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Cytosine methylation of an Sp1 site contributes to organ-specific and cell-specific regulation of expression of the lung epithelial gene t1alpha.

ABSTRACT: Several recent observations have suggested that cytosine methylation has a role in the in vivo transcriptional regulation of cell-specific genes in normal cells. We hypothesized that methylation regulates T1alpha, a gene expressed primarily in lung in adult rodents. In fetuses T1alpha is expressed in several organs, including the entire nervous system, but during development its expression is progressively restricted to lung alveolar type I epithelial cells, some osteoblasts and choroid plexus. Here we report that T1alpha is methylated at a key Sp1 site in the proximal promoter in cells and organs, including brain, where no gene expression is detectable. Conversely, in T1alpha-expressing cells, these sites are not methylated. In embryonic brain T1alpha is unmethylated and expressed; in adult brain the gene is methylated and not expressed. In lung epithelial cell lines, methylation of the T1alpha promoter in vitro decreases expression by approx. 50% (the maximum suppression being 100%). Analysis of mutated promoter constructs indicates that a single Sp1 site in the proximal promoter provides all or most of the methylation-sensitive gene silencing. We conclude that, in addition to regulation by transcription factors, cytosine methylation has a role in the complex expression patterns of this gene in intact animals and primary cells.

SUBMITTER: Cao YX

PROVIDER: S-EPMC1221323 | biostudies-other | 2000 Sep

REPOSITORIES: biostudies-other

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Project description:Methylation of cytosine in DNA at position C5 increases the rate of C→T mutations in bacteria and eukaryotes. Methylation at the N4 position, employed by some restriction-modification systems, is not known to increase the mutation rate. Here, I report that a Salmonella enterica Type III restriction-modification system that includes a cytosine-N4 methyltransferase causes an enormous increase in the rate of mutation of the methylated cytosines, which occur at the overlined C in the motif CACC̅GT Mutations consist mainly of C→A transversions, the rate of which is increased ∼500-fold by the restriction-modification system. The rate of C→T transitions is also increased and somewhat exceeds that at C5-methylated cytosines in Dcm sites. Two other Salmonella N4 methyltransferases investigated do not have such dramatic effects, although in one case there is a modest increase in C→A mutations along with an increase in C→T mutations. The sensitivity of the C→A rate to orientation with respect to both DNA replication and transcription is higher at hypermutable sites than at other cytosines, suggesting a fundamental mechanistic difference between hypermutation and ordinary mutation.IMPORTANCE Mutation produces the raw material for adaptive evolution but also imposes a burden because most mutations are deleterious. The rate of mutation at a particular site is affected by a variety of factors. In both prokaryotes and eukaryotes, methylation of C at the C5 position, a naturally occurring DNA modification, greatly increases the rate of C→T mutation. A distinct C modification that occurs in prokaryotes, methylation at N4, is not known to increase mutation rate. Here, I report that a bacterial restriction-modification system, found in some Salmonella bacteria, increases the rate of C→A mutation by a factor of 500 at sites that it methylates at N4. This rate increase is much greater than that caused by C5 methylation. Although fewer than 1 in 1,600 positions analyzed are methylation sites, over 10% of all mutations occur at these sites. Like other examples of extremely high mutation rate, whether naturally occurring or the result of laboratory mutation, this phenomenon may shed light on the mechanism of mutation in general.

Project description:BackgroundThe family of MAGE genes is well known due to the majority of MAGE genes expressing specifically in tumor tissues while restrictedly in normal tissues. MAGE-D4 is one of the MAGE family and considered as a promising target for glioma immunotherapy because of its overexpression in glioma and restricted expression in normal tissues. Whereas the mechanism of MAGE-D4 heterogeneous expression in glioma has not yet been elucidated. In this study, the transcriptional regulation mechanism of MAGE-D4 in glioma is focused from the perspectives of promoter methylation and SP1.MethodsDual-luciferase reporter assay was performed to identify the core promoter of MAGE-D4 gene. Mass spectrometry was applied to quantify the methylation status of MAGE-D4 promoter in 50 glioma and 9 normal brain tissues. The influence of methylation and SP1 on MAGE-D4 transcriptional activity was evaluated by dual-luciferase reporter assay, qRT-PCR, western blot and ChIP-qPCR. Decitabine, an epigenetic drug, was used to treat the glioma cells. Then the treated cells were evaluated the influence of demethylation on SP1 binding to MAGE-D4 promoter.ResultsThe -358 to +172 bp region was identified as the core promoter of MAGE-D4 gene which demonstrated hypomethylated and negative correlation between methylation level and MAGE-D4 mRNA expression in glioma tissues. For single CpG unit analysis, 8 CpG units (CpG unit 1, 2, 3, 4, 5, 6, 9 and 12) in MAGE-D4 core promoter showed hypomethylated in glioma and the methylation level of CpG unit 6 was positively associated with the prognosis of glioma patients. Furthermore, the methylation level of CpG unit 1 and 6 was negative negatively correlated with MAGE-D4 mRNA expression. Then, the results demonstrated that the promoter activity of MAGE-D4 was decreased by methylation in glioma cell lines. In addition, SP1 can binds directly to the MAGE-D4 promoter leading to up-regulation of MAGE-D4 mRNA through activation of its promoter. Finally, demethylation of MAGE-D4 promoter could benefit the SP1 binding and resulting co-activation of MAGE-D4 promoter by demethylation and SP1 in glioma cell lines.ConclusionThese findings indicate that the synergies of promoter hypomethylation and SP1 up-regulated MAGE-D4 transcription in glioma, which implies a potential approach to resolve the heterogeneous expression of MAGE-D4 in order to establish foundation for the MAGE-D4 based glioma therapy.

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Cytosine methylation of an Sp1 site contributes to organ-specific and cell-specific regulation of expression of the lung epithelial gene t1alpha.

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