Mutations and epimutations in mammalian cells

Early studies on heritable variation in cultured mammalian cells suggested that both mutation and epigenetic events might be involved. The importance of mutations has subsequently been fully documented, but only recently has an alternative form of inheritance been uncovered. This is based on the post-synthetic methylation of cytosine in regulatory regions of genes. The pattern of methylation is heritable, and in almost all cases studied, methylation of a region is associated with lack of gene expression. Such silent genes can be reactivated by the powerful demethylating agent 5-azacytidine (5-aza-CR). Changes in heritable DNA methylation which alter phenotype are referred to as epimutations. It now seems very likely that the well known ‘functional hemizygosity’ in CHO cells and other near diploid cell lines is due to the existence of one active and one silent gene at many autosomal loci. It is clear that permanent cell lines inactive genes by de novo methylation, whereas normal diploid cells do not have this activity. This has important implications for our understanding of cellular transformation, tumor progression, and the increase in chromosome number frequently associated with these cellular changes. It is likely that both mutations and epimutations are important in the emergence of fully transformed tumorigenic cells. Agents which increase or reduce DNA methylation in cells can be regarded as epimutagens, although in many cases the mechanisms of inducing hypo- or hyper-methylation are not understood. Two exceptions are 5-aza-CR which inhibits the normal DNA maintenance methylase activity, and 5-methyldeoxycytidine triphosphate which is incorporated into cellular DNA following electroporation and has been shown to silence genes.     

Evidence for allelic exclusion in Chinese hamster ovary cells.

Earlier results suggested that the functional hemizygosity of genes in pseudodiploid Chinese hamster ovary (CHO) cells is due to the silencing of one allele by DNA methylation. From this one could make a strong prediction that we have now been able to confirm by genetic experiments, using thymidine kinase (TK) alleles. TK- mutants induced by ethylmethane sulphonate (EMS) were all revertible to TK+ at high frequency by the demethylating agent 5-azacytidine (5-aza-CR). This revertibility was due to reactivation of a silent nonmutant TK allele. Further mutagenesis by EMS yielded TK- derivatives that were no longer revertible by 5-aza-CR; these are assumed to have mutations in both alleles. TK- cells were also transfected with equine herpes virus TK+ DNA, and the TK+ derivatives were shown to be markedly less stable than cells with the normal TK+ gene. CHO cells lack metallothionein activity (sensitive to cadmium), and also require proline for growth, because genes have become silenced during the establishment of the cell line. In both cases 5-aza-CR reactivates these genes to give the cadmium resistant and proline independent phenotypes. Long-term experiments with reactivants in the absence of selection showed that the genes become silent, presumably as a result of de novo methylation. A strain resistant to cytosine arabinoside (araCR) was also resistant to 5-azadeoxycytidine (5-aza-CdR), but not to 5-aza-CR, which would be expected if the araCR strain lacked deoxycytidine kinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA hypomethylation of karyoplasts for bovine nuclear transplantation

The objective of this research was to evaluate if DNA hypomethylation in cells used as karyoplasts would improve development of bovine nuclear transplantation (NT) embryos. DNA from serum-fed (SF), serum-starved (SS), and 1, or 5 microM 5-azacytidine (5-aza-CR) treated cells was digested with a methylation sensitive enzyme, and evaluated for DNA methylation. A significant reduction in DNA methylation was observed in cells cultured for 48 or 72 hr in SS medium as well as in cells cultured for 48 hr in the presence of 5 microM 5-aza-CR when compared to cells cultured in SF medium. All other comparisons contained no significant differences when compared to controls. When donor cells were cultured in 5-aza-CR, SF, or SS treatment media for 48 hr, no significant difference was observed (P = 0.06) in blastocyst development rates after NT. One embryo produced by donor cells treated with 5-aza-CR established a pregnancy. Four pregnancies resulted from embryos produced by SS donor cell NT and 3 resulted from embryos produced by SF donor cell NT. Supplementation of the donor cell culture medium with 5-aza-CR was not beneficial for increasing blastocyst rate or establishing pregnancy after NT.     

