Vol. 265, No. 9, Issue of March 25, pp. 4639-4843, 1990 Printed in U.S.A.

DNA Methylation and Collagen IV Gene Expression in F9 Teratocarcinoma Cells*

(Received for publication, August 16, 1989)

Peter D. BurbeloS, S. Horikoshi, and Y. Yamada From the Laboratory of Developmental Biology and Anomalies, National Institute of Dental Research, National Institutes if Health, Bethesda, Ma&-znd 20892

Although undifferentiated F9 teratocarcinoma cells express low levels of mRNAs for collagen IV, previous transient transfection experiments using an al(IV) col- lagen gene promoter-enhancer-CAT construct re- vealed a high level of transcriptional activity in these cells. In this report, we find that when this construct is introduced stably into undifferentiated F9 terato- carcinoma cells, expression does not occur unless the cells are treated with retinoic acid and dibutyryl- CAMP. Such activation mimics the endogenous gene activity. Treatment of the cells containing the inte- grated construct with S-azacytidine, an agent which prevents DNA methylation, also activates transcrip- tion and acts synergistically with retinoic acid and CAMP. Analysis of DNA isolated from F9 teratocarci- noma cells revealed that there was a specific demeth- ylation of the DNA within the 5’-flanking region of the collagen IV genes following treatment with retinoic acid and CAMP. These results suggest that during dif- ferentiation, DNA demethylation may play an impor- tant role in transcriptional regulation of the collagen IV genes.

Collagen IV, the major structural component of basement membranes, is composed of two (ul(IV) and one a2(IV) chains. The genes for the two polypeptides exist in a head to head arrangement on opposite strands of DNA, separated by 130 base pairs, in both the mouse (Burbelo et al., 1988; Kaytes et al., 1988) and the human (Poschl et al., 1988, Soininen et al., 1988). The close proximity and arrangement of the two col- lagen IV genes on the same chromosome distinguishes these genes from those of other collagen types which are dispersed throughout the genome (Solomon et al., 1985). In addition, both the al(IV) and aS(IV) chain genes share a bidirectional promoter which utilizes an enhancer element within the first intron of the al(IV) collagen gene for efficient transcriptional activity (Burbelo et al., 1988).

The transcriptional regulation of these and other basement membrane component genes is often studied in F9 teratocar- cinema stem cells. These cells differentiate into parietal en- doderm-like cells in the presence of both retinoic acid and dibutyryl-CAMP (Strickland et al., 1980) and show a striking coordinate and increased synthesis of basement membrane proteins including collagen IV (Kleinman et al., 1987; Kurki- nen et al., 1983; Marotti et al., 1985; Wang and Gudas, 1983)

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Postdoctoral Fellow supported by Juvenile Diabetes Foundation International. To whom correspondence should be addressed.

and laminin (Carlin et al., 1983; Kleinman et al., 1987; Wang and Gudas, 1983). Thus, F9 teratocarcinoma cells provide a model system for studying the regulation of these genes. Induction of the mRNA for these genes requires approxi- mately 18-24 h and is blocked by the addition of cyclohexi- mide indicating that protein synthesis is required (Gudas and Wang, 1986).

The promoter-enhancer constructs for the al(IV) collagen chain gene are not promiscuously expressed in all cells, but rather only in cells producing the protein (Killen et al., 1988). Undifferentiated F9 teratocarcinoma cells express very low levels of collagen IV; however, they show a high level of transcriptional activity of the promoter-enhancer-CAT’ con- struct in transient assays (Killen et al., 1988). One possible explanation is that undifferentiated F9 cells contain the trans- acting factors that activate both the promoter and enhancer regions for collagen IV genes, but do not bind to the regulatory regions of the endogenous gene because of their methylation state and/or chromatin structure. To investigate this possi- bility, we have integrated stably cul(IV) chain promoter-en- hancer CAT constructs into undifferentiated F9 teratocarci- noma cells and found that they are not transcribed in these cells. Following treatment of the cells with retinoic acid and dibutyryl-CAMP, however, there was a marked stimulation of transcriptional activity. We have also found that the 5’- flanking DNA of the al(IV) gene was demethylated during differentiation of F9 cells. These results indicate that the methylation state and/or chromatin structure of the collagen IV genes, rather than the production of trans-acting factors, may play a key role in the activation of collagen IV genes in this system.

