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Molecular Cancer Research 4:655-665 (2006)
© 2006 American Association for Cancer Research

Signaling and Regulation

ZNF652, A Novel Zinc Finger Protein, Interacts with the Putative Breast Tumor Suppressor CBFA2T3 to Repress Transcription

Raman Kumar1, Jantina Manning1, Hayley E. Spendlove3, Gabriel Kremmidiotis4, Ross McKirdy1, Jaclyn Lee1, David N. Millband1, Kelly M. Cheney1, Martha R. Stampfer5, Prem P. Dwivedi2, Howard A. Morris2 and David F. Callen1

1 Breast Cancer Genetics Group, Dame Roma Mitchell Cancer Research Laboratories, Department of Medicine, University of Adelaide and Hanson Institute; 2 Endocrine Bone Laboratory, Hanson Institute, Adelaide, South Australia, Australia; 3 Department of Laboratory Genetics, Women's and Children's Hospital, North Adelaide, South Australia, Australia; 4 Bionomics, Ltd., Thebarton, South Australia, Australia; and 5 Lawrence Berkeley National Laboratory, Berkeley, California

Requests for reprints: David F. Callen, Breast Cancer Genetics Group, Dame Roma Mitchell Cancer Research Laboratories, Hanson Institute, Institute of Medical and Veterinary Science, Frome Road, Adelaide, SA 5000, Australia. Phone: 61-8-8222-23145; Fax: 61-8-8222-3217. E-mail: david.callen{at}imvs.sa.gov.au.


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The transcriptional repressor CBFA2T3 is a putative breast tumor suppressor. To define the role of CBFA2T3, we used a segment of this protein as bait in a yeast two-hybrid screen and identified a novel uncharacterized protein, ZNF652. In general, primary tumors and cancer cell lines showed lower expression of ZNF652 than normal tissues. Together with the location of this gene on the long arm of chromosome 17q, a region of frequent loss of heterozygosity in cancer, these results suggest a possible role of ZNF652 in tumorigenesis. In silico analysis of this protein revealed that it contains multiple classic zinc finger domains that are predicted to bind DNA. Coimmunoprecipitation assays showed that ZNF652 strongly interacts with CBFA2T3 and this interaction occurs through the COOH-terminal 109 amino acids of ZNF652. In contrast, there was a weak interaction of ZNF652 with CBFA2T1 and CBFA2T2, the other two members of this ETO family. Transcriptional reporter assays further confirmed the strength and selectivity of the ZNF652-CBFA2T3 interaction. The transcriptional repression of growth factor independent-1 (GFI-1), a previously characterized ETO effector zinc finger protein, was shown to be enhanced by CBFA2T1, but to a lesser extent by CBFA2T2 and CBFA2T3. We therefore suggest that each of the various gene effector zinc finger proteins may specifically interact with one or more of the ETO proteins to generate a defined range of transcriptional repressor complexes. (Mol Cancer Res 2006;4(9):655–65)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
Tumor growth, characterized by unchecked cell division, results from both the overexpression of growth-promoting oncogenes and the reduced expression of growth-inhibiting tumor suppressor genes. These genes often encode proteins that are components of coactivator and corepressor complexes involved in the regulation of genes critical for cell division. Identification and functional characterization of oncogenes and tumor-suppressor genes continues to be the key to an understanding of the molecular mechanisms of cancer. Detailed genetic and cytogenetic analyses of breast tumors and breast cancer cell lines have shown that there is frequent loss of heterozygosity at 16q24.3 (1, 2), suggesting that this band is the likely location of one or more tumor-suppressor genes. Subsequent expression studies of genes located at 16q24.3 identified CBFA2T3 (also called MTG16) as a potential breast tumor-suppressor gene (3). Molecular and cell biology assays showed CBFA2T3 to have characteristics consistent with a tumor suppressor because expression was significantly reduced in primary breast tumors and in the breast tumor cell lines 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, ETO) and CBFA2T2 (MTGR1), form the small "ETO" family (6), the terminology referring to the Eight-Twenty-One translocation associated with CBFA2T1. The ETO proteins show the highest homology within four NHR domains, originally identified in the Drosophila melanogaster protein Nervy (7). The ETO family members function as transcriptional repressors by forming complexes with the transcriptional corepressors N-CoR, SMRT, mSin3A, and recruit histone deacetylases (HDAC). There are some differences between the ETO proteins in these associations, e.g., CBFA2T1 and CBFA2T2, but not CBFA2T3, which associate with mSin3A (8, 9). The greater differences occur with HDAC interactions; CBFA2T1 interacts with HDAC1 to HDAC3, CBFA2T2 interacts only with HDAC3, whereas CBFA2T3 associates with HDAC1, HDAC2, HDAC3, HDAC6, and HDAC8 (8, 9). The ETO proteins possess two atypical conserved zinc-finger motifs (-C-x-x-C-7x-C-x-x-C- and -C-x-x-x-C-7x-H-x-x-x-C-) that are involved in interaction with proteins but not DNA (10, 11). The gene specificity of ETO-based repressor complexes is determined by the recruitment of proteins that can directly bind to the promoters of target genes. The DNA-binding partners of the ETO proteins include C2H2 zinc finger transcription factors BCL6 (12) and PLZF (13), which interact with CBFA2T1, and growth factor independent-1 (GFI-1), which interacts with either CBFA2T1 or CBFA2T3 (14). Therefore, a major mechanism whereby the ETO proteins impart their normal function is by transcriptional repression of diverse classes of genes through their interaction with different DNA-binding zinc finger proteins. In addition to interactions with zinc finger–binding proteins, CBFA2T1 and CBFA2T2 have been shown to form complexes with the E-box–binding protein HEB, a basic-helix-loop-helix transcription factor, and these complexes mediate the roles of HEB in hematopoiesis (15).