Effect of hydrocortisone on methylation and molecular population of DNA in rat liver]

The content of 5-methyl cytosine in rat liver DNA increases 1,7-fold 8 hours after intraperitoneal injection of hydrocortisone (5 mg per 100 g animal weight). The content of GC, physicochemical parameters (Tm, delta T, etc.) and DNA renaturation pattern did not show any changes. No changes were observed in the pattern of H3-thymidine incorporation into rat liver DNA: after hydrocortisone injection the radioactivity was found to be equally distributed in all isolated sequences of DNA, differing in the degree of reiteration (specific radioactivities of these DNA, fractions are very similar). Thus, the molecular population of DNA in liver cells remains unchanged, which suggests that the hormone-induced increase in the 5-methyl cytosine content is due to a change in the DNA methylation level. The methyation level of unique sequences (COt greater than 600), i. e. that of structural genes, does not undergo any essential changes. The reversible methylation of DNA regulated by hormones seems to be one of the mechanisms controlling gene activity.     

Gene silencing and endogenous DNA methylation in mammalian cells

It is known that transformed mammalian cells can spontaneously inactivate genes at low frequency by the de novo methylation of promoter sequences. It is usually assumed that this is due to DNA methyl transferase activity, but an alternative possibiity is that 5-methyldCTP is present in these cells and can be directly incorporated into DNA. The ongoing repair of DNA containing 5-methylcytosine will produce 5-methyldeoxycytidine monophosphate (5-methyldCMP), so the question arises whether this can be phosphorylated to 5-methyldCTP. We have tested this using three strains of CHO cells with different levels of 5-methyldCMP deaminase activity. That with the lowest enzyme activity, designated HAM⁻, has previously been shown to incorporate tritium labelled 5-methyldeoxycytidine into 5-methylcytosine in DNA, with a greater amount of label in thymine. This strain is phenotypically unstable producing cells resistant to bromodeoxyuridine (BrdU) and 6-thioguanine (6-TG) at high frequency. In contrast, the strain with the highest 5-methyldCMP deaminease, designated HAM⁺, is extremely stable, and the starting strain K1 HAM^s1 is intermediate between the HAM⁻ and HAM⁺ phenotypes. We have also shown that human diploid fibroblast strain MRC-5 has a phenotype like HAM⁺, whereas its SV40 transformed derivative, MRC-5V2 resembles HAM⁻ in having low 5-methyl dCMP deaminase activity, and is phenotypically unstable with regard to 6-TG resistance. It seems that 5-methyldCMP deaminase can be down-regulated in transformed cells, and this can promote de novo methylation by incorporation of 5-methyldCTP derived from 5-methyldCMP.     

5-Azacytidine permits gene activation in a previously noninducible cell type

We previously reported that silent muscle genes in fibroblasts could be activated following fusion with muscle cells to form heterokaryons. This activation did not require changes in chromatin structure involving significant DNA synthesis. We report here that muscle gene activation was never observed when HeLa cells were used as the nonmuscle fusion partner. However, if HeLa cells were treated with 5-azacytidine (5-aza-CR) prior to fusion, muscle gene expression was induced in the heterokaryons. The genes for both an early (5.1H11 cell surface antigen) and a late (MM-creatine kinase) muscle function were activated, but were frequently not coordinately expressed. These results suggest that the expression of two muscle genes, which is usually sequential, is not interdependent. Furthermore, changes induced by 5-aza-CR, presumably in the level of DNA methylation, are required for muscle genes in HeLa cells to be expressed in response to putative trans-acting regulatory factor(s) present in muscle cells.     

Expression of asparagine independence in variants of ICR 2A haploid frog cells

Properties of the change from asparagine dependence (asn-) to independence (asn+) were investigated in the androgenetic haploid frog cell line ICR 2A. Two types of asn+ variants arose spontaneously during culture. Glutamine-dependent asparagine synthetase (AS) activity, found to be deficient in asn- cells, was repressed by asparagine in one type of variant and expressed constitutively in the other. No quantitative differences in AS-specific DNA sequences or changes in ploidy were evident between asn+ and asn- cells. The asn+ frequency in ICR 2A populations, not dramatically influenced by chemical mutagens, was increased 130-fold by exposure to 5-azacytidine. The methylation of CCGG sequences at the 5' end of the AS structural gene was found to be reduced equally in both types of asn+ variant. These results indicate that decreased DNA methylation is essential but not necessarily sufficient for the expression of AS activity in this frog cell system.     

