©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of the DNA Methyltransferase by the Ras-AP-1 Signaling Pathway (*)

(Received for publication, August 25, 1994; and in revised form, November 14, 1994)

Julie Rouleau (§) A. Robert MacLeod Moshe Szyf (¶)

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

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Using deletion analysis and site-specific mutagenesis to map the 5` regulatory region of the DNA methyltransferase (MeTase) gene, we show that a 106-bp sequence (at -1744 to -1650) bearing three AP-1 sites is responsible for induction of DNA MeTase promoter activity. Using transient cotransfection chloramphenicol acetyltransferase assays in P19 cells, we show that the DNA MeTase promoter is induced by c-Jun or Ha-Ras but not by a dominant negative mutant of Jun, ∂9. The activation of the DNA MeTase promoter by Jun is inhibited in a ligand dependent manner by the glucocorticoid receptor. Stable expression of Ha-Ras in P19 cells results in induction of transcription of the DNA MeTase mRNA as determined by nuclear run-on assays and the steady state levels of DNA MeTase mRNA as determined by an RNase protection assay. These experiments establish a potential molecular link between nodal cellular signaling pathways and the control of expression of the DNA MeTase gene. This provides us with a possible molecular explanation for the hyperactivation of DNA MeTase in many cancer cells and suggests that DNA MeTase is one possible downstream effector of Ras.


INTRODUCTION

The establishment of a pattern of DNA methylation of CpG dinucleotide sequences is critical for normal development of vertebrates(1) . It is well established that regulated changes in the pattern of DNA methylation occur during development (2, 3) and cellular differentiation(4) , and that aberrant changes in the pattern of methylation occur during cellular transformation(5, 6, 7) . We have previously suggested that one mode of control of the pattern of methylation is at the level of regulation of the DNA methyltransferase (MeTase) (^1)gene which encodes the activity that catalyzes the transfer of methyl groups to DNA(8, 9) . If the level of DNA MeTase activity in the cell is an important determinant of DNA methylation patterns, then it should be coordinated with cell growth and development. As the 5` region of the DNA MeTase was recently cloned(10) , we can now address the question whether it contains such regulatory elements.

Many cancer cell lines express high levels of DNA MeTase activity (11) and human colon carcinoma cell lines have been shown to express dramatically higher levels of DNA MeTase mRNA than normal cells in a manner that correlates with the state of malignancy of the cell(12) . While some sites have been shown to be hypomethylated in cancer cells(5) , critical regions of the genome are hypermethylated, such as CpG islands(13) , candidate tumor suppressor genes(14) , cell type-specific genes(15) , genes responsible for terminal differentiation such as MyoD(16) , and areas of the genome associated with tumor suppression(17) . Several lines of evidence suggest that this methylation plays an important role in the inactivation of genes in tumor cells and the process of cellular transformation: (a) in vitro methylation of CpG islands results in inactivation of these genes following transfection (18) and (b) treatment of cells with the methylase inhibitor 5-azacytidine (14, 19) or expression of an antisense to the DNA MeTase mRNA results in reactivation of this class of genes (20) . Recent experiments have shown that forced expression of an endogenous DNA MeTase cDNA in NIH 3T3 cells leads to their transformation (21) and expression of an antisense to the DNA MeTase in the adrenocortical carcinoma cell line Y1, or treatment of Y1 cells with 5-azacytidine leads to reversal of transformation. (^2)The molecular mechanisms responsible for the increased DNA methylation activity in the process of cellular transformation are unknown. One possible explanation is that the regulatory elements of the gene are responsive to some oncogenic signaling pathways (23) in addition to the cell cycle regulation of DNA MeTase gene expression which occurs mainly at the posttranscriptional level(24) .

The DNA MeTase 2-kb 5` upstream region bears one consensus AP-1 site located at -1744 and six AP-1 sites with one or two mismatches(10) . This study tests the hypothesis that overexpression of signaling pathways leading to activation of Jun (25, 26, 27) can result in hyperinduction of the DNA MeTase promoter.


