(2008) 27, 5260–5266 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $30.00 www.nature.com/onc SHORT COMMUNICATION and the leukemia-associated PLZF factor interact to repress target promoters

K Petrie1,8, F Guidez2,8, J Zhu3, L Howell1, G Owen4, YP Chew5, S Parks2, S Waxman6, J Licht7, S Mittnacht5 and A Zelent1

1Section of Haemato-Oncology, Institute of Research, Sutton, UK; 2Department of Medical and Molecular Genetics, King’s College, London, UK; 3Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Second Medical University, Shanghai, China; 4Department of Physiology, Faculty of Biological Sciences, Catholic University of Chile, Santiago, Chile; 5Cancer Research UK Centre for Cell and Molecular Biology, The Institute of Cancer Research, London, UK; 6Division of Hematology and Medical Oncology, Mount Sinai School of Medicine, NY, USA and 7Division of Hematology/Oncology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

Translocations of the retinoic acid -a (RARa) The PLZF (promyelocytic leukemiazinc-finger) gene locus with the promyelocytic leukemia zinc-finger (PLZF) was originally identified as a result of the t(11;17) or PML lead to expression of oncogenic PLZF– translocation with the -a (RARa) RARa or PML–RARa fusion , respectively. locus that gives rise to the PLZF–RARa fusion These fusion oncoproteins constitutively repress RARa oncoprotein in acute promyelocytic leukemia (APL). target genes, in large part through aberrant recruitment of PLZF is amember of the BTB/POZ–ZF family of multiprotein co-repressor complexes. PML and PML– transcriptional repressors, which also includes BCL6, RARa have previously been shown to associate with the LRF, Kaiso and FAZF (Kelly and Daniel, 2006). These retinoblastoma (Rb) tumour suppressor protein in its proteins are characterized by the presence of a conserved hypophosphorylated state. Here, we demonstrate that N-terminal poxvirus and zinc-finger (POZ) domain that PLZF also interacts with Rb in vitro and in vivo. The mediates protein–protein interactions and transcrip- interaction between PLZF and Rb is mediated through the tional repression, as well as C-terminal Kru¨ ppel-type Rb pocket and the region of PLZF that lies between its zinc-fingers that define sequence-specific DNA binding. transcriptional repression (poxvirus and zinc-finger, The t(11;17) translocation is the second most common POZ) and DNA-binding (zinc-finger) domains. In addi- involving the RARa gene, with the majority of APL tion, Rb can simultaneously interact with PLZF and the cases possessing the PML–RARa associated t(15;17). -inducing , suggesting Several other very rare translocations involving that these proteins can exist in the same multiprotein RARa have also been discovered (Zelent et al., 2001). complex. In contrast to the interaction of Rb with PML or Although both PLZF–RARa and PML–RARa E2F1, the PLZF–Rb interaction is not dependent on fusion proteins constitutively repress RARa target hypophosphorylation of Rb. These data are supported by genes, only PML–RARa associated APL responds immunoprecipitation analysis, which indicates remarkably well to treatment with all-trans-retinoic acid that PLZF associates with the region of CDC6, (ATRA) and has served as a paradigm for differentia- a known /Rb target gene. Co-expression of PLZF tion therapy (Warrell, 1997). The lack of ATRA and Rb results in enhancement of transcriptional repression responsiveness in PLZF–RARa associated APL com- of PLZF and E2F/Rb target genes, indicating functional pared to PML–RARa is due to distinct interactions of co-operation between the two proteins. Both PLZF and Rb the fusion oncoprotein with deacetylase have been shown to function in stem cells and taken (HDAC)-containing co-repressor complexes, which are together these data suggest that interactions between mediated via the PLZF moiety (Hong et al., 1997; PLZF and Rb could be important in biology. Grignani et al., 1998; Guidez et al., 1998; He et al., 1998; Oncogene (2008) 27, 5260–5266; doi:10.1038/onc.2008.159; Lin et al., 1998). published online 26 May 2008 Mice lacking PLZF (also known as ZNF145) exhibit defects in patterning of the limb and axial skeleton Keywords: PLZF; Rb; E2F1 (Barna et al., 2000), although a hematopoietic pheno- type, if any exists, has not yet been described. However, studies of PLZF expression and function in cultured Correspondence: Dr A Zelent, Section of Haemato-Oncology, murine and human hematopoietic cells suggest that it Institute of Cancer Research, 15 Cotswold Road, Sutton SM2 inhibits differentiation (Shaknovich et al., 1998) and 5NG, UK. may play a role in maintenance of the hematopoietic E-mail: [email protected] stem cell pool (Reid et al., 1995; Licht et al., 1996). 8These authors contributed equally to this work and should be Moreover, anumber of studies haveindicatedthatcell- considered joint first authors. Received 28 January 2008; accepted 19 March 2008; published online cycle regulators (Yeyati et al., 1999; McConnell et al., 26 May 2008 2003) and Hox genes are major targets of this PLZF interactions with K Petrie et al 5261 transcription factor (Barna et al., 2000, 2002; Ivins et al., role as a transcriptional repressor, Rb assembles multi- 2003). protein complexes containing the SWI/SNF The retinoblastoma (Rb) tumour suppressor protein remodelling machinery and epigenetic modifiers such as connects the cell-cycle and transcriptional control HDACs, the SUV39H1 lysine and the of genes (Giacinti and Giordano, 2006). Thus, it plays a DNMT1 DNA methyltransferase (Macaluso et al., critical role in the inhibition of cellular proliferation, a 2006). Rb has also recently been shown to recruit the fact underscored by its inactivation in many human arginine methyltransferase PRMT2, which repressed tumours. Major targets of Rb are genes required for E2F1 transcriptional activity in a Rb-dependent manner cell-cycle progression that are under the control of the (Yoshimoto et al., 2006). E2F/DP family of transcription factors (Giacinti and Given the documented interaction of PML and PML– Giordano, 2006). In addition to E2F family members, RARa with Rb (Alcalay et al., 1998; Labbaye et al., more than 100 proteins have been found to associate 1999; Khan et al., 2001), we sought to investigate with Rb either directly or as part of a complex, and whether PLZF and PLZF–RARa also associated many of these interacting proteins also have transcrip- with this transcriptional repressor. Pull-down assays tional regulatory roles (Morris and Dyson, 2001). In its using bacterially expressed GST–Rb or GST–PLZF and