EXPERIMENTAL PROCEDURES

Cell Culture and DNA Transfections-The F9 teratocarcinoma cells were maintained in culture in the undifferentiated state as described previously (Killen et al., 1988). Differentiation of the cells to parietal endoderm-like cells was initiated by addition of all-trans retinoic acid (lo-’ M) plus dibutyryl-CAMP (10m3 M) (Strickland et al., 1980).

The otl(IV) promoter-CAT construct (p47A) and the al(W) pro- moter-enhancer-CAT construct (~48) have been described previously (4). Transient transfection into undifferentiated F9 teratocarcinoma cells was performed as described previously (Burbelo et al., 1988). Briefly, cells (250.000 cells) were transfected with 5 ue of nlasmid DNA-6y the calcihm phosphate method (Graham ani ian ‘der Eb, 1973). Eighteen hours later, the medium was changed, and the cells were incubated either in normal medium or in the presence of dibu- tyryl-CAMP and retinoic acid. Forty eight hours after the medium was changed, the cells were harvested and assayed for CAT activity (Gorman et al., 1982).

For stable transfection, undifferentiated F9 cells were cotrans- fected with 2 pg of the CAT plasmids and 18 pg of RSVneo using the calcium phosphate method (Graham and Van der Eb, 1973). Colonies

1 The abbreviations used are: CAT, chloramphenicol acetyltrans- ferase; kb, kilobase(

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DNA Methylation and Collagen IV Expression

expressing the neo gene were isolated based on their resistance to the antibiotic G418. Resistant colonies were pooled (>2OO/transfection), expanded in mass cultures in the presence of G418 (350 rg/ml), and used for experiments. Some cells (250,000) were treated for 48 h with retinoic acid and dibutyryl-CAMP and then assayed for CAT activity. Other cells were treated with 5-azacytidine (lo-’ M) for 24 h after which medium was changed and maintained drug-free for 24 h.

Individual clones of cells containing the stably transfected al(W) chain gene promoter-enhancer-CAT construct were also obtained by the limited dilution method. CAT activity was determined in cells which had been cultured with and without retinoic acid and dibutyryl- CAMP for 48 h. The copy number of the collagen IV promoter- enhancer-CAT construct in the individual clones was determined by dot hybridization using a RsaI fragment from the coding region of the CAT gene (Brinst.er et al., 1985). CAT activity is expressed as raw counts or as a percentage of total chloramphenicol converted to acetylated derivatives.

DNA Meth$ation Pattern-High-molecular weight cellular DNA was prepared from both undifferentiated and differentiated F9 tera- tocarcinoma cells by the procedure of Gross-Bellard et al. (1973). Aliquots (5 rg) of DNA were digested with HindIII, XhoI HpaII, and/ or MspI restriction endonucleases (5 units/,ug of DNA) for 18 h at 37 “C. DNA samples were electrophoresed on 1% agarose gel and transferred to nitrocellulose by the method of Southern (1975). The probe to the 5’-end of the collagen IV genes was a 0.72-kb XbaI-NcoI DNA fragment containing the bidirectional promoter region, part of the first exon of the nl(IV) collagen, and the first exon, intron, and 20 base pairs of untranslated sequence from the second exon of the (uB(IV) collagen gene. This fragment was labeled by the random prime method (Feinberg and Vogelstein, 1982) and used as probe.

