Genomic DNA Demethylation Research Articles

Redox and epigenetic regulation of the APE1 gene in the hippocampus of piglets: The effect of early life exposures.

Oxidative stress via redox reactions can regulate DNA repair pathways. The base excision repair (BER) enzyme apurinic/apyrimidinic endonuclease 1 (APE1) is a key player in the redox regulation of DNA repair. Environmental factors can alter the methylation of DNA repair genes, change their expression and thus modulate BER activity and susceptibility to oxidative DNA damage. Therefore, we hypothesized that epigenetic modifications play a role in the redox regulation of APE1 in hippocampi of newborns and investigated the effect of supplementation of pregnant sows with a diet enriched in antioxidants and other nutrients on oxidative stress, DNA methylation and DNA repair in their offspring. High levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and low levels of glutathione were detected in control piglets after birth compared with supplemented piglets, indicating the presence of oxidative stress. In control animals, this oxidative stress was associated with genomic DNA demethylation, decreased APE1 promoter methylation, increased APE1 expression and with slightly but not statistically significant increased BER-related DNA incision activity. Supplementation of piglets with antioxidants and other nutrients significantly lowered 8-oxodG levels compared to control animals, which was accompanied by overall lower APE1 promoter methylation and enhanced APE1 expression at day 7-28 after birth in supplemented piglets, although DNA incision activity was not significantly different between groups. Preliminary attempts to study the interaction between redox and epigenetic regulatory mechanisms revealed an inverse correlation between APE1 expression and methylation of CpG-sites 11 and 13 in the promoter region, which according to Genomatix "MatInspector" are located in the core binding sites of redox-sensitive transcription factors. We are the first to study methylation of the APE1 promoter and its role in mediating the functional effects of redox reactions induced by oxidative stress. Epigenetic and redox mechanisms may interact in regulating APE1-related DNA repair processes, involving redox-sensitive TFs.

Genetic and epigenetic changes in fibrosis-associated hepatocarcinogenesis in mice.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers and is rising in incidence worldwide. The molecular mechanisms leading to the development of HCC are complex and include both genetic and epigenetic events. To determine the relative contribution of these alterations in liver tumorigenesis, we evaluated epigenetic modifications at both global and gene specific levels, as well as the mutational profile of genes commonly altered in liver tumors. A mouse model of fibrosis-associated liver cancer that was designed to emulate cirrhotic liver, a prevailing disease state observed in most humans with HCC, was used. Tumor and nontumor liver samples from B6C3F1 mice treated with N-nitrosodiethylamine (DEN; a single ip injection of 1 mg/kg at 14 days of age) and carbon tetrachloride (CCl4; 0.2 ml/kg, 2 times/week ip starting at 8 weeks of age for 14 weeks), as well as corresponding vehicle control animals, were analyzed for genetic and epigenetic alterations. H-ras, Ctnnb1 and Hnf1α genes were not mutated in tumors in mice treated with DEN+CCl4 . In contrast, the increased tumor incidence in mice treated with DEN+CCl4 was associated with marked epigenetic changes in liver tumors and nontumor liver tissue, including demethylation of genomic DNA and repetitive elements, a decrease in histone 3 lysine 9 trimethylation (H3K9me3) and promoter hypermethylation and functional downregulation of Riz1, a histone lysine methyltransferase tumor suppressor gene. Additionally, the reduction in H3K9me3 was accompanied by increased expression of long interspersed nucleotide elements 1 and short interspersed nucleotide elements B2, which is an indication of genomic instability. In summary, our results suggest that epigenetic events, rather than mutations in known cancer-related genes, play a prominent role in increased incidence of liver tumors in this mouse model of fibrosis-associated liver cancer.

