The kinetics of DNA methylation in cultures of a mouse adrenal cell line

Direct measurements of the methylation of newly-synthesized DNA were made in cultures of a clonal mouse adrenal cortex cell line, Y129OS3, by (1) following the incorporation of radioactivity from methionine-(methyl)-C¹⁴ into a segment of DNA which had been density-labeled with bromouracil and (2) labeling DNA cytosine with C¹⁴-deoxycytidine and then following the appearance of radioactivity in DNA 5-methylcytosine. The results establish that during exponential growth the DNA of this cell line is methylated entirely within a few minutes of its synthesis. Using the second technique described above accurate, sensitive measurements of DNA methylation levels can be made by comparing radioactivity in 5-methylcytosine to radioactivity in cytosine plus 5-methylcytosine. In this cell line 5-methylcytosine accounts for 4.3 ± 0.2% of the DNA cytosine. Some apparent contradictions between these results and those of other workers are discussed.     

Characterization of DNA methylation in the rat

In the rat, differentiation and cell proliferation both affect DNA methylation. We studied 5-methylcytosine at the inner cytosine of the sequence C-C-G-G, a common methylation site, using endonuclease MspI (which cleaves C-C-G-G- and C-^mC-G-G), and its isoschizomer HpaII (which cleaves only C-C-G-G). DNA from all tissues and cell lines studied was methylated at C-C-G-G, at levels ranging from 45 to 80%, but the methylation sites were not distributed uniformly. Our analysis suggests a model in which cells contain variable amounts of three DNA methylation states, averaging 30–40, 70–80 and 95–100% methylation, respectively. One biological parameter that alters methylation is the prolferative state of the cell. We observed that NRK, a non-transformed cell line, increased its DNA methylation from 45 to 67% when monolayer cultures became confluent and non-dividing. We also observed that a class of repetitive DNA was completely methylated in DNA from all sources except a transformed cell line.     

Characterization of DNA methylation in the rat

In the rat, differentiation and cell proliferation both affect DNA methylation. We studied 5-methylcytosine at the inner cytosine of the sequence C-C-G-G, a common methylation site, using endonuclease MspI (which cleaves C-C-G-G- and C-mC-G-G), and its isoschizomer HpaII (which cleaves only C-C-G-G). DNA from all tissues and cell lines studied was methylated at C-C-G-G, at levels ranging from 45 to 80%, but the methylation sites were not distributed uniformly. Our analysis suggests a model in which cells contain variable amounts of three DNA methylation states, averaging 30-40, 70-80 and 95-100% methylation, respectively. One biological parameter that alters methylation is the proliferative state of the cell. We observed that NRK, a non-transformed cell line, increased its DNA methylation from 45 to 67% when monolayer cultures became confluent and non-dividing. We also observed that a class of repetitive DNA was completely methylated in DNA from all sources except a transformed cell line.     

Methylation of parental and progeny DNA strands in Physarum polycephalum

Although 5-methylcytosine comprises 4 to 8% of the cytosine residues in the major nuclear DNA of Physarum polycephalum (Evans &; Evans, 1970), only 1 % of the cytosine residues of progeny DNA become methylated during replication. Further methylation occurs during the same and subsequent mitotic cycles, so that 6 to 7 cycles after its synthesis, 5-methylcytosine comprises 5 to 7% of the DNA-cytosine residues of a single generation of DNA. The extent of methylation occurring during the S period has been measured by the determination of the specific activity of the precursor (S-adenosylmethionine) and the product (DNA-5-methylcytosine) and by comparison of the radioactivity in DNA-cytosine and DNA-5-methylcytosine after incorporation of [¹⁴C]deoxycytidine. Continuing methylation of parental DNA has been shown, by density shift experiments and by the conversion of prelabeled DNA-cytosine to DNA-5-methylcytosine. The DNA-5-methylcytosine once formed was found to be stable.     

