In vitro methylation of the promoter and enhancer of Proα1(I) collagen gene leads to its transcriptional inactivation

James P. Thompson, Carl P. Simkevich, Maureen A. Holness, Andrew Kang, Rajendra Raghow

Research output: Contribution to journalArticle

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Abstract

We created pCOL-KT, a plasmid construct in which the promoter/enhancer of human Proα1(I) gene is linked to the chloramphenicol acetyl transferase reporter gene. The Proα1(I) promoter/enhancer in pCOL-KT was methylated in vitro and tested for transcriptional activity by transient expression analysis. Methylation of the construct with bacterial methylases reduced transcriptional activity about 25-fold. Site-specific methylation of eight potential canonical sites of eukaryotic methylation within the promoter greatly reduced transcriptional activity. Chromatin conformation of the transfected pCOL-KT DNA was analyzed by nuclease sensitivity. Although both methylated and unmethylated transfected DNA had increased susceptibility to DNase I compared with the endogenous gene, the methylated transfected DNA showed increased resistance to nuclease when compared with unmethylated transfected DNA, indicating that the methylation of the DNA alters the chromatin conformation. We also tested the ability of a human rhabdomyosarcoma cell line that does not express type I collagen to support transcription from an exogenously added Proα1(I) promoter/enhancer. The transformed cell line is able to support transcription from the Proα1(I) promoter/enhancer. Treatment of the transformed cell line with 5-azacytidine, a potent inhibitor of DNA methylation, resulted in transcriptional activation of the Proα1(I) gene. These findings, along with the extreme methylation sensitivity of the Proα1(I) promoter and enhancer, suggest that DNA methylation may be an important mechanism of transcriptional inactivation of interstitial collagen genes.

Original languageEnglish (US)
Pages (from-to)2549-2556
Number of pages8
JournalJournal of Biological Chemistry
Volume266
Issue number4
StatePublished - Feb 5 1991
Externally publishedYes

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Methylation
DNA Methylation
Collagen
Genes
Transformed Cell Line
DNA
Chromatin
Cells
Transcription
Conformations
Azacitidine
Rhabdomyosarcoma
Deoxyribonuclease I
Chloramphenicol
Transferases
Collagen Type I
Reporter Genes
Transcriptional Activation
Plasmids
Cell Line

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

In vitro methylation of the promoter and enhancer of Proα1(I) collagen gene leads to its transcriptional inactivation. / Thompson, James P.; Simkevich, Carl P.; Holness, Maureen A.; Kang, Andrew; Raghow, Rajendra.

In: Journal of Biological Chemistry, Vol. 266, No. 4, 05.02.1991, p. 2549-2556.

Research output: Contribution to journalArticle

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