PML (ΔCC)

Pre-bleach Bleach 1s 5s

5’-PAX5

Pre-bleach Bleach 1s 5s

3’-PML

Pre-bleach Bleach 40s 80s

1.2 1 0.8 PAX5 0.6 PAX5-PML 0.4 PAX5-PML(ΔCC) 0.2 5’-PAX5 Relative fluorescence 3’-PML 0 0 8 24 32 40 56 80 96 16 48 64 72 88 160 168 176 184 192 104 112 120 128 136 144 152 Time after bleach (Seconds) Figure 5 FRAP analysis of YFP-tagged PAX5–PML, PAX5–PML(DCC), PAX5, 50-PAX5 and 30-PML in transfected live HeLa cells. (a) Images show single z-sections and were obtained before bleaching and at the indicated time points after. The white rectangles represent the bleaching area within nucleus. (b) Fluorescence recovery of curves after photobleaching of PAX5–PML, PAX5– PML(DCC), PAX5, 50-PAX5 and 30-PML in HeLa cells. The initial fluorescence at the end of the photobleaching was normalized to zero and final fluorescence at equilibrium was designated as 1. Data presented at each point are the mean fluorescence within the photo-bleached area of 20 cells. proteins. As shown in Figure 4b and Supplementary typical POD (PML oncogenic domain) structure found Figure 5, all of these YFP-tagged proteins were localized in wild-type PML (Koken et al., 1994). predominantly in the nucleus. Although PAX5, 50-PAX5 and PAX5–PML(DCC) predominantly exhibited a diffuse granular pattern, a microspeckled distribution in the The intranuclear mobility of PAX5–PML is decreased background of diffuse granular pattern within the nucleus compared with wild-type PAX5 has been found following the expression of YFP– To study the intranuclear mobility of PAX5–PML PAX5–PML. In addition, YFP–30–PML, was shown a and compare its mobility with wild-type PAX5, we

Oncogene PAX5–PML in ALL JJ Qiu et al 974

Table 1 Fluorescence recovery t1/2 of YFP–PAX5–PML, YFP– the intranuclear mobility reduction of PAX5–PML PAX5–PML(DCC), YFP–PAX5, YFP–50–PAX5 and YFP–30–PML chimeric protein, which was identified from two proteins acute lymphoblastic leukemia (ALL) patients (Nebral

No Construct t1/2 et al., 2007).