Direct detection of methylation in genomic DNA

The identification of methylated sites on bacterial genomic DNA would be a useful tool to study the major roles of DNA methylation in prokaryotes: distinction of self and nonself DNA, direction of post-replicative mismatch repair, control of DNA replication and cell cycle, and regulation of gene expression. Three types of methylated nucleobases are known: N⁶-methyladenine, 5-methylcytosine and N⁴-methylcytosine. The aim of this study was to develop a method to detect all three types of DNA methylation in complete genomic DNA. It was previously shown that N⁶-methyladenine and 5-methylcytosine in plasmid and viral DNA can be detected by intersequence trace comparison of methylated and unmethylated DNA. We extended this method to include N⁴-methylcytosine detection in both in vitro and in vivo methylated DNA. Furthermore, application of intersequence trace comparison was extended to bacterial genomic DNA. Finally, we present evidence that intrasequence comparison suffices to detect methylated sites in genomic DNA. In conclusion, we present a method to detect all three natural types of DNA methylation in bacterial genomic DNA. This provides the possibility to define the complete methylome of any prokaryote.     

Differential methylation of mitochondrial and nuclear DNA in cultured mouse, hamster and virus-transformed hamster cells. In vivo and in vitro methylation

Information has been lacking as to whether mitochondrial DNA of animal cells is methylated. The methylation patterns of mitochondrial and nuclear DNAs of several mammalian cell lines have therefore been compared by four methods: (1) in vivo transfer of the methyl group from [methyl-³H]methionine; (2) in vivo incorporation of [³²P]orthophosphate and a combination of (1) and (2); (3) in vivo incorporation of [³H]deoxycytidine; (4) in vitro methylation of DNAs with ³H-labeled S-adenosylmethionine as methyl donor and DNA methylase preparations from L cell nuclei. The cell lines were mouse L cells, $\text{BHK21}\text{C13}$, $\text{C13}\text{B4}$ (baby hamster kidney cells transformed by the Bryan strain of Rouse sarcoma virus), and PyY (BHK cells transformed by polyoma virus). DNA bases were separated chromatographically, using 5-methylcytosine, 6-methylaminopurine and, in some cases, 7-methylguanine as markers.Mitochondrial DNA was found to be significantly less methylated than nuclear DNA with respect to 5-methylcytosine in all cell types studied and by all methods used. The relative advantages and disadvantages of each method have been discussed. The level of 5-methylcytosine in mitochondrial DNA as compared with that in nuclear DNA was estimated as one-fourth to one-fourteenth in various cell lines. The estimated 5-methylcytosine content per circular mitochondrial DNA molecule (mol. wt 10 × 10⁶) was about 12 methylcytosine residues for L cells and 24, 30 and 36 methylcytosine residues for BHK, B4 and PyY cells, respectively. Relative to cytosine residues, the estimate was one 5-methylcytosine per 500 cytosine residues of mitochondrial DNA and one 5-methylcytosine per 36 cytosine residues of nuclear DNA from L-cells. The values for methylcytosine of mitochondrial DNA are presumed to be maximal. PyY cells as compared with other cells had the highest methylcytosine content of both mitochondrial and nuclear DNA as estimated by method (3). No methylation of nuclear DNA was observed in confluent L cells.Evidence for the presence of DNA methylase activity associated with mitochondrial fractions was obtained. This activity could be distinguished from other cellular DNA methylase activity by differential response to mercaptoethanol. Radioactivity from ³H-labeled S-adenosylmethionine was found only in 5-methyl-cytosine of DNA.     

Hypomethylation of repair patches in HeLa cells

In HeLa cells, under conditions where normal semiconservative synthesis is suppressed by hydroxyurea, the excision repair process after irradiation by UV results in a small amount of incorporation of nucleotides into nonreplicated DNA. By labelling the cytosine moieties of these repair patches, and measuring the ratio between cytosine and 5-methylcytosine, we have found that the level of methylation of cytosine in repair patches five hours after UV-irradiation of the cells is about half of that observed in normal semiconservatively synthesized DNA.     

DNA methylation in xeroderma pigmentosum

DNA methylation was examined in xeroderma pigmentosum (XP) cells. The amount of 5-methylcytosine (mC) in DNA from XP cells was about 70% of that in DNA from normal controls. Southern hybridization analysis showed that the HLA-DR alpha gene in XP lymphocyte B cells was differently methylated from normals, but its expression was apparently unaffected. The methylation of dihydrofolate reductase, a housekeeping gene, was the same as in controls. The revertants to UV resistance from XP fibroblasts recovered a methylation level close to that of normal cells. Results suggested that XP DNA was undermethylated non-randomly, and that DNA methylation might be associated with DNA repair function.     

