Human ZNF268 gene is a typical Krüppel-associated box/ C2H2 zinc finger gene whose homolog has been found only in higher mammals and not in lower mammals such as mouse. Its expression profiles have suggested that it plays a role in the differentiation of blood cells during early human embryonic development and the pathogenesis of leukemia. To gain additional insight into the molecular mechanisms controlling the expression of the ZNF268 gene and to provide the necessary tools for further genetic studies of leukemia, we have mapped the 5-end of the human ZNF268 mRNA by reverse transcription-PCR and primer extension assays. We then cloned the 5-flanking genomic DNA containing the putative ZNF268 gene promoter and analyzed its function in several different human and mouse tissue culture cell lines. Interestingly, our studies show that the ZNF268 gene lacks a typical eukaryotic promoter that is present upstream of the transcription start site and directs a basal level of transcription. Instead, the functional promoter requires an essential element that is located within the first exon of the gene. Deletion and mutational analysis reveals the requirement for a cAMP response-element-binding protein (CREB)-binding site within this element for promoter function. Gel mobility shift and chromatin immunoprecipitation assays confirm that CREB-2 binds to the site in vitro and in vivo. Furthermore, overexpression of CREB-2 enhances the promoter activity. These results demonstrate that the human ZNF268 gene promoter is atypical and requires an intragenic element located within the first exon that mediates the effect of CREB for its activity.The first zinc finger protein containing the Krüppel-associated box (KRAB 4 -containing proteins) was discovered in 1991by Bellefroid et al. (1) and was found to be down-regulated during in vitro terminal differentiation of human myeloid cells. KRAB-containing zinc finger genes represent a subfamily within a large family of zinc finger genes, and they typically act as transcriptional repressors (2). They make up approximately one-third (290) of the 799 different zinc finger proteins present in the human genome, and as a result, this group of proteins is the largest single subfamily of transcriptional regulators in mammals. Many genes encoding KRAB-containing proteins are arranged in clusters, but others occur individually throughout the genome (3). This family of genes has been shown to be involved in diverse developmental and pathological processes (4 -9). By studying the molecular basis of human embryonic development, we have previously constructed a cDNA library from RNA isolated from 3-to 5-week-old human fetuses. Screening of this library led us to isolate the human zinc finger 268 protein gene, which is a typical KRAB-containing zinc finger protein gene (10, 11). More importantly, expression analysis has implicated a role for ZNF268 in embryogenesis (10, 11).By using a recombinant expression cloning (SEREX) approach to identify tumor-associated antigens in chronic lymphocytic leukemia...

Abstract. The ZNF268 gene was originally isolated from an early human embryo cDNA library. Several different transcripts have been isolated for the ZNF268 gene and developmental expression studies suggest that ZNF268 plays a role in the development of human fetal liver and the differentiation of blood cells. In our effort to study the functions of ZNF268 in different organs during development and in pathogenesis, we have now identified 3 novel splicing isoforms, ZNF268e, ZNF268f and ZNF268g, in human fetal tissues and human tumor derived cell lines. The 8 alternatively spliced mRNAs discovered so for are predicted to encode 3 protein isoforms. Expression analysis showed that different mRNA isoforms have different expression profiles. In particular, ZNF268c mRNA was detected only in tumor cells, and ZNF268f appeared to be tissue-specific. By Western blot analysis, all 3 ZNF268 protein isoforms, ZNF268a, ZNF268b1 and ZNF268b2, were expressed in tumor cell lines, while only two protein products, ZNF268b1 and ZNF268b2, were detected in human fetal tissues. Subcellular localization analysis showed that ZNF268a and ZNF268b2 distributed diffusely throughout the cell, while ZNF268b1 mainly localized in the cytoplasm. Moreover, using a CAT reporter system fused to the Gal4 DNA binding domain of the ZNF268 gene, the ZNF268a and b2 activated the CAT reporter gene expression, while the KRAB domain, corresponding to the ZNF268b1 repressed the reporter gene expression. Taken together, our results showed that multiple ZNF268 splicing products encode multiple ZNF268 protein isoforms with different subcellular localization, and that the ZNF268 gene may function as a transcriptional activator in the growth and differentiation of cells in development and/or pathogenesis.

Expression of the human T-cell leukemia virus type 1 (HTLV-1) oncoprotein Tax is correlated with cellular transformation, contributing to the development of adult T-cell leukemia. In this study, we investigated the role of Tax in the regulation of the ZNF268 gene, which plays a role in the differentiation of blood cells and the pathogenesis of leukemia. We demonstrated that ZNF268 mRNA was repressed in HTLV-1-infected cells. We also showed that stable and transient expression of HTLV-1 Tax led to repression of ZNF268. In addition, by using reporter constructs that bear the human ZNF268 promoter and its mutants, we showed that Tax repressed ZNF268 promoter in a process dependent on a functional cAMP-responsive element. By using Tax, cAMP-responsive element-binding protein (CREB)-1, CREB-2, and their mutants, we further showed that Tax repressed ZNF268 through the CREB/activating transcription factor pathway. Electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated the formation of the complex of Tax⅐CREB-1 directly at the cAMP-responsive element both in vitro and in vivo. These findings suggest a role for ZNF268 in aberrant T-cell proliferation observed in HTLV-1-associated diseases.

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