Abstract
Ectopic expression of DNA methyltransferase 1 (DNMT1) has been proposed to play an important role in cancer. dnmt1 mRNA is undetectable in growth-arrested cells but is induced upon entrance into the S phase of the cell cycle, and until now, the mechanisms responsible for this regulation were unknown. In this report, we demonstrate that the 3'-untranslated region (3'-UTR) of the dnmt1 mRNA can confer a growth-dependent regulation on its own message as well as a heterologous beta-globin mRNA. Our results indicate that a 54-nucleotide highly conserved element within the 3'-UTR is necessary and sufficient to mediate this regulation. Cell-free mRNA decay experiments demonstrate that this element increases mRNA turnover rates and does so to a greater extent in the presence of extracts prepared from arrested cells. A specific RNA-protein complex is formed with the 3'-UTR only in growth-arrested cells, and a UV cross-linking analysis revealed a 40-kDa protein (p40), the binding of which is dramatically increased in growth-arrested cells and is inversely correlated with dnmt1 mRNA levels as cells are induced into the cell cycle. Although ectopic expression of human DNMT1 lacking the 3'-UTR can transform NIH-3T3 cells, inclusion of the 3'-UTR prevents transformation. These results support the hypothesis that deregulated expression of DNMT1 with the cell cycle is important for cellular transformation.
Highlights
The pattern of methylation is faithfully maintained during cell division by the enzyme DNA methyltransferase 1 (DNMT1),1 the maintenance DNA methyltransferase, [4] which catalyzes the transfer of a methyl group from S-adenosyl-methionine to the 5-position of the cytosine ring [5, 6]
Addition of serum-containing medium induced entry of the cells into the S phase of the cell cycle, which peaked 24 h poststimulation (S ϭ 51%). dnmt1 mRNA was barely detectable in arrested cells and was induced late in G1 (8 –12 h), reaching its maximum levels at the G1-S boundary (20 h) (Fig. 1, B and C). dnmt1 mRNA remained elevated throughout S, and began to decrease as cells continued into G2-M
Because hyperactivation of DNMT1 has been implicated in cellular transformation [14], understanding how this enzyme is regulated with cell growth is of obvious interest
Summary
The pattern of methylation is faithfully maintained during cell division by the enzyme DNMT1,1 the maintenance DNA methyltransferase, [4] which catalyzes the transfer of a methyl group from S-adenosyl-methionine to the 5-position of the cytosine ring [5, 6]. It stands to reason that the mechanisms involved are linked to basic control points of the cell cycle, and potentially linked to cellular transformation To test this hypothesis, the factors responsible for regulation of dnmt expression with the cell cycle must be identified. Regulation of mRNA stability by AU-rich elements (AREs) is an important mechanism involved in orchestrating the expression of critical genes in development [32, 33] and cell cycle [34, 35], early response genes involved in cellular growth such as c-myc [36] and c-fos [37], and cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) [38] and interleukin 3 [39]. We demonstrate that the 3Ј-UTR influences the cellular changes observed upon overexpression of DNMT1 in NIH-3T3 cells, providing a link between dnmt regulation, cell cycle, and oncogenesis
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