Regulation of innate immune responses by DAI (DLM-1/ZBP1) and other DNA-sensing molecules

Abstract

DNA, whether it is microbe-derived or host-derived, evokes immune responses when exposed to the cytosol of a cell. We previously reported that DNA-dependent activator of IFN regulatory factors (DAI), also referred to as DLM-1/ZBP1, functions as a DNA sensor that activates the innate immune system. In the present study, we examined the regulation of the complex DNA-sensing system by DAI and other molecules. We first show that DAI directly interacts with DNA in vitro and that it requires three DNA-binding domains for full activation in vivo. We also show that the artificially induced dimerization of DAI results in the DNA-independent activation of type I IFN genes, thereby providing a better understanding for the molecular basis of DAI activation. Furthermore, we provide evidence for the presence of additional DNA sensors, either positively or negatively regulating cytosolic DNA-mediated innate immune responses. These results in toto provide insights into the mechanism of DAI activation and reveal the complex regulatory mechanisms underlying DNA-mediated protective and pathologic immune responses.

Fig. 1.

Recognition of DNAs by DAI protein. (a) Schematic illustration of DAI (DLM-1/ZBP1; DAI-WT) and its deletion mutants. Zα and Zβ DNA-binding domain (pink) and DNA-binding domain D3 (orange) are shown. DAI-ΔN2 is a deletion mutant of all three DNA-binding domains (14). (b) Coomassie brilliant blue (CBB) staining of GST or GST-DAI recombinant proteins. (c) Binding analysis of DAI protein with B-DNA. Recombinant DAI protein was incubated with biotin-conjugated B-DNA and with streptavidin (SA)-conjugated magnetic beads in the absence or presence of unconjugated B-DNA, poly(dG-dC)·poly(dC-dG), ISD, or poly(rI:rC) (0, 0.5, and 5.0 μg/ml; wedge above gels). DAI protein was analyzed by immunoblotting with anti-DAI antibody. (d) DNA length-dependent type I IFN production. L929 cells were transiently transfected with a plasmid vector for Renilla siRNA (Control) or for siRNA targeting DAI (siDAI#1). IFN-β mRNA induction was analyzed by qRT-PCR. Data are mean ± SD (n = 3). ND, not detected. *, P < 0.01 siDAI#1 versus control. (e) DNA pull-down analysis of DAI and DAI mutants. HEK293T cells were transiently transfected with plasmid for HA-tagged DAI-WT or deletion mutants as in a, and whole-cell lysate was incubated with biotin-conjugated B-DNA and SA-conjugated magnetic beads. (Left) Bound protein was analyzed by immunoblotting with anti-HA antibody. (Right) Input protein used for this assay is shown. (f) Type I IFN gene induction by DAI and DAI mutants. L929 cells that retrovirally expressed mock (Control), DAI-WT, or deletion mutants of DAI as in a were stimulated with 6 μg/ml B-DNA for 9 h. Induction of IFN-β mRNA was analyzed by qRT-PCR. Data are mean ± SD (n = 3). *, P < 0.01 DAI-WT or mutants versus control. (g) Interaction of DAI-WT and DAI mutants with IRF3. DAI-WT or deletion mutant of DAI as in a was transiently expressed in L929 cells with FLAG-tagged IRF3. Cells were stimulated with B-DNA for 2 h and analyzed by immunoprecipitation with anti-FLAG antibody, followed by immunoblotting with anti-HA (Upper) and anti-FLAG (Lower) antibodies.

Fig. 2.

Dimerization/oligomerization of DAI activates immune response. (a) Dimer/oligomer formation of DAI by B-DNA stimulation. HA-tagged DAI (HA-DAI) and FLAG-tagged DAI (FLAG-DAI) were transiently coexpressed in L929 cells. The cells were stimulated with 6 μg/ml B-DNA for the indicated periods and analyzed by immunoprecipitation with anti-FLAG antibody, followed by immunoblotting with anti-HA (Upper) and anti-FLAG (Lower) antibodies. (b) Dimerization of DAI induces type I IFN production. HA-DAI or Fv (Fv-vector) or Fv-fused DAI (Fv-DAI) was transiently expressed in L929 cells. The cells were treated with AP20187 for the indicated periods, and the expressions of IFN-β (Top), IFN-α4 (Middle), and GAPDH (Bottom) mRNAs were assessed by RT-PCR. Results from B-DNA treatment on L929 cells are also shown. (c) DAI-WT, DAI-A1, or DAI-A2 was transiently expressed in L929 cells with either FLAG-TBK1 (Upper) or FLAG-IRF3 (Lower). These cells were stimulated with B-DNA and analyzed by immunoprecipitation with anti-FLAG antibody, followed by immunoblotting with anti-HA and anti-FLAG antibodies. The relative band intensities depicted in the graphs are of B-DNA-treated samples measured by a densitometer normalized to those of unstimulated sample.

Fig. 3.

Redundancy in the cytosolic DNA-sensing system. (a) L929 cells were transiently transfected with a plasmid vector for Renilla siRNA (Control) or for siRNA-targeting DAI (siDAI#1), stimulated with 6 μg/ml B-DNA for the indicated periods. (Left) Immunoblots with anti-DAI (Upper) and anti-β-actin (Lower) are shown. (Right) The induction of IFN-β mRNA in siRNA-expressing L929 cells after 6 h of treatment of B-DNA was assessed by qRT-PCR. Data are mean ± SD (n = 3). *, P < 0.01 siDAI#1 versus control. (b) MEFs were transiently transfected with Renilla siRNA (Control) or siDAI#1 plasmid vector, stimulated with B-DNA. (Left) Immunoblots with anti-DAI (Upper) and anti-β-actin (Lower) are shown. (Center and Right) The induction of IFN-β mRNA in siRNA-expressing MEFs after B-DNA stimulation (Center) or 10 μg/ml of ISD stimulation (Right) was assessed by qRT-PCR. Data are mean ± SD. (n = 3). *, P < 0.001 siDAI#1 versus control.

Fig. 4.

Negative regulation of DNA-sensing system. (a and b) MEFs that retrovirally expressed mock (Control, white bars), E3L (a) (black bars), or ADAR1 (b) (black bars) were stimulated with 6 μg/ml B-DNA for the indicated periods. Induction of IFN-β mRNA was analyzed by qRT-PCR. *, P < 0.01; E3L versus control (a) or ADAR1 versus control (b). Data are mean ± SD (n = 3). (c) Adar1^flox/+ and Adar1^flox/− MEFs were stimulated with B-DNA (Left) for the indicated periods or infected with 1 moi of HSV-1 (Right) for 18 h. Induction of IFN-β mRNA was analyzed by qRT-PCR. *, P < 0.01 Adar1^flox/−-adeno-Cre versus Adar1^flox/+-adeno-Cre. Data are mean ± SD (n = 3).