Cell cycle modulation of cyclin A expression is due to the periodic relief of a transcriptional repression mediated by a bipartite negative DNA regulatory region. The 5′ element (Cell Cycle Responsive Element: CCRE; cell Cycle Dependent Element: CDE) is clearly occupied in a cyclic manner in vivo, whereas the 3′ element, whose sequence is shared by B-myb, cdc25C and cdc2 genes (cell Cycle gene Homology Region: CHR), is involved in more subtle interactions. Mutation of either element results in complete deregulation of cyclin A promoter activity. Whereas some reports claim that E2F/DP can bind to the CCRE/CDE, the nature of the protein(s) interacting with the CHR is unknown. In the present work we have characterized an activity present in quiescent cells and absent in cells blocked in S phase, which binds specifically to cyclin A CHR, but not to B-myb, or to cdc25C, or to cdc2 CHRs. A 90 kD protein, named CHF (cyclin A CHR binding factor), has been identified through preparative electrophoresis and UV crosslinking experiments. In order to address in more functional terms the binding of CHF to cyclin A CHR, we developed in vitro and in vivo oligonucleotide competition assays. Both in vitro transcription and in vivo microinjection experiments demonstrate that a functional difference exists between the composite CCRE/CDE-CHR repressor regions of cell cycle regulated genes such as cyclin A and cdc25C.
CHF: a novel factor binding to cyclin A CHR corepressor element
Philips, A.; Chambeyron, S.; Lamb, N.; Vie, A.; Blanchard, J. M.
1999-11-04 / vol 18 / pages 6222-32
Humans; Animals; Mice; *Transcription, Genetic; Molecular Sequence Data; Base Sequence; Binding Sites; Electrophoresis, Polyacrylamide Gel; *Regulatory Sequences, Nucleic Acid; Binding, Competitive; G0 Phase; *Promoter Regions (Genetics); Macromolecular Substances; Cyclin A/*genetics; Cell Cycle/*genetics; Chromatography, Gel; Hydroxyurea/pharmacology; Microinjections; Molecular Weight; S Phase/drug effects; T-Lymphocytes/drug effects/*metabolism; Transcription Factors/*isolation & purification/metabolism