Inhibition of Histone Deacetylase Activity Suppresses IFN-γ Induction of Tripartite Motif 22 via CHIP-Mediated Proteasomal Degradation of IRF-1

Bo Gao, Yaxin Wang, Wei Xu, Shangshan Li, Qiao Li and Sidong Xiong
J Immunol July 1, 2013, 191 (1) 464-471; DOI: https://doi.org/10.4049/jimmunol.1203533

Abstract

Tripartite motif (TRIM)22 plays an important role in IFN-mediated antiviral activity. We previously demonstrated that IFN regulatory factor (IRF)-1 was crucial for basal and IFN-induced TRIM22 transcription via binding to a novel cis-element named 5′ extended IFN-stimulating response element. In this study, we investigated the role of histone deacetylase (HDAC) activity in TRIM22 induction by IFN-γ and its underlying mechanism. We found that the HDAC activity, especially that conferred by HDAC6, was required for IFN-γ–induced TRIM22 transcription. Importantly, inhibition of HDAC activity by trichostatin A (TSA) enhanced the hyperacetylation of heat shock protein (HSP)90 and suppressed its chaperone activity for IRF-1. Further study showed that TSA treatment promoted the proteasomal degradation of IRF-1 protein via enhancing the association of IRF-1 with the ubiquitin E3 ligase carboxyl terminus of Hsc70-interacting protein. Moreover, carboxyl terminus of Hsc70-interacting protein was found to be involved in the TSA-mediated inhibitory effect on IFN-γ induction of TRIM22 as well as other IRF-1–dependent IFN-stimulated genes. This study may provide novel insight into the role of HDAC activity in the transcriptional control of IFN-stimulated gene induction.

Introduction

Tripartite motif (TRIM)22 is a member of the TRIM family of proteins that are involved in diverse biological processes, including cell differentiation, apoptosis, transcriptional regulation, and immune signaling pathways (1–5). Many TRIM proteins are upregulated in response to IFN stimulation (6) and are revealed to play important roles in antiviral immune responses (4, 5, 7–10). TRIM22 was originally identified as an IFN-inducible gene in Daudi cells (11) and was recently found to be implicated in IFN-mediated antiretroviral activity in recent years (12, 13). We previously demonstrated that TRIM22 was one of the most strongly induced TRIMs by IFNs in HepG2 cells and could inhibit the replication of hepatitis B virus efficiently (14). Additionally, TRIM22 was identified as a RING finger E3 ubiquitin ligase (15) and was involved in the activation of NF-κB (16) in our previous study.

With the aim of elucidating the molecular mechanism of TRIM22 induction by IFNs, we previously identified a novel cis-element named 5′ extended IFN-stimulating response element in the TRIM22 promoter region that was crucial for IFN-γ–induced TRIM22 expression. It was IFN regulatory factor (IRF)-1, not STAT1, that bound to this cis-element and played a critical role in the IFN-γ induction of TRIM22 (17). Furthermore, it was revealed that the association of IRF-1 with 5′ extended IFN-stimulating response element was also important for TRIM22 induction by IFN-α, as well as for the basal TRIM22 expression (17). Our more recent investigation demonstrated that chromatin remodeling enzyme BRG1 was critical for TRIM22 induction by IFN-γ via controlling IRF-1 recruitment (18), further establishing the central role of IRF-1 in IFN-γ–induced TRIM22 expression. However, the molecular mechanisms of TRIM22 induction by IFNs still await further study.

Except for BRG1, which uses the energy of ATP hydrolysis to alter nucleosome structure or position, histone acetylases and histone deacetylases (HDACs) also play important roles in chromatin remodeling and gene expression by covalently modifying histones. Activation of histone acetylases or inhibition of HDACs leads to the hyperacetylation of histones and thus allows chromatin to assume a more open state permitting transcriptional activators to form a preinitiation complex (19–21). Of note, histone acetylases and HDACs can also target nonhistone proteins to alter their functions (22). Although HDAC activity is commonly associated with transcriptional repression, accumulating evidence demonstrates that it is essential for the transcriptional activation of IFN-stimulated genes (ISGs) and for host antiviral immune responses (23–29). Additionally, HDAC activity is also required for the gene expression induced by other cytokines, such as IL-2 and TNF-α (30, 31). However, not all inducible genes require HDAC function for activation. For instance, the expression of TGF-β–responsive genes is enhanced rather than suppressed by cotreatment with the HDAC inhibitor trichostatin A (TSA) (32), and IFN induction of some genes whose transcription required STAT1 homodimers binding to the γ-activated sequence (GAS) elements, such as IRF-1 and ICAM-1, was not altered by inhibition of HDAC activity (27, 33).

In the present study, we demonstrate that HDAC activity, especially that conferred by HDAC6, was required for IFN-γ–induced TRIM22 transcription. Further study showed that inhibition of HDAC activity by TSA led to the hyperacetylation of heat shock protein (HSP)90, shifted the chaperone association of IRF-1 from HSP90 to HSP70, and thus led to the proteasomal degradation of IRF-1 protein. Importantly, TSA treatment was revealed to facilitate the degradation of IRF-1 protein via enhancing the interaction between IRF-1 and ubiquitin E3 ligase carboxyl terminus of Hsc70-interacting protein (CHIP), and CHIP was implicated in the TSA-mediated inhibitory effect on the induction of TRIM22 as well as other IRF-1–dependent ISGs by IFN-γ.

Materials and Methods

Cell culture and stimulation

HepG2, Huh7, and HeLa cells were maintained in DMEM supplemented with 10% FBS and antibiotics. Where indicated, cells were stimulated with IFN-γ (PeproTech, Rocky Hill, NJ) at 1000 U/ml, TSA (Sigma-Aldrich, St. Louis, MO) at 300 ng/ml, and suberoylanilide hydroxamic acid (SAHA; Sigma-Aldrich) at 2 uM.

Western blot

Western blot was performed as described previously (14, 17). Abs against IRF-1 (SC-497), CHIP (SC-133066), HSP90 (SC-69703), and HSP70 (SC-32239) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); Abs against ubiquitin (05-1307) and acetyl-lysine (05-515) were obtained form Millipore (Billerica, MA); and Abs against phospho-Stat1 (9171) and the total form of Stat1 (9172) were obtained from Cell Signaling Technology (Beverly, MA).

