Array-based genomic resequencing of human leukemia.

To identify oncogenes in leukemias, we performed large-scale resequencing of the leukemia genome using DNA sequence arrays that determine approximately 9 Mbp of sequence corresponding to the exons or exon-intron boundaries of 5648 protein-coding genes. Hybridization of genomic DNA from CD34-positive blasts of acute myeloid leukemia (n=19) or myeloproliferative disorder (n=1) with the arrays identified 9148 nonsynonymous nucleotide changes. Subsequent analysis showed that most of these changes were also present in the genomic DNA of the paired controls, with 11 somatic changes identified only in the leukemic blasts. One of these latter changes results in a Met-to-Ile substitution at amino-acid position 511 of Janus kinase 3 (JAK3), and the JAK3(M511I) protein exhibited transforming potential both in vitro and in vivo. Further screening for JAK3 mutations showed novel and known transforming changes in a total of 9 out of 286 cases of leukemia. Our experiments also showed a somatic change responsible for an Arg-to-His substitution at amino-acid position 882 of DNA methyltransferase 3A, which resulted in a loss of DNA methylation activity of >50%. Our data have thus shown a unique profile of gene mutations in human leukemia.

Xenopus eggs express an identical DNA methyltransferase, Dnmt1, to somatic cells.

In mouse, an oocyte-specific short isoform of DNA methyltransferase-1 (Dnmt1) lacking amino terminal 118 amino acid residues exists and plays a crucial role in maintaining the methylation state of imprinted genes during early embryogenesis [Howell et al. (2001) Cell 104, 829-838]. To address the question of whether or not Xenopus oocyte expresses such a short isoform, we raised monoclonal antibodies against the amino-terminal portion of Xenopus Dnmt1. Two of the isolated monoclonal antibodies, 3C6 and 4A8, were determined to recognize (1-32) and (115-126) of Xenopus Dnmt1, respectively. The amounts of Dnmt1 in Xenopus eggs were determined to be similar, 10.0 2.5, 8.0 0.8, and 8.2 0.2 ng per egg with monoclonal antibodies 3C6 and 4A8, and polyclonal antibodies, respectively. This indicated that Dnmt1 in Xenopus mature eggs had an identical amino-terminal sequence to the amino acid sequence deduced from the cDNA. Together with the fact that Dnmt1 in A6 cells immuno-reacted with all the monoclonal antibodies isolated and with the polyclonal antibodies, we concluded that Dnmt1 expressed in Xenopus mature eggs possesses an identical amino-terminal sequence to that in somatic cells. Immuno-purified Xenopus Dnmt1 in mature eggs showed similar specific activity to that in proliferating A6 cells and that of mouse recombinant Dnmt1.

Enzymatic properties of de novo-type mouse DNA (cytosine-5) methyltransferases.

We have purified GST-fused recombinant mouse Dnmt3a and three isoforms of mouse Dnmt3b to near homogeneity. Dnmt3b3, an isoform of Dnmt3b, did not have DNA methylation activity. Dnmt3a, Dnmt3b1 or Dnmt3b2 showed similar activity toward poly(dG-dC)-poly(dG-dC) for measuring de novo methylation activity, and toward poly(dI-dC)-poly(dI-dC) for measuring total activity. This indicates that the enzymes are de novo-type DNA methyltransferases. The enzyme activity was inhibited by NaCl or KCl at concentrations >100 mM. The kinetic parameter, K(m)(AdoMet), for Dnmt3a, Dnmt3b1 and Dnmt3b2 was 0.4, 1.2 and 0.9 microM when poly(dI-dC)-poly(dI-dC) was used, and 0.3, 1.2 and 0.8 microM when poly(dG-dC)-poly(dG-dC) was used, respectively. The K(m)(DNA) values for Dnmt3a, Dnmt3b1 and Dnmt3b2 were 2.7, 1.3 and 1.5 microM when poly(dI-dC)-poly(dI-dC) was used, and 3.5, 1.0 and 0.9 microM when poly(dG-dC)-poly(dG-dC) was used, respectively. For the methylation specificity, Dnmt3a significantly methylated CpG >> CpA. On the other hand, Dnmt3b1 methylated CpG > CpT >/= CpA. Immuno-purified Dnmt3a, Myc-tagged and overexpressed in HEK 293T cells, methylated CpG >> CpA > CpT. Neither Dnmt3a nor Dnmt3b1 methylated the first cytosine of CpC.

Expression of DNA methyltransferase (Dnmt1) in testicular germ cells during development of mouse embryo.

