863

CBFA2T3

ETO2|MTG16|MTGR2|ZMYND4;core-binding factor, runt domain, alpha subunit 2; translocated to, 3;CBFA2T3;core-binding factor, runt domain, alpha subunit 2; translocated to, 3

MTG8-related gene 2|MTG8-related protein 2|myeloid translocation gene 8 and 16b|myeloid translocation gene on chromosome 16 protein|protein CBFA2T3|zinc finger MYND domain-containing protein 4

16q24

protein-coding

Numerous cytogenetic and molecular studies of breast cancer have identified frequent loss of heterozygosity (LOH) of the long arm of human chromosome 16. On the basis of these data, the likely locations of breast cancer tumor suppressor genes are bands 16q22.1 and 16q24.3. We have mapped the CBFA2T3 (MTG16) gene, previously cloned as a fusion partner of the AML1 protein from a rare (16;21) leukemia translocation, to the 16q24.3 breast cancer LOH region. The expression of CBFA2T3 was significantly reduced in a number of breast cancer cell lines and in primary breast tumors, including early ductal carcinomas in situ, when compared with nontransformed breast epithelial cell lines and normal breast tissue. Reintroduction of CBFA2T3 into different breast tumor derived cell lines with decreased expression of this gene reduced colony growth on plastic and in soft agar. CBFA2T3 was shown to function as a transcriptional repressor when tethered to the GAL4 DNA-binding domain in a reporter gene assay and, therefore, has the potential to be a transcriptional repressor in normal breast epithelial cells. Taken together, these findings suggest that CBFA2T3 is a likely candidate for the breast cancer tumor suppressor gene that is the target for the frequent 16q24 LOH in breast neoplasms.

Loss of heterozygosity (LOH) of chromosome 16q24.3 is a common genetic alteration observed in invasive ductal and lobular breast carcinomas. We constructed a physical map and generated genomic DNA sequence data spanning 2.4 Mb in this region. Detailed in silico and in vitro analyses of the genomic sequence data enabled the identification of 104 genes. It was hypothesized that tumor-suppressor genes would exhibit marked mRNA expression variability in a panel of breast cancer cell lines as a result of downregulation due to mutation or hypermethylation. We examined the mRNA expression profiles of the genes identified at 16q24.3 in normal breast, a normal breast epithelial cell line, and several breast cancer cell lines exhibiting 16q24.3 LOH. Three of the genes, CYBA, Hs.7970, and CBFA2T3, exhibited variability ten times higher than the baseline. The possible role of these genes as tumor suppressors is discussed.

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.