CBFA2T1 is the best-characterized member of the ETO family due to its involvement in the t(8;21) translocation with RUNX1 (previously called AML1) that generates a RUNX1-CBFA2T1 gene fusion, the major cause of acute myeloid leukemia (16-18). It has been suggested that the normal function of RUNX1 is to regulate genes that are critical for hematopoiesis (19). The RUNX1-CBFA2T1 fusion product disrupts this regulation, thereby promoting progression to leukemia (20, 21). In patients with therapy-related acute myeloid leukemia, RUNX1 can also be involved in a translocation, t(16;21)(q24;q22), with CBFA2T3 (22). Targeted disruption of Cbfa2t1 in mouse reveals a critical role in gut development (23), whereas disruption of the mouse Cbfa2t2 shows a role in maintenance of the secretory cell lineage in the small intestine (9). Because the ETO proteins may be functionally redundant, the observed phenotypes of these mice may reflect those tissues that only express a single member of the ETO family.

To further define the role of CBFA2T3 as a tumor suppressor, we used a yeast two-hybrid screen to identify CBFA2T3-interacting proteins from a breast expression library. From this screen, a previously uncharacterized zinc finger protein ZNF652 was identified and shown to be a specific DNA-binding partner of CBFA2T3. We show that ZNF652 has a role in tumorigenesis and that ETO proteins selectively interact with the DNA-binding proteins ZNF652 and GFI-1.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Identification and Analysis of ZNF652
A yeast two-hybrid screen of a breast cDNA library was used to identify novel proteins that were part of the CBFA2T3 repressor complex. As the full-length CBFA2T3 autoactivated the reporter genes in this assay, CBFA2T3142-567 (amino acids 142-567 of the CBFA2T3b open reading frame) was used as bait. A number of cDNA clones encoding different candidate interacting proteins were identified in this screen. Atrophin-1, one of the most frequently represented proteins, validated our screening procedure as it has been reported to interact with CBFA2T3 in a similar screen where atrophin-176-1438 was used as a bait (24). In addition to atrophin-1, a cDNA encoding the 61 COOH-terminal amino acids of a previously uncharacterized protein ZNF652 was identified.

The 5,428 bp ZNF652 cDNA sequence (National Center for Biotechnology Information accession no. NM_014897) located at chromosome band 17q21.32 encodes a predicted 606-amino-acid protein with the presence of seven zinc finger motifs located in the central region of the protein. These classic C2H2 zinc finger motifs, comprising two conserved cysteine and histidine residues, conform to the consensus CX2CX12HX3H sequence and three of the zinc fingers are joined by part or all of a consensus TGEKP linker sequence. These motifs are common to zinc finger proteins involved in DNA binding (25, 26), suggesting that ZNF652 is most likely a DNA-binding protein.

Expression of ZNF652
The variation of ZNF652 expression in different normal tissues and the corresponding matched tumors was determined by probing a cDNA-profiling array with the 5' 738 bp of the ZNF652 open reading frame (Fig. 1A and B ). The hybridization signals were normalized against the expression of the housekeeping gene ubiquitin. There was a large variation in the level of ZNF652 expression among different nonmalignant human tissues, with the highest average expression in the normal breast, vulva, prostate, and pancreas. Compared with normal tissues, cancers of breast (one sample with no change, one with overexpression, and the remaining eight showed 40% down-regulation), vulva (average 69% down-regulation), prostate (three of four samples showed 34% down-regulation), and pancreas (53% down-regulation) showed reduced levels of ZNF652 expression. When averaged over all samples, the tumors showed a 34% down-regulation in ZNF652 expression compared with their matched normal tissues.


Figure 1
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  		FIGURE 1. Expression of ZNF652 is reduced in cancer tissues. A. Cancer Profiling Array II was probed with radiolabeled PCR fragment (nucleotides 1-738) of ZNF652 or a housekeeping ubiquitin cDNA. Levels of ZNF652 hybridization signals from normal and tumor samples of different tissues relative to the respective ubiquitin signals were assayed and plotted. B. Part of the cancer-profiling array showing normal (N) and tumor (T) breast, vulva, and liver samples probed with ZNF652 (ZNF) or ubiquitin (UBI) radiolabeled DNA.

 
An affinity-purified rabbit anti-ZNF652 polyclonal antibody was generated that specifically detected the endogenous ZNF652 protein (Fig. 2A ). This antibody was used for Western blot analysis of protein lysates from finite lifespan human mammary epithelial cells, nonmalignant immortalized mammary epithelial cells, and breast cancer cell lines. A specific band of 85 kDa was detected in all cell lines (Fig. 2B). The level of ZNF652 expression was similar in nonmalignant immortalized and estrogen receptor–positive breast tumor cell lines, but was reduced in three of four estrogen receptor–negative breast tumor cell lines.


Figure 2
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  		FIGURE 2. Affinity-purified rabbit polyclonal anti-ZNF652497-606 antibody specifically detects the ZNF652 protein and ZNF652 levels vary among different breast cancer cell lines. A. Western blots showing that anti-ZNF652497-606 antibody specifically detect the ZNF652 protein. Western blots carrying ZR75-1 nuclear extracts (lanes 1, 3, 5, and 7) and protein lysates from HEK293T cells transfected with pCMV-HA-ZNF652 (lanes 2, 4, 6, and 8) were probed with various primary (shown below each panel) and appropriate HRP-conjugated secondary antibodies. B. Western blot carrying protein from various breast cell lines was probed with anti-ZNF652497-606 antibody. Protein from pCMV-HA-ZNF652–transfected HEK293T cells showed a single band at 85 kDa. ß-actin was used as a loading control.

