DNA methylation. The effect of minor bases on DNA-protein interactions

Adams, Roger

doi:10.1042/bj2650309

Cited by 183 publications

(103 citation statements)

References 134 publications

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“…In such cases, the lack of its expression in the extrahepatic tissues could be due to situations known to stably repress the transcription of many tissue-specific genes, such as DNA methylation or genome organization (i.e. compaction of the chromatin) (46,47). By analyzing a series of 5Ј-end deletions, two major regulatory regions have been identified, a promoter proximal region (Ϫ193 bp relative to the transcription start site) and a far upstream area extending nucleotides Ϫ1251 to Ϫ958.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Rat Liver-specific Methionine Adenosyltransferase Gene Promoter

Álvarez

Sánchez-Góngora²,

Mingorance

et al. 1997

Journal of Biological Chemistry

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Methionine adenosyltransferase is a ubiquitous enzyme that catalyzes the only known route of biosynthesis of S-adenosylmethionine, the major methyl group donor in cell metabolism. In mammals, two different methionine adenosyltransferases exist: one is confined to the liver, and the other one is distributed in extrahepatic tissues. In the present study, we report the cloning of the 5 -flanking region of liver-specific methionine adenosyltransferase gene from rat. Two closely spaced sites for transcriptional initiation were identified by primer extension analysis. The major transcription start site was determined to be 29 nucleotides downstream from the putative TATA box. Transient transfection assays of constructs containing sequentially deleted 5 -flanking sequences fused to the luciferase gene showed that rat hepatic methionine adenosyltransferase promoter was able to efficiently drive reporter expression not only in liver-type cells (rat hepatoma H35 cells and human hepatoblastoma HepG2 cells) but also in Chinese hamster ovary cells. Two regions spanning nucleotides ؊1251 to ؊958 and ؊197 to ؉65 were found to be crucial for the promoter efficiency. The distal upstream region contains elements that positively regulate promoter activity in H35 and HepG2 cells but are ineffective in Chinese hamster ovary cells. Eight protein binding sites were characterized in both regions by DNase I footprinting analysis. Two of these elements, sites A and B, located in the distal region, were found to be essential for the regulation of promoter activity. Electrophoretic mobility shift assays and competition experiments showed that site A is recognized by an NF1 protein. Site B was able to interact with a member of HNF-3 family when nuclear extracts from rat liver and H35 cells were used in the in vitro assay, but an additional binding activity to an NHF1-like protein was obtained with the hepatoma cell extracts. It is suggested that this differential binding can contribute to the cell specificity of promoter function.

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Section: Discussionmentioning

confidence: 99%

Characterization of Rat Liver-specific Methionine Adenosyltransferase Gene Promoter

Álvarez

Sánchez-Góngora²,

Mingorance

et al. 1997

Journal of Biological Chemistry

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“…In land plants, cytosine methylation at CpHpG sites (where H equals A, T, or C) is faithfully inherited by daughter cells in addition to the symmetric CpG sites (Tariq and Paszkowski 2004;Chan et al 2005). In A. thaliana, .30% of total C's in the genome are methylated (Adams 1990), while in the case of C. reinhardtii (Hattman et al 1978) and its closely related multicellular relative, Volvox carteri (Babinger et al 2001), the percentages of cytosine methylation are only 0.7 and 1.1%, respectively. In V. carteri, limited CpG methylation undoubtedly contributes to gene silencing 1 of transgenes and endogenous transposons (Babinger et al 2001(Babinger et al , 2007.…”

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confidence: 99%

Unstable RNAi Effects Through Epigenetic Silencing of an Inverted Repeat Transgene in Chlamydomonas reinhardtii

2008

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RNA interferences in the unicellular green alga, Chlamydomonas reinhardtii, can be silenced. We have used the silencing of a transgene (aadA) that confers resistance to spectinomycin to investigate the mechanisms responsible for silencing by an artificial inverted repeat (IR) of the aadA gene. The IR construct provided strong silencing, but the RNAi efficiency varied among subclones of a single RNAitransformed strain with successive cell divisions. Northern blot analyses revealed an inverse correlation between the copy number of the hairpin RNA and the spectinomycin resistance of the subclones. There is an inverse correlation between the efficiency of RNAi and the frequency of methylated CpG (*CpG) in the silenced region. No significant methylated cytosine was observed in the target aadA gene, which suggests the absence of RNA-directed DNA methylation in trans. Several experiments suggest the existence of an intrinsic IR sequence-dependent but a transcription-independent DNA methylation system in C. reinhardtii. The correlation between the *CpG levels and the IR transcript implies the existence of IR DNA-dependent DNA methylation. Treatment of RNAi-induced cells with a histone deacetylase inhibitor, Trichostatin A, rapidly increased the amount of the hairpin RNA and suggests that transcription of the silencer construct was repressed by *CpG-related silencing mechanisms.

