©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ras Induces a General DNA Demethylation Activity in Mouse Embryonal P19 Cells (*)

Moshe Szyf(§), Johanne Theberge, and Vera Bozovic

From the (1) Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec H3G 1Y6, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We demonstrate that expression of v-Ha-ras in mouse embryonal P19 cells results in genome-wide demethylation. Analysis of the pattern of methylation of specific genes reveals that different types of genes are demethylated in the ras transfectants: skeletal muscle specific genes, a gene specifically expressed in the adrenal cortex (c21), ubiquitous genes, and exogenously introduced sequences. Transient transfection and in vitro demethylation assays reveal that the ras transfectants express high levels of a general DNA demethylation activity. This demonstrates that the general DNA demethylation activity in mouse embryonal cells is controlled by an important cellular signal transducer and that DNA demethylase is a potential downstream effector of Ras.


INTRODUCTION

DNA methylation patterns are a hallmark of vertebrate genomes. The cytosine moieties residing in the dinucleotide sequence CpG are generally methylated in vertebrate genomes; however, some CpGs are not methylated. The distribution of nonmethylated cytosines is tissue- and site-specific, resulting in a pattern of methylation (1, 2) . It is clear that the pattern of methylation is established by a sequence of methylation and demethylation events (3, 4) . Although it was originally suggested that DNA demethylation is accomplished by a passive loss of methyl groups during replication (5) , it is now clear that an active process of demethylation occurs in embryonal cells (6) , in differentiating cell lines (7, 8, 9) , and in response to estrogen treatment (10) . Whereas site-specific hypomethylation occurs during development, it is unclear whether the demethylation machinery per se is site-specific or whether site specificity is determined by other signals. A critical question for understanding the biological role of demethylation is whether it is an indirect result of other site-specific events, such as transcriptional activation, or whether mammalian cells bear a demethylation activity that is independently programmed with development. If demethylation is a programmed event, then the question is whether it is regulated by central cellular control points.

We have recently shown that mouse embryonal teratocarcinoma P19 cells express a general demethylase activity. This activity was partially purified and was shown to demethylate DNA in the absence of exogenous dCTP, suggesting that it does not act via an excision repair mechanism.() To address the question of whether this activity is regulated by cellular signaling pathways, we introduced exogenous v-Ha-ras into P19 cells by DNA-mediated gene transfer. The involvement of signaling pathways triggered by Ras in differentiation of P19 cells has been studied before. Whereas both Src (11) and Jun, a downstream effector of Ras (12) , can induce the differentiation of P19 cells, forced expression of oncogenic Ras does not induce or inhibit differentiation of P19 cells (13) . Ras might still induce some events that are part of the differentiation program. General genome-wide demethylation is one of the events associated with induction of differentiation of embryonal carcinoma cells (14) . The fact that Ras does not induce full differentiation or cellular transformation of P19 cells will enable one to differentiate between events that are consequences of differentiation and those that are specifically triggered by Ras.


