A novel regulatory element in the dnmt1 gene that responds to co-activation by Rb and c-Jun Andrew Slack a,1 , Marc Pinard a,1 , Felipe D. Araujo b , Moshe Szyf a, * a Department of Pharmacology and Therapeutics, McGill University, 3655 Drummond Street, Montreal PQ H3G 1Y6V, Canada b Department of Biochemistry and McGill Cancer Center, 3655 Drummond Street, Montreal PQ H3G 1Y6V, Canada Received 18 December 2000; received in revised form 19 February 2001; accepted 6 March 2001 Received by E. Boncinelli Abstract Rb, c-Jun and dnmt1 play critical roles in the process of cellular differentiation. We demonstrate that a regulatory region of murine dnmt1 contains an element which is responsible for transactivation by Rb and c-Jun in P19 embryocarcinoma cells which is not observed in Y1 adrenocarcinoma cells. During differentiation of P19 cells, the induction of Rb and c-Jun coincides with an increase of dnmt1 mRNA. Using linker scanning mutagenesis we identify the element that is responsible for this activation to be a non-canonical AP-1 site. Our data is an example of how a proto-oncogene activates its downstream effectors by recruiting a tumor suppressor. This interaction of Rb and a proto- oncogene might play an important role in differentiation. The responsiveness of dnmt1 to this type of signal is consistent with an important role for regulated expression of dnmt1 during cellular differentiation. q 2001 Published by Elsevier Science B.V. All rights reserved. Keywords: dnmt1; DNA methylation; Rb; c-Jun; Transactivation 1. Introduction The retinoblastoma tumor-suppressor protein Rb inhibits cell proliferation by repressing a subset of genes that are controlled by the E2F family of transcription factors, which are involved in progression from the G1 to the S-phase of the cell cycle. Rb, which is recruited to target promoters by E2F1, represses transcription by masking the E2F1 transac- tivation domain and by inhibiting surrounding enhancer elements, an active repression that controls cell cycle progression (Weinberg, 1995; Weintraub et al., 1995). Rb has also been shown to be essential in the differentiation of a number of cell types (Ookawa et al., 1997; Kobayashi et al., 1998). c-Jun is a nuclear proto-oncogene that activates tran- scription of its target genes by forming homodimeric or heterodimeric AP-1 complexes with the c-fos gene products. Although c-Jun has been demonstrated to have well-de®ned roles in cellular proliferation (Johnson et al., 1993) and transformation (Hartl and Vogt, 1992), other data has also supported a crucial role for c-Jun in differentiation (de Groot et al., 1990a). Original data pointed towards opposite roles for Rb, a tumor suppressor which arrests mitosis and Jun, a proto- oncogene with a pro-mitogenic effect (for reviews, see (Kaelin, 1999; Wisdom, 1999)). Interestingly, however, recent evidence has demonstrated Rb-mediated enhance- ment of the transactivation of genes containing AP-1 recog- nition elements via formation of a Rb-c-Jun protein complex (Nead et al., 1998; Nishitani et al., 1999). These Rb-c-Jun complexes were shown to be present in terminally differen- tiating keratinocytes (Nead et al., 1998). This type of inter- action might be a paradigm that operates during cellular differentiation. Substantial evidence has demonstrated that the DNA 5- cytosine methyltransferase (dnmt1) is a critical downstream effector of the Ras oncogenic signaling pathway. Dnmt1 is transcriptionally up-regulated by the Ras/AP-1 pathway (MacLeod et al., 1995; Rouleau et al., 1995). K-ras muta- tions in colon cancer cell lines, colon cancers and adenomas have been shown to be directly correlated with hypermethy- lation and silencing of the tumor suppressor gene p16 (Guan et al., 1999). Ex vivo tumorigenesis of the Y1 adrenal-carci- noma line, in which K- Ras is ampli®ed, is inhibited by expressed dnmt1 antisense (MacLeod and Szyf, 1995) and Gene 268 (2001) 87±96 0378-1119/01/$ - see front matter q 2001 Published by Elsevier Science B.V. All rights reserved. PII: S0378-1119(01)00427-9 www.elsevier.com/locate/gene Abbreviations: dnmt1, DNA Methyltransferase; RA, retinoic acid; EMSA, electrophoretic mobility shift assay, CAT; chloramphenicol acet- yltransferase, GST; glutathione S-transferase, RB; retinoblastoma suscept- ibility gene product, LSM; linker scanning mutagenesis, RIPA buffer; RadioImmunoProtectionAssay buffer * Corresponding author. Tel.: 11-514-398-7107; fax: 11-514-398-6690. E-mail address: mszyf@pharma.mcgill.ca (M. Szyf). 1 These authors contributed equally to this article.