The Journal of Experimental Medicine
IN Cell Analyzer 2000
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© The Rockefeller University Press, 0022-1007/1999/12/1595/ $5.00
The Journal of Experimental Medicine, Volume 190, Number 11, December 6, 1999 1595-1604

Original Article

Methylation-Dependent Gene Silencing Induced by Interleukin 1β via Nitric Oxide Production

Abdelkrim Hmadchaa, Francisco J. Bedoyaa, Francisco Sobrinoa, and Elizabeth Pintadoa

a Departamento de Bioquímica Médica y Biología Molecular, Facultad de Medicina y Hospital Universitario Virgen Macarena, Universidad de Sevilla, 41009 Sevilla, Spain
Departamento de Bioquímica, Médica y Biología Molecular, Facultad de Medicina, Avda. Sánchez Pizjuán, 4, 41009 Sevilla, Spain.34-95-490-704134-95-455-9852

elizabet{at}cica.es

Interleukin (IL)-1β is a pleiotropic cytokine implicated in a variety of activities, including damage of insulin-producing cells, brain injury, or neuromodulatory responses. Many of these effects are mediated by nitric oxide (NO) produced by the induction of NO synthase (iNOS) expression. We report here that IL-1β provokes a marked repression of genes, such as fragile X mental retardation 1 (FMR1) and hypoxanthine phosphoribosyltransferase (HPRT), having a CpG island in their promoter region. This effect can be fully prevented by iNOS inhibitors and is dependent on DNA methylation. NO donors also cause FMR1 and HPRT gene silencing. NO-induced methylation of FMR1 CpG island can be reverted by demethylating agents which, in turn, produce the recovery of gene expression. The effects of IL-1β and NO appear to be exerted through activation of DNA methyltransferase (DNA MeTase). Although exposure of the cells to NO does not increase DNA MeTase gene expression, the activity of the enzyme selectively increases when NO is applied directly on a nuclear protein extract. These findings reveal a previously unknown effect of IL-1β and NO on gene expression, and demonstrate a novel pathway for gene silencing based on activation of DNA MeTase by NO and acute modification of CpG island methylation.

Key Words: interleukin 1β • nitric oxide • FMR1 • CpG island methylation • gene repression

Abbreviations used in this paper: ActD, actinomycin D; AMT, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine; AzadC, 5-aza-2'-deoxycytidine; β-ME, β-mercaptoethanol; DNA MeTase, DNA methyltransferase; DTT, dithiothreitol; EIT, S-ethylisothiourea; FMR1, fragile X mental retardation 1 gene; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GSH, glutathione; HK, hexokinase; HPRT, hypoxanthine phosphoribosyltransferase; iNOS, inducible NOS; LDH, lactate dehydrogenase; L-NIL, L-N6-(1-iminoethyl)-lysine; NF-{kappa}B, nuclear factor {kappa}B; NMA, N-methyl arginine; NO, nitric oxide; NOS, NO synthase; PK, pyruvate kinase; RIN, RINm5F; RT, reverse transcription; SIN, 3-morpholinosydnonimine hydrochloride; SNAP, S-nitroso-N-acetylpenicillamine; SNP, sodium nitroprusside; SSPE, saline-sodium phosphate-EDTA; TSA, trichostatin A.

© 1999 The Rockefeller University Press


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