Abstract
The human dihydrofolate reductase (DHFR) gene was found to be undermethylated only in its 5' promoter region; the remaining CCGG residues in the 30-kilobase (kb) DHFR gene were insensitive to digestion by HpaII. Each of 27 CpG residues that were part of an HpaII or HhaI cutting site within a 1.1-kb segment of the DHFR gene promoter region were found to be unmethylated. All 80 copies of the DHFR gene in methotrexate-resistant HeLa cell line exhibited this pattern of undermethylation of only the promoter region. This same region was shown to be DNase I hypersensitive in chromatin from normal cells and from those cells in which the DHFR gene was amplified. Again, all copies of the amplified gene exhibited DNase I hypersensitivity of the promoter region. The remainder of the 30-kb DHFR gene is both completely methylated and insensitive to DNase I digestion. Detailed mapping of the DNase I-hypersensitive region revealed four strong cutting sites within a 500-base pair segment immediately upstream from the DHFR coding sequence and a weak cutting site within intron I. Two of the strong DNase I cutting sites in chromatin were also sensitive to S1 nuclease nicking when this DNA fragment was part of supercoiled plasmid DNA. Promoter undermethylation and DNase I hypersensitivity, features previously shown for specialized and inducible genes, have now been shown to be characteristic of the constitutively expressed DHFR gene. That these features characterize all copies of the amplified DHFR gene in a methotrexate-resistant cell line suggest that all gene copies are transcriptionally active.
Highlights
Cutting sites within a 500-base pair segment immedi- The Dihydrofolate reductase (DHFR) gene is among the class of genes, often referred ately upstream from theDHFR coding sequenceand a weak cutting site within intron I
The DHFR gene is constitutively expressed at bosyl transferase and the mouse DHFR genes is found only low levels in all cells
We have recently cloned and character- at the5’ ends; the remainder of the genes are fully methylated ized the functional human DHFR gene and three intronless a t the sites assayed with methyl-sensitive restriction endo
Summary
The structural characteristics of the human DHFR gene in Methylation Pattern of theHumanDHFR Genes-The normal cells, we investigated the statuosf the multiple DHFR methylation pattern of specific sites within the DHFR gene gene copies in cells resistant to MTXby virtue of amplifica- was assayed using the methylation-sensitiveenzyme, HpaII, tion of the DHFRgene. Southern Blot Analysis-Restriction endonuclease digestion of genomic DNA was performed at anenzyme to DNA ratio of 5 units/pg tively (Fig. 1A) Each of these bands is cut once or several times with MspI whereas the mobility of these bands is not changed by double digestion with HpaII. These results indicate that all of the HpaII sites within these fragments are methylatedontheirinternal cytosine residues. DNA sequencing of the 1.8-kg EcoRI fragment that contains the 5' end of the DHFR gene (Fig. 2 0 ) [1].Double digestion with EcoRI and either MspI or HpaIIreleased a 0.7-kb fragment thathybridized to the5' flanking probe, indicating that theHpaIIsite closest to the 5' end of the 1.8-kbEcoRI. Double digestion with EcoRI and either MspI or HpaII yielded a 0.15-kb fragment that annealed to thisprobe, indicating that the HpaII sitcelosest
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