Semiquantitative RT-PCR and quantitative real-time RT-PCR

Semiquantitative and quantitative RT-PCR analysis were carried out as previously described (14, 17). Primers used in this study are shown as follows: TRIM22, forward, 5′-ACCAAACATTCCGCATAAAC-3′, reverse, 5′-GTCCAGCACATTCACCTCAC-3′; IRF-1, forward, 5′-TATCGAGGAGGTGAAAGACC-3′, reverse, 5′-TGCATGTAGCCTGGAACTG-3′; B7-H1, forward, 5′- CTACCTCTGGCACATCCTCC-3′, reverse, 5′-CCTTCCTCTTGTCACGCTCA-3′; ISG20, forward, 5′-CTGCCGTAAGGAAGCCATCA-3′, reverse, 5′- GGTCCCAACATAATCACAAGC-3′; GBP-1, forward, 5′- GAGAACACTAATGGGCGACTG-3′, reverse, 5′-TAGCCTGCTGGTTGATGGT-3′; GAPDH, forward, 5′-ATCCCATCACCATCTTCCAG-3′, reverse, 5′-GA GTCCTTCCACGATACCAA-3′. The semiquantitative RT-PCR primers used for determining the knockdown efficiency of each HDAC were supplied by Santa Cruz Biotechnology (the amplicon size is between 400 and 600 bp).

Plasmid transfection and luciferase reporter gene assay

Plasmid transfection into HepG2 cells was carried out as described previously (14, 17). For the reporter gene assay, cells were transfected with a TRIM22 promoter-dependent luciferase reporter plasmid (pLuc-160). Twenty-four hours after transfection, cells were harvested and the luciferase activity in the cell lysates was determined with the Luciferase Reporter Assay System (Promega). In all transfection assays, pCMV-β-gal was cotransfected to normalize the transfection efficiency.

Immunoprecipitation

Immunoprecipitation was performed as previously described (15). Briefly, cells were lysed in RIPA buffer (50 mM Tris [pH 8.0], 280 mM NaCl, 0.5% Nonidet P-40, 2 mM EDTA, 10% glycerol, 1 mM DTT) supplemented with a protease inhibitor mixture (Roche). Lysates were sonicated 10 times on ice for 5 s with 10-s breaks between each sonication interval. After preclearing with protein A/G-agarose beads for 2 h at 4°C, lysates were then incubated with indicated Abs at 4°C overnight, followed by incubation with protein A/G-agarose beads for 2 h. The beads were washed five times with lysis buffer, and the immunoprecipitated proteins were then subjected to Western blot analysis.

Small interfering RNA assay

The small interfering RNAs (siRNAs) targeting HDAC1–10, CHIP, HSP70, and the scrambled siRNA were purchased from Santa Cruz Biotechnology. The cells were transfected with 100 nM each siRNA using Lipofectamine 2000 according to the manufacturer’s instructions.

Statistics

Results are reported as means ± SD of one representative experiment or SEM for pooled experiments. A t test was applied to comparisons between groups; a p value <0.05 was considered statistically significant.

Results

HDAC activity is required for IFN-γ–induced TRIM22 transcription

To investigate the role of HDAC activity in IFN-γ–induced TRIM22 expression, HepG2 cells were treated with IFN-γ in the absence or presence of TSA. TRIM22 expression was then examined at protein or mRNA level by Western blot and RT-PCR, respectively. Results showed that TSA inhibited IFN-γ–induced TRIM22 expression efficiently at indicated time points both at the protein and mRNA levels (Fig. 1A, 1B). We further investigated the effect of TSA on IFN-γ–induced transcriptional activity of TRIM22 gene by transfecting a TRIM22 promoter-dependent luciferase reporter plasmid (pLuc-160) into HepG2 cells. As shown in Fig. 1C, TSA treatment inhibited IFN-γ–induced TRIM22 promoter activity significantly in HepG2 cells.

FIGURE 1.
FIGURE 1.

HDAC activity is required for IFN-γ–induced TRIM22 expression at the transcriptional level. (A) HepG2 cells were treated with 1000 U/ml IFN-γ in the absence or presence of 300 ng/ml TSA at indicated time points. TRIM22 protein expression level was determined by Western blot. Lower panel, The immunoblots from three independent experiments were scanned and subjected to densitometric analysis. The density value from the cells stimulated by IFN-γ for 24 h in the absence of TSA was set to 100%. Data are shown as means ± SEM of five samples pooled from three independent experiments. *p < 0.05. (B) Cells were treated as in (A). TRIM22 mRNA levels were determined by quantitative RT-PCR. (C) Cells were transfected with the TRIM22 reporter plasmid pLuc-160. Twenty-four hours after transfection, cells were treated with IFN-γ with or without TSA, followed by measuring the luciferase activity in the cell lysates. (D) Effect of TSA on IFN-γ–induced TRIM22 promoter activity in Huh7 or HeLa cells. As in (C), except that Huh7 or HeLa cells, rather than HepG2 cells, were used. (E) Effect of SAHA on IFN-γ–induced TRIM22 promoter activity in HepG2 cells. As in (C), but with SAHA. Data in (B)–(E) are shown as mean ± SD of triplicates and are representative of three independent experiments. *p < 0.05.

To exclude the possibility that our observation is restricted to a single cell line, the effect of TSA on IFN-γ–induced TRIM22 transcription was assessed in other cell lines, such as Huh7 and HeLa cells. Fig. 1D shows that TSA treatment strongly repressed IFN-γ–induced TRIM22 promoter activity in both Huh7 and HeLa cells. To confirm that the effect of TSA was through inhibition of HDAC activity, a structurally distinct HDAC inhibitor SAHA was tested for its effect on TRIM22 transcriptional activity in HepG2 cells. Results showed that treatment of cells with SAHA also efficiently inhibited IFN-γ–induced TRIM22 promoter activity (Fig. 1E).

Taken together, these data indicated that HDAC activity was required for inhibiting IFN-γ–induced TRIM22 expression at the transcriptional level.

