Transcription of Human Zinc Finger ZNF268 Gene Requires an Intragenic Promoter Element

Abstract

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...

Human T-cell Leukemia Virus Type 1 Oncoprotein Tax Represses ZNF268 Expression through the cAMP-responsive Element-binding Protein/Activating Transcription Factor Pathway

“…In the absence of Tax, dominant negative CREB-1 had little effect (a small, nonsignificant increase in Fig. 7B) on the ZNF268 promoter, consistent with the lack of effect of CREB-1 on this promoter in HeLa cells (27) (see "Discussion" for a possible explanation). This result suggests that CREB-1 plays an important role in the repression of ZNF268 gene by Tax.…”

“…Chromatin Immunoprecipitation (ChIP)-The assay was done as previously described (27). Monolayer of HEK293 cells (80% confluent) were incubated for 24 h after transfection and then were serumstarved for 24 h. Formaldehyde was added to the culture medium to a final concentration of 1%.…”

“…pGL3(Ϫ37/ϩ938)-p53-mut (ϩ596 to ϩ621), pGL3(Ϫ37/ϩ938)-Ets-mut (ϩ606 to ϩ631), pGL3(Ϫ37/ϩ938)-CREB-mut (ϩ724 to ϩ749), pGL3(Ϫ37/ ϩ938)-AP1-mut (ϩ722 to ϩ746), and pGL3(Ϫ37/ϩ938)-C/ EBP-mut (ϩ728 to ϩ752) were constructed by using the overlapping extension PCR method with pGL3(Ϫ37/ϩ938) plasmid as described previously (27). pcDNA-Tax expresses the wild-type Tax, M22 expresses a Tax mutant that can activate CREB/ATF but not NF-B, and M47 expresses a Tax mutant that can activate NF-B but not CREB/ATF (29).…”

“…Plasmid Construction and Cell Culture-The sequence from Ϫ37 to ϩ938 containing the intragenic promoter element and a series of truncation mutants of the human ZNF268 promoter were inserted into the promoterless luciferase expression vector pGL3 (Promega) (27). pGL3(Ϫ37/ϩ938)-p53-mut (ϩ596 to ϩ621), pGL3(Ϫ37/ϩ938)-Ets-mut (ϩ606 to ϩ631), pGL3(Ϫ37/ϩ938)-CREB-mut (ϩ724 to ϩ749), pGL3(Ϫ37/ ϩ938)-AP1-mut (ϩ722 to ϩ746), and pGL3(Ϫ37/ϩ938)-C/ EBP-mut (ϩ728 to ϩ752) were constructed by using the overlapping extension PCR method with pGL3(Ϫ37/ϩ938) plasmid as described previously (27).…”

“…Analysis of the ZNF268 gene promoter shows that the ZNF268 gene utilizes an intragenic promoter element to control its transcription, similar to some other genes involved in the diseases development and progression, such as WT-1. In addition, in HeLa cells, CREB-2 has been shown to play an important role during the regulation of ZNF268 expression (27).…”