Elevation of dCTP pools in xeroderma pigmentosum variant human fibroblasts alters the effects of DNA repair arrest by arabinofuranosyl cytosine

DNA excision repair inhibition by arabinofuranosyl cytosine (ara-C) or by ara-C/hydroxyurea (HU) was measured in log phase and confluent cultures of normal and xeroderma pigmentosium (XP)-variant human fibroblasts following insult by ultraviolet (UV) light (20 J/m²). Repair inhibition was determined by measuring the accumulation of DNA single-strand breaks/10⁸ daltons following cell culture exposure to ara-C or ara-C/HU in a series of 3 hr. pulses up ro 24 hr. after UV insult. Both normal and XP-variant derived cells showed a wide range of sensitivity to ara-C in log phase cells (0.2–9.4 breaks/10⁸ daltons DNA), although strand break accumulation was constant for each specific cell line. The same cells were more sensitive to ara-C/HU with a 2–14 fold increase in DNA strand breaks depending upon the cell line assayed. In confluent cultures of normal cells, maximum sensitivity to ara-C and ara-C/HU was achieved with similar levels of repair inhibition observed (16.1 and 16.5 breaks/10⁸ daltons, respectively). The same level of repair inhibition was observed in confulent XP-variants receiving ara-C/HU, but was reduced by 62–68% in cells treated with ara-C alone. Ara-C repair arrest was more rapidly reversed by competing concentrations of exogenous deoxycytidine (dCyd) in XP-variant compared to normal cells, especially in confluent cell cultures. In ara-C/HU treated cells, the level of dCyd reversal was reduced in the XP-variant when compared to cells exposed to ara-C alone. However, the same addition of HU had relatively little effect on dCyd reversal in normal cells. The measurements of dNTP levels indicate an elevated level of intracellular deoxycytosine triphosphate in XP-variant vs normal cells. The implications of these results are discussed as they relate to possible excision repair anomalies in the XP-variant.Abbreviations ara-C arabinofuranosul cytosine - dCTP deoxycytosine triphosphate - dCyd deoxycytidine - dNTP deoxynucleoside triphosphate - dT thymidine - HU hydroxyurea - XP xeroderma pigmentosium This research was sponsored jointly by the National Cancer Institute under Interagency Agreement #40-5-63, and the Office of Health and Environment Research, U. S. Department of Energy, under Contract W-7405-eng-26 with the Union Carbide Corporation.     

Repair methylation of parental DNA in synchronized cultures of Novikoff hepatoma cells.

Parental and filial DNA strands were isolated from a Novikoff rat hepatoma cell line, synchronized by S-phase arrest with excess thymidine, that had completed up to one round of DNA replication in the presence of (14-C-methyl)methionine and (6-3-H) bromodeoxyuridine. Both strands were methylated, the proportion of total methyl label in parental DNA increasing slightly with time in S-phase. The studies were repeated with (14-C-methyl)methionine and (3-H)deoxycytidine to determine if parental methylation occurred on extant or repair-inserted cytosine residues. Both (14-C) and (3-H) were found in parental DNA. The (14-C)/(3-H) ration of parental DNA-5-methylcytosine was about twice that in filial DNA while the (3-H) data showed twice the concentration of 5-methylcytosine in parental compared to filial DNA. Thus parental methylation occurred on repair-inserted cytosine residues and resulted in overmethylation. That the DNA damage and repair was due to 5-phase arrest was shown by repeating the studies using a sequential mitotic-G1 arrest method. With this method little (14-C) or (3-H) was found in parental DNA. We conclude that S-phase arrest leads to DNA damage and repair with subsequent overmethylation of repair-inserted cytosines; that sequential mitotic-G1 arrest minimizes DNA damage; and, that the latter technique, suitable for synchronization of large quantities of cells, may prove useful in relatively artifact-free studies of eukaryotic DNA replication.     