MATERIALS AND METHODS

Stable Transfection and Analysis of Genomic DNA Methylation

The pZEMras expression vector bearing a 2.3-kb BamHI-EcoRI fragment encoding v-Ha-ras from the murine mammary tumor virus v-Ha-ras plasmid (15) or the pZEM control vector (16) was cointroduced into P19 cells with pUCSVneo (1 µg) as a selectable marker by DNA-mediated gene transfer using the calcium phosphate protocol (17) . G418-resistant cells were cloned and propagated in selective medium and analyzed for expression of the transfected DNA by Southern and Northern blot analysis (17) . DNA was extracted from three P19 PZEM control transfectants (1, 2, and 3) and three P19 lines transfected with pZEMras and subjected to a nearest neighbor analysis as described previously (8) . 2 µg of DNA were incubated at 37 °C for 15 min with 0.1 unit of DNase and 2.5 µl of [-P]dGTP (3000 Ci/mmol from Amersham). 2 units of Kornberg DNA polymerase (Boehringer Mannheim) were then added, and the reaction was incubated for an additional 25 min at 30 °C. 50 µl of water were then added to the reaction mixture, and the unincorporated nucleotides were removed by spinning through a Microspin S-300 HR column (Pharmacia Biotech Inc.). The labeled DNA (20 µl) was digested with 70 µg of micrococcal nuclease (Pharmacia) in the manufacturer's recommended buffer for 10 h at 37 °C. Equal amounts of radioactivity were loaded on TLC phosphocellulose plates (Merck), and the 3`-mononucleotides were separated by chromatography in one dimension (isobutyric acid:HO:NHOH in the ratio 66:33:1). The chromatograms were exposed to XAR film (Eastman Kodak), and the autoradiograms were scanned by scanning laser densitometry (Scanalytics one-dimensional analysis). Spots corresponding to cytosine and 5-methylcytosine-dimensional were quantified, and the percentage of nonmethylated CpG dinucleotide sequences was calculated. To study demethylation of specific genes, genomic DNA (10 µg) was extracted from three pZEM (1, 2, and 3) and three pZEMras (6, 11, and 17) transfectants and subjected to digestion with HindIII (Boehringer Mannheim, 25 units) followed by digestion with either 25 units of MspI(M), which is insensitive to CCGG methylation, or HpaII(H), which is sensitive to methylation, for 8 h at 37 °C. At least three different DNA preparations isolated from three independent passages were assayed per transfectant to exclude variability between different DNA samples and verify the stability of DNA methylation patterns. A representative experiment is presented.

Transient Transfection and Nuclear DNA Extraction

pMetCAT+ plasmid was methylated in vitro by incubating 20 µg of plasmid with 10 units of SssI CpG DNA methyltransferase (27) (New England Biolabs Inc.) in a buffer recommended by the manufacturer. To verify that the plasmids were fully methylated, we repeated the methylation reaction (after full protection from HpaII had been observed) for two additional times. Methylated pMetCAT+ DNA (1-10 µg) (physical map in Fig. 3A, bottom panel) was introduced into P19 cells (8 10/well in a six-well tissue culture dish (Nunc)) by DNA-mediated gene transfer using Lipofectin (Life Technologies, Inc.) according to the manufacturer's recommendations. We used different concentrations of DNA/transfection mix to exclude differences in transfected plasmid concentration as an explanation for the observed differences in the level of DNA demethylation (1-10 µg). To avoid contamination of the transfected DNA with non-nuclear plasmid DNA, we first isolated the nuclei. Cells were dislodged by scraping in phosphate-buffered saline buffer, nuclei were isolated as described previously, and DNA was extracted (18) .


Figure 3: Demethylation of transiently transfected pMetCAT+ plasmid in P19 pZEM and pZEMras transfectants. P19, PZEM1, and PZEMras 6 and 11 transfectants were transiently transfected with methylated pMetCAT plasmid as described under ``Materials and Methods.'' 10 µl of nuclear DNA prepared from the transiently transfected cells (1 and 3 µg of plasmid/transfectant) were subjected to HpaII and MspI digestion, Southern blot transfer, and hybridization to pOCAT (A) or Met 5` probes (B). Nontransfected DNA is fully methylated as indicated in the first pair of lanes. The physical map of the transfected pMetCAT+ plasmid is presented in the bottom panel of A. The CAT sequence is hatched, the herpesvirus tk gene is shaded, the DNA methyltransferase 5` region is indicated as a line, the exons are filled boxes, and the AP-1 sites are shaded ovals. HpaII sites (H), the BamHI site (BHI), and the HindIII site (HIII) are shown. The expected HpaII fragments are indicated under the physical map. C, Northern blot analysis. RNA was prepared from the cytosolic fraction made from pZEM (lines 1, 2, and 3) and pZEMras transfectants and subjected to Northern blot analysis and hybridization with a P-labeled ras probe (0.7-kb HindIII-PstI fragment (15)) according to standard procedures (17). The viral ras directs a 2-kb transcript that is easily distinguishable from the shorter endogenous ras messages (1-1.4 kb). D, DpnI cleavage of transiently transfected pMetCAT DNA. SssI-methylated (M) and nonmethylated (NM) pMetCAT DNA and DNA prepared from control P19 and pZEM and pZEMras transfectants transiently transfected with pMetCAT (3 µg) were subjected to digestion with DpnI (Boehringer Mannheim), Southern blotting, and hybridization with a POCAT probe (indicated in the bottom panel). The bottom panel is a physical map of the pMetCAT construct; the DpnI sites analyzed by the POCAT probe are indicated (D). The first DpnI in the CAT region of the plasmid is designed to be located 3 base pairs apart from a HpaII site. The sequence of the combined site is presented relative to the physical map. The arrows indicate the methylated C or A, and the HpaII and DpnI recognition sites are underlined. E, an autoradiogram like the one shown in A was scanned (Masterscan Scanalytics), and the intensity of hybridization to pOCAT of fragments corresponding to nonmethylated HpaII sites and methylated HpaII sites was determined. The ratio of nonmethylated to methylated molecules was then determined. The first bar (shaded) in each set indicates cells transfected with 1 µg of plasmid, and the second bar (hatched) indicates transfectants initially transfected with 3 µg of DNA.



