Reduced DNA Methylation Levels Research Articles

Alteration of DNA methylation levels in MRL lupus mice.

Recent reports suggest that DNA methylation is involved in the cause of autoimmune disease. We investigated the alteration of DNA methylation levels in lupus strains of mice, MRL/lpr as a model, which develop an age-dependent lymphadenopathy and autoimmune disease. DNA methylation levels of thymus and axillary lymph nodes in 20-week-old MRL/lpr mice, which are in an autoimmune disease state, were lower than those of 4-week-old MRL/lpr mice with no symptoms as yet. No significant changes were observed in MRL/+ strain mice, which seemed normal at least 20 weeks, while DNA methylation levels in the spleen of both strains of mice increased significantly from the age of 4 to 20 weeks. However, no significant changes of DNA methylation levels in peripheral blood were observed with ageing in MRL strains. Moreover, we clarified that administration of 5-azacytidine had a strong effect on longer survival of MRL/lpr mice and reduced DNA methylation levels in the axillary lymph nodes and spleen. The possible relevance of DNA methylation levels to the progression of autoimmune disease is discussed.

38 EFFECTS OF TRICHOSTATIN A ON DNA METHYLATION IN CLONED BOVINE EMBRYOS

Recently, the efficiency of full-term development of somatic cloned mouse embryos was significantly increased by treatment with trichostatin A (TSA), an inhibitor of histone deacetylase (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089). We have shown that TSA treatment improved the rate of development of the cloned bovine embryos to the blastocyst stage (Iwamoto et al. 2007 Reprod. Fertil. Dev. 19, 142 abst). Higher levels of DNA methylation have been shown in early cloned bovine embryos than in in vitro-fertilized (IVF) embryos (Dean et al. 2001 Proc. Nat. Acad. Sci. USA 98, 13734–13738; Santos et al. Curr. Biol. 13, 1116–1121). In this study, we examined the effects of TSA on DNA methylation levels in cloned bovine embryos by immunostaining with an antibody to 5-methyl cytosine (5-MeC). Bovine fibroblasts were cultured under serum starvation (0.4% FCS) for 7 days before they were used as donor cells. The cells were electrofused with bovine enucleated matured oocytes, and activated with a calcium ionophore and cycloheximide. Atotal of 131 cloned embryos were produced. The NT embryos were exposed to 0 (control) and 50 nmTSA from the start of activation to 48 h post-activation (hpa). They were then cultured in an mSOF medium. At 60 hpa, only embryos developed to the 8-cell stage were used for assessment of DNA methylation levels. Sixteen TSA-treated, 22 non-treated, and 19 IVF embryos were immunostained with 5-MeC antibody. For quantitative analysis of the DNA methylation levels, 5-MeC signals in the fluorescent images were determined using an image analyzer system (Aqua Cosmos; Hamamatsu Photonics, Shizuoka, Japan). The data were analyzed with Tukey-Kramer post hoc test for multiple comparisons following ANOVA. Relative levels of DNA methylation of TSA-treated cloned and IVF embryos did not differ (P > 0.05), but were lower than those of non-treated cloned embryos (P < 0.05). The results indicate that TSA treatment of cloned bovine embryos leads to a reduction of DNA methylation levels of their genome. The data suggest that the TSA treatment decreased the DNA methylation levels of cloned bovine embryos to the levels of IVF embryos, resulting in improved blastocyst development of the cloned embryos.

