TI - Dnmt1 core promoter activity in oncogenic transformed cells . AB - As shown in Figure 1C , Dnmt1 mRNA increased approximately 2-fold in transformed cells . To examine whether this accumulation of Dnmt1 mRNA reflects altered promoter activity , we transfected the luciferase reporter construct containing the Dnmt1 core promoter ( 300-luc in Fig 3A ) into transformed 3Y1 cells . All transformed cells showed atleast 30 times more transctiptional activity than parental 3Y1 cells (Fig 4A) , indicating that the promoter activity is increased in the transformed cells . E2F trans-activity is inactivated by binding to Rb , but it is activated by releasing Rb on PHOSphorylation of Rb by Rb kinases ( 24 ) . The Rb-E2F complex is also disrupted by SV40 large T antigen or expression of a pocket domain mutant form of Rb which cannot bind to E2F ( 24 ) . To determine whether up-regulation of Dnmt1 mRNA is dependent on E2F binding to site A and site C in the promoter , we measured luciferase activity of mutated Dnmt1 core promoters in SV-3Y1 cells which have been transformed with SV40 (Fig 4B) . In SV-3Y1 cells , the wild-type Dnmt1 promoter had 30-fold greater promoter activity than parental 3Y1 cells (Fig 4A) . The constructs containing mutations in site A (300A) , site C ( 300C ) or in both ( 300AC ) showed lower promoter activity than the wild-type construct ( 300 ) in SV-3Y1 cells (Fig 4B) . These results indicate that Dnmt1 transcription may be activated by SV40 large T antigen through sites A and C . Therefore , transcription of Dnmt1 is likely activated by E2F transcription factors binding to sites A and C in the promoter , and both putative E2F binding sites A and C are required to enhance promoter activity in SV-3Y1 cells . These observations are unlikely to result from differences between species , because the core promoter region is highly conserved across mouse and rat Dnmt1 (Fig 4C) . Interestingly , the sequence of site C in mouse Dnmt1 matches exactly with that of site C in rat , but the sequence of site A differs between mouse and rat . Taken together , our findings suggest that Dnmt1 transcription is controlled through two cis-elements in both normal and transformed cells . It is possible , however , that factors other than E2F are responsible for activation of Dnmt1 expression through these cis-elements .