p53 acts as a co-repressor to regulate K14 expressionduring epidermal cell differentiation

• Main Authors: HSIN I-LUN, 辛翌綸
• Other Authors: 趙壯飛
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/75225077070333416123

碩士 === 國防醫學院 === 生物及解剖學研究所 === 96 === The p53 protein is an important transcription factor which plays a central role in cell cycle regulation and cell proliferation. During epidermal cell differentiation, the p53 protein level is decreased but the activity of p53 is increased. The Keratin 14, an epidermal basal cell marker, is also decreased during epidermal cell differentiation. Because of the similar downregulation of p53 and K14, we hypothesize that the p53 might regulate K14 in the normal epidermal cell differentiation. In our previous study, the K14 promoter activity was downregulated after co-transfected p53 with of full-length K14-p2000 or K14-p269 to H1299 cells(a p53 null cells). Because there is no p53 whole binding site on the K14 promoter, the p53 regulated K14 might through binding to other transcription factor as a co-repressor. To evaluate this hypothesis we constructed the serial deletion mutants of K14-p269 to search the response elements. We found the deletion mutant K14-p160 still could be repressed by p53. It had been known that p53 could bind sp1 to repress promoter activity of several genes. We found the mutant K14-p160, with sp1 binding-site deletion, could truly reverse the repression of p53. Furthermore, the mutant p53(△293-393) which did not interact with sp1 was also no longer to repress the activity of K14-p160. Therefore, we demonstrated that p53 as a co-repressor to downregulate K14 expression through associated with the transcription factor sp1. In order to investigate p53 downregulate endogenous K14, the C9 keratinocyte cell line with endogenous p53, △Np63 and K14 was used as the model system. We used TPA-induced differentiation of treated C9 cells to decrease the activation of K14 by △Np63. The RT-PCR analysis demonstrated over-expressing of p53 could repress K14 mRNA level in differentiated C9 cells. Finally, we used DNA affinity precipitation assay (DAPA) to confirm the p53 could interact with sp1 to bind to sp1 binding-site at K14-p160.