In Vitro Demethylation Assay

A P-labeled, fully methylated dCpG substrate was prepared as follows. 100 ng of a double-stranded (mdC-dG) oligodimer (Pharmacia) were denatured by boiling, which was followed by partial annealing at room temperature. The complementary strand was extended with the Klenow fragment (Boehringer Mannheim) using mdCTP (Boehringer Mannheim) and [-P]dGTP (100 µCi, 3000 Ci/mmol), and the unincorporated nucleotides were removed by chromatography through a NAP-5 column (Pharmacia). Nuclear extracts were prepared from logarithmic cultures of pZEM (lines 1, 2, and 3) and pZEMras transfectants (lines 6, 11, and 17) as described previously and incubated with 1 ng of fully methylated CpG substrate for 30 min at 37 °C in a buffer containing 10 mM Tris-HCl, pH 7.6, 25% glycerol (v/v), and 5 mM EDTA. The reacted DNA was purified by phenol/chloroform extraction and subjected to micrococcal nuclease digestion to 3` mononucleotides, TLC (using polyethyleneimine thin layer chromatography, Brinkman), and autoradiography as described above.


RESULTS

Exogenous Expression of Ras Induces Genomic DNA Demethylation

P19 cells were cotransfected with pUCSVneo and either pZEMras (15) or pZEM (16) as a control. G418-resistant colonies were isolated, and cells bearing the pZEM and pZEMras plasmids were identified by Northern and Southern blot analyses (data not shown). (Fig. 2F is a Southern blot demonstrating the presence of the pZEM and pZEMras constructs, and the Northern blot in Fig. 3indicates the presence of the v-Ha-ras message in the selected transfectants.) Three transfectants/plasmid were randomly selected for our studies. The general level of CpG methylation in the ras transfectants and controls was determined by nearest neighbor analysis (8) . The results presented in Fig. 1show a slight reduction in total DNA methylation in the ras transfectants. The DNA methyltransferase promoter bears AP-1 sites (19) and has been shown by us to be inducible with Ras or Jun (20) . The general DNA methylation level might therefore reflect a balance of methylation and demethylation events induced by Ras.


