NF-Y-mediated trans-activation of the human thymidine kinase promoter is closely linked to activation of cyclin-dependent kinase.
Transcriptional activation is important for the elevated expression of human thymidine kinase (hTK) in tumor cells. Here, we used TK(-133/+33)-luciferase reporter gene construct and bandshift assay to study the cis-elements involved in transcriptional activation of the hTK promoter. We found that two CCAAT boxes at -71/-67 and -40/-36 and Sp1 binding site located at -118/-113 were critical for maximal expression of the hTK promoter activity. As Sp1-mediated activation of the hTK promoter was not detectable for the promoter construct with double mutations at two CCAAT boxes, we proposed that NF-Y binding to the hTK promoter sequence is a requisite step for the functional interaction with Sp1. Here, we further showed that the hTK promoter activity was reduced in HeLa cells transfected with p16 or p21, both of which are inhibitors of cyclin-dependent kinases (CDKs). Inhibition of the hTK promoter activity by p16 could be abrogated by overexpression of cyclin A, indicating that the cyclin A activating event is more directly involved in transcriptional activation of the hTK promoter. We thus proposed that NF-Y-mediated activation of the hTK promoter is closely linked to the activation of CDK2/cyclin A pathway.
Chang
Z F
ZF
Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China.
Huang
D Y
DY
Hu
S F
SF
D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D019925:Cyclin A / Q000494:pharmacology; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000494:pharmacology; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter; D018922:HL-60 Cells; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D013937:Thymidine Kinase / Q000235:genetics*; D013997:Time Factors; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
D022762:CCAAT-Enhancer-Binding Proteins; C493956:CDKN1A protein, human; D019925:Cyclin A; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D015534:Trans-Activators; D008156:Luciferases; D013937:Thymidine Kinase; D018844:Cyclin-Dependent Kinases