A comparative analysis of developmental profiles for DNA methylation in 5-azacytidine-induced early-flowering flax lines and their control

Earlier experiments demonstrated that DNA from young plants of 5-azacytidine-induced flax (Linum usitatissimum) lines that flower earlier-than-normal is hypomethylated relative to DNA from their control lines and detected differences in methylation level between plants sampled at different ages, which suggested that the methylation level in flax changes during development. To investigate this possibility, and its potential impact on the difference in methylation level between early-flowering and control lines, developmental profiles were established for the cytosine methylation levels in DNA from post-germination seedlings and from the shoot tips of main stems and the cotyledons sampled throughout vegetative phase. The methylation profiles for two early-flowering lines and their control lines were compared. The methylation profiles were then compared to profiles for DNA content, tissue weight and chlorophyll content (green tissues); these additional parameters provided information on tissue status in terms of cell division, tissue expansion and/or photosynthetic maturity. With one exception, methylation levels were either static or increased with plant age and/or tissue maturity; the highest methylation levels were seen in senescent cotyledons. Although DNA from immature plants or tissues of the early-flowering lines was usually hypomethylated, the hypomethylation was not always apparent in tissues from older plants.     

5-azacytidine-induced tumorous transformation and DNA hypomethylation in Nicotiana tissue cultures

The phenomenon of habituation is considered in plant tissue cultures to be a real process of chemical tumorogenesis; the cultures acquire the capacity of autonomous growth in a hormone-free medium under the influence of a variety of chemical and physical agents. Treatments with 5-azacytidine (AzaC) of in vitro cultured cells of the Nicotiana glauca x N. langsdorffii nontumorous hybrid (NNT) during the culture cycle led to the induction of a habituated phenotype. The repetitive DNA sequences showed a significant lower level of endogenous methylation in the treated cells in comparison with the normal ones. It is worth noting that it was impossible until now to habituate this strain by conventional methods and that the treatments were effective only in the first 5 days of subculturing; various evidence (cytological and biochemical) pointed out a phenomenon of DNA amplification, occurring in the same period. Moreover, analysis of DNA from control and treated cells shows the induction of variations in the endogenous methylation pattern by AzaC in a critical period of cell culture. These results suggest that demethylation can act as a switch from hormone-dependent to autonomous proliferation by activation of genes coding for or regulating the synthesis of growth factors.     

DNA hypomethylation and unusual chromosome instability in cell lines from ICF syndrome patients

The ICF syndrome (immunodeficiency, centromeric region instability, facial anomalies) is a unique DNA methylation deficiency disease diagnosed by an extraordinary collection of chromosomal anomalies specifically in the vicinity of the centromeres of chromosomes 1 and 16 (Chr1 and Chr16) in mitogen-stimulated lymphocytes. These aberrations include decondensation of centromere-adjacent (qh) heterochromatin, multiradial chromosomes with up to 12 arms, and whole-arm deletions. We demonstrate that lymphoblastoid cell lines from two ICF patients exhibit these Chr1 and Chr16 anomalies in 61% of the cells and continuously generate 1qh or 16qh breaks. No other consistent chromosomal abnormality was seen except for various telomeric associations, which had not been previously noted in ICF cells. Surprisingly, multiradials composed of arms of both Chr1 and Chr16 were favored over homologous associations and cells containing multiradials with 3 or >4 arms almost always displayed losses or gains of Chr1 or Chr16 arms from the metaphase. Our results suggest that decondensation of 1qh and 16qh often leads to unresolved Holliday junctions, chromosome breakage, arm missegregation, and the formation of multiradials that may yield more stable chromosomal abnormalities, such as translocations. These cell lines maintained the abnormal hypomethylation in 1qh and 16qh seen in ICF tissues. The ICF-specific hypomethylation occurs in only a small percentage of the genome, e.g., ICF brain DNA had 7% less 5-methylcytosine than normal brain DNA. The ICF lymphoblastoid cell lines, therefore, retain not only the ICF-specific pattern of chromosome rearrangements, but also of targeted DNA hypomethylation. This hypomethylation of heterochromatic DNA sequences is seen in many cancers and may predispose to chromosome rearrangements in cancer as well as in ICF.     

