02003 Oxfoi-d University Press Nucleic Acids Research Supplement No. 3 307-308 Control elements of Dnmfl gene are regulated in cell-cycle dependent manner Shotaro Kishikawa, Takehide Murata, Hideyo Ugai, Takahito Yamazaki and Kazunari K. Yokoyama Gene Engineering Division, Department of Biological Systems, RlKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, lbaraki 305-0074, Japan ABSTRACT fistone is a central component of nucleosome and is The Dnmtl gene is constitutively expressed and is regulated by the modification induced by histone required for the maintenance of global acetyltransferase (HAT) and histone methylation after DNA replication. We deacetyltransferase (HDAC). Binding of investigated here the effects of histone deacetylase transcription factors to DNA results to recruit HATS (HDAC) inhibitor and DNA demetylation agent that lead to the acetylation of core histone, to on promoter activity of mouse Dnmtl gene in enhance nucleosomal relaxation, and subsequently somatic cells. The promoter activity of Dnmtl to induce transcription. On the other hand, several gene was increased approximately 2-fold in the transcription factors associated with HDACs, caused treatment of cells by Tricostatin A (TSA) at 1 x to stabilize nucleosomal structures and repress the lo-' M, as compared with that without treatment transcription (3). Thus, histone acetylation and of TSA. By contrast, treatment with histone deacetylation are known to regulate the 5-azacytidne (Sua-C) did not affect the promoter transcriptional activity in eukaryotic cells. activity of the Dnmtl gene. This result indicates We have studied the regulation of gene expression the Dnmtl gene is possibly regulated by histone of the mouse Dnmtl gene in somatic cells. acetylation. We also examined the expression Previously, we reported to identify a cis-element that levels of Dnmtl gene and of its control elements appeared to be functional in somatic cells and Spl like Spl, Sp3 and p300 by the chromatin and Sp3 played critical roles in the regulation of the immunoprecipitation and Western blot analysis. Dnmtl gene (4). Moreover, the p300 seemed to be The expression of Dnmtl gene is observed at early associated with Sp3 and regulated Sp3-mediated S phase. S p l is recruited mainly at the G1 activation of Dnmtl gene promoter. In this study phase and Sp3 is recruited at the early S phase. we have constructed stable transformed cells that p300 is also obviously recruited at the second S was introduced a luciferase reporter plasmid with a phase. These data indicated that the regulators minimum promoter of the Dnmtl gene. We have of Dnmtl gene were controlled in cell-cycle examined the effects of HDAC inhibitor Tricostatin dependent manner. A (TSA) and/or DNA demetylation agent 5-azacytidine (5-aza-C) in the promoter activity of INTRODUCTION Dnmtl gene. When the cells was treated with TSA, The five lstinct families of genes for DNA the reporter of Dnmtl gene promoter was activated, metyltransferases, designated Dnmt 1, Dnmt2, however the treatment with 5-aza-C did not. Thus, Dnmt3a, Dnmt3b and DnmtL, have been we speculate that the transcription of the Dnmtl characterized in mammalian cells (1). The Dnmtl gene seems to be regulated by histone acetylation. has been known that it is constitutively expressed In addition, we have measured the level of Dnmtl, and is required for the maintenance of global Spl, Sp3 and p300 using the chromatin methylation after DNA replication. At least, three immunoprecipitation and Western blot analysis. initiation sites of transcription have been found in These results indicate that the expression of Dnmtl the promoter of Dnmtl gene, namely, an gene and the recruited expression of Spl, Sp3 and oocyte-specific site, a somatic cell-specific site and a p300 are coordinately regulated in a cell-cycle spamatocyte-specific site (2). specific manner. 308 Nucleic Acids Reseurch Supplement N o . 3 M.W. Marker 1 Positive Control 1 1 3 6 9 12 15 18 21 (hr) IgG SPl SP3 p300 Pol I1 Fig. 1. Chromatm immunoprecipitationanalysis of occupancy of the Dnmtl promoter by Spl, Sp3 and p300. Cross-linked chromatin was isolated from NIH3T3 cells after starvation of serum. The extracts of cross-linked chromatin was precipitated with specific antibohes against Spl, Sp3, p300, Pol I1 and unspeclfc antibodies (control) and amplified the binding site of the Dfimtl promoter by specific primers. Primers for the GAPDH gene were used as a control. RESULTS AND DISCUSSION REFERENCES To study the effects of histone acetylation and DNA 1. Bestor, T, H, (2000)Hum.Mol.Genet.9,2395-2402. methylation on the promoter activity of Dnmtl gene, 2. Robertson, K. D. (2001) Oncogene20,3139-3 155. the transformed were treated with 3. Berger, S. L. (2002) Curr. Opin. Genet. Dev. 12, various concentration of TSA and 5-aza-C for 48 h. 142-148. Promoter activity was determined by the reporter luciferase assay. The reporter activity of the 4. fishkawa, S., Murata, T., kmura, H., Shiota, K Dnmtl -luciferase construct was increased and Yokoyama, K. K. (2002) Eur. J. Biochem.. approximately two-fold in the case of the treatment 269, 2961-2970. with TSA at 1 x 10-*M as compared with that of non-treatment. By contrast, 5-am-C treatment did not affect the promoter activity of the Dnmtl gene. These results indicated that the transcription of the Dnmtl gene is possibly regulated by histone acetylation, not by the DNA methylation. We also examined the effects of transcription factor Sp 1, Sp3 and p300 coactivator to be recruited to the elements of the Dnmfl promoter. Using the chromatin immunoprecipitation, we found the following observations. (1) Variation of the expression levels of these factors during the cell cycle indicates the presence of the cell cycle controlled mechanism of the expression of each factor. (2) Spl is recruited at G1 phase and early S phase and Sp3, however, is recruited at G1 phase. (3) p300 is recruited at S phase of the second cell cycle. The cooperation of these factors was obvious since the induced expression of Dnmtl gene was found at S phase. Previously the transcription factors Spl and Sp3 are the family of the house keeping genes. Thus, it is surprising that the expression level of Spl and Sp3 are changed during the cell cycle. We are now undertaken to study what residue of Lysine of the histone tail could be acetylated in Sp3-mediated regulation to control the gene expression of Dnmtl gene. How the acetylated histone controls the formation of euchromatin? The molecular mechanism of histone acetylation and deacetylation also remains to be clarified.