| Embryogenesis is a complex process that requires accurate temporal and spatial regulation of gene expression and function. Alteration of this regulation may lead to congenital abnormality and inheritable genetic diseases. Although various animal models have provided some information of early mammalian development, vital differences between human and other animals argue for studies on human development.In an effort to identify and functional characterize genes important for human development, we previously constructed a cDNA library from 3-week-old human embryos. By using a differential hybridization strategy, we were able to isolate, from this library, many genes expressed in the early human embryos, among which, a novel zinc finger gene, ZNF268.To determine whether ZNF268 is involved in human embryogenesis, we first analyzed its expression by Northern blot hybridization on RNA from human embryos and adult human tissues. The ZNF268 probe detected an mRNA of 3.8 kb in length in human embryos. While the mRNA was present in all embryos analyzed, but no ZNF268 mRNA was detectible in all adult human tissuesanalyzed. Those imply ZNF268 play a role specifically during early embryogenesis.To investigate the expression of ZNF268 protein, we first generated an anti~ZNF268 antibody specifically. Western blot analysis indicated that ZNF268 was expressed in the fetal liver, but not in the heart, kidney, brain and spleen. To further investigate ZNF268 expression in development, immunohistochemical analysis was carried on tissue sections of human embryos of various ages. Again, the anti-ZNF268 antibody specifically detected the protein in the liver of a human embryo as early as 5 week old but not in other areas of the embryo. The protein level in the liver hepatocytes remained high up to 4 month old but drastically reduced by 6 month old and to non-detectible levels by 7 months. These results suggest that ZNF268 is involved in the development of fetal liver in early human fetuses. In addition, Immunohistochemistry analysis also indicated that ZNF268 expression was found in the hematopoietic stem/progenitor cells. 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.The immunohistochemical analysis of ZNF268 expression in embryonic liver also revealed, at high resolution, that the protein was localized in thecytoplasm of hepatocytes through out development (5 weeks to 4 months). To further investigate the subcelluar distribution of ZNF268 protein, we expressed the fused EGFP zinc finger protein in COS7 cells. Examination result showed that ZNF268 was predominantly, if not exclusively, a cytoplamic protein, belonging to the small group of cytoplamic zinc figure proteins but distinct from other krtlppel-Iike zinc finger proteins.To examine whether the ZNF268 KRAB possesses transcriptional represser activity, KRAB-A, KRAB-B and KRAB-AB were fused in-frame to the GAL4 expression vector pBXGl respectively. Then they were co-expressed with reporter plasmid pG5SV-BCAT which containing five GAL4-binding elements. Both KRAB-AB and KRAB-A decreased the expression level of the reporter gene in a dose-dependent manner. The KRAB-B suppressed transcription of the reporter gene not as significant as the other two; KRAB-AB repressed the reporter gene distinctly than KRAB-A. In conclusion, KRAB domain of ZNF268 has transcription repressing activity, and ZNF268 may act as a represser.To study the interact protein of ZNF268, we constructed the "Bait" with ful 1 cDNA of ZNF268, and screened in the human liver cDNA library of 18-24 weeks. Now we identified a interact protein AHSG (alpha2-Heremans Schmid Glycoprotein). AHSG mayplay roles in the development of liver, brain and may involve in bone mineralization and in the immune response. The interaction between ZNF268 and AHSG indicate that AHSG, recruiting ZNF268, play a role in early liver development. Otherwise, AHSG also expressed serum, bone marrow hemopoieti...
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