1 YFP–PAX5–PML 18.51±4.24 s 2 YFP–PAX5–PML(DCC) 2.19±0.71 s 3 YFP–PAX5 1.75±0.52 s 4 YFP–50–PAX5 1.61±0.37 s Discussion 5 YFP–30–PML 35.66±3.94 s In the present study, we have demonstrated that one Po0.001for1versus2,3,4,or5;Po0.001 for 5 versus 2, 3, of lymphoid leukemia-associated proteins, PAX5–PML or 4; P ¼ 0.579 for 2 versus 4; P ¼ 0.442 for 2 versus 3; P ¼ 0.137 for 3versus4. chimeric oncogenic protein, could bind to PAX5 response-element as homodimer to inhibit the transacti- vation of PAX5 target-genes, presumedly, and disrupt the normal PAX5 function in the development of B- performed fluorescent recovery after photobleach lymphocyte for leukemogenesis. (FRAP) on live HeLa cells transfected with YFP-tagged The protein-protein interactions by homo/oligodimer- constructs (Figure 5 and Table 1). A rectangular region ization are widely used for regulation of various in the nucleus was bleached by applying maximal laser biological processes including the pathogenesis of acute power for approximately 1 s. In all FRAP experiments, leukemia (So et al., 2003b; So and Cleary, 2004). The only cells expressing low levels of protein were examined one well-known oncogenic mechanism, by chromosomal to avoid artifacts of overexpression as previously translocations that result in fusion with heterologous described by us (Dong et al., 2004; Qiu et al., 2006). protein partners providing dimerization-motif, is the Figure 5 shows images taken right before bleaching (pre- homo-dimerization, which could modulate its biological bleach), immediately after bleaching (bleach) and at activities including enzymatic activation and signal several time points after bleaching. Following photo- transduction (for example, BCR–ABL fusion protein), bleaching of live HeLa cells transfected with YFP- gene transcriptional regulation and nuclear mobility (for PAX5, fluorescence recovery occurred rapidly with a example, AML1–ETO and PML–RARa leukemic half-maximal fluorescence recovery time (t1/2)of chimeric proteins) (So and Cleary, 2004). We and others 1.75±0.52 s (Table 1). In comparison with wild-type have recently shown in acute leukemias that AML1 and PAX5, 50-PAX5, which deleted the C-terminal region of RARa homo-oligomers result in constitutive transcrip- wild-type PAX5 had similar mobility of t1/2 of tion repression activity, while homodimeric MLL 1.61±0.37 s. Remarkably, FRAP of PAX5–PML re- chimeras act as constitutive transcriptional activators vealed a recovery t1/2 of 18.51±4.24 s, which is 10.5-fold (Dong et al., 2003; So et al., 2003b; So and Cleary, slower than PAX5 (Figure 5 and Table 1). In addition, 2004). Indeed, like acute promyelocytic leukemia fusion 30-PML, which lacking 43 amino acid in the N-terminal proteins, PAX5–PML homodimer mediated through the region of PML, was shown the slowest intranuclear CC domain of PML, repressed and deregulated PAX5 mobility with t1/2 of 35.66±3.94 s. downstream target gene transcriptional activities, possi- bly through the abnormal interaction with both its transcriptional coregulators and its target-sequence The role of the CC domain in the reduction of intranuclear following introduction of PML sequence into PAX5– mobility of PAX5–PML PML fusion protein, which is from two B-cell acute We recently reported that, in addition to being essential leukemia patients (Nebral et al., 2007). This is reminis- for other oncogenic functions, the CC dimerization cent of PML–RARa from t(15;17) translocation identi- domain of NuMA–RARa, PML–RARa and NHR2 fied in acute promyelocytic leukemia patients, in which dimerization domain of AML1–ETO was responsible the homodimerization through the identical CC domain for the reduced intranuclear mobility of NuMA–RARa, of PML, could increase the stability and stoichiometry PML–RARa and AML1–ETO, respectively (Dong of its association with nuclear receptor corepressor– et al., 2004; Qiu et al., 2006; Huang et al., 2008). histone deacetylase complex and result in aberrant In this study, we next examined whether or not the repression of RARa target genes, that was linked to dimerization domain (CC-domain) in PAX5–PML its leukemogenesis (Melnick and Licht, 1999). We also contributes to its mobility reduction within the nucleus. noticed that other PAX5-fusion proteins from acute For this purpose, one PAX5–PML mutant, in which the lymphoblastic leukemia (ALL) patients, like PAX5– dimerization domain (CC) has been deleted, was FOXP1 and PAX5–EVT6, have also possessed the created, and subjected to FRAP analysis (Figure 5). dimerization motif in the moiety of their partner Indeed, this mutant has fast intranuclear mobility with a (Mullighan et al., 2007). PAX5–FOXP1 retains two CC recovery t1/2 ¼ 2.19±0.71 s, which is 8.5-fold faster than domains, which have potential dimerizational function. PAX5–PML but very close to that of wild-type PAX5 The dimerization pointed domain (also called HLH (Figure 5 and Table 1). Therefore, similar to its domain) of ETV6 has been demonstrated to directly contribution to other activities of PAX5–PML, the CC contribute ETV6-AML1 dimerization and interactions of dimerization domain of PML also has a key role in the chimeric product with wild-type ETV6. Therefore, this

