©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Epigenetic Gene Inactivation Induced by a Cis-acting Methylation Center (*)

(Received for publication, October 18, 1994; and in revised form, November 3, 1994)

Padmaja Mummaneni (1) Kimberly A. Walker (1) Peggy L. Bishop (1) Mitchell S. Turker (1) (2)(§)

From the  (1)Department of Pathology, Markey Cancer Center and the (2)Department of Microbiology and Immunology, University of Kentucky, Lexington, Kentucky 40536

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In this report we test the hypothesis that a cis-acting methylation center can induce epigenetic gene inactivation. The cis-acting element used is an 838-base pair fragment that was shown previously to provide a de novo methylation signal (Mummaneni, P., Bishop, P. L., and Turker, M. S.(1993) J. Biol. Chem. 268, 552-558). Its normal location is approximately 1.3 kilobase pairs upstream of the mouse aprt (adenine phosphoribosyltransferase) gene. To determine if the methylation center could induce inactivation of the aprt gene, a plasmid construct was created in which the methylation center was moved next to the aprt promoter. Transfection experiments demonstrated inactivation of the aprt gene on the hybrid construct. The inactivation event was shown with a Southern blot analysis to correlate with hypermethylation and to be reversible by treatment with 2-deoxy-5`-azacytidine, a demethylating agent. Interestingly, gene inactivation induced by the methylation center required truncation of the aprt promoter. The results demonstrate that epigenetic gene inactivation can be induced by a DNA methylation center.


INTRODUCTION

De novo DNA methylation in mammalian cells is a developmentally controlled process that is involved in X chromosome inactivation (1) and genomic imprinting(2) . Promoter region methylation is associated with transcriptional inactivation of both unique (3) and repetitive sequences(4) . High levels of aberrant DNA methylation have been reported in human cancers for autosomal regions that contain tumor suppressor genes (5) and have been specifically demonstrated for the retinoblastoma (6) and Wilm's tumor (7) gene promoters. Aberrant methylation of the fmr-1 gene has been demonstrated in the fragile X syndrome(8) .

The signals for de novo methylation in mammalian cells have not been identified(9) . One hypothesis is that this event occurs by default throughout most of the genome with specific protection of CpG-rich sequences known as CpG islands(10) . The promoter regions of many mammalian genes are CpG islands, and a sequence that can protect CpG islands from de novo methylation has been reported(11) . An alternative, though not exclusive, hypothesis is that de novo methylation can be signaled by cis-acting methylation centers (12) . Such centers have been described recently in regions upstream of the mouse aprt (adenine phosphoribosyltransferase) gene (13) and the rat alphafetoprotein gene(14) . Methylation of a CpG site in exon 7 of the p53 tumor suppressor gene has also been attributed to a cis-acting sequence(15) . Although specific functions have not been ascribed to methylation centers, they clearly represent DNA regions that have a higher probability for being methylated than neighboring DNA regions. Moreover, methylation of linked DNA fragments can be dependent upon the presence of a methylation center(13, 14, 15) .

We have reported previously that the mouse aprt gene will inactivate spontaneously in P19 embryonal carcinoma cells via an epigenetic event associated with hypermethylation of the CpG-rich promoter region(16) . This is a low frequency event that occurs approximately in one cell in a million. We have also reported that a cis-acting methylation center is located upstream of the mouse aprt gene and that it accounts for a specific methylation pattern that is found in this region(13) . The location of a methylation center approximately 1.3 kbp (^1)upstream of the mouse aprt gene led us to hypothesize that this cis-acting element is responsible for the aberrant inactivation event. In this report we test a plasmid construct, in which the methylation center fragment is juxtaposed with the aprt promoter, for epigenetic gene inactivation. Gene inactivation associated with hypermethylation was found to occur when the plasmid construct was stably transfected into the P19 embryonal carcinoma cell line.


EXPERIMENTAL PROCEDURES

Preparation of Constructs

An 838-bp fragment separating the H1a and H1 HpaII/MspI sites in the pSam6.3 plasmid (see Fig. 1) was isolated with an MspI digest. Based on work from a previous study, this fragment is termed a methylation center (MC)(13) . After filling in the ends of the MC fragment with Klenow enzyme, the fragment was ligated into the SmaI site of the pGem7Z vector (Promega, Madison, WI) multicloning site region to create a plasmid termed pGem7Z-MC. This process converted the H1a and H1 sites to SmaI sites, which contain within them HpaII/MspI sites. To reclone the MC fragment (see below), it was removed from the pGem7Z-MC plasmid with an XhoI and HindIII digest (these restriction sites bracket the SmaI site) and filled in with Klenow enzyme. In a separate set of plasmid constructions, a 1.7-kbp EcoRI/HindIII fragment was removed from the pSam3.1 plasmid (Fig. 1) and cloned into compatible EcoRI and HindIII sites in the multicloning site region of pGem7Z. Unidirectional deletions of 664 and 751 bp were made beginning from the EcoRI site of the 1.7-kbp insert with the Erase-a-Base kit (Promega). The blunt end-prepared MC fragment (see above) was cloned into these deletion constructs to create preliminary versions of 664MC and 751MC (see Fig. 2). DNA sequencing was used to confirm the correct orientation of the MC fragment. Next, a 1.3-kbp HindIII fragment containing exons 3-5 of the aprt gene (Fig. 1) was cloned into the preliminary versions of 664MC and 751MC to restore the aprt gene coding region. These final versions of 664MC and 751MC are shown in Fig. 2. Finally, the MC fragments were removed from the 664MC and 751MC constructs to create the 664 and 751 constructs, respectively (Fig. 2).


