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Recruitment of histone deacetylase 3 to the interferon-A gene promoters attenuates interferon expression.

Génin P, Lin R, Hiscott J, Civas A - PLoS ONE (2012)

Bottom Line: Analysis of chromatin-protein association by Chip-QPCR demonstrated that recruitment of interferon regulatory factor (IRF)3 and IRF7, as well as TBP correlated with enhanced histone H3K9 and H3K14 acetylation, whereas recruitment of HDAC3 correlated with inhibition of histone H3K9/K14 acetylation, removal of IRF7 and TATA-binding protein (TBP) from IFN-A promoters and inhibition of virus-induced IFN-A gene transcription.Additionally, HDAC3 overexpression reduced, and HDAC3 depletion by siRNA enhanced IFN-A gene expression.Furthermore, activation of IRF7 enhanced histone H3K9/K14 acetylation and IFN-A gene expression, whereas activation of both IRF7 and IRF3 led to recruitment of HDAC3 to the IFN-A gene promoters, resulting in impaired histone H3K9 acetylation and attenuation of IFN-A gene transcription.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique-FRE3235, Paris Descartes University, Paris, France.

ABSTRACT

Background: Induction of Type I Interferon (IFN) genes constitutes an essential step leading to innate immune responses during virus infection. Sendai virus (SeV) infection of B lymphoid Namalwa cells transiently induces the transcriptional expression of multiple IFN-A genes. Although transcriptional activation of IFN-A genes has been extensively studied, the mechanism responsible for the attenuation of their expression remains to be determined.

Principal findings: In this study, we demonstrate that virus infection of Namalwa cells induces transient recruitment of HDAC3 (histone deacetylase 3) to IFN-A promoters. Analysis of chromatin-protein association by Chip-QPCR demonstrated that recruitment of interferon regulatory factor (IRF)3 and IRF7, as well as TBP correlated with enhanced histone H3K9 and H3K14 acetylation, whereas recruitment of HDAC3 correlated with inhibition of histone H3K9/K14 acetylation, removal of IRF7 and TATA-binding protein (TBP) from IFN-A promoters and inhibition of virus-induced IFN-A gene transcription. Additionally, HDAC3 overexpression reduced, and HDAC3 depletion by siRNA enhanced IFN-A gene expression. Furthermore, activation of IRF7 enhanced histone H3K9/K14 acetylation and IFN-A gene expression, whereas activation of both IRF7 and IRF3 led to recruitment of HDAC3 to the IFN-A gene promoters, resulting in impaired histone H3K9 acetylation and attenuation of IFN-A gene transcription.

Conclusion: Altogether these data indicate that reversal of histone H3K9/K14 acetylation by HDAC3 is required for attenuation of IFN-A gene transcription during viral infection.

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Histone H3K9/K14 acetylation pattern associated with the IFN-A gene promoters during virus infection.(A) Acetylated histone H3K9, acetylated H3K14 and histone H3 associated with the promoter and the coding region of the IFN-A2 gene were determined by quantitative ChIP assays in Namalwa B cells infected by Sendai virus. Mean and standard deviation values of enrichment fold was calculated as described in Materials and Methods, with p values of statistical significance lesser than 0.01 indicated by **. These values derived from two replicate samples in two representative experiments. Anti-acetylated histone H3K9 and anti-acetylated H3K14 antibodies used in these experiments have shown to be highly specific for these acetylation sites [47], [48]. Anti-rabbit IgG serum was used as control of ChIP experiments. (B) Enrichment folds for the recruitment of GCN5 and PCAF to the IFN-A2 promoter were determined as described in Figure 2. Anti-goat IgG serum was used as control of ChIP experiments.
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pone-0038336-g003: Histone H3K9/K14 acetylation pattern associated with the IFN-A gene promoters during virus infection.(A) Acetylated histone H3K9, acetylated H3K14 and histone H3 associated with the promoter and the coding region of the IFN-A2 gene were determined by quantitative ChIP assays in Namalwa B cells infected by Sendai virus. Mean and standard deviation values of enrichment fold was calculated as described in Materials and Methods, with p values of statistical significance lesser than 0.01 indicated by **. These values derived from two replicate samples in two representative experiments. Anti-acetylated histone H3K9 and anti-acetylated H3K14 antibodies used in these experiments have shown to be highly specific for these acetylation sites [47], [48]. Anti-rabbit IgG serum was used as control of ChIP experiments. (B) Enrichment folds for the recruitment of GCN5 and PCAF to the IFN-A2 promoter were determined as described in Figure 2. Anti-goat IgG serum was used as control of ChIP experiments.