Figure 2: The state of methylation of specific genes in ras transfected and control pZEM transfectants. DNA prepared from three independent pZEM and pZEMras transfectants was loaded in the lanes presented in the figure in the following order from left to right: pZEM 1, 2, and 3 and pZEMras 6, 11, and 17. The digested DNA was size-fractionated on a 1.5% agarose gel, Southern blot transferred onto Hybond-N+ in 0.4 M NaOH, and hybridized with the respective P-labeled DNA probe according to standard protocols (17). The filters were stripped of radioactivity by boiling the filter for 15 min in 0.2 SSC, 1% sodium dodecyl sulfate buffer and rehybridized with other probes. A, c21 3.8. A 3.8-kb BamHI fragment containing the coding portions of the c21 gene was used as a probe. The physical map of the gene is presented in the bottom panel. The locations of HpaII sites are indicated as H. The HpaII fragments that are expected if the CCGG sites are not methylated are indicated relative to the physical map. The partial 1.9-kb fragment observed in the ras transfectants is indicated (1.9*). The locations of the exons are indicated as closed boxes, and the transcription initiation site is indicated by an arrow. The probe used is shown as a thickline relative to the physical map. B, acetylcholine receptor subunit promoter. The 1-kb HindIII (HIII) fragment 5` region probe (22) is indicated as a thick line in the bottom panel relative to the location of the HpaII sites (H). The expected HpaII fragments from a nonmethylated versus a methylated gene are indicated under the physical map. C, thy-1. The probe used is a HpaII-XbaI 214-base pair fragment located upstream from the first exon as shown in the bottom panel relative to a physical map of the region. The expected HpaII (H) fragments are indicated. The partially methylated fragment is indicated as an asterisk relative to the autoradiogram. The internal site shows some residual resistance to MspI; we have observed this before (25) with multiple DNA preparations. D, quantification of de novo methylation of the thy-1HpaII site in ras transfectants. The autoradiogram shown in E was scanned using Masterscan (Scanalytics one-dimensional analysis), and the ratio of the methylated 0.8-kb HpaII fragment to the nonmethylated 0.6-kb fragment was determined. The bars for pZEM 1, 2, and 3 and Ras 6, 11, and 17 are presented (from left to right). E, 5` region of DNA methyltransferase. The physical map of the DNA methyltransferase 5` region is indicated. Filled boxes are exons, the arrows indicate the transcription initiation sites, and the shadedovals indicate the location of the AP-1 recognition sequences. The probe is a 1.3-kb BamHI (BHI)-HindIII (HIII) fragment indicated as a thick line, and the HindIII-HpaII fragments expected for a methylated or a nonmethylated HpaII (H) site are indicated relative to the physical map. F, 3` region of HGH. The state of methylation of the transfected plasmid was studied using a 0.77-kb probe encoding the 3` HGH sequence included in the expression vector as indicated (thick line) in the bottompanel. The physical map and the expected HpaII (H) fragments (as well as the observed partial HpaII fragments, which are indicated as asterisks next to the autoradiogram, for the pZEMras) for both the pZEM and pZEMras plasmids are shown. Metallothionein sequences are lightly shaded, v-Ha-ras sequences are darkly shaded, and HGH sequences are hatched.




Figure 1: State of genomic methylation of P19 pZEM and P19 ras transfectants. The state of methylation of genomic DNA of P19 pZEM and P19 ras transfectants was determined as described under ``Materials and Methods.'' Results are presented as the averages of three independent transfectants/group ± S.D. The experiment was repeated three times. The statistical significance of the difference between the pZEM and pZEM ras groups was determined by a t test (p < 0.01).



State of Methylation of Specific Genes

To determine whether Ras induces demethylation or hypermethylation in P19 cells and whether these changes are general or targeted at a certain class of genes, we analyzed the state of methylation of specific genes using MspI/HpaII restriction enzyme analysis, Southern blotting, and hybridization with specific probes. Five classes of genes were selected: (a) a gene that is expressed in skeletal muscles (skeletal muscles are one of the prominent cell types into which P19 cells can differentiate (21) ), the acetylcholine receptor chain (22) ; (b) a gene that is specifically expressed in the adrenal cortex but is normally heavily methylated and repressed in P19 cells, steroid 21-hydroxylase (c21) (23) ; (c) a gene that is specifically expressed in P19 cells, thy-1 (24); (d) a gene that is ubiquitously expressed in all dividing tissues, the 5` region of the DNA methyltransferase (19) ; and (e) the 3` region of the human growth hormone gene, which was exogenously introduced into P19 cells (16) (exogenously transfected DNA is methylated de novo in mouse embryonal cells (25) ). DNA was prepared from exponentially growing cells, digested with HindIII and either MspI or HpaII, and sequentially hybridized with the different gene-specific probes indicated in Fig. 2. The pattern of methylation observed per each transfectant and the P19 controls were stable (data not shown), suggesting that, once forced expression of Ras triggers a change in DNA methylation, the new state of methylation is maintained.