Lower Levels of Transgene Silencing in Roots is Associated with Reduced DNA Methylation Levels at Non-Symmetrical Sites but not at Symmetrical Sites

Transgene transcripts were recently shown to accumulate at higher levels in roots, relative to leaves, of silenced-transgenic Nicotiana benthamiana plants and to be inversely related with the accumulation of small interfering RNAs (siRNAs), suggesting that RNA silencing is less active in roots than in leaves (Andika et al., 2005. Mol. Plant-Microbe Interact. 18: 194). Here we show that the lower transgene RNA silencing activity in roots was associated with lower transgene methylation levels at non-symmetrical CpNpN context but not at symmetrical CpG or CpNpG context in three sets of transformant plants with different exogenous genes. In contrast, such a difference between roots and leaves was not observed for the Tnt1 retrotransposon: no Tnt1 transcript was detected in roots or in leaves of N. benthamiana, while equal levels of Tnt1-derived siRNA accumulation and Tnt1 methylation were found. From our data and previously reported information, we suggest that roots have less of an activity that acts at the step of generation of siRNAs.

Opposing effects of DNA hypomethylation on intestinal and liver carcinogenesis

Genome-wide DNA hypomethylation and concomitant promoter-specific tumor suppressor gene hypermethylation are among the most common molecular alterations in human neoplasia. Consistent with the notion that both promoter hypermethylation and genome-wide hypomethylation are functionally important in tumorigenesis, genetic and/or pharmacologic reduction of DNA methylation levels results in suppression or promotion of tumor incidence, respectively, depending on the tumor cell type. For instance, DNA hypomethylation promotes tumors that rely predominantly on loss of heterozygosity (LOH) or chromosomal instability mechanisms, whereas loss of DNA methylation suppresses tumors that rely on epigenetic silencing. Mutational and epigenetic silencing events in Wnt pathway genes have been identified in human colon tumors. We used Apc(Min/+) mice to investigate the effect of hypomethylation on intestinal and liver tumor formation. Intestinal carcinogenesis in Apc(Min/+) mice occurs in two stages, with the formation of microadenomas leading to the development of macroscopic polyps. Using Dnmt1 hypomorphic alleles to reduce genomic methylation, we observed elevated incidence of microadenomas that were associated with LOH at Apc. In contrast, the incidence and growth of macroscopic intestinal tumors in the same animals was strongly suppressed. In contrast to the overall inhibition of intestinal tumorigenesis in hypomethylated Apc(Min/+) mice, hypomethylation caused development of multifocal liver tumors accompanied by Apc LOH. These findings support the notion of a dual role for DNA hypomethylation in suppressing later stages of intestinal tumorigenesis, but promoting early lesions in the colon and liver through an LOH mechanism.

The Role of DNA Methylation in Plant Development

DNA methylation has been implicated in a wide range of biological processes including gene regulation (Razin and Cedar 1991; Finnegan et al. 1993), determination of chromatin structure (Lewis and Bird 1991), timing of DNA replication (Lewis and Bird 1991), genomic imprinting (Sasaki et al. 1993), increasing the spontaneous mutation rate (Coulondre et al. 1978; Duncan and Miller 1980), as the basis for epigenetic phenomena (Holliday 1993), and as a defense mechanism to protect against foreign or invading DNA (Bird 1986; Bestor 1990; Doerfler 1991). Despite this, the existence of organisms such as yeast and Drosophila that lack DNA methylation has led to debate about the role of DNA methylation. There can be no doubt that DNA methylation plays a fundamental role in mouse development following the establishment of a targeted mutation of the DNA methyltransferase gene in the germ line of transgenic mice. Embryos that were homozygous for the mutation had methylation levels reduced to about 30% of wild-type levels. These embryos were stunted and delayed in development compared to their normal siblings and spontaneously aborted in mid-gestation. Although the underlying cause for the embryo-lethal phenotype has not been determined, it has been proposed that inappropriate gene expression resulting from the reduced level of DNA methylation is involved (Li et al. 1992). In contrast, mutants of Neurospora crassa that abolish all detectable methylation showed only minor changes in vigor and growth morphology, suggesting that DNA methylation is not absolutely essential for development of this organism (Foss et al. 1993). This...

Mutations affecting the biosynthesis of S-adenosylmethionine cause reduction of DNA methylation in Neurospora crassa.