The role of HDAC isoforms in TRIM22 transcriptional activity in response to IFN-γ stimulation

To determine which HDAC isoform might be involved in IFN-γ–induced transcriptional activity of TRIM22 gene, we cotransfected HepG2 cells with pLuc-160 and siRNA for different HDAC isoforms (HDAC1–10). Forty-eight hours after transfection, cells were stimulated with IFN-γ for another 24 h, followed by the detection of luciferase activity in the cell lysates. As shown in Fig. 2A, knockdown of HDAC6 by siRNA markedly inhibited IFN-γ–induced TRIM22 transcriptional activity. It was also found that knockdown of HDAC1 and HDAC3 showed inhibitory effects on IFN-γ induction of TRIM22, whereas other HDACs appeared not involved in this event (Fig. 2A). As a control experiment, the knockdown efficiency of each HDAC isoform was investigated. Fig. 2B shows that HDAC mRNAs were specifically reduced by their respective siRNAs in HepG2 cells.

FIGURE 2.
FIGURE 2.

The role of HDAC isoforms in TRIM22 transcriptional activity in response to IFN-γ stimulation. (A) HepG2 cells were transfected with pLuc-160 together with siRNA for HDAC1–10. Forty-eight hours later, transfected cells were treated with IFN-γ for another 24 h, followed by measuring the luciferase activity in the cell lysates. The values are expressed as percentage of IFN-γ–treated scrambled control. Data are shown as means ± SD of triplicates and are representative of three independent experiments. *p < 0.05. (B) At 72 h after control or HDAC siRNA transfection, HDAC mRNA expression levels were determined by semiquantitative RT-PCR with primers supplied by Santa Cruz Biotechnology (amplicon size is between 400 and 600 bp). Lower panel, The PCR product bands were scanned and relative densities were presented in the bar graph as a percentage of the control siRNA value. Data are means ± SEM of six samples pooled from three independent experiments. *p < 0.05.

Inhibition of HDAC activity leads to the hyperacetylation of HSP90 and abrogates HSP90 chaperone activity for IRF-1

It is well known that HDAC6, and in some cases HDAC1, is crucial for the deacetylation of HSP90 (34–37), and IRF-1 has been demonstrated to be a client protein of HSP90 (38). We therefore wondered whether HDAC activity is required for HSP90 chaperone activity for IRF-1, thus contributing to IFN-γ–induced TRIM22 expression. Fig. 3A shows that inhibition of HDAC activity by TSA led to the hyperacetylation of HSP90 significantly in HepG2 cells. We next examined the contribution of HDAC activity to the association of IRF-1 with HSP90 or HSP70 by immunoprecipitation assay. As shown in Fig. 3B, TSA treatment significantly decreased the binding of HSP90 to IRF-1, whereas it enhanced the association of IRF-1 with HSP70. Importantly, TSA treatment was found to inhibit IFN-γ–induced IRF-1 protein expression at various time points (Fig. 3C). We further investigated the effect of TSA on IFN-γ-induced IRF-1 transcription. Time course analysis showed that TSA treatment had no significant effect on IFN-γ–induced IRF-1 mRNA (Fig. 3D), and it also did not alter the IFN-γ–induced STAT1 tyrosine phosphorylation, which is well established to be crucial for IRF-1 transcription (Fig. 3E). Further analysis using an IRF-1 GAS-dependent reporter plasmid showed that TSA treatment did not affect IRF-1 promoter activity induced by IFN-γ (Fig. 3F). Together, these data indicated that inhibiting HDAC activity by TSA suppressed IFN-γ–induced IRF-1 expression mainly at the posttranslational level.

FIGURE 3.
FIGURE 3.

Inhibition of HDAC activity leads to the hyperacetylation of HSP90 and abrogates HSP90 chaperone activity for IRF-1. (A) HepG2 cells were treated with or without TSA for 12 h. The HSP90 was immunoprecipitated with anti-HSP90 and probed with anti-Ack. Lower panel, Quantitative analysis of the effect of TSA on HSP90 acetylation. The value from TSA-treated cells was set to 100%. (B) HepG2 cells were treated with IFN-γ in the absence or presence of TSA for 6 h. Coimmunoprecipitation assays were performed with anti–IRF-1 for pulldown and anti-HSP90, anti-HSP70, or anti–IRF-1 for detection. Right panel, Quantitative analysis of the effect of TSA on the association of IRF-1 with HSP90 or HSP70. The values were normalized to total HSP90 or HSP70 in the lysate and the precipitated IRF-1. (C) HepG2 cells were treated with IFN-γ in the absence or presence of TSA at indicated time points. The IRF-1 protein expression was determined by Western blot. Lower panel, Quantitative analysis of the effect of TSA on the IFN-γ–induced IRF-1 protein levels. The value from the cells stimulated by IFN-γ in the absence of TSA was set to 100%. Data are shown as means ± SEM of four (A) or five (B, C) samples pooled from three independent experiments. *p < 0.05. (D) HepG2 cells were treated as in (C). IRF-1 mRNA levels were determined by quantitative RT-PCR. Data are shown as mean ± SD of triplicates and are representative of three independent experiments. (E) After serum starvation for 24 h, HepG2 cells were treated with IFN-γ at indicated time points in the absence or presence of TSA. Phosphorylated and total STAT1 were detected by Western blot. Lower panel, Quantitative analysis of the effect of TSA on the IFN-γ–induced tyrosine phosphorylation of STAT1. The value from cells stimulated by IFN-γ for 2 h in the absence of TSA was set to 100%. Data are shown as means ± SEM of four samples pooled from three independent experiments. (F) HepG2 cells were transfected with IRF-1-GAS element–dependent reporter plasmids. The reporter activity was detected as in Fig. 1C. Data are shown as means ± SD of triplicates and are representative of three independent experiments.

Inhibition of HDAC activity promotes IRF-1 protein degradation via the ubiquitin/proteasome pathway

It is now known that IRF-1 is a short-lived protein, and its degradation occurs mainly through the ubiquitin/proteasome pathway (39, 40). We thus examined the effect of the proteasome inhibitor MG132 on TSA-mediated degradation of IRF-1 protein. Fig. 4A shows that MG132 treatment reversed the inhibitory effect of TSA on IRF-1 protein expression, indicating that the effect of TSA on the degradation of IRF-1 protein was via the proteasome pathway. To determine the effect of TSA on IRF-1 ubiquination, we performed a coimmunoprecipitation assay with anti–IRF-1 for pull-down and anti-ubiquitin for detection. As shown in Fig. 4B, TSA treatment promoted the ubiquitination of IFN-γ–induced IRF-1 protein significantly, and cotreatment of HepG2 cells with TSA and MG132 further augmented this event. Taken together, these data indicated that inhibiting HDAC activity by TSA led to IRF-1 protein degradation via the proteasome/ubiquitin pathway.

