Abstract 144 Cardiac Development and Gene Regulation Platform, Sunday, 5/2

Efforts toward myocardial regeneration have been limited by the inability of post-neonatal myocytes to enter mitosis and complete the cell cycle. We recently identified human Cdc5 (hCdc5) as the first mammalian transcription factor to positively regulate G2 progression and mitotic entry ( Bernstein et al. (1997) J Biol Chem 272:5833; Bernstein et al. (1998) J Biol Chem 273:4666). hCdc5 contains tandem repeats of a helix-turn-helix DNA binding domain, similar to that seen in Myb-family members, however, the binding specificity of hCdc5 and Cdc5-like proteins is distinct. Aside from its role in mitotic entry, little else is known about the downstream effectors of hCdc5 and its homologs. We have expressed the amino terminus of hCdc5, including the DNA binding domain, as a His6-fusion protein in E. coli, and demonstrated that this region dimerizes under physiological conditions. Using the recombinant protein, we have performed cyclic amplification and selection of potential DNA binding sites from a pool of random oligonucleotides and have identified consensus sequences which bind hCdc5 through its helix-turn-helix domain, as confirmed by electrophoretic mobility supershift assay. Concurrently, we have devised a genetic screen in yeast to identify human genomic sequences that interact with the amino terminus of hCdc5, and have found some of these to contain the consensus sequences identified from the random oligomer pool. These studies provide the first step toward identifying downstream targets of the Cdc5 family, and ultimately may provide reagents with which to manipulate the cell cycle in non-dividing myocytes.