Mentions: Genome-wide analyses of histone acetylation have demonstrated that the acetylation of individual lysines in histone H3 and H4 tails and more specifically histone H3 acetylation at lysines K9 and K14 correlates with the active state of gene transcription [55], [56], [57]. To further understand the involvement of histone acetylation in regulating IFN-A gene expression, the modulation of histone H3K9 and H3K14 acetylation at the IFN-A promoters was examined in SeV-infected Namalwa B cells. At 6–12 h p.i, robust H3K9 and H3K14 acetylation was detected, with H3K9 acetylation levels 3- to 5-fold higher than those detected earlier (4 h) or later (14–16 h) times after infection (Fig. 3Aand Fig. S2A). Virus-infection increased H3K14 acetylation by 2- to 3-fold compare to the constitutive levels detected in uninfected cells. ChIP experiments with histone H3 antibodies indicated that nucleosome occupancy remained stable in the promoter region (Fig. 3A), thus confirming that promoter-specific histone H3K9 and H3K14 acetylation was modulated during virus infection. Accordingly, histone acetylation of the IFN-A promoters was barely detectable in uninfected Namalwa B cells at different time points (Fig. S3A). Low levels of histone H3 acetylation were observed within the coding region of IFN-A genes, suggesting that virus-infection induced local acetylation at the IFN-A gene promoter regions (Fig. 3A). As a negative control, virus infection did not induce c-fos gene expression and did not modify histone H3 marks associated with the c-fos promoter (data not shown). Negative controls also included PCR amplifications performed with the chromatin extracts in the absence of antibodies or with chromatin precipitated in the presence of irrelevant antisera (Fig. S3B). H3K9 and H3K14 acetylation associated with IFN-A gene promoters strongly decreased at 14–16 h p.i (Fig. 3A and Fig. S2A). Furthermore, inhibition of H3K9 and H3K14 acetylation correlated with removal of IRF7 and TBP from the IFN-A promoters and inhibition of transcription (seeFigs. 1and2). These results indicated that inhibition of SeV-induced IFN-A gene transcription was related to inhibition of histone H3K9/K14 acetylation in the IFN-A gene promoters.


Recruitment of histone deacetylase 3 to the interferon-A gene promoters attenuates interferon expression.

Génin P, Lin R, Hiscott J, Civas A - PLoS ONE (2012)

Histone H3K9/K14 acetylation pattern associated with the IFN-A gene promoters during virus infection.(A) Acetylated histone H3K9, acetylated H3K14 and histone H3 associated with the promoter and the coding region of the IFN-A2 gene were determined by quantitative ChIP assays in Namalwa B cells infected by Sendai virus. Mean and standard deviation values of enrichment fold was calculated as described in Materials and Methods, with p values of statistical significance lesser than 0.01 indicated by **. These values derived from two replicate samples in two representative experiments. Anti-acetylated histone H3K9 and anti-acetylated H3K14 antibodies used in these experiments have shown to be highly specific for these acetylation sites [47], [48]. Anti-rabbit IgG serum was used as control of ChIP experiments. (B) Enrichment folds for the recruitment of GCN5 and PCAF to the IFN-A2 promoter were determined as described in Figure 2. Anti-goat IgG serum was used as control of ChIP experiments.
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getmorefigures.php?uid=PMC3369917&req=5