Characterization of long patch excision repair of DNA in ultraviolet-irradiated Escherichia coli: An inducible function under rec-lex control

Summary Excision repair in ultraviolet-irradiated wild-type Escherichia coli produces a bimodal distribution of repair patch sizes in the DNA. Approximately 99% of the repair events result in short patches of 20–30 nucleotides produced by a constitutive repair system. The remaining 1% result in patches which are at least 1,500 nucleotides in length. This long patch repair is shown to be a damage-inducible process under control of the rec-lex regulatory circuit. The kinetics of the two processes differ; short patch synthesis begins immediately after irradiation and is virtually completed prior to synthesis of the majority of the long patches. Long patch repair synthesis is a linear function of UV dose up to a plateau at 60 J/m², and hence each long patch event is the consequence of a single UV-induced lesion. Long patch repair does not appear to be nessarily error-prone, since no alteration in repair synthesis occurs as a result of a mutation umuC ^- which renders cells nonmutable by UV. Evidence is presented suggesting that DNA polymerase I is responsible for both long and short patch synthesis in wild type cells under inducing conditions. In the absence of polymerase I the constitutive patch size averages 80–90 nucleotides, and this distribution is unchanged by induction.     

Methylation pattern of fish lymphocystis disease virus DNA.

The content and distribution of 5-methylcytosine in DNA from fish lymphocystis disease virus was analyzed by high-pressure liquid chromatography, nearest-neighbor analysis, and with restriction endonucleases. We found that 22% of all C residues were methylated, including methylation of the following dinucleotide sequences: CpG to 75%, CpC to ca. 1%, and CpA to 2 to 5%. Comparison of relative digestion of viral DNA with MspI and HpaII indicated that CCGG sequences were almost completely methylated at the inner C. The degree of methylation of GCGC was much lower. The methylation pattern of fish lymphocystis disease virus DNA differed from that of the host cell DNA.     

An AP Endonuclease Functions in Active DNA Demethylation and Gene Imprinting in Arabidopsis

Active DNA demethylation in plants occurs through base excision repair, beginning with removal of methylated cytosine by the ROS1/DME subfamily of 5-methylcytosine DNA glycosylases. Active DNA demethylation in animals requires the DNA glycosylase TDG or MBD4, which functions after oxidation or deamination of 5-methylcytosine, respectively. However, little is known about the steps following DNA glycosylase action in the active DNA demethylation pathways in plants and animals. We show here that the Arabidopsis APE1L protein has apurinic/apyrimidinic endonuclease activities and functions downstream of ROS1 and DME. APE1L and ROS1 interact in vitro and co-localize in vivo. Whole genome bisulfite sequencing of ape1l mutant plants revealed widespread alterations in DNA methylation. We show that the ape1l/zdp double mutant displays embryonic lethality. Notably, the ape1l^+/−zdp^−/− mutant shows a maternal-effect lethality phenotype. APE1L and the DNA phosphatase ZDP are required for FWA and MEA gene imprinting in the endosperm and are important for seed development. Thus, APE1L is a new component of the active DNA demethylation pathway and, together with ZDP, regulates gene imprinting in Arabidopsis.     

Arabidopsis DEMETER-LIKE proteins DML2 and DML3 are required for appropriate distribution of DNA methylation marks

Cytosine DNA methylation is a stable epigenetic mark for maintenance of gene silencing across cellular divisions, but it is a reversible modification. Genetic and biochemical studies have revealed that the Arabidopsis DNA glycosylase domain-containing proteins ROS1 (REPRESSOR OF SILENCING 1) and DME (DEMETER) initiate erasure of 5-methylcytosine through a base excision repair process. The Arabidopsis genome encodes two paralogs of ROS1 and DME, referred to as DEMETER-LIKE proteins DML2 and DML3. We have found that DML2 and DML3 are 5-methylcytosine DNA glycosylases that are expressed in a wide range of plant organs. We analyzed the distribution of methylation marks at two methylated loci in wild-type and dml mutant plants. Mutations in DML2 and/or DML3 lead to hypermethylation of cytosine residues that are unmethylated or weakly methylated in wild-type plants. In contrast, sites that are heavily methylated in wild-type plants are hypomethylated in mutants. These results suggest that DML2 and DML3 are required not only for removing DNA methylation marks from improperly-methylated cytosines, but also for maintenance of high methylation levels in properly targeted sites.     