Oncogene PAX5–PML in ALL JJ Qiu et al 975 domain may also mediate the homodimer formation needs further study. In addition, the secondary mole- within PAX5–ETV6 fusion protein. cular mutation(s) may cooperate with PAX5–PML The reduction of intranuclear mobility of PAX5– event to give a complete transformation of normal PML is also linked to its homodimerization formation lymphoid cell to leukemic cells. Taken together, our in our study. In our previous studies (Dong et al., 2004; results indicate that homodimerized DNA-binding, the Qiu et al., 2006), the decreasing intranuclear mobility of reduction of intranuclear mobility and the inhibition of leukemia-related fusion proteins resulting from chro- PAX5 target gene expression by PAX5–PML homo- mosomal translocation in acute myeloid leukemia dimeric chimeric oncogenic protein are indispensable for (AML), namely PML-RARa or AML1-ETO, has been the functional activities of PAX5–PML, which may demonstrated compared with wild-type RARa or account for its blockage of lymphoid cell differentiation AML1, respectively. The CC domain of PML, like in and potential leukemogenesis. PML-RARa oncogenic chimeric protein (Huang et al., 2008), was also shown to be responsible for the reduction of intranuclear mobility of PAX5–PML, Materials and methods implying its importance for this effect. It was reported that the formation of nuclear PML-RARa homodimeric Constructs complexes through the PML CC domain is crucial for PML (in pSG5 expression vector, Stratagene, La Jolla, CA, the ability of PML-RARa to mediate retinoic acid USA) has been described previously (de The et al., 1991). The response in APL cells (Grignani et al., 1999). This raised PAX5 (in pKW2T vector) and CD19-luciferase reporter vector were kindly provided by Dr Busslinger from Research Institute the possibility that the CC domain, which mediates of Molecular Pathology, Vienna, Austria (Adams et al., 1992). homodimerization, may itself be sufficient for the The pSG5-PAX5 construct was directly cloned from PAX5 (in activity of PAX5–PML. Presumedly, without CC pKW2T). The hRAG2-luciferase reporter vector was made by dimerization domain in PAX5–PML chimeric protein, PCR amplification of human RAG2 promoter/enhancer some interacting protein(s) or structure would be lost, sequence into pGL3-Basic vector (Promega, Madison, WI, which might be important for maintenance of PAX5– USA) (Lauring and Schlissel, 1999). PAX5–PML was con- PML functional structure for its oncogenic transforma- structed in the pSG5 expression vector by fusing human PAX5 tion. Identification of these potential protein(s) or and PML sequences using PCR approach based on functional structure, which is responsible for both the published sequence (Nebral et al., 2007). A similar PCR interaction with CC domain of PAX5–PML and the approach was also applied to construct pSG5–PAX5– PML(DCC) in which the dimerization domain (CC domain) reduced intranuclear mobility of PAX5–PML, will be of PML is deleted (Figure 1a) (Huang et al., 2008), and pSG5– important to further understand the mechanism of 50–PAX5 expression vector. The cDNA sequences for PAX5– PAX5–PML-mediated leukemogenesis. The reduction of PML, PAX5–PML(DCC), PAX5 from its pSG5 vectors were intranuclear mobility of PAX5–PML would reflect its cloned into MSCV-puro vector (Clontech, Mountain View, longer stay in the chromatin area of PAX5 targeting gene or CA, USA) (Qiu et al., 2010b), to make MSCV–PAX5–PML, new chromatin regulatory area independent of PAX5, MSCV–PAX5–PML(DCC), MSCV–PAX5 expression vectors, which results in deregulation of PAX5 targeting gene and respectively. The PAX5–PML, PAX5–PML(DCC), PAX5 or would also create new targeting genes which are not PML open reading sequence were also put into pBD-S5 vector controlled by wild-type PAX5 B-cell transcriptional factor. or pAD-S5 vector (Qiu et al., 2010b), to make BD–PAX5– In this study, we have shown that PAX5–PML PML, AD–PAX5–PML, AD–PAX5–PML(DCC), AD–PAX5 and AD–PML vectors for the mammalian two-hybrid assay. chimeric protein was localized to the nucleus and was The EYFP, GFP-LacR and ChFP (for cherry fluorescent protein) able to suppress the transcriptional activities of key sequence (Qiu et al., 2007), were obtained by standard PCR PAX5 target genes in our luciferase and quantitative methods and subsequently cloned into the N-terminal region of real-time PCR experiments, including CD19, Rag2 and cDNA sequences of PAX5–PML, PAX5–PML(DCC), PAX5, Blk genes (Cobaleda et al., 2007). This is in agreement PML, 50-PAX5 or 30-PML in pSG5 expression vectors to make with the similar results of Kawamata et al. (2008), using YFP–PAX5–PML, YFP–PAX5–PML(DCC), YFP–PAX5, NALM-6 cell line with ectopic expression of PAX5- YFP–50–PAX5, YFP–30–PML, GFP–LacR–PAX5–PML, C20orf112 chimeric oncogenic protein. Therefore, ChFP–PAX5–PML, ChFP–PAX5–PML(DCC), ChFP–PAX5 PAX5–PML from (9;15) translocation in B-cell acute and ChFP–PML expression vectors, respectively. All constructs leukemia may account for the blockage of leukemic were confirmed by DNA sequencing and/or immunobloting. cells at the pre-B-cell differentiation stage, giving that wild-type PAX5 is essential for B-cell differentiation Primary BM cell isolation and retroviral transduction (Nutt et al., 1999; Mikkola et al., 2002). The mechanism The murine BM cells were harvested from long bones of 4- to involved in this process appears to be, at least in part, 10-week-old wild-type C57BL/6 mice, as previously described through a dominant-negative effect of PAX5–PML on (Zeisig et al., 2007; Kwok et al., 2009; Qiu et al., 2010b). the wild-type PAX5, in which PAX5–PML behaves like The viral supernatants were collected 48 h after transfection of an altered PAX5 to interfere with normal B-lymphoid GP2-293 cells (Clontech) and used to infect the purified murine hematopoietic stem/progenitors cells that were positively cell differentiation. We also noticed that PAX5–PML selected for c-Kit expression by magnetic activated cell sorting could interact with wild-type PML in the present study. (Miltenyi Biotec., Auburn, CA, USA) (Kwok et al., 2009; Qiu However, the involvement and/or disruption, or not, et al., 2010b). After spinoculation by centrifugation at 800 g of PML function by PAX5–PML in the pathogenesis of for 2 h at 32 1C, transduced BM cells were plated in 1% B-cell acute lymphoblastic leukemia is not clear, which lymphoid conditioned methylcellulose contained an Iscove’s

