Induction of flowering by 5-azacytidine in some plant species: relationship between the stability of photoperiodically induced flowering and flower-inducing effect of DNA demethylation

The flower-inducing effect of 5-azacytidine, a DNA demethylating reagent, was examined in several plant species with a stable or unstable photoperiodically induced flowering state under non-inductive photoperiodic conditions. The long day plant Silene armeria , whose flowering state is stable and the short day plant Pharbitis nil , whose flowering state is unstable were induced to flower by 5-azacytidine under a non-inductive condition. Thus, the replacement of photoinduction by 5-azacytidine treatment is not specific to Perilla frutescens . On the other hand, 5-azacytidine did not induce flowering in Xanthium strumarium whose flowering state is stable and Lemna paucicostata whose flowering state is unstable. Thus, epigenetics caused by DNA demethylation may be involved in the regulation of photoperiodic flowering irrespective of the stability of the photoperiodically induced flowering state.     

Epigenetic regulation of photoperiodic flowering

The cytidine analogue 5-azacytidine, which causes DNA demethylation, induced flowering in the non-vernalization-requiring plants Perilla frutescens var. crispa, Silene armeria and Pharbitis nil (synonym Ipomoea nil) under non-inductive photoperiodic conditions, suggesting that the expression of photoperiodic flowering-related genes is regulated epigenetically by DNA methylation. The flowering state induced by DNA demethylation was not heritable. Changes in the genome-wide methylation state were examined by methylation-sensitive amplified fragment length polymorphism analysis. This analysis indicated that the DNA methylation state was altered by the photoperiodic condition. DNA demethylation also induced dwarfism, and the induced dwarfism of P. frutescens was heritable.Key words: 5-azacytidine, DNA methylation, photoperiodic flowering, epigenetics, methylation-sensitive amplified fragment length polymorphism, CpG island, dwarfism     

The effect of day length, vernalization and DNA demethylation on the flowering time in Arabidopsis thaliana

We studied the effect of three factors on the induction of flowering in Arabidopsis thaliana , i.e. vernalization, day length and DNA demethylation. Seven natural late flowering genotypes and 13 late flowering mutants were used in the experiments. The effect of the vernalization and the short day (SD) was uniform in all genotypes used, resulting in shortening (vernalization) or extension of the period before the appearance of the first flower primordia. On the other hand, the effect of the demethylating agent (5-azacytidine [5-azaC]) was not uniform in the genotypes used. In all natural late genotypes (except Lu-1 ), the shortening of the flowering time (FT) after 5-azaC treatment was observed. On the contrary, only five mutants – dl , pm , M63 , M73 and fca-1 – showed a shortening of the FT, while in the majority of the late flowering mutants, no significant response (earlier flowering) was found. The different response to the vernalization and demethylation treatment in late flowering mutants shows the possibility of two different pathways leading to the flowering, both of which are regulated by DNA demethylation. The different response of natural and induced late flowering genotypes after 5-azaC treatment shows that genes that play a role in flower development are of a different nature.     

Correspondence between effects of 5-azacytidine on SCE formation, cell cycling and DNA methylation in Chinese hamster cells

The effects of 5-azacytidine (5-Aza-C), alone and in combination with mitomycin C, were measured on sister-chromatid exchange (SCE) formation and DNA methylation in different genomic regions of Chinese hamster ovary cells and in Chinese hamster cells containing amplified, dihydrofolate reductase sequences and resistant to methotrexate. 5-Aza-C, when present for the penultimate preharvest cell cycle, induced SCEs in a manner consistent with a directly measured reduction in deoxycytosine methylation in cellular DNA. At higher 5-Aza-C concentrations, cell cycling was inhibited and both SCE induction and DNA demethylation tended to level off. Under appropriate conditions, 5-Aza-C also potentiated the induction of SCEs by mitomycin C. 5-Aza-C-induced DNA demethylation could also be detected in the vicinity of different DNA sequences with the use of comparative HpaII/MspI digestion, DNA blotting, and molecular probes. The efficiency of an individual demethylation event in inducing SCE induction appeared to be very low, compared with alkylating agents such as 8-methoxypsoralen, suggesting that SCE induction by 5-Aza-C might be an indirect effect from long range changes induced in cellular DNA or chromatin conformation.     