The DNA methylation pattern is reprogrammed in embryonic germ cells. In female germ cells, the short-form DNA methyltransferase Dnmt1, which is an alternative isoform specifically expressed in growing oocytes, plays a crucial role in maintaining imprinted genes. To evaluate the contribution of Dnmt1 to the DNA methylation in male germ cells, the expression profiles of Dnmt1 in embryonic gonocytes were investigated. We detected a significant expression of Dnmt1 in primordial germ cells in 12.5-14.5 day postcoitum (dpc) embryos. The expression of Dnmt1 was downregulated after 14.5 dpc after which almost no Dnmt1 was detected in gonocytes prepared from 18.5 dpc embryos. The short-form Dnmt1 also was not detected in the 16.5-18.5 dpc gonocytes. On the other hand, Dnmt1 was constantly detected in Sertoli cells at 12.5-18.5 dpc. The expression profiles of Dnmt1 were similar to that of proliferating cell nuclear antigen (PCNA), a marker for proliferating cells, suggesting that Dnmt1 was specifically expressed in the proliferating male germ cells. Inversely, genome-wide DNA methylation occurred after germ cell proliferation was arrested, when the Dnmt1 expression was downregulated. The present results indicate that not Dnmt1 but some other type of DNA methyltransferase contributes to the creation of DNA methylation patterns in male germ cells.

Maintenance-type DNA methyltransferase is highly expressed in post-mitotic neurons and localized in the cytoplasmic compartment.

Maintenance-type DNA methyltransferase (Dnmt1) is usually down-regulated in non-proliferating cells. In the present study, we detected significant expression of Dnmt1 protein in adult mouse brain where the majority of the cells are in a post-mitotic state. A significant amount of Dnmt1 protein was fractionated into the post-nuclear fraction for both cerebrum and cerebellum. The Dnmt1 in this fraction was enzymatically active. An immunofluorescence study revealed that Dnmt1 protein was mainly expressed in neurons and seemed to be localized in the cytoplasmic compartment. Primary culturing of neurons confirmed the expression and localization of Dnmt1 in the cytoplasmic compartment. The findings that the Dnmt1 transcript in the brain utilized the somatic-type exon and that the apparent size of the Dnmt1 protein in the cytoplasm was identical to that in proliferating culture cells indicate that the cytoplasmic Dnmt1 in neurons was of the somatic-type.

Isolation of the novel cDNA of a gene of which expression is induced by a demethylating stimulus.

We have isolated a novel cDNA clone, named AZ2, from a cDNA library of mRNA prepared from C3H10T1/2 cells that had been transiently exposed to 5-azacytidine, a potent inhibitor of DNA methyltransferase. The elucidated nucleotide sequence revealed that the 5' region of the cDNA was rich in the CpG sequence. The AZ2 cDNA contained a 1215-nucleotide open reading frame, and the expected amino acid sequence had a molecular mass of 46090. The amount of the transcript increased on 5-azacytidine treatment of C3H10T1/2 cells, and the transcript was significantly expressed in mouse testis, brain, lung, kidney, heart and ovary. Specific antibodies raised against a fusion protein including glutathione S-transferase revealed a band of an approximately 48kDa translation product for testis, brain, lung, and cultured cells that ectopically expressed the AZ2 protein. The AZ2 protein was mainly localized in the cytoplasm. The amino-terminal part of the AZ2 protein was homologous to the previously reported TANK (Cheng and Baltimore, 1996. Genes Dev. 10, 963-973) and I-TRAF (Rothe, 1996. Proc. Natl. Acad. Sci. USA 93, 8241-8246), which participate in the signal transduction cascade from the tumor necrosis factor-receptor to the transcription factor, NFkappaB. Overexpression of AZ2 inhibited TNF alpha mediated NFkappaB activation. AZ2 could be a component of a regulator of the NFkappaB activation cascade.

Xenopus maintenance-type DNA methyltransferase is accumulated and translocated into germinal vesicles of oocytes.

In vertebrates, DNA methylation plays an important role in the regulation of gene expression and embryogenesis. DNA methyltransferase, which catalyzes the introduction of a methyl group at the 5th position of cytosine in the CpG sequence, is highly accumulated in mouse oocytes and is excluded from nuclei [Carlson et al. (1992) Genes Dev. 6, 2536-2541]. In this study, we examined the expression level and localization of Xenopus DNA methyltransferase in oocytes during oogenesis. The DNA methyltransferase protein was detectable in stage III oocytes and increased thereafter, until the oocytes had matured. The rate of DNA methyltransferase synthesis rapidly increased after stage IV oocytes. Different from in mouse oocytes, DNA methyltransferase was equally distributed in the nuclear and post-nuclear fractions, in stage VI oocytes. DNA methyltransferase translocated into nuclei was uniformly localized in the nuclear matrix, and the accumulated DNA methyltransferase in stage VI nuclei had DNA methylation activity.