 
ZNF652 Interacts Specifically with CBFA2T3
To confirm the interaction of the COOH terminus of ZNF652 with CBFA2T3 observed in yeast, coimmunoprecipitation assays were done in HEK293T mammalian cells transiently expressing hemagglutinin (HA)-ZNF652 and myc-CBFA2T3. HA-ZNF652 was clearly detected in complexes immunoprecipitated with anti-myc antibody that binds to myc-CBFA2T3 (Fig. 3A, lane 4 and B, lane 7 ). This interaction was specific to CBFA2T3 as coimmunoprecipitation of the related proteins CBFA2T2 and CBFA2T1 showed only a weak interaction with ZNF652 (Fig. 3A, lanes 5 and 6, respectively). Negative controls confirmed the specificity of the interactions (not shown). Repeated attempts to perform reciprocal coimmunoprecipitations were unsuccessful, possibly due to masking of the HA epitope in the HA-ZNF652 protein.


Figure 3
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  		FIGURE 3. ZNF652 interacts strongly and specifically with CBFA2T3 through its COOH terminus. A. Immunoprecipitation of transfected ZNF652 and ETO proteins in HEK293T cells showing specific CBFA2T3 interaction. Constructs expressing myc-tagged ETO and HA-tagged ZNF652 proteins were used. Proteins were coimmunoprecipitated (IP) with anti-myc antibody and Western blotted (WB) with anti-HA or anti-myc antibodies (lanes 4-6). All proteins were detected at predicted sizes in the input (lanes 1-3). Nonspecific antibody coimmunoprecipitations showed no HA-ZNF652 bands (not shown). B. ZNF652 interacts with CBFA2T3 through its COOH terminus. Bar diagram, full-length HA-ZNF652 and four regions of the HA-ZNF652 used in immunoprecipitations. Proteins were coimmunoprecipitated with anti-myc antibody and Western blotted with anti-HA or anti-myc antibodies. All proteins were detected at predicted sizes in the input (lanes 1-6). Only the HA-ZNF652 proteins carrying the COOH-terminal 109 amino acids coimmunoprecipitated with CBFA2T3 (lanes 7, 9, and 11). Nonspecific antibody coimmunoprecipitations showed no HA-ZNF652 bands (not shown). C. ZNF652 contains a proline- and histidine-rich region. The COOH-terminal 109 amino acids of ZNF652 (beginning at amino acid 498) contains a proline-rich region (blocked sequence) and a histidine-rich region (underlined sequence). M, markers.

 
To map the region of ZNF652 that interacts with CBFA2T3, plasmid constructs expressing various HA-tagged regions of ZNF652 were generated (Fig. 3B, bars on right). Each of these constructs was included in a coimmunoprecipitation assay with CBFA2T3. Only ZNF652, ZNF652243-606, and the COOH-terminal ZNF652498-606 proteins coimmunoprecipitated with CBFA2T3 (Fig. 3B, lanes 7, 9, and 11). Negative controls confirmed the specificity of the interactions (not shown). These results indicate that the COOH-terminal 109-amino-acid region of ZNF652 (amino acids 498-606) interacts with CBFA2T3. This interaction was apparently stabilized in the presence of the zinc finger region because there was a more intense immunoprecipitated band observed with ZNF652243-606 (compare lanes 9 and 11, Fig. 3B). These observations were also consistent with the finding that in the yeast two-hybrid screen, the identified ZNF652 cDNA encoded the COOH-terminal region of the ZNF652 protein. In silico analysis of this COOH terminus did not identify any typical motifs associated with protein binding, although a proline- and histidine-rich region was present (Fig. 3C).

ZNF652 Function Is Dependent on CBFA2T3
The breast cancer cell line MCF-7 (an estrogen receptor–positive breast tumor cell line) expresses comparatively higher levels of CBFA2T3 message, whereas in SKBR3 (an estrogen receptor–negative breast tumor cell line) the levels are very low (ref. 5 and unpublished Western blot data). It was of interest to determine if ectopic expression of ZNF652 in these cell lines resulted in different phenotypic effects because cell lines with low levels of CBFA2T3 (e.g., SKBR3) would be predicted to show a reduced effect of ZNF652 ectopic expression compared with a cell line expressing higher levels of CBFA2T3 (e.g., MCF-7). Accordingly, the effect of retroviral-mediated expression of ZNF652 on the colony formation and proliferation ability of MCF-7 and SKBR3 were compared with the effects of retroviral-mediated expression of the ETO proteins. Controls were empty vector and expression of p53 (Fig. 4A ). Ectopic expression of CBFA2T3 in SKBR3 cells resulted in a greater reduction (relative to the vector control) in number of colonies compared with its expression in MCF-7 cells (Fig. 4A), consistent with results obtained previously (4). However, when the cell lines were transduced with retrovirus expressing ZNF652, the reduction in colony numbers was greater in MCF-7 than SKBR3, opposite to the effects of CBFA2T3 ectopic expression. Similar findings were seen in proliferation assays (Fig. 4B). Compared with the empty vector, ectopic expression of ZNF652 resulted in a minimal reduction in proliferation of SKBR3, whereas proliferation was markedly reduced in cells ectopically expressing CBFA2T3. In MCF-7, the effect was reversed with ectopic expression of CBFA2T3 showing no effect on proliferation compared with empty vector, whereas expression of ZNF652 reduced proliferation (Fig. 4B). For both SKBR3 and MCF-7, ectopic expression of CBFA2T1 reduced cell proliferation. Ectopic expression of CBFA2T2 reduced MCF-7 proliferation but had no effect on SKBR3. Therefore, the finding that ectopic expression of ZNF652 has a more pronounced effect in the presence of CBFA2T3 (i.e., in MCF-7 compared with SKBR3) suggests that both proteins are required to functionally interact to mediate their role as transcriptional repressors.