DNA demethylation induced by 5-azacytidine does not affect fragile X expression.

Experiments were performed to determine the role of DNA demethylation in fragile X expression. Fragile X positive lymphoblastoid cells were treated with 5-azacytidine and harvested for analysis of fragile X expression both directly following treatment and after a recovery period in the absence of the drug. The effectiveness of 5-azacytidine treatment in inducing DNA demethylation was concurrently monitored by analysis of methylation changes at random autosomal loci in isolated DNA from treated cells. Under conditions where 5-azacytidine was found to inhibit fragile X expression, no DNA demethylation was observed. At the time when demethylation did occur, fragile X expression was not affected. These results strongly indicate that DNA demethylation is not involved in fragile X expression.     

Effect of anti-DNA methylation drugs on growth, level of methylated DNA, peroxidase activity and ethylene production of Bryonia dioica internodes

A previous paper (Galaud et al. 1993. Physiol. Plant. 87: 25–30) reported a rapid demethylation of DNA after mechanical rubbing of young Bryonia dioica internodes, leading to growth inhibition. In order to assess an eventual causative role of DNA demethylation in the mechanisms underlying thigmomorphogenesis, the effects of the anti-DNA methylation drugs, 5-azacytosine (5-azacyt) and 5-azacytidine (5-azacyd) were investigated. Treatment with 5-azacyd induced a decrease of the methylated DNA level. This was accompanied by an increase of peroxidase activity and of ethylene production. On the contrary, 5-azacyt had no significant effect on growth, peroxidase activity and ethylene production. The use of 5-azacyd as an inducer of DNA demethylation is discussed in relation to the control of gene expression     

The mouse L-histidine decarboxylase gene: structure and transcriptional regulation by CpG methylation in the promoter region

To investigate the regulation of mouse L-histidine decarboxylase (HDC) gene expression, we isolated genomic DNA clones encoding HDC. Structural analysis revealed that the mouse HDC gene was composed of 12 exons, spanning approximately 24 kb. Northern blotting analysis indicated that, among the cell lines examined, a high level of HDC gene expression was restricted to mature mast cell lines and an erythroblastic cell line. The gene was induced strongly in the mouse immature mast cell line P815 after incubation in the peritoneal cavity of BDF1 mice. We observed that the promoter region was demethylated in the HDC-expressing cell lines and in induced P815 cells. Interestingly, forced demethylation by 5-azacytidine (5-azaC) treatment induced high expression of HDC mRNA in P815 cells. The activity of a mouse HDC promoter-reporter construct stably transfected in P815 cells was repressed by in vitro patch-methylation. This low promoter activity of the patch-methylated reporter construct was restored after 5-azaC treatment, which demethylated the patch-methylated promoter. These results indicate that DNA methylation state of the promoter region controls HDC gene expression.     

Direct visualization of the sites of DNA methylation in human,and mosquito chromosomes

Human and mosquito fixed chromosomes were digested with restriction endonucleases that are inhibited by the presence of 5-methylcytosine in their restriction sites (Hha I, Hin PI, Hpa II), and with endonucleases for which cleavage is less dependent on the state of methylation (Taq I, Msp I). Methylation-dependent enzymes extracted low DNA amounts from human chromosomes, while methylation-independent enzymes extracted moderate to high amounts of DNA. After DNA demethylation with 5-azacytidine the isoschizomers Hpa II (methylation-dependent) and Msp I (methylation-independent) extracted 12-fold and 1.4-fold amounts of DNA from human chromosomes, respectively. These findings indicate that human DNA has a high concentration of Hpa II and Msp I restriction sites (CCGG), and that the internal C of this sequence is methylated in most cases, while the external cytosine is methylated less often. All the enzymes tested released moderate amounts of DNA from mosquito chromosomes whether or not the DNA was demethylated with 5-azacytidine. Hpa II induced banding in the centromere chromosome regions. After demethylation with 5-azacytidine this banding disappeared. Mosquito DNA has therefore, moderate to high frequencies of nonmethylated CpG duplets. The only exception is the centromeric DNA, in which the high levels of C methylation present produce cleavage by Hpa II and the appearance of banding. Centromere regions of human chromosomes 1 have a moderately low concentration of Hpa II-Msp I restriction sites.     