MATERIALS AND METHODS

Cell Culture and CAT Assays

P19 cells (from Dr. McBurney) were plated at a density of 8 times 10^4/well in a six-well tissue culture dish (Nunc) and transiently transfected with the appropriate amounts of plasmid DNA using the calcium phosphate precipitation method as described previously(10, 28) . CAT assays were performed in triplicate as described previously(20, 29) . For stable transfections, (1 times 10^6) P19 cells were plated on a 150-mm dish (Nunc) 15 h before transfection. The pZEM expression vector bearing a 2.3 BamHI-EcoRI fragment encoding v-Ha-ras from the MMTV-v-Ha-ras plasmid or the pZEM control vector (10 µg) (30) were cointroduced into P19 cells with pUCSVneo (1 µg) as a selectable marker by DNA mediated gene transfer using the calcium phosphate protocol(28) . G418-resistant cells were cloned and propagated in selective medium (0.5 mg/ml G418, Life Technologies, Inc.).

Plasmid Construction

The physical maps of the deletion constructs are presented in Fig. 3and the sequences deleted in the different constructs are listed in Table 1. The construction of pMet CAT+(-2.3) and pMet CAT+ ∂1 was described previously(10) . To generate pMet CAT+ ∂6, pMet CAT+ ∂7 M/Wt and the pMet CAT+ ∂8 Wt/M, the sequence encoding -1.75 to -1.65 of the DNA MeTase 5` (10) was amplified using the 5` oligo containing the AP-1 site at -1744 (5`-AATGCAGCATGACTCATGCT-3`) and a 3` oligo encoding the AP-1 site at -1650 (5`-AGGAGCTGTCAGTCAGGGTC-3`). To generate the M/Wt version, the 5` oligo was the same except that the sequence encoding the AP-1 site (underlined in the sequence presented above) included in the oligo was mutated to TGAGGTCA. To generate Wt/M the 3` oligo was mutated so that the antisense 3` AP-1 site was changed to TGCCTCA. The fragment was amplified from 10 ng of SKmet+ (10) using Hot tub (Amersham Corp.) and its recommended buffer using the following cycle: 1) 95 °C 5 min, 2) 45 °C 1 min, 3) 95 °C 1 min, 4) 55 °C 1 min, 5) 72 °C 0.5 min, followed by 20 cycles of steps 3-5. The 0.1-kb polymerase chain reaction fragment was subcloned into PCRII (InVitrogen), verified by sequencing, excised by XbaI-HindIII, and inserted upstream to -0.39 in pMetCAT as indicated in Fig. 3and Table 1. The expression vectors encoding the human glucocorticoid receptor (HGCRC) (31) c-jun (RSV-cJun)(26) , ∂9 v-jun(32) were previously described. The jun constructs were kindly provided by Dr. M. Karin. To construct pZEM, we subcloned a 2.3 EcoRI-BamHI fragment encoding v-Ha-ras from the MMTV-v-Ha-ras construct (33) into the BglII site of pZEM (30) after transforming the EcoRI site into a BamHI site using a BamHI linker (Life Technologies, Inc.).


Figure 3: Deletion analysis of the DNA MeTase 5` region. The physical maps of the different deleted pMetCAT+ constructs are shown in the right panel relative to the original pMetCAT+ construct. Regions deleted are indicated by a broken line. The boundaries of the deleted sequence are indicated relative to the transcription initiation site and listed in Table 1. The first number indicates the 5` of the deleted sequence and the second number indicates the 3` boundary. Exons are indicated by filled boxes, CAT sequence is hatched, transcription initiation sites are indicated by horizontal arrows, shaded ovals indicate AP-1 sites (forming DNA protein interactions, Fig. 4), filled ovals are mismatched AP-1 sites. HIII, HindIII recognition site; BII, BglII; RI, EcoRI; RV, EcoRV; BHI/NI is an original NaeI site that was modified by linkers to a BamHI site, BHI, BamHI; S3AI, SauIIIAI. M/Wt, the AP-1 site at -1750, is mutated to TGAGGCA but the one at -1644 is wild type. Wt/M, the AP-1 site at -1750 is wild type but the 3` site is mutated as above. The different constructs (10 µg) were transfected in triplicate into P19 cells with either 4 µg of SK plasmid (Control) or 4 µg of RSV-c-jun (+Jun), harvested after 48 h, and CAT activity was determined as described above. Each experiment was repeated twice using different plasmid preparations. The results are presented as averages of three determinations ±S.D.