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“…The DMI might play an analogous role in mammalian cells where nuclear structure is required for site-specific initiation of DNA replication (20,21). In fact, site specificity is established during the middle of G 1 phase in each cell cycle (22), about the time when DNA methyltransferase activity appears and then increases during S phase (23)(24)(25). Therefore, it was imperative to confirm the existence of a DMI at a well characterized mammalian replication origin and to determine whether or not a DMI appears at a specific time during the cell division cycle.…”

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confidence: 99%

Absence of an Unusual “Densely Methylated Island” at the Hamster dhfr ori-β

Rein

Natale

Gärtner

et al. 1997

Journal of Biological Chemistry

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An unusual "densely methylated island" (DMI), in which all cytosine residues are methylated on both strands for 127-516 base pairs, has been reported at mammalian origins of DNA replication. This report had far-reaching implications in understanding of DNA methylation and DNA replication. For example, since this DMI appeared in about 90% of proliferating cells, but not in stationary cells, it may regulate origin activation. In an effort to confirm and extend these observations, the DMI at the well characterized ori-␤ locus 17 kilobases downstream of the dhfr gene in chromosomes of Chinese hamster ovary cells was checked for methylated cytosines in genomic DNA. The methylation status of this region was examined in randomly proliferating and stationary cells and in cell populations enriched in the G 1 , S, or G 2 ؉ M phases of their cell division cycle. DNA was subjected to 1) cleavage by methylation-sensitive restriction endonucleases, 2) hydrazine modification of cytosines followed by piperidine cleavage, and 3) permanganate modification of 5-methylcytosines ( m C) followed by piperidine cleavage. The permanganate reaction is a novel method for direct detection of m C residues that complements the more commonly used hydrazine method. These methods were capable of detecting m C in 2% of the cells. At the region of the proposed DMI, only one mC at a CpG site was detected. However, the ori-␤ DMI was not detected in any of these cell populations using any of these methods.DNA methylation at CpG dinucleotides in mammalian cells has been implicated as an important component of such pivotal processes as transcription (1), imprinting (2), recombination (3), carcinogenesis (4), development (5), and replication timing (6). CpG methylation is carried out by a methyltransferase that is associated with replication foci in the nucleus (7). In general, this enzyme methylates specifically hemimethylated CpG dinucleotides, although de novo methylation of unmethylated CpG dinucleotides can occur but at a low rate (8). In addition, this enzyme occasionally methylates cytosines in sequences other than CpG (9, 10). However, given the specificity of the mammalian methyltransferase, this non-target methylation should be of little significance in vivo because the pattern of non-CpG methylation will not be maintained during the subsequent round of DNA replication.Recently, an unusual form of DNA methylation has been implicated as an important component of mammalian origins of DNA replication. A densely methylated island (DMI) 1 has been reported at three different mammalian origins of DNA replication (11, 12). These DMIs were found only in proliferating cell populations and consisted of a 127-, 258-, or 516-bp stretch of DNA in which all cytosines were methylated, regardless of their sequence context (12). The discovery of DMIs has several important implications. First, this unprecedented methylation pattern suggested that a second (or modified) methylation enzyme exists in mammalian cells. Interestingly, limited proteolysis of the mamm...

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DNA methylation. The effect of minor bases on DNA-protein interactions

Cited by 183 publications

References 134 publications

Characterization of Rat Liver-specific Methionine Adenosyltransferase Gene Promoter

Characterization of Rat Liver-specific Methionine Adenosyltransferase Gene Promoter

Unstable RNAi Effects Through Epigenetic Silencing of an Inverted Repeat Transgene in Chlamydomonas reinhardtii

Absence of an Unusual “Densely Methylated Island” at the Hamster dhfr ori-β

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