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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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Inhibitory effect of HDAC3 on IFN-A gene transcription.(A) The effect of HDACs class I (1, 2 and 3) on IFN-A gene transcription was determined in HEK293-TLR3 cells transfected with pcDNA3-IRF7A together with an HDAC-encoding plasmid added in 2-fold increasing amounts, as indicated in Materials and Methods. After 24 h of expression, the inhibition fold was calculated from IFN-A mRNA levels determined by RT-QPCR in HDAC-expressing cells in comparison to control cells transfected with pcDNA3. HDAC expression determined by anti-flag immunoblotting of the cell lysates is shown in the insets. (B) HDAC3 expression was specifically inhibited for 40 h by siRNA in HEK293-TLR3 cells expressing TRAF3 and IRF7A. IFN-A gene expression was determined after HDAC3 depletion by RT-QPCR following 8 h of infection by Sendai virus and compared to the expression determined in cells transfected with scrambled siRNA oligonucleotides. (C) Silencing efficiency was confirmed by RT-QPCR and Western blot analyses using total RNA and proteins extracted from cells transfected with siRNA oligonucleotides specific for HDAC3 or a mixture of scrambled (sc) siRNAs. (D) Histone H3K9 acetylation associated with the IFN-A2 gene promoter was determined in control and HDAC3-depleted cells by quantitative ChIP assays as described in Materials and Methods. Anti-rabbit IgG was used to determine non-specific binding. (E) IFN-B gene expression was determined by RT-QPCR following 8 h of infection by Sendai virus in HEK293-TLR3 cells expressing TRAF3 and IRF7A and transfected with scrambled siRNA or siRNA oligonucleotides specific for HDAC3.
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pone-0038336-g006: Inhibitory effect of HDAC3 on IFN-A gene transcription.(A) The effect of HDACs class I (1, 2 and 3) on IFN-A gene transcription was determined in HEK293-TLR3 cells transfected with pcDNA3-IRF7A together with an HDAC-encoding plasmid added in 2-fold increasing amounts, as indicated in Materials and Methods. After 24 h of expression, the inhibition fold was calculated from IFN-A mRNA levels determined by RT-QPCR in HDAC-expressing cells in comparison to control cells transfected with pcDNA3. HDAC expression determined by anti-flag immunoblotting of the cell lysates is shown in the insets. (B) HDAC3 expression was specifically inhibited for 40 h by siRNA in HEK293-TLR3 cells expressing TRAF3 and IRF7A. IFN-A gene expression was determined after HDAC3 depletion by RT-QPCR following 8 h of infection by Sendai virus and compared to the expression determined in cells transfected with scrambled siRNA oligonucleotides. (C) Silencing efficiency was confirmed by RT-QPCR and Western blot analyses using total RNA and proteins extracted from cells transfected with siRNA oligonucleotides specific for HDAC3 or a mixture of scrambled (sc) siRNAs. (D) Histone H3K9 acetylation associated with the IFN-A2 gene promoter was determined in control and HDAC3-depleted cells by quantitative ChIP assays as described in Materials and Methods. Anti-rabbit IgG was used to determine non-specific binding. (E) IFN-B gene expression was determined by RT-QPCR following 8 h of infection by Sendai virus in HEK293-TLR3 cells expressing TRAF3 and IRF7A and transfected with scrambled siRNA or siRNA oligonucleotides specific for HDAC3.

Mentions: In the same experimental model, HDAC3 overexpression produced a 2–4 fold dose-dependent inhibition of IRF7-mediated IFN-A gene expression without altering expression of either IRF3 or IRF7 (Fig. 6A). Other HDACs tested did not exert similar effects (Fig. S4). Conversely to the inhibitory effect of HDAC3 overexpression, silencing of HDAC3 expression by siRNA led to a 2-3-fold decrease in HDAC3 mRNA and protein levels and enhanced virus-induced IFN-A gene expression by 3- to 5-fold compared to cells transfected with control siRNA (Fig. 6B-C). Also, a significant 1.5 fold increase (p<0.01) in histone H3K9 acetylation of the IFN-A2 promoter was quantified in cells transfected with siRNA targeting HDAC3 (Fig. 6D). Since a knockdown screen performed in poly(I).poly(C)-treated cells showed that HDACs affected negatively (HDAC1 and HDAC8) or positively (HDAC6) the activity of a IFN-B promoter construct in reporter assays [64], we tested the effect of HDAC3 knockdown on SeV-induced IFN-B expression. Depletion of HDAC3 slightly enhanced SeV-induced IFN-B gene transcription by less than 2.5 fold (Fig. 6E) and overexpression of HDAC3 did not affect virus-induced IFN-B gene transcription (data not shown). These observations suggested that HDAC3 did not play a critical role in IFN-B gene regulation following virus infection.