Figure 4: Binding of the AP-1 transcription complex to sequences in the DNA MeTase 5` upstream region. A, A 21-mer double-stranded oligomer, Met 5` AP-1, encoding the sequence: 5`-AATGCAGCATGACTCATGCT-3` located at(-1753, -1734) in the DNA MeTase 5` region (the putative AP-1 recognition sequence is underlined) was end-labeled with [-P]ATP (Amersham Corp.) and incubated with 10 µg of P19 nuclear extract. For competition experiments, an excess of nonlabeled double-stranded oligomers was used. The free oligomer (gray arrow) and the bound AP-1 complex (black arrow) were separated on a native polyacrylamide gel. An excess of labeled substrate was used for all assays. The following competitors were used: (a), Met 3` AP-1 is located at -299 to -275 in the DNA MeTase 5` region and encodes the sequence 5`-GTTTTGAGGCAGGATTTTGA-3` (the underlined sequence contains one mismatch with the consensus AP-1 recognition sequence); (b) AP-1 encodes a consensus AP-1 sequence (5`-CGCTTGATGAGTCAGCCGGAA-3`) (Promega); (c) Sp1 encodes the binding recognition sequence of the transcription factor Sp-1 (5`-GATCGATCGGGGCGGGGCGATC-3`). B, an in vitro DNase footprinting assay was performed on an end labeled 0.6-kb BamHI-Sau3AI fragment containing the 5` AP-1 region of the DNA MeTase gene (see Fig. 3) which was incubated with 1 footprint unit (fpu) of a bacterially expressed c-Jun protein (Promega). The AP-1-reacted fragment and a naked (nonreacted) control were subjected to cleavage by DNase I (Boehringer Mannheim) which was followed by electrophoresis and autoradiography. An M13 sequencing reaction (Pharmacia) was used as as a size marker ladder. The regions that are protected from DNase I cleavage by AP-1 are indicated. The footprints at -1744 to -1738 (TGACTCA) and -1650-1644 (TGACTGA) match the AP-1 consensus recognition sequence. The footprint at -1718 to -1708 (TGGACGGCTTT) does not correspond to the consensus AP-1 recognition sequence.



Gel Retardation Assays

Pairs of complementary 21-mer oligodeoxynucleotides were annealed and 0.4 µg of the double-stranded oligodeoxynucleotides were end-labeled with [-P]ATP (Amersham Corp.) using T4 polynucleotide kinase (Boehringer Mannheim) and purified by excision from an 8% polyacrylamide gel(28) . Binding assays were performed by incubating 1 ng (10^3 cpm) of an end-labeled oligomer with 3 µg of crude nuclear extract from P19 cells (24) as described by Ausubel et al.(28) .

DNase Footprinting Assays

DNase footprinting reactions were performed as recommended by Promega and as described in Ausubel et al.(28) . A 648-bp BamHI-Sau3AI fragment containing four putative AP-1 binding sites (at -1744, -1650, -1547, and -1514) (10) was cloned at the BamHI site of the pGEM3 vector (Promega), digested with SmaI (Boehringer Mannheim), treated with calf intestinal phosphatase (Boehringer Mannheim), end-labeled with [P]ATP (Amersham Corp.) using T4 polynucleotide kinase (Boehringer Mannheim), and then subjected to EcoRI digestion. The radiolabeled fragment (2 times 10^4 dpm) was incubated with 1 footprint unit of the cloned and bacterially expressed c-Jun (Promega), and the binding reaction was subjected to DNase I digestion (0.15 unit) for 3 min at room temperature.

RNase Protection Assays

RNA was prepared from exponentially growing cells using standard protocols. RNase protection assays were performed as described in reference 10 using a 0.7-kb HindIII-BamHI fragment (-0.39 to +0.318) as a riboprobe (probe A in Rouleau et al.(10) ).

Nuclear Run-on Assays

Nuclei were prepared from 3 times 10^6 exponentially growing pZEM and pZEM transfected P19 cells and were incubated with [alpha-P]UTP (800 Ci/mmol) as described previously(24) . The transcribed P-labeled RNA (1 times 10^6 dpm/sample) was hybridized with pSKMet5` plasmid (10) and SK plasmid as a control that were immobilized onto a Hybond-N+ filter as described previously(24) .