FIGURE 4.
FIGURE 4.

TSA treatment promotes IRF-1 protein degradation via the ubiquitin/proteasome pathway. (A) HepG2 cells were treated with IFN-γ in the presence of TSA, MG132, or TSA plus MG132. The expression of IRF-1 was determined by Western blot. Lower panel, Quantitative analysis of the effect of proteasomal inhibitor MG132 on the TSA-mediated degradation of IRF-1 protein induced by IFN-γ. The value from cells treated with IFN-γ in the absence of TSA was set to 100%. (B) HepG2 cells were treated as in (A), and cell lysates were immunoprecipitated with anti–IRF-1 and blotted with anti-ubiquitin. Lower panel, Quantitative analysis of the effect of TSA on the ubiquitination of IFN-γ–induced IRF-1 protein. The value from cells treated with IFN-γ plus TSA was set to 100%. Data in this figure are shown as means ± SEM of five samples pooled from three independent experiments. *p < 0.05.

Inhibition of HDAC activity promotes IRF-1 ubiquitination via enhancing the interaction between IRF-1 and ubiquitin E3 ligase CHIP

The chaperone-associated ubiquitin ligase CHIP is reported to interact with IRF-1 and has been identified as a ubiquitin ligase for IRF-1 (41). However, CHIP was revealed to have a positive effect on IRF-1 protein expression in unstressed cells, and only when the association of CHIP with IRF-1 was increased under some stressful conditions could CHIP target IRF-1 for proteasomal degradation (41). To assess a possible regulatory role for CHIP in TSA-mediated IRF-1 degradation, cells were subjected to coimmunoprecipitation experiments with anti–IRF-1 for pull-down and with anti-CHIP or anti–IRF-1 for detection. Fig. 5A shows that upon TSA treatment, although the immuoprecipitated IRF-1 was decreased markedly and the expression level of CHIP in the cell lysates was not altered, there were more CHIP proteins presented in the immunocomplex, indicating that the association of CHIP with IRF-1 was increased significantly by TSA treatment.

FIGURE 5.
FIGURE 5.

TSA accelerates the degradation of IRF-1 protein via enhancing the interaction between IRF-1 and ubiquitin E3 ligase CHIP. (A) HepG2 cells were treated with IFN-γ for 6 h with or without TSA, and cell lysates were immunoprecipitated with anti–IRF-1 and blotted with anti-CHIP or anti–IRF-1. Right panel, The CHIP/IRF-1 interaction was quantified and values were normalized to total CHIP in the lysate and the precipitated IRF-1. (B) HepG2 cells were transfected with empty vector or CHIP expression plasmids. Twenty-four hours after transfection, cells were treated with IFN-γ for 6 h in the absence or presence of TSA. The IRF-1 protein expression was determined by Western blot. Right panel, Quantitative analysis of the effect of CHIP overexpression on TSA-mediated effect on IFN-γ–induced IRF-1 protein expression. The value from empty vector-transfected cells stimulated by IFN-γ in the absence of TSA was set to 100%. (C) HepG2 cells were transfected with control or CHIP-specific siRNA. At 72 h after transfection, cells were treated with IFN-γ for 6 h in the absence or presence of TSA. The expression levels of CHIP, IRF-1, or actin were determined by Western blot. Right panel, Quantitative analysis of the effect of CHIP knockdown on TSA inhibition of IRF-1 protein expression. The value from control siRNA-transfected cells stimulated by IFN-γ in the absence of TSA was set to 100%. (D) HepG2 cells were treated as in (C), and cell lysates were immunoprecipitated with anti–IRF-1 and blotted with anti-ubiquitin or anti–IRF-1. Right panel, Quantitative analysis of the effect of CHIP knockdown on TSA-mediated IRF-1 ubiquitination. The value from control siRNA-transfected cells treated with IFN-γ plus TSA was set to 100%. The effect of CHIP knockdown on the TSA-mediated effect on IRF-1 ubiquitination is shown. Data in this figure are shown as means ± SEM of four (B) or five (A, C, D) samples pooled from three independent experiments. *p < 0.05.

To investigate the role of CHIP in TSA-mediated

downregulation of IRF-1 protein induced by IFN-γ, we transfected a CHIP expression plasmid into HepG2 cells, followed by the treatment of IFN-γ in the absence or presence of TSA. We found that although CHIP overexpression had little effect on the IFN-γ–induced IRF-1 protein expression (Fig. 5B, compare lane 2 with lane 5), it significantly potentiated TSA-induced IRF-1 degradation (Fig. 5B, compare lane 3 with lane 6). To further verify the role of CHIP in TSA-mediated downregulation of IRF-1 protein, we knocked down the expression of CHIP in HepG2 cells with siRNA. Compared with control siRNA, CHIP siRNA treatment strongly attenuated TSA-mediated IRF-1 degradation (Fig. 5C, compare lane 3 with lane 6). The above results showed that TSA treatment enhanced the IRF-1 degradation in a proteasome/ubiquitin-dependent pathway. We thus further investigated whether CHIP was involved in TSA-mediated IRF-1 ubiquitination by immunoprecipitation with anti–IRF-1 and immunoblotting with anti-ubiquitin. As shown in Fig. 5D, knockdown of CHIP also attenuated TSA-mediated IRF-1 ubiquitination significantly.

Collectively, the above data indicated that TSA treatment enhanced the interaction between CHIP and IRF-1, thus promoting TSA-induced IRF-1 ubiquitination and degradation.

