Activation of the innate immunity in Drosophila by endogenous chromosomal DNA that escaped apoptotic degradation - PubMed (original) (raw)

Activation of the innate immunity in Drosophila by endogenous chromosomal DNA that escaped apoptotic degradation

Naomi Mukae et al. Genes Dev. 2002.

Abstract

Apoptotic cell death is accompanied by degradation of chromosomal DNA. Here, we established in Drosophila a null mutation in the gene for inhibitor of caspase-activated DNase (ICAD) by P-element insertion. We also identified a loss-of-function mutant in Drosophila for DNase II-like acid DNase. The flies deficient in the ICAD gene did not express CAD, and did not undergo apoptotic DNA fragmentation during embryogenesis and oogenesis. In contrast, the deficiency of DNase II enhanced the apoptotic DNA fragmentation in the embryos and ovary, but paradoxically, the mutant flies accumulated a large amount of DNA, particularly in the ovary. This accumulation of DNA in the DNase II mutants caused the constitutive expression of the antibacterial genes for diptericin and attacin, which are usually activated during bacterial infection. The expression of these genes was further enhanced in flies lacking both dICAD and DNase II. These results indicated that CAD and DNase II work independently to degrade chromosomal DNA during apoptosis, and if the DNA is left undigested, it can activate the innate immunity in Drosophila.

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Figures

Figure 1

Establishment of a dICAD mutant by the insertion of a P-element. (A) The dICAD chromosomal gene locus and the position of P-element in each Drosophila strain are schematically shown. The dICAD gene consists of four exons and is depicted as boxes in which the open and filled areas represent the noncoding and coding regions, respectively. The Drosophila l(2)k00617 line contains a copia element in intron 1 of the dICAD gene. Positions of the P-element are shown at the top in kb, starting from the 5′ end of the dICAD gene. (B) Southern hybridization analysis of the Drosophila strains carrying the P-element. Genomic DNAs (10 μg) from P(l(2)k00617) (lane 1), P(l(2)k00617)-P(228) (lane 2), P(228) (lane 3), P(228)-P(16-11) (lane 4), and P(16–11) (lane 5) were digested with _Eco_RI (lanes 1–5), and separated by electrophoresis on a 0.8% agarose gel. Hybridization was carried out with a 32P-labeled 1.0-kb DNA fragment carrying the P-element sequence as a probe. DNA was also prepared from P(16-11) (lane 6); P(icad), which carries two P-elements at 16-11 and at the 5′ noncoding region of the dICAD gene (lane 7); and the revertant of P(icad) (lane 8) in which the P-element at the 5′ noncoding region was removed by excision. DNAs were digested with _Xba_I and subjected to Southern hybridization as above. (C) Northern hybridization analysis. Poly(A) RNA (2 μg) prepared from adult flies of the wild-type (lane 1), dicad mutant (lane 2), and its excision revertant was analyzed by Northern hybridization with dICAD (left panel) or dCAD (right panel) cDNA as the probe. In the lower panels, the membranes used for hybridization were stained with methylene blue. (D) Western blot analysis. The cell lysates (30 μg of protein) prepared from adult wild-type (lane 1), dicad mutant (lane 2), and its excision revertant (lane 3) flies were subjected to Western blot analysis using a rabbit anti-dICAD (left panel) or anti-dCAD (right panel) antibody. The relative molecular masses of the standard proteins are shown in kD at left. The positions of dICAD and dCAD are indicated by arrows. The bands indicated by asterisks appeared to be nonspecific.

Figure 1

Figure 2

Identification of a Drosophila mutant in the DNase II gene. (A) The amino acid sequence of Drosophila DNase II and its mutant. The amino acid sequences of Drosophila DNase II (D), murine DNase II (M), and human DNase II (H) are aligned to give maximum homology by introducing several gaps (−). The amino acid residues that are conserved in all three proteins are shown in bold, and sets of three residues regarded as favored substitutions are indicated by underlines. Three histidine residues conserved in Drosophila, human, and mouse DNase II are indicated by asterisks. The serine residue (S) at amino acid position 223 of Drosophila DNase II was replaced with asparagine (N) in the _DNase-1lo_mutant. (B) Expression of dDNase II in COS cells. COS cells were transfected with the expression vector (lane 1), the expression vector carrying the Flag-tagged wild-type Drosophila DNase II cDNA (lane 2), or the Flag-tagged S223N mutant DNase II cDNA (lane 3). The cell lysates (40 μg of protein) from the transfected cells were separated by electrophoresis on a 10%–20% gradient polyacrylamide gel, and analyzed by Western blot using an anti-Flag antibody. Molecular masses of marker proteins are shown in kD at left. (C) Lack of DNase activity in the mutant DNase II. COS cells were transfected with the empty vector (lanes 2–4), or the expression vector for the wild-type DNase II (lanes 5–7), or its S223N mutant (lanes 8–10). The cell lysates (2 mg of protein) were applied to M2 agarose beads (30 μL bed-volume), and the Flag-tagged DNase II was eluted with 90 μL of PBS containing 100 μg/mL Flag peptide. Using aliquots of 1.0 μL (lanes 2,5,8), 3.0 μL (lanes 3,6,9), and 9.0 μL (lanes 4,7,10) of the eluate, the acid DNase activity was determined with 1.0 μg of plasmid DNA as a substrate. In lane 1, the plasmid DNA was incubated in the assay buffer without the eluate.

Figure 2

Figure 3

The apoptotic DNA fragmentation in Drosophila embryos and ovaries. Genomic DNA was prepared from the 7–11 h embryos (lanes 2–5) or ovaries of day eight adult female flies (lanes 6–9) of the wild-type (CS; lanes 2,6), _dicad_-mutant (lanes 3,7), _ddnase II_-mutant of DNase-1lo(lanes 4,8), and the double mutant for dicad and ddnase II (double; lanes 5,9). An aliquot of DNA (40 ng) was subjected to LM-PCR, and the products were separated by electrophoresis on a 1.0% agarose gel. In lane 1, the LM-PCR was carried out without genomic DNA. The lower panel shows the control PCR for the reaper gene, confirming the equivalent amounts of DNA used for LM-PCR. The molecular mass size marker DNAs were run in lane M, and their sizes are indicated in kb at left.

Figure 4

Accumulation of DNA in ovaries. Ovaries from the adult flies (8 d) of wild-type (A,B), _dicad_-mutant (C,D), _ddnase II_-mutant (E,F), the heterozygous mutant line DNase-1lo(ddnase II)/Df(3R)P14 (G,H), and the double mutant for dICAD and dDNase II (I,J) were stained with acridine orange as described in Materials and Methods.

Figure 5

Expression of antibacterial peptide genes. (A) Constitutive activation of antibacterial peptide genes. Poly(A) RNA (3 μg) from the adult Drosophila flies at day eight of the wild-type (WT), _ddicad_-mutant (dicad), DNase-1lo (ddnase II), and the double mutant for dICAD and dDNase II (double) was subjected to Northern hybridization using cDNA for diptericin, attacin