In vivo structure of the cell cycle-regulated human cdc25C promoter

The cdc25C promoter is regulated during the cell cycle by the transcription al repressor CDF-1 that inhibits the activation function of upstream transc riptional activators, most notably the nuclear factor Y/CAAT box binding fa ctor (NF-Y/CBF), In this report a detailed analysis of the in vivo structur e of the cdc25C promoter was made. Micrococcus nuclease and methidiumpropyl -EDTA footprinting strongly suggest that the proximal promoter encompassing the cell cycle-dependent element/cell cycle genes homology region and the upstream NF-Y sites is organized in a positioned nucleosome throughout the cell cycle. Furthermore, structural perturbations were detected by DNase I, phenanthroline copper, and KMnO4 footprinting at the NF-Y binding sites in vivo, which is in agreement with the reported property of NF-Y to bend DNA in vitro. Similar results were obtained with the structurally and function ally related cyclin A promoter. The structural perturbations seen in DNase I and phenanthroline copper footprints were less pronounced in G(0) cells w hen compared with cycling cells. This presumably reflects a weakened in viv o interaction of NF-Y with its cognate DNA element in G(0). It is likely th at these structural perturbations, together with the reported ability of NF -Y to recruit histone acetyl transferase activity, contribute to an opened chromatin structure as a prerequisite for optimal regulation through activa tion and repression.