Figure 1: The pSam6.3 and pSam3.1 constructs. The closed boxes represent the aprt gene exons, and the bubble figures represent HpaII/MspI restriction sites. Restriction sites shown are EcoRI (E), HindIII (H), PstI (P), and SphI (S). The downstream HindIII site represents a restriction site found in the vector sequence. The EcoRI site in pSam3.1 is an artificial site. The relative position of this site on pSam6.3 is shown with E. The locations of the M1 and M3 fragments are shown below the pSam3.1 construct, and the location of the MC fragment is shown on the pSam6.3 construct. The 1.8- and 3.3-kbp lines represent hybridization bands for the D3 cell line that are discussed in the text and shown in Fig. 4. The broken lines represent bacterial vector DNA.




Figure 2: Deletion constructs used in this study. The methods used to make these constructs are described under ``Experimental Procedures.'' The large closed boxes represent the aprt gene exons, and the open box represents the MC fragment. The small closed boxes represent the Sp1 binding sites that comprise the aprt promoter. These binding sites are not drawn to scale. The M1 fragment was isolated from pSam3.1 with an EcoRI/MspI digest as shown in Fig. 1. The bubble figures represent some of the HpaII/MspI sites found on the constructs (see Fig. 1for sites that are not shown). The restriction site shown (P) is PstI. The 2.1- and 1.8-kbp lines represent hybridization bands for the transfected 751MC construct (see text and Fig. 4). The broken lines represent bacterial vector DNA.




Figure 4: Hypermethylation accompanies inactivation of the 751MC construct in transfectants. DNA preparations were digested with PstI and HpaII, separated on a 1.2% agarose gel by electrophoresis, and blot hybridized to P-labeled M3 probe. The approximate molecular sizes of the hybridization bands are indicated. See text for details.



Transfection Experiments

The conditions for transfection of DNA into cultured embryonal carcinoma cells via electroporation were as described previously(17) . The plasmid constructs were linearized with ScaI prior to transfection. A single ScaI site is present in the vector DNA. The recipient cell line used for these experiments, DelTG3, was derived from the P19 embryonal carcinoma cell line. The DelTG3 cell line lacks both aprt alleles and their respective upstream regions.

Southern Blot Analysis

The Southern blot analysis was performed as described previously(18) . The copy number of integrated constructs for each cell line was determined by digesting genomic DNA with HindIII and blot hybridizing to the pSam3.1 insert. With this method, each integrated construct yields a 1.3-kbp hybridization band representing the fragment separating the two HindIII sites in the constructs used (Fig. 1) and a second hybridization band of variable size representing the region upstream of the intronic HindIII site. Each independently integrated construct gives a single variable hybridization band allowing a determination of the number of integrants/transfectant (i.e. copy number).

Adenine Phosphoribosyltransferase Assay and Autoradiography of [^3H]Adenine Accumulation

These procedures were performed as described previously(19) .

Accession Number

The sequence for the region upstream of the mouse aprt gene has the accession number l15342.


RESULTS

To determine if the MC fragment could induce gene inactivation, the 664MC and 751MC constructs were made (Fig. 2). The 664MC construct contains the MC fragment positioned 5` to the four Sp1 binding sites that comprise the aprt promoter. In the 751MC construct, two Sp1 binding sites were deleted, and the MC fragment was inserted 5` to the two remaining Sp1 binding sites. It has been shown by others that deletion of the two 5` Sp1 binding sites does not affect aprt expression(20) . The MC fragments were then removed from 664MC and 751MC to create 664 and 751, respectively (Fig. 2).

