Reactivation of the methylation-inhibited late E2A promoter of adenovirus type 2 by a strong enhancer of human cytomegalovirus

VIROLOGY

166, 166-174

(1988)

Reactivation of the Methylation-Inhibited Type 2 by a Strong Enhancer

Late E2A Promoter of Adenovirus of Human Cytomegalovirus

DAGMAR KNEBEL-MijRSDORF,* SABINE ACHTEN,* KLAUS-DIETER LANGNER,*f’ RijDlGER RijGER,V BERNHARD FLECKENSTEIN,t AND WALTER DOERFLER**3 *Institute of Genetics, University of Cologne, Cologne, Germany, and tlnstitut fiir Klinische and Molekulare Virologie der Universitgt Erlangen-Nijmberg, Erlangen, Germany Received March 18, 1988; accepted May 23, 1988 Promoter inactivation by sequence-specific methylation was demonstrated by using a construct which contained the late E2A promoter of adenovirus type 2 (Ad2) DNA and the prokaryotic gene for chloramphenicol acetyltrsnsferase (CAT) as indicator. After the in vitro methylation of 5’-CCGG-3’ sequences at positions -215, f6, and f24 relative to the cap site of the promoter, the construct was inactive upon transfection into mammalian cells. The same pAd2E2ALCAT construct was active in the unmethylated form. Promoter inactivation could be overcome when the strong immediate early enhancer of human cytomegalovirus DNA, which lacked 5’-CCGG-3’ sites, was inserted into the construct either in a position immediately antecedent to the promoter or in a location several thousand nucleotides remote from it. Reactivation of the 5’-CCGG-3’ methylated pAd2E2AL-CAT construct entailed initiation of transcription at the authentic cap site of the late E2A promoter and maintenance of methylation at least during the duration of the transient expression experiment. Reactivation of the methylated late E2A promoter had also been demonstrated by the transactivating 289 amino acid protein which was encoded in the ElA region of adenoviruses (B. Weisshaar et a/., 1988,J. Mol. Biol. 202, 255-270). Thus there are several ways in which a methylated and silenced promoter can be reactivated 0 1998Academic Press, Inc. in mammalian cells.

dimensional structure of an operative promoter has been elucidated. In the El A promoter of adenovirus type 12 (Ad1 2) DNA, methylation of 5’-CCGG-3’ or 5’-GCGC-3’ sequences upstream of the TATA signal inactivates or inhibits the promoter (Kruczek and Doerfler, 1983). The methylation of other nucleotide sequences in the ElA promoter distributed over a region of about 500 nucleotides can either inactivate this promoter, even when IV’-methyldeoxyadenosine has been introduced, or remain without effect depending on the specific sequences which have been modified (Knebel and Doerfler, 1986). The late E2A promoter of adenovirus type 2 (Ad2) DNA has been extensively studied for inactivation by methylation. The methylation of three 5’-CCGG3’ sequences inactivates or reduces the activity of this promoter in transient expression experiments both after microinjection into Xenopus laevis oocytes (Langner eta/., 1984) and upon transfection into mammalian cells (Langner et al., 1986). The introduction of eleven 5-methyldeoxycytidine residues at the same sequences in the 3’structural region of the E2A gene has no effect on its activity. The late E2A promoter is also inactivated by methylation of the three 5’-CCGG-3’sequences when it has subsequently been genomically fixed by integration together with an indicator gene in the DNA of mammalian cells (Mtiller and Doerfler, 1987).

INTRODUCTION Results from numerous eukaryotic systems lend support to the notion that sequence-specific promoter methylations cause the inactivation or inhibition of eukaryotic promoters (Doerfler, 1981, 1983; Doerfler et al., 1985, 1988, for reviews). The promoter sequences, whose methylation is decisive in the shut-down of promoter function, are apparently not localized at unique and highly characteristic distances from landmark sequences in the promoter, like the TATA signal or the cap site. The essential cytidine residues have to be determined experimentally for each promoter. A functional promoter probably requires a certain DNA structure rather than a unique nucleotide sequence. This complex structure is facilitated by multiple DNA-protein and protein-protein interactions. It is conceivable that promoter methylations interfere in an as yet unknown manner with some of these interactions, and in different promoters specific proteins interact with nucleotide sequences which are distinct for each promoter. A functionally meaningful pattern of promoter methylations associated with promoter inactivations may therefore become discernible only after the three’ Present address: Behring Werke, Marburg, Germany. * Present address: Boehringer, Tutzing, Germany. 3 To whom requests for reprints should be addressed. 0042-6822188

$3.00

Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

166

HCMV

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ABROGATES

Similar conclusions on the methylation sensitivity of promoters have been deduced from results on the yglobin promoter (Busslinger et a/., 1983; Murray and Grosveld, 1987) as well as on the thymidine kinase gene and adenosylphosphoribosyltransferase gene promoters (Keshet et a/., 1985). For the thymidine kinase gene a methylation-sensitive site has also been detected in the 3’structural part of the gene. This latter finding further strengthens the notion that complex arrangements of nucleotide sequences govern promoter activity. Methylations at certain sequences that flank a gene may or may not influence promoter activity depending on the regulatory significance of these sequences. Promoter inactivation by sequence-specific methylation seems to function as a reversible inhibitory signal. A flexible mechanism, that is associated with long-term gene inactivation, may permit reactivation either by double-stranded demethylation or by the transient release of the transcriptional block. Such a release has recently been demonstrated (Langner et al., 1986; Weisshaar et a/., 1988). The inhibitory function of the three methylated sequences in the late E2A promoter can be abrogated by the simultaneous expression of the 289 amino acid polypeptide which is encoded in the ElA region of the Ad2 genome. This protein is a well characterized trans-activator of gene activity and reactivates the methylated late E2A promoter (Langner eta/,, 1986) without demethylating both strands and by allowing the reinitiation of transcription at the authentic cap site of the late E2A promoter (Weisshaar et a/., 1988). In the present report, another mechanism for promoter reactivation will be investigated. A strong transcription enhancer has been identified upstream of the major immediate early (IE-1) gene of human cytomegalovirus (HCMV) (Boshart eta/., 1985). Enhancers are &-acting, eukaryotic genetic signals which stimulate the expression of neighboring genes independent of orientation and precise location relative to the gene. Enhancers have first been detected in viral genomes and later close to many viral and nonviral eukaryotic genes (for review, Weiher et a/., 1983). The HCMV enhancer which extends up to about -530 nucleotides upstream of the cognate promoters consists of a complex array of nonabutting repeat sequences that constitute protein binding sites (Ghazal et a/., 1987). This enhancer has little cell type or species preference and is more active than other viral or cellular enhancers previously described (Boshart et a/., 1985; Foecking and Hofstetter, 1986). Since detailed information on the methylation sensitivity of the late E2A promoter of Ad2 is available (Vardimon et al., 1982; Langner et a/., 1984, 1986; Mijller and Doerfler, 1987; Hoeveler and Doerfler, 1987;

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Weisshaar et al., 1988), we have used this promoter in combination with an indicator gene, the prokaryotic gene for chloramphenicol acetyltransferase (CAT), in the plasmid pSVO-CAT (Gorman et a/., 1982). In the present report the HCMV enhancer will be shown to override the incapacitating effect of site-specific methylation in the late E2A promoter of Ad2 DNA and restore its activity even though it has remained methylated during reactivation. Transcription is reinitiated inside the methylated late E2A promoter at the authentic cap site in these experiments. MATERIALS

AND METHODS

Most of the methods employed in this study belong to the standard repertoire of molecular biology and have been described elsewhere. HeLa cells were cultured as monolayers in plastic flasks in Dulbecco’s modified medium containing 10% fetal calf serum. Cells were transfected with plasmid constructs by the Ca*+ phosphate method (Graham and van der Eb, 1973). Activity assays for CAT were performed as described (Gorman et a/., 1982; Kruczek and Doerfler, 1983). Other standard techniques of molecular biology were employed in Southern blotting (Southern, 1975) nick-translation (Rigby et al., 1977) DNA-DNA and DNA-RNA hybridization experiments (Wahl et a/., 1979) and the determination of nucleotide sequences (Sanger et a/., 1977). In some experiments, nucleotide sequences were determined in plasmid DNA preparations by using oligodeoxynucleotide primers (Wallace eta/., 1981). Construction of pAd2E2AL-CAT containing the HCMV enhancer locations

plasmids in different

In previously published work, different parts of the late E2A promoter were used, i.e., the HindIll-EcoRI (nucleotides 26,369 to 25,633 in the Ad2 DNA sequence, Roberts eta/., 1986), the HindIll-Kpnl (nucleotides 26,369 to 25,881) or the HindIll-Asp718 fragment. The Kpnl and Asp71 8 sites were identical, except that the two endonucleases cleaved the 5’-GGTACC-3’ sequence differently. The HCMV enhancer was cloned into the plasmid containing the HindlllAsp71 8 fragment of the late E2A promoter of Ad2 DNA and the CAT gene. The HCMV sequence was inserted in one instance in a position immediately preceding the late E2A promoter, in another construction into the only BarnHI site in the vector at a location quite remote from the late E2A promoter. Construction maps are illustrated in Fig. 1.

KNEBEL-MCjRSDORF

168

ET AL m

E2A promoter

m

KMV

m

CAT-gene

enhancer

Hindlll

hnlll \

/

pAd2 EPAL(Hind-Asp)-CAT

pSVO-CAT

c\

Hindlll

\

E2A promoter

E2A promoter

FIG. 1. Schemes which detail construction plans of the late EZA promoter-HCMV enhancer-CAT constructs, pAd2E2AL-HCMV-CAT pAd2E2AL-HCMV(Bam)-CAT. Experimental details are described in the text. The /-/pall sites in the late E2A promoter are designated byo.

(i) The HCMV enhancer in the immediate vicinity of the late E2A promoter (Fig. 1): pAd2E2AL-HCMV-CAT. The pAd2-(Hind-Eco) construct carrying the EcoRIHindIll promoter fragment was linearized with HindIll, and the staggered termini were filled in with Klenow polymerase (Klenow et al., 1971). The termini were furnished with BamHl linkers, and the DNA was subsequently cut with BarnHI and EcoRI. The generated

and

BarnHI-EcoRI fragment of the late E2A promoter was purified by gel electrophoresis and ligated into the corresponding sites in the pUC8 vector which had been previously cut with ,!!coRI and BarnHI. The new construct was designated pAL5 (Fig. 1). Plasmid pRR46/2 contained an HCMV enhancer fragment of 311 bp which corresponded to the IE-1 upstream sequence between nucleotides -222 and -532 entirely compris-

HCMV

ENHANCER

ABROGATES

ing the HCMV-specific sequence of recombinant virus C4 (Boshart et al., 1985). The enhancer subfragment was shortened with nuclease Ba/31 and was cloned into plasmid pUC8 using synthetic HindIll (upstream) and BamHl (downstream) linkers. As indicated in Fig. 1, pRR4612, the addition of a BamHl linkerto nucleotide -222 of the HCMV enhancer fragment, happened to generate a new /-/pall site which was ascertained by resequencing this particular segment. By using BamHl linkers, the 311-bp fragment was ligated into the BamHl site of plasmid pAL5 creating the construct pBEB (Fig. 1). In this construct, one BamHl site had become deleted during the cloning procedure. Finally, pBEB DNA was cut with Asp7 18, the recessed termini were filled in with Klenow polymerase, and HindIll linkers were added. HindIll cleavage then excised the HCMV enhancer-late E2A promoter assembly which was ligated into the HindIll site of the pSVO-CAT construct The final product was termed pAd2E2ALHCMV-CAT (Fig. 1). (ii) The 311 -bp HCMV enhancer fragment was also ligated into the BamHl site of the pAd2E2AL-(HindAsp)-CAT construct (Fig. 1). The pRR46/2 plasmid was cut with HindIll, the termini were completed with Klenow polymerase, BamHl linkers were added, and the HCMV enhancer was completely excised with BamHI. The fragment was ligated into the BamHl site of the pAd2E2AL-(Hind-Asp)-CAT construct. Both orientations of the enhancer were generated. The new construct was named pAd2E2AL-HCMV(Bam)-CAT (Fig. 1). The plasmid preparations were propagated in Escherichiacolistrain HBl Ol/Xand purified by standard dyebuoyant density gradient centrifugation. Isolation

of RNA from transfected

cells

HeLa cells were transfected with 10 pg of the enhancer constructs. At 45-48 hr after transfection, total RNA was isolated by the guanidinium isothiocyanate method (Chirgwin et al., 1979). Sl protection

assay

The assay was performed according to the method of Berk and Sharp (1977). RNA pretreated with RNasefree DNase (Boehringer) was hybridized to a singlestranded DNA probe which was prepared as follows. An amount of 2 pg of the single-stranded M 13 DNA clone containing the HindIll-Kpnl fragment of the late EZA promoter of Ad2 DNA (gift of Ursula Lichtenberg) was incubated with 5 pmol of the standard M 13 sequencing primer (17-mer, Biolabs) in 10 PI of 7 mM Tris-HCI, pH 7.7, 7 mlVI MgCI,, 50 mn/r NaCl for 5 min at 90” and was allowed to cool to room temperature.

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After the addition of 1 ~1 of 10 ml\/l dlTP, 1 ~1of 10 mM dGTP, 1 ~1of 10 mn/l dCTP, 4 ~1of [(u-32P]dATP (40 &i, 300 Ci/mmol), and 5 units of Klenow polymerase in 10 ~1 of 7 mM Tris-HCI, pH 7.5, 7 mn/r MgC&, 50 ml\/l NaCI, 100 mlVI dithiothreitol, the primer-template mixture was incubated for 20 min at 37”. Subsequently, 1 ~1 of 10 ml\/l dATP was added, and incubation was continued for 20 min at 37”. Incubation was extended for another 45 min after the addition of 20 units of Pvull. The labeled single-stranded DNA fragment was isolated after separation on a 5% polyacrylamide gel containing 8 n/l urea. The RNA (5 pg) was annealed to a 404-bp DNA probe (Fig. 3a) (100,000 cpm) at 52” and subsequently treated with 800 units of Sl nuclease (Boehringer) in 280 mM NaCI, 50 mMsodium acetate, pH 4.6,4.5 mn/l ZnSO, for 1 hr at 37”. After heating, the hybrids were analyzed by electrophoresis on a 6% polyacrylamide gel containing 8 M urea. The 71- and 69-bp signals (cf. Fig. 3b) were due to the late E2A DNA promoter fragment protected by RNA. In a further set of experiments, RNA was hybridized to a double-stranded DNA probe, terminally labeled by [T-~‘P]ATP and polynucleotide kinase. The RNA (5 pg) was annealed to the 381 nucleotide Asp718-Pvull fragment (Fig. 3a) (6000 cpm) at 52” and subsequently treated with 200 units of Sl nuclease in 200 mltl NaCI, 30 mM sodium acetate, pH 4.6, 1 mM ZnSO, for 30 min at 30”. RESULTS Experimental

plan

The late E2A promoter of Ad2 DNA is inactivated or inhibited by the methylation of three specific 5’-CCGG3’ sequences at positions -2 15, +6, and +24 relative to the cap site of the promoter. The effect of the strong enhancer from HCMV (Boshart et a/., 1985) was tested in the construct that harbors the 5’-CCGG-3’ methylated late E2A promoter. In one construct, this enhancer was placed in a position immediately preceding the E2A promoter, in another construct in a more remote location, i.e., the BamHl site in the plasmid vector sequence of the construct (Fig. 1). The HCMV enhancer lacked a 5’-CCGG-3’ site in its essential sequences. The plasmid part of the construct contained several Hpall sites including the one that had been haphazardly generated at the site of transition between plasmid and HCMV sequences (see nucleotide sequence in pRR4612, Fig. 1). The two different enhancer constructs in the 5’-CCGG-3’ methylated or the unmethylated form of the pAd2E2AL-CAT construct were separately transfected into human HeLa cells growing in culture and CAT activities were determined in cell

KNEBEL-MijRSDORF

170

% acetylated Cpl pAd2 ESAL(Hind-Asp)-CAT

23.6

pAd2 ESAL(Hind-Asp)-CAT, Hpa II methylated

5.1

pAd2 EZAL-HCMV-CAT

ET AL.

extracts which were prepared 45 to 48 hr after transfection. Reaction kinetics of extracts prepared from cells which had been transfected with the unmethylated or the methylated construct were compared. The continued presence of methylated 5’-CCGG-3’ sequences was ascertained by restriction analysis and Southern blotting experiments. Total RNAs were extracted from cells which had been transfected with the unmethylated or the 5’-CCGG-3’ methylated construct. The E2A-specific RNA sequences were mapped at the appropriate initiation site in the late E2A promoter. Reactivation of the 5’-CCGG-3’ methylated E2A promoter in HeLa ceils by the HCMV enhancer immediately preceding or more remote from the E2A promoter

________

-_------

________-

--

a]lZh *

Incubation

time (min.)

FIG. 2. Reactivation of the 5’.CCGG-3’ methylated constructs pAd2E2AL-HCMV-CAT and pAd2E2AL-HCMV(Bam)-CAT. (a) Basic CATexperiment. HeLa cells were transfected with 10 pg of CATconstructs as indicated. Extracts of the transfected cells were prepared 48 hr later and assayed for CAT activity by standard procedures (60 min incubation). The CAT construct carried the HindIll-Asp71 8 fragment of the late E2A promoter (cf. Materials and Methods) as an unmethylated or a 5’-CCGG-3’ methylated promoter. The autoradiogram demonstrates the conversion of ‘Wabeled chloramphenicol (CAM) to the acetylated forms. The results were quantitated by determining the 14C activities in different locations on the thin-layer silica plate by scintillation counting. The percentages of acetylated CAM are indicated in the ordinate on the right. (b, c) Reaction kinetics during a 40-min incubation period. Experimental procedures are described in (a), except that constructs were transfected which carried

In a series of experiments, the 5’-CCGG-3’ methylated or unmethylated pAd2E2AL(Hind-Asp)-CAT construct was transfected into HeLa cells. This construct contained the HCMV enhancer in the two locations described above (Fig. 1) or was devoid of the HCMV enhancer sequence. The HCMV enhancer in the BarnHI site of the plasmid part of the construct was tested in two different orientations. The results of an exemplary set of CAT experiments with extracts prepared at 48 hr after transfection are presented in Fig. 2a. It was apparent that the pAd2E2AL-(Hind-Asp)-CAT construct without the HCMV enhancer had significant activity in HeLa cells (23.6% conversion in the experiment shown) which was reduced to 5.1% conversion when the construct was 5’-CCGG-3’ methylated (cf. also Langner et al., 1986). The activity of the unmethylated constructs was dramatically increased in the presence of the HCMV enhancer as demonstrated in the kinetic experiments. HeLa cell extracts were prepared at 12, 24, or 48 hr after transfection with different constructs in the methylated or unmethylated configuration, and the kinetics of the conversion of 14C-labeled chloramphenicol to acetylated forms were determined. (Figs. 2b, c). The constructs carrying the HCMV enhancer in the BarnHI site in either orientation consistently had similar activity levels. Another striking result was apparent from the data shown in Figs. 2b, c. The methylated constructs also exhibited significant activity levels at different times after transfection. These activity levels

the pAd2E2AL-HCMV-CAT (b) or the pAd2E2AL-HCMV(Bam)-CAT (c)enhancers as indicated. Percentages of acetylated CAM were determined as described in (a). Extracts of cells transfected with the unmethylated (-) or the methylated (---) construct were prepared 12, 24, and 48 hr after transfection as designated and CAT reaction kinetics were determined for each time point.

HCMV pAd2 ESAL- (Hind-Asp)-

8

E2A

pBR322 -s--------

A

ENHANCER

ABROGATES

CAT

IFbb *

A

PVUII I---.

Asp710 404nt 69 resp. 71 nt 301 nt

L @

404

12

34

0

73 resp. 75 nt

1234567

-

FIG. 3. Initiation of transcription at the authentic late E2A cap site in the fully methylated constructs containing the HCMV enhancer sequences. Analyses of 32P-labeled promoter DNAfragments which were protected from degradation by Sl nuclease after annealing to RNA preparations as described under (b) and (c). (a) Map of the late E2A promoter fragment. The single-stranded DNA fragment of 404 bp was prepared as described under Materials and Methods. This fragment was used for the Sl protection experiment shown in (b). In the experiment shown in (c) the double-stranded Asp718-Pvull fragment labeled at the Asp7 18 site by [-r-32P]ATP and polynucleotide kinase was used. The cap sites are indicated by -. and the three 5’-CCGG-3’ sequences by o. (b) In the annealing experiments for DNA protection, the following RNA preparations, which were isolated 48 hr after transfection, were employed. (3) RNA from HeLa cells transfected with the unmethylated pAd2E2AL-HCMV(Bam)CAT construct or (4) with the 5’~CCGG-3’ methylated construct. (1) The single stranded 404-bp probe, (2) the terminally 32P-labeled, denatured //pall fragments of pUC8 DNA were coelectrophoresed as size markers. The 404.bp fragment and the protected 69/7 1-bp fragments are indicated by arrows. (c) In the annealing experiments for DNA protection, the following RNA preparations, which were isolated 48 hr after transfection, were employed: (2) RNA from HeLa cells transfected with the unmethylated pAd2E2AL-HCMV(Bam)CAT construct, (3) RNA from cells transfected with the construct used in (2) that was 5’-CCGG-3’ methylated, (4) RNA from HeLa cells transfected with the unmethylated pAd2E2AL-HCMV-CAT construct, (5) RNA from cells transfected with the construct used in (4) that was 5’.CCGG-3’ methylated, and (6) tRNA were hybridized to the double-stranded probe. In (1, 7) marker DNAs [see lane (2) in (b)] were coelectrophoresed. In the experiments 5 pg of RNA and 6000 cpm of the double-stranded DNA fragment (a) were annealed at 52”. The 381-bp fragment used in the RNA annealing reaction and the protected 73/75-bp fragments are indicated. The difference in the sizes of the protected fragments in (b) and (c)was due to the different sizes of the DNAfragments used for protection. The high molecularweight bands in lanes 2 to 6 are due to reannealed DNA. The RNA

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were only slightly lower, if at all, than those of the unmethylated constructs, in particular at late times of incubation when the reaction approached saturation (Figs. 2b, c). High levels of activity were obtained with the methylated pAd2E2AL-CAT DNA when the HCMV enhancer was located in a position immediately preceding that of the promoter in the construct (Fig. 2b). With the HCMV enhancer inserted in the BarnHI site, the activity reached similar levels (Fig. 2~). These data were reproduced eight times in independent experiments which yielded very similar results. Moreover, the efficiency of transfection was assessed in different experiments. At 48 hr after transfection with the late E2A promoter-CAT gene construct, the total intranuclear DNA was extracted and cleaved with Hoall or Nlspl, and the fragments were separated by gel electrophoresis and blotted. The construct-specific fragments were visualized by hybridization with 32P-labeled construct probe. The data, as exemplified in Fig. 4, indicated that the amounts of DNA introduced into HeLa cells in different transfection experiments were comparable. The results were interpreted to indicate that the HCMV enhancer increased the activity of the pAd2E2ALCAT construct in HeLa cells very significantly and simultaneously reduced or even eliminated the inhibitory effect of 5’-CCGG-3’ methylation in this construct. The question arose whether, in the methylated constructs, the authentic site of transcription initiation in the late E2A promoter was utilized or whether alternate initiation sites were activated by the enhancer sequence. Sl nuclease analyses of RNAs synthesized in HeLa cells on 5’-CCGG-3’ methylated or unmethylated pAd2E2AL-(Hind-Asp)-CAT constructs containing the HCMV enhancer In the 5’.CCGG-3’ methylated construct containing the HCMV enhancer in the two different locations (Fig. l), it needed to be ascertained whether transcription was initiated at the authentic late E2A cap site. The HindIll-Kpnl fragment from the late E2A promoter of Ad2 DNA as an Ml 3 clone was used as singlestranded hybridization probe (Fig. 3b). RNA, which was isolated from HeLa cells at 48 hr after transfection with different constructs, was annealed to DNA fragments. The hybrids were treated with Sl nuclease (cf. Materials and Methods), and the protected DNA fragments were resolved by polyacrylamide gel electrophoresis.

preparations in (c) were derived from the (48 hr) transfection experiments presented in Fig. 2. Sizes of DNA fragments are indicated in base pairs (bp).

172

KNEBEL-MCjRSDORF

Irrespective of whether the unmethylated or the 5’CCGG-3’ methylated pAd2E2AL-CAT construct with the HCMV enhancer in the two different locations was transfected, transcription started at the same E2A promoter site as documented by the sizes of the protected 69- and 71-bp fragments (Fig. 3b). As demonstrated earlier (Langner et a/., 1984, 1986), these fragment sizes corresponded to the authentic cap sites in the late E2A promoter(Baker and Ziff, 1981). Additional hybrid DNA signals were not apparent. Transfer RNA used as a negative control did not protect the late E2A promoter fragment from S 1 nuclease degradation. The amounts of RNA produced in HeLa cells transfected with the methylated construct appeared to be only slightly lower, since the signals elicited by the protected fragment were slightly reduced in RNA preparations isolated from HeLa cells which had been transfected with the methylated constructs (Fig. 3b). In the Sl experiments a considerable excess of probe DNA was employed. In an additional set of experiments, the Asp718Pvull fragment (Fig. 3a) was employed as doublestranded probe (Fig. 3~). The amounts of stable RNAs derived from the pAd2E2AL-HCMV(Bam)-CAT (Fig. 3c, lanes 2,3) or the pAd2E2AL-HCMV-CAT (Fig. 3c, lanes 4, 5) in the unmethylated (Fig. 3c, lanes 2, 4) or the 5’-CCGG-3’ methylated (Fig. 3c, lanes 3, 5) form were compared by Sl protection analyses. Striking differences were not found, and this finding was consistent with the data presented in Fig. 2. It was concluded that the increased CAT activity after transfection with methylated constructs, which contained the HCMV enhancer in two different locations, was not due to the selection of surrogate initiation sites for transcription. Transcription was initiated efficiently at the authentic late E2A promoter site, even when this promoter was methylated. There was no evidence for usage of additional or surrogate promoter sites in the enhancer or even in the plasmid part of the construct (cf. Langner et a/., 1984) when the methylated constructs were reactivated in the presence of the HCMV enhancer sequences. The methylated pAd2E2AL-(Hind-Asp)-CAT constructs with the HCMV enhancers remained methylated during the 48-hr transfection period The DNAs extracted from HeLa cells at 48 hr after transfection with the unmethylated or the 5’-CCGG-3’ methylated pAd2E2AL-(Hind-Asp)-CAT constructs carrying the HCMV enhancer as indicated in Fig. 4 were cleaved with Hoall or Ivlspl. The fragments were separated by electrophoresis on a 1% agarose gel, transferred to nitrocellulose filters (Southern, 1975) and hy-

ET AL.

8

1234 bp

2061-

620 457 -

FIG. 4. Stability of the pattern of methylation of the transfected CAT constructs containing the HCMV enhancer sequences. As described in the text, DNAs from cells transfected with the constructs detailed below were isolated 48 hr aftertransfection and analyzed by cleavage with Hoall (1, 3) or Mspl (2, 4). In (a) cells were transfected with the pAd2E2AL-HCMV-CAT construct and in (b) with the pAd2E2AL-HCMV(Bam)-CAT construct. Constructs were unmethylated in 1 and 2 and 5’CCGG-3’ methylated in 3 and 4. The sizes of DNA fragments shown in the autoradiograms are indicated in bp.

bridized to the 32P-labeled pAd2E2AL-HCMV-CATconstruct. The autoradiograms in Fig. 4 demonstrated that the 5’-CCGG-3’ methylated construct carrying the HCMVenhancer in the promoter adjacent position (Fig. 4a) or in the plasmid BarnHI site (Fig. 4b) remained /-@all cleavage resistant during the 48-hr period of the experiment. Hence the high activity levels of the methylated promoter construct could not be attributed to active demethylation of the promoter, at least not in both DNA complements. It was concluded that the presence of the strong HCMV enhancer immediately adjacent to or several thousand base pairs remote from the 5’-CCGG-3’methylated late E2A promoter reactivated the methylationinhibited promoter without leading to demethylation in both strands. As shown in the preceding section, transcription was initiated in the reactivated, methylated promoter at the authentic E2A cap site. DISCUSSION A coherent concept of how promoter activity in higher eukaryotes is regulated is only gradually emerging. The tools of molecular biology have rendered a number of approaches amenable to study promoter function. In keeping with the multifaceted analytical procedures, a number of factors have been investigated which seem to influence promoter activity. DNA-protein interactions have long been cherished as a very important factor. There is abundant evidence for

HCMV

ENHANCER

ABROGATES

the notion that the binding of several, perhaps many, specific proteins to specific promoter sequences does in fact play a decisive role in modulating activity levels of eukaryotic promoters. Then, there is the much less well-definable supposition that the three-dimensional structure of DNA-protein complexes has a major part in determining if and how transcription is to proceed. Only limited possibilities exist to modify DNA. The best studied modification is the methylation of cytidine residues. It has been demonstrated for a number of viral and nonviral eukaryotic genes that the methylation of specific sequences in these promoters leads to inactivation or inhibition. The positions of the decisive & quences, whose methylation entails promoter inactivation, cannot be predicted yet and appear to be different from promoter to promoter. This-to the systematic mind-apparent inconsistency may have to do with the complex structure of an active promoter. Sites which are decisive for methylation inactivation, and which seem to be in disarray might be in harmonious positions if one knew about the details of that complex structure. Moreover, in different promoters different specific proteins can bind and it depends on the protein-DNA interaction whether it is positively or negatively affected by sequence-specific methylations in a particular promoter sequence. An inhibitory regulation signal has to be reversible. A methyl group once attached may be stable for a long time. Possibilities for active demethylation do perhaps exist (Razin eta/., 1986). On the other hand, there may be more expedient and refined ways to counteract transiently the inactivating function of a methylated cytidine. We have shown previously that the 289-amino acid protein encoded in the ElA region of adenovirus DNA and a Vans-activator par excellence can reactivate a methylation-inactivated promoter by reinitiating transcription at the authentic start site and without demethylation of both complements (Langner et al., 1986; Weisshaar et al., 1988). A similar reactivation has been shown for the methylation-inactivated ElA promoter of adenovirus type 12 DNA (Kruczek and Doerfler, 1983), in frog virus 3-infected mammalian cells (Thompson et al., 1986). In the present report, we have adduced evidence that there is still another path by which a methylationinhibited promoter can be reactivated. A strong enhancer inserted immediately adjacent to or several thousand nucleotides remote from the methylated promoter reactivates this promoter with the same functional implications as mentioned above, i.e., the usage of authentic cap sites and the lack of demethylation. Aside from Vans-activators and enhancers, there may be additional mechanisms in a cell that could achieve similar reactivations. It is, therefore, not surprising that

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in the past some investigators reported activity even for methylated promoters. The methylation signal is not a simple one but has to be interpreted in a general functional context and after the detailed structural and functional analyses of a given promoter. Perhaps there is a hierarchy of events in promoter regulation, and the methylation of specific nucleotides is a single but decisive step in a series of events leading to promoter inactivation. Trans-activators and enhancers have been known for some time to be intricately interwoven in these events. It has now been shown that both functional principles can also help to reactivate a methylation-inhibited promoter. ACKNOWLEDGMENTS We thank Hanna Mansi-Wothke for media preparation, Petra Bijhm for excellent editorial work, and Birgit Schmitz for reliable technical assistance. This research was supported by the Deutsche Forschungsgemeinschaft through SFB74-Cl and Ru 336/l-4. Note added in proof. Upon co-transfection of HeLa cells with the pAd2E2AL promoter-CAT gene assembly and the strong HCMV enhancer on separate plasmids. the enhancing effect on CAT expression was not observed. The same observation was made with the 5’CCGG-S’methylated pAd2E2AL-CAT construct.

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