Human Dbf4/ASK promoter is activated through the Sp1 and MluI cell-cycle box (MCB) transcription elements.
Dbf4 is the regulatory subunit of Cdc7 kinase, which is essential for entry into and traversing through S phase. The level of Dbf4, which is critical for the activation of Cdc7, is regulated by transcription and protein degradation. To gain a better understanding as to how the transcription of human Dbf4 (HuDbf4) is regulated, we have cloned and characterized its promoter. We found that HuDbf4 core promoter is localized within (-)211 to -285 of the translation start-codon. This 75 bp DNA segment contains, among others, a putative MluI Cell-cycle Box (MCB). A point mutation within the MCB dramatically reduced the promoter activity. This is the first example that an MCB element plays an essential role in the activation of a core promoter in mammalian cells. The auxiliary elements required for the full promoter activity are present within 162-bp upstream from the core promoter (i.e., -286/-447). A point mutation within the Sp1 element at -353/-361 resulted in a decrease of promoter activity to the basal level, while the deletion of the putative HES-1 at -326/-331 dramatically increased the promoter activity. Taken together, our data suggests that the MCB element is essential for the core promoter activation, while the Sp1 positive regulator and the HES-1 repressor coordinately determine the efficiency of the HuDbf4 promoter. We have also found: (i) that the major transcription initiations occur at -220, -235 and -245; (ii) that HuDbf4 gene consists of 12 exons, which spread over a 33-kb region.
Wu
Xing
X
Northeastern Ontario Regional Cancer Centre, Sudbury, Ontario P3E 5J1, Canada.
Lee
Hoyun
H
D001426:Bacterial Proteins / Q000378:metabolism; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites; D018797:Cell Cycle Proteins; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005091:Exons / Q000235:genetics; D015723:Gene Library; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D016296:Mutagenesis; D016366:Open Reading Frames; D017354:Point Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D021122:Protein Subunits; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism; D000072056:Transcription Factor HES-1; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D001426:Bacterial Proteins; D051792:Basic Helix-Loop-Helix Proteins; D018797:Cell Cycle Proteins; D004247:DNA; D018076:DNA, Complementary; D015252:Deoxyribonucleases, Type II Site-Specific; D018398:Homeodomain Proteins; D021122:Protein Subunits; D012097:Repressor Proteins; D016329:Sp1 Transcription Factor; D000072056:Transcription Factor HES-1; D014157:Transcription Factors; C485678:DBF4 protein, human; C489734:Hes1 protein, mouse; C078493:HES1 protein, human; C035041:endodeoxyribonuclease MluI