RESULTS

Previously, we have shown that the DNA segment between +2.7 and +5.0 kb in the first intron of the al(IV) collagen gene contains enhancer activity which is essential for the cell- specific activation of both the al(IV) and cuB(IV) collagen genes (Burbelo et al., 1988; Killen et al., 1988). CAT activity of transiently transfected F9 teratocarcinoma cells is shown in Fig. 1A. A P-actin CAT construct, which served as control, displayed a similar high level of activity in both cell types regardless of whether the cells were treated with retinoic acid plus CAMP or not (Fig. IA, lanes C and D). The promoter- CAT construct for the cul(IV) collagen gene was found to be inactive under both conditions as shown previously (Fig. IA, lanes A and B). The (ul(IV) chain gene promoter-enhancer- CAT construct exhibited a high level of activity in both cell types (Fig. IA, lanes E and F). These transfection experiments and experiments utilizing shortened constructs from the en- hancer region of collagen IV have been performed over 10 times and always show enhancer activity in both cell types. These data suggested that additional regulatory mechanisms were responsible for inhibiting collagen IV transcription in undifferentiated F9 cells.

To evaluate the possibility that the methylation state of the DNA and/or chromatin structure was important in tran- scriptional regulation of the collagen IV genes, we established lines of F9 cells containing integrated copies of al(IV) chain gene-CAT constructs. Undifferentiated F9 teratocarcinoma cells were co-transfected with either the al(IV) promoter- CAT or (ul(IV) promoter-enhancer-CAT construct and RSVneo. After 4 weeks, pooled cells (from approximately 200 G418-resistant colonies) were examined for CAT gene expres- sion before and after treatment with retinoic acid and dibu- tyryl-CAMP. The control P-actin CAT construct again dis- played a similar high level of activity independent of whether the cells were induced to differentiate with retinoic acid and CAMP (Fig. lB, lanes C and D). The al(IV) promoter con- struct lacking the enhancer was inactive when integrated stably (Fig. lB, lanes A and B), as found in the transient transfection experiments. The promoter-enhancer-CAT con- struct was not active in undifferentiated F9 cells (Fig. lB,

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36.1 FIG. 1. A, transient transfection of the al(IV) chain gene-CAT

plasmids in F9 teratocarcinoma cells. Undifferentiated F9 cells were transfected with 5 pg of plasmid DNA. After 18 h, the DNA was removed and the cells were untreated (-) or treated with dibutyryl- CAMP and retinoic acid (+). 48 h later, CAT activity was determined. Lanes A and B represent the (ul(IV) promoter-CAT-construct - and +; lanes E and F represent the al(W) promoter-enhancer-CAT construct - and +; and lanes C and D are @actin-CAT construct - and +. The percent acetylation is shown below the corresponding picture of the autoradiograph. Data represent greater than 10 inde- pendent transfections. B, stable integration of the al(IV) chain gene- CAT plasmids in F9 teratocarcinoma cells. The CAT constructs were cotransfected with RSVneo into undifferentiated F9 cells as described under “Experimental Procedures.” Cells resistant to G418 were ob- tained approximately 4 weeks later. Colonies were pooled for each of the constructs and expanded in culture. Cells were then either un- treated (-) or treated (+) with retinoic acid and dibutyryl-CAMP for 48 h. Cells were harvested and CAT activity measured. Lanes A and B represent the al(W) promoter-CAT construct - and +; lanes E and F represent the cul(IV) promoter-enhancer-CAT construct - and +; and lanes C and D are fl-actin-CAT construct - and +. The percent acetylation is shown below the corresponding picture of the autora- diograph. Data represent greater than three replications.

lane E); however, it could be activated by treatment of the cells with retinoic acid plus CAMP (Fig. lB, lane F). Dibu- tyryl-CAMP alone was ineffective in activating the collagen IV constructs (Fig. 2, lane B), whereas retinoic acid induced transcription (Fig. 2, lane C) and acted synergistically with CAMP (Fig. 2, lane D). The addition of a calcium-phosphate DNA precipitate to the undifferentiated cell constructs did not result in an activation of CAT activity (data not shown). This ruled out the possibility that the collagen IV constructs were being activated by the transfection procedure, as has been shown for other genes (Pine et al., 1988).