Hydroquinone Increases 5-Hydroxymethylcytosine Formation through Ten Eleven Translocation 1 (TET1) 5-Methylcytosine Dioxygenase

DNA methylation regulates gene expression throughout development and in a wide range of pathologies such as cancer and neurological disorders. Pathways controlling the dynamic levels and targets of methylation are known to be disrupted by chemicals and are therefore of great interest in both prevention and clinical contexts. Benzene and its metabolite hydroquinone have been shown to lead to decreased levels of DNA methylation, although the mechanism is not known. This study employs a cell culture model to investigate the mechanism of hydroquinone-mediated changes in DNA methylation. Exposures that do not affect HEK293 cell viability led to genomic and methylated reporter DNA demethylation. Hydroquinone caused reactivation of a methylated reporter plasmid that was prevented by the addition of N-acetylcysteine. Hydroquinone also caused an increase in Ten Eleven Translocation 1 activity and global levels of 5-hydroxymethylcytosine. 5-Hydroxymethylcytosine was found enriched at LINE-1 prior to a decrease in both 5-hydroxymethylcytosine and 5-methylcytosine. Ten Eleven Translocation-1 knockdown decreased 5-hydroxymethylcytosine formation following hydroquinone exposure as well as the induction of glutamate-cysteine ligase catalytic subunit and 14-3-3σ. Finally, Ten Eleven Translocation 1 knockdown decreased the percentage of cells accumulating in G2+M following hydroquinone exposure, indicating that it may have a role in cell cycle changes in response to toxicants. This work demonstrates that hydroquinone exposure leads to active and functional DNA demethylation in HEK293 cells in a mechanism involving reactive oxygen species and Ten Eleven Translocation 1 5-methylcytosine dioxygenase.

Axin gene methylation status correlates with radiosensitivity of lung cancer cells

BackgroundWe previously reported that Axin1 (Axin) is down-regulated in many cases of lung cancer, and X-ray irradiation increased Axin expression and inhibited lung cancer cells. The mechanisms, however, were not clear.MethodsFour lung cancer cell lines were used to detect the methylation status of Axin with or without X-ray treatment. Real-time PCR was used to quantify the expression of Axin, and western blot analysis was applied to measure protein levels of Axin, β-catenin, Cyclin D1, MMP-7, DNMTS, MeCP2 and acetylated histones. Flow cytometric analysis, colony formation assay, transwell assay and xenograft growth experiment were used to study the biological behavior of the cells with hypermethylated or unmethylated Axin gene after X-ray treatment.ResultsHypermethylated Axin gene was detected in 2 of 4 cell lines, and it correlated inversely with Axin expression. X-ray treatment significantly up-regulated Axin expression in H446 and H157 cells, which possess intrinsic hypermethylation of the Axin gene (P<0.01), but did not show up-regulation in LTE and H460 cells, which have unmethylated Axin gene. 2Gy X-ray significantly reduced colony formation (from 71% to 10.5%) in H157 cells, while the reduction was lower in LTE cells (from 71% to 20%). After X-ray irradiation, xenograft growth was significantly decreased in H157 cells (from 1.15 g to 0.28 g) in comparison with LTE cells (from 1.06 g to 0.65 g). Significantly decreased cell invasiveness and increased apoptosis were also observed in H157 cells treated with X-ray irradiation (P<0.01). Down-regulation of DNMTs and MeCP2 and up-regulation of acetylated histones could be detected in lung cancer cells.ConclusionsX-ray-induced inhibition of lung cancer cells may be mediated by enhanced expression of Axin via genomic DNA demethylation and histone acetylation. Lung cancer cells with a different methylation status of the Axin gene showed different radiosensitivity, suggesting that the methylation status of the Axin gene may be one important factor to predict radiosensitivity of the tumor.

Interplay between Menin and K-Ras in Regulating Lung Adenocarcinoma

MEN1, which encodes the nuclear protein menin, acts as a tumor suppressor in lung cancer and is often inactivated in human primary lung adenocarcinoma. Here, we show that the inactivation of MEN1 is associated with increased DNA methylation at the MEN1 promoter by K-Ras. On one hand, the activated K-Ras up-regulates the expression of DNA methyltransferases and enhances the binding of DNA methyltransferase 1 to the MEN1 promoter, leading to increased DNA methylation at the MEN1 gene in lung cancer cells; on the other hand, menin reduces the level of active Ras-GTP at least partly by preventing GRB2 and SOS1 from binding to Ras, without affecting the expression of GRB2 and SOS1. In human lung adenocarcinoma samples, we further demonstrate that reduced menin expression is associated with the enhanced expression of Ras (p < 0.05). Finally, excision of the Men1 gene markedly accelerates the K-Ras(G12D)-induced tumor formation in the Men1(f/f);K-Ras(G12D/+);Cre ER mouse model. Together, these findings uncover a previously unknown link between activated K-Ras and menin, an important interplay governing tumor activation and suppression in the development of lung cancer.

