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Persistent prion infection disturbs the function of Oct-1, resulting in the down-regulation of murine interferon regulatory factor-3.

Homma T, Ishibashi D, Nakagaki T, Fuse T, Sano K, Satoh K, Atarashi R, Nishida N - Sci Rep (2014)

Bottom Line: We further investigated promoter activity of 5'- flanking region of murine IRF-3 using a luciferase reporter system and found that the nucleotides -119 to -1 were indispensable for the promoter activity.Intriguingly, Oct-1 protein was significantly reduced in prion-infected cells and mice brains compared with uninfected groups.Taken together, we concluded that prion infection could interfere in the function of Oct-1, resulting in the down-regulation of IRF-3.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan [2].

ABSTRACT
As a prompt response against invasion of various viruses, interferon regulatory factor-3 (IRF-3) is initially phosphorylated to become activated and upregulates mainly Type I Interferons (IFN-I) in most cell types. We previously reported that IRF-3-dependent host innate immune responses partially interfere in infection of prions. Here, we found that stable infection of prion suppressed IRF-3 gene-expression. The decreased promoter activity of IRF-3 was significantly restored along with treatment of anti-prion drugs in the prion-infected cells, suggesting that infection of prion directly influence the regulation of IRF-3 transcription. We further investigated promoter activity of 5'- flanking region of murine IRF-3 using a luciferase reporter system and found that the nucleotides -119 to -1 were indispensable for the promoter activity. Within this region, mutations in the Oct-1 binding site significantly reduced the promoter activity and chromatin immunoprecipitation (ChIP) assay revealed that Oct-1 indeed binds to the region. In addition, overexpression of Oct-1 increased the promoter activity of IRF-3. Intriguingly, Oct-1 protein was significantly reduced in prion-infected cells and mice brains compared with uninfected groups. Taken together, we concluded that prion infection could interfere in the function of Oct-1, resulting in the down-regulation of IRF-3.

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Exogenous Oct-1 regulates murine IRF-3 promoter activity.(a) N2a58 cell lysate was subjected to ChIP assay using anti-Oct-1 (IP: Oct-1) or normal rabbit IgG (negative control) antibodies. After the DNA was purified, the promoter region (nt -119 to -1) was amplified by PCR (see “Materials and Methods” in detail). (b) N2a cells were co-transfected with either pGL3 -119/-1 or pGL3 -119/-1 (M1) and either pcDNA Oct-1-HA (Oct-1) or the empty plasmid (mock). Results were normalized to the control renilla luciferase activity, and the value of (pGL3 -119/-1 + mock) was set to 100%. Results representing the mean ± SD, n = 4. P values were determined by one-way ANOVA followed by Tukey's multiple comparison (***: P < 0.001, n.s.: not significant).
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f4: Exogenous Oct-1 regulates murine IRF-3 promoter activity.(a) N2a58 cell lysate was subjected to ChIP assay using anti-Oct-1 (IP: Oct-1) or normal rabbit IgG (negative control) antibodies. After the DNA was purified, the promoter region (nt -119 to -1) was amplified by PCR (see “Materials and Methods” in detail). (b) N2a cells were co-transfected with either pGL3 -119/-1 or pGL3 -119/-1 (M1) and either pcDNA Oct-1-HA (Oct-1) or the empty plasmid (mock). Results were normalized to the control renilla luciferase activity, and the value of (pGL3 -119/-1 + mock) was set to 100%. Results representing the mean ± SD, n = 4. P values were determined by one-way ANOVA followed by Tukey's multiple comparison (***: P < 0.001, n.s.: not significant).

Mentions: To confirm that Oct-1 binds to the endogenous murine IRF-3 promoter, chromatin immunoprecipitation (ChIP) assay was performed. PCR analysis revealed that the Oct-1 antibody precipitated the promoter region (nt -119 to -1) from N2a58 cells (Fig. 4a), while the negative control (anti-rabbit IgG) did not exhibit the DNA binding activity. These data suggest that endogenous Oct-1 binds to the promoter region in vivo. Furthermore, to investigate whether the promoter activity is affected by Oct-1, pcDNA Oct-1-HA and pGL3 -119/-1 were co-transfected into N2a cells. Ectopically Oct-1 overexpression significantly increased the original promoter activities (pGL3 -119/-1 + mock vs pGL3 -119/-1 + pcDNA Oct-1-HA), while Oct-1 expression had no effect on the mutated promoter activities [pGL3 -119/-1 (M1) + mock vs pGL3 -119/-1 (M1) + pcDNA Oct-1-HA], indicating exogenous Oct-1 functions in the promoter region (Fig. 4b).


