title: Positive and negative regulation of the human thymidine kinase promoter mediated by CCAAT binding transcription factors NF-Y/CBF, dbpA, and CDP/cut.
abstract: The proximal CCAAT element located 38 bp upstream of the transcription initiation site contributes to the human thymidine kinase (htk) promoter activity, because site-directed mutagenesis of a 10-bp region containing this CCAAT motif (TKC1) reduced the promoter activity by 55%. Through binding site competitions and antigenic cross-reactivity, the major factor that binds TKC1 from both HeLa and hamster nuclear extracts is identified as NF-Y/CBF. In serum-stimulated cells, the binding of NF-Y/CBF to TKC1 increased gradually, reaching a plateau at the S phase. In cell transfection assays, a dominant-negative mutant of NF-Y/CBF inhibited the htk promoter in a dosage-dependent manner, providing direct evidence that NF-Y/CBF is required for maximal htk promoter activity. Recently, it has been demonstrated that the site occupied by NF-Y/CBF also binds the serum-inducible dbpA and dpbB. We show here that recombinant dbpA interacts with the htk promoter, and overexpression of dbpA can stimulate htk promoter activity mediated through TCK1. In contrast, CDP/cut, the CCAAT displacement protein with known repressor property, binds the htk promoter through both the proximal and distal CCAAT elements. Our discovery that CDP/cut binds the htk promoter primarily in quiescent cells and that overexpression of CDP/cut inhibits htk promoter activity provides an explanation for the reported dramatic increase in htk promoter activity in serum-starved cells when both CCAAT elements were mutated. Thus, a combination of suppression in quiescent cells and activation in serum-stimulated cells mediated through various CCAAT-binding proteins may account in part for the induction of htk promoter activity as quiescent cells reenter the cell cycle.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern California School of Medicine, Los Angeles 90033-0800, USA.
authors.forename: E C
authors.initials: EC
authors.lastname: Kim
authors.forename: J S
authors.initials: JS
authors.lastname: Lau
authors.forename: S
authors.initials: S
authors.lastname: Rawlings
authors.forename: A S
authors.initials: AS
authors.lastname: Lee
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D016466:CHO Cells; D002352:Carrier Proteins; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000378:metabolism; D018398:Homeodomain Proteins; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism*; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; C080632:CUX1 protein, human; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C094867:YBX3 protein, human; D013937:Thymidine Kinase; C092717:thymidine kinase 1