A 63-base pair DNA segment containing an Sp1 site but not a canonical E2F site can confer growth-dependent and E2F-mediated transcriptional stimulation of the human ASK gene encoding the regulatory subunit for human Cdc7-related kinase.
Cdc7-Dbf4 kinase complexes, conserved widely in eukaryotes, play essential roles in initiation and progression of the S phase. Cdc7 kinase activity fluctuates during cell cycle, and this is mainly the result of oscillation of expression of the Dbf4 subunit. Therefore, it is crucial to understand the mechanisms of regulation of Dbf4 expression. We have isolated and characterized the promoter region of the human ASK gene encoding Dbf4-related regulatory subunit for human Cdc7 kinase. We have identified a 63-base pair ASK promoter segment, which is sufficient for mediating growth stimulation. This minimal promoter segment (MP), containing an Sp1 site but no canonical E2F site, can be activated by ectopic E2F expression as well. Within the 63-base pair region, the Sp1 site as well as other elements are essential for stimulation by growth signals and by E2F, whereas an AT-rich sequence proximal to the coding region may serve as an element required for suppression in quiescence. Gel shift assays in the presence of an antibody demonstrate the presence of E2F1 in the protein-DNA complexes generated on the MP segment. However, the complex formation on MP was not competed by a DHFR promoter fragment, known to bind to E2F, nor by a consensus E2F binding oligonucleotide. Gel shift assays with point mutant MP fragments indicate that a non-canonical E2F site in the middle of this segment is critical for generation of the E2F complex. Our results suggest that E2F regulates the ASK promoter through an atypical mode of recognition of the target site.
Yamada
Masayuki
M
Department of Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan.
Sato
Noriko
N
Taniyama
Chika
C
Ohtani
Kiyoshi
K
Arai
Ken-ichi
K
Masai
Hisao
H
D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D017931:DNA Primers; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D004789:Enzyme Activation; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D021122:Protein Subunits; D015183:Restriction Mapping; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D018797:Cell Cycle Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D021122:Protein Subunits; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D004247:DNA; D008156:Luciferases; C068619:CDC7 protein, human; D017346:Protein Serine-Threonine Kinases