@Copyright 1989 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163-4992/89/1512--0021502.00

Promoter Inactivation or Inhibition by Sequence-Specific Methylation and Mechanisms of Reactivation

WALTER DOERFLER, 1'* ARND HOEVELER, l BERND WEISSHAAR, 1 PAWEL DOBRZANSKI, 1

DAGMAR KNEBEL, ~ KLAUS-DIETER LANGNER, 2 SABINE ACHTEN, ~ AND ULRICH MCILLER 3

1 Institute of Genetics, University of Cologne, Weyertal 121,D-5000 Cologne 41, Germany; 2 Present address: Behring Werke, Marburg, Germany; and 3 Present address: Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, NJ

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ABSTRACT

In studies on adenovirus promoters, predominantly on the late E2A promoter of adenovirus type 2 (Ad2), we have demonstrated by a number of experimental approaches that the sequence-specific methylation of three 5'-CCGG-3' sequences inactivates this promoter. Recently, we have developed a cell-free transcription system in which the methylation-inactivation of eukaryotic promoters can be studied in detail. It has also been shown that methylation-caused promoter inactivation can be reversed by the 289 amino acid EIA protein of Ad2 or of adenovirus type 5. In the presence of this protein with a transac- tivating effect, transcription is initiated at the authentic cap site of the methylated late E2A promoter. A similar reactivation of the methylated late E2A promoter can also be effected by a cis-acting genetic element, i.e., the strong enhancer of human cytomegalovirus. Further studies will be directed toward the biochemical mechanisms of promoter silencing by sequence-specific methylations.

*Author to whom all correspondence and reprint requests should be addressed.

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Index Entries: Adenovirus promoter; human cytomegalovirus; adenovirus type 2; promoter methylation and gene inactivation; cell- free transcription system; reactivation of methylation-inhibited pro- moter; EIA protein of adenovirus type 2; enhancer of human cyto- megalovirus

INTRODUCTION

The regulation of promoter activity in eukaryotic cells is subject to several regulatory factors, among which highly specific DNA-protein in- teractions seem to assume a central role. In addition, sequence-specific promoter methylations have been shown to lead to the long-term inactiva- tion of eukaryotic promoters (1-3). It is plausible to propose that sequence- specific promoter methylations interfere positively or negatively with the binding of specific cellular proteins and exert their promoter inactivating function in this way. There is, however, no proof for this notion as yet. It is an open question at the time of this writing whether, and if so, how, a methylated cytidine residue in DNA can be removed, and, hence, its in- activating function be reversed. DNA replication and the concomitant inhibition of DNA methyltransferase activities or active demethylation by a mechanism involving excision-repair of methylated nucleotides (4) are theoretically possible, but unproven mechanisms.

In this laboratory, adenovirus promoters have been used as model systems in studies on the importance of sequence-specific methylations for promoter activity. These results have been published previously and summarized in reviews (1,2,5-7). Most of the more recent work has been performed with the late E2A promoter of human adenovirus type 2 (Ad2). Initially, we demonstrated that the methylation of the 14 5"CCGG-3' (HpaII) sequences in the E2A gene correlates perfectly with the inactive state of this promoter in Ad2-transformed hamster cells that carry parts of the Ad2 genome in an integrated form. In a cell line in which these sites are unmethylated, the E2A gene is expressed (8). In order to distinguish between the two alternative interpretations that DNA methylation could be cause or consequence of gene inactivation, experiments on in vitro methylated genes have been carried out. The results of these experiments will be briefly summarized.

SYNOPSIS OF PREVIOUS RESULTS

After the in vitro methylation of all 14 5'-CCGG-3' sequences in the E2A gene of Ad2 DNA and microinjection into nuclei of Xenopus laevis oocytes, the E2A gene is inactive, the unmethylated gene is, however, actively transcribed (9). When the 5'-CCGG-3' methylated E2A gene is coinjected with unmethylated histone genes from sea urchin, the E2A gene is silent, and the histone genes are expressed (9).

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Reactivation of a Methylation-lnactivated Promoter 23

In later experiments, we have shown that it is sufficient for E2A gene inactivation to 5"CCGG-3' methylate the three sites in the late E2A pro- moter region and to leave the eleven sequences in the 3' part of the gene unmethylated. When the 1i 3' sequences are methylated and the three promoter sites are left unmethylated, the construct is expressed after microinjection into Xenopus laevis oocytes (10).

The late E2A promoter of Ad2 DNA contains three HpaII methylatable 5'-CCGG-3' sequences at positions -215, + 6, and + 24 (numbers refering to the 3' C residues) relative to the cap site of the gene. This promoter has been fused to the prokaryotic gene for chloramphenicol acetyltransferase (CAT), and the activity of the construct has been tested after transfection into mammalian cells. The methylated construct is inactive, the unmethyl- ated construct is transcribed, and transcription is initiated at the authentic cap site of this promoter (11). Similar results have been adduced for the HpaII (two sites) or the HhaI (seven sites) methylated EIA promoter of adenovirus type 12 (Ad12) DNA (12).

The late E2A promoter-CAT gene construct in the unmethylated or in the 5'-CCGG-3' methylated form has also been genomically fixed in BHK21 hamster cells by cotransfection of the gene for neomycin phosphotrans- ferase. The inheritance of methylation patterns as well as promoter activi- ties have been tested in many of the established cell lines that had been selected by G418 resistance. Only those cell lines have been used for the analyses that carry the late E2A promoter-CAT gene assembly in a non- rearranged form. When the late E2A promoter has been methylated, the gene is inactive, and the unmethylated promoter mediates CAT expres- sion. There have been a few cell lines in which the previously methylated promoter has become partly demethylated. The E2A promoter has then been found active (13).

The DNAs of the baculovirus Autographa californica Nuclear Polyhedro- sis Virus (AcNPV) and of its host Spodoptera frugiperda are not detectably methylated (14). The promoter of the late-expressed pl0 gene in AcNPV DNA has been fused to the CAT gene. This construct is active in AcNPV- infected Spodoptera frugiperda insect cells. When the pl0 promoter is in vitro methylated at one 5'-CCGG-3' sequence in the promoter and at two such sites in the 5' part of the coding sequence, the construct is inactivated in AcNPV-infected Spodoptera frugiperda cells. Thus, the methylation sig- nal is recognized in insect cells, even though this signal does not seem to be utilized in Spodoptera frugiperda cells (15).

The data briefly summarized here and the results of work on other eukaryotic genes (1-3) support the notion that sequence-specific promoter methylations can lead to the inactivation of the promoter. The sequences whose methylation causes inactivation appear to be different in different promoters. We have been interested in the biochemical mechanisms by which the methylation signals cause promoter inactivation and have again used the late E2A promoter of Ad2 DNA for model investigations. The most likely way by which promoter methylations might affect promoter

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activity is by the positive or negative interference with specific DNA- protein interactions. In the late E2A promoter of Ad2 DNA, at least six different sites have been identified that bind to cellular proteins. When the three 5"CCGG-3' sequences in the late E2A promoter are methylated, striking differences in the binding of cellular proteins have not been de- tected by the migration delay assay or by the DNaseI footprinting tech- nique (16). Only recently, distinct differences in protein binding in a syn- thetic oligodeoxynucleotide encompassing nucleotides + 37 to - 13 of the late E2A promoter have been seen when the unmethylated DNA and the sequence methylated at two 5'-CCGG-3' sites have been compared by the migration delay technique (17).

STUDIES ON PROMOTER INACTIVATION OR INHIBITION BY METHYLATION IN A CELL FREE SYSTEM

Studies on the biochemical mechanism by which sequence-specific promoter methylations lead to gene inactivation have necessitated the development of a cell free transcription system that can respond to pro- moter methylations by inactivation. Such systems have hitherto not been available. In nuclear extracts from HeLa cells the late E2A promoter of adenovirus type 2 (Ad2), which has been methylated at the three 5 '-CCGG- 3' sequences at nucleotides -215, +6, and +24, or the HpaII or HhaI methylated major late promoter (MLP) exhibit strikingly lower activity than the unmethylated construct or no activity at all. Circular templates have to be used, the DNA concentration has to be kept at a critical level, and the protein concentration in the cell free extract has to be high (18). We are currently pursuing the hypothesis that cellular factors are crucial in recognizing methylated promoters and in effecting their inactivation. The in vitro transcription system will, hopefully, help to isolate and char- acterize these cellular proteins.

REACTIVATION OF THE METHYLATION-SILENCED LATE E2A PROMOTER

The 2 8 9 Amino Acid E 1A Prote in of Ad2

It has now been demonstrated that the inactivation of the late pro- moter of Ad2 DNA can be reversed by transactivating functions that are encoded in the 13S messenger RNA of the EIA region of Ad2 DNA. The reactivation of a methylation-inactivated eukaryotic promoter by transac- tivating functions has general significance in that the value of a regulatory signal can be fully realized only by its controlled reversibility. It has been demonstrated in transient expression experiments that the 5'-CCGG-3' methylated late E2A promoter is reactivated in cell lines constitutively ex- pressing the E1 region of Ad2 or of adenovirus type 5 (Ad5) DNA (11,19).

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Reactivation of a Methylation-lnactivated Promoter 25

The reactivation leads to transcriptional initiation at the authentic cap site of the late E2A promoter and has not been associated with promoter demethylation, at least not in both DNA complements. Reactivation of the methylation-inactivated E2A promoter can also be demonstrated in two BHK21 cell lines (mc14 and mc20) which carry the late E2A promoter- chloramphenicol-acetyltransferase (CAT) gene assembly in an integrated form. In these cell lines the late E2A promoter is methylated and the CAT gene is not expressed (13).

By transfection experiments of cell lines mc14 and mc20 the reactivat- ing functions have been shown to reside in the pAd2EIA-13S cDNA clone of Ad2 DNA. The pAd2EIA-12S cDNA clone or the pAd2EIB clone show no reactivating potential. These findings implicate the EIA 289 amino acid protein of Ad2, a well known transactivator, as the reactivating function of the endogenous, previously dormant late E2A promoter-CAT gene assembly (19). The methylated promoter is not demethylated, at least not in both complements, and it has been shown that reactivation of the meth- ylated promoter entails transcriptional initiation at the authentic late E2A cap site. Since EIA and EIB jointly have a more pronounced effect, it is conceivable that genes in both regions can act together in the abrogation of the inhibitory effect of promoter methylations in the late E2A promoter (19).

The Strong Enhancer of Human Cytomegalovirus

Promoter inactivation by sequence-specific methylation seems to function as a reversible inhibitory signal. In fact, a flexible mechanism that has been shown to be associated with long-term gene inactivation has to allow for permanent reactivation, perhaps by double-stranded demethylation, as well as for the transient release of the transcriptional block. Such a release has recently been demonstrated (11,19). A second mechanism of reactivation will now be presented.

In the genome of human cytomegalovirus a strong transcription en- hancer has been identified upstream of an immediate early viral gene (20). Enhancers are cis-acting, eukaryotic genetic signals that stimulate the ex- pression of neighboring genes independent of orientation and precise location relative to the gene. Enhancers have first been detected in viral genomes, later close to many viral and nonviral eukaryotic genes (21).

Can a strong enhancer reactivate a promoter that has been shut off by site-specific methylation? Since detailed information on the methylation sensitivity of the late E2A promoter of Ad2 is available (10,11,13,18,19), we have used this promoter in combination with an indicator gene, the gene for chloramphenicol acetyltransferase (CAT) in the plasmid pSVO- CAT (22), in these studies. It has been demonstrated that the cytomegalo- virus enhancer can override the incapacitating effect of site-specific methy- lations in the late E2A promoter of Ad2 DNA and restore its activity even though it remains methylated during reactivation. Transcription is reiniti- ated inside the methylated late EZA promoter at the authentic cap site (23).

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26 Doerfler et al.

CONCLUSION

Studies on the mechanism of promoter inactivation or inhibition by sequence-specific methylations will continue to occupy our interest. It will be of prime importance to identify the proteins that are responsible for recognizing specific methylated cytidine residues in a promoter, and either bind at such a site or positively or negatively influence the binding of other proteins at such sites. Either type of interference with protein- DNA binding by a methyl group is conceivable and compatible with the demonstrated inactivating effects. The possibility also exists that 5-methyl cytidine does not intercept protein-DNA binding directly but rather af- fects protein-protein interactions of host proteins with a protein-promoter complex. Obviously, much further work will be required to understand these complicated interactions.

We have also initiated studies to determine which of the two DNA strands in the late E2A promoter need be modified in order to effect pro- moter inactivation. Moreover, much interest will also be directed toward patterns of methylation in active and inactive promoters in living cells, and toward the generation and stability of these patterns. We will also apply the results of this theoretical groundwork to the comparative study of patterns of methylation in normal human and tumor cells.

ACKNOWLEDGMENTS

We thank Petra BOhm for excellent editorial work and Hanna Mansi- Wothke for the preparation of media and of cells. This research was sup- ported by the Deutsche Forschungsgemeinschaft through SFB74-C1, by the Fonds der Chemischen Industrie and by a donation of the Hoechst AG.

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