Analyses of the genomic methylation status of the human cyclin A1 promoter by a novel real-time PCR-based methodology.
The role of CpG methylation in the regulation of tissue-specific gene expression is highly controversial. Cyclin A1 is a tissue-specifically expressed gene that is strongly methylated in non-expressing tumor cell lines. We have established a novel real-time PCR method to quantitate genomic CpG methylation of the cyclin A1 promoter. Genomic DNA samples from different human organs were treated with bisulfite and amplified with methylation-specific primers and with primers amplifying methylated as well as non-methylated DNA. PCR product quantitation was obtained by using a fluorogenic probe labeled with FAM and TAMRA. These analyses demonstrated that the human cyclin A1 promoter was methylated in kidney, colon, spleen, testis, and small intestine, but not in brain, liver, pancreas, or heart. Expression of cyclin A1 was predominantly found in testis. Low level expression of cyclin A1 was present in spleen, prostate, leukocytes, colon, and thymus. Taken together, our data provide evidence that CpG methylation patterns of the human cyclin A1 promoter in human organs do not generally correlate with cyclin A1 gene expression in vivo.
Müller-Tidow
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Department of Medicine, Hematology and Oncology, University of Münster, Domagkstr. 3, 48129 Münster, Germany. muellerc@uni-muenster.de
Bornemann
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Diederichs
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Westermann
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Klümpen
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Zuo
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Wang
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Berdel
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Serve
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D003106:Colon / Q000378:metabolism; D018899:CpG Islands / Q000235:genetics; D019925:Cyclin A / Q000235:genetics*; D056750:Cyclin A1; D019175:DNA Methylation; D018076:DNA, Complementary / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007421:Intestine, Small / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013154:Spleen / Q000378:metabolism; D013447:Sulfites / Q000378:metabolism; D013737:Testis / Q000378:metabolism; D013997:Time Factors; D014018:Tissue Distribution; D020298:U937 Cells
C503745:CCNA1 protein, human; D019925:Cyclin A; D056750:Cyclin A1; D018076:DNA, Complementary; D013447:Sulfites