Effect of 5-Azacytidine on the Methylation Aspects of NMDA Receptor NR2B Gene in the Cultured Cortical Neurons of Mice

Our previous study revealed that the exposure of the drug 5-Azacytidine and ethanol to the cultured cortical neurons of mice causes demethylation of cytosine residues in the CpG island of the NMDA receptor NR2B gene (Marutha Ravindran and Ticku, Mol Brain Res 121:19-27, 2004). In the present study, we further analyzed methylation in the CpG island with various concentration frame and time frame of exposure of the cultured cortical neurons with 5-azacytidine to identify whether methylation in the NR2B gene is site specific or region specific. Methylation was studied by digesting the genomic DNA with methylation sensitive HpaII, MspI, AciI or HhaI enzyme following the exposure of cultured cortical neurons of mice with 5-azacytidine by performing PCR and Southern hybridization. We observed demethylation of DNA at 1, 3 and 5 μM concentrations of 5-azacytidine in the regions (5982–6155), (6743–7466) and at 3 and 5 μM concentrations of 5-azacytidine used in the region (6477–6763). Similarly in the time frame study with 5-azacytidine, demethylation of DNA was observed at 24 h and 36 h of incubation with 5-azacytidine in the regions (5982–6155), (6743–7466) and at 36 h of incubation with 5-azacytidine used in the region (6477–6763). Our experimental results demonstrate that the methylation in the CpG islands of the NR2B gene may not be site specific or region specific in the cultured cortical neurons of mice.     

Opposing effects of reduced DNA methylation on flowering time in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Demethylation of DNA promotes flowering in plants from the vernalization-responsive ecotype C24 of Arabidopsis thaliana (L.) Heynh., but delays flowering in the ecotype Landsberg erecta which is not responsive to vernalization. To investigate these contrasting effects of low methylation we have monitored flowering times and expression of two repressors of flowering, FLC and FWA, in low-methylation plants from three late-flowering mutants in the ecotype Landsberg erecta. Demethylation of DNA decreased FLC expression in the vernalization-responsive mutants, but was not associated with a promotion of flowering; rather, in some lines, demethylation delayed flowering. The opposing effects of demethylation could be explained by its differential effect on the expression of two repressors of flowering. FLC was down-regulated in plants with low methylation, promoting flowering, while FWA was activated in response to demethylation, which probably delays the transition to flowering. Expression of the FWA gene did not delay flowering in plants of ecotype C24; our data suggest that the FWA protein of C24 may be non-functional.     

5-Azacytidine-induced demethylation of DNA to senescent level does not block proliferation of human fibroblasts.

IMR-90 human diploid fibroblasts (HDF) lose from 30-50% of their genomic 5-methyldeoxycytidine (5mdC) during the cellular aging process. In contrast, immortal SV40-transformed IMR-90 maintain a constant level of 5mdC in culture. Precrisis SV40-transformed HDF (AG3204) represent a stage in between normal cell aging and immortalization because these cells still have a finite proliferative lifespan, but it is longer than that of normal HDF and ends in cell death rather than in G1-arrest. We find that AG3204 cells continue to lose from 12-33% of their 5mdC after a population has become 99% positive for SV40 T-antigen. Both IMR-90 cells and AG3204 cells have similar levels of 5mdC (average of 2.25%) at the end of lifespan. We investigated whether this level of 5mdC is an absolute block to further proliferation by treating IMR-90 and AG3204 cells with 5-azacytidine (5azaC) to reduce their 5mdC levels below the terminal level normally achieved at end of lifespan. We find that both IMR-90 and AG3204 cells undergo extensive proliferation with subterminal levels of 5mdC and that the lifespans of both cell types are shortened by 5azaC treatment. These studies indicate that random genomic DNA demethylation to a specific level of 5mdC is not a direct cause of finite proliferative lifespan. However, the correlation between accelerated DNA demethylation and accelerated aging still suggests that these two phenomena are related. Two ways to explain this relationship are: (1) DNA demethylation during aging is not random, and/or (2) both DNA demethylation and other independent aging processes cooperate to produce finite lifespan. In both cases, accelerated random DNA demethylation could accelerate aging, but not necessarily in direct relationship to the final genomic level of 5mdC achieved during the normal aging process.     