Individual clones from the pooled cells containing stably

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DNA Methylation and Collagen IV Expression 4841

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FIG. 2. Effect of CAMP and retinoic acid on the stably in- tegrated collagen IV promoter-enhancer-CAT constructs in F9 teratocarcinoma cell. Pooled undifferentiated F9 cells contain- ing the stably integrated rul(IV) promoter-enhancer-CAT constructs were untreated (lane A), treated only with CAMP (lane B), treated only with retinoic acid (lane C), or treated with both retinoic acid and CAMP (lane D). The level of CAT expression shown here parallels the endogenous levels of al(W) chain mRNA in F9 cells treated in this manner. The percent acetylation is shown below the correspond- ing picture of the autoradiograph. Data represent greater than three replications.

TABLE I Characterization of clones containing the cul(IV) promoter-enhancer-

CAT construct Individual clones from the pool of cells containing the stably

integrated cul(IV) promoter-enhancer-CAT construct were obtained. The copy number of the collagen IV promoter-enhancer construct in the individual clones was determined by dot hybridization to cellular DNA. CAT activity of the individual clones was measured with (+) and without (-) differentiation by retinoic acid and CAMP. CAT activity was quantitated by counting radioactive material in the appropriate region of the thin layer chromatograms.

IV construct (Fig. 3, lane C) and had a marked synergistic effect when added with retinoic acid and CAMP (Fig. 3, lane D).

The methylation pattern of the 5’-end of the collagen IV genes was analyzed in both undifferentiated and differentiated F9 cell DNA using the two isoschizomers MspI and HpaII. Both MspI and HpaII recognize the sequence CCGG, although MspI will not cut this sequence when the 5’ cytosine is methylated, whereas HpaII will not cut when either cytosine is methylated. No difference in the methylation pattern be- tween the undifferentiated and differentiated F9 cells was detected using MspI (Fig. 4, lanes 1 and 9). The methylation

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lagen IV promoter-enhancer-CAT construct in F9 teratocar- cinema cell. Pooled cells containing the stably integrated (ul(IV) promoter-enhancer-CAT construct were untreated (lane A), treated with retinoic acid and CAMP (lane B), treated with 5-azacytidine (lane C), or treated with 5-azacytidine plus retinoic acid and CAMP (lane D). The percent acetylation is shown below the corresponding picture of the autoradiograph. The data represent greater than three replications.

Che CAT copy number

F9 - F9 + IIlC%3Se CAT CAT following

activity activity differentiation KB 1 2 3 4 5 6 7 8 9 101112

-fold A 9 1,921 34,617 18 B 48 5,090 30,130 6 E 11 435 15,095 35 F 12 6,987 193,609 28 H 14 1,036 11,554 11 I 48 4,133 317,775 77 K 9 435 331,130 762

integrated collagen IV promoter-enhancer-CAT constructs were also analyzed for CAT gene expression and copy number (Table I). Nine of eleven clones were found to have the CAT gene, whereas only two clones contained just the RSVneo gene. All nine clones showed a marked increase in CAT activity following retinoic acid and dibutyryl-CAMP treat- ment suggesting that the site of integration was not critical for the activation of these constructs. However, the number of integrated copies did not correlate directly with the level of activity observed following retinoic acid and dibutyryl- CAMP treatment.

The difference in transcriptional activity seen between transient and stable transfections could be due to differences in the methylation state in the enhancer and/or promoter. The pooled clones containing the integrated promoter-en- hancer-CAT construct were therefore treated with 5-azacyti- dine, which blocks DNA methylation of replicating cells. 5- Azacytidine treatment increased transcription of the collagen

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FIG. 4. Methylation pattern of the 5’-end of the collagen IV genes during F9 teratocarcinoma cell differentiation. DNA was isolated from both undifferentiated or differentiated F9 cells (14). DNA was digested with the various restriction enzymes, electropho- resed, and blot-hybridized with a probe to the 5’.end of the collagen IV genes as described under “Experimental Procedures.” The size of the fragments are shown in kilobases. Differentiated F9 cell DNA is represented by lanes I-6, whereas undifferentiated F9 cell DNA is represented by lanes 7-12. Lanes 1 and 9 were digested with MapI; lanes 2 and 8 were digested with MspIIHindIII; lanes 3 and 7 were digested with MspI/XhoI; lanes 4 and 12 were digested with HpaII; lanes 5 and I1 were digested with HpaII/HindIII; and lanes 6 and 10 were digested with HpaII/XhoI.

