The role of NF‐Y in the regulation of Op18 expression

Op18 (oncoprotein 18/Stathmin) is an important mitotic regulator that is highly expressed in many cancers. It functions to destabilize microtubules. Anti‐sense mediated down‐regulation of Op18 has been shown to inhibit cellular proliferation. However, the transcriptional mechanisms responsible for its normal regulation and for its high level of expression in proliferating cells remain poorly understood. In this study we have characterized transcription factor binding sites for the ubiquitously expressed CCAAT‐binding factor NF‐Y. The Op18 gene contains four putative NF‐Y sites located at −980 (site 1), −745 (site 2), −599 (site 3) and −65 (site 4) relative to the transcription start site. NF‐Y has been implicated in the regulation of cell cycle controlled genes. We have used promoter‐reporter assays and EMSA analysis to functionally examine each of these putative NF‐Y sites. Mutagenesis studies indicate that site 1 and 3 are repressive sites, whereas site 2 and 4 are stimulatory. EMSA analysis shows that only sites 3 and 4 are competent to bind NF‐Y. Functionally, site 3 is a repressive site and site 4 is a stimulatory site. Mutagenesis of both sites 3 and 4 yield a reporter activity that falls between the individual mutations, suggesting that they perform opposing functions. EMSA competition analysis indicates that the stimulatory site 4 is a high affinity site as compared to site 3. These results suggest that NF‐Y can function as both an activator and a repressor of Op18 expression.