The expression of DNMT1, the major maintenance DNA methyltransferases, is critical in coordinating DNMT1 activity with biological processes and therefore must be tightly regulated in the cell cycle. Here, we report p21(WAF1) as a novel upstream regulator of DNMT1 expression. Ectopic expression of p21(WAF1) or TSA-mediated p21(WAF1) induction inhibits DNMT1 at the transcriptional level, and this observation consistently coincides with a reduction in p300. Furthermore, siRNA-mediated p300 knockdown significantly abolishes DNMT1 mRNA levels, demonstrating the dependence of DNMT1 expression on p300. Consistent with this, p300 enhances transactivation of DNMT1 promoter 340 bp upstream of the initiation start site harboring the E2F1 and Sp1/3 binding sites. Collectively, we identified p300 as a crucial transcription regulator for DNMT1. We proposed that the reduction in p300 following p21(WAF1) up-regulation contributes to DNMT1 down-regulation. This novel p21(WAF1)-p300-DNMT1 pathway may play a pivotal role to ensure regulated DNMT1 expression and DNA methylation in mammalian cell division. (C) 2009 Elsevier Inc. All rights reserved.