Regulation of the catalase gene promoter by Sp1, CCAAT-recognizing factors, and a WT1/Egr-related factor in hydrogen peroxide-resistant HP100 cells.
Reactive oxygen species play a critical role in the onset of apoptosis induced by various extracellular stimuli, including ionizing radiation. Therefore active regulation of reactive oxygen species-metabolizing enzymes may be one response to an apoptotic stimulus. In this regard, HP100 cells, H(2)O(2)-resistant variants derived from human leukemia HL60 cells, display an interesting phenotype in which the activity of catalase is constitutively high, whereas its mRNA is reduced after X-ray irradiation. In the present study, we investigated the molecular mechanisms underlying this phenomenon. By combining analyses from nuclear run-on, reporter gene transient transfection, genomic footprinting, site-directed mutagenesis, electrophoretic mobility shift analysis, and Western blotting experiments, we found that constitutively elevated catalase expression is strongly regulated at the transcriptional level by both Sp1 and CCAAT-recognizing factors and that much higher levels of nuclear Sp1 and NF-Y are present in HP100 nuclei as compared with HL60 nuclei. In addition, we demonstrated an X-ray-inducible association of a WT1/Egr-related factor with an overlapping Sp1/Egr-1 recognition sequence located within the core promoter of the catalase gene. This association may lead to inactivation of the promoter by disturbing or competing with the transactivating ability of Sp1.
Nenoi
M
M
Radiation Hazards Research Group, National Institute of Radiological Sciences, 9-1, Anagawa-4-chome, Inage-ku, Chiba 263-8555, Japan. m_nenoi@nirs.go.jp
Ichimura
S
S
Mita
K
K
Yukawa
O
O
Cartwright
I L
IL
D001483:Base Sequence; D023081:CCAAT-Binding Factor / Q000502:physiology*; D002374:Catalase / Q000096:biosynthesis / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology*; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D051766:Early Growth Response Protein 1; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D020868:Gene Silencing / Q000502:physiology / Q000528:radiation effects; D005809:Genes, Regulator / Q000235:genetics; D018922:HL-60 Cells / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity*; D017874:Immediate-Early Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*; D015533:Transcriptional Activation / Q000502:physiology; D025721:WT1 Proteins
D023081:CCAAT-Binding Factor; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D025721:WT1 Proteins; D006861:Hydrogen Peroxide; D002374:Catalase