Activation of the p21(WAF1/CIP1) promoter independent of p53 by the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) through the Sp1 sites

Suberoylanilide hydroxamic acid (SAHA) is a novel histone deacetylase inhib itor with high potency in inducing differentiation of cultured murine eryth roleukemia cells. We have recently demonstrated that SAHA induces cell cycl e arrest and apoptosis in human breast cancer cells, accompanied by up-regu lation of the cyclin-dependent kinase inhibitor, p21(WAF1/CIP1) via a p53-i ndependent mechanism. In this study, we used p21 gene expression as a model system to elucidate the molecular mechanism(s) underlying SAHA-mediated ge ne activation. Treatment of human breast cancer cell line MICF7 cells with SAHA induced p21 mRNA as a consequence of an immediate-early gene activatio n. Moreover, SAHA activated the p21 promoter primarily through two Spl site s located at -82 and -69 relative to the transcription start site. Furtherm ore, Spl and Sp3 proteins were the major factors binding to the Spl site of the p21 promoter. However, SAHA did not alter their DNA binding activities , suggesting that SAHA mediates p21 promoter activity by a mechanism other than altering the DNA binding activities of Spl and Sp3, Further studies us ing the GAL4 luciferase assay system demonstrated that both GAL4-Sp1 and GA L4-Sp3 fusion proteins supported SAHA-mediated gene activation from a promo ter driven by five GAL4 DNA binding sites, and that GAL4-Sp3 fusion protein ,vas suppressive in the absence of SAHA treatment. Collectively, our result s suggest that SAHA activates the p21 promoter through the Spl sites, and t hat both Spl and Sp3 proteins can mediate SAHA-induced gene activation.