Cellular differentiation, cytidine analogs and DNA methylation

The nucleoside analog 5-azacytidine (5-aza-CR) induced marked changes in the differentiated state of cultured mouse embryo cells and also inhibited the methylation of newly synthesized DNA. The DNA strand containing 5-aza-CR remained undermethylated in the round of DNA synthesis following analog incorporation. The extent of inhibition of DNA modification and induction of muscle cells in treated cultures were dependent on the 5-aza-CR concentration over a narrow dose range. Experiments with the restriction enzyme Hpa II, which is sensitive to cytosine methylation in the sequence CCGG, demonstrated that the DNA synthesized in 5-aza-CR-treated cultures was maximally undermethylated 48 hr after treatment. Three other analogs of cytidine, containing a modification in the 5 position of the pyrimidine ring [5-aza-2'-deoxycytidine(5-aza-CdR), pseudoisocytidine (psi ICR) and 5-fluoro-2'-deoxycytidine(FCdR)] also induced the formation of muscle cells and inhibited DNA methylation. In contrast, 1-beta-D-arabinofuranosylcytosine (araC) and 6-azacytidine (6-aza-CR) did not inhibit DNA methylation or induce muscle formation, whereas 5-6-dihydro-5-azacytidine (dH-aza-CR) was a poor inducer of muscle cells and a poor inhibitor of DNA methylation. These results provide experimental evidence for a role for DNA modification in differentiation, and suggest that cytidine analogs containing an altered 5 position perturb previously established methylation patterns to yield new cellular phenotypes.     

Enhanced production of hepatitis B virus surface antigen in mouse C127 cell on a bovine papillomavirus-metallothionein vector

We have constructed a recombinant plasmid pCPS12 containing the hepatitis B viral surface antigen (HBsAg) gene linked to the mouse metallothionein promoter on a BPV-pML2 vector. Two stable clones S12-8 and S12-2, obtained by transfection of the mouse C127 cells with pCPS12 propagated in dam+ dcm+ and dam- dcm- Escherichia coli respectively, exhibited different types of response to 5-azacytidine (5-aza-CR) and cadmium (Cd) induction. In S12-8, the productivity of HBsAg was enhanced by 5-aza-CR or 5-aza-CR plus Cd, but not by Cd alone. In S12-2, the expression of HBsAg was not affected by 5-aza-CR but was induced by Cd in the presence or absence of 5-aza-CR. This suggests that methylation may be important in controlling the HBsAg expression and the inducibility of Cd in the transfectants.     

Strong effects of 5-azacytidine on the in vitro lifespan of human diploid fibroblasts

Azacytidine (5-aza-CR) and azadeoxycytidine (5-aza-CdR) are known to inhibit the methylation of cytosine (5-mC) in DNA, and their effects on the long-term growth of human fibroblasts, strain MRC-5, have been examined. A single treatment with either analogue initially inhibits growth, but the cells recover to normal morphology, growth rate and cell density at confluence. However, a memory of the treatment is retained, since the cells' subsequent lifespan is considerably reduced in comparison with controls, and the terminal stages of growth are indistinguishable from senescent cultures of untreated cells. The effect of 5-aza-CR or 5-aza-CdR does not appear to be closely related to the concentration used, or to the length of treatment up to about half-way through the total lifespan. Sequential doses have cumulative effects on longevity. There is evidence that the pattern of 5-mC in mammalian DNA is inherited via cell division; therefore, a reduction in 5-mC induced by a pulse treatment of 5-aza-CR or 5-aza-CdR will be transmitted to all descendants. The results are consistent with independent observations that the level of 5-mC declines continually during the serial subculture of human diploid cells. The analogues would be expected to precipitate this decline and thereby advance the physiological age of the culture. The results provide support for the view that the random loss of methyl groups in DNA may eventually have deleterious consequences, such as aberrant epigenetic changes in gene expression.     

