DAXX is a new AIRE-interacting protein

Abstract

The AIRE protein plays a remarkable role as a regulator of central tolerance by controlling the promiscuous expression of tissue-specific antigens in thymic medullary epithelial cells. Defects in the AIRE gene cause the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, a rare disease frequent in Iranian Jews, Finns, and Sardinian population. To this day, the precise function of the AIRE protein in regulating transcription and its interacting proteins has yet to be entirely clarified. The knowledge of novel AIRE interactors and their precise role will improve our knowledge of its biological activity and address some of the foremost autoimmunity-related questions. In this study, we have used a yeast two-hybrid system to identify AIRE-interacting proteins. This approach led us to the discovery of a new AIRE-interacting protein called DAXX. The protein is known to be a multifunctional adaptor with functions both in apoptosis and in transcription regulation pathways. The interaction between AIRE and DAXX has been validated by in vivo coimmunoprecipitation analysis and colocalization study in mammalian cells. The interaction has been further confirmed by showing in transactivation assays that DAXX exerts a strong repressive role on the transcriptional activity of AIRE.

FIGURE 1.

AIRE interacts with DAXX in yeast two-hybrid assay. Left panel, schematic representation of AIRE and DAXX constructs. A and I, full-length AIRE and DAXX. B, AIRE construct used to screen human thymus library. C–H, AIRE constructs used to map interaction domains. L, DAXX fragment that interacts with AIRE in yeast two-hybrid assay. M, DAXX construct used to map interaction domain. Right panel, quantitative two hybrid assay. α-Galactosidase units detected by the quantitative assay are shown as gray bars. The constructs used in each assay correspond to the aligned left panel. Plus and minus symbols indicate the transformant capabilities to grow on high stringency medium through ADE and HIS reporter gene activation. CC, coiled coil; NLS, nuclear localization signal; PST, Pro/Ser/Thr.

FIGURE 2.

Coprecipitation of DAXX with AIRE in COS-1 cells. A, coprecipitation of transfected DAXX with transfected HA-AIRE and HA-AIRE-(1–161) in COS cells. Cells were transfected with full-length HA-AIRE and HA-AIRE (aa 1–161). IP was performed from lysates using antibodies against HA (lanes 1 and 2) or beads only (lane 3). Upper panel shows the coprecipitated DAXX of the HA-AIRE IPs. 2nd and 3rd panel from the top show the relative amount of transfected DAXX and transfected HA-AIRE. WB, Western blot. T.L., total lysate. B, coprecipitation of transfected DAXX and HA-AIRE constructs in COS cells. Cells were transfected with HA-AIRE and DAXX. Lanes 1 and 2 of the top panel show DAXX amounts of anti-HA immunoprecipitations performed on cell lysates transfected with HA-AIRE constructs lacking C-terminal region; lanes 3 and 4 of the same panel show DAXX amount of anti-HA IPs performed on cell lysates transfected with HA-AIRE constructs lacking N-terminal region. 2nd and 3rd panel from the top show the transfected amount of DAXX and HA-AIRE respectively. C, coprecipitation of endogenous DAXX with transfected V5-AIRE in COS cells. Cells were transfected with V5-AIRE, and IP was performed from lysates using antibodies against V5 (lane 2) or beads only (lane 1). Upper panel shows the coprecipitated endogenous DAXX of the V5-AIRE IP. 2nd panel from the top shows the immunoprecipitated V5-AIR, and lower panels show anti-V5-AIRE expression and endogenous DAXX levels on total lysates.

FIGURE 3.

HA-AIRE (aa 1–100) is ubiquitinylated in COS-1 cells. COS-1 cells were transfected with HA-AIRE (aa 1–100). After 24 h from transfection cells were treated with 2 and 5 μm MG132 (lanes 2 and 3) or left untreated (lane 1). Upper panel shows anti-HA-AIRE Western blotting (WB). Bottom panel shows the relative amounts of α-actin.

FIGURE 4.

Colocalization of GFP-AIRE with DAXX. AIRE and DAXX expression constructs were transiently cotransfected in HeLa cells and then immunostained with Texas Red anti-rabbit secondary antibody. Nuclei were visualized by 4′,6-diamidino-2-phenylindole staining (data not shown).

FIGURE 5.

Transactivation properties of AIRE are negatively regulated by DAXX. A and B, activation of pMG3-insulin/Luc reporter gene in transfected HeLa (A) and COS-7 cells (B). HeLa and COS-7 cells were cotransfected with a pMG3-insulin/Luc firefly luciferase reporter, a control Renilla luciferase reporter, equal amounts of pEF5HA-AIRE, and increasing amounts of DAXX. Cells were cultured for 24 h and then lysed, and luciferase activity was measured on cell lysates. The average in ratio between firefly and Renilla luciferase activities of lysates was plotted versus AIRE and DAXX expression in same lysates as assessed by anti-HA and anti-DAXX blots.

FIGURE 6.

Homodimerization inhibition assay. DAXX has no influence on AIRE homodimer formation. COS-1 cells were transfected with V5-AIRE, HA-AIRE, Xpress-DAXX, and IPs were performed from lysates using antibodies against V5 (lanes 2–5) or beads only (lane 1). Upper panel shows anti AIRE-HA blot (homodimer formation). 2nd panel from top shows V5-AIRE. 3rd panel shows endogenous and transfected DAXX levels (lanes 1–5). Lower panels show anti-HA and α-actin blots on total lysates (T.L.). WB, Western blot.

FIGURE 7.

Presence of TSA abolishes the negative influence operated by DAXX protein on the transactivation properties of AIRE. Activation of pMG3-insulin/Luc reporter in transfected HeLa. HeLa cells were cotransfected with a pMG3-insulin/Luc firefly luciferase reporter, a control Renilla luciferase reporter, equal amounts of pEF5HA-AIRE, and an increasing amounts of DAXX. Cells were left untreated for 5 h after transfection and then cultured for 24 h in presence of TSA (100 ng/ml). Cells were then lysed, and luciferase activity was measured on cell lysates. The average in ratio between firefly and Renilla luciferase activities of lysates was plotted versus AIRE and DAXX expression in same lysates as assessed by anti-HA and anti-DAXX blots.

FIGURE 8.

Coprecipitation of endogenous HDAC1 and HDAC2 with transfected HA-AIRE in COS cells. COS-1 cells were transfected with full-length HA-AIRE. IPs were performed from lysates using antibodies against HDAC1 and HDAC2. Upper panel shows the coprecipitated HA-AIRE of anti-HDAC1 IP; bottom panel shows the coprecipitated HA-AIRE of anti-HDAC2 IP. LT, total lysate.

FIGURE 9.

Hypothetical model of action of AIRE-DAXX interaction. The figure shows the proposed working model, which is summarized as follows. A, AIRE-PHD1 binds H3K4me0 and recruits H3K4 histone methyltransferase, which establishes the H3K4me3 mark, characteristic of a transcriptional active chromatin state. B and C, complex DAXX-HDAC-H3K4HDM represses AIRE activity by the simultaneous event of histone deacetylation and Lys-4 demethylation. Subsequently epigenetic repression markers as H3K4me0, H3K27me3, and deacetylation occur. D, complex DAXX-HDAC-H3K4HDM is removed, and AIRE inhibits H3K27 histone methyltransferase. The positive epigenetic markers such as H3K27me0, H3K4me3, and acetylation take place.