ZNF652, A Novel Zinc Finger Protein, Interacts with the Putative Breast Tumor Suppressor CBFA2T3 to Repress Transcription Raman Kumar,1 Jantina Manning,1 Hayley E. Spendlove,3 Gabriel Kremmidiotis,4 Ross McKirdy,1 Jaclyn Lee,1 David N. Millband,1 Kelly M. Cheney,1 Martha R. Stampfer,5 Prem P. Dwivedi,2 Howard A. Morris,2 and David F. Callen1 1Breast Cancer Genetics Group, Dame Roma Mitchell Cancer Research Laboratories, Department of Medicine, University of Adelaide and Hanson Institute; 2Endocrine Bone Laboratory, Hanson Institute, Adelaide, South Australia, Australia; 3Department of Laboratory Genetics, Women’s and Children’s Hospital, North Adelaide, South Australia, Australia; 4Bionomics, Ltd., Thebarton, South Australia, Australia; and 5Lawrence Berkeley National Laboratory, Berkeley, California Abstract gene effector zinc finger proteins may specifically The transcriptional repressor CBFA2T3is a putative interact with one or more of the ETO proteins to generate breast tumor suppressor. To define the role of CBFA2T3, a defined range of transcriptional repressor complexes. we used a segment of this protein as bait in a yeast (Mol Cancer Res 2006;4(9):655–65) two-hybrid screen and identified a novel uncharacterized protein, ZNF652. In general, primary tumors and cancer Introduction cell lines showed lower expression of ZNF652 than Tumor growth, characterized by unchecked cell division, normal tissues. Together with the location of this gene results from both the overexpression of growth-promoting on the long arm of chromosome 17q, a region of frequent oncogenes and the reduced expression of growth-inhibiting loss of heterozygosity in cancer, these results suggest tumor suppressor genes. These genes often encode proteins that In silico a possible role of ZNF652 in tumorigenesis. are components of coactivator and corepressor complexes analysis of this protein revealed that it contains multiple involved in the regulation of genes critical for cell division. classic zinc finger domains that are predicted to bind Identification and functional characterization of oncogenes and DNA. Coimmunoprecipitation assays showed that tumor-suppressor genes continues to be the key to an ZNF652 strongly interacts with CBFA2T3and this understanding of the molecular mechanisms of cancer. Detailed interaction occurs through the COOH-terminal 109 genetic and cytogenetic analyses of breast tumors and breast amino acids of ZNF652. In contrast, there was a weak cancer cell lines have shown that there is frequent loss of interaction of ZNF652 with CBFA2T1 and CBFA2T2, the heterozygosity at 16q24.3 (1, 2), suggesting that this band is the other two members of this ETO family. Transcriptional likely location of one or more tumor-suppressor genes. reporter assays further confirmed the strength Subsequent expression studies of genes located at 16q24.3 and selectivity of the ZNF652-CBFA2T3interaction. identified CBFA2T3 (also called MTG16) as a potential breast The transcriptional repression of growth factor tumor-suppressor gene (3). Molecular and cell biology assays independent-1 (GFI-1), a previously characterized ETO showed CBFA2T3 to have characteristics consistent with a effector zinc finger protein, was shown to be enhanced tumor suppressor because expression was significantly reduced by CBFA2T1, but to a lesser extent by CBFA2T2 and in primary breast tumors and in the breast tumor cell lines CBFA2T3. We therefore suggest that each of the various MDA-MB-468 and MDA-MB-231(4, 5). In addition, ectopic expression of CBFA2T3 in breast cancer cell lines inhibited both the ability to form colonies on plastic and anchorage- independent growth on soft agar (4). CBFA2T3, together with the homologues CBFA2T1 (MTG8, Received 11/27/05; revised 6/22/06; accepted 7/5/06. ETO) and CBFA2T2 (MTGR1), form the small ‘‘ETO’’ family Grant support: National Health and Medical Research Council of Australia grant (6), the terminology referring to the Eight-Twenty-One 207703, and Susan G. Komen Breast Cancer Foundation (D.F. Callen); U.S. Department of Energy under contract no. DE-AC03-76SF00098 (M.R. Stampfer); translocation associated with CBFA2T1. The ETO proteins Cancer Council of South Australia (H.A. Morris); Faculty of Health Sciences and show the highest homology within four NHR domains, Department of Medicine, University of Adelaide; and the Hanson Institute, originally identified in the Drosophila melanogaster protein Institute of Medical and Veterinary Science. The costs of publication of this article were defrayed in part by the payment of Nervy (7). The ETO family members function as transcriptional page charges. This article must therefore be hereby marked advertisement in repressors by forming complexes