CHIP is involved in the TSA-mediated inhibitory effect on the expression of TRIM22 as well as other IRF-1–dependent ISGs in response to IFN-γ

Our previous data demonstrated that IRF-1 was crucial for transcriptional induction of TRIM22 by IFN-γ, and the above data demonstrate that CHIP was required for TSA-mediated IRF-1 degradation. We therefore further investigated whether CHIP was involved in TSA-mediated inhibition of IFN-γ–induced transcriptional activity of TRIM22 gene. We downregulated CHIP expression in HepG2 cells by siRNA and then tested the effect of TSA on IFN-γ–induced TRIM22 promoter activity using the TRIM22 luciferase reporter construct (pLuc-160). We found that CHIP knockdown markedly decreased TSA-mediated inhibition of TRIM22 promoter activity (Fig. 6A). Further analysis showed that siRNA-mediated CHIP downregulation also significantly reversed TSA inhibition of IFN-γ–induced TRIM22 mRNA expression (Fig. 6B, compare lane 3 with lane 6).

FIGURE 6.
FIGURE 6.

CHIP is involved in the TSA-mediated downregulation of TRIM22 as well as other IRF-1–dependent ISGs induced by IFN-γ. (A) Control or CHIP knockdown cells were transfected with reporter plasmid pLuc-160. Twenty-four hours after transfection, cells were then treated with IFN-γ with or without TSA, followed by measuring the luciferase activity in the cell lysate. (B) Control or CHIP knockdown cells were treated with IFN-γ with or without TSA for 24 h. TRIM22 mRNA levels were determined by quantitative RT-PCR. (C) HepG2 cells were treated with IFN-γ in the absence or presence of TSA for the indicated time points. The mRNA expression of B7-H1, ISG20, and GBP-1 was determined by quantitative RT-PCR. (D) Control or CHIP knockdown cells were treated with IFN-γ with or without TSA for 12 h. The mRNA expression of B7-H1, ISG20, and GBP-1 was determined by quantitative RT-PCR. All data in this figure are shown as means ± SD of triplicates and are representative of three independent experiments. *p < 0.05.

IRF-1 is known to play a crucial role in IFN induction of many ISGs. It is therefore of interest to know whether our study represents a general mechanism for the induction of IRF-1–dependent ISGs. We first determined the role of HDAC activity in the induction of several well-characterized IRF-1–dependent ISGs, such as B7-H1 (42), ISG20 (43), and GBP-1 (44). We found that all of these IRF-1–dependent ISGs were subjected to the inhibitory of TSA (Fig. 6C). Importantly, further study showed that, similar to its role in TRIM22 expression, CHIP was also involved in the TSA inhibition of these IRF-1–dependent ISGs (Fig. 6D).

Discussion

Many TRIM members are regulated in response to IFN stimulation, supporting their role in antiviral and immunomodulatory activity. Given the importance of TRIM22 in IFN-mediated antiviral activity and the importance of HDAC activity in ISGs induction, we want to know whether the IFN induction of TRIM22 is subjected to the regulation of HDAC activity.

In present investigation, HDAC activity, especially that conferred by HDAC6, was demonstrated to be crucial for TRIM22 transcription by IFN-γ. Many reports indicate that HDAC6 is crucial for the deacetylation of HSP90, and IRF-1 was recently revealed to be a client protein of HSP90. It is therefore of interest to know whether inhibiting HDAC activity will lead to the hyperacetylation of HSP90 in HepG2 cells, and thus abrogate the chaperone activity of HSP90 for IRF-1. Our results showed that inhibition of HDAC activity by TSA indeed significantly upregulated the hyperacetylation of HSP90, shifted the chaperone association of IRF-1 from HSP90 to HSP70, and suppressed IRF-1 protein expression significantly.

Sakamoto et al. (27) reported that treatment of cells with TSA had little effect on IFN-β–induced IRF-1 expression. Because the mechanism whereby IFN-γ induces IRF-1 expression is similar to that of IFN-β (both IFN-γ and IFN-β induce the tyrosine phosphorylation of STAT1 and lead to the formation of STAT1 homodimers that bind to GAS element to induce the transcription of IRF-1), the data presented by Sakamoto et al. seemed to be in conflict with our results. However, they only determined the effect of TSA on IRF-1 expression at the transcriptional level in response to IFN-β stimulation. In fact, we had investigated the effect of TSA on IFN-γ–induced IRF-1 transcription and found that TSA treatment had little effect on this event, as evidenced by the slight alteration in IFN-γ–induced IRF-1 mRNA, STAT1 tyrosine phosphorylation, and IRF-1 promoter activity following TSA treatment. We also examined the effect of TSA on IFN-β–induced IRF-1 expression in HepG2 cells and found that TSA treatment significantly inhibited IFN-β–induced IRF-1 protein expression, whereas it had no significant effect on IRF-1 mRNA expression induced by IFN-β (data not shown). Collectively, these data indicated that TSA exerted its suppressive effect on IFN-induced IRF-1 expression mainly at the protein level. Although most current data support the notion that the requirement of HDAC activity for ISG induction is mainly at the epigenetic transcriptional level, our data argue that the role of HDAC in ISG induction at the protein level should also be taken into account.

Because HDAC activity was shown to be required for IFN-γ–induced IRF-1 protein expression, but not its transcription, we further investigated its underlying mechanism. Our results showed that treatment of HepG2 cells with TSA facilitated IRF-1 degradation by the ubiquitin/proteasome pathway. Similar to our results, inhibition of HDAC activity by TSA also alters the function of several other important transcription factors, such as estrogen receptor α and steroidogenic factor 1, through targeting them for proteasomal degradation (45, 46). CHIP was identified as an E3 ligase for IRF-1 as well as a number of HSP70-associated proteins, such as ErbB2 and PTEN (47, 48); however, it was reported that CHIP acted as the chaperone for IRF-1 in unstressed cells. Only under special conditions in which the interaction between CHIP and IRF-1 gets enhanced will CHIP target IRF-1 for ubiquitination and degradation (41). We therefore first determined the effect of TSA on the association between CHIP and IRF-1 by coimmunoprecipitation assays. Results showed that TSA treatment indeed potentiated the interaction between IRF-1 and CHIP significantly. Importantly, further studies revealed that CHIP overexpression enhanced, whereas CHIP knockdown attenuated, TSA-mediated IRF-1 degradation. Although Narayan et al. (41) reported that CHIP could bind IRF-1 directly, their data did not rule out the possibility that CHIP interacts with IRF-1 through HSP70. Because our data showed that TSA treatment enhanced the interaction between HSP70 and IRF-1 markedly (Fig. 2B), we examined the role of HSP70 in TSA-mediated enhancement of IRF-1/CHIP interaction. We found that knockdown of HSP70 significantly attenuated TSA-mediated enhancement of CHIP/IRF-1 interaction and IRF-1 degradation (data not shown).

