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Retinoblastoma Protein and the Leukemia-Associated PLZF Transcription Factor Interact to Repress Target Gene Promoters

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Retinoblastoma Protein and the Leukemia-Associated PLZF Transcription Factor Interact to Repress Target Gene Promoters Oncogene (2008) 27, 5260–5266 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $30.00 www.nature.com/onc SHORT COMMUNICATION Retinoblastoma protein and the leukemia-associated PLZF transcription factor interact to repress target gene 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 Cancer 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 receptor-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 genes lead to expression of oncogenic PLZF– translocation with the retinoic acid receptor-a (RARa) RARa or PML–RARa fusion proteins, 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). E2F1 S phase-inducing transcription factor, 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 chromatin immunoprecipitation analysis, which indicates remarkably well to treatment with all-trans-retinoic acid that PLZF associates with the promoter region of CDC6, (ATRA) and has served as a paradigm for differentia- a known E2F/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 histone 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 stem cell 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 retinoblastoma protein 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 nucleosome The retinoblastoma (Rb) tumour suppressor protein remodelling machinery and epigenetic modifiers such as connects the cell-cycle clock and transcriptional control HDACs, the SUV39H1 lysine methyltransferase 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.
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