with the transcriptional accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: David F. Callen, Breast Cancer Genetics Group, Dame corepressors N-CoR, SMRT, mSin3A, and recruit histone Roma Mitchell Cancer Research Laboratories, Hanson Institute, Institute of Medical deacetylases (HDAC). There are some differences between and Veterinary Science, Frome Road, Adelaide, SA 5000, Australia. Phone: 61-8- the ETO proteins in these associations, e.g., CBFA2T1and 8222-23145; Fax: 61-8-8222-3217. E-mail: [email protected]. Copyright D 2006 American Association for Cancer Research. CBFA2T2, but not CBFA2T3, which associate with mSin3A doi:10.1158/1541-7786.MCR-05-0249 (8, 9). The greater differences occur with HDAC interactions; Mol Cancer Res 2006;4(9). September 2006 655 Downloaded from mcr.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. 656 Kumar et al. CBFA2T1interacts with HDAC1to HDAC3, CBFA2T2 CBFA2T3 in a similar screen where atrophin-176-1438 was used interacts only with HDAC3, whereas CBFA2T3 associates as a bait (24). In addition to atrophin-1, a cDNA encoding the with HDAC1, HDAC2, HDAC3, HDAC6, and HDAC8 (8, 9). 61COOH-terminal amino acids of a previously uncharacterized The ETO proteins possess two atypical conserved zinc-finger protein ZNF652 was identified. motifs (-C-x-x-C-7x-C-x-x-C- and -C-x-x-x-C-7x-H-x-x-x-C-) The 5,428 bp ZNF652 cDNA sequence (National Center that are involved in interaction with proteins but not DNA (10, for Biotechnology Information accession no. NM_014897) 11). The gene specificity of ETO-based repressor complexes is located at chromosome band 17q21.32 encodes a predicted determined by the recruitment of proteins that can directly bind 606-amino-acid protein with the presence of seven zinc finger to the promoters of target genes. The DNA-binding partners of motifs located in the central region of the protein. These the ETO proteins include C2H2 zinc finger transcription factors classic C2H2 zinc finger motifs, comprising two conserved BCL6 (12) and PLZF (13), which interact with CBFA2T1, and cysteine and histidine residues, conform to the consensus growth factor independent-1(GFI-1),which interacts with CX2CX12HX3H sequence and three of the zinc fingers are either CBFA2T1or CBFA2T3 (14).Therefore, a major joined by part or all of a consensus TGEKP linker sequence. mechanism whereby the ETO proteins impart their normal These motifs are common to zinc finger proteins involved in function is by transcriptional repression of diverse classes of DNA binding (25, 26), suggesting that ZNF652 is most likely genes through their interaction with different DNA-binding zinc a DNA-binding protein. finger proteins. In addition to interactions with zinc finger– binding proteins, CBFA2T1and CBFA2T2 have been shown to form complexes with the E-box–binding protein HEB, a basic- Expression of ZNF652 helix-loop-helix transcription factor, and these complexes The variation of ZNF652 expression in different normal mediate the roles of HEB in hematopoiesis (15). tissues and the corresponding matched tumors was determined ¶ CBFA2T1 is the best-characterized member of the ETO by probing a cDNA-profiling array with the 5 738 bp of the family due to its involvement in the t(8;21) translocation with ZNF652 open reading frame (Fig. 1A and B). The hybridization RUNX1 (previously called AML1) that generates a RUNX1- signals were normalized against the expression of the CBFA2T1 gene fusion, the major cause of acute myeloid housekeeping gene ubiquitin. There was a large variation in leukemia (16-18). It has been suggested that the normal the level of ZNF652 expression among different nonmalignant function of RUNX1 is to regulate genes that are critical for human tissues, with the highest average expression in the hematopoiesis (19). The RUNX1-CBFA2T1 fusion product normal breast, vulva, prostate, and pancreas. Compared with disrupts this regulation, thereby promoting progression to normal tissues, cancers of breast (one sample with no change, leukemia (20, 21). In patients with therapy-related acute one with overexpression, and the remaining eight showed 40% myeloid leukemia, RUNX1 can also be involved in a down-regulation), vulva (average 69% down-regulation), translocation, t(16;21)(q24;q22), with CBFA2T3 (22). Targeted prostate (three of four samples showed 34% down-regulation), disruption of Cbfa2t1 in mouse reveals a critical role in gut and pancreas (53% down-regulation) showed reduced levels of development (23), whereas disruption of the mouse Cbfa2t2 ZNF652 expression. When averaged over all samples, the shows a role in maintenance of the secretory cell lineage in the tumors showed a 34% down-regulation in ZNF652 expression small intestine (9). Because the ETO