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4842 DNA Methylation and Collagen IV Expression

pattern in differentiated F9 cells cut with MspI and HpaII (Fig. 4, lanes 1 and 4) was not identical. These results indi- cated that the CCGG site of the 1.8-kb fragment (Fig. 4, lone 1) has its internal cytosine methylated but not its external cytosine, since MspI was able to cut and HpaII was unable to cut. Mapping of this site by double digestion with MspI/ Hind111 and MspI/XhoI indicated this site was located 1.7 kb upstream of the start of transcription of the cul(IV) gene corresponding to the third intron of the a2(IV) collagen gene. Interestingly, HpaII digestion showed significant differences between the methylation pattern of the two cell types. The appearance of additional high molecular weight bands of approximately 5.2 and 2.6 kb in the undifferentiated F9 cell DNA cut with HpaII (Fig. 4, lane 12) compared with a single band of 2.3 kb in differentiated F9 cells (Fig. 4, lane 4) suggested that DNA within the collagen IV genes is demeth- ylated following differentiation. Mapping of the two sites which become demethylated in F9 cells following differentia- tion indicated they were located -2.2 and -2.5 kb upstream from the start of transcription of the (ul(IV) collagen gene corresponding to within the third intron of the a2(IV) gene. Thus, a positive correlation emerges between the degree of demethylation of DNA within the 5’-flanking region of the (Al gene and the increased transcriptional activity of the (Al collagen chain gene.

DISCUSSION

DNA methylation appears to be an important mechanism which regulates gene expression. With some exceptions, the transcriptional activation of certain genes correlates with the hypomethylation of certain CpG residues in the regulatory elements of cells which express the genes (see Bird, 1986; Cedar, 1988). Using direct genomic sequencing of regulatory regions, specific CpG residues have been identified which are methylated in nonexpressing cells, but are demethylated when the gene is actively transcribed (Becker et al., 1986; Saluz et al., 1986). Furthermore, 5azacytidine, a potent demethylating agent, has been shown to activate cellular genes in replicating cells by inhibiting the conservative methylation reaction (Charache et al., 1983; Desimone et al., 1982; Jones, 1985; Jones and Taylor, 1980; Ley et al., 1982).

The expression of collagen IV appears to be highly regulated both spatially and temporally during development. The expression of both the al(IV) and (u2(IV) chain genes requires both a bidirectional promoter and a shared enhancer located in the first intron of the al(IV) gene (Burbelo et al., 1988). Transient transfection experiments with a collagen IV pro- moter-enhancer-CAT construct generally reveal a cell-specific expression in cells, which correlates with their ability to produce collagen IV (Killen et al., 1988).’ One exception to this cell-specific expression is found in undifferentiated F9 cells, which show a high level of activity in the transient assay but show very low levels of collagen IV mRNA. In contrast, the integration of the collagen IV promoter-enhancer-CAT constructs in undifferentiated cells extinguishes expression, whereas treatment of these cells with retinoic acid induces a high level of CAT expression. The inactivation of the collagen IV promoter-enhancer-CAT construct by stable integration is not novel, since other foreign genes introduced into both cells and transgenic mice have also been shown to become methylated and inactive upon genomic integration (Jaenisch et al., 1985; Jahner et al., 1982; Palmiter et al., 1982; Steward et al., 1982). Unlike these genes, the stably integrated collagen IV promoter-enhancer-CAT construct was able to be reacti-

’ P. D. Burbelo, unpublished data.

vated upon treatment with retinoic acid, paralleling the in- duction of endogenous levels of collagen IV mRNA. Although 5-azacytidine has many effects including the alteration of differentiation, the activation of the stably integrated collagen IV CAT constructs by this agent suggested that the methyl- ation state might be important in regulating collagen IV genes during differentiation by altering the methylation state of the promoter.