Effect of aphidicolin on DNA methyltransferase in the nucleus.

Methylation of cytosine in the genomic DNA plays an important role in mammalian embryogenesis. DNA methyltransferase activity, which contributes mainly to the maintenance of the methylation pattern during proliferation, is under the control of the cell cycle, its activity being higher in the S phase than in the other phases (Adams, R.L.P., 1990, Biochem. J. 265, 309-320). In the present study, we examined how DNA methyltransferase is regulated in the cells arrested at S phase by aphidicolin treatment. The activity and protein levels of DNA methyltransferase in the nuclei were kept constant in proliferating mouse erythroleukemia cells, and increased about twofold after 6 h incubation in the presence of aphidicolin. This increase of DNA methyltransferase levels by aphidicolin treatment was positively correlated with the cell population at S phase. De novo synthesis of DNA methyltransferase protein was increased by the treatment. In addition, the relative half life of pulse labeled DNA methyltransferase was prolonged by aphidicolin treatment. Both increase in synthesis and prolongation of half life of DNA methyltransferase in S phase contributed to the increase of the activity and the protein levels by aphidicolin treatment. Prolongation of half life was abolished by cycloheximide, suggesting that newly synthesized protein(s) with a short half life participated in the degradation of DNA methyltransferase.

Regulation and function of DNA methylation in vertebrates.

In vertebrates, genomic DNA is often methylated at the 5th position of cytosine in the sequence of CpG, and this is the only chemical modification that genomic DNA of vertebrates allows under physiological conditions. During evolution, vertebrates acquired CpG methylation as a new tool for controlling gene expression in addition to the varieties of transcription factors. In mammals, the methylation pattern of genomic DNA is erased and reset in germ line and at the early stage of embryogenesis. Maintenance-type methylation activity ensures clonal transmission of the lineage-specific methylation pattern in somatic cells. The methylation pattern is dynamic and changes during cell differentiation. Prior to the expression of tissue-specific genes, specific sites of the promoters are demethylated. In general, the methylation of a gene suppresses its expression. However, not much is known about the mechanisms that regulate the methylation state and the gene expression by DNA methylation.

A novel DNA binding protein that recognizes the methylated c-Myc binding motif.

We detected a novel nuclear protein, MMBP-3, that bound to the c-Myc binding motif (CACGTG) in which deoxycytidine in the CpG sequence was methylated. MMBP-3 was partially purified by chromatography on heparin-agarose and hydroxyapatite, followed by affinity adsorption to a matrix coupled to the methylated binding motif. Its binding to the methylated c-Myc binding motif was specific, although it also recognized the unmethylated motif weakly. MMBP-3 was further found to recognize only one of two differently hemimethylated forms of the double-stranded c-Myc binding motif. MMBP-3 activity was detected in proliferating C2C12 and C3H/10T1/2 cells, and down regulated when the growth of these cells was inhibited. We propose that MMBP-3 plays a role in regulating the c-Myc function by recognizing the methylation state of the c-Myc binding motif in a growth-dependent manner.

Identification of two novel mouse nuclear proteins that bind selectively to a methylated c-Myc recognizing sequence.

The c-Myc recognizes the sequence CACGTG (Blackwell, T. K., Kretzner, L., Blackwood, E.M., Eisenman, R. N., and Weintraub, H. (1990) Science 250, 1149-1151), and its binding is inhibited by methylation of the core CpG (Prendergast, G. C. and Ziff, E. B. (1991) Science 251, 186-189). We identified two novel nuclear proteins, MMBP-1 and MMBP-2, that bound specifically and under physiological salt condition to the c-Myc binding motif of which cytidine in the CpG sequence was methylated. MMBP-1 was about 42 kD and MMBP-2 was about 63 kD. MMBP-1 was found in specific cells, while MMBP-2 was found in all the cell lines tested, suggesting that MMBP-1 may modulate the role of MMBP-2 in tissue specific manner. We propose that the two proteins play a role in the regulation of c-Myc function through stabilizing or destabilizing the methylation state of the c-Myc binding motif.

Contractile activity and fluorescence changes in fluo-3-loaded isolated ventricular myocytes.

Isolated rat ventricular myocytes were loaded with fluo-3 which did not result in a loss of beating activity, in order to record the changes in the fluorescence and contractile parameters. Changes in the beating activity induced by various reagents in perfusing medium were related to variations in the peak intensity of fluorescence time course and possibly to changes in myofibrillar ATPase activity. Isoproterenol stimulated, whereas 2,3-butanedione monoxime (BDM) and a medium with a low Ca2+ concentration suppressed the contractile activity by increasing and reducing the Ca2+ transient, respectively, and, in the presence of BDM, a decrease in the maximum ATPase activity also contributed the suppressive effect.