As IRF-1 was previously demonstrated to be crucial for IFN-γ–induced TRIM22 expression and CHIP was revealed to be responsible for TSA-mediated IRF-1 degradation, we further investigated whether CHIP contributed to TSA inhibition of IFN-γ–induced TRIM22 transcription. We found that CHIP knockdown significantly attenuated TSA inhibition of TRIM22 transcriptional activity in response to IFN-γ stimulation. We further determined the role of CHIP in IFN-γ induction of other IRF-1–dependent ISGs, such as B7-H1, ISG20, and GBP-1. We found that CHIP also contributed to TSA inhibition of these IRF-1–dependent ISGs. Because IRF-1 was involved in the induction of many ISGs, this study may play a contributing role for elucidating the molecular mechanism of TSA-mediated effects on IFN induction of ISGs. Additionally, besides its role in the IFN-responsive system, IRF-1 is also involved in other physiological processes, including antiviral immunity and tumor suppression (49). These findings might thus be useful for illuminating these IRF-1–dependent bioprocesses.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by the National Natural Science Foundation of China (Grants 81072428, 30890141, and J1210041), the Major State Basic Research Development Program of China (Grant 2013CB530501), the Program for Changjiang Scholars and Innovative Research Team in University (Grant IRT1075), the Priority Academic Program Development of Jiangsu Higher Education Institutions, the Jiangsu “Pan-Deng” Project (Grant BK2010004), the Natural Science Foundation of the Jiangsu Higher Education Institutions (Grant 11KJA180003), the Qing Lan Project of the Jiangsu Higher Education Institutions, and by Science and Technology Commission of Shanghai Municipality Grant 09JC1401800.

  • Abbreviations used in this article:

    CHIP
    carboxyl terminus of Hsc70-interacting protein
    GAS
    γ-activated sequence
    HDAC
    histone deacetylase
    HSP
    heat shock protein
    IRF
    IFN regulatory factor
    ISG
    IFN-stimulated gene
    SAHA
    suberoylanilide hydroxamic acid
    siRNA
    small interfering RNA
    TRIM
    tripartite motif
    TSA
    trichostatin A.

  • Received December 27, 2012.
  • Accepted May 3, 2013.

