Selective transcription of p53 target genes by zinc finger-p53 DNA binding domain chimeras
Selective transcription of p53 target genes by zinc finger-p53 DNA binding domain chimeras
复制标题
DOI：
10.1016/j.bbaexp.2004.09.011
复制
发表时间：
2004-11-24
期刊：
影响因子：
--
通讯作者：
Juliano, RL
中科院分区：
文献类型：
--
作者：
Falke, D;Fisher, MH;Juliano, RL
活性p53刺激许多关键基因的转录，包括促凋亡基因bax以及细胞周期调节因子p21。在这项研究中，我们构建了新的嵌合锌指-p53 DNA结合域（DBD）转录因子的设计结合到特定的p53调控基因的启动子。为了选择性地增加Bax的表达，我们将靶向Bax启动子中的序列的预先选择的三锌指（Zif）肽与最小的p53 DBD偶联。这种嵌合蛋白可以增加报告基因转录从一个最小的bax启动子（高达10倍），但不是从一个最小的p21启动子在p53缺陷的Saos-2细胞。然而，携带较长p53 DBD的融合蛋白显示出完全不同的选择性和效力。因此，Zif-p53 DBD嵌合体含有N和C-末端延伸的最小DBD可以增加转录驱动的最小p21启动子高达800倍。这些嵌合体优选最小p21启动子高达500倍以上的最小bax启动子。此外，在表达p21选择性Zif-p53 DBD嵌合体的细胞中内源性p21信使和蛋白水平增加，并且嵌合蛋白的表达导致部分细胞周期停滞。细胞分级实验表明，Zifs增强了Zif-p53 DBD嵌合体的核定位。这些研究表明，有可能产生能够强烈和选择性地激活p53下游基因的嵌合转录因子。(C)2004 Elsevier B.V.保留所有权利。
Active p53 stimulates the transcription of a number of key genes, including the pro-apoptotic gene bax, as well as p21, a cell cycle regulator. In this study we constructed novel chimeric zinc finger-p53 DNA binding domain (DBD) transcription factors designed to bind to the promoters of specific p53 regulated genes. In order to selectively increase the expression of Bax, we coupled a pre-selected three-zinc finger (Zif) peptide targeted to a sequence in the bax promoter to a minimal p53 DBD. This chimeric protein could increase reporter gene transcription from a minimal bax promoter (up to 10-fold) but not from a minimal p21 promoter in p53-deficient Saos-2 cells. However, fusion proteins carrying longer p53 DBDs displayed entirely different selectivity and potency. Thus, Zif-p53 DBD chimeras containing Nand C-terminal extensions of the minimal DBD could increase transcription driven by a minimal p21 promoter up to 800-fold. These chimeras preferred the minimal p21 promoter up to 500-fold over the minimal bax promoter. Additionally, endogenous p21 message and protein levels were increased in cells expressing the p21 selective Zif-p53 DBD chimera and expression of the chimeric proteins resulted in partial cell cycle arrest. Cell fractionation experiments indicated that the Zifs enhanced nuclear localization of the Zif-p53 DBD chimera. These studies suggest that it is possible to create chimeric transcription factors able to strongly and selectively activate genes downstream of p53. (C) 2004 Elsevier B.V. All rights reserved.