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

Weißhaar, Bernd; Langner, Klaus-Dieter; Jüttermann, Ruth; Müller, Ulrich; Zock, Christiane; Klimkait, Thomas; Doerfler, Walter

doi:10.1016/0022-2836(88)90456-1

Cited by 65 publications

(31 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The methylated nucleoside 5-methyl-2'-deoxycytidine (m'dC) has been recognized as a genetic signal that can lead to promoter inhibition or inactivation, provided it is present in specific promoter sequences (2)(3)(4). It (5,6), or in cis, like the strong enhancer from human cytomegalovirus (7). It has been suggested that transient demethylations can occur (8,9).…”

mentioning

confidence: 99%

Genomic sequencing reveals a 5-methylcytosine-free domain in active promoters and the spreading of preimposed methylation patterns.

Tóth

Lichtenberg

Doerfler

1989

Proc. Natl. Acad. Sci. U.S.A.

105

View full text Add to dashboard Cite

Previous work demonstrated an inverse correlation between methylation at the three 5'-CCGG-3' sequences in positions +24, +6, and -215 relative to the cap site of the late E2A promoter of adenovirus type 2 (Ad2) DNA and its activity. In the study presented here, we used the genomic sequencing method to detect 5-methyl-2'-deoxycytidine (m5dC) residues in 5'-CG-3' sequences other than the 5'-CCGG-3' (Hpa II) sites. The patterns of methylation in all 5'-CG-3' sequences over a region of about 180 base pairs required for gene activity in the late E2A promoter of integrated Ad2 DNA were determined in cell lines that carry this promoter in an active or inactive state. In cell lines HE1 and uc2, the late E2A promoter is active and all thirteen 5'-CG-3' sequences between positions +24 and -160 are unmethylated. In cell line HE2, the same promoter is permanently shut off and all 5'-CG-3' sequences are methylated in both strands. Thus, the inverse correlation Is perfect in these cell lines over a region of about 180 base pairs in the late E2A promoter. The same promoter segment was analyzed in cell lines mc23 and mc4O, in which a late E2A promoter-chloramphenicol acetyltransferase (CAT) gene construct had been genomically fixed after in vitro 5'-CCGG-3' methylation and subsequent transfection. In cell line mc23, the preimposed methylation pattern was stable and the CAT gene was inactive. Genomic sequencing confirmed the presence of msdC in the 5'-CCGG-3' sequences and revealed the spreading of methylation to neighboring 5'-CG-3' sequences along the entire promoter. Some of these sites were hemimethylated. In cell line mc4O, several of the 120 integrated copies became demethylated in positions +24 and +6, but the promoter was methylated in some of the copies upstream of position -50. Cell line mc4O expressed the CAT gene.The regulation of promoter activity is governed by the interaction of specific promoter sequence motifs with regulatory proteins whose binding can entail positive or negative regulatory effects (for review, see ref. 1). While this aspect of promoter modulation has been studied in detail, much less is known about the role of the three-dimensional structure of promoter-protein complexes in the regulation of gene activity. The repertoire of modifications on the DNA side of the binary DNA-protein system is very limited. The methylated nucleoside 5-methyl-2'-deoxycytidine (m'dC) has been recognized as a genetic signal that can lead to promoter inhibition or inactivation, provided it is present in specific promoter sequences (2)(3)(4) HEl, HE2, and HE3 (20,21) and the BHK21 hamster cell lines uc2, mc23, and mc4O, which were generated by fixing the unmethylated (uc) or the 5'-CCGG-3'-methylated (mc) pAd2E2AL-CAT construct in the cellular genome by cotransfection with and selection for the Abbreviations: m5dC, 5-methyl-2'-deoxycytidine; Ad2, adenovirus type 2; CAT, chloramphenicol acetyltransferase.

show abstract

mentioning

confidence: 99%

Genomic sequencing reveals a 5-methylcytosine-free domain in active promoters and the spreading of preimposed methylation patterns.

Tóth

Lichtenberg

Doerfler

1989

Proc. Natl. Acad. Sci. U.S.A.

105

View full text Add to dashboard Cite

show abstract

“…[1][2][3][4][5][6]. Adenovirus promoters have been used as models to study the mechanism of promoter inactivation (7)(8)(9)(10)(11)(12)(13)(14)(15). The development of de novo patterns of DNA methylation in transformed cell lines is characterized by the ordered, nonrandom spreading of methylation (16)(17)(18); By using restriction enzyme and Southern blotting analyses (19), as well as the very exact genomic sequencing procedure (17,20,21), we have studied human DNA methylation in segments and in the promoter regions of the tumor necrosis factor (TNF) genes a (cachectin) and 8 (lymphotoxin) (22)(23)(24)(25)(26)(27)(28)(29).…”

mentioning

confidence: 99%

Interindividual concordance of methylation profiles in human genes for tumor necrosis factors alpha and beta.