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.     

Modulation of human nucleotide excision repair by 5-methylcytosines

Previous reports showed that methylated CpG sites are primary targets of bulky lesions induced by UV radiation, benzo[a]pyrene (B[a]P), or other environmental genotoxic agents. This study was performed to determine whether the repair of DNA damage formed preferentially at CpG dinucleotides is sensitive to 5-methylcytosine substitutions. Reactivation assays using UV- or B[a]P diol epoxide-damaged shuttle vectors established that human nucleotide excision repair enzymes are able to process fully methylated target DNA molecules. Repair reactions in human cell extracts suggested that 5-methylcytosines modulate local repair efficiency in a seemingly unpredictable manner. In fact, excision of the predominant (+)-trans-anti-B[a]P-dG adduct situated in a mutational hot spot sequence (codon 273 of the p53 gene) was stimulated by CpG methylation. Interestingly, excision activity was increased by a single 5-methylcytosine residue flanking the adduct in the damaged strand, but the same stimulatory effect was also induced by a single 5-methylcytosine residue located opposite the adduct in the undamaged strand. No such stimulation was observed when the (+)-trans-anti-B[a]P-dG lesion was placed in a different site containing a sequence of contiguous guanines, and strong inhibition was detected when a representative of the rare (+)-cis-anti-B[a]P-dG isomer was tested in the same assay. These results raise the possibility that 5-methylcytosines in CpG dinucleotides modulate not only the distribution of bulky DNA lesions but, at least in some cases, also the kinetics of subsequent excision repair reactions. This study confirms that the efficiency of bulky lesion repair is determined by the configuration of base pairs at damaged sites.     

Alterations of DNA methylation patterns in germ cells and Sertoli cells from developing mouse testis

In situ alterations of DNA methylation were studied between 14 d postcoitum and 4 d postpartum in Sertoli cells and germ cells from mouse testis, using anti-5-methylcytosine antibodies. Compared to cultured fibroblasts, Sertoli cells display strongly methylated juxtacentromeric heterochromatin, but hypomethylated chromatids. Germ cells always possess hypomethylated heterochromatin, whereas their euchromatin passes from a demethylated to a strongly methylated status between days 16 and 17 postcoitum. This hypermethylation occurs in the absence of DNA replication, germ cells being blocked in the G(0)-G(1) phase from day 15 postcoitum to birth. The DNA hypermethylation of germ cells is maintained until birth and could be visualized on both chromatids of metaphase chromosomes at the first postpartum cell division. Subsequently, the DNA hypermethylation is lost semiconservatively, being replaced by a methylation pattern recalling the typical fibroblast pattern. These alterations of DNA methylation follow a strict chronology, are chromosome structure and cell-type dependent, and may underlie profound changes of genome function.     

Distribution of DNA damage in chromatin and its relation to repair in human cells treated with 7-bromomethylbenz(a) anthracene.

We have examined the relationship between the distribution of DNA damage and repair in chromatin from confluent human fibroblasts treated with the carcinogen 7-bromomethylbenz (a) anthracene. Analysis of staphylococcal nuclease (SN)4 digestion kinetics and gel electrophoresis revealed that more total damage occurs in nucleosome core DNA (approximately 80-85% of chromatin DNA) than in SN sensitive DNA (APPROXIMATELY15-20%). Furthermore, over a 24 hr period, damage is removed at about the same rate from these two regions. In contrast, virtually all of the nucleotides incorporated during repair synthesis are initially SN sensitive even when measured at 12 hr after damage. With time many repair-incorporated nucleotides become SN resistant and coelectrophorese with nucleosome core DNA. To explain these data we propose a model whereby excision repair occurs in both linker and core DNA; however, in core DNA the repair process induces conformational changes resulting in temporarily increased SN sensitivity; subsequently, rearrangement occurs and results in the re-establishment of native or near-native nucleosome conformation and SN resistance.