Figure 4
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  		FIGURE 4. ZNF652 and CBFA2T3 functionally interact in vivo. Breast cancer cell lines MCF-7 and SKBR3 transduced with retroviruses expressing ZNF652 or ETO proteins. A. Colony formation of transduced cell lines. Cells were plated 24 hours following transduction, grown for 2 weeks in G418-supplemented medium, and the number of colonies were scored. Columns, number of colonies relative to the control vector. B. Growth curves of transduced cell lines. Growth assays were initiated from cultures after selection in G418-supplemented medium for 6 days following transduction with retroviruses expressing either the ETO proteins, ZNF652, p53, or empty vector.

 
ZNF652 Transcriptional Repression Is Specifically Enhanced by CBFA2T3
To assess the effect of ZNF652 on transcription, dual-luciferase reporter assays were done in Chinese hamster ovary (CHO) cells. A reference level of expression was observed in cells transfected with the pGL2-GAL4-TK-luciferase plasmid (Fig. 5A ). Transfection of increasing amounts of plasmid expressing GAL4-ZNF652 caused a significant (P < 0.05) exponential decrease in luciferase expression (Fig. 5A). Expression of a known strong repressor of transcription, GAL4-NK10 (27), validated the system as it caused a significant decrease in luciferase expression. These results indicated that ZNF652 functions as a repressor of transcription.


Figure 5
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  		FIGURE 5. ZNF652 transcriptional repression is enhanced in the presence of CBFA2T3. A. Increasing expression of ZNF652 caused an exponential decrease in the level of luciferase reporter gene expression. Dual-luciferase reporter assays were done to assess the effect of ZNF652 on transcription. CHO cells were transfected with the various combinations of plasmid constructs indicated below each column. One hundred nanograms of pGL2-GAL4-TK-luciferase provided a reference level of expression. The positive control NK10 caused a strong repression. The amount of DNA in each transfection was supplemented with empty vector to a total of 100 ng. B. ZNF652 transcriptional repression is enhanced primarily by CBFA2T3. Dual-luciferase reporter assays were done as in A. Treatments included expression of increasing amounts of each of the ETO proteins to assess the effect of ZNF652 and ETO family members on transcription. One hundred fifty nanograms of pGL2-GAL4-TK-luciferase provided a reference level of luciferase expression. The amount of DNA in each transfection was supplemented with empty vector to a total of 150 ng. There was no reduction of luciferase expression when each of the ETO proteins was expressed in the presence of the pGL2-GAL4-TK-luciferase (data not shown).

 
Because ZNF652 interacts with CBFA2T3, it was predicted that the repressive effect of ZNF652 would be enhanced in the presence of CBFA2T3. To test this hypothesis, dual-luciferase reporter assays were done in CHO cells simultaneously transfected with plasmids expressing GAL4-ZNF652 and myc-CBFA2T3 (Fig. 5B). Two different amounts of myc-CBFA2T3–expressing plasmid were used to achieve a dose-dependent indication of the effects of this protein on luciferase expression. The results confirmed that GAL4-ZNF652 alone decreased luciferase reporter gene expression and that increasing amounts of myc-CBFA2T3 expression caused a further dose-dependent decrease in expression (Fig. 5B). Therefore, CBFA2T3 further enhances the transcriptional repression caused by ZNF652.

To test if this repression was specifically caused by CBFA2T3, or would also be displayed by the other ETO family members, dual-luciferase reporter assays were done using CHO cells simultaneously transfected with plasmids expressing GAL4-ZNF652 and either CBFA2T1 or CBFA2T2 (Fig. 5B). For each protein, cells were transfected with 50 or 100 ng plasmid. In the presence of GAL4-ZNF652, increasing amounts of CBFA2T2 did not result in repression of luciferase activity, CBFA2T1. There was a significant decrease in luciferase reporter gene expression caused by simultaneous transfection of constructs expressing ZNF652 and CBFA2T1 at 50 ng, and this decrease was not altered by an increasing amount (100 ng) of CBFA2T1 expressing plasmid. Therefore, compared with CBFA2T3, only CBFA2T1 weakly enhances the transcriptional repression of ZNF652.

GFI-1 Transcriptional Repression Is Specifically Enhanced by CBFA2T1
Because we showed that ZNF652 repression is enhanced selectively by CBFA2T3, we investigated for similar selectivity in the interactions between ETO proteins and another zinc finger protein. CBFA2T1 and CBFA2T3 have previously been shown to interact with GFI-1, a zinc-finger transcription repressor (14). We recently showed that GFI-1 represses 25-hydroxyvitamin D 1{alpha}-hydroxylase (CYP27B1) expression in human prostate cancer cells, and that this repression is mediated through GFI-1–specific, DNA-binding sequences within the CYP27B1 promoter (28). Reporter assays were done to investigate if the ETO proteins differ in their ability to further repress the GFI-1–mediated transcriptional repression from the CYP27B1 promoter. For these assays, three firefly luciferase gene constructs containing CYP27B1 promoter sequences were used. The first construct, designated pCYP27B1WT(–1200)-Luc, contained a natural GFI-1–binding sequence located at –1161/–1138; the second construct, pCYP27B1Enh(–997)-Luc, was deleted for this GFI-1–binding sequence; and the third construct, pCYP27B1mGFI1(–1200)-Luc, contained a mutated GFI-1–binding sequence. The reporter assays were done in LNCaP cells that express low to medium levels of endogenous GFI-1 (data not shown). Compared with pCYP27B1Enh(–997)-Luc, pCYP27B1WT(–1200)-Luc showed repression in luciferase activity resulting from endogenous GFI-1 and possibly ETO proteins. This repression was further significantly reduced in a dose-dependent manner by ectopic expression of GFI-1 (Fig. 6A ). Ectopic expression of CBFA2T1 also significantly repressed luciferase expression from the pCYP27B1WT(–1200)-Luc construct in a dose-dependent manner. In contrast, there was a minor repression in luciferase expression in cells transfected with comparable quantities of CBFA2T2- or CBFA2T3-expressing constructs. Western blot analysis on LNCaP cells transfected with equal amounts of either of the three ETO plasmids showed lower levels of ectopic CBFA2T1 expression than either CBFA2T2 or CBFA2T3 (data not shown). Therefore, the specific transcriptional repression caused by CBFA2T1 is likely to be more pronounced than was actually observed.