RESULTS

c-Jun and Ha-Ras Transactivate the DNA MeTase Promoter

To test the hypothesis that the DNA MeTase promoter is activated by high levels of AP-1 activity we cotransfected a fusion construct expressing the CAT reporter gene under the direction of 2 kb of the DNA MeTase 5` upstream region (pMetCAT+) containing the minimal promoter and the AP-1 recognition sequences (see physical map in Fig. 3)(10) , with expression vectors encoding the protooncogene c-jun (RSV-c-jun) (26) into P19 cells. Recent results have demonstrated that activated Ras indirectly induces transcription of genes that are regulated by promoters that contain AP-1 recognition sequences and that this induction is mediated by activation of c-Jun by phosphorylation(26) . To test the hypothesis that activation of the Ras signaling pathway can induce DNA MeTase promoter activity, we cotransfected an expression vector encoding the Ha-Ras protein (pZEM) and pMetCAT+. To rule out variability in culture conditions and transfection efficiencies as an explanation for the induction of pMetCAT+ by Jun and Ras, we performed multiple experiments (each different experiment was performed in triplicate) at different times using different cultures of P19 cells. In each experiment an increasing dose of either pZEM or RSV-c-jun (2-10 µg) was used, the maximal fold induction was determined and the average fold induction for all experiments was calculated. The results shown in Fig. 1A demonstrate that both Jun and Ras can induce the DNA MeTase promoter (10-30-fold for Jun and 8-15-fold for Ras) from very low basal levels (the results are of statistical significance as determined by a t test, p < 0.001). This induction of pMetCAT+ is not a nonspecific enhancement of transfection efficiency or transcription from plasmid DNA because all transfections had equal amounts of total plasmid DNA.


Figure 1: Fos and Ras induce the mouse DNA MeTase promoter. A, P19 cells, an embryonic carcinoma cell line(22) , were transfected with 10 µg of pMetCAT+ (see Fig. 3for physical map) and increasing concentrations (1-10 µg) of expression vectors expressing c-jun (RSV-c-jun) (29) or Ha-ras (pZEM). Cells were harvested after 48 h and CAT activity was determined as described under ``Materials and Methods.'' Each experiment was performed in triplicate. Twenty-two independent experiments using independent cultures of P19 cells at different times were performed for the Jun expression vector and eleven experiments with the Ha-ras. The maximal fold induction above the values obtained with pMetCAT+ was determined per construct for each experiment. Each value is presented as mean of the values obtained in all experiments ±S.D. The difference between Jun- and Ras-induced pMetCAT + expression and the noninduced control is highly significant p < 0.001. B, P19 cells were transfected with either pMetCAT+ (4 µg) and SK-, pMetCAT+ (4 µg) and RSV-cJun (4 µg), or pMetCAT+ (4 µg) and pZEM (4 µg) and an increasing concentrations of a vector encoding the transdominant negative mutant of Jun ∂9-v-Jun which was kindly provided by Dr. B. Wasylik(32) . The total concentration of plasmid DNA was kept constant by adding SK plasmid to the transfection mix. The values are presented as an average of three determinations ±S.D.



To demonstrate that this transactivation of the DNA MeTase promoter by Ras and Jun is dependent on functional Jun activity rather than a nonspecific effect, we cotransfected pMetCAT+ (4 µg/ml) and increasing concentrations of a transdominant negative mutant of Jun, ∂9 which lacks the transactivation domain of Jun but expresses the DNA binding domain(32) . The total concentration of transfected plasmid DNA was maintained constant by adding SK plasmid DNA. As demonstrated in Fig. 1B, ∂9 cannot transactivate the DNA MeTase promoter. Coexpression of ∂9 with Jun (4 µg/ml) results in a dose-dependent inhibition of transactivation by Jun as expected. When ∂9 is coexpressed with Ras (4 µg/ml), the induction of the DNA MeTase promoter by Ras is repressed even at low concentrations of the ∂9 plasmid. This demonstrates that both Ras and Jun induce the DNA MeTase by a common pathway.

The Glucocorticoid Receptor Inhibits the Transactivation of the DNA MeTase Promoter by Jun