Notch signaling genes: Myogenic DNA hypomethylation and 5-hydroxymethylcytosine

Notch intercellular signaling is critical for diverse developmental pathways and for homeostasis in various types of stem cells and progenitor cells. Because Notch gene products need to be precisely regulated spatially and temporally, epigenetics is likely to help control expression of Notch signaling genes. Reduced representation bisulfite sequencing (RRBS) indicated significant hypomethylation in myoblasts, myotubes, and skeletal muscle vs. many nonmuscle samples at intragenic or intergenic regions of the following Notch receptor or ligand genes: NOTCH1, NOTCH2, JAG2, and DLL1. An enzymatic assay of sites in or near these genes revealed unusually high enrichment of 5-hydroxymethylcytosine (up to 81%) in skeletal muscle, heart, and cerebellum. Epigenetics studies and gene expression profiles suggest that hypomethylation and/or hydroxymethylation help control expression of these genes in heart, brain, myoblasts, myotubes, and within skeletal muscle myofibers. Such regulation could promote cell renewal, cell maintenance, homeostasis, and a poised state for repair of tissue damage.     

Sequencing of two transgenic early-flowering poplar lines confirmed vector-free single-locus T-DNA integration

Transgenic Research - Next-generation sequencing (NGS) approaches are attractive alternatives to the PCR-based characterisation of genetically modified plants for safety assessment and labelling...     

DNA hypomethylation and human diseases

Changes in human DNA methylation patterns are an important feature of cancer development and progression and a potential role in other conditions such as atherosclerosis and autoimmune diseases (e.g., multiple sclerosis and lupus) is being recognised. The cancer genome is frequently characterised by hypermethylation of specific genes concurrently with an overall decrease in the level of 5 methyl cytosine. This hypomethylation of the genome largely affects the intergenic and intronic regions of the DNA, particularly repeat sequences and transposable elements, and is believed to result in chromosomal instability and increased mutation events. This review examines our understanding of the patterns of cancer-associated hypomethylation, and how recent advances in understanding of chromatin biology may help elucidate the mechanisms underlying repeat sequence demethylation. It also considers how global demethylation of repeat sequences including transposable elements and the site-specific hypomethylation of certain genes might contribute to the deleterious effects that ultimately result in the initiation and progression of cancer and other diseases. The use of hypomethylation of interspersed repeat sequences and genes as potential biomarkers in the early detection of tumors and their prognostic use in monitoring disease progression are also examined.     

Albumin and alpha-fetoprotein gene transcription in rat hepatoma cell lines is correlated with specific DNA hypomethylation and altered chromatin structure in the 5'' region.

We examined DNA methylation and DNase I hypersensitivity of the alpha-fetoprotein (AFP) and albumin gene region in hepatoma cell lines which showed drastic differences in the level of expression of these genes. We assayed for methylation of the CCGG sequences by using the restriction enzyme isoschizomers HpaII and MspI. We found two methylation sites located in the 5' region of the AFP gene and one in exon 1 of the albumin gene for which hypomethylation is correlated with gene expression. Another such site, located about 4,000 base pairs upstream from the AFP gene, seems to be correlated with the tissue specificity of the cells. DNase I-hypersensitive sites were mapped by using the indirect end-labeling technique with cloned genomic DNA probes. Three tissue-specific DNase I-hypersensitive sites were mapped in the 5' flanking region of the AFP gene when this gene was transcribed. Similarly, three tissue-specific DNase I-hypersensitive sites were detected upstream from the albumin gene in producing cell lines. In both cases, the most distal sites were maintained after cessation of gene activity and appear to be correlated with the potential expression of the gene. Interestingly, specific methylation sites are localized in the same DNA region as DNase I hypersensitive sites. This suggests that specific alterations of chromatin structure and changes in methylation pattern occur in specific critical regulatory regions upstream from the albumin and AFP genes in rat hepatoma cell lines.     