pone-0038336-g003: Histone H3K9/K14 acetylation pattern associated with the IFN-A gene promoters during virus infection.(A) Acetylated histone H3K9, acetylated H3K14 and histone H3 associated with the promoter and the coding region of the IFN-A2 gene were determined by quantitative ChIP assays in Namalwa B cells infected by Sendai virus. Mean and standard deviation values of enrichment fold was calculated as described in Materials and Methods, with p values of statistical significance lesser than 0.01 indicated by **. These values derived from two replicate samples in two representative experiments. Anti-acetylated histone H3K9 and anti-acetylated H3K14 antibodies used in these experiments have shown to be highly specific for these acetylation sites [47], [48]. Anti-rabbit IgG serum was used as control of ChIP experiments. (B) Enrichment folds for the recruitment of GCN5 and PCAF to the IFN-A2 promoter were determined as described in Figure 2. Anti-goat IgG serum was used as control of ChIP experiments.
Mentions: Genome-wide analyses of histone acetylation have demonstrated that the acetylation of individual lysines in histone H3 and H4 tails and more specifically histone H3 acetylation at lysines K9 and K14 correlates with the active state of gene transcription [55], [56], [57]. To further understand the involvement of histone acetylation in regulating IFN-A gene expression, the modulation of histone H3K9 and H3K14 acetylation at the IFN-A promoters was examined in SeV-infected Namalwa B cells. At 6–12 h p.i, robust H3K9 and H3K14 acetylation was detected, with H3K9 acetylation levels 3- to 5-fold higher than those detected earlier (4 h) or later (14–16 h) times after infection (Fig. 3Aand Fig. S2A). Virus-infection increased H3K14 acetylation by 2- to 3-fold compare to the constitutive levels detected in uninfected cells. ChIP experiments with histone H3 antibodies indicated that nucleosome occupancy remained stable in the promoter region (Fig. 3A), thus confirming that promoter-specific histone H3K9 and H3K14 acetylation was modulated during virus infection. Accordingly, histone acetylation of the IFN-A promoters was barely detectable in uninfected Namalwa B cells at different time points (Fig. S3A). Low levels of histone H3 acetylation were observed within the coding region of IFN-A genes, suggesting that virus-infection induced local acetylation at the IFN-A gene promoter regions (Fig. 3A). As a negative control, virus infection did not induce c-fos gene expression and did not modify histone H3 marks associated with the c-fos promoter (data not shown). Negative controls also included PCR amplifications performed with the chromatin extracts in the absence of antibodies or with chromatin precipitated in the presence of irrelevant antisera (Fig. S3B). H3K9 and H3K14 acetylation associated with IFN-A gene promoters strongly decreased at 14–16 h p.i (Fig. 3A and Fig. S2A). Furthermore, inhibition of H3K9 and H3K14 acetylation correlated with removal of IRF7 and TBP from the IFN-A promoters and inhibition of transcription (seeFigs. 1and2). These results indicated that inhibition of SeV-induced IFN-A gene transcription was related to inhibition of histone H3K9/K14 acetylation in the IFN-A gene promoters.

Bottom Line: Analysis of chromatin-protein association by Chip-QPCR demonstrated that recruitment of interferon regulatory factor (IRF)3 and IRF7, as well as TBP correlated with enhanced histone H3K9 and H3K14 acetylation, whereas recruitment of HDAC3 correlated with inhibition of histone H3K9/K14 acetylation, removal of IRF7 and TATA-binding protein (TBP) from IFN-A promoters and inhibition of virus-induced IFN-A gene transcription.Additionally, HDAC3 overexpression reduced, and HDAC3 depletion by siRNA enhanced IFN-A gene expression.Furthermore, activation of IRF7 enhanced histone H3K9/K14 acetylation and IFN-A gene expression, whereas activation of both IRF7 and IRF3 led to recruitment of HDAC3 to the IFN-A gene promoters, resulting in impaired histone H3K9 acetylation and attenuation of IFN-A gene transcription.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique-FRE3235, Paris Descartes University, Paris, France.

ABSTRACT

Background: Induction of Type I Interferon (IFN) genes constitutes an essential step leading to innate immune responses during virus infection. Sendai virus (SeV) infection of B lymphoid Namalwa cells transiently induces the transcriptional expression of multiple IFN-A genes. Although transcriptional activation of IFN-A genes has been extensively studied, the mechanism responsible for the attenuation of their expression remains to be determined.

Principal findings: In this study, we demonstrate that virus infection of Namalwa cells induces transient recruitment of HDAC3 (histone deacetylase 3) to IFN-A promoters. Analysis of chromatin-protein association by Chip-QPCR demonstrated that recruitment of interferon regulatory factor (IRF)3 and IRF7, as well as TBP correlated with enhanced histone H3K9 and H3K14 acetylation, whereas recruitment of HDAC3 correlated with inhibition of histone H3K9/K14 acetylation, removal of IRF7 and TATA-binding protein (TBP) from IFN-A promoters and inhibition of virus-induced IFN-A gene transcription. Additionally, HDAC3 overexpression reduced, and HDAC3 depletion by siRNA enhanced IFN-A gene expression. Furthermore, activation of IRF7 enhanced histone H3K9/K14 acetylation and IFN-A gene expression, whereas activation of both IRF7 and IRF3 led to recruitment of HDAC3 to the IFN-A gene promoters, resulting in impaired histone H3K9 acetylation and attenuation of IFN-A gene transcription.

Conclusion: Altogether these data indicate that reversal of histone H3K9/K14 acetylation by HDAC3 is required for attenuation of IFN-A gene transcription during viral infection.

Show MeSH
Related in: MedlinePlus