Figure 6
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  		FIGURE 6. CBFA2T1 specifically represses transcription through its interaction with GFI-1 from the CYP27B1 promoter. A. CBFA2T1 causes specific exponential decrease in the level of luciferase reporter gene expression from the CYP27B1 promoter sequences carrying a GFI-1 binding site. Dual-luciferase reporter assays were done to assess the effect of ETO proteins on GFI-1–mediated transcription. LNCaP cells were transfected with the various combinations of plasmid constructs as indicated below each column. To transfect comparable amounts of DNA, plasmids were supplemented with empty vector. B. GFI-1 repression was specifically enhanced in the presence of CBFA2T1. LNCaP cells were transfected with pCYP27B1WT(–1200)-Luc and various other constructs as indicated below each column. C. CBFA2T1-3 did not repress transcription from the construct carrying the CYB27B1 promoter with a mutated GFI-1 DNA-binding sequence. T1 to T3, CBFA2T1, CBFA2T2, and CBFA2T3. pCYP27B1WT(–1200) contains the 1,200 bp of the CYP27B1 promoter that includes the consensus GFI-1–binding site. This binding site is deleted in pCYP27B1Enh(–997)-Luc and mutated in the pCYP27B1mGFI1(–1200)-Luc construct.

 
To determine if ectopic expression of GFI-1 further enhanced the repression of CBFA2T1, reporter activation of pCYP27B1WT(–1200)-Luc was assayed in cells transfected with plasmid constructs expressing both GFI-1 and either of the three ETO proteins (Fig. 6B). Ectopic expression of both GFI-1 and either of the three ETO proteins resulted in a decrease in the luciferase activity of 54% (CBFA2T1), 27% (CBFA2T2), and 37% (CBFA2T3). These results suggest that the GFI-1 and CBFA2T1 interaction is functionally more significant than the interactions of GFI-1 with the other two members of the ETO family.

To confirm that CBFA2T1 mediates its specific repressive effect through GFI-1 bound to specific sequences within the pCYP27B1 promoter, reporter assays were done using the pCYP27B1mGFI1(–1200)-Luc construct. Reporter activity in LNCaP cells transfected with plasmid containing CYP27B1 promoter with a mutated GFI-1 binding site was >2-fold higher than in the cells with the wild-type sequence and this did not change in the presence of GFI-1 or the ETO proteins (Fig. 6C). These results confirm that the repression of transcription by CBFA2T1 in cells transfected with pCYP27B1WT(–1200)-Luc is mediated through sequence-specific binding of the endogenous GFI-1 at the CYP27B1 promoter.


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
Studies of CBFA2T1 and CBFA2T3 have centered on their involvement in the t(8;21) and t(16;21) translocations that are associated with acute myeloid leukemia (29). These translocations generate fusion proteins containing the DNA-binding region of the RUNX1 transcriptional activator and the majority of the transcriptional repressor proteins CBFA2T1 or CBFA2T3. The fusion proteins result in the repression of genes normally activated by the RUNX1 protein. All three ETO proteins have been shown to be transcriptional repressors in reporter assays, and their in vivo function is likely to be mediated by the recruitment of various corepressors, including N-CoR, mSin3A, and HDACs (4, 8, 18, 20). Although ETO proteins are very similar in their amino acid sequences, and similar regions of the proteins are responsible for their repressor function, the proteins do exhibit some differences in their ability to interact with these corepressors (8).

ETO proteins have been shown to localize to the nucleus, function as transcriptional repressors, and can homodimerize and heterodimerize to generate a diversity of repressor complexes (30, 31). The normal physiologic roles of the ETO proteins are beginning to emerge, although their functions are likely to be broader than presently defined. CBFA2T1 is a regulator of early adipogenesis (32) and targeted disruption of Cbfa2t1 in the mouse shows a role in gut development (23). The role of CBFA2T2 is obscure although disruption of this gene in mice suggests that Cbfa2t2 maintains the secretory cell lineage in the small intestine (9). CBFA2T3 has been shown to coordinate cellular proliferation and differentiation during erythropoiesis (33) and has a role as a human breast tumor suppressor (4). It is also clear that as ETO family members do not have the ability to directly bind DNA, they initiate repression of their respective target genes by interacting with various DNA-binding proteins and by recruitment of corepressors to the transcriptional repressor scaffold. For example, it has been shown that CBFA2T1 interacts with PLZF, GFI-1, BCL6, and HEB, and CBFA2T3 interacts with GFI-1 to repress the transcription of various genes (12-15). The interaction of ETO proteins with such sequence-specific, DNA-binding proteins is essential for their transcriptional repressor functions.

