Study On The Clone And Function Analysis Of Two Novel ZNF268 Isoforms

We previously constructed a cDNA library from 3-week-old human embryos, and isolated a novel Cys₂/His₂ zinc finger gene, ZNF268, from this library. To understand the function of ZNF268, we analyzed its expression by Northern blot hybridization and Western blot analysis on RNA from human embryos and adult human tissues, the data show ZNF268 was expressed specially in the fetal liver.To investigate the expression of ZNF268 protein, Immunohistochemistry analysis was used to check the expression of ZNF268 protein by the the specific antigen of ZNF268 in 3 5 weeks human embryo, and the ZNF268 protein expressed in the blood island in yolk sac of 3 week embryo, in dissociated cells of 5 week human embryo heart and dissociated cells of 5 week human embryo liver sinusoidal endothelial cells. The cells who expressed the ZNF268 was checked by monoclonal antigen of CD34 and CD38. They all showed CD34⁺ and CD38^- which suggested they are hematopoietic stem/progenitor cells. In general, zinc finger proteins are involved in the binding of transcriptionfactors to their cognate DNA recognition site, resulting in the specific expression during cell differentiation and development. Consequently, expression pattern is an important clue for predicting the possible function of novel zinc finger genes. This make a suggestion that ZNF268 may involve in the development of the blood cells in early human embryo and may have a function in early human hematopoiesis.We analyzed its expression by RT-PCR on total RNA from twenty adult human. RNA was isolated from the whole blood using Trizol LS; one step RT-PCR was used; PCR was then performed. PCR products were cloned into T-vector and sequenced. We got four clones. We identified two novel forms of ZNF268 — ZNF268c(NCBI access number: DQ057356) , ZNF268d(NCBI access number: DQ057357), the other two were ZNF268a and ZNF268b which were pubiliced. We compared the genomic sequence to the cDNA sequence from the NCBI database. All exon/intron boundaries of ZNF268 gene obeyed the classic GT/AT rule. Compared with the published forms of ZNF268— ZNF268a and ZNF268b, ZNF268c was absent exonlV and exonVI; ZNF268d was absent in exonlll, exonlV, and exonVI. ExonlV encodes the Krueppel-associated A box, which is proved to be a strong transcriptional repressor element in ZNF268 gene. Multiple mRNAs is generated from ZNF268 gene, which in turn can be translated into a group of diverse proteins with different roles and structures. Inherited and acquired changes in pre-mRNA splicing have been documented to play a significant role in human disease development and many cancer-associated genes are regulated by alternative splicing. Understanding the process of aberrant splicing and the detailed characterization of the splice variants may prove crucial toour understanding of malignant transformation. Although the functions of ZNF268c and ZNF268d, it is possible that they regulate the activity of ZNF268 proteins by competing for the same binding protein, thereby reversing the positive or negative regulation of the transcriptional activity.