One important characteristic of AP-1 induced genes is the modulation of this induction by the glucocorticoid receptor(35, 36) . Nuclear receptors such as the glucocorticoid receptor serve as modulators of differentiation and cellular growth in many systems, for example, the well characterized antimitotic and cytotoxic effect of glucocorticoid on T cells(37) . Several groups have recently established that nuclear receptors can modulate the activity of AP-1 complexes by binding overlapping AP-1 recognition sequences (38) or by protein-protein interactions with Jun(35, 36) . These interactions have been postulated to explain the antimitotic effects of glucocorticoids. We tested the hypothesis that the glucocorticoid receptor can modulate the transactivation of DNA MeTase by AP-1 in a ligand dependent manner. We transiently transfected pMetCAT+ (10 µg/ml) with the glucocorticoid receptor expression vector HGCRC (10 µg/ml) in the presence or absence of inducing concentrations of Jun (4 µg/ml) and in the presence or absence of 0.1 µM dexamethasone. Fig. 2is an average of three independent experiments that were performed at different times using different cultures of P19 cells. As observed in Fig. 2, dexamethasone per se does not affect either the basal level of activity of the DNA MeTase promoter or its induction by Jun, most probably because of lack of expression of the glucocorticoid receptor in P19 cells. Cotransfection of the glucocorticoid receptor has a limited inhibitory effect on the expression of the basal and Jun-induced promoter activity resulting most probably from low concentrations of dexamethasone in the the growth medium. However, a significant (p < 0.05) inhibition of DNA MeTase promoter activity is observed in the presence of both the glucocorticoid receptor and its ligand demonstrating that the glucocorticoid receptor inhibits the expression of the DNA MeTase promoter in a ligand dependent manner. This demonstrates that the inhibition is not a nonspecific effect of the glucocorticoid plasmid or dexamethasone. This experiment therefore suggests that transactivation of DNA MeTase by Jun can be modulated by the glucocorticoid receptor and that the DNA MeTase can be responsive to both the Ras signaling pathway as well as to nuclear receptors.


Figure 2: Inhibition of Jun-dependent induction of the DNA MeTase promoter by the glucocorticoid receptor. A, Ten µg of pMetCAT+ were cotransfected in triplicate into P19 cells with either SK plasmid (pMetCAT+), SK plasmid and 10 µg of the human glucocorticoid receptor expression vector (HGCRC)(27) , RSV-c-jun (+Jun), or RSV-c-jun and the HGCRC (as indicated in the figure). One set of transfectants was treated with 0.1 µM of dexamethasone (Sigma) for 24 h before harvesting (+dex) and CAT activity was determined in extracts prepared from the different transfected cells 48-h posttransfection as described under ``Materials and Methods.'' The fold induction over pMetCAT+ (10 µg with SK-) was determined. The experiment was repeated three different times using three different cultures of P19 cells. The values are presented as an average of three independent experiments ±S.D. The statistical significance of the difference between Jun and Jun + HGCRC transfectants and Jun as well as the statistical difference between dexamethasone treated HGCRC + Jun and Jun transfectants was determined using a t test, p < 0.05.



Deletion Mapping of AP-1 Responsive Elements

To delineate the sequences responsible for regulation of the DNA MeTase by AP-1 and to determine whether induction of DNA MeTase promoter by AP-1 is mediated by direct interaction of AP-1 with the DNA MeTase 5` region, we carried out a deletion analysis of the 2-kb 5` upstream region of the DNA MeTase (Fig. 3). The different deletion constructs (10 µg) were cotransfected with either SK plasmid or RSV-cJun (4 µg). AP-1 inducibility is lost when the 5` region including all the potential AP-1 sites upstream to the minimal promoter at -0.18 is deleted. Very limited activity is observed when only one nonconsensus AP-1 site at -290 is present in the construct (-0.39), however, a deletion of most of the 1-kb sequence between -1.3 and -0.39 is still inducible with Jun suggesting that AP-1 inducibility is encoded between -2 and -1.35. The region contained between -2 and -1.35 does not confer inducibility in our assay when it is inserted in the antisense orientation. When both this region and the promoter are inserted in the antisense orientation to CAT, no CAT activity is detected supporting the conclusion that the activity detected in our assays is directed by the DNA MeTase promoter. Four potential AP-1 sites are located in the sequence contained between -2 and -1.35. The AP-1 site at -1744 (TGACTCA) is a consensus site, the site at -1650 TGACTGA and 1547 (TGACTCT) bears one mismatch and the sites at -1514 bear two mismatches with the consensus site. To test the hypothesis that AP-1 inducibility is encoded by the 5` consensus AP-1 site at -1744 and -1650 we amplified the sequence contained between -1744 and -1650 using the primers described under ``Materials and Methods'' and inserted it upstream to -0.39 (Fig. 3). The 118-bp fragment encoding the two 5` AP-1 sites confers full inducibility with Jun upon the DNA MeTase promoter. The 5` AP-1 site plays a more significant role in Jun inducibility since introduction of two mismatches (TGAGGCA) into the 5` AP-1 site (M/Wt) inhibits the inducibility of the construct while introduction of the same mutation to the 3` site does not have the same effect (Wt/M). It is interesting to note that constructs bearing deletions of the sequence between -1.3 and -0.39 express a higher basal DNA MeTase promoter activity than the nondeleted control. This might suggest the presence of a cis-repressor recognition sequence encoded in this region. This repression of transactivation of DNA MeTase promoter by endogenous AP-1 might be an additional mechanism through which DNA MeTase gene expression is controlled and maintained at a basal level under normal conditions. In summary, the deletion experiments demonstrate that the control of the DNA MeTase by AP-1 is mediated by an interaction between Jun (AP-1) and the AP-1 site(s) located at -1744 to -1650 upstream to the transcription initiation site.