Because CBFA2T3 was shown to be a putative tumor suppressor in breast cancer (4), we screened a normal breast cDNA library with the 142 to 567 amino acids of CBFA2T3 to identify interacting proteins. From this yeast two-hybrid screen, the novel protein ZNF652 was identified. A Northern blot probed with a radiolabeled ZNF652 fragment showed that this gene is ubiquitously expressed in human tissues (34). These observations were confirmed by the results from a cancer-profiling array (Fig. 1). Of particular interest was the consistent reduction in ZNF652 expression observed among different types of tumors, and this was more pronounced in the breast, prostate, vulva, and pancreas. It is noteworthy that ZNF652 is located on chromosome 17q21.32 at 44.7 Mb (build 35.1 of the Human Genome, National Center for Biotechnology Information), 6.3 Mb distal to BRCA1. Chromosome 17 is frequently involved in breast tumor–restricted loss of heterozygosity. A large consortium study of 1,280 breast carcinomas showed loss of heterozygosity averaged 31% at various 17q markers, including those flanking the location of ZNF652 (35). However, there was no compelling evidence of peaks in loss of heterozygosity frequency on the long arm that would support, or refute, ZNF652 as a potential tumor suppressor in breast cancer. The levels of expression of ZNF652 protein in breast cancer cell lines may suggest a down-regulation in estrogen receptor–negative cell lines (Fig. 2B), although additional studies are required to confirm this finding.

Based on the presence of seven classic (C2H2) zinc finger domains, together with the linkers with consensus sequence TGEKP joining three of these zinc fingers, ZNF652 is predicted to bind DNA (25, 36) and, therefore, be the DNA-binding component of a CBFA2T3 regulatory complex. The interaction of ZNF652 and CBFA2T3 was confirmed by coimmunoprecipitation in HEK293T mammalian cells (Fig. 3A and B; ref. 34). The interaction of ZNF652 and CBFA2T3 occurs through the 109 amino acids at the COOH terminus of ZNF652 (Fig. 3B). This is consistent with the finding that the initial cDNA clone identified in the yeast two-hybrid screen encoded the 61 COOH-terminal amino acids of the ZNF652 protein. Furthermore, this interaction is apparently stabilized by the presence of the zinc finger region. A detailed protein database search does not detect significant homology of this region of ZNF652 to any other known proteins. However, further in silico analysis of this region of ZNF652 revealed proline- and histidine-rich regions (Fig. 3C) that are thought to mediate protein-protein interactions critical for the activation, inactivation, and functioning of some proteins (37, 38). These results suggest that ZNF652 is a specific DNA-binding partner of the CBFA2T3 regulatory complex.

Functional assays in the breast cancer cell lines MCF-7 and SKBR3 provided evidence that in vivo the function of ZNF652 is enhanced in the presence of CBFA2T3 (Fig. 4). The effect of ZNF652 on reducing both the clonability and cell proliferation of these cell lines was greater in MCF-7 compared with SKBR3. These effects inversely correlated to endogenous CBFA2T3 expression, which is high in MCF-7 and low in SKBR3 (ref. 5 and unpublished Western blot results). The expression level of ZNF652 in MCF-7 and SKBR3 is comparable (Fig. 2B). These studies in breast tumor cells suggest that ZNF652 function is largely dependent on the presence of CBFA2T3, and support an in vivo interaction of these two proteins. Ectopic expression of CBFA2T1 and CBFA2T2 had less pronounced effects on the growth of SKBR3 but a more significant effect in MCF-7. This may be due to the presence of additional ETO DNA-binding partners in MCF-7 cells.

Transcriptional reporter assays expressing GAL4-DBD fused to ZNF652 showed that increasing amounts of ZNF652 caused an exponential decrease in the level of luciferase reporter gene expression and indicates that ZNF652 functions as an independent repressor of transcription (Fig. 5A). Similarly, BCL6, PLZF, and GFI-1, the other zinc finger proteins that interact with the ETO family of proteins, have also been shown to be independent repressors of transcription (12-14). Transcriptional reporter assays also showed that the repressor activity caused by ZNF652 was enhanced in the presence of CBFA2T3 (Fig. 5B). A similar scenario was seen in studies on transcriptional repression mediated by BCL6 (12) and PLZF (13), where the presence of CBFA2T1 enhanced their repression. It is not known whether these zinc finger proteins can function in vivo to repress transcription in the absence of the scaffold ETO proteins. We suggest that ZNF652 is a putative breast cancer tumor suppressor because CBFA2T3 has this role (4), and the two proteins form a transcriptional repressor complex. In tumors, it is proposed that the normal transcriptional repressor function of the CBFA2T3 complex can be circumvented by down-regulation, e.g., by loss of heterozygosity and methylation (1, 5), of either CBFA2T3 or ZNF652 expression.

Although the zinc finger proteins BCL6 and PLZF have been shown to interact with CBFA2T1, and GFI-1 with both CBFA2T1 and CBFA2T3 (12-14), there has been no evidence to suggest that the different zinc finger proteins specifically interact with particular ETO family members. However, coimmunoprecipitations done on transiently expressed proteins in mammalian cells provide evidence that ZNF652 strongly and specifically interacts with CBFA2T3, but weakly with CBFA2T1 and CBFA2T2 (Fig. 3A). It was of interest to determine if this observed selectivity of ZNF652 interactions with ETO proteins was also reflected in a functional specificity. This was indeed the case because in luciferase reporter assays, CBFA2T3 augmented the repression of ZNF652 but this was reduced with CBFA2T1 and CBFA2T2. To investigate if the functional selectivity between ETO proteins and their interacting zinc finger proteins is a more general property, we determined the repressor activity of GFI-1 from the CYP27B1 promoter. Coexpression of GFI-1 and CBFA2T1 resulted in a higher level of repression than coexpression of GFI-1 with either CBFA2T2 or CBFA2T3 (Fig. 6A and B). Taken together, these observations suggest that particular combinations of ETO proteins and specific DNA-binding transcription factors interact to achieve maximal levels of target gene repression in vivo. This novel finding implies that the ETO family of proteins generates a diverse group of complexes, with variation in both the architecture and the recruitment of the gene effector zinc finger proteins. We therefore suggest that each of the various gene effector zinc finger proteins specifically interacts with one or more of the ETO proteins to generate a defined range of transcriptional repressor complexes that control the expression of specific set of genes. The genes controlled by the ZNF652-CBFA2T3 complex are likely to have a role in the tumorigenesis of breast and other tissues.