Impact of DNA methyltransferase inhibitor 5-azacytidine on cardiac development of zebrafish in vivo and cardiomyocyte proliferation,apoptosis, and the homeostasis of gene expression in vitro

Cardiac development is a peculiar process involving coordinated cellular differentiation, migration, proliferation, and apoptosis. DNA methylation plays a key role in genomic stability, tissue-specific gene expression, cell proliferation, and apoptosis. Hypomethylation in the global genome has been reported in cardiovascular diseases. However, little is known about the impact and specific mechanism of global hypomethylation on cardiomyocytes. In the present study, we explored the impact of DNA methyltransferase inhibitors 5-azacytidine on cardiac development. In vivo experiment showed that hypomethylation of zebrafish embryos with 5-azacytidine exposure significantly reduced survival, induced malformations, and delayed general development process. Furthermore, zebrafish embryos injected with 5-azacytidine developed pericardial edema, ventricular volume reduction, looping deformity, and reduction in heart rate and ventricular shortening fraction. Cardiomyocytes treated with 5-azacytidine in vitro decreased proliferation and induced apoptosis in a concentration-dependent manner. Furthermore, 5-azacytidine treatment in cardiomyocytes resulted in 20 downregulated genes expression and two upregulated genes expression in 45 candidate genes, which indicated that DNA methylation functions as a bidirectional modulator in regulating gene expression. In conclusion, these results show the regulative effects of the epigenetic modifier 5-azacytidine in cardiac development of zebrafish embryos in vivo and cardiomyocyte proliferation and apoptosis and the homeostasis of gene expression in vitro, which offer a novel understanding of aberrant DNA methylation in the etiology of cardiovascular disease.     

5-Azacytidine increases the activity of neomycin phosphotransferase II in transgenic Nicotiana tabacum: A posttranslational mechanism may play a role

In previous studies, tobacco protoplasts were transformed with the bacterial gene encoding neomycin phosphotransferase II (NPT II). Transformed calluses lost neomycin phosphotransferase II activity after several subcultures. Treatment of calluses with 5-azacytidine, a demethylating agent, restored enzyme activity, suggesting that methylation of npt II sequences might be responsible for loss of NPT II activity. Studies presented here were designed to test that hypothesis. Results indicated that the effect of 5-azacytidine could not be blocked by the DNA replication inhibitor, hydroxyurea, nor by the 5-azacytidine analogue, cytidine as would be expected with a DNA demethylation mechanism. The level of NPT II mRNA was not increased by 5-azacytidine. Treatment with cycloheximide, a protein synthesis inhibitor, had no effect on 5-azacytidine-increased NPT II activity. There was no increase of NPT II protein caused by 5-azacytidine, whereas 5-azacytidine increased activity of NPT II. In contrast, the auxin 2,4-D increased both the NPT II protein and activity. Assays for malate dehydrogenase demonstrated that the effect of 5-azacytidine and hydroxyurea on NPT II was not due to an overall effect on callus metabolism. In vitro studies involving standard bacterial NPT II enzyme and crude extracts from untreated and 5-azacytidine- or hydroxyurea-treated calluses showed that the activity of NPT II added to the untreated extracts was lower than the activity of NPT II added to the extracts from calluses treated with 5-azacytidine or hydroxyurea, indicating that there was an unknown factor (or factors) in callus extracts which affected the activity of NPT II and itself was affected by 5-azacytidine and hydroxyurea treatment. These results suggested that one effect of 5-azacytidine in increasing NPT II activity was posttranslational.Abbreviations ELISA enzyme-linked immunosorbent assay - NOS nopalene synthase - nos DNA segment encoding NOS - NPT II neomycin phosphotransferase - npt II DNA segment encoding NPT II - PAGE polyacrylamide gel electrophoresis     