The Involvement of 5-Hydroxymethylcytosine in Active DNA Demethylation in Mice1

Active DNA demethylation occurs after a sperm enters an egg. However, the mechanisms for the active DNA demethylation remain poorly understood. Ten-eleven translocation enzymes were recently shown to catalyze the conversion of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Thus, we decided to investigate the role of 5hmC in active demethylation. We analyzed the methylation and hydroxymethylation status in metaphase II oocytes as well as 1-cell stage and cleavage stage embryos. In zygotes, 5hmC was mainly detected in the paternal pronucleus and it increased from the pronuclear-2 (PN2) to PN5 stages, an indication that 5hmC was involved in paternal genomic DNA demethylation. Bisulfite-sequencing PCR and qGluMS-PCR (DNA glucosylation and digestion before quantitative PCR) results showed that a large reduction of methylcytosine and hydroxymethylcytosine in LINE1 (long interspersed nuclear element 1) occurred between the 4- and 8-cell stages, which indicates that demethylation potentially occurred after the 4-cell stage. We then microinjected mouse zygote with plasmids that were methylated in vitro by SssI methylase and analyzed for the hydroxymethylation status of the plasmids promoter region. We found that the rapid onset of expression of the unmethylated plasmids in mouse embryos happened in <12 h, but the expression of methylated plasmids was delayed until 50 h when most embryos were at the 8-cell stage. Quantitative GluMS-PCR results suggested that 5hmC was present in the plasmid's promoter region at the MspI site where the active demethylation occurred. Our results demonstrate that 5hmC is involved in active demethylation in mice.

Hypoxia Induces Genomic DNA Demethylation through the Activation of HIF-1α and Transcriptional Upregulation of MAT2A in Hepatoma Cells

Hypoxia-inducible factor 1 (HIF-1) emerges as a crucial player in tumor progression. However, its role in hepatocellular carcinoma (HCC), especially its relation with global DNA methylation patterns in HCC under hypoxic tumor microenvironment is not completely understood. Methionine adenosyltransferase 2A (MAT2A) maintains the homeostasis of S-adenosylmethionine (SAM), a critical marker of genomic methylation status. In this study, we investigated the link between HIF-1α and MAT2A as a mechanism responsible for the change in genomic DNA methylation patterns in liver cancer under hypoxia conditions. Our results showed that hypoxia induces genomic DNA demethylation in CpG islands by reducing the steady-state SAM level both in vitro and in vivo. In addition, HIF-1α and MAT2A expression is correlated with tumor size and TNM stage of liver cancer tissues. We further showed that hypoxia-induced MAT2A expression is HIF-1α dependent and requires the recruitment of p300 and HDAC1. We also identified an authentic consensus HIF-1α binding site in MAT2A promoter by site-directed mutagenesis, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay. Taken together, we show for the first time that hypoxia induces genomic DNA demethylation through the activation of HIF-1α and transcriptional upregulation of MAT2A in hepatoma cells. These findings provide new insights into our understanding of the molecular link between genomic DNA methylation and tumor hypoxia in HCC.

Prenatal Tobacco Smoke Exposure and Genomewide Methylation in Adulthood

Abstract Genomic DNA demethylation, including demethylation of repetitive elements (which comprise 45% of the human genome), has been linked to increased risk of breast and other cancers. Genomic DNA methylation can be altered prenatally and throughout life and may be a mechanism through which the environment alters disease risk. There is evidence that prenatal tobacco smoke exposure has a persistent impact on genomic DNA methylation; however, no study to date has assessed the association between prenatal smoke exposure and adult repetitive element methylation. We measured repetitive element methylation of Alu, LINE-1, and Sat2 using MethyLight in 92 members of the New York Women's Birth Cohort, a follow-up of former female participants of the New York site of the U.S. National Collaborative Perinatal Project (mean age at blood draw = 43.5, SD = 1.8). Prenatal smoke exposure was reported prospectively. We estimated associations using multivariable linear regression, and used the natural log of Alu, LINE-1, and Sat2 methylation level. Thirty-one (36%) participants were exposed to prenatal smoke. These participants were more likely to smoke at the time of interview (P &amp;lt; 0.01). Prenatal smoke exposure was inversely associated with genomic DNA methylation of Sat2 and Alu, adjusted for age, childhood environmental tobacco smoke exposure (ETS) and adult smoking status (exposed vs. unexposed to prenatal smoke: Sat2: β = −0.20, 95% CI = −0.39, −0.02; Alu: β = −0.09, 95% CI = −0.26, 0.08). In multivariable models, childhood ETS had a positive, borderline significant association with Sat2 methylation (β = 0.17, 95% CI −0.02, 0.37). If replicated in larger studies, these results suggest that prenatal smoke exposure may have a persistent impact on genomic DNA demethylation of Sat2 and Alu in adulthood, and thus may be a pathway through which prenatal smoke exposure impacts adult disease. Results are strengthened by the fact that prenatal smoke exposure data were collected prospectively in the early 1960s, before there was a stigma associated with maternal smoking during pregnancy. These results are consistent with the one study assessing this relation in children. Further studies are needed to confirm this finding and to investigate the underlying biological mechanism.

