CHF: a novel factor binding to cyclin A CHR corepressor element

Cell cycle modulation of cyclin A expression is due to the periodic relief of a transcriptional repression mediated by a bipartite negative DNA regula tory region. The 5' element (Cell Cycle Responsive Element: CCRE; cell CYcl e Dependent Element: CDE) is clearly occupied in a cyclic manner in vivo, w hereas the 3' element, whose sequence is shared by B-myb, cdc25C and cdc2 g enes (cell Cycle gene Homology Region: CHR), is involved in more subtle int eractions. Mutation of either element results in complete deregulation of c yclin 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 u nknown. In the present work me have characterized an activity present in qu iescent cells and absent in cells blocked in S phase, which binds specifica lly 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 th rough 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 tha t a functional difference exists between the composite CCRE/CDE-CHR repress or regions of cell cycle regulated genes such as cyclin A and cdc25C.