The c21 gene is heavily methylated in P19 cells and the pZEM transfectants, showing a high molecular band at 9 kb() following HpaII digestion, whereas the MspI pattern reflects the expected fragments at 1, 0.9, 0.8, 0.7, 0.4, and 0.15-0.2 kb (Fig. 2A). All three ras transfectants show a diminution of the 9-kb, high molecular weight fragment following HpaII digestion and a distinct partially methylated HpaII fragment migrating as a 1.9-kb fragment (see physical map in Fig. 2A for possible origin of this fragment). In addition to the site-specific demethylation at the sites flanking the 1.9-kb partial HpaII fragment, some of the fully hypomethylated low molecular fragments are now observed at 0.8, 0.7, 0.4, and 0.15 kb. The demethylation of c21, which is not expressed in P19 cells (data not shown), suggests that this demethylation is not an effect of induction of gene expression.

We analyzed the state of methylation of a gene that is specifically expressed in myogenic lineages. The acetylcholine receptor chain 5` region 1.3-kb HindIII fragment bears one CCGG site (physical map in Fig. 2B). The majority of the population of pZEM control transfectants is methylated at this site as indicated by the relative abundance of the 1-kb fully methylated HindIII fragment. In the ras transfectants, most of these sites are demethylated as implied by the presence of the 0.3- and 0.7-kb HpaII fragments (Fig. 2B). The myoD and myogenin genes are also heavily methylated in pZEM controls and demethylated in ras transfectants (data not shown).

thy-1 is expressed in P19 cells (24) but not in a number of other cell types. The HpaII sites located in a CpG island upstream from the transcription initiation site of thy-1 are hypomethylated in P19 pZEM transfectants as well as P19 cells as demonstrated by the presence of the 0.6-kb HpaII fragment. One of these HpaII sites (site 2) undergoes de novo methylation in the ras 6 and 11 transfectants as indicated by the relative intensification of the 0.8-kb partially methylated HpaII fragment (Fig. 2C; see also the results of a densitometric analysis presented in Fig. 2D). These results imply that even under conditions where general genomic demethylation is taking place, some sites will remain selectively methylated or even undergo de novo methylation.

The DNA methyltransferase gene is expressed in all proliferating cells and in P19 cells (19) . The 5` region of the DNA methyltransferase bears a HpaII site, which is located in proximity to a cluster of AP-1 sites 1.7 kb upstream of the transcription initiation site (physical map in Fig. 2E). Demethylation of this site will result in a 1.3-kb fragment following HpaII and HindIII digestion. The majority of P19 cells is methylated at this HpaII site as indicated by the presence of the hypermethylated 4-kb HindIII fragment in all three pZEM transfectants. A large fraction of the population of ras transfectants is hypomethylated at this site as indicated by the relative diminution of the 4-kb fragment and the increase in the relative intensity of the unmethylated fragment.

We determined the state of methylation of the 3` region of human growth hormone (HGH) cDNA included in the pZEM and pZEMras construct. Both constructs include the same promoter and 3` sequences and should express similar transcriptional activity (both constructs are expressed in nonembryonal cells, data not shown). Whereas the transfected pZEM is heavily methylated in the control pZEM transfectants, the HGH 3` region is markedly hypomethylated in all ras transfectants (Fig. 2F) as indicated by the presence of the expected HpaII low molecular fragments at 0.7, 0.5, and 0.3 kb. Some low level of de novo methylation is observed in the ras transfectants (Fig. 2F, compare the MspI and HpaII lanes; the partially methylated fragments are indicated by asterisks). This marked difference between P19 and ras transfectants suggests that de novo methylation is inhibited by v-Ha-Ras. There are three possible explanations for the inhibition of de novo methylation in ras transfectants: Ras might inhibit de novo methylation per se; maintenance methylation might be inhibited; and de novo methylation might be intact, but demethylation activity is induced in ras transfectants. Similarly, the genome-wide demethylation observed in ras transfectants can be explained by inhibition of maintenance methylation, de novo methylation, or activation of demethylation. Alternatively, genome-wide demethylation might be caused by wide changes in chromatin structure altering the accessibility of DNA to the methylation machinery.