DNA demethylation induced by the methyl-CpG-binding domain protein MBD3

The methyl-CpG binding domain protein MBD3 has been shown to be essential for embryonic development and differentiation, and to act by suppressing gene expression through the recruitment of co-repressor complexes. We have recently shown that MBD3 is also involved in maintaining the demethylated and active state of rRNA genes, and that depletion of MBD3 results in hypermethylation of rRNA promoters. The possibility that MBD3 could also trigger DNA demethylation of RNA polymerase II-transcribed genes has not been addressed. In this study we used a gain-of-function approach to examine whether MBD3 expression alters DNA methylation states in a living cell and whether it has specific targets for DNA methylation or demethylation in the genome. We used a combination of methylated DNA immunoprecipitation (mDIP) and hybridization to a human promoter tiling microarray to examine the landscape of DNA methylation patterns in response to MBD3 overexpression. We demonstrate that MBD3 induces genomic DNA demethylation and that it has specific targets in the genome with which it associates. Demethylation is localized to promoter regions with intermediate CpG density, and promoters with predicted transcription factor binding sites for NF-Y were significantly affected. These data demonstrate a causal relationship between MBD3 and DNA demethylation of genomic targets in cells.

Activity-Dependent Suppression of Miniature Neurotransmission through the Regulation of DNA Methylation

DNA methylation is an epigenetic mechanism that plays a critical role in the repression of gene expression. Here, we show that DNA methyltransferase (DNMT) inhibition in hippocampal neurons results in activity-dependent demethylation of genomic DNA and a parallel decrease in the frequency of miniature EPSCs (mEPSCs), which in turn impacts neuronal excitability and network activity. Treatment with DNMT inhibitors reveals an activity-driven demethylation of brain-derived neurotrophic factor promoter I, which is mediated by synaptic activation of NMDA receptors, because it is susceptible to AP-5, a blocker of NMDA receptors. The specific effect of DNMT inhibition on spontaneous excitatory neurotransmission requires gene transcription and is occluded in the absence of the transcriptional repressor methyl-CpG-binding protein 2 (MeCP2). Interestingly, enhancing excitatory activity, in the absence of DNMT inhibitors, also produces similar decreases in DNA methylation and mEPSC frequency, suggesting a role for DNA methylation in the control of homeostatic synaptic plasticity. Furthermore, adding excess substrate for DNA methylation (S-adenosyl-L-methionine) rescues the suppression of mEPSCs by DNMT inhibitors in wild-type neurons, as well as the defect seen in MeCP2-deficient neurons. These results uncover a means by which NMDA receptor-mediated synaptic activity drives DNA demethylation within mature neurons and suppresses basal synaptic function.

Functional Diversity of DNA Methyltransferase Inhibitors in Human Cancer Cell Lines