Physical Interaction between AP-1 Sites in the DNA MeTase Promoter and AP-1 Transactivation Complex

To determine whether the transactivation of the DNA MeTase by AP-1 involves direct interaction of AP-1 with the AP-1 recognition sequences in the DNA MeTase 5` upstream region, we performed gel retardation and DNase footprinting assays. Nuclear extracts from P19 cells contain a measurable level of DNA-binding activity interacting with a P-labeled oligomer from the 5` MeTase region(-1753, -1734) containing an AP-1 recognition sequence from -1744, -1738 (Met5` AP-1) (Fig. 4A). This DNA-protein complex can be competed out with a 10-fold excess of a nonlabeled oligomer encoding a previously characterized consensus AP-1 recognition sequence (AP1) (Fig. 4A), with an excess of nonlabeled Met5` AP-1 oligomer but not with an oligonucleotide sequence containing an AP-1 recognition sequence with two mismatches located at -505-499 (Met 3` AP1) or an oligomer encoding the recognition sequence of the transcription factor Sp1 (Fig. 4A). This demonstrates that the AP-1 site at -1744-1738 can specifically interact with the AP-1 complex. This DNA-protein complex could be competed with an excess of nonlabeled AP-1 but not with Sp1 or Met3` AP-1.

To visualize the specific molecular interactions between Jun and the AP-1 binding region of DNA MeTase 5` upstream sequences, we incubated a 648 bp fragment containing these sequences with c-Jun protein and subjected the bound products to a DNase I footprint analysis as described in materials and methods (Fig. 4B). Two potential AP-1 sites (TGACTCA (-1744, -1738, and TGACTGA(-1650, -1644)) were protected from DNase cleavage as indicated in Fig. 4B. These experiments have therefore established that the AP-1 sites in the 5` of the DNA MeTase gene are functional AP-1 sites in vitro.

Exogenous Expression of Ras Induces Transcription of the Endogenous DNA MeTase

To determine whether induction of the Ras signaling pathway can induce the expression of the endogenous DNA MeTase gene, we introduced either pZEM or pZEM into P19 cells by DNA mediated gene transfer. G418-resistant colonies were propagated and Ras expressing and pZEM colonies were identified. Three colonies bearing exogenous pZEM or pZEM were randomly selected. The pZEM transfectants express exogenous Ras as indicated by the Northern blot presented in Fig. 5A. To determine whether Ras induces transcription of DNA MeTase, we prepared nuclei from exponentially growing P19 pZEM control transfectants and P19 lines transfected with Ha-ras and subjected them to a run-on transcription assay using [alpha-P]UTP. Equal amounts of labeled RNA were hybridized with either immobilized SK (as a control) or SKmet5`. The hybridized filters were exposed to autoradiography after stringent washing. The results presented in Fig. 5B show that the two pZEM transfectants tested express higher levels of DNA MeTase transcript than the pZEM control. To determine the levels of steady state DNA MeTase mRNA, we resorted to RNase protection assays using a 720-bp riboprobe encoding the first two exons of the DNA MeTase and the transcription initiation site(10) . This probe displays a group of fragments at 89-99 bp representing the two exons and two initiation sites. Equal amounts of RNA (as determined by hybridization with an 18 S oligonucleotide probe) prepared from three independent transfectants with pZEM and three independent pZEM transfectants were subjected to a RNase protection assay as described in Rouleau et al. (10) (Fig. 5C). The results were quantified by densitometry and the quantification is presented in Fig. 5D. The results presented in Fig. 5D demonstrate that the ras transfectants express higher levels of DNA MeTase mRNA than the pZEM controls (10-20-fold). Two transfectants express high levels of mRNA that is initiated at multiple sites in addition to the standard sites observed with other cell lines and the controls(10) . To quantify this difference in transcription rate, three independent pZEM transfectants and three independent pZEM transfectants were subjected to a runon analysis, the level of DNA MeTase transcript was quantified by densitometry (Masterscan, Scanalytics) and the results were presented as an average of the three independent transfectants (Fig. 5D). These results demonstrate that the pZEM transfectants express 3-fold higher levels of DNA MeTase transcript than the controls. The higher levels of induction of steady state levels of mRNA relative to the change in the transcription rate might either suggest that Ras affects the stability of the DNA MeTase mRNA in addition to its rate of transcription, or alternatively, it might reflect differences in the experimental procedures used to measure these two parameters. In summary, the results presented in this section demonstrate that transcription of the endogenous P19 DNA MeTase mRNA is induced by forced expression of Ras. The effects that this induction might have on DNA methylation and DNA MeTase activity is, however, complex since Ras induces an increase in demethylase activity (^3)in parallel to its induction of transcription of DNA MeTase.


