E2F SITE ACTIVATES TRANSCRIPTION IN FISSION YEAST SCHIZOSACCHAROMYCES-POMBE AND BINDS TO A 30-KDA TRANSCRIPTION FACTOR

The mammalian transcription factor E2F binds to several cellular prote ins including Rb, p107, cyclin A, cyclin E, and p33cdk2 protein kinase in a stage-specific manner during cell cycle. Its recognition sequenc e, TTTCGCGC, is present in two of the human adenovirus early promoters and in several promoters of cellular genes whose products are implica ted in the control of cell proliferation. These observations suggest t hat E2F may play an important role in cell-cycle regulation and prompt ed us to ask whether E2F-like activities are present in yeast. We foun d that the E2F motif can function as an activating sequence in Schizos accharomyces pombe when cloned upstream of a reporter gene. Consistent with this, the expression of adenovirus E2 promoter in S. pombe was d ependent on both E2F motifs of this promoter. A protein, spE2F, that b inds to the E2F site was partially purified from S. pombe using DNA-af finity chromatography. The binding specificity of this protein was com pared to that of human E2F using a number of mutant E2F sites as compe titors. These studies showed that spE2F recognizes a sequence closely related to the E2F site. Ultraviolet cross-linking and Southwestern bl ot studies indicated that the molecular size of spE2F is 30 kDa. Previ ous studies have shown that a cis-acting element, ACGCGTNA, also calle d MluI cell cycle box, or MCB, is critical for the regulated expressio n of cell cycle related genes both in fission and budding yeast. In S. pombe, the cdc10 gene product binds to this element and controls the cell cycle related genes. Electrophoretic mobility shift assays and mo lecular size determination studies indicated that spE2F is different f rom that encoded by cdc10. Thus, our studies suggest that spE2F is a n ovel transcription factor. We discuss these results in light of recent observations about the periodically expressed genes involved in the c ell cycle progression in yeast.