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A novel pathway links oxidative stress to loss of insulin growth factor-2 (IGF2) imprinting through NF-κB activation.

Yang B, Wagner J, Damaschke N, Yao T, Wuerzberger-Davis SM, Lee MH, Svaren J, Miyamoto S, Jarrard DF - PLoS ONE (2014)

Bottom Line: Assays identify a recruitment of the canonical pathway proteins NF-κB p65 and p50 to the CTCF promoter associated with the co-repressor HDAC1 explaining gene repression.We conclude CTCF plays a central role in mediating the effects of NF-κB activation that result in altered imprinting both in vitro and in vivo.This novel finding connects inflammation found in aging prostate tissues with the altered epigenetic landscape.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America.

ABSTRACT
Genomic imprinting is the allele-specific expression of a gene based on parental origin. Loss of imprinting(LOI) of Insulin-like Growth Factor 2 (IGF2) during aging is important in tumorigenesis, yet the regulatory mechanisms driving this event are largely unknown. In this study oxidative stress, measured by increased NF-κB activity, induces LOI in both cancerous and noncancerous human prostate cells. Decreased expression of the enhancer-blocking element CCCTC-binding factor(CTCF) results in reduced binding of CTCF to the H19-ICR (imprint control region), a major factor in the allelic silencing of IGF2. This ICR then develops increased DNA methylation. Assays identify a recruitment of the canonical pathway proteins NF-κB p65 and p50 to the CTCF promoter associated with the co-repressor HDAC1 explaining gene repression. An IκBα super-repressor blocks oxidative stress-induced activation of NF-κB and IGF2 imprinting is maintained. In vivo experiments using IκBα mutant mice with continuous NF-κB activation demonstrate increased IGF2 LOI further confirming a central role for canonical NF-κB signaling. We conclude CTCF plays a central role in mediating the effects of NF-κB activation that result in altered imprinting both in vitro and in vivo. This novel finding connects inflammation found in aging prostate tissues with the altered epigenetic landscape.

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An IκBα super-repressor blocks NF-κB activity and IGF2 LOI.(A) IκBα super repressor blocks oxidative stress-induced NF-κB activity. An IκBα super-repressor was utilized that contains mutant IκBα resistant to phosphorylation and degradation, thus blocking canonical NF-κB activation. Stable integration of this super-repressor or empty vector control was performed in PPC1 and 9E6/E7. These cells were then transduced with an NF-κB reporter for 48 hr and then treated with H2O2 for 6 hr. Values are expressed as mean+/−S.D. of three independent experiments. ** P<0.01 (t-test). (B) IκBα super-repressor inhibits oxidative stress-induced decreases in CTCF protein expression. Western blot of protein lysates were analyzed and quantitated. (C) IκBα super-repressor inhibits oxidative stress-induced decreases in CTCF mRNA expression. The mRNA levels of CTCF were measured using RT-qPCR in the cells. (D) IκBα super-repressor blocks loss of IGF2 imprinting. FLuPE analysis of RNA was performed to evaluate allele-specific expression.
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pone-0088052-g003: An IκBα super-repressor blocks NF-κB activity and IGF2 LOI.(A) IκBα super repressor blocks oxidative stress-induced NF-κB activity. An IκBα super-repressor was utilized that contains mutant IκBα resistant to phosphorylation and degradation, thus blocking canonical NF-κB activation. Stable integration of this super-repressor or empty vector control was performed in PPC1 and 9E6/E7. These cells were then transduced with an NF-κB reporter for 48 hr and then treated with H2O2 for 6 hr. Values are expressed as mean+/−S.D. of three independent experiments. ** P<0.01 (t-test). (B) IκBα super-repressor inhibits oxidative stress-induced decreases in CTCF protein expression. Western blot of protein lysates were analyzed and quantitated. (C) IκBα super-repressor inhibits oxidative stress-induced decreases in CTCF mRNA expression. The mRNA levels of CTCF were measured using RT-qPCR in the cells. (D) IκBα super-repressor blocks loss of IGF2 imprinting. FLuPE analysis of RNA was performed to evaluate allele-specific expression.

Mentions: It was then determined whether IGF2 LOI induced by H2O2 is dependent on the activation of NF-κB signaling. To specifically inactivate canonical NF-κB signaling, a retroviral construct harboring super-repressor IκBα mutant (or control) was stably transfected into PPC1 and 9E6/E7 cells. The stable cell lines were then transiently transduced with the NF-κB-dependent luciferase reporter gene for 48 hr and subsequently treated with H2O2. NF-κB reporter activity was induced in PPC1 (14 fold) and 9E6/E7 (2.3 fold) (Fig. 3A) in empty vector control lines. NFκB activity was not significantly altered in the super-repressor stable cells indicating effective blocking of NF-κB.