Transient Transfection Demethylation Assay

To address the question of whether Ras induces a general demethylation activity in P19 cells, we utilized a transient demethylation assay. We have previously shown that P19 cells cannot methylate transiently introduced exogenous DNA de novo, but they possess an activity that demethylates in vitro methylated plasmid DNA. Therefore, a transient transfection demethylation assay can measure the level of demethylation activity in the cell without confounding de novo methylation. We utilized the previously described pMetCAT+ plasmid as a substrate (19) . This plasmid bears the 2-kb DNA methyltransferase 5` region inserted in front of the bacterial CAT gene from pOCAT and a viral herpes tk gene (26) . The HpaII site residing 5` of the DNA methyltransferase fragment (-1931) (Fig. 3A, physical map) is methylated in P19 cells and demethylated in ras transfectants (Fig. 2E). The cat portion of the plasmid is a bacterial sequence that does not have a homologue in the P19 genome and could be used to measure nonspecific demethylation.

The plasmid was methylated in vitro with SssI methylase (27) , which methylates all CpG sites, and introduced into P19 cells, pZEM, and PZEMras transfectants by DNA-mediated gene transfer. Each experiment was repeated twice, and each transfection was done in triplicate. The autoradiogram in Fig. 3 (A and B) presents one representative experiment for each condition. DNA was prepared from nuclei purified from the different transfectants 48 h after transfection and was subjected to either HpaII or MspI digestion. Following agarose gel fractionation and Southern blot transfer, the filters were sequentially hybridized with either a CAT probe (Fig. 3A) or a methyltransferase 5` probe (Fig. 2B). The presence of mRNA encoding v-Ha-Ras in the ras transfectants was verified by subjecting RNA made from the cytosolic fraction to Northern blot analysis and hybridization with a ras probe (Fig. 3C).

To determine that loss of methylation of the transiently transfected plasmids does not result from replication in the absence of methylation, DNA prepared from the transient transfectants was subjected to digestion with DpnI. DpnI cleaves DNA only when its GATC sites are methylated on both strands. Because bacterial cells but not mammalian cells bear the methylase required for methylating GATC sites, sensitivity to DpnI implies that the plasmid did not replicate in the mammalian cell. As observed in Fig. 3D, the transiently transfected plasmid maintained its sensitivity to DpnI digestion. Especially instructive is an engineered site in the plasmid (Fig. 3D, bottom panel, for localization and description) where the DpnI cleavage site is located 3 base pairs apart from the HpaII cleavage site. Whereas >95% of the molecules are demethylated at this site in the ras transfectants as indicated by the sensitivity to HpaII cleavage (Fig. 3B, the 2.3-kb HpaII fragment), >95% of the transfected molecules are still sensitive to DpnI cleavage of this site (Fig. 3D, see bottom panel for location of the site), implying that the adenine residue is still methylated on both strands. This excludes replication and repair as a mechanism for demethylation. This does not exclude, however, very short patch excision repair (less than 3 bases) as a possible mechanism, as has been previously suggested (28).

The autoradiograms were scanned, and the fraction of molecules that were fully demethylated at each point was determined (Fig. 3E). The transfection efficiency was similar in the different transfectants as determined by hybridization with a plasmid probe and normalizing to the level of nuclear DNA at each point (data not shown). As observed in Fig. 3, the levels of demethylation of both the exogenous Met 5` region and the bacterial CAT sequences are increased severalfold (10-fold in ras 6 to 100-fold in the ras 11 transfectant) over the rate of demethylation observed in control lines. The fact that demethylation of murine, bacterial, and herpesvirus sequences is observed is consistent with the hypothesis that v-Ha-ras expression induces the expression of a general demethylation activity.