Figure 1 In vitro mapping of the interaction domain between retinoblastoma (Rb) and promyelocytic leukemia zinc-finger (PLZF). (a, left panel) Rb was expressed in bacteria as a GST fusion and tested in pull-down assays for in vitro interaction with 35S-methionine (35S) labelled E2F1, PML or PLZF as indicated. (a, right panel) PLZF was expressed in bacteria as a GST fusion and tested in pull-down assays for in vitro interaction with 35S–Rb. (b) GST fusions of different regions of Rb (as indicated) were used in pull-down assays for in vitro interaction with 35S–PLZF. (c) Different regions of PLZF (as indicated) were in vitro translated and 35S-methionine labelled. These test proteins were then used in pull-down assays with GST–Rb. Pull-downs with GST protein alone are shown as negative controls. Inputs correspond to 10% of 35S-methionine labelled protein as indicated. All GST fusion proteins were prepared using standard procedures (Dong et al., 1996). 35S-methionine-labelled proteins were synthesized in vitro using acoupled reticulocyte lysate system (TNT, Promega). In vitro interaction pull-down assays were performed as previously described (Guidez et al., 1998). Mammalian cDNA expression vectors for PML, PLZF, 50PLZF (encoding amino acids 1–455 of PLZF, but lacking the last seven C-terminal zinc-fingers), poxvirus and zinc-finger (POZ) domain (encoding the N-terminal amino acids 1–120 of PLZF) and the GST– PLZF plasmid have been previously described (Chen et al., 1994; Dong et al., 1996; Guidez et al., 1998). Expression vectors for E2F1 (Hsieh et al., 1997), Rb (Chew et al., 1998) and GST–Rb (full-length and deletions) (Zarkowska and Mittnacht, 1997) have also been previously described.