Results using a luciferase reporter system provide an insight into the interactions of ZNF652 that may be occurring in vivo. However, confirmation of these results using reporter-vector constructs carrying the actual DNA-binding sequence of ZNF652 upstream of the luciferase gene will be an important next step. We are currently working toward identifying the ZNF652 DNA-binding sequences.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
In silico Analysis
ZNF652 mRNA sequence was obtained from the National Center for Biotechnology Information (accession no. NM_014897).6 Protein domain searches were done using ExPASY-PROSITE,7 and protein homology searches were done using Basic Alignment Search Tools.6

Cell Lines and Antibodies
184B and 48RS are finite lifespan human mammary epithelial cell strains derived from two different individuals' reduction mammoplasty tissues, whereas 184A1 is a nonmalignant immortally transformed cell line derived from normal specimen 184 (39). HEK293T (human embryonic kidney), CHO, and all other cell lines were purchased from the American Type Culture Collection (Manassas, VA) and grown in the recommended medium at 37°C in 5% CO2. Antibodies used were rat anti-HA (Roche Diagnostics, Indianapolis, IN), mouse anti-myc (Santa Cruz Biotechnology, Santa Cruz, CA), horseradish peroxidase (HRP)–conjugated secondary antibody (rabbit anti-rat-IgG-HRP; DakoCytomation, Carpinteria, CA), sheep anti-mouse IgG-HRP, and donkey anti-rabbit IgG-HRP (Amersham Biosciences, Piscataway, CA). A rabbit polyclonal anti-ZNF652 antibody was generated. A ZNF652 DNA fragment expressing amino acids 497 to 606 was cloned in-frame into the bacterial expression vector pET14b (Novagen, Madison, WI). The 6x His-ZNF652497-606 protein induced from BL21pLys bacteria harboring the expression construct was purified on Ni-NTA superflow beads (Qiagen, Hilden, Germany) and injected into White New Zealand rabbits. The rabbit polyclonal antibody was affinity-purified using bacterially expressed GST-ZNF652497-606 protein conjugated to CNBr-activated Sepharose 4B matrix (Amersham Biosciences).

Plasmids
All plasmids were constructed with the complete open reading frame of human genes unless specified otherwise. For coimmunoprecipitation assays, the plasmid construct expressing myc-CBFA2T3b (smaller isoform of CBFA2T3) was as reported earlier (4), and CBFA2T1 and CBFA2T2 open reading frames were cloned in-frame into the BglII site of pCMV-myc (BD Biosciences, San Jose, CA). To generate constructs expressing various regions of the ZNF652 protein, the relevant fragments of the cDNA were PCR amplified from the existing construct using primers with sequence extensions that created BamHI restriction sites, and cloned in-frame into the BglII restriction site of pCMV-HA (BD Biosciences). Constructs generated were as follows: ZNF652 (complete open reading frame, amino acids 1-606), ZNF6521-246 (amino acids 1-246), ZNF652243-606 (amino acids 243-606), ZNF652243-491 (amino acids 243-491), and ZNF652498-606 (amino acids 498-606). Constructs with various gene fragments in pLNCX2 were used for retroviral experiments. For luciferase reporter assays, ZNF652 or amino acids 1 to 90 of a known mouse zinc finger repressor NK10 protein (4, 27) were cloned in-frame with the region expressing the GAL4 DNA-binding domain in pM (BD Biosciences). To generate pGL2-GAL4-TK-luciferase construct, 5' and 3' ends of the XbaI-BamHI fragment carrying 5x GAL4 DNA-binding sequences from pG5CAT (Promega, Madison, WI) were filled-in using Klenow DNA polymerase and cloned at blunted XhoI site of pGL2-TK-luciferase. Constructs used for GFI-1–mediated repression assays were pCYP27B1WT(–1200)-Luc that contained a 1,244 bp region of the CYP27B1 promoter (–1200 together with 44 bp of CYP27B1 5' untranslated region), pCYP27B1Enh(–997)-Luc that was deleted for the region containing the GFI-1 consensus binding site, and pCYP27B1mGFI (-1200)-Luc that contained mutated GFI-1 binding site (28). The construct expressing FLAG-GFI-1 was kindly provided by H.L. Grimes (Institute for Cellular Therapeutics, University of Louisville, Louisville, KY). The Renilla luciferase vector (pRL-TK) was from Promega. For repression assays, the NH2 terminus myc-tagged ETO family members were cloned into pcDNA3.1 (Invitrogen, Carlsberg, CA) at XbaI/EcoRI sites for CBFA2T3b, ApaI/KpnI sites for CBFA2T2, and XbaI/HindIII sites for CBFA2T1. DNA was prepared and validated using standard molecular biology techniques (40).

Yeast Two-Hybrid Assay
Display GREEN basic yeast two-hybrid kit (Display Systems Biotech, Copenhagen, Denmark) was used to perform a yeast two-hybrid screen. DNA sequence encoding CBFA2T3142-567 was PCR amplified with the two primers (the reverse primer was designed with a 5'-end extension to generate a sequence encoding a FLAG-tag at the COOH terminus) and cloned in-frame with the lexA DNA-binding protein in the pdisplayBAIT vector. Human breast cDNA Hybrid Hunter library (Invitrogen) cloned in pYESTrp2 vector was screened using pdisplayBAIT-CBFA2T3142-567 and pdisplayREPORTER vectors transformed into yeast H strain (MAT{alpha} trp1 his3 ura3 leu2 6 LexAops-LEU2).