In Vitro Demethylation Assays

To further determine whether Ras induces a demethylation activity that is independent of DNA replication, we determined whether nuclear extracts prepared from ras transfectants bear a higher demethylase activity than extracts prepared from control P19 cells. We synthesized a fully methylated P-labeled double-stranded poly-mdCpG oligonucleotide in vitro using mdCTP and [-P]dGTP as described in the legend to Fig. 4. 1 ng of the methylated substrate was incubated with 10 µg of 0.4 M NaCl nuclear extracts prepared from three pZEM and three pZEMras transfectants for 30 min at 37 °C in the absence of exogenous dNTPs. The reacted DNA was digested to 3` mononucleotides with micrococcal nuclease and subjected to thin layer chromatography to separate methylated and nonmethylated 3` P-labeled dCMPs. The autoradiograms were scanned, and the relative intensity of demethylated dCMP was determined. As observed in Fig. 4, the extracts prepared from ras P19 transfectants bear a measurable level of an active demethylase, whereas this activity is barely detectable under these conditions in the control transfectants.


Figure 4: In vitro demethylation assay. P-Labeled, fully methylated dCpG substrate was incubated (for 0.5 h) with nuclear extracts (10 µg) prepared from P19pZEM and pZEMras transfectants. The relative abundance of mdCMP and dCMP was determined after subjecting the reacted DNA to micrococcal nuclease digestion and chromatographic separation as described under ``Materials and Methods.'' The top panel shows a representative chromatogram. The autoradiograms were scanned, spots corresponding to dCMP and mdCMP were quantified, and the percentage of nonmethylated dCMP was calculated. The bottom panel presents results obtained for extracts prepared from three different transfectant lines/plasmid.




DISCUSSION

Although it is well established that demethylation of specific sites in DNA is associated with development (1, 2, 3, 4) , it is unclear whether site-specific demethylation events that have been extensively documented are side effects of readjustments of chromatin structure or whether they reflect an independent cellular process. In this report we show that genome-wide demethylation events could be programmed by an important cellular signaling pathway and that it can occur independent of the full manifestations of either differentiation or cellular transformation. The fact that ras transfectants demethylate transiently transfected exogenous DNA encoding a number of different sequences is consistent with the hypothesis that ras induces a general demethylation activity. These conclusions are further supported by the observation that nuclear extracts prepared from ras transfectants bear a measurable CpG demethylating activity. The chemical nature of the demethylation reaction is still unclear and will be determined in future experiments.

Is this demethylation related to site-specific demethylation of CpG islands that occurs in embryonal cells? How can induction of a general demethylation activity lead to a specific pattern of methylation? We do not have the data to answer this question, but it has been previously hypothesized that the final demethylation pattern will reflect an interplay between a general demethylase(s) and local site-specific signals (29) . Another interesting observation is that expression of ras results in some limited de novo methylation of specific sites (Fig. 2C). The fact that both methylation and demethylation can be induced by Ras might be relevant to the question of how genome-wide hypomethylation (30) and hyperactivation of DNA methyltransferase can coexist (31, 32) in cancer cells as has been previously suggested (29) . The fact that general demethylation could be induced by an important cellular signal transducer raises the interesting possibility that the control of the availability of a demethylase might be an important site through which the state of methylation of the genome might be regulated.


FOOTNOTES

*
This work was supported by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Research Scientist of the National Cancer Institute of Canada. To whom correspondence should be addressed: Dept. of Pharmacology and Therapeutics, McGill University, 3655 Drummond St., Montreal, Quebec H3G 1Y6, Canada. Tel.: 514-398-7107; Fax: 514-398-6690; E-mail: MCMS@MUSICA.McGILL.CA.

M. Szyf, J. Theberge, and V. Bozovic, unpublished data.

The abbreviations used are: kb, kilobase(s) pair(s); HGH, human growth hormone; c21, steroid 21-hydroxylase.


ACKNOWLEDGEMENTS

We thank Dr. Olson for the myogenin cDNA and Dr. Gardner for the acetylcholine receptor ( subunit) promoter probe. The excellent technical help of Anik Boudreau is sincerely acknowledged. We thank Dr. Gary Tanigawa for critical reading of the manuscript.


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