Characterization of DNA methylation change in stem cell marker genes during differentiation of human embryonic stem cells

Pluripotent human embryonic stem cells (hESCs) have the distinguishing feature of innate capacity to allow indefinite self-renewal. This attribute continues until specific constraints or restrictions, such as DNA methylation, are imposed on the genome, usually accompanied by differentiation. With the aim of utilizing DNA methylation as a sign of early differentiation, we probed the genomic regions of hESCs, particularly focusing on stem cell marker (SCM) genes to identify regulatory sequences that display differentiation-sensitive alterations in DNA methylation. We show that the promoter regions of OCT4 and NANOG, but not SOX2, REX1 and FOXD3, undergo significant methylation during hESCs differentiation in which SCM genes are substantially repressed. Thus, following exposure to differentiation stimuli, OCT4 and NANOG gene loci are modified relatively rapidly by DNA methylation. Accordingly, we propose that the DNA methylation states of OCT4 and NANOG sequences may be utilized as barometers to determine the extent of hESC differentiation.     

Naturally occurring endo-siRNA silences LINE-1 retrotransposons in human cells through DNA methylation

Long interspersed nuclear element 1 (LINE-1) retrotransposons are mutagens that are capable of generating deleterious mutations by inserting themselves into genes and affecting gene function in the human genome. In normal cells, the activity of LINE-1 retrotransposon is mostly repressed, maintaining a stable genome structure. In contrast, cancer cells are characterized by aberrant expression of LINE-1 retrotransposons, which, in principle, have the potential to contribute to genomic instability. The mechanistic pathways that regulate LINE-1 expression remain unclear. Using deep-sequencing small RNA analysis, we identified a subset of differentially expressed endo-siRNAs that directly regulate LINE-1 expression. Detailed analyses suggest that these endo-siRNAs are significantly depleted in human breast cancer cells compared with normal breast cells. The overexpression of these endo-siRNAs in cancer cells markedly silences endogenous LINE-1 expression through increased DNA methylation of the LINE-1 5′-UTR promoter. The finding that endo-siRNAs can silence LINE-1 activity through DNA methylation suggests that a functional link exists between the expression of endo-siRNAs and LINE-1 retrotransposons in human cells.     

DNA methylation at promoter regions regulates the timing of gene activation in Xenopus laevis embryos

The levels of genomic DNA methylation in vertebrate species display a wide range of developmental dynamics. Here, we show that in contrast to mice, the paternal genome of the amphibian, Xenopus laevis, is not subjected to active demethylation of 5-methyl cytosine immediately after fertilization. High levels of methylation in the DNA of both oocyte and sperm are maintained in the early embryo but progressively decline during the cleavage stages. As a result, the Xenopus genome has its lowest methylation content at the midblastula transition (MBT) and during subsequent gastrulation. Between blastula and gastrula stages, we detect a loss of methylation at individual Xenopus gene promoters (TFIIIA, Xbra, and c-Myc II) that are activated at MBT. No changes are observed in the methylation patterns of repeated sequences, genes that are inactive at MBT, or in the coding regions of individual genes. In embryos that are depleted of the maintenance methyltransferase enzyme (xDnmt1), these developmentally programmed changes in promoter methylation are disrupted, which may account for the altered patterns of gene expression that occur in these embryos. Our results suggest that DNA methylation has a role in regulating the timing of gene activation at MBT in Xenopus laevis embryos.     

DNA methylation as a regulatory mechanism in rat gamma-crystallin gene expression.

We have investigated the methylation state of the rat gamma-crystallin genes in DNA from lens cells at different developmental stages as well as from kidney and heart cells. A clear correlation between the extent of demethylation of the promoter and 5' gene regions and the expression of these genes was observed. No change in the methylation state of the far upstream or 3' regions of the genes was seen. The demethylation of the promoter region was shown to occur during the differentiation from the lens epithelial to the lens fiber cell. The effect of cytosine methylation on gamma-crystallin promoter activity was tested by measuring gamma-crystallin promoter/chloramphenicol acetyltransferase fusion gene expression after in vitro primed repair synthesis of the promoter region in the presence of either dCTP or 5mdCTP. The hemimethylated promoter was no longer capable of promoting high CAT activity after introduction into lens-like cells. Taken together, our data suggest that DNA demethylation may be the determining step in the developmental stage-specific expression of the rat gamma-crystallin genes.