Transcriptional repression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 gene mediated by cis elements present in the 3'-untranslated region.
The cyclin-dependent kinase inhibitor p21WAF1/CIP1 plays a major role in the induction of G1 cell cycle arrest following DNA damage and is known to be regulated by p53-dependent and -independent pathways. Here, we show that p21WAF1/CIP1 transcription is also regulated, independently of p53, by the cis elements that are located downstream of the transcription start site. A cDNA fragment of approximately 180 bp, located 260 bases 3' to the translation termination codon of p21WAF1/CIP1 cDNA, was cloned in both the sense and antisense orientations downstream of the CMV promoter, upstream of the SV40 promoter, and both upstream and downstream of the p21WAF1/CIP1 promoter in the plasmids carrying the luciferase reporter gene. The constructs were transiently transfected in human breast carcinoma cells MCF-7 and MDA-MB-468 and a Syrian hamster smooth muscle cell line DDT1MF2 and were found to elicit 2-3-fold or higher repression of luciferase activities. By using overlapping deletions of the above 180-bp fragment, we identified a 48-bp subfragment that contains putative cis element(s) that participate in the transcriptional repression of the p21WAF1/CIP1 gene. The overlapping subfragments bind, in vitro, to specific proteins present in the nuclear extracts of MDA-MB-468 and DDT1MF2 cells. We, therefore, propose that additional mechanism(s) exist that regulate expression of the cellular p21WAF1/CIP1 and may contribute to p21WAF1/CIP1-dependent control of the cell cycle.
Rishi
A K
AK
Department of Medicine, University of Maryland School of Medicine, Baltimore 21201, USA.
Hsu
C K
CK
Li
X S
XS
Hussain
A
A
Gerald
T M
TM
Fontana
J A
JA
D000818:Animals; D006224:Cricetinae; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics*; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins; D012333:RNA, Messenger / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics* / Q000502:physiology; D013539:Simian virus 40 / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D018076:DNA, Complementary; D009687:Nuclear Proteins; D012333:RNA, Messenger; D012097:Repressor Proteins; D008156:Luciferases