Oncogene PLZF interactions with retinoblastoma protein K Petrie et al 5262

Figure 2 In vitro analysis of interactions between retinoblastoma (Rb) and promyelocytic leukemia zinc-finger (PLZF). Adenovirus E1aprotein but not E2F1 compete with PLZF for binding to Rb. ( a) The interaction between GST–Rb and 35S–PLZF was competed with adenovirus E1a, a peptide containing the LxCxE motif, and a control peptide consisting of the same amino acids as the LxCxE peptide but in arandomorder (SCR). ( b) Competition assays were performed on the interaction between GST–Rb and 35S–E2F1 with increasing amounts of E1a (left panel) or 35S–PLZF (right panel). Amounts of in vitro translated protein or peptide used to compete interactions are as indicated. Hypophosphorylation of Rb is not required for interaction with PLZF.(c) Unphosphorylated GST–Rb and phosphorylated GST–RbP proteins were used to pull down 35S-methionine labelled PLZF or E2F1 as indicated. GST–Rb fusion proteins were phosphorylated in vitro with a combination of cyclinD–cdk4 and cyclinE–cdk2 as described (Zarkowska and Mittnacht, 1997). Pull-downs with GST protein alone are shown as negative controls. Inputs correspond to 10% of 35S-methionine labelled protein as indicated. Experimental procedures and plasmids used are as described in Figure 1, and the expression vector for E1a has been previously described (Yavuzer et al., 1995).

35S-methionine in vitro translated test proteins showed although multiple contacts between PLZF and the Rb that PLZF associates with GST–Rb (Figure 1a left pocket cannot be ruled out. panel, lane 6) and vice versa (Figure 1a right panel, lane Oncoproteins from DNA tumour viruses such as 3). Moreover, at least in this context, the strength of adenovirus E1a contain an LxCxE sequence, which they interaction between PLZF and Rb was greater than that use to bind Rb and inhibit its function (Felsani et al., of PML and Rb (Figure 1a left panel, lane 4). The 2006). Cellular proteins such as HDAC1 and HDAC2 interaction between Rb and PLZF involves the Rb also contain an LxCxE-like sequence, which mediates pocket (Figure 1b left panel, lane 5 and 1b right panel, their interaction with Rb (Morris and Dyson, 2001). The lane 4) and a minimal region of PLZF located between in vitro interaction between Rb and PLZF was competed amino acids 115–455 (Figure 1c). This region encom- by E1a (Figure 2a, lanes 3 and 4), but not a peptide passes the acidic and proline-rich motifs and the first containing the LxCxE motif (Figure 2a, lanes 5 and 6). two zinc-fingers but lies outside of the POZ domain and Interestingly, although E1a displaces Rb from E2F1 does not contain the last seven C-terminal zinc-fingers, (Figure 2b left panel, lanes 3 and 4), titration with