Cancer Profiling
Cancer Profiling Array II (BD Biosciences) was probed with a PCR product (nucleotides 1-738) of ZNF652. The probe was labeled with [{alpha}-32P]dCTP using the Megaprime DNA labeling system (Amersham Biosciences). Hybridization and washing conditions were as the manufacturer instructed. The membrane was stripped and then probed with the radiolabeled housekeeping ubiquitin gene probe supplied with the kit. The hybridization signals were quantified using a PhosporImager (Molecular Dynamics), digitized using ImageJ 1.33u (NIH) software, and normalized for ubiquitin expression.

Retoviral-Mediated Expression Studies
Various relevant constructs in the pLNCX2 vector were used to generate retrovirus and transduce MCF-7 and SKBR3 cells as previously described (41). Following transduction, cells were plated in six-well plates, selected for 2 weeks in G-418–containing medium, and the number of colonies were scored. Results are presented as the mean of three replicates ± SE. Proliferation assays were determined on transduced cells selected for 6 days in the presence of G418. These cells were plated at 10% to 20% confluence in 96-well plates and at various times assayed by incubating cells with the CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the instructions of the manufacturer. Results are presented as the mean relative luminescence units of three replicates.

Coimmunoprecipitations
Approximately 1 x 106 HEK293T cells were transiently transfected with 4 µg of the each relevant plasmid using LipofectAMINE 2000 (Invitrogen), according to the instructions of the manufacturer. After 24 hours, cells were harvested and lysed in 50 mmol/L Tris-HCl (pH 8), 150 mmol/L NaCl, and 1% Triton X-100 supplemented with complete protease inhibitor cocktail (Roche Diagnostics). Lysates were sonicated and cell debris was removed by centrifugation at 16,000 x g. Supernatants were incubated with 500 ng mouse monoclonal 9E10 anti-myc antibody (Roche Diagnostics) overnight at 4°C, followed by the addition of 15 µL sheep anti-mouse IgG Dynabeads (Invitrogen). Beads were washed with lysis buffer, twice with wash buffer [50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS], and then twice with low salt buffer [20 mmol/L Tris-HCl (pH 7.5)]. Proteins were eluted with 50 µL of 1x SDS protein-loading buffer [0.0625 mol/L Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 5% ß-mercaptoethanol] for 5 minutes at 95°C. Inputs and coimmunoprecipitates were subjected to SDS-PAGE and transferred to a nitrocellulose membrane (Hybond C-Extra, Amersham Biosciences).

Western Blotting
For extracting proteins, cells from different cell lines were collected at 80% confluence; lysed in 50 mmol/L Tris-HCl (pH 7.5), 250 mmol/L NaCl, 1% Triton X-100, 1 mmol/L EDTA, 50 mmol/L NaF, 0.1 mmol/L Na3VO4, 1 mmol/L DTT, and 1x protease inhibitors (Roche Diagnostics) on ice for 15 minutes; clarified; and then assayed using BCA protein assay reagent kit (Pierce, Rockford, IL). Twenty-five micrograms protein from each sample were used for Western blotting analysis. For detecting endogenous ZNF652 in different cancer cell lines, affinity-purified rabbit polyclonal anti-ZNF652497-606 antibody and donkey anti-rabbit IgG-HRP were used. Equal loading of different proteins was confirmed by probing the same membrane with mouse anti-ß-actin antibody (Sigma-Aldrich, St Louis, MO). For analyzing the immunoprecipitates, Western blots carrying inputs and immunoprecipitated samples were first incubated with primary antibody (rat anti-HA or mouse anti-myc) followed by appropriate HRP-conjugated antibodies. Proteins were visualized by enhanced chemiluminescence kit (Amersham Biosciences).

Luciferase Assays
Approximately 2 x 105 CHO cells were transiently transfected using LipofectAMINE 2000 with 700 ng pGL2-GAL4-TK-luciferase, constructs expressing varying amounts of GAL4-ZNF652 and/or myc-CBFA2T1, -T1 or -T3, and 50 ng pRL-TK plasmid (Promega) as an internal transfection control. Cells were harvested after 16 hours, lysed, and assayed using the dual-luciferase reporter assay system (Promega). Firefly luciferase values were normalized to Renilla luciferase activity and expressed as relative luciferase units. Each treatment was done in triplicate and assays were repeated at least thrice. The data presented is of a representative experiment with ±SE of mean of three replicates. For GFI-1 reporter assays, LNCaP cells (1.5 x 105) were seeded into 24-well plates and transfections were carried out in triplicate. Each transfection was done with 200 ng pCYP27B1WT(–1200)-Luc, pCYP27B1Enh(–997)-Luc, or pCYP27B1mGFI(–1200)-Luc together with varying amounts of constructs expressing FLAG-GFI-1 and/or either of the three ETO proteins and 50 ng pRL-TK plasmid. Cells were harvested and analyzed as above.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Grant support: National Health and Medical Research Council of Australia grant 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); Cancer Council of South Australia (H.A. Morris); Faculty of Health Sciences and Department of Medicine, University of Adelaide; and the Hanson Institute, Institute of Medical and Veterinary Science.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

6 http://www.ncbi.nlm.nih.gov. Back

7 http://www.expasy.org/prosite. Back

Received 11/27/05; revised 6/22/06; accepted 7/ 5/06.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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