The ability of each of the four constructs to express the aprt gene was tested by transfecting them into the DelTG3 cells and selecting for stable aprt expression with medium containing azaserine and adenine (AzA medium)(19) . Fig. 3demonstrates little difference in the transfection efficiencies for the 664, 664MC, and 751 constructs; a reduction of approximately 20% was observed for the 751 construct. In contrast, a reduction in the transfection efficiency of approximately 4-fold was found for the 751MC construct as compared with the 751 construct. This difference in transfection efficiencies for the 751 and 751MC constructs was observed consistently in a total of seven paired tests (p value = 0.0002). The significant reduction in transfection efficiency for the 751MC construct suggested that it was frequently inactivated when transfected into the DelTG3 cells. To determine if the difference in the behavior of the 664MC and 751MC constructs was due to a distance effect, a 91-bp insert was placed into 751MC between the Sp1 binding sites and the methylation center. This insert is the multiple cloning site from the pGem7Z vector used in these experiments (see ``Experimental Procedures''). Fig. 3demonstrates that the transfection efficiency for this new construct, termed 751-91-MC (construct not shown), was essentially the same as that seen for 751MC. As an additional control, the MC fragment was replaced by the M1 fragment, which is located at the 5` end of pSam3.1 (Fig. 1) to create 751M1 (Fig. 2). The transfection efficiency for the 751M1 construct was essentially equal to that observed for the 751 construct (Fig. 3). This result suggests that a DNA fragment taken from the region between the MC fragment and the aprt promoter did not affect expression of the aprt gene.


Figure 3: Transfection efficiencies for the deletion constructs. 5 µgs of plasmid DNA were linearized with ScaI and transfected into DelTG3 cells by electroporation. Expression of the plasmid DNA was selected with AzA medium. Each result represents the average of two transfections.



To directly test for inactivation of the 751MC construct in the DelTG3 cells, a second set of transfections was performed in which the 664MC, 751, and 751MC constructs were each co-transfected with the bacterial neo gene. G418-resistant transfectants were isolated and analyzed with a Southern blot analysis to identify those transfectants that had integrated intact copies of the 664MC, 751, or 751MC constructs. The copy number ranged from 1 to 3 integrants/transfectant. As shown in Table 1, 3 of 3 transfectants containing the 664MC construct and 6 of 6 transfectants containing the 751 construct were found to express significant levels of the adenine phosphoribosyltransferase enzyme. Identical results were found for the pSam3.1 and pSam6.3 constructs (Fig. 1) when they were co-transfected with the neo gene (data not shown). In contrast, 5 of 7 transfectants containing 1-3 copies of the 751MC construct failed to express a detectable amount of adenine phosphoribosyltransferase enzyme. A sixth transfectant, 751MC-119 (Table 1), expressed only a low level of the enzyme. This experiment confirmed that the aprt gene in the 751MC construct was inactivated frequently when transfected into the DelTG3 cells and that inactivation was due to the presence of the MC fragment.



Gene inactivation associated with hypermethylation can be reversed by treating cells with 2-deoxy-5`-azacytidine (5aCdr)(21) . In previous work we have shown that 5aCdr caused high frequency reactivation of the endogenous mouse aprt gene when it was hypermethylated. A nonexpressed aprt allele that was not hypermethylated was unaffected by the 5aCdr treatment(22) . To determine if inactivation of the transfected 751MC construct was associated with hypermethylation, two of the 751MC transfectants containing nonexpressed constructs were treated with 5aCdr and analyzed for reacquisition of adenine phosphoribosyltransferase expression by monitoring [^3H]adenine incorporation with autoradiography. Table 2demonstrates that approximately 40-50% of the treated cells incorporated [^3H]adenine as compared with 0% of the untreated cells. In an attempt to isolate cells containing a reactivated 751MC construct for further analysis, 5aCdr-treated cells were plated in the presence of AzA medium. It was possible to expand AzA-selected clones from the 5aCdr-treated 751MC-302 transfectant for further analysis. The adenine phosphoribosyltransferase specific activities for two of these reactivants, 751MC-302-R2 and -R4, are shown in Table 1. Autoradiography showed that all 751MC-302-R4 cells had incorporated [^3H]adenine (Table 2).



To confirm hypermethylation of the inactive 751MC construct in transfectants at the molecular level, a Southern blot analysis was performed on DNA preparations isolated from some of the transfected cell lines listed in Table 1. The DNA preparations were digested with HpaII and PstI. One PstI site is located in the aprt gene, and a second site is located in the MC fragment (Fig. 2). These PstI sites are separated by a distance of 2.1 kbp on the 751MC construct. A 120-bp CpG island fragment ( Fig. 1and Fig. 2, M3), which separates the H3 and H4 HpaII/MspI sites, was used as the probe. Fig. 4shows that DNA preparations from transfectants expressing aprt initially (751-12, 751MC-105) and those reactivated with 5aCdr (751MC-302-R2 and -R4) were unmethylated at the H3 and H4 sites. This was shown by the presence of a 120-bp hybridization band in the HpaII digestion lanes and the absence of additional hybridization bands. In contrast, the 120-bp hybridization band was markedly reduced in the DNA preparations from nonexpressing 751MC transfectants (751MC-301 and -302), and strong hybridization bands of 2.1 and 1.8 kbp were noted. These hybridization bands were also noted in the DNA preparation from the 751MC-119 cell line, but in this case the hybridization signals were weak. The 2.1-kbp hybridization band results from methylation of the H1-H11 HpaII sites, and the 1.8-kbp hybridization band results from methylation of the H1-H9 HpaII/MspI sites (Fig. 2). As a control, a DNA preparation from the D3 cell line was examined (Fig. 4). The D3 cell line contains a single inactive copy of the endogenous aprt gene(22) . The 3.3-kbp hybridization band observed for this cell line represents methylation of the H1-H11 HpaII/MspI sites for the endogenous aprt gene (Fig. 1). The 1.8-kbp hybridization band contains methylated H1-H6 or H7 HpaII/MspI sites.


DISCUSSION

We have demonstrated that a cis-acting methylation center can induce inactivation of a linked mammalian gene (i.e. aprt) that contains a CpG-rich promoter. To the best of our knowledge this is the first such demonstration, and it provides a model for epigenetic gene inactivation associated with de novo DNA methylation. We expected the mouse aprt gene promoter to be sensitive to methylation-associated gene inactivation because the endogenous gene can inactivate spontaneously in an event associated with hypermethylation(16) . Although the aprt promoter is comprised solely of Sp1 binding sites, which are not inhibited by DNA methylation in vitro(23) , in vivo footprinting has shown that Sp1 binding does not occur at the four Sp1 binding sites in the D3 cell line(24) . This cell line contains a hypermethylated aprt gene (Fig. 4). The lack of Sp1 binding to the aprt promoter in the D3 cell line suggests that methylation of the promoter region, rather than the Sp1 binding sites themselves, is responsible for the inactivation events, perhaps through alteration of the chromatin structure. We note that the aprt promoter region in the D3 cells is in a closed chromatin conformation(22) . Important additional experiments will be to compare and contrast the ability of the methylation center used in this study to inactivate methylation-sensitive and -insensitive promoters(25) .

It will also be important to determine why the inactivation event was blocked when the intact aprt promoter (i.e. with four Sp1 binding sites) was present. The possibility that inactivation of the 664MC construct does not occur because the methylation center is positioned 87 bp from the two Sp1 binding sites found in the 751MC construct appears unlikely. This statement is based on the observation that a 91-bp insert placed between the methylation center and the two Sp1 binding sites in the 751MC construct did not increase the transfection efficiency (Fig. 3). Therefore, the additional 87-bp fragment that is present in 664MC may block the inactivation event in a sequence-specific fashion. This fragment contains the two 5` Sp1 binding sites. We speculate that the presence of four Sp1 binding sites is necessary to block gene inactivation even though only two of these sites are required for high level gene expression. If so, the aprt promoter may have evolved to contain four Sp1 binding sites as a mechanism to block the effect of the upstream methylation center. Consistent with this notion are recent reports that Sp1 binding sites in both the mouse (26) and hamster (27) aprt gene promoters are capable of blocking methylation of their respective CpG islands. The approximate location of the mouse aprt CpG island can be seen in Fig. 1as a cluster of HpaII/MspI sites (H3-H8). We note that in previous work we were able to methylate these sites by moving the CpG island into the methylation center region(13) . In this case only a single Sp1 binding site remained attached to the CpG island. The work presented here has shown that the presence of two Sp1 binding sites is also insufficient to block the methylation signal indicating that at least three Sp1 binding sites are necessary. Such a result was shown by Macleod et al.(26) , although they did not consider a nonconsensus Sp1 binding site that was also on the fragment they tested. This is the 5`-most Sp1 binding site shown in Fig. 2for the 664 and 664MC constructs. We consider this as an Sp1 binding site in our analysis based on in vitro(20) and in vivo(24) footprinting data. The latter data, however, indicate that it is a relatively weak binding site. Whether this binding site plays a role in blocking the methylation signal remains to be determined, although it clearly cannot block the signal on its own(26) .

In conclusion, we have shown that juxtaposition of a mammalian methylation center with a CpG-rich gene promoter can induce epigenetic gene inactivation. This result offers a potential molecular explanation for physiological and/or aberrant epigenetic gene inactivation in mammalian cells. The ability to induce epigenetic gene inactivation intracellularly at a very high frequency by providing a cis-acting methylation signal may provide a convenient system to study methylation-associated inactivation for a variety of mammalian genes.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants AG08199 and CA56383. 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.

§
To whom reprint requests should be addressed. Tel.: 606-257-4951; Fax: 606-323-2094.

(^1)
The abbreviations used are: kbp, kilobase pair(s); bp, base pair(s); MC, methylation center; 5aCdr, 2-deoxy-5`-azacytidine.


ACKNOWLEDGEMENTS

We thank M. Pieretti, N. Khattar, and K. Albers for a critical reading of this manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.