| Summary: | 碩士 === 臺北醫學大學 === 細胞及分子生物研究所 === 92 === Notch receptors are evolutionally highly conserved and play important roles in modulating cell fate decisions throughout the development from invertebrate to vertebrate species. Four homologues of Notch receptors have been identified in human and aberrant Notch signaling is associated with a number of human diseases. There is a consensus model of Notch signaling pathway containing multiple proteolytic steps. After ligand binding, Notch receptors are cleaved to release the intracellular domains of Notch receptors. The intracellular domains, the activated form of Notch receptors, are then translocated into nuclei and interact with other transcriptional machineries to regulate the expression of cellular genes.
In the previous data of Dr. Yeh and our lab, the proteomic study of GST-Notch1-Ank domain(GST-N1IC-Ank) pull down associated with cell lysate of erythrleukemia K562 cell, several zinc finger proteins were identified. Here, we wish to identify one of these zinc finger proteins, ZNF74, and characterize its biological function. The ZNF74 belongs to the family of Cys2-His2 type zinc finger proteins, it contains a KRAB repression domain at the amino- terminal region and 12 zinc finger motifs at carboxyl terminus. It has been showed that ZNF74 is localized to nucleus and the KRAB domain exhibits transcription repressor activity, whereas the Cys2-His2 zinc finger motifs recognized DNA sequence.
In this study, we designed to confirm the association between N1IC and ZNF74. The specific primers were synthesized and the RT-PCR result has shown that the ZNF74 transcript is splicing isoform I in the K562 cell. The mammalian expressing vector harboring HA-ZNF74 were transfected into HEK293 cells expressing N1IC, the cell lysate was prepared and immunoprecipitated by anti-HA or anti-N1-C-terminus antibody. The experiment showed that ZNF74 can associate with N1IC. For further dissecting the domains of ZNF74 associate with N1IC, we constructed different N- or C-terminal truncated ZNF74 fused to MBP protein for MBP pull down assay. The data showed that the zinc finger domain of ZNF74 is sufficient for binding with Ankyrin domain (Ank) of N1IC. The last part of this thesis, we tried to use SELEX (systematic evolution of ligands by exponential enrichment ) to find the target oligonucleotides binding sequence of ZNF74 protein. Due to the small sample size, the alignment data were complicated and difficult for interpretation. In conclusion, we still need more efforts to characterize the biological function of ZNF74 in K562 cell and to elucidate it’s relationship with Notch signaling pathway.
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