Oncogene PLZF interactions with retinoblastoma protein K Petrie et al 5263

Figure 3 In vivo analysis of interactions between retinoblastoma (Rb) and promyelocytic leukemia zinc-finger (PLZF). Rb and PLZF co-immunoprecipitate in vivo.(a) Rb and PLZF were transiently transfected (GenePorter, Genlantis, San Diego, CA, USA) into Rb- null C33A cervical carcinoma cells (also lacking PLZF expression) and co-immunoprecipitated (Catch and Release; Millipore, Billerica, MA, USA) using rabbit polyclonal anti-PLZF (H300, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-Rb (C-15, Santa Cruz Biotechnology) antibodies. C33A cells are human papillomavirus negative (HPV–) and do not express the E7 oncoprotein, which could interfere with the properties of pRb (Crook et al., 1991). Rabbit immunoglobulin G (IgG) was used as a negative control. Samples containing co-immunoprecipitated Rb and PLZF were resolved on a 10% SDS polyacrylamide gel and detected by immunoblotting as previously described (Petrie et al., 2003) with mouse monoclonal anti-PLZF (D-9, Santa Cruz Biotechnology) and anti-Rb (G3–245, BD Biosciences, San Jose, CA, USA) antibodies as indicated. Rb and PLZF co-localize. (b) C33A cells were transiently transfected (Lipofectamine 2000; Qiagen, Valencia, CA, USA) with PLZF and Rb (Chen et al., 1994; Chew et al., 1998). After fixation with 4% paraformaldehyde, cells were stained with To-pro-3 iodide (Invitrogen, Carlsbad, CA, USA, stained blue) and rabbit polyclonal anti-PLZF (H300; Santa Cruz Biotechnology, shown in green) and mouse monoclonal anti-Rb (G3–245; BD Biosciences; shown in red) antibodies. Endogenous Rb and PLZF co-immunoprecipitate from KG-1 cells. (c) Endogenous Rb and PLZF were co-immunoprecipitated from KG-1 cells and blotted as described in (a). Inputs correspond to 10% of the cell lysate volume used for immuniprecipitation. Rabbit IgG was used as a negative control. PLZF is recruited to E2F/Rb target genes. (d) KG-1 cells were subjected to chromatin immunoprecipitation analysis as previously described (Guidez et al., 2005). Soluble chromatin preparations were immunoprecipitated with mouse monoclonal anti-PLZF antibody (Koken et al., 1997) and analysed by semiquantitative PCR. The following oligonucleotide primers were used: CDC6 Fwd 50-GCAGTGAGAAGGGGAACCACA-30; CDC6 Rev 50-AACAAACTGCACAGCGGCAGC-30; CCNA2 Fwd 50-CGTCGGGCCCTAAATCCTACC-30 and CCNA2 Rev 50- GAGGAGGTTGCGAAAGGCGCA-30. The number of cycles was determined empirically to give results that fall within the linear range of this particular PCR assay. Anti-GAL4 (DBD) antibody (RK5C1; Santa Cruz Biotechnology) was used as a negative control. Aliquots of soluble chromatin were also analysed before immunoprecipitation (Input). KG-1 cells were maintained in Iscove’s modified Dulbecco’s medium with 10% fetal calf serum and 1.5 g/l sodium bicarbonate and C33A cells in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum; both media were supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin. increasing amounts of PLZF had no effect on E2F1 of Rb by cyclinE/cdk2 and cyclinD/ binding to Rb, and a GST–Rb fusion was able to pull cdk4 slightly reduced, but did not abolish interactions down both PLZF and E2F1 as a complex (Figure 2b with PLZF (Figure 2c, lane 3). right panel, lanes 4–6). Given that the association of Rb Having characterized the in vitro interaction between with E2F proteins is governed by the phosphorylation Rb and PLZF, we extended this study including in vivo status of Rb, we tested whether this relationship also experiments. Consistent with the in vitro data, Rb and existed for the interaction between Rb and PLZF. We PLZF co-immunoprecipitated (Figure 3a) and co- found that in contrast to E2F1 (Figure 2c, lane 6), localized (Figure 3b) when expressed in Rb null C33A

