Abstract
The methylation-sensitive restriction endonucleases HpaII and HhaI and the methylation-insensitive enzyme MspI were used to examine the methylation status of the 5' end of the human apolipoprotein B gene in cells in which the gene is transcriptionally active, such as HepG2 and CaCo-2 cells, and in HeLa cells, which do not express the gene. Several CCGG and GCGC sequences present in the region from -899 to +121 relative to the transcriptional start of the gene were undermethylated in HepG2 and CaCo-2 cells but methylated in HeLa cells. Because the region between -899 and +121 had previously been shown to exhibit tissue-specific promoter activity and to contain many DNaseI- and micrococcal nuclease-hypersensitive sites (Levy-Wilson, B., Fortier, C., Blackhart, B.D., and McCarthy, B.J. (1988) Mol. Cell. Biol. 8, 71-80), the current results suggest a correlation between undermethylation and nuclease sensitivity at the 5' end of the apolipoprotein B gene and expression of the gene.
Highlights
The methylation-sensitive restriction endonucleases Up11 and Hhal and the methylation-insensitive enzyme MspI were used to examine the methylation status of the 5’ end of the human apolipoprotein B gene in cells in which the gene is transcriptionally active, such as HepG2 and CaCo-2 cells, and in HeLa cells, which do not express the gene.Several CCGG and GCGC sequences present in the region from -899 to +121 relative to the transcriptional start of the gene were undermethylated in HepGZ and CaCo-2 cells but methylated in HeLa cells
To achieve a more comprehensive understanding of the controlfactors involved inapoB gene regulation, we have examined the extentof DNA methylation in the 5‘ end of the gene
Because methylation of cytosines is the only postsynthetic modification that hasbeen detected in the DNA of high eucaryotes, it has been the basis of several proposed mechanisms of gene activity and cellular differentiation [10]
Summary
Apolipoprotein B PromRoetegrion quots containing 10 pgof DNA digested either with PuuII or with HindIII and PstI and subsequently with either HpaII, MspI,or HhaI were electrophoresed in 1.5 or 1.8% agarose gels, transferred by Southernblotting onto nitrocellulose filters, and hybridized with various fragments from the 5' end of the human apoB gene, as shown in the corresponding figures.
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