Figure 5: Transcription of the endogenous DNA MeTase gene is induced by forced expression of exogenous Ha-ras. P19 cells were stably transfected with either (10 µg) pZEM or (10 µg) pZEM Ha-ras encoding the Ha-ras oncogene which contains activation mutations in codon 12 (Gly to Arg) and 59 (Ala to Thr) and (1 µg) pUCSVneo as a selectable marker. G418 (0.5 mg/ml) resistant colonies were selected and propagated. Preliminary Southern blot analysis using a P-labeled Ha-ras probe (0.7 kb HindIII-PstI fragment) has been performed to identify transfectants and three randomly picked positive clones per each transfected plasmid were used for our further studies. A, Northern blot analysis using a ras probe. The ras transfected panels were exposed for a short time to enable their presentation alongside with the nontransfected controls. The viral ras directs a 2-kb transcript that is easily distinguishable from the shorter endogenous ras messages (1-1.4 kb). As observed in the figure (left panel) the ras transfectants express large amounts of ras that is probably initiated at multiple sites in addition to the viral initiation site. The filter was stripped from the radioactivity and rehybridized with an 18 S rRNA-specific P-labeled oligonucleotide (45) (bottom panel). B, P-labeled RNA (1 times 10^6 dpm) transcribed in nuclei prepared from ras-transfected P19 cells (lanes 11 and 17) and pZEM control (lane 1) was hybridized with filter immobilized SKmet5` (10) containing a DNA MeTase genomic fragment encoding the 5` transcribed regions of the gene and SK plasmid as a negative control. C, RNase protection assay of DNA MeTase mRNA. RNA prepared from three P19 pZEM transfectants (lanes 6, 11, and 17) and pZEM control transfectants was subjected to an RNase protection assay using a 700-bp riboprobe (probe A) (10) encoding the DNA MeTase genomic sequence from -0.39 to +318. The major bands representing the two major initiation sites are indicated (92 and 90). The first exon will give a 99-bp protected fragment. Additional minor initiation sites are markedly represented in the two ras transfectants that express significant levels of DNA MeTase. The experiment has been repeated twice with similar results. D, quantification of transcription rate and steady state mRNA levels in Ras transfectants versus controls. 1) Run on assays: a run on assay similar to the one presented in B was performed on nuclei prepared from three independent ras transfectants (6, 11, 17) and three control transfectants pZEM (lanes 1, 2, and 3). The intensity of the signal obtained following hybridization of the labeled RNA with SKmet5` was determined by densitometry (Scanalytics Masterscan) and the average for the three lines is presented with the S.D. 2) mRNA- quantification of the signal obtained in the autoradiogram presented in C at the 92-, 90-, and 99-bp fragments hybridizing with DNA MeTase mRNA. The average for the three ras-transfected and the three pZEM-transfected lines is presented with the S.D.




