Differential role for Sp1/Sp3 transcription factors in the regulation of the promoter activity of multiple cyclin-dependent kinase inhibitor genes

Cyclin-dependent kinase inhibitors play a significant role in cell cycle pr ogression and in cellular differentiation and their expression is regulated in different cellular settings. CC-rich regions in the promoter sequences of the cyclin-dependent kinase inhibitor genes p15INK4B and p21CIP1/WAF1 me diate the transcriptional response of these genes to extracellular stimuli. Similar CC-rich sequences in the promoter of the p15INK4A and p16INK4B gen e can be targeted for transcriptional inactivation by methylation of cytosi ne residues. CC-rich regions represent putative target sites for binding of the ubiquitously expressed Spl and Sp3 transcription factors. Using a comb ination of functional and biochemical studies, we analyzed the potential ro le of the Spl and Sp3 factors in the regulation of CDKI p15, p16, and p21 p romoter activities. Using transient reporter gene assays, we determined tha t Spl is a strong activator of these promoters, whereas Sp3 functions as a weak transactivator. We have identified multiple protein-binding sites in t he proximal promoter sequences of these genes by footprinting analysis. Som e of these sites are bound by Spl and Sp3, as demonstrated by gel-shift exp eriments using Sp1/Sp3-specific antibodies, permitting the demonstration th at a differential role exists for Spl and Sp3 in the regulation of the acti vity of these promoters. (C) 2000 Wiley-Liss, Inc.