Sp1 and Sp3 activate p21 (WAF1/CIP1) gene transcription in the Caco-2 colon adenocarcinoma cell line Andrei L Gartel* ,1 , Eugene Goufman 1 , Feridoon Najmabadi 1 and Angela L Tyner 1 1 Department of Molecular Genetics, University of Illinois at Chicago, 900 S. Ashland Avenue, Chicago, Illinois, IL 60607, USA The CDK inhibitor p21 WAF1/CIP1 is a negative regulator of the cell cycle, and its expression is induced during terminal dierentiation in vitro and in vivo. Expression of p21 is controlled at the transcriptional level by both p53-dependent and -independent mechanisms. Our pre- vious studies established that p21 is expressed in the Caco-2 adenocarcinoma cell line, and its expression is induced by a p53-independent mechanism during dier- entiation of these cells. Here we have found that transcription of p21 in Caco-2 cells is controlled primarily by the transcription factors Sp1 and Sp3 through two Sp1 binding sites, Sp1-1 and Sp1-2, located between 7119 and 7114 bp and between 7109 and 7104 bp of the p21 promoter, respectively. Sp1 and Sp3 binding to the p21 promoter increased during Caco-2 cell dierentiation, while the absolute level of Sp1 did not change and the absolute level of Sp3 increased approximately twofold. Transfection experiments in the SL2 Drosophila cell line that lacks endogenous Sp3 activity demonstrated that Sp1 transactivates the p21 promoter primarily through the Sp1-2 site, while Sp3 acts through the Sp1-1 site. In these cells Sp3 is a stronger transactivator of the p21 promoter than Sp1. Our data suggest that induction of p21 transcription during Caco-2 dierentiation is modulated by Sp1/Sp3 interactions with the p21 promoter. Oncogene (2000) 19, 5182 ± 5188. Keywords: p21; Caco-2; promoter; Sp1; Sp3 Introduction p21 WAF1/CIP1 binds and inhibits cyclin/cyclin-dependent kinase (Cdk) complexes resulting in cell cycle arrest (El-Deiry et al., 1993; Harper et al., 1993; Xiong et al., 1993; Jiang et al., 1994; Noda et al., 1994). Regulation of p21 transcription is controlled by both p53- dependent and -independent pathways (reviewed in Gartel and Tyner, 1999). DNA damage activates p21 transcription in a p53-dependent manner in most tissues through two p53 binding sites within the p21 promoter (Macleod et al., 1995). Cultured p21-de®cient mouse embryonic ®broblasts were compromised in their ability to undergo G1 arrest in response to DNA damage (Brugarolas et al., 1995; Deng et al., 1995). G1 arrest coincides with terminal dierentiation, and p21 expression is activated by p53-independent mechanisms during terminal dierentiation in a number of cell types (Jiang et al., 1994; Steinman et al., 1994; Halevy et al., 1995; Macleod et al., 1995; Parker et al., 1995; Gartel et al., 1996a) (reviewed in Gartel et al., 1996b; Gartel and Tyner, 1998). The region between 7119 basepairs (bp) and the start of transcription of the human p21 gene contains six Sp1 binding sites (Sp1-1 to Sp1-6), and plays a major role in the regulation of p21 transcription (Figure 1c) (reviewed in Gartel and Tyner, 1999). Sp1 is a member of a multigene family that binds DNA through C-terminal zinc-®nger motifs. Sp2, Sp3, and Sp4 share extensive structural and sequence homology with Sp1 (Kennett et al., 1997). Sp1 mediates induction of the p21 gene through the Sp1-1 and Sp1-2 sites in response to phorbol ester (PMA) and okadaic acid (Biggs et al., 1996). The Sp1-3 site in the p21 promoter has been shown to be required for p21 induction by transforming growth factor-b (TGF-b) (Datto et al., 1995), Ca 2+ (Prowse et al., 1997), butyrate (Nakano et al., 1997), lovastatin (Lee et al., 1998), NGF (Billon et al., 1999) and the histone deacetylase inhibitor, trichostatin A (TSA) (Sowa et al., 1997). Sp3, but not Sp1, has been shown to mediate transactivation of the p21 promoter by TSA (Sowa et al., 1999). It appears that TSA relieves histone deacetylase 1 (HDAC1)-mediated transcriptional repression that targets Sp3 on the p21 promoter (Doetzlhofer et al., 1999). TGF-b, TSA and butyrate inhibit proliferation and induce G1 cell cycle arrest via p21 activation in various cell types (Datto et al., 1995; Nakano et al., 1997; Sowa et al., 1997). Interaction of Sp1 with Smad proteins mediates induction of the p21 promoter by TGF-b in human hepatoma cells (Li et al., 1998; Moustakas and Kardassis, 1998). The transcriptional co-activators p300 and CBP (CREB-binding protein), which possess histone acetyl transferase activity, cooperate with Sp1/Sp3 to induce expression from the p21 promoter after dierent stimuli. For example addition of nerve growth factor (NGF) to PC12 cells induces neuronal dierentiation and p21 expression (Billon et al., 1996; Yan and Zi, 1997). NGF induces p21 transcription through regula- tion of p300 transcriptional coactivator activity and p300 cooperates with Sp1 for transactivation of the p21 promoter (Billon et al., 1999). Induction of the p21 promoter during Ca 2+ -induced dierentiation of cul- tured mouse keratinocytes is also dependent on p300 (Missero et al., 1995). Since Sp3 has been shown to be responsible for induction of the p21 promoter by calcium (Prowse et al., 1997), it is tempting to speculate that p300 interacts with Sp3 for induction of the p21 promoter. The progesterone receptor (PR) has been found in complexes with CBP/p300 and Sp1 that are required for induction of the p21 promoter by progesterone via the Sp1-3 and Sp1-4 sites of the p21 promoter (Owen et al., 1998). c-Jun can physically Oncogene (2000) 19, 5182 ± 5188 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc *Correspondence: AL Gartel Received 9 November 1999; revised 4 August 2000; accepted 31 August 2000