Kochanek

Tóth

Dehmel

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The DNA in mammalian genomes is characterized by complex patterns of DNA methylation that reflect the states of all genetic activities of that genome. The modified nucleotide 5-methyldeoxycytidine (5mdC) can affect the interactions of specific proteins with DNA sequence motifs. The most extensively studied effect of sequence-specific methylations is that of the long-term silencing of eukaryotic (mammalian) promoters. We have initiated studies on the methylation status of parts of the human genome to view patterns of DNA methylation as indicators for genetic activities. In this report, analyses using both restriction enzyme-Southern blotting and the very precise genomic sequencing technique have been done. The genes for tumor necrosis factors (TNF) a and ,B-in particular, their 5'-upstream and promoter regions-have been investigated in DNA isolated from human lymphocytes, granulocytes, and sperm. The results are characterized by a remarkable interindividual concordance of DNA methylation in specific human cell types. The patterns are identical in the DNA from one cell type for different individuals even of different genetic origins but different in the DNA from different cell types. As an example, in the DNA from human granulocytes of 15 different individuals (ages 20-48 yr, both sexes), 5mdC residues have been localized by the genomic sequencing technique in three identical sequence positions in the 5'-upstream region and in one downstream position of the gene encoding TNF-a. The promoter of this gene is free of 5mdC, and TNF-a is expressed in human granulocytes. The TNF-,B promoter is methylated in granulocytes from 9 different individuals, and TNF-fl is not expressed. In human lymphocytes, the main source of TNF-fB the TNF-,B promoter is free of 5mdC residues. All 5'-CG-3' sites studied in the TNF-a and -fl genes are methylated in DNA from human sperm. In human cell lines HL-60, Jurkat, and RPMI 1788, the extent ofDNA methylation in TNF-a and -,B genes has also been studied. Subsequently, lymphocytes and granulocytes were fractionated on a Ficoll-Paque gradient. Granulocytes were recovered from the pellet and freed from contaminating erythrocytes by lysing the latter in 0.83% NH4C1. The resulting granulocyte preparation was >95% pure, as determined in a Pappenheimstained (32) cell smear. The lymphocyte preparation was >90o lymphocytes, 5-10%o monocytes, and <3% granulocytes. (ii) Human cell lines HL-60, a human promyelocytic leukemia cell line; Jurkat, a T-cell line; and RPMI 1788, an IgM-secreting B-lymphoblastoid cell line, were propagated in RPMI 1640 medium (GIBCO-BRL) supplemented with 10-20%o fetal bovine serum. (iii) Human sperm cells were washed, and the DNA was extracted after dithiothreitol treatment (33). The nuclear DNA from the different cell preparations was extracted by standard protocols (34).Isolation of Cellular RNA. Granulocytes were purified >99%. Granulocytes or the cells from established cell lines were kept in culture for 5 hr in the absence or presence of 8 nM PMA (phorbol 12-my...

show abstract

“…1A, Table I, left side). This promoter has been shown to be inhibited by sequence-specific methylation (46,47). The S clones have SVluc with the SV40 early promoter 5Ј of the luciferase coding sequence and at the 3Ј-end the SV40 enhancer element containing the 72-and 21-bp repeats (Fig.…”

Section: Homologous Recombinant and Randomlymentioning

confidence: 99%

Factors Affecting de Novo Methylation of Foreign DNA in Mouse Embryonic Stem Cells

Hertz¹,

Schell

Doerfler

1999

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Integration of foreign DNA into an established host genome can lead to changes in methylation in both the inserted DNA and in host sequences and potentially alters transgene and cellular transcription patterns. This work addresses the questions of what factors influence de novo methylation, and whether the integration site or inserted DNA can affect de novo methylation. Homologous recombination was used to integrate foreign DNA into a specific gene, B lymphocyte kinase (BLK), in mouse embryonic stem (ES) cells. Two plasmids were chosen for integration; one contained the adenovirus type 2 E2AL promoter upstream of the luciferase reporter gene, and the second carried the early SV40 promoter. The methylation patterns were analyzed using HpaII and MspI restriction endonucleases for both homologously recombined and randomly integrated foreign DNA in the ES cell clones.Upon homologous reinsertion of the BLK gene into the genome of mouse ES cells, methylation patterns in this gene were reestablished. In DNA segments adjoined to the BLK gene, the de novo patterns of DNA methylation depended on the viral sequences in these clones and on the locations of the inserts, i.e. on whether the insertions resulted from homologously recombined or randomly integrated foreign DNA. In homologously recombined DNA, sequences carrying the adenovirus type 2 promoter were heavily methylated, and those with an SV40 promoter and an SV40 enhancer element remained unmethylated or hypomethylated. Upon removal of the enhancer element, these inserted constructs also became heavily methylated. In addition, all randomly integrated constructs were heavily methylated independently of the promoter and enhancer element present in the construct. These results indicate that modes and sites of integration as well as the inserted nucleotide sequence, possibly promoter strength, are factors affecting de novo methylation.

show abstract

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

Cited by 65 publications

References 34 publications

Genomic sequencing reveals a 5-methylcytosine-free domain in active promoters and the spreading of preimposed methylation patterns.

Genomic sequencing reveals a 5-methylcytosine-free domain in active promoters and the spreading of preimposed methylation patterns.

Interindividual concordance of methylation profiles in human genes for tumor necrosis factors alpha and beta.

Factors Affecting de Novo Methylation of Foreign DNA in Mouse Embryonic Stem Cells

Contact Info

Product

Resources

About