Oncogene PLZF interactions with retinoblastoma protein K Petrie et al 5264 Lastly, we analysed the consequences of the PLZF– Rb interaction for transcriptional regulation and found that co-expression with PLZF enhanced the ability of Rb to repress E2F1/DP1 mediated activation of a luciferase reporter containing an E2F1 binding site corresponding to the one found in the c- gene (Figure 4a) (Mudryj et al., 1990). Also, co-expression with Rb increased PLZF directed repression of the Hoxb2 r3/r5 enhancer, which does not contain an E2F binding site (Figure 4b). As both PLZF and Rb effectively repress transcription from their respective reporters when transfected alone, enhanced repression arising from their co-expression may represent a reciprocal fine-tuning of the transcriptionally repressive activities of PLZF and Rb/E2F complexes. Of note, the Rb interaction region is retained in the PLZF–RARa oncoprotein (data not shown), suggesting that recruitment of Rb-containing complexes to RARa target genes and/or delocalization of Rb may be a common feature in APL with both t(15;17) and t(11;17) translocations. Within the hematopoietic system, PLZF is expressed with a punctate nuclear distribution in CD34 þ progenitors, but not differentiated cells (Reid et al., 1995; Dai et al., 2002), and knockout mice for PLZF and its closely related homologue FAZF (or PLZF2) both reportedly show a disruption of the balance between quiescent and cycling CD34 þ cells (Piazza Figure 4 Co-expression of retinoblastoma (Rb) and promyelocy- et al., 2004). Moreover, PLZF null mice (and also a tic leukemia zinc-finger (PLZF) enhances transcriptional repression model harbouring a PLZF mutation) display an age- of PLZF and E2F1/Rb target genes. C33A cells were transiently dependent loss of self-renewal in spermatagonal stem transfected (Lipofectamine 2000, Qiagen) with luciferase reporters cells, likely due to uncontrolled exit from quiescence and containing (a) three copies of an E2F consensus binding sequence along with mammalian expression plasmids encoding PLZF, erosion of the stem cell pool (Buaas et al., 2004; Costoya phosphorylation-deficient Rb protein (RbNPC) or E2F1 and DP1 et al., 2004). These data support the notion that PLZF as indicated (Chen et al., 1994; Chew et al., 1998) or (b) aPLZF– serves as a growth suppressor in stem cells and are responsive Hoxb2 r3/r5 enhancer luciferase reporter (Ivins et al., consistent with its recruitment to the promoter of the 2003) along with PLZF and RbNPC as indicated. Cells were harvested 36 h after transfection and luciferase assays performed CDC6 gene, which is expressed selectively in proliferat- as previously described (Petrie et al., 2003). Error bars represent ing but not quiescent cells (Yan et al., 1998). standard deviations from three independent experiments each Although Rb is involved in multiple aspects of stem performed in triplicate. Tests for statistical significance were cell biology (Galderisi et al., 2006), its contribution in performed using the Mann–Whitney test (Prism 5, GraphPad early hematopoiesis remains unclear. For example, it Software Inc.) and asterisks (***) indicate a P-value of 0.0001. o has been reported that Rb is dispensable for self-renewal and multilineage differentiation of adult hematopoietic stem cells (Walkley and Orkin, 2006). However, Rb has also been shown to determine the choice between cervical carcinoma cells from co-transfected expression neutrophilic and monocytic commitment of CD34 þ vectors. The proteins were also co-immunoprecipitated progenitors (Bergh et al., 1999) and play a role in the from CD34 þ KG-1 cells (Figure 3c), in which they are biology of early erythroid progenitor cells, where endogenously expressed, indicating that the PLZF/Rb erythropoietin induces hyperphosphorylation of Rb complex exists under physiological conditions in vivo. (Sivertsen et al., 2006). Taken together, these data Given the in vivo association of Rb and PLZF, we provide a plausible scenario in which interactions examined whether PLZF could be recruited to Rb target between PLZF and Rb play a role in stem/progenitor gene promoters. As expected, PLZF was found to cell biology (such as lineage commitment, differentiation associate with the promoter of CCNA2, which encodes and/or self renewal, for example). Moreover, recent (Figure 3d, left panel). Although CCNA2 is a results indicate that, apart from their generic transcrip- known PLZF target gene whose promoter contains both tional activity through canonical E2F sites, the Rb- PLZF and E2F binding sites (Yeyati et al., 1999), PLZF binding E2Fs can act in member-specific and cell was also recruited to the promoter of CDC6 (Figure 3d, lineage-selective ways. This is facilitated through selec- right panel), which does not contain a PLZF consensus tive partnering with other transcription factors, which in binding site but is a known E2F/Rb target gene (Yan turn bring promoters with combinatorial binding sites et al., 1998). under E2F and, hence, Rb control (Schlisio et al., 2002;

Oncogene PLZF interactions with retinoblastoma protein K Petrie et al 5265 Giangrande et al., 2004; Korenjak et al., 2004). Thus, Acknowledgements the biological significance of the presence of PLZF in a complex with E2F proteins and Rb merits further We acknowledge support from the Leukaemia Research Fund investigation. It is interesting to speculate that PLZF of Great Britain (AZ and FG), the Samuel Waxman Cancer could modulate the activities of E2F proteins in Research Foundation, the Kay Kendall Leukaemia Fund hematopoietic stem cells and/or progenitors through (KP), Cancer Research UK (SM) and the National Institute of Health (CA59936, AZ, JL and SW). We also thank Drs Angela combinatorial DNA binding or serve to ‘lock down’ Barrett and Manuel Boix-Chornet for critical reading of the repression mediated through canonical E2F sites. article.

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