Protein-DNA Interactions in the Transcriptional Regulation of Eukaryotic Genes

• Main Authors: Yee Guang-Yau, 易光燿
• Other Authors: Kuang-Yu Hu
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/42651704630628579724

碩士 === 國防醫學院 === 生物化學研究所 === 86 === Thymidine kinase, a crucial enzyme in the salvage pathway of thymidine triphosphate formation, is involved in the process of DNA replication. The level of thymidine kinase(TK) is known to be increased at the G1/S phase of the cell cycle. Transcriptional activation is important for the increase of human thymidine kinase(hTK) expression at the G1/S phase of the cell cycle . In this study, in order to examine the DNA sequence responsible for the transcriptional activation of hTK gene in young cells during growth stimulation and transcriptional repression in old ones, the interaction between nuclear protein factors and hTK promoter was examined by in vivo footprinting in young and old IMR-90 cells, respectively. Results from in vivo footprinting of hTK promoter in young and old IMR-90 cells showed that NF-Y binding sites could be one of key cis-elements responsible for transcriptional activation of hTK gene. In addition, in vivo footprints of old IMR- 90 cells on the DNA sequence between two adjacent NF-Y binding sites, CCAAT boxes, were apparently different from those of young cells. Thus, CCAAT-binding proteins might be associated ageing. On the other hand, in order to understand the molecular basis underlying the E1A-mediated transcriptional repression of the c-erbB-2 overexpression in human cancer cells, we compared the nuclear factor-binding patterns of c-erbB-2 promoter in vivo in cell lines with and without exogenous E1A expression. However, we did not observe any difference of nuclear factor-binding in vivo of E1A-non-expressing cells versus E1A-expressing cells. Therefore, E1A might repress the transcriptional overexpression of c-erbB-2 gene through protein-protein interactions with some endogenous factors. Furthermore, in order to show the correlation between the distribution of Sp1 elements and the demethylation patterns in the CpG islands of the human globin gene, we have analyzed the methylation patterns of human globin promoter and its interaction with Sp1 factor by genomic sequencing and in vivo footprinting, respectively. HeLa cells with high passage number didn't show significant protein- DNA interactions at Sp1 binding sites within the human globin gene as K562 cells did and its genomic DNA was highly methylated downstream of the cap site (+1). This indicated that Sp1 sites in human adult globin gene promoters might correlate to demethylation of erythroid-specific CpG islands.