Macronuclear DNA demethylation is involved in the encystment process of the ciliate Colpoda inflata.

Ciliate encystment is an eukaryotic cell differentiation process which involves a specific gene expression, to form the resting stage. In this study, we investigate, for first time, the DNA methylation pattern changes during encystment in the ciliate Colpoda inflata, and the 5-azacytidine effect on growing cells and encystment. Results indicate that 5-methylcytosine is present in macronuclear DNA of this ciliate and the 5-azacytidine treatment induces encystment in growth conditions. From restriction enzyme digestion and 5-azacytidine experiments, we conclude that a specific DNA demethylation is probably involved in the encystment gene expression of this ciliate.     

Macronuclear DNA demethylation is involved in the encystment process of the ciliate Colpoda inflata.

Ciliate encystment is an eukaryotic cell differentiation process which involves a specific gene expression, to form the resting stage. In this study, we investigate, for first time, the DNA methylation pattern changes during encystment in the ciliate Colpoda inflata, and the 5-azacytidine effect on growing cells and encystment. Results indicate that 5-methylcytosine is present in macronuclear DNA of this ciliate and the 5-azacytidine treatment induces encystment in growth conditions. From restriction enzyme digestion and 5-azacytidine experiments, we conclude that a specific DNA demethylation is probably involved in the encystment gene expression of this ciliate.     

DNA 去甲基化机制的研究进展*

DNA甲基化作为动植物体内一种重要的表观遗传修饰形式,在调控基因表达、维持基因组的稳定性等方面发挥重要的生物学作用。固有DNA甲基化水平和模式的变化会导致生物的表型异常甚至死亡。而5-甲基胞嘧啶的水平和模式是由DNA甲基化和去甲基化共同决定的。DNA去甲基化可以分为主动去甲基化与被动去甲基化,而基因组甲基化模式的形成主要依赖于主动去甲基化。本文综述了生物体内DNA主动去甲基化五种潜在机制:DNA转葡糖基酶参与的碱基切除修复途径、脱氨酶参与的碱基切除修复途径、核苷酸切除修复途径、氧化作用去甲基化与水解作用去甲基化。     

DNA methylation is involved in maintenance of an unusual expression pattern of an introduced gene

In one of 30 transgenic tobacco (Nicotiana tabacum) plants, the expression of an introduced β-glucuronidase (GUS) gene driven by the cauliflower mosaic virus 35S promoter, was found to be repressed as the plant matured, whereas the endogenous GUS activity was unaffected. Plants grown from seeds or regenerated from leaf discs derived from this plant showed a similar temporal pattern of expression. Suspension-cultured cells established from nonexpressing leaves did not express the introduced gene. In these cells, the silent gene could be reactivated by treatment for 5 or 10 days with 5-azacytidine. Overall, demethylation of the genome preceded recovery of the enzyme activity. The increase in the fraction of reactivated cells progressed in two phases. Up to 8 weeks after starting the 5-azacytidine treatment, approximately 2 to 4% of the cells were expressing GUS, followed by a dramatic increase of GUS-expressing cells. Thirteen weeks after starting the 5-azacytidine treatment, the fraction of GUS-expressing cells amounted to 80%. At this time, the original overall level of DNA methylation was reestablished. The degree of DNA demethylation, as well as the magnitude of reactivation, was dependent on the duration of the 5-azacytidine treatment. These results demonstrate that DNA methylation appears to be involved in the regulation of the introduced GUS gene and that this development-dependent pattern of expression can be inherited.