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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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Mutation analysis of murine IRF-3 promoter.(a) Nucleotides sequences of the promoter region (nt −119 to −1) and the putative binding sites for the transcription factors are indicated on the sequence. (b) Nucleotide sequences of the original and mutated (M1 and M2) Oct-1 binding sites. (c) The promoter activity of N2a cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M1). (d) The promoter activity of 3T3 cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M1). (e) The promoter activity of N2a cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M2). (f) The promoter activity of 3T3 cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M2). (g) Nucleotide sequences of the original and mutated (M3) E2F1 binding sites. (h) The promoter activity of N2a cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M3). (i) The promoter activity of 3T3 cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M3). Results were normalized to the co-expressed renilla luciferase activity, and the value of the original activity was set to 100%. Results represent the mean ± SD, n = 4. P values were determined by Student's-t test (***: P < 0.001; **: P < 0.01).
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f3: Mutation analysis of murine IRF-3 promoter.(a) Nucleotides sequences of the promoter region (nt −119 to −1) and the putative binding sites for the transcription factors are indicated on the sequence. (b) Nucleotide sequences of the original and mutated (M1 and M2) Oct-1 binding sites. (c) The promoter activity of N2a cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M1). (d) The promoter activity of 3T3 cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M1). (e) The promoter activity of N2a cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M2). (f) The promoter activity of 3T3 cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M2). (g) Nucleotide sequences of the original and mutated (M3) E2F1 binding sites. (h) The promoter activity of N2a cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M3). (i) The promoter activity of 3T3 cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M3). Results were normalized to the co-expressed renilla luciferase activity, and the value of the original activity was set to 100%. Results represent the mean ± SD, n = 4. P values were determined by Student's-t test (***: P < 0.001; **: P < 0.01).

Mentions: We identified putative transcription factor binding sites in nt -119 to -1 with the software TFSEARCH ver.1.3 (http://www.cbrc.jp/research/db/TFSEARCH.html) and found that this region contains a potential Oct-1 binding site (5′-ATTTGCAT-3′, nt -42 to -35) and an acute myeloid leukemia 1 protein (AML1) binding site (5′- TGCGGT-3′, nt -49 to -44). In addition, an E2F transcription factor 1 (E2F1) binding site (5′-TTTCCCAC-3′, nt -116 to -109) was also conserved in murine (Fig. 3a).


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)

Mutation analysis of murine IRF-3 promoter.(a) Nucleotides sequences of the promoter region (nt −119 to −1) and the putative binding sites for the transcription factors are indicated on the sequence. (b) Nucleotide sequences of the original and mutated (M1 and M2) Oct-1 binding sites. (c) The promoter activity of N2a cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M1). (d) The promoter activity of 3T3 cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M1). (e) The promoter activity of N2a cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M2). (f) The promoter activity of 3T3 cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M2). (g) Nucleotide sequences of the original and mutated (M3) E2F1 binding sites. (h) The promoter activity of N2a cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M3). (i) The promoter activity of 3T3 cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M3). Results were normalized to the co-expressed renilla luciferase activity, and the value of the original activity was set to 100%. Results represent the mean ± SD, n = 4. P values were determined by Student's-t test (***: P < 0.001; **: P < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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f3: Mutation analysis of murine IRF-3 promoter.(a) Nucleotides sequences of the promoter region (nt −119 to −1) and the putative binding sites for the transcription factors are indicated on the sequence. (b) Nucleotide sequences of the original and mutated (M1 and M2) Oct-1 binding sites. (c) The promoter activity of N2a cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M1). (d) The promoter activity of 3T3 cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M1). (e) The promoter activity of N2a cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M2). (f) The promoter activity of 3T3 cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M2). (g) Nucleotide sequences of the original and mutated (M3) E2F1 binding sites. (h) The promoter activity of N2a cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M3). (i) The promoter activity of 3T3 cells transiently transfected with pRL-, and pGL3 -119/-1 or pGL3 -119/-1 (M3). Results were normalized to the co-expressed renilla luciferase activity, and the value of the original activity was set to 100%. Results represent the mean ± SD, n = 4. P values were determined by Student's-t test (***: P < 0.001; **: P < 0.01).
Mentions: We identified putative transcription factor binding sites in nt -119 to -1 with the software TFSEARCH ver.1.3 (http://www.cbrc.jp/research/db/TFSEARCH.html) and found that this region contains a potential Oct-1 binding site (5′-ATTTGCAT-3′, nt -42 to -35) and an acute myeloid leukemia 1 protein (AML1) binding site (5′- TGCGGT-3′, nt -49 to -44). In addition, an E2F transcription factor 1 (E2F1) binding site (5′-TTTCCCAC-3′, nt -116 to -109) was also conserved in murine (Fig. 3a).

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