References

  1. ↵
    1. Meroni G.,
    2. G. Diez-Roux
    . 2005. TRIM/RBCC, a novel class of “single protein RING finger” E3 ubiquitin ligases. Bioessays 27: 1147–1157.
    OpenUrlCrossRefPubMed
    1. Reymond A.,
    2. G. Meroni,
    3. A. Fantozzi,
    4. G. Merla,
    5. S. Cairo,
    6. L. Luzi,
    7. D. Riganelli,
    8. E. Zanaria,
    9. S. Messali,
    10. S. Cainarca,
    11. et al
    . 2001. The tripartite motif family identifies cell compartments. EMBO J. 20: 2140–2151.
    OpenUrlAbstract
    1. Hatakeyama S.
    2011. TRIM proteins and cancer. Nat. Rev. Cancer 11: 792–804.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Ozato K.,
    2. D. M. Shin,
    3. T. H. Chang,
    4. H. C. Morse III.
    . 2008. TRIM family proteins and their emerging roles in innate immunity. Nat. Rev. Immunol. 8: 849–860.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Jefferies C.,
    2. C. Wynne,
    3. R. Higgs
    . 2011. Antiviral TRIMs: friend or foe in autoimmune and autoinflammatory disease? Nat. Rev. Immunol. 11: 617–625.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Carthagena L.,
    2. A. Bergamaschi,
    3. J. M. Luna,
    4. A. David,
    5. P. D. Uchil,
    6. F. Margottin-Goguet,
    7. W. Mothes,
    8. U. Hazan,
    9. C. Transy,
    10. G. Pancino,
    11. S. Nisole
    . 2009. Human TRIM gene expression in response to interferons. PLoS ONE 4: e4894.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Nisole S.,
    2. J. P. Stoye,
    3. A. Saïb
    . 2005. TRIM family proteins: retroviral restriction and antiviral defence. Nat. Rev. Microbiol. 3: 799–808.
    OpenUrlCrossRefPubMed
    1. Stremlau M.,
    2. C. M. Owens,
    3. M. J. Perron,
    4. M. Kiessling,
    5. P. Autissier,
    6. J. Sodroski
    . 2004. The cytoplasmic body component TRIM5α restricts HIV-1 infection in Old World monkeys. Nature 427: 848–853.
    OpenUrlCrossRefPubMed
    1. Wolf D.,
    2. S. P. Goff
    . 2007. TRIM28 mediates primer binding site-targeted silencing of murine leukemia virus in embryonic cells. Cell 131: 46–57.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Uchil P. D.,
    2. A. Hinz,
    3. S. Siegel,
    4. A. Coenen-Stass,
    5. T. Pertel,
    6. J. Luban,
    7. W. Mothes
    . 2013. TRIM protein-mediated regulation of inflammatory and innate immune signaling and its association with antiretroviral activity. J. Virol. 87: 257–272.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Tissot C.,
    2. N. Mechti
    . 1995. Molecular cloning of a new interferon-induced factor that represses human immunodeficiency virus type 1 long terminal repeat expression. J. Biol. Chem. 270: 14891–14898.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Barr S. D.,
    2. J. R. Smiley,
    3. F. D. Bushman
    . 2008. The interferon response inhibits HIV particle production by induction of TRIM22. PLoS Pathog. 4: e1000007.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Singh R.,
    2. G. Gaiha,
    3. L. Werner,
    4. K. McKim,
    5. K. Mlisana,
    6. J. Luban,
    7. B. D. Walker,
    8. S. S. Karim,
    9. A. L. Brass,
    10. T. Ndung’u,
    11. CAPRISA Acute Infection Study Team
    . 2011. Association of TRIM22 with the type 1 interferon response and viral control during primary HIV-1 infection. J. Virol. 85: 208–216.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Gao B.,
    2. Z. Duan,
    3. W. Xu,
    4. S. Xiong
    . 2009. Tripartite motif-containing 22 inhibits the activity of hepatitis B virus core promoter, which is dependent on nuclear-located RING domain. Hepatology 50: 424–433.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Duan Z.,
    2. B. Gao,
    3. W. Xu,
    4. S. Xiong
    . 2008. Identification of TRIM22 as a RING finger E3 ubiquitin ligase. Biochem. Biophys. Res. Commun. 374: 502–506.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Yu S.,
    2. B. Gao,
    3. Z. Duan,
    4. W. Xu,
    5. S. Xiong
    . 2011. Identification of tripartite motif-containing 22 (TRIM22) as a novel NF-κB activator. Biochem. Biophys. Res. Commun. 410: 247–251.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Gao B.,
    2. Y. Wang,
    3. W. Xu,
    4. Z. Duan,
    5. S. Xiong
    . 2010. A 5′ extended IFN-stimulating response element is crucial for IFN-γ-induced tripartite motif 22 expression via interaction with IFN regulatory factor-1. J. Immunol. 185: 2314–2323.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Wang Y.,
    2. B. Gao,
    3. W. Xu,
    4. S. Xiong
    . 2011. BRG1 is indispensable for IFN-γ-induced TRIM22 expression, which is dependent on the recruitment of IRF-1. Biochem. Biophys. Res. Commun. 410: 549–554.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Fry C. J.,
    2. C. L. Peterson
    . 2001. Chromatin remodeling enzymes: who’s on first? Curr. Biol. 11: R185–R197.
    OpenUrlCrossRefPubMed
    1. Christova R.,
    2. T. Jones,
    3. P. J. Wu,
    4. A. Bolzer,
    5. A. P. Costa-Pereira,
    6. D. Watling,
    7. I. M. Kerr,
    8. D. Sheer
    . 2007. p-STAT1 mediates higher-order chromatin remodelling of the human MHC in response to IFNγ. J. Cell Sci. 120: 3262–3270.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Bhaumik S. R.,
    2. E. Smith,
    3. A. Shilatifard
    . 2007. Covalent modifications of histones during development and disease pathogenesis. Nat. Struct. Mol. Biol. 14: 1008–1016.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Glozak M. A.,
    2. N. Sengupta,
    3. X. Zhang,
    4. E. Seto
    . 2005. Acetylation and deacetylation of non-histone proteins. Gene 363: 15–23.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Shakespear M. R.,
    2. M. A. Halili,
    3. K. M. Irvine,
    4. D. P. Fairlie,
    5. M. J. Sweet
    . 2011. Histone deacetylases as regulators of inflammation and immunity. Trends Immunol. 32: 335–343.
    OpenUrlCrossRefPubMed
    1. Chang H. M.,
    2. M. Paulson,
    3. M. Holko,
    4. C. M. Rice,
    5. B. R. Williams,
    6. I. Marié,
    7. D. E. Levy
    . 2004. Induction of interferon-stimulated gene expression and antiviral responses require protein deacetylase activity. Proc. Natl. Acad. Sci. USA 101: 9578–9583.
    OpenUrlAbstract/FREE Full Text
    1. Nusinzon I.,
    2. C. M. Horvath
    . 2003. Interferon-stimulated transcription and innate antiviral immunity require deacetylase activity and histone deacetylase 1. Proc. Natl. Acad. Sci. USA 100: 14742–14747.
    OpenUrlAbstract/FREE Full Text
    1. Nusinzon I.,
    2. C. M. Horvath
    . 2005. Histone deacetylases as transcriptional activators? Role reversal in inducible gene regulation. Sci. STKE 2005: re11.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Sakamoto S.,
    2. R. Potla,
    3. A. C. Larner
    . 2004. Histone deacetylase activity is required to recruit RNA polymerase II to the promoters of selected interferon-stimulated early response genes. J. Biol. Chem. 279: 40362–40367.
    OpenUrlAbstract/FREE Full Text
