Limits...
Methylation-dependent gene silencing induced by interleukin 1beta via nitric oxide production.

Hmadcha A, Bedoya FJ, Sobrino F, Pintado E - J. Exp. Med. (1999)

Bottom Line: The effects of IL-1beta 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-1beta 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.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica Médica y Biología Molecular, Facultad de Medicina y Hospital Universitario Virgen Macarena, Universidad de Sevilla, 41009 Sevilla, Spain.

ABSTRACT
Interleukin (IL)-1beta 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-1beta 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-1beta 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-1beta 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.

Show MeSH

Related in: MedlinePlus

HPRT expression in RIN and Jurkat T cells assessed by RT-PCR. (a) RIN cells unstimulated and treated for 24 h with IL-1β (25 U/ml) in the absence or presence of AzadC (2 μg/ml). (b) Similar protocol used with Jurkat T cells stimulated with 100 μM SIN. (c) RT-PCR of FMR1 and HPRT from fresh peripheral lymphocytes incubated for 24 h with SIN (100 μM) in the absence or presence of AzadC (2 μg/ml). RT-PCR of GAPDH was used as control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2195731&req=5

Figure 6: HPRT expression in RIN and Jurkat T cells assessed by RT-PCR. (a) RIN cells unstimulated and treated for 24 h with IL-1β (25 U/ml) in the absence or presence of AzadC (2 μg/ml). (b) Similar protocol used with Jurkat T cells stimulated with 100 μM SIN. (c) RT-PCR of FMR1 and HPRT from fresh peripheral lymphocytes incubated for 24 h with SIN (100 μM) in the absence or presence of AzadC (2 μg/ml). RT-PCR of GAPDH was used as control.

Mentions: Given that the inhibitory effect of NO on FMR1 expression can be explained by activation of DNA MeTase and methylation of the CpG island, we explored if a similar action is exerted on other genes, such as HPRT, known to contain a CpG island in the promoter region. Fig. 6 shows that exposure of RIN and Jurkat T cells to IL-1β (a) and NO donors (b), respectively, resulted in abolishment of HPRT expression. In both cases, demethylation with AzadC produced recovery of gene expression. NO-dependent methylation of CpG islands observed in transformed cells (such as RIN and Jurkat T cells) was also clearly apparent in fresh human lymphocytes. Fig. 6 c illustrates the silencing of FMR1 and HPRT genes by NO donors and the reversion of this effect by AzadC. The expression of genes such as GAPDH or Na/K ATP-ase, which do not contain CpG-rich promoters, is unaffected by NO (Fig. 6, a–c; see also Fig. 1Fig. 2Fig. 3).


Methylation-dependent gene silencing induced by interleukin 1beta via nitric oxide production.

Hmadcha A, Bedoya FJ, Sobrino F, Pintado E - J. Exp. Med. (1999)

HPRT expression in RIN and Jurkat T cells assessed by RT-PCR. (a) RIN cells unstimulated and treated for 24 h with IL-1β (25 U/ml) in the absence or presence of AzadC (2 μg/ml). (b) Similar protocol used with Jurkat T cells stimulated with 100 μM SIN. (c) RT-PCR of FMR1 and HPRT from fresh peripheral lymphocytes incubated for 24 h with SIN (100 μM) in the absence or presence of AzadC (2 μg/ml). RT-PCR of GAPDH was used as control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195731&req=5

Figure 6: HPRT expression in RIN and Jurkat T cells assessed by RT-PCR. (a) RIN cells unstimulated and treated for 24 h with IL-1β (25 U/ml) in the absence or presence of AzadC (2 μg/ml). (b) Similar protocol used with Jurkat T cells stimulated with 100 μM SIN. (c) RT-PCR of FMR1 and HPRT from fresh peripheral lymphocytes incubated for 24 h with SIN (100 μM) in the absence or presence of AzadC (2 μg/ml). RT-PCR of GAPDH was used as control.
Mentions: Given that the inhibitory effect of NO on FMR1 expression can be explained by activation of DNA MeTase and methylation of the CpG island, we explored if a similar action is exerted on other genes, such as HPRT, known to contain a CpG island in the promoter region. Fig. 6 shows that exposure of RIN and Jurkat T cells to IL-1β (a) and NO donors (b), respectively, resulted in abolishment of HPRT expression. In both cases, demethylation with AzadC produced recovery of gene expression. NO-dependent methylation of CpG islands observed in transformed cells (such as RIN and Jurkat T cells) was also clearly apparent in fresh human lymphocytes. Fig. 6 c illustrates the silencing of FMR1 and HPRT genes by NO donors and the reversion of this effect by AzadC. The expression of genes such as GAPDH or Na/K ATP-ase, which do not contain CpG-rich promoters, is unaffected by NO (Fig. 6, a–c; see also Fig. 1Fig. 2Fig. 3).

Bottom Line: The effects of IL-1beta 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-1beta 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.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica Médica y Biología Molecular, Facultad de Medicina y Hospital Universitario Virgen Macarena, Universidad de Sevilla, 41009 Sevilla, Spain.

ABSTRACT
Interleukin (IL)-1beta 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-1beta 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-1beta 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-1beta 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.

Show MeSH
Related in: MedlinePlus