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)

Inhibitory effect of HDAC3 on IFN-A gene transcription.(A) The effect of HDACs class I (1, 2 and 3) on IFN-A gene transcription was determined in HEK293-TLR3 cells transfected with pcDNA3-IRF7A together with an HDAC-encoding plasmid added in 2-fold increasing amounts, as indicated in Materials and Methods. After 24 h of expression, the inhibition fold was calculated from IFN-A mRNA levels determined by RT-QPCR in HDAC-expressing cells in comparison to control cells transfected with pcDNA3. HDAC expression determined by anti-flag immunoblotting of the cell lysates is shown in the insets. (B) HDAC3 expression was specifically inhibited for 40 h by siRNA in HEK293-TLR3 cells expressing TRAF3 and IRF7A. IFN-A gene expression was determined after HDAC3 depletion by RT-QPCR following 8 h of infection by Sendai virus and compared to the expression determined in cells transfected with scrambled siRNA oligonucleotides. (C) Silencing efficiency was confirmed by RT-QPCR and Western blot analyses using total RNA and proteins extracted from cells transfected with siRNA oligonucleotides specific for HDAC3 or a mixture of scrambled (sc) siRNAs. (D) Histone H3K9 acetylation associated with the IFN-A2 gene promoter was determined in control and HDAC3-depleted cells by quantitative ChIP assays as described in Materials and Methods. Anti-rabbit IgG was used to determine non-specific binding. (E) IFN-B gene expression was determined by RT-QPCR following 8 h of infection by Sendai virus in HEK293-TLR3 cells expressing TRAF3 and IRF7A and transfected with scrambled siRNA or siRNA oligonucleotides specific for HDAC3.
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pone-0038336-g006: Inhibitory effect of HDAC3 on IFN-A gene transcription.(A) The effect of HDACs class I (1, 2 and 3) on IFN-A gene transcription was determined in HEK293-TLR3 cells transfected with pcDNA3-IRF7A together with an HDAC-encoding plasmid added in 2-fold increasing amounts, as indicated in Materials and Methods. After 24 h of expression, the inhibition fold was calculated from IFN-A mRNA levels determined by RT-QPCR in HDAC-expressing cells in comparison to control cells transfected with pcDNA3. HDAC expression determined by anti-flag immunoblotting of the cell lysates is shown in the insets. (B) HDAC3 expression was specifically inhibited for 40 h by siRNA in HEK293-TLR3 cells expressing TRAF3 and IRF7A. IFN-A gene expression was determined after HDAC3 depletion by RT-QPCR following 8 h of infection by Sendai virus and compared to the expression determined in cells transfected with scrambled siRNA oligonucleotides. (C) Silencing efficiency was confirmed by RT-QPCR and Western blot analyses using total RNA and proteins extracted from cells transfected with siRNA oligonucleotides specific for HDAC3 or a mixture of scrambled (sc) siRNAs. (D) Histone H3K9 acetylation associated with the IFN-A2 gene promoter was determined in control and HDAC3-depleted cells by quantitative ChIP assays as described in Materials and Methods. Anti-rabbit IgG was used to determine non-specific binding. (E) IFN-B gene expression was determined by RT-QPCR following 8 h of infection by Sendai virus in HEK293-TLR3 cells expressing TRAF3 and IRF7A and transfected with scrambled siRNA or siRNA oligonucleotides specific for HDAC3.
Mentions: In the same experimental model, HDAC3 overexpression produced a 2–4 fold dose-dependent inhibition of IRF7-mediated IFN-A gene expression without altering expression of either IRF3 or IRF7 (Fig. 6A). Other HDACs tested did not exert similar effects (Fig. S4). Conversely to the inhibitory effect of HDAC3 overexpression, silencing of HDAC3 expression by siRNA led to a 2-3-fold decrease in HDAC3 mRNA and protein levels and enhanced virus-induced IFN-A gene expression by 3- to 5-fold compared to cells transfected with control siRNA (Fig. 6B-C). Also, a significant 1.5 fold increase (p<0.01) in histone H3K9 acetylation of the IFN-A2 promoter was quantified in cells transfected with siRNA targeting HDAC3 (Fig. 6D). Since a knockdown screen performed in poly(I).poly(C)-treated cells showed that HDACs affected negatively (HDAC1 and HDAC8) or positively (HDAC6) the activity of a IFN-B promoter construct in reporter assays [64], we tested the effect of HDAC3 knockdown on SeV-induced IFN-B expression. Depletion of HDAC3 slightly enhanced SeV-induced IFN-B gene transcription by less than 2.5 fold (Fig. 6E) and overexpression of HDAC3 did not affect virus-induced IFN-B gene transcription (data not shown). These observations suggested that HDAC3 did not play a critical role in IFN-B gene regulation following virus infection.

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