Research Article ZNF703 in luminal B breast cancer utilized a pEGFP-C1 vector that produces only a GFP protein (Fig 2A). Cellular fractionation showed that GFP-ZNF703 localized both to the nucleus and the cytoplasm (Supplementary Fig 4). Utilizing GFP expression, we monitored ZNF703 localization in the MCF7 GFP-ZNF703 cell line. GFP-ZNF703 was mainly localized in the nuclear matrix in subcellular dot- like structures (Fig 2A). To avoid potential artefactual subcellular location of ZNF703 due to the fusion protein product with GFP, we conﬁrmed that endogenous ZNF703 is present in the same structures (Supplementary Fig 5A). The size and the morphology of these nuclear structures are reminiscent of promyelocytic leukaemia (PML), oncogenic domains (PODs) and RNA splicing bodies (SBs), which contain different sets of proteins (Dyck et al, 1994; Spector et al, 1991). To examine the potential overlap of ZNF703 localization with these nuclear structures, MCF7 GFP-ZNF703 cells were subjected to immuno- ﬂuorescence staining with an antibody against PML and to DNase 1 or RNase A treatment. The staining pattern of PML did not overlap with that of ZNF703 (Supplementary Fig 5B). RNase A treatment did not affect ZNF703 nuclear structures (Fig 2B) suggesting that ZNF703 bodies are distinct from both PODs and SBs. In contrast, DNase 1 treatment completely disorganized ZNF703 nuclear localization (Fig 2B). Although ZNF703 protein has only one zinc ﬁnger motif (Nakamura et al, 2004), proscribing a direct DNA-binding, our results suggest that ZNF703 is part of nuclear structures that directly interact with DNA. ZNF703 is part of a nuclear repressor complex We next sought to identify proteins that associate with ZNF703 in the nucleus. SDS-polyacrylamide gel electrophoresis (SDS- PAGE) analysis revealed three potential ZNF703-interacting proteins (Fig 2C). Their identity was determined by mass spectrometry (MS). All peptides identiﬁed by MS analysis corresponded to three different proteins: DCAF7, HSP60 and PHB2 (Prohibitin 2) (Supplementary Table 7). Co-immunopre- cipitation experiments conﬁrmed the interaction of ZNF703 with HSP60 and DCAF7 in HCC1500 and MDA-MB-134 (endogenous conditions) and in MCF7 GFP-ZNF703 (Fig 2C and Supplemen- tary Fig 6). Moreover, DCAF7 and PHB2 co-localized with ZNF703 in the dot-like nuclear structures (Fig 2D). Despite the interaction between ZNF703 and HSP60, these two proteins did not co-localize in the nuclear matrix. HSP60 was only present in the cytoplasm and may interact with the cytoplasmic form of ZNF703 (Supplementary Fig 5C) and the ZNF703-HSP60 protein complex may have a distinct function from that of the ZNF703- DCAF7-PHB2 complex. PHB2 (also called REA for repressor of ER activity) is a nuclear co-repressor of ER activity in mammary gland development (Mussi et al, 2006; Umanskaya et al, 2007). Prohibitin mediates transcriptional repression through recruit- ment of additional nuclear co-repressors (NCOR1/NCOR2) and HDACs (Kurtev et al, 2004; Wang et al, 2002). We stained MCF7 GFP-ZNF703 cells with anti-NCOR2 antibody. The staining pattern of NCOR2 overlapped completely with the ZNF703 nuclear structures detected by GFP ﬂuorescence (Fig 2D). Interestingly, when we questioned the multi-experiment matrix (MEM) web interface (http://biit.cs.ut.ee/mem/) for ZNF703 Figure 2. Identification and characterization of ZNF703 nuclear domain. A. Immunoblotting and immunofluorescence analyses of MCF7 GFP-ZNF703 and MCF7 GFP breast cancer cell lines. Immunoblotting with anti-ZNF703 and anti-GFP antibodies identifies GFP-ZNF703 fusion protein with a molecular weight of 83 kDa. a-tubulin protein expression is shown as a control. Immunofluorescence reveals nuclear dot-like structures in MCF7 GFP-ZNF703 cells, whereas GFP is detected in all subcellular structures in MCF7 GFP cells. B. GFP-ZNF703 interacts with DNA in the nucleus. MCF7 GFP-ZNF703 cells non-treated (control) or treated with RNAse A or DNAse I were processed for immunofluorescence with anti-GFP antibody. The GFP- ZNF703 signal in the nucleus is perturbed only when cells are treated with DNAse. C. MCF7 cells expressing GFP and GFP-ZNF703 proteins were subjected to immunoprecipitation with anti-GFP antibody (IP GFP) or beads only (No Ab), separated by SDS-PAGE, and stained with colloidal Coomassie blue. Three co-precipitated proteins were identified by mass spectrometry: HSP60 (accession number, NP 955472); DCAF7 (NP 005819) and PHB2 (NP 001138303), respectively. The lane on the left contains molecular weight standards (MW). The interaction between ZNF703 and HSP60 or DCAF7 was confirmed by ZNF703-immunoprecipitation, followed by Western blot analysis using anti-HSP60 or anti-DCAF7 antibody. Input represents whole-cell extract from HCC1500 and MDA-MB-134. D. MCF7 cells overexpressing GFP-ZNF703 were fixed and immunostained with anti-DCAF7, anti-PHB2 or anti-SMRT/NCOR2 antibody. GFP-ZNF703 (green) co-localizes in the nucleus with DCAF7, PHB2 and SMRT/NCOR2 (red). Nuclei are counterstained with Hoechst (blue). 156 2011 EMBO Molecular Medicine EMBO Mol Med 3, 153–166 www.embomolmed.org