A temperature-sensitive methionine auxotroph of Neurospora crassa was found in a collection of conditional mutants and shown to be deficient in DNA methylation when grown under semipermissive conditions. The defective gene was identified as met-3, which encodes cystathionine-gamma-synthase. We explored the possibility that the methylation defect results from deficiency of S-adenosylmethionine (SAM), the presumptive methyl group donor. Methionine starvation of mutants from each of nine complementation groups in the methionine (met) pathway (met-1, met-2, met-3, met-5, met-6, met-8, met-9, met-10 and for) resulted in decreased DNA methylation while amino acid starvation, per se, did not. In most of the strains, including wild-type, intracellular SAM peaked during rapid growth (12-18 h after inoculation), whereas DNA methylation continued to increase. In met mutants starved for methionine, SAM levels were most reduced (3-11-fold) during rapid growth while the greatest reduction in DNA methylation levels occurred later. Addition of 3 mM methionine to cultures of met or cysteine-requiring (cys) mutants resulted in 5-28-fold increases in SAM, compared with wild-type, at a time when DNA methylation was reduced approximately 40%, suggesting that the decreased methylation during rapid growth in Neurospora is not due to limiting SAM. DNA methylation continued to increase in a cys-3 mutant that had stopped growing due to methionine starvation, suggesting that methylation is not obligatorily coupled to DNA replication in Neurospora.

Arabidopsis thaliana DNA methylation mutants.

Three DNA hypomethylation mutants of the flowering plant Arabidopsis thaliana were isolated by screening mutagenized populations for plants containing centromeric repetitive DNA arrays susceptible to digestion by a restriction endonuclease that was sensitive to methylated cytosines. The mutations are recessive, and at least two are alleles of a single locus, designated DDM1 (for decrease in DNA methylation). Amounts of 5-methylcytosine were reduced over 70 percent in ddm1 mutants. Despite this reduction in DNA methylation levels, ddm1 mutants developed normally and exhibited no striking morphological phenotypes. However, the ddm1 mutations are associated with a segregation distortion phenotype. The ddm1 mutations were used to demonstrate that de novo DNA methylation in vivo is slow.

Clonal analysis of the malignant properties of B16 melanoma cells treated with the DNA hypomethylating agent 5-azacytidine

The role of DNA methylation in the generation of tumor cell variants with altered growth behavior has been investigated. Cultures of the clonally heterogeneous B16 melanoma cell line and a clonal population (B16-CL) derived from it were treated with the DNA hypomethylating agent, 5-azacytidine (5-Aza-CR). The tumorigenic and metastatic properties of (sub)clones isolated from these cultures before and after drug treatment were assayed by injection via multiple routes into syngeneic C57BL/6 mice using a range of cell doses. The rate of tumor growth was monitored following intrafootpad (i.f.p.) injection and the tumor incidence was calculated from the frequency of tumor formation at i.f.p. and supraclavicular subcutaneous (s.c.) sites. Formation of both spontaneous (i.f.p., s.c. inoculations) and experimental (intravenous (i.v.) inoculation) metastatic potential was also investigated. The most consistent effect of 5-Aza-CR was the introduction of heterogeneity with respect to the tumorigenic phenotype. The effect of 5-Aza-CR treatment on metastatic behavior was variable. The majority of tumor cell variants that arose following 5-Aza-CR treatment displayed decreased malignant potential and reduced DNA methylation levels relative to untreated control cells, but the correlation was not absolute. The decreases in DNA methylation levels induced by 5-Aza-CR were unstable and began to rebound within 1 week of drug treatment. The results of the current study indicate that although 5-Aza-CR can introduce significant shifts in the malignant properties of treated cells, the direction and magnitude of the induced alterations are not predictable and are influenced by a variety of experimental parameters including the starting tumor cell population, route of tumor cell inoculation, and the drug treatment protocol. In addition, because DNA methylation levels can rebound rapidly (days) it is difficult to correlate changes in this parameter with the observed alterations in malignancy, which can only be assessed in long-term biological assays (weeks).