Persistent prion infection disturbs the function of Oct-1, resulting in the down-regulation of murine interferon regulatory factor-3.

Homma T, Ishibashi D, Nakagaki T, Fuse T, Sano K, Satoh K, Atarashi R, Nishida N - Sci Rep (2014)

Exogenous Oct-1 regulates murine IRF-3 promoter activity.(a) N2a58 cell lysate was subjected to ChIP assay using anti-Oct-1 (IP: Oct-1) or normal rabbit IgG (negative control) antibodies. After the DNA was purified, the promoter region (nt -119 to -1) was amplified by PCR (see “Materials and Methods” in detail). (b) N2a cells were co-transfected with either pGL3 -119/-1 or pGL3 -119/-1 (M1) and either pcDNA Oct-1-HA (Oct-1) or the empty plasmid (mock). Results were normalized to the control renilla luciferase activity, and the value of (pGL3 -119/-1 + mock) was set to 100%. Results representing the mean ± SD, n = 4. P values were determined by one-way ANOVA followed by Tukey's multiple comparison (***: P < 0.001, n.s.: not significant).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4126003&req=5

f4: Exogenous Oct-1 regulates murine IRF-3 promoter activity.(a) N2a58 cell lysate was subjected to ChIP assay using anti-Oct-1 (IP: Oct-1) or normal rabbit IgG (negative control) antibodies. After the DNA was purified, the promoter region (nt -119 to -1) was amplified by PCR (see “Materials and Methods” in detail). (b) N2a cells were co-transfected with either pGL3 -119/-1 or pGL3 -119/-1 (M1) and either pcDNA Oct-1-HA (Oct-1) or the empty plasmid (mock). Results were normalized to the control renilla luciferase activity, and the value of (pGL3 -119/-1 + mock) was set to 100%. Results representing the mean ± SD, n = 4. P values were determined by one-way ANOVA followed by Tukey's multiple comparison (***: P < 0.001, n.s.: not significant).
Mentions: To confirm that Oct-1 binds to the endogenous murine IRF-3 promoter, chromatin immunoprecipitation (ChIP) assay was performed. PCR analysis revealed that the Oct-1 antibody precipitated the promoter region (nt -119 to -1) from N2a58 cells (Fig. 4a), while the negative control (anti-rabbit IgG) did not exhibit the DNA binding activity. These data suggest that endogenous Oct-1 binds to the promoter region in vivo. Furthermore, to investigate whether the promoter activity is affected by Oct-1, pcDNA Oct-1-HA and pGL3 -119/-1 were co-transfected into N2a cells. Ectopically Oct-1 overexpression significantly increased the original promoter activities (pGL3 -119/-1 + mock vs pGL3 -119/-1 + pcDNA Oct-1-HA), while Oct-1 expression had no effect on the mutated promoter activities [pGL3 -119/-1 (M1) + mock vs pGL3 -119/-1 (M1) + pcDNA Oct-1-HA], indicating exogenous Oct-1 functions in the promoter region (Fig. 4b).

Bottom Line: We further investigated promoter activity of 5'- flanking region of murine IRF-3 using a luciferase reporter system and found that the nucleotides -119 to -1 were indispensable for the promoter activity.Intriguingly, Oct-1 protein was significantly reduced in prion-infected cells and mice brains compared with uninfected groups.Taken together, we concluded that prion infection could interfere in the function of Oct-1, resulting in the down-regulation of IRF-3.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan [2].

ABSTRACT
As a prompt response against invasion of various viruses, interferon regulatory factor-3 (IRF-3) is initially phosphorylated to become activated and upregulates mainly Type I Interferons (IFN-I) in most cell types. We previously reported that IRF-3-dependent host innate immune responses partially interfere in infection of prions. Here, we found that stable infection of prion suppressed IRF-3 gene-expression. The decreased promoter activity of IRF-3 was significantly restored along with treatment of anti-prion drugs in the prion-infected cells, suggesting that infection of prion directly influence the regulation of IRF-3 transcription. We further investigated promoter activity of 5'- flanking region of murine IRF-3 using a luciferase reporter system and found that the nucleotides -119 to -1 were indispensable for the promoter activity. Within this region, mutations in the Oct-1 binding site significantly reduced the promoter activity and chromatin immunoprecipitation (ChIP) assay revealed that Oct-1 indeed binds to the region. In addition, overexpression of Oct-1 increased the promoter activity of IRF-3. Intriguingly, Oct-1 protein was significantly reduced in prion-infected cells and mice brains compared with uninfected groups. Taken together, we concluded that prion infection could interfere in the function of Oct-1, resulting in the down-regulation of IRF-3.

Show MeSH
Related in: MedlinePlus