Site-directed mutagenesis of the ATM promoter: consequences for response to proliferation and ionizing radiation.
Although ATM, the protein defective in ataxia-telangiectasia (A-T), is activated primarily by radiation, there is also evidence that expression of the protein can be regulated by both radiation and growth factors. Computer analysis of the ATM promoter proximal 700-bp sequence reveals a number of potentially important cis-regulatory sequences. Using nucleotide substitutions to delete putative functional elements in the promoter of ATM, we examined the importance of some of these sites for both the basal and the radiation-induced activity of the promoter. In lymphoblastoid cells, most of the mutations in transcription factor consensus sequences [Sp1(1), Sp1(2), Cre, Ets, Xre, gammaIre(2), a modified AP1 site (Fse), and GCF] reduced basal activity to various extents, whereas others [gammaIre(1), NF1, Myb] left basal activity unaffected. In human skin fibroblasts, results were generally the same, but the basal activity varied up to 8-fold in these and other cell lines. Radiation activated the promoter approximately 2.5-fold in serum-starved lymphoblastoid cells, reaching a maximum by 3 hr, and all mutated elements equally blocked this activation. Reduction in Sp1 and AP1 DNA binding activity by serum starvation was rapidly reversed by exposure of cells to radiation. This reduction was not evident in A-T cells, and the response to radiation was less marked. Data provided for interaction between ATM and Sp1 by protein binding and co-immunoprecipitation could explain the altered regulation of Sp1 in A-T cells. The data described here provide additional evidence that basal and radiation-induced regulation of the ATM promoter is under multifactorial control.
Gueven
Nuri
N
Queensland Cancer Fund Research Laboratory, The Queensland Institute of Medical Research, Royal Brisbane Hospital, Brisbane, Queensland, Australia.
Keating
Karen
K
Fukao
Toshiyuki
T
Loeffler
Heidi
H
Kondo
Naomi
N
Rodemann
H Peter
HP
Lavin
Martin F
MF
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D001665:Binding Sites / Q000235:genetics / Q000528:radiation effects; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000235:genetics / Q000528:radiation effects; D002460:Cell Line; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D005720:Gamma Rays; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D016297:Mutagenesis, Site-Directed / Q000235:genetics* / Q000528:radiation effects*; D011401:Promoter Regions, Genetic / Q000235:genetics* / Q000528:radiation effects*; D011485:Protein Binding / Q000235:genetics / Q000528:radiation effects; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics / Q000528:radiation effects; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000528:radiation effects; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D014709:Vero Cells
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D016329:Sp1 Transcription Factor; D018808:Transcription Factor AP-1; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases