Reactivation of the methylation-inactivated late E2A promoter of adenovirus type 2 by E1A (13 S) functions

Bernd Weisshaar, Klaus Dieter Langner, Ruth Jüttermann, Ulrich Mueller, Christiane Zock, Thomas Klimkait, Walter Doerfler

Research output: Contribution to journalArticle

Abstract

The inactivating effect of sequence-specific promoter methylations was extensively studied by using the late E2A promoter of adenovirus type 2 (Ad2) DNA. The modification of the three 5′ CCGG 3′ sequences at nucleotides +24, +6 and -215, relative to the cap site in this promoter, sufficed to silence the gene in transient expression either in Xenopus laevis oocytes or in mammalian cells, and after the fixation of the E2A promoter-chloramphenicol-acetyltransferase (CAT) gene construct in the genome of hamster cells. It will now be demonstrated that the inactivation of the late promoter of Ad2 DNA can be reversed by transactivating functions that are encoded in the 13 S messenger RNA of the E1A region of Ad2 DNA. The reactivation of a methylation-inactivated eukaryotic promoter by transactivating functions has general significance in that the value of a regulatory signal can be fully realized only by its controlled reversibility. It was demonstrated in transient expression experiments that the 5′ CCGG 3′-methylated late E2A promoter was at least partly reactivated in cell lines constitutively expressing the E1 region of Ad2 or of adenovirus type 5 (Ad5) DNA. The reactivation led to transcriptional initiation at the authentic cap sites of the late E2A promoter and was not associated with promoter demethylation, at least not in both DNA complements. Reactivation of the methylation-inactivated E2A promoter could also be demontrated in two BHK21 cell lines (mc14 and mc20), which carried the late E2A promoter-CAT gene assembly in an integrated form. In these cell lines the late E2A promoter was methylated and the CAT gene was not expressed. By transfection of cell lines mc14 and mc20, the reactivating functions were shown to reside in the pAd2E1A-13 S cDNA clone of Ad2 DNA. The pAd2E1A-12 S cDNA clone or the pAd2E1B clone showed no reactivating function. These findings implicated the E1A 289 amino acid residue protein of Ad2, a well-known transactivator, as the reactivating function of the endogenous, previously dormant, late E2A promoter-CAT gene assembly. The methylated promoter was not demethylated, at least not in both complements, and it was shown that reactivation of the methylated promoter entailed transcriptional initiation at the authentic late E2A cap site. Since E1A and E1B jointly had a more pronounced effect, it was conceivable that genes in both regions acted together in the abrogation of the inhibitory effect of promoter methylations in the late E2A promoter.

Original languageEnglish (US)
Pages (from-to)255-270
Number of pages16
JournalJournal of Molecular Biology
Volume202
Issue number2
DOIs
StatePublished - Jul 20 1988
Externally publishedYes

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Adenoviridae
Methylation
Chloramphenicol O-Acetyltransferase
DNA
Genes
Cell Line
Clone Cells
Complementary DNA
Trans-Activators
Xenopus laevis
Cricetinae
Oocytes
Transfection
Genome
Amino Acids
Messenger RNA
Proteins

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Reactivation of the methylation-inactivated late E2A promoter of adenovirus type 2 by E1A (13 S) functions. / Weisshaar, Bernd; Langner, Klaus Dieter; Jüttermann, Ruth; Mueller, Ulrich; Zock, Christiane; Klimkait, Thomas; Doerfler, Walter.

In: Journal of Molecular Biology, Vol. 202, No. 2, 20.07.1988, p. 255-270.

Research output: Contribution to journalArticle

Weisshaar, Bernd ; Langner, Klaus Dieter ; Jüttermann, Ruth ; Mueller, Ulrich ; Zock, Christiane ; Klimkait, Thomas ; Doerfler, Walter. / Reactivation of the methylation-inactivated late E2A promoter of adenovirus type 2 by E1A (13 S) functions. In: Journal of Molecular Biology. 1988 ; Vol. 202, No. 2. pp. 255-270.
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abstract = "The inactivating effect of sequence-specific promoter methylations was extensively studied by using the late E2A promoter of adenovirus type 2 (Ad2) DNA. The modification of the three 5′ CCGG 3′ sequences at nucleotides +24, +6 and -215, relative to the cap site in this promoter, sufficed to silence the gene in transient expression either in Xenopus laevis oocytes or in mammalian cells, and after the fixation of the E2A promoter-chloramphenicol-acetyltransferase (CAT) gene construct in the genome of hamster cells. It will now be demonstrated that the inactivation of the late promoter of Ad2 DNA can be reversed by transactivating functions that are encoded in the 13 S messenger RNA of the E1A region of Ad2 DNA. The reactivation of a methylation-inactivated eukaryotic promoter by transactivating functions has general significance in that the value of a regulatory signal can be fully realized only by its controlled reversibility. It was demonstrated in transient expression experiments that the 5′ CCGG 3′-methylated late E2A promoter was at least partly reactivated in cell lines constitutively expressing the E1 region of Ad2 or of adenovirus type 5 (Ad5) DNA. The reactivation led to transcriptional initiation at the authentic cap sites of the late E2A promoter and was not associated with promoter demethylation, at least not in both DNA complements. Reactivation of the methylation-inactivated E2A promoter could also be demontrated in two BHK21 cell lines (mc14 and mc20), which carried the late E2A promoter-CAT gene assembly in an integrated form. In these cell lines the late E2A promoter was methylated and the CAT gene was not expressed. By transfection of cell lines mc14 and mc20, the reactivating functions were shown to reside in the pAd2E1A-13 S cDNA clone of Ad2 DNA. The pAd2E1A-12 S cDNA clone or the pAd2E1B clone showed no reactivating function. These findings implicated the E1A 289 amino acid residue protein of Ad2, a well-known transactivator, as the reactivating function of the endogenous, previously dormant, late E2A promoter-CAT gene assembly. The methylated promoter was not demethylated, at least not in both complements, and it was shown that reactivation of the methylated promoter entailed transcriptional initiation at the authentic late E2A cap site. Since E1A and E1B jointly had a more pronounced effect, it was conceivable that genes in both regions acted together in the abrogation of the inhibitory effect of promoter methylations in the late E2A promoter.",
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AU - Zock, Christiane

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N2 - The inactivating effect of sequence-specific promoter methylations was extensively studied by using the late E2A promoter of adenovirus type 2 (Ad2) DNA. The modification of the three 5′ CCGG 3′ sequences at nucleotides +24, +6 and -215, relative to the cap site in this promoter, sufficed to silence the gene in transient expression either in Xenopus laevis oocytes or in mammalian cells, and after the fixation of the E2A promoter-chloramphenicol-acetyltransferase (CAT) gene construct in the genome of hamster cells. It will now be demonstrated that the inactivation of the late promoter of Ad2 DNA can be reversed by transactivating functions that are encoded in the 13 S messenger RNA of the E1A region of Ad2 DNA. The reactivation of a methylation-inactivated eukaryotic promoter by transactivating functions has general significance in that the value of a regulatory signal can be fully realized only by its controlled reversibility. It was demonstrated in transient expression experiments that the 5′ CCGG 3′-methylated late E2A promoter was at least partly reactivated in cell lines constitutively expressing the E1 region of Ad2 or of adenovirus type 5 (Ad5) DNA. The reactivation led to transcriptional initiation at the authentic cap sites of the late E2A promoter and was not associated with promoter demethylation, at least not in both DNA complements. Reactivation of the methylation-inactivated E2A promoter could also be demontrated in two BHK21 cell lines (mc14 and mc20), which carried the late E2A promoter-CAT gene assembly in an integrated form. In these cell lines the late E2A promoter was methylated and the CAT gene was not expressed. By transfection of cell lines mc14 and mc20, the reactivating functions were shown to reside in the pAd2E1A-13 S cDNA clone of Ad2 DNA. The pAd2E1A-12 S cDNA clone or the pAd2E1B clone showed no reactivating function. These findings implicated the E1A 289 amino acid residue protein of Ad2, a well-known transactivator, as the reactivating function of the endogenous, previously dormant, late E2A promoter-CAT gene assembly. The methylated promoter was not demethylated, at least not in both complements, and it was shown that reactivation of the methylated promoter entailed transcriptional initiation at the authentic late E2A cap site. Since E1A and E1B jointly had a more pronounced effect, it was conceivable that genes in both regions acted together in the abrogation of the inhibitory effect of promoter methylations in the late E2A promoter.

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