Selective transcription of p53 target genes by zinc finger-p53 DNA binding domain chimeras.
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 N- and 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.
Falke
D
D
Department of Pharmacology, CB# 7365, School of Medicine, University of North Carolina, Chapel Hill, North Carolina, 27599-7365, USA.
Fisher
M H
MH
Juliano
R L
RL
D000818:Animals; D001859:Bone Neoplasms / Q000378:metabolism / Q000473:pathology; D019556:COS Cells; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D002467:Cell Nucleus; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins / Q000235:genetics*; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D012516:Osteosarcoma / Q000378:metabolism / Q000473:pathology; D011401:Promoter Regions, Genetic; D021381:Protein Transport; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism*; D016335:Zinc Fingers / Q000235:genetics*; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D008156:Luciferases