Analysis of the methylation state of the endogenous colla- gen IV genes revealed a higher level of methylation in the 5’- flanking region of the al(IV) collagen gene corresponding to the third intron of the (uZ(IV) collagen gene in nonexpressing, undifferentiated F9 cells compared to expressing, differen- tiated F9 cells. Methylation analysis using a probe covering the active region of the collagen IV enhancer within the first intron of the (ul(IV) revealed no differences between undif- ferentiated and differentiated F9 cells, although sequence analysis revealed very few MspI/HpaII sites (data not shown). Since not all CpGs are accessible to methylation sensitive restriction analysis, it is possible that greater changes in the methylation state of the bidirectional promoter and shared enhancer exist, which cannot be detected with this method- ology. For example, the mouse and human collagen IV bidi- rectional promoter is 23 and 30% CpG-rich, respectively, yet contains no 5’-CCGG-3’ sites for methylation analysis by MspI/HpaII. Sequence analysis of the collagen IV bidirec- tional promoter shows a large dyad of symmetry containing a sequence which resembles a potential retinoic acid-responsive sequence (Umesono et al., 1988). One possibility is that during F9 teratocarcinoma cell differentiation, a retinoic acid-recep- tor complex might have a greater affinity for this binding site than the DNA methylase resulting in the general hypometh- ylation of the DNA in the promoter region. These results suggest that retinoic acid may mediate some of its effects by altering the methylation state. Two other hormones, estrogen (Jost et al., 1984; Saluz et aZ., 1986) and glucocorticoid (Mer- mod et al., 1983; Saluz et al., 1986), have also been shown to both induce gene activation via DNA demethylation.

The mechanism by which the methylation state controls collagen IV gene expression is intriguing. It is possible that DNA methylation may produce inactive chromatin structures (Groudine et al., 1981; Michalowsky and Jones, 1989). For example, in vitro methylation of DNA prior to integration has been shown to block expression (Busslinger et al., 1983; Keshet et al., 1985; Stein et al., 1982; Yisraeli et al., 1986) and product DNase I-insensitive structures characteristic of in- active genes (Keshet et al., 1986). Furthermore, in the case of the human dihydrofolate reductase promoter, the methylation state and chromatin structure of the regulatory DNA was found to be an intrinsic property of the DNA (Shimada et al., 1987). Thus, methylation of the sites within the 5’-flanking region of the al(IV) collagen gene in undifferentiated F9 teratocarcinoma cells may produce transcriptionally inactive chromatin structures at the 5’-end of the collagen IV genes. Alternatively, methylation of certain cis-acting elements within the promoter and/or enhancer, not detected by the methylation sensitive enzymes MspI/EZpaII, may interfere with the binding of some trans-acting factors. As has been recently shown in vitellogenin gene expression, a ubiquitous factor (NH-2) displayed altered binding depending on the methylation state (Feavers et al., 1987). The presence of a CAMP-like responsive element within the collagen IV enhancer3 may also be a potential target of methylation regulated control. CpG methylation of the CAMP-responsive

3P. D. Burkelo, L. Bruggeman, P. Klotman, G. Gabriel, and Y. Yamada, Mol. Cell. Biol., submitted for publication.

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DNA Methylation and Collagen IV Expression 4843

element has been shown to abolish specific factor binding and transcriptional activation (Iguchi-Ariga and Schaffner, 1989). Future experiments using genomic sequencing will be useful in identifying whether sites within the promoter and/or en- hancer of the collagen IV genes show altered methylation during F9 teratocarcinoma cell differentiation.

Acknowledgments-We thank Drs. B. Clement, H. K. Kleinman, P. Klotman, G. R. Martin, and B. Weeks for critical reading of the manuscript.

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P D Burbelo, S Horikoshi and Y YamadaDNA methylation and collagen IV gene expression in F9 teratocarcinoma cells.

1990, 265:4839-4843.J. Biol. Chem. 

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