Involvement of Oct-1 in the regulation of CDKN1A in response to clinically relevant doses of ionizing radiation.
CDKN1A is a cyclin-dependent kinase inhibitor that plays a critical role in cell cycle checkpoint regulation. It is transcriptionally induced by TP53 (p53) following exposure to ionizing radiation (IR). Induction of CDKN1A after irradiation is closely related to IR-sensitivity of tumor cells, but the underlying mechanisms remain obscure because conventional reporter gene systems respond poorly to IR unless hyperlethal doses are used. Here, we performed a promoter analysis of the CDKN1A gene following irradiation with clinically relevant doses of IR using the adeno-associated virus-mediated reporter system which we have recently shown to be highly responsive to IR. We demonstrate that there are regulatory elements at -1.1 kb, -1.4 kb, and -1.8 kb, and deletion of these elements attenuate induction of the CDKN1A gene promoter in response to 0.2-2.0 Gy of IR. EMSA and ChIP assays showed that Oct-1 binds constitutively to the elements at -1.1 kb and -1.8 kb. Functional involvement of Oct-1 was confirmed by RNA interference targeting the Oct-1 gene, which suppressed both the basal and IR-inducible components of the CDKN1A expression. Thus, our results reveal that Oct-1 is crucial to the TP53-mediated regulation of the CDKN1A gene promoter following exposure to clinically relevant doses of IR.
Nenoi
Mitsuru
M
Radiation Effect Mechanisms Research Group, National Institute of Radiological Sciences, 9-1, Anagawa-4-chome, Inage-ku, Chiba 263-8555 Japan. m_nenoi@nirs.go.jp
Daino
Kazuhiro
K
Nakajima
Tetsuo
T
Wang
Bing
B
Taki
Keiko
K
Kakimoto
Ayana
A
D000230:Adenocarcinoma / Q000473:pathology; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics* / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D004307:Dose-Response Relationship, Radiation; D005260:Female; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D050811:Octamer Transcription Factor-1 / Q000378:metabolism / Q000502:physiology*; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011829:Radiation Dosage; D011839:Radiation, Ionizing; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D050759:Cyclin-Dependent Kinase Inhibitor p21; D050811:Octamer Transcription Factor-1; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; D008156:Luciferases