[Identification of two new p53 target genes through implementation of the modified chromatin immunoprecipitation method and inverse PCR].
Direct target loci for the transcription factor p53 were identified through the employment of a combination of a modified version of chromosomal immunoprecipitation and inverse PCR. Irradiation of Hela cells to drive DNA damage response was followed by sequential chromosomal immunoprecipitation utilizing antibodies which recognize the large subunit of RNA polymerase II and p53. Inverse PCR with degenerate oligonucleotides specific for the p53 binding site was subsequently performed on immunoprecipitated DNA and fragments containing putative p53 target genes were subcloned and sequenced. Two sequences were identified which contain near-consensus p53 binding sites as well as recognition sites for the core transcriptional machinery including RNA polymerase II and Sp1. Cotransfections of vectors containing these sequences linked to a reporter with p53 expression vectors resulted in stimulation of transcription. Application of the technology described herein may result in the identification of target loci for a wide variety of transcription factors.
Burgess
R
R
TransGenetics Incorporated, University of California at San Diego, La Jolla, California, USA.
Luniak
V
V
Noskin
L
L
Giliano
N
N
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002843:Chromatin / Q000235:genetics / Q000302:isolation & purification*; D004249:DNA Damage / Q000528:radiation effects; D006367:HeLa Cells / Q000528:radiation effects; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011233:Precipitin Tests / Q000379:methods*; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D002843:Chromatin; D016329:Sp1 Transcription Factor; D016159:Tumor Suppressor Protein p53; D012319:RNA Polymerase II