Sequence-specific methylation in a downstream region of the late E2A promoter of adenovirus type 2 DNA prevents protein binding

Hermann, Ralf; Hoeveler, Arnd; Doerfler, Walter

doi:10.1016/0022-2836(89)90340-9

Cited by 38 publications

(18 citation statements)

References 11 publications

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“…Regardless of the detailed mechanism, key players for the maintenance of the inactive state are likely to be DNA methylation and nucleosome formation, as it has been shown that both cytosine methylation (Becket et al 1987;Kovesdi et al 1987;Watt and Molloy 1988;IguchiAriga and Schaffner 1989;Hermann et al 1989;Comb et al 1990) and nucleosomes (Pifia et al 1990;Wolffe 1990;Archer et al 1991) interfere with the binding of transcription factors. However, DNA methylation does not prevent the binding of some factors.…”

Section: The Maintenance Of X Chromosome Inactivation and Chromatin Amentioning

confidence: 99%

Chromatin differences between active and inactive X chromosomes revealed by genomic footprinting of permeabilized cells using DNase I and ligation-mediated PCR.

Pfeifer

Riggs²

1991

Genes Dev.

189

121

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Ligation-mediated polymerase chain reaction (LMPCR) provides adequate sensitivity for nucleotide-level analysis of single-copy genes. Here, we report that chromatin structure can be studied by enzyme treatment of permeabilized cells followed by LMPCR. DNase I treatment of lysolecithin-permeabilized cells was found to give very clear footprints and to show differences between active and inactive X chromosomes (Xa and Xi, respectively 1 at the human X-linked phosphoglycerate kinase (PGK-11 locus. Beginning 380 bp upstream and continuing 70 bp downstream of the major transcription start site of PGK-1, we analyzed both strands of this promoter and CpG island and discovered the following: {1) The transcriptionally active Xa in permeabilized cells has several upstream regions that are almost completely protected on both strands from DNase I nicking. {2) Nuclei isolated in polyamine-containing buffers lack these footprints, suggesting that data from isolated nuclei can be flawed; other buffers are less disruptive. (3) The Xa has no detectable footprints at the transcription start and HIP1 consensus sequence. {4) The heterochromatic and transcriptionally inactive Xi has no footprints but has two regions showing increased DNase I sensitivity at 10-bp intervals, suggesting that the DNA is wrapped on the surface of a particle; one nucleosome-sized particle seems to be positioned over the transcription start site and another is centered -260 bp upstream. (51 Potassium permanganate and micrococcal nuclease {MNase) studies indicate no melted or otherwise unusual DNA structures in the region analyzed, and MNase, unlike restriction endonuclease MspI, does cut within the positioned particles on the Xi. Results are discussed in the context of X chromosome inactivation and the maintenance of protein and DNA methylation differences between euchromatin and facuhative heterochromatin at CpG islands.

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Section: The Maintenance Of X Chromosome Inactivation and Chromatin Amentioning

confidence: 99%

Chromatin differences between active and inactive X chromosomes revealed by genomic footprinting of permeabilized cells using DNase I and ligation-mediated PCR.

Pfeifer

Riggs²

1991

Genes Dev.

189

121

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“…The lack of transcriptional activity has been associated with methylation of cytosine residues within the promoter region of a gene (8,9). DNA-binding proteins, including transcription factors, can be sensitive to the presence of methylated cytosines in DNA (10), and this may be what prevents transcription.…”

mentioning

confidence: 99%

DNA methylation, vernalization, and the initiation of flowering.

Burn

Bagnall

Metzger

et al. 1993

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

289

181

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Late-flowering ecotypes and mutants of Arabidopsis thalana and the related crucifer Thlaspi arvense flower early after cold treatment (vernalization). Treatment with the DNA demethylating agent 5-azacytidine induced nonvernalized plants to flower significantly earlier than untreated controls. Cytidine at similar concentrations had no effect on time to flower. In contrast, late-flowering mutants that are insensitive to vernalization did not respond to 5-azacytidine treatment. Normal flowering time was reset in the progeny of plants induced to flower early with 5-azacytidine, paralleling the lack of inheritance of the vernalized condition. Arabidopsis plants, either cold-treated or 5-azacytidine-treated, had reduced levels of 5-methylcytosine in their DNA compared to nonvernalized plants. A Nicotiana plumbaginifolia cell line also showed a marked decrease in the level of 5-methylcytosine after treatment with either 5-azacytidine or low temperature. We suggest that DNA methylation provides a developmental control preventing early flowering in Arabidopsis and Thlaspi ecotypes. Vernalization, through its general demethylating effect, releases the block to flowering initiation. We propose that demethylation of a gene critical for flowering permits its transcription. We further suggest, on the basis of Thlaspi data, that the control affects transcription ofkaurenoic acid hydroxylase, a key enzyme in the gibberellic acid biosynthetic pathway.In many plants the transition of the shoot apical meristem from the vegetative to the flowering state occurs in response to environmental cues. Day length is one such cue, some plants requiring short, others long, days to initiate flowering. The site of perception of day length is the leaf, where a signal is generated and subsequently translocated to the shoot apex, where a new pattern of cell division forms the flowering meristem that subsequently produces the inflorescence (1).Exposure to low temperature can induce flowering in many temperate monocots and dicots, the cold treatment being termed vernalization (1). Experiments involving cooling treatments localized to parts of plants have shown that the shoot apical meristem is itself a site of perception of the low-temperature treatment (2, 3). Wellensiek (4) provided evidence that cell division during vernalization is necessary for thermoinduction in Lunaria annua and that flowering structures are ultimately derived from the mitotically active cells that were subjected to vernalizing temperatures. He concluded that thermoinduction is a cell-autonomous process that is mitotically propagated.The observation that tissues other than the shoot apical meristem also have the capacity to support thermoinductive responses to vernalization is consistent with the concept of mitotic transmission. Shoots regenerated from leaf cuttings of both L. annua (4) and Thlaspi arvense (3) exhibited a developmental state identical to that of plants from which they were obtained: flowering shoots developed from leaves of cold-treated plants, where...

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“…[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.

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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

Sequence-specific methylation in a downstream region of the late E2A promoter of adenovirus type 2 DNA prevents protein binding

Cited by 38 publications

References 11 publications

Chromatin differences between active and inactive X chromosomes revealed by genomic footprinting of permeabilized cells using DNase I and ligation-mediated PCR.

Chromatin differences between active and inactive X chromosomes revealed by genomic footprinting of permeabilized cells using DNase I and ligation-mediated PCR.

DNA methylation, vernalization, and the initiation of flowering.

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

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