Gamma interferon triggers interaction between ICSBP (IRF-8) and TEL, recruiting the histone deacetylase HDAC3 to the interferon-responsive element - PubMed (original) (raw)

Gamma interferon triggers interaction between ICSBP (IRF-8) and TEL, recruiting the histone deacetylase HDAC3 to the interferon-responsive element

Takeshi Kuwata et al. Mol Cell Biol. 2002 Nov.

Abstract

ICSBP (IRF-8) is a transcription factor of the IRF family expressed only in the immune system. It is induced in macrophages by gamma interferon (IFN-gamma) and contributes to macrophage functions. By interacting with Ets family protein PU.1, ICSBP binds to the IRF/Ets composite element and stimulates transcription. ICSBP binds to another DNA element, the IFN-stimulated response element (ISRE), a common target of the IRF family. Limited knowledge as to how ICSBP and other IRF proteins regulate ISRE-dependent transcription in IFN-gamma-activated macrophages is available. By mass-spectrometric analysis of ISRE-bound proteins in macrophages, we identified TEL, another Ets member, as a factor recruited to the element in an IFN-gamma-dependent manner. In vitro analysis with recombinant proteins indicated that this recruitment is due to a direct interaction between ICSBP and TEL, which is enhanced by the presence of ISRE. Significantly, the interaction with TEL in turn resulted in the recruitment of the histone deacetytase HDAC3 to the ISRE, causing increased repression of IFN-gamma-mediated reporter activity through the ISRE. This repression may provide a negative-feedback mechanism operating after the initial transcriptional activation by IFN-gamma. By associating with two different Ets family proteins, ICSBP exerts a dual function in IFN-gamma-dependent gene regulation in an immune system-specific manner.

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Figures

FIG. 1.

FIG. 1.

IFN-γ-dependent recruitment of TEL to the ISRE. (A) Three copies of the wild-type (WT) and mutant (Mut) ISRE immobilized to magnetic beads were incubated with nuclear extracts from RAW cells treated with or without IFN-γ (100 U/ml) for 12 h. Bound materials were separated by SDS-8% PAGE and visualized by silver staining. Bands 1 and 2 and band 3 (arrowheads) were identified as TEL and ICSBP, respectively (Table 1). (B) Immobilized wild-type ISREs were incubated with 100 μg of extracts from RAW cells treated with IFN-γ for 12 h, and bound proteins were separated by SDS-10% PAGE and detected by immunoblotting. Input represents 5% of total extracts. ∗, nonspecific protein that did not bind to the ISRE. (C) The wild-type and mutant ISREs were immobilized to the beads, and the binding of the indicated factors was tested by immunoblotting as for panel B. Input represents 5% of total extracts.

FIG. 2.

FIG. 2.

ICSBP-dependent recruitment of recombinant TEL to the ISRE. (A) Immobilized wild-type ISREs were incubated with 20 ng of the indicated recombinant proteins for 1 h at 4°C, and bound proteins were detected by immunoblot analysis. Input represents 10% of the total reaction mixture. (B) Immobilized wild-type (WT) and mutant (Mut) ISREs (M2) were first incubated with 50 ng of the indicated recombinant proteins for 1 h at 4°C, washed, and then incubated with the second protein under the same conditions. Bound proteins were detected as for panel A. Input represents 5% of total extracts.

FIG. 3.

FIG. 3.

Domains required for TEL-ICSBP interaction. (A) (Top) Diagram of TEL deletion mutants and summary of ICSBP binding. (Bottom) Coomassie blue staining of GST-TEL fusions and binding of 35S-labeled ICSBP. Input represents 10% of total reaction. WT, wild type. (B) (Top) Diagram of ICSBP deletion mutants and summary of TEL binding. (Bottom) 35S-lableled ICSBP deletion mutants were incubated with control GST or GST-full-length TEL, and bound materials were detected by autoradiography. Input represents 10% of the reaction mixture.

FIG. 4.

FIG. 4.

Increased repression of ISRE-reporter activity by TEL and ICSBP. (A) RAW cells were transfected with 0.4 μg of ISRE-luciferase reporter along with 0.4 μg of pcx-ICSBP and/or pcDNA-TEL and the Renilla plasmid (5 ng) for 12 h. The total amount of transfected DNA was adjusted with appropriate empty vector to 1.205 μg. Cells were then treated with or without IFN-γ for 12 h. Luciferase activity was normalized by the Renilla internal control. Values represent the averages of three determinations ± standard deviations (SD). (B) RAW cells were transfected with ISRE-reporter along with pcx-ICSBP and the indicated TEL deletion mutants and the Renilla control as for panel A. Cells were treated with IFN-γ for 12 h, and luciferase activity was measured as for panel A. Values represent the averages of three determinations ± SD.

FIG. 5.

FIG. 5.

Reduced 2′,5′-OAS transcript expression by TEL and ICSBP. (A) 32D cells were stably transfected with the control vector or full-length TEL, and appropriate clones were isolated. The TEL clone was transduced with control (TEL) or ICSBP retrovirus vector (TEL + ICSBP). Cells were treated with IFN-γ (100 U/ml) for 6 h. Expression of TEL and ICSBP proteins was detected by immunoblot analysis using 100 μg of nuclear extracts. (B) 2′,5′-OAS mRNA levels in indicated 32D clones treated with IFN-γ (100 U/ml) for the indicated periods were measured by real-time reverse transcription-PCR. Values represent relative mRNA levels normalized by HPRT.

FIG. 6.

FIG. 6.

IFN-γ-dependent recruitment of HDAC3 to the ISRE. (A and B) The immobilized wild-type (WT) and mutant (Mut) ISREs were incubated with 100 μg of extracts from RAW cells treated with IFN-γ (100 U/ml) (A and B) or IFN-α/β (1,000 U/ml) (B) for 12 h, and bound proteins were detected by immunoblotting. Input represents 5% of total extracts. (C) Binding of indicated factors was tested with 100 μg of extracts from RAW cells treated for the indicated times. Input represents 10% of total extracts.

FIG. 7.

FIG. 7.

A model for interaction of ICSBP with TEL. ICSBP interacts with Ets family proteins TEL and PU.1 in IFN-γ-stimulated macrophages. The interaction with TEL occurs on the ISRE, while that with PU.1 requires the EICE. The two partners bind to the elements in opposite orientations: on the ISRE, ICSBP binds to the 5′ site and TEL binds to the 3′ site, while on the EICE, PU.1 binds the 5′ site and ICSBP binds to the 3′ site. ICSBP-TEL complex formation may be a late event that occurs following transcriptional activation by IRF-1, as it recruits HDAC3 and represses ISRE-dependent transcription. This repression may be part of a negative-feedback mechanism. On the other hand, the ICSBP-PU.1 complex activates EICE-dependent transcription presumably by recruiting the histone acetylase (HAT) CBP/p300. This model illustrates a dual role of ICSBP that is acquired by the interaction with two different Ets family partners, permitting immune cell-specific gene regulation by IFN-γ.

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