A novel pathway links oxidative stress to loss of insulin growth factor-2 (IGF2) imprinting through NF-κB activation.

Yang B, Wagner J, Damaschke N, Yao T, Wuerzberger-Davis SM, Lee MH, Svaren J, Miyamoto S, Jarrard DF - PLoS ONE (2014)

An IκBα super-repressor blocks NF-κB activity and IGF2 LOI.(A) IκBα super repressor blocks oxidative stress-induced NF-κB activity. An IκBα super-repressor was utilized that contains mutant IκBα resistant to phosphorylation and degradation, thus blocking canonical NF-κB activation. Stable integration of this super-repressor or empty vector control was performed in PPC1 and 9E6/E7. These cells were then transduced with an NF-κB reporter for 48 hr and then treated with H2O2 for 6 hr. Values are expressed as mean+/−S.D. of three independent experiments. ** P<0.01 (t-test). (B) IκBα super-repressor inhibits oxidative stress-induced decreases in CTCF protein expression. Western blot of protein lysates were analyzed and quantitated. (C) IκBα super-repressor inhibits oxidative stress-induced decreases in CTCF mRNA expression. The mRNA levels of CTCF were measured using RT-qPCR in the cells. (D) IκBα super-repressor blocks loss of IGF2 imprinting. FLuPE analysis of RNA was performed to evaluate allele-specific expression.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3928145&req=5

pone-0088052-g003: An IκBα super-repressor blocks NF-κB activity and IGF2 LOI.(A) IκBα super repressor blocks oxidative stress-induced NF-κB activity. An IκBα super-repressor was utilized that contains mutant IκBα resistant to phosphorylation and degradation, thus blocking canonical NF-κB activation. Stable integration of this super-repressor or empty vector control was performed in PPC1 and 9E6/E7. These cells were then transduced with an NF-κB reporter for 48 hr and then treated with H2O2 for 6 hr. Values are expressed as mean+/−S.D. of three independent experiments. ** P<0.01 (t-test). (B) IκBα super-repressor inhibits oxidative stress-induced decreases in CTCF protein expression. Western blot of protein lysates were analyzed and quantitated. (C) IκBα super-repressor inhibits oxidative stress-induced decreases in CTCF mRNA expression. The mRNA levels of CTCF were measured using RT-qPCR in the cells. (D) IκBα super-repressor blocks loss of IGF2 imprinting. FLuPE analysis of RNA was performed to evaluate allele-specific expression.
Mentions: It was then determined whether IGF2 LOI induced by H2O2 is dependent on the activation of NF-κB signaling. To specifically inactivate canonical NF-κB signaling, a retroviral construct harboring super-repressor IκBα mutant (or control) was stably transfected into PPC1 and 9E6/E7 cells. The stable cell lines were then transiently transduced with the NF-κB-dependent luciferase reporter gene for 48 hr and subsequently treated with H2O2. NF-κB reporter activity was induced in PPC1 (14 fold) and 9E6/E7 (2.3 fold) (Fig. 3A) in empty vector control lines. NFκB activity was not significantly altered in the super-repressor stable cells indicating effective blocking of NF-κB.

Bottom Line: Assays identify a recruitment of the canonical pathway proteins NF-κB p65 and p50 to the CTCF promoter associated with the co-repressor HDAC1 explaining gene repression.We conclude CTCF plays a central role in mediating the effects of NF-κB activation that result in altered imprinting both in vitro and in vivo.This novel finding connects inflammation found in aging prostate tissues with the altered epigenetic landscape.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America.

ABSTRACT
Genomic imprinting is the allele-specific expression of a gene based on parental origin. Loss of imprinting(LOI) of Insulin-like Growth Factor 2 (IGF2) during aging is important in tumorigenesis, yet the regulatory mechanisms driving this event are largely unknown. In this study oxidative stress, measured by increased NF-κB activity, induces LOI in both cancerous and noncancerous human prostate cells. Decreased expression of the enhancer-blocking element CCCTC-binding factor(CTCF) results in reduced binding of CTCF to the H19-ICR (imprint control region), a major factor in the allelic silencing of IGF2. This ICR then develops increased DNA methylation. Assays identify a recruitment of the canonical pathway proteins NF-κB p65 and p50 to the CTCF promoter associated with the co-repressor HDAC1 explaining gene repression. An IκBα super-repressor blocks oxidative stress-induced activation of NF-κB and IGF2 imprinting is maintained. In vivo experiments using IκBα mutant mice with continuous NF-κB activation demonstrate increased IGF2 LOI further confirming a central role for canonical NF-κB signaling. We conclude CTCF plays a central role in mediating the effects of NF-κB activation that result in altered imprinting both in vitro and in vivo. This novel finding connects inflammation found in aging prostate tissues with the altered epigenetic landscape.

Show MeSH
Related in: MedlinePlus