Inhibitory effect of caffeine on 5-azacytidine-induced digital malformations in the rat

The effect of caffeine on 5-azacytidine (5-AC)-induced digital malformations in rat fetuses was investigated. Caffeine suppressed all types of digital defects in the fore- and hindlimbs except for syndactyly induced by 1.0 mg/kg of 5-AC; it was still effective when administered 24 hours after 5-AC treatment. However, fetal mortality increased as the frequency of malformations decreased. While the malformation results support the view that caffeine inhibits the processes leading to malformation expression, the relation between its suppressive effect on malformations and its enhancing effect on fetal mortality is unclear.     

DNA hypomethylation in ethionine-induced rat preneoplastic hepatocyte nodules

DNA from hepatocyte nodules induced in rats with dietary DL-ethionine and from the surrounding non-nodular liver contained less 5-methyldeoxycytidine per deoxycytidine when compared with that from normal adult liver. The degree of apparent hypomethylation, 37% in nodules and 20% in the surrounding liver, decreased somewhat (29% and 16% respectively) at 2 weeks after terminating the exposure to ethionine. Nodules and surrounding liver, like normal liver, responded to partial hepatectomy with a decrease in the 5-methyldeoxycytidine level at 24 hrs and a return to the level at the time of partial hepatectomy by 38 hrs. These findings indicate the need for careful control of cell proliferation in comparing the levels of a post-replicative DNA modification, methylation, in proliferating and non-proliferating cell populations. These findings also suggest that a portion of the hypomethylation in preneoplastic nodules may be due to a bona fide decrease in the level of cytosine methylation in the parental strand of DNA. This hypomethylation could be one basis for the altered gene expression in hepatocyte nodules, possible precursors for liver cancer.     

DNA global hypomethylation in EBV-transformed interphase nuclei.

In tumors, DNA is often globally hypomethylated compared to DNA extracted from normal tissues. This observation is usually made after extraction and exhaustive digestion of DNA followed by analysis of nucleosides by chromatography or digestion with restriction enzymes, gel analysis, and hybridization. This approach provides an average value which does not give information on the various cell subpopulations included in heterogeneous samples. Therefore an immunochemical technique was set up with the aim of demonstrating, in a population of mixed cells, the possibility of detecting the presence of individual nuclei containing hypomethylated DNA, on a cell-by-cell basis. Monoclonal antibodies to 5-methylcytidine were used to label cells grown in vitro. Under appropriate fixation and permeabilization conditions, interphase nuclei were labeled. Quantitative differences in the labeling were detected between Epstein-Barr virus-transformed cells and normal peripheral blood monocytes by flow cytometry analysis. Similar differences were observed by fluorescence microscopy. Both results were confirmed by Southern transfer and hybridization of DNA fragments generated by restriction enzyme digestion. This observation, which is in accordance with the occurrence of global DNA hypomethylation in tumors as established by chromatography, opens the field for the analysis of fresh tumor samples by flow cytometry and microscopy.     

Mechanisms of peroxisome proliferator-induced DNA hypomethylation in rat liver

Genomic hypomethylation is a consistent finding in both human and animal tumors and mounting experimental evidence suggests a key role for epigenetic events in tumorigenesis. Furthermore, it has been suggested that early changes in DNA methylation and histone modifications may serve as sensitive predictive markers in animal testing for carcinogenic potency of environmental agents. Alterations in metabolism of methyl donors, disturbances in activity and/or expression of DNA methyltransferases, and presence of DNA single-strand breaks could contribute to the loss of cytosine methylation during carcinogenesis; however, the precise mechanisms of genomic hypomethylation induced by chemical carcinogens remain largely unknown. This study examined the mechanism of DNA hypomethylation during hepatocarcinogenesis induced by peroxisome proliferators WY-14,643 (4-chloro-6-(2,3-xylidino)-pyrimidynylthioacetic acid) and DEHP (di-(2-ethylhexyl)phthalate), agents acting through non-genotoxic mode of action. In the liver of male Fisher 344 rats exposed to WY-14,643 (0.1% (w/w), 5 months), the level of genomic hypomethylation increased by approximately 2-fold, as compared to age-matched controls, while in the DEHP group (1.2% (w/w), 5 months) DNA methylation did not change. Global DNA hypomethylation in livers from WY-14,643 group was accompanied by the accumulation of DNA single-strand breaks, increased cell proliferation, and diminished expression of DNA methyltransferase 1, while the metabolism of methyl donors was not affected. In contrast, none of these parameters changed significantly in rats fed DEHP. Since WY-14,643 is much more potent carcinogen than DEHP, we conclude that the extent of loss of DNA methylation may be related to the carcinogenic potential of the chemical agent, and that accumulation of DNA single-strand breaks coupled to the increase in cell proliferation and altered DNA methyltransferase expression may explain genomic hypomethylation during peroxisome proliferator-induced carcinogenesis.     

