The inhibition of cyclin B1 gene transcription in quiescent NIH3T3 cells is mediated by an E-box.
Cyclin Bl plays an important role in cell proliferation. Its expression is tightly regulated at the mRNA and protein levels during the cell cycle and is found to be deregulated in various malignancies. To enlighten the signalling pathways which lead to the cell cycle dependent expression of the cyclin B1 gene, we studied its transcriptional regulation in quiescent and proliferating NIH3T3 cells. We previously showed that the transcriptional activity of the cyclin B1 promoter decreases in quiescent cells. Here, we map a quiescence-responsive element of the human cyclin B1 promoter to an E-box sequence, CACGTG, which spans positions -124/-119. Nuclear proteins protect this sequence in a DNase I digestion assay and bind, in electromobility shift assays, an oligonucleotide spanning positions -133/-110. Max-specific antibodies block the DNA-binding activity of protein complexes to this probe. A mutation in the E-box core sequence abolishes the decrease in transcription that occurs during quiescence. Finally, we find that over-expression of Max protein in proliferating cells specifically inhibits cyclin B1 promoter activity through this E-box. Moreover, Max over-expression in proliferating NIH3T3 cells leads to down-regulation of the endogenous cyclin B1 protein. In conclusion, these data support a model whereby E-box-binding proteins mediate the decrease in the transcriptional activity of the cyclin B1 promoter observed in quiescent cells and suggest that Max contributes to this response.
Farina
A
A
Laboratorio di Oncogenesi Molecolare, CRS-IRE, Rome, Italy.
Gaetano
C
C
Crescenzi
M
M
Puccini
F
F
Manni
I
I
Sacchi
A
A
Piaggio
G
G
D016475:3T3 Cells / Q000166:cytology / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D002455:Cell Division / Q000502:physiology; D019926:Cyclin B; D056744:Cyclin B1; D016213:Cyclins / Q000096:biosynthesis* / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D051379:Mice; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014157:Transcription Factors; D014158:Transcription, Genetic
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; C534699:CCNB1 protein, human; C534700:Ccnb1 protein, mouse; D019926:Cyclin B; D056744:Cyclin B1; D016213:Cyclins; D004268:DNA-Binding Proteins; C499330:MAX protein, human; C067850:Myc associated factor X; D009687:Nuclear Proteins; D014157:Transcription Factors; C068422:Max protein, mouse