Oncogene PAX5–PML in ALL JJ Qiu et al 976 modified Dulbecco’s medium-based Methocult (Methocult previously reported for luciferase assay or immunoblotting M3231; StemCell Technologies, Vancouver, BC, Canada) analysis (Qiu et al., 2007). Antibodies used in immunoblotting supplemented with 100 ng/ml mouse stem cell factor and study were PAX5 rabbit polyclonal antibody (Millipore, 20 ng/ml each of mouse interleukin 7 (IL-7), mouse Flt3 ligand Billerica, MA, USA). Luciferase assays were performed in (PeproTech Inc., Rocky Hill, NJ, USA) in the presence of cells transiently co-transfected with plasmids expressing appropriate drug selection (1 mg/ml puromycin, Sigma, St Louis, PAX5-related proteins, a luciferase reporter gene (CD19- MO, USA) (So et al., 2003a; Kawamata et al., 2008). luciferase reporter gene, human RAG2-luciferase reporter Immunophenotypic analysis was performed by fluorescence- gene or 4 Â UAS–TK-luciferase reporter construct) and a activated cell sorter using fluorophore-conjugated antibodies to b-galactosidase vector, which was used as internal control. Each c-kit, CD3e, B220 and Mac-1/CD11b (Biolegend, San Diego, point was the mean of three to four independent experiments. CA, USA), respectively, as described previously (So et al., 2003a; Zeisig et al., 2007; Kwok et al., 2009; Qiu et al., 2010b). Gel shift DNA-binding assays The annealed PAX5 probes were end-labeled at its 50-end with Quantitative real-time reverse trancriptase–PCR analysis (g-32P)ATP and subsequently were purified using MicroSpin Total RNA was extracted from transduced BM cells using G-25 Columns (GE healthcare Life Sciences, Piscataway, NJ, RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and treated USA). For DNA-protein binding reactions, we used a DNA- with DNA free (Ambion, Austin, TX, USA) to remove any binding buffer containing 13 mM HEPES, pH 8.0, 65 mM KCl, DNA contamination. cDNA was synthesized using TaqMan 1mM DTT, 0.14 mM EDTA and 10% glycerol in the presence Reverse Transcription kit (Applied Biosystems, Foster City, of 50 ng/ml poly (dI-dC) and labeled 1.0 ng double-strand CA, USA) according to the manufacturer’s instructions. synthesized oligonucleotides containing an PAX5-binding site Quantitative PCR was performed on an ABI7000 using SYBR (Adams et al., 1992; Qiu et al., 2010b). The reaction mixtures Green PCR Master Mix (Applied Biosystems) (Qiu et al., of a 20 ml final volume were incubated for 30 min at 4 1C and 0 0 2003). CD19 (5 -ACGTGAAGGTCATTGCAAGGT-3 and subsequently for 30 min at room temperature followed by 0 0 0 5 -TCCATCCACCAGTTCTCAACAG-3 ), Blnk (5 -AATGA electrophoresis on 5% non-denaturing polyacrylamide gels 0 0 AGGTGGAATAATGGACAAGA-3 and 5 -CCCTTCGAG equilibrated in 0.25 Â TBE and analyzed by a PhosphorImager 0 0 GAACACTTGGA-3 ), Rag2 (5 -AAAGGATTCAGAGAGG (GE healthcare Life Sciences). We performed gel shift assay 0 0 GATAAGCA-3 and 5 -GCTTGTGGATGTGAAATACTCT with the whole-cell extract from 293T cells with transient- 0 0 TTCT-3 ), Mb1/CD79a (5 -TCTTCTTGTCATACGCCTGT expression of PAX5–PML, PAX5–PML(DCC) or PAX5. For 0 0 0 TTG-3 and 5 -AAGTTCACCGTCAGGGATGGT-3 ), Blk supershift assays, the reaction mixture was included with 1 mgof 0 0 0 (5 -TTGCCCCACCATCTCCTAAC-3 and 5 -AAACAGAG PAX5 goat polyclonal antibody (N-19, Santa Cruz Biotechnology, 0 0 CCACCACAAAACG-3 ), Atp1b1 (5 -GGCAGGACCGGTG Santa Cruz, CA, USA) or PML rabbit polyclonal antibody 0 0 0 GTAGTT-3 and 5 -AGCCAGGCAGCCATAAAATATC-3 ), (H238, Santa Cruz Biotechnology) incubated for 10 min at 0 0 0 Nedd9 (5 -AACGCTCCTCAAGAGAAAATGTG-3 and 5 -CA 4 1C before the addition of radiolabeled probes. CTCAGGGACGTTGTCATACAA-30) primers were used to amplify the mouse Pax5 target genes, CD19,Blnk,Rag2,Mb1/ CD79a, Blk, Atp1b1 and Nedd9 cDNA, respectively. The disso- Fluorescent microscopy and fluorescence recovery after ciationofSYBRGreenlabeledcDNAwasperformedafterPCR photobleaching to monitor the specificity of the amplification. Data were analyzed A03_1 cells and HeLa cells were seeded onto coverslips coated by a comparative relative quantification method using the with poly-D-lysine (Sigma) in 24-well cell culture plate at a formula 2ÀDDCt. The fold change of mRNA level was normalized concentration of 104 cells/well at 24 h before transfection. Cells with the Ct (threshold cycle) value of b2-microglobulin used as an with transient expression of GFP–LacR–tagged plus ChFP– endogenous control and was calculated in relation to the Ct value tagged constructs (in A03_1 cell) and YFP–PAX5–PML, of cells transduced with MSCV vector used as a calibrator. The YFP–PAX5–PML(DCC), YFP–PAX5, YFP–50–PAX5 or relative mRNA level of the calibratorwassetas1.Thepresented YFP–30–PML (in HeLa and 293T cell) were fixed and decon- data correspond to the mean±s.d from three independent volution microscopy was performed as described previously reactions. (Qiu et al., 2006, 2007, 2010a). FRAP analysis of the mobility of fluorescence-tagged proteins within nucleus has been described extensively by us (Dong et al., Cell culture 2004; Qiu et al., 2006; Huang et al., 2008). Briefly, 24 h before 293T cells were cultured in Dulbecco’s modified Eagle medium transfection, cells were plated onto Delta T dishes (Bioptechs, supplemented with 10% fetal bovine serum and HeLa cells were Butler, PA, USA) at a concentration of 2 Â 105 cells per dish. maintained in Opti-MEM I media (Invitrogen, Grand Island, Transient expression of YFP-tagged plasmids, including PAX5– NY, USA) with 4% fetal bovine serum (Qiu et al., 2006, 2007). PML, PAX5–PML(DCC), PAX5, 50-PAX5 or 30-PML, was A03_1 cell line, which contains integrated lac operator (lacO) accomplished using GeneJuice transfection reagent. The sub- heterochromatic array arranged in a globular structure useful for sequent FRAP experiments were performed on Zeiss LSM 510 studying protein–protein interaction in the context of chromatin, confocal microscopes using the laser set a wavelength of 514 nm was cultured in F-12 Ham’s medium (without hypoxanthine for YFP at maximum power for 50 iterations of a box and thymidine) (Invitrogen) with 0.3 mM methotrexate and 10% representing B20% of the nuclear volume. A minimum of 20 dialyzed fetal bovine serum (HyClone, Logan, UT, USA) (Qiu cells for each construct were examined. The fluorescence et al., 2007). Cells were replenished with fresh medium and intensities of regions of interest were obtained using LSM510 serum 12 h before all experiments. software, and data were exported to Excel for analysis.

Cell transfections, immunoblotting and reporter gene assays GeneJuice transfection reagent (Novagen, San Diego, CA, USA) was applied for transient transfection of 293T, HeLa, Conflict of interest A03_1 cells according to the manufacturer’s instructions. At 48 h after transfection, cells were lysed in lysis buffer as The authors declare no conflict of interest.

Oncogene PAX5–PML in ALL JJ Qiu et al 977 Acknowledgements Illinois Champagne-Urbana) for the A03_1 cell line, and Dr CWE So (King’s College London, London, UK) for critical We thank Dr M Busslinger (Research Institute of Molecular reading of the manuscript. This work was supported in part, Pathology, Vienna, Austria) for the CD19-luciferase and by funds from the Albert and Margaret alkek Foundation and PAX5 expression vectors, Dr AS Belmont (University of Leukemia Research Foundation (LRF).

References

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