DISCUSSION

This report addresses the hypothesis that the activity of DNA MeTase is regulated by nodal cellular signaling pathways. While this fact by itself is not sufficient to demonstrate that regulation of DNA MeTase is an important cellular control point, it is consistent with that hypothesis(23, 39) . We demonstrate in this study that when Ras-Jun signaling pathway is activated by either expressing high levels of exogenous Ras or Jun, the DNA MeTase promoter is significantly induced ( Fig. 1and Fig. 3), that this activation is mediated by AP-1 recognition sequences in the 5` region of the gene (-175-1640) and that it is inhibited by the glucocorticoid receptor in a ligand dependent manner (Fig. 2). This inhibition is most probably mediated by protein-protein interactions with AP-1 as has been shown for other AP-1 induced promoters since the DNA MeTase 5` region does not contain a full glucocorticoid recognition site(10) . Forced expression of an exogenous Ha-ras induces transcription and an increase in the steady state levels of endogenous DNA MeTase mRNA (Fig. 5). Induction of Jun appears to be associated with induction of differentiation in P19 cells(40) . Since Ras has multiple effects on P19 cells it is hard to assess the relative effect of increased DNA MeTase activity on DNA methylation patterns. Our ras transfectants induce high levels of demethylase activity^3 which complicates the analysis of the effects that induction of DNA MeTase has on the pattern of methylation. Whereas other cell systems should be sought to study the effects of an increase in Ras activity on DNA methylation, P19 cells are an excellent model to dissect AP-1 and glucocorticoid responsiveness of the exogenous promoter because they do not express high levels of c-jun(41) . Using other cell systems to study the effects induction of Ras and Jun might have on DNA methylation, we have recently shown that inhibition of the Ras-Jun pathway in an adrenocortical tumor cell line Y1 results in inhibition of DNA methylation activity and reduction in methylation levels of CpG dinucleotides. (^4)What is the significance of regulation of DNA MeTase by Ras and Jun? One documented instance when DNA MeTase is markedly up-regulated is cancer; many cancer cell lines and colon cancer cells in vivo express significantly higher levels of DNA MeTase(11, 12) . In this report we suggest a possible molecular route through which oncogenic pathways can lead to induction of DNA MeTase. DNA MeTase exhibits very low constitutive levels of expression ( Fig. 1and Fig. 3). This basal level of expression is responsive to the proliferative state of the cell by a posttranscriptional up-regulation as we have previously shown(24) . However, in addition to this basal control it is possible that cellular signaling systems such as the one induced by Ras can induce high activities of DNA MeTase in a programmed manner at distinct sites and times in development. When these signaling pathways are aberrantly up-regulated as happens in many cancer cells, DNA MeTase promoter is induced, possibly resulting in elevation of DNA MeTase (11, 12) and the hypermethylation of specific genomic regions in cancer cells(17) . While it is clear that factors other than the level of activity of DNA MeTase must play a role in shaping the methylation pattern of the genome(43) , the level of DNA MeTase activity is most probably an important factor. It is possible however that in addition to AP-1, other pathways control DNA MeTase gene expression. What are the changes in DNA methylation that are critical for cellular transformation? Several mechanisms have previously been suggested such as silencing of tumor suppressor genes (13, 16) , an increase in spontaneous mutations resulting from deamination of methylated cytosines (44) or altered regulation of replication(23) . In contrast to the induction of cellular proliferation by Ras in many cell types, Ras can induce the differentiation of PC12 cells(42) . Induction of DNA MeTase might play an important role in these processes as well. Although additional experiments are required to determine whether DNA MeTase plays a critical role in the transformation process as a downstream effector of Ras, the results presented in this report establish a molecular link between cellular signaling pathways and the machinery responsible for controlling the pattern of modification of the genome.


FOOTNOTES

*
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.

§
Funded by a fellowship from Fonds de Formation des Chercheurs et d'aide à la Recherche.

Research Scientist of the National Cancer Institute of Canada (NCIC) and is supported by the NCIC. To whom correspondence should be addressed: Dept. of Pharmacology and Therapeutics, McGill University, 3655 Drummond St., Montreal, PQ, Canada H3G 1Y6. Tel.: 514-398-7107; Fax: 514-398-6690; mcms{at}musica.mcgill.ca.

(^1)
The abbreviations used are: MeTase, methyltransferase; CAT, chloramphenicol acetyltransferase; bp, base pair(s); kb, kilobase(s).

(^2)
R. A. MacLeod and M. Szyf, submitted for publication.

(^3)
M. Szyf, unpublished results.

(^4)
R. A. MacLeod, J. Rouleau, and M. Szyf, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. Gary Tanigawa, Marc Pinard, and Shyam Ramchandani for a critical analysis of the manuscript. V. Bozovic and J. Theberge are acknowledged for excellent technical assistance. We thank Drs. M. Karin, B. N. Sonenberg, and M. Nemer for the expression vectors.


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