Human DNA sequences exhibiting gamete-specific hypomethylation.

Three human DNA sequences have been cloned from DNA regions which are strikingly undermethylated in sperm, highly methylated in adult somatic tissues, and methylated to an intermediate extent in tissues of extraembryonic origin. It is proposed that some such DNA sequences may function specifically early in embryogenesis or during gametogenesis. They may be subsequently extensively methylated in the embryonic cell lineage and methylated to a lesser extent in extraembryonic tissues in order to allow embryogenesis to proceed.     

Curcumin is a potent DNA hypomethylation agent

Molecular docking of the interaction of curcumin and DNMT1 suggested that curcumin covalently blocks the catalytic thiolate of C1226 of DNMT1 to exert its inhibitory effect. This was validated by showing that curcumin inhibits the activity of M. SssI with an IC₅₀ of 30 nM, but no inhibitory activity of hexahydrocurcumin up to 100 μM. In addition, curcumin can induce global DNA hypomethylation in a leukemia cell line.     

Spontaneous and 5-azacytidine-induced reexpression of ornithine carbamoyl transferase in hepatoma cells.

Rat hepatoma cells that do not synthesize the hepatic enzyme ornithine carbamoyl transferase spontaneously give rise to producing cells at a low frequency. Reexpression of this differentiation trait is strongly increased by 5-azacytidine treatment, suggesting that hypermethylation plays a critical role in the impaired expression of the ornithine carbamoyl transferase gene in hepatoma cells.     

Causes and consequences of DNA hypomethylation in human cancer.

While specific genes are hypermethylated in the genome of cancer cells, overall methylcytosine content is often decreased as a consequence of hypomethylation affecting many repetitive sequences. Hypomethylation is also observed at a number of single-copy genes. While global hypomethylation is highly prevalent across all cancer types, it often displays considerable specificity with regard to tumor type, tumor stage, and sequences affected. Following an overview of hypomethylation alterations in various cancers, this review focuses on 3 hypotheses. First, hypomethylation at a single-copy gene may occur as a 2-step process, in which selection for gene function follows upon random hypo methylation. In this fashion, hypomethylation facilitates the adaptation of cancer cells to the ever-changing tumor tissue microenvironment, particularly during metastasis. Second, the development of global hypomethylation is intimately linked to chromatin restructuring and nuclear disorganization in cancer cells, reflected in a large number of changes in histone-modifying enzymes and other chromatin regulators. Third, DNA hypomethylation may occur at least partly as a consequence of cell cycle deregulation disturbing the coordination between DNA replication and activity of DNA methyltransferases. Finally, because of their relation to tumor progression and metastasis, DNA hypomethylation markers may be particularly useful to classify cancer and predict their clinical course.     

Extensive maternal DNA hypomethylation in the endosperm of Zea mays

A PCR-based genomic scan has been undertaken to estimate the extent and ratio of maternally versus paternally methylated DNA regions in endosperm, embryo, and leaf of Zea mays (maize). Analysis of several inbred lines and their reciprocal crosses identified a large number of conserved, differentially methylated DNA regions (DMRs) that were specific to the endosperm. DMRs were hypomethylated at specific methylation-sensitive restriction sites upon maternal transmission, whereas upon paternal transmission, the methylation levels were similar to those observed in embryo and leaf. Maternal hypomethylation was extensive and offers a likely explanation for the 13% reduction in methyl-cytosine content of the endosperm compared with leaf tissue. DMRs showed identity to expressed genic regions, were observed early after fertilization, and maintained at a later stage of endosperm development. The implications of extensive maternal hypomethylation with respect to endosperm development and epigenetic reprogramming will be discussed.