    1. Vlasáková J.,
    2. Z. Nováková,
    3. L. Rossmeislová,
    4. M. Kahle,
    5. P. Hozák,
    6. Z. Hodny
    . 2007. Histone deacetylase inhibitors suppress IFNα-induced up-regulation of promyelocytic leukemia protein. Blood 109: 1373–1380.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Icardi L.,
    2. K. De Bosscher,
    3. J. Tavernier
    . 2012. The HAT/HDAC interplay: multilevel control of STAT signaling. Cytokine Growth Factor Rev. 23: 283–291.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Koyama Y.,
    2. M. Adachi,
    3. M. Sekiya,
    4. M. Takekawa,
    5. K. Imai
    . 2000. Histone deacetylase inhibitors suppress IL-2-mediated gene expression prior to induction of apoptosis. Blood 96: 1490–1495.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Furumai R.,
    2. A. Ito,
    3. K. Ogawa,
    4. S. Maeda,
    5. A. Saito,
    6. N. Nishino,
    7. S. Horinouchi,
    8. M. Yoshida
    . 2011. Histone deacetylase inhibitors block nuclear factor-κB-dependent transcription by interfering with RNA polymerase II recruitment. Cancer Sci. 102: 1081–1087.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Su G. H.,
    2. T. A. Sohn,
    3. B. Ryu,
    4. S. E. Kern
    . 2000. A novel histone deacetylase inhibitor identified by high-throughput transcriptional screening of a compound library. Cancer Res. 60: 3137–3142.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Hashioka S.,
    2. A. Klegeris,
    3. P. L. McGeer
    . 2012. The histone deacetylase inhibitor suberoylanilide hydroxamic acid attenuates human astrocyte neurotoxicity induced by interferon-γ. J. Neuroinflammation 9: 113.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Kovacs J. J.,
    2. P. J. Murphy,
    3. S. Gaillard,
    4. X. Zhao,
    5. J. T. Wu,
    6. C. V. Nicchitta,
    7. M. Yoshida,
    8. D. O. Toft,
    9. W. B. Pratt,
    10. T. P. Yao
    . 2005. HDAC6 regulates Hsp90 acetylation and chaperone-dependent activation of glucocorticoid receptor. Mol. Cell 18: 601–607.
    OpenUrlCrossRefPubMed
    1. Rao R.,
    2. W. Fiskus,
    3. Y. Yang,
    4. P. Lee,
    5. R. Joshi,
    6. P. Fernandez,
    7. A. Mandawat,
    8. P. Atadja,
    9. J. E. Bradner,
    10. K. Bhalla
    . 2008. HDAC6 inhibition enhances 17-AAG–mediated abrogation of hsp90 chaperone function in human leukemia cells. Blood 112: 1886–1893.
    OpenUrlAbstract/FREE Full Text
    1. Kim I. A.,
    2. M. No,
    3. J. M. Lee,
    4. J. H. Shin,
    5. J. S. Oh,
    6. E. J. Choi,
    7. I. H. Kim,
    8. P. Atadja,
    9. E. J. Bernhard
    . 2009. Epigenetic modulation of radiation response in human cancer cells with activated EGFR or HER-2 signaling: potential role of histone deacetylase 6. Radiother. Oncol. 92: 125–132.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Zhou Q.,
    2. A. T. Agoston,
    3. P. Atadja,
    4. W. G. Nelson,
    5. N. E. Davidson
    . 2008. Inhibition of histone deacetylases promotes ubiquitin-dependent proteasomal degradation of DNA methyltransferase 1 in human breast cancer cells. Mol. Cancer Res. 6: 873–883.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Narayan V.,
    2. M. Eckert,
    3. A. Zylicz,
    4. M. Zylicz,
    5. K. L. Ball
    . 2009. Cooperative regulation of the interferon regulatory factor-1 tumor suppressor protein by core components of the molecular chaperone machinery. J. Biol. Chem. 284: 25889–25899.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    1. Nakagawa K.,
    2. H. Yokosawa
    . 2000. Degradation of transcription factor IRF-1 by the ubiquitin-proteasome pathway. The C-terminal region governs the protein stability. Eur. J. Biochem. 267: 1680–1686.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Pion E.,
    2. V. Narayan,
    3. M. Eckert,
    4. K. L. Ball
    . 2009. Role of the IRF-1 enhancer domain in signalling polyubiquitination and degradation. Cell. Signal. 21: 1479–1487.
    OpenUrlCrossRefPubMed
  41. ↵
    1. Narayan V.,
    2. E. Pion,
    3. V. Landré,
    4. P. Müller,
    5. K. L. Ball
    . 2011. Docking-dependent ubiquitination of the interferon regulatory factor-1 tumor suppressor protein by the ubiquitin ligase CHIP. J. Biol. Chem. 286: 607–619.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Lee S. J.,
    2. B. C. Jang,
    3. S. W. Lee,
    4. Y. I. Yang,
    5. S. I. Suh,
    6. Y. M. Park,
    7. S. Oh,
    8. J. G. Shin,
    9. S. Yao,
    10. L. Chen,
    11. I. H. Choi
    . 2006. Interferon regulatory factor-1 is prerequisite to the constitutive expression and IFN-γ-induced upregulation of B7-H1 (CD274). FEBS Lett. 580: 755–762.
    OpenUrlCrossRefPubMed
  43. ↵
    1. Gongora C.,
    2. G. Degols,
    3. L. Espert,
    4. T. D. Hua,
    5. N. Mechti
    . 2000. A unique ISRE, in the TATA-less human Isg20 promoter, confers IRF-1-mediated responsiveness to both interferon type I and type II. Nucleic Acids Res. 28: 2333–2341.
    OpenUrlAbstract/FREE Full Text
  44. ↵
    1. Briken V.,
    2. H. Ruffner,
    3. U. Schultz,
    4. A. Schwarz,
    5. L. F. Reis,
    6. I. Strehlow,
    7. T. Decker,
    8. P. Staeheli
    . 1995. Interferon regulatory factor 1 is required for mouse Gbp gene activation by γ interferon. Mol. Cell. Biol. 15: 975–982.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    1. Chen W. Y.,
    2. J. H. Weng,
    3. C. C. Huang,
    4. B. C. Chung
    . 2007. Histone deacetylase inhibitors reduce steroidogenesis through SCF-mediated ubiquitination and degradation of steroidogenic factor 1 (NR5A1). Mol. Cell. Biol. 27: 7284–7290.
    OpenUrlAbstract/FREE Full Text
  46. ↵
    1. Alao J. P.,
    2. E. W. Lam,
    3. S. Ali,
    4. L. Buluwela,
    5. W. Bordogna,
    6. P. Lockey,
    7. R. Varshochi,
    8. A. V. Stavropoulou,
    9. R. C. Coombes,
    10. D. M. Vigushin
    . 2004. Histone deacetylase inhibitor trichostatin A represses estrogen receptor α-dependent transcription and promotes proteasomal degradation of cyclin D1 in human breast carcinoma cell lines. Clin. Cancer Res. 10: 8094–8104.
    OpenUrlAbstract/FREE Full Text
  47. ↵
    1. Xu W.,
    2. M. Marcu,
    3. X. Yuan,
    4. E. Mimnaugh,
    5. C. Patterson,
    6. L. Neckers
    . 2002. Chaperone-dependent E3 ubiquitin ligase CHIP mediates a degradative pathway for c-ErbB2/Neu. Proc. Natl. Acad. Sci. USA 99: 12847–12852.
    OpenUrlAbstract/FREE Full Text
  48. ↵
    1. Ahmed S. F.,
    2. S. Deb,
    3. I. Paul,
    4. A. Chatterjee,
    5. T. Mandal,
    6. U. Chatterjee,
    7. M. K. Ghosh
    . 2012. The chaperone-assisted E3 ligase C terminus of Hsc70-interacting protein (CHIP) targets PTEN for proteasomal degradation. J. Biol. Chem. 287: 15996–16006.
    OpenUrlAbstract/FREE Full Text
  49. ↵
    1. Tamura T.,
    2. H. Yanai,
    3. D. Savitsky,
    4. T. Taniguchi
    . 2008. The IRF family transcription factors in immunity and oncogenesis. Annu. Rev. Immunol. 26: 535–584.
    OpenUrlCrossRefPubMed
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