DNA methyltransferase inhibitors represent promising new drugs for cancer therapies. The first of these compounds (5-azacytidine, Vidaza) has recently been approved as an antitumor agent, and others are presently in various stages of their preclinical or clinical development. Most of the archetypal inhibitors have been established and characterized in different experimental systems, which has thus far precluded their direct comparison. We have now established defined experimental conditions that allowed a comparative analysis of the six most widely known DNA methyltransferase inhibitors: 5-azacytidine (5-aza-CR), 5-aza-2'-deoxycytidine (5-aza-CdR), zebularine, procaine, (-)-epigallocatechin-3-gallate (EGCG), and RG108. Of these, 5-aza-CR, 5-aza-CdR, zebularine, and EGCG were found to exhibit significant cytotoxicity in human cancer cell lines. 5-aza-CdR and EGCG were also found to be genotoxic, as evidenced by the induction of micronuclei. In addition, 5-aza-CR, 5-aza-CdR, zebularine, and RG108 caused concentration-dependent demethylation of genomic DNA, whereas procaine and EGCG failed to induce significant effects. Finally, the experiments in cancer cell lines were complemented by a cell-free in vitro assay with purified recombinant DNA methyltransferase, which indicated that RG108 is the only drug capable of direct enzyme inhibition. These results show a substantial diversity in the molecular activities of DNA methyltransferase inhibitors and provide valuable insights into the developmental potential of individual drugs.

Fractionated Low-Dose Radiation Exposure Leads to Accumulation of DNA Damage and Profound Alterations in DNA and Histone Methylation in the Murine Thymus

Thymus, an important component of hematopoietic tissue, is a well-documented "target" of radiation carcinogenesis. Both acute and fractionated irradiation result in a high risk of leukemia and thymic lymphoma. However, the exact mechanisms underlying radiation-induced predisposition to leukemia and lymphoma are still unknown, and the contributions of genetic and epigenetic mechanisms in particular have yet to be defined. Global DNA hypomethylation is a well-known characteristic of cancer cells. Recent studies have also shown that tumor cells undergo prominent changes in histone methylation, particularly a substantial loss of trimethylation of histone H4-Lys20 and demethylation of genomic DNA. These losses are considered a universal marker of malignant transformation. In the present study, we investigated the effect of low-dose radiation exposure on the accumulation of DNA lesions and alterations of DNA methylation and histone H4-Lys20 trimethylation in the thymus tissue using an in vivo murine model. For the first time, we show that fractionated whole-body application of 0.5 Gy X-ray leads to decrease in histone H4-Lys20 trimethylation in the thymus. The loss of histone H4-Lys20 trimethylation was accompanied by a significant decrease in global DNA methylation as well as the accumulation of DNA damage as monitored by persistence of histone gammaH2AX foci in the thymus tissue of mice exposed to fractionated irradiation. Altered DNA methylation was associated with reduced expression of maintenance (DNMT1) and, to a lesser extent, de novo DNA methyltransferase DNMT3a in exposed animals. Expression of another de novo DNA methyltransferase DNMT3b was decreased only in males. Irradiation also resulted in approximately 20% reduction in the levels of methyl-binding proteins MeCP2 and MBD2. Our results show the involvement of epigenetic alterations in radiation-induced responses in vivo. These changes may play a role in genome destabilization that ultimately leads to cancer.

DNA methyltransferase-3a interacts with p53 and represses p53-mediated gene expression

Genome stability maintenance is regulated by both genetic and epigenetic mechanisms. DNA methylation is the predominant epigenetic mechanism in regulation of gene expression and in suppression of mobile DNA elements from random integration in the genome. The importance of DNA methylation in tumorigenesis has been demonstrated in cancer cells which harbor global genomic DNA hypomethylation and regional hypermethylation at CpG islands of tumor suppressor genes. DNA methylation is mediated by a class of DNA methyltransferases (Dnmts) involved in de novo methylation of genomic DNA and in the maintenance of DNA methylation patterns during replication. Global genomic DNA demethylation induced by 5-Aza-deoxycytidine activates the p53 signaling pathway and induces apoptosis, suggesting that DNA methylation mediated by Dnmts is associated with p53 signaling in maintaining genome stability. In this report, we show that Dnmt3a interacts with p53 directly and represses p53-mediated transactivation of the p21 gene. It was found that trans-repression by Dnmt3a does not require the methyltransferase activity implying that transcriptional repression does not involve promoter silencing through DNA methylation by Dnmt3a. Finally, the activity of Dnmt3a in vivo was demonstrated when this enzyme was overexpressed in a breast cell line in which Dnmt3a repressed p21 upregulation following DNA damage. The results presented in this study provide new understanding of tumor promotion as mediated by Dnmt3a through its interaction with p53, and suppression of the p53-mediated transcription of tumor suppressor genes. Given that the expression of Dnmts is increased in certain cancers, it is likely that increased Dnmts could block the transactivation function of p53 following its induction by chemotherapeutic drugs resulting in chemoresistance. The use of a DNA methyltransferase inhibitor would therefore restore the p53 tumor suppression function and the utilization of such an inhibitor in combination with DNA damage agents might be an effective therapy for certain cancers.

Epigenetic Reactivation of Tumor Suppressor Genes by a Novel Small-Molecule Inhibitor of Human DNA Methyltransferases

DNA methylation regulates gene expression in normal and malignant cells. The possibility to reactivate epigenetically silenced genes has generated considerable interest in the development of DNA methyltransferase inhibitors. Here, we provide a detailed characterization of RG108, a novel small molecule that effectively blocked DNA methyltransferases in vitro and did not cause covalent enzyme trapping in human cell lines. Incubation of cells with low micromolar concentrations of the compound resulted in significant demethylation of genomic DNA without any detectable toxicity. Intriguingly, RG108 caused demethylation and reactivation of tumor suppressor genes, but it did not affect the methylation of centromeric satellite sequences. These results establish RG108 as a DNA methyltransferase inhibitor with fundamentally novel characteristics that will be particularly useful for the experimental modulation of epigenetic gene regulation.

DNA methylation and Lamarckian inheritance

Jean Baptiste de Lamarck (1744-1829) maintained that characteristics that were acquired during an organism's lifetime are passed on to its offspring. This theory, known as Lamarckian inheritance, was later completely discredited. However, recent progress in epigenetics research suggests it needs to be reexamined in consideration of DNA methylation. In this article, I summarize our observations, which support Lamarckian inheritance. Initial experiments indicate that (1) artificially induced demethy-lation of rice genomic DNA results in heritable dwarfism, and (2) cold stress induces extensive demethy-lation in somatic cells of the maize root. Based on these results, I propose the hypothesis that traits that are acquired during plant growth are sometimes inherited by their progeny through persistent alteration of the DNA methylation status.

Genomic Organization of the MouseAZ1Gene That Encodes the Protein Localized to Preacrosomes of Spermatids

The AZ1 protein is localized to the preacrosome region of spermatids. Previous developmental studies suggested thatAZ1gene transcription begins in pachytene spermatocytes and that expression of the gene is induced in cultivated fibroblasts on treatment with 5-azacytidine, which is known to lead to the demethylation of genomic DNA. In the present study, we cloned the gene coding AZ1 cDNA and elucidated its genomic organization. AZ1 cDNA was encoded by 25 exons distributed within 25 kb of genomic DNA. The transcription started at nucleotide positions −312 and −311 of the cDNA, with the A of the ATG codon specifying the initiator methionine assigned as nucleotide position +1. Exon 1 and a part of intron 1 of the AZ1 gene constituted a typical CpG island, and an about 0.2-kb sequence of the 5′-proximal region exhibited a G + C content and CpG ratio higher than the averages for the mammalian genomic sequence.

Toxicity of 5-aza-2'-deoxycytidine to mammalian cells is mediated primarily by covalent trapping of DNA methyltransferase rather than DNA demethylation.

The deoxycytidine analog 5-aza-2'-deoxycytidine (5-azadCyd) has been widely used as a DNA methylation inhibitor to experimentally induce gene expression and cellular differentiation. Prior to the availability of mutant mice with altered DNA methyltransferase levels, treatment of cells with drugs has been the only means to experimentally manipulate the level of genomic DNA methylation in mammalian cells. Substitution of DNA with 5-azadCyd leads to covalent trapping of the enzyme, thereby depleting the cells of enzyme activity and resulting in DNA demethylation. 5-AzadCyd or 5-azacytidine treatment causes multiple changes in treated cells, including activation of silent genes, decondensation of chromatin, and induction of cellular differentiation, all of which are believed to be consequences of drug-induced demethylation. 5-AzadCyd is highly toxic in cultured cells and animals and is utilized as a potent antitumor agent for treatment of certain human cancers. It has been postulated that the toxicity of the drug in mammalian cells is also due to its inhibition of DNA methylation. The chemistry of the methylation reaction is consistent, however, with an alternative mechanism: the cytotoxic effect of 5-azadCyd may be directly mediated through the covalent binding of DNA methyltransferase to 5-azadCyd-substituted DNA. We have tested this possibility by using embryonic stem cells and mice with reduced levels of DNA methyltransferase due to a targeted mutation of the gene. When exposed to 5-azadCyd mutant embryonic stem cells or embryos were significantly more resistant to the toxic effects of the drug than wild-type cells and embryos, respectively. These results strongly suggest that the cellular DNA methyltransferase itself, rather than the secondary demethylation of genomic DNA, is the primary mediator of 5-azadCyd cytotoxicity. In light of our results, some conclusions from previous studies using 5-azadCyd in order to experimentally manipulate cellular methylation levels may have to be reassessed. Also, our data make clear predictions for cancer treatment: tumor cells with elevated DNA methyltransferase levels would be expected to be susceptible to treatment with 5-azadCyd, whereas tumors with reduced levels of the enzyme would be resistant.

A novel ras-related rgp1 gene encoding a GTP-binding protein has reduced expression in 5-azacytidine-induced dwarf rice

Exposure of normal, tall rice (Oryza sativa) seedlings to 5-azacytidine, a powerful inhibitor of DNA methylation in vivo, induced both demethylation of genomic DNA and dwarf plants. Genes that had been affected by treatment were identified by differential screening of a cDNA library, and a ras-related gene, rgp1, was subsequently isolated. The cDNA of rgp1 was found to encode a deduced protein sequence of 226 amino acids with a relative molecular mass of 24850, which was most closely related to the ras-related ypt3 protein of fission yeast, Schizosaccharomyces pombe. The rgp1 protein, expressed in transformed Escherichia coli, clearly showed GTP-binding activity. During seedling rgp1 expression was first observed 14 days after germination, reaching a maximum level between 28 and 42 days, and gradually decreased thereafter until 63 days when it attained the same level of expression as in 14-day-old seedlings. Expression of rgp1 was found to be markedly reduced throughout the growth period of both 5-azacytidine-induced dwarf plants and their progenies, relative to levels in untreated tall control plants. These results suggest that expression of rgp1 may be influenced, either directly or indirectly, by DNA methylation, and that the rgp1 protein may play an important role in plant growth and development.

A single treatment of rice seedlings with 5-azacytidine induces heritable dwarfism and undermethylation of genomic DNA

A single exposure of germinated rice seeds (Oryza sativa) to either of the DNA demethylating agents 5-azacytidine (azaC) or 5-azadeoxycytidine (azadC) induced dwarf plants. At maturity, seeds treated with azaC exhibited normal morphological characteristics in comparison with untreated controls except that their height (total stem length) was reduced by about 15%. The M1 progeny, obtained by self-fertilization of an azaC-induced dwarf plant, segregated into dwarf (35%) and apparently tall types (65%). The M2 progenies, obtained by self-fertilization of dwarf M1 plants, were also dwarf, while those from tall M1 plants were only tall. Genomic DNA isolated from mature leaves of azaC-treated seeds showed about a 16% reduction in the 5-methylcytosine (m5C) content in comparison with DNA from untreated samples. A similar reduction in the m5C content was also observed in the M1 and M2 progenies. Thus, both undermethylation and dwarfism induced by azaC treatment were heritable. The results suggest that azaC induced demethylation of genomic DNA, which caused an altered pattern of gene expression and consequently a reduction in plant stem length.

Correlation between DNA undermethylation and dwarfism in maize

The relationship between DNA methylation and dwarfism in maize ( Zea mays) was examined. The amount of 5-methylcytosine (m 5C) in the DNA of a single-gene dwarf mutant, d5, was found to be about 8% lower than that of its tall, near-isogenic counterpart. In order to establish whether DNA undermethylation was at all correlated with dwarfism, germinated seeds of a common tall strain (honey bantum) were treated for 16 h with 5-azacytidine (azaC), a strong inhibitor of DNA methylation in vivo. After the chemical had been removed by repeated washing, seedlings were planted and the growth rate was measured. At maturity, a 28% reduction in the total stem length of treated plants was observed in comparison with untreated controls. Genomic DNA, isolated from an azaC-treated plant, showed about an 8% reduction in m 5C content. Southern hybridization analysis also showed demethylation in repeated sequences. Therefore, azaC treatment was found to induce both dwarfism and DNA demethylation. Although the direct relationship between demethylation of genomic DNA and dwarfism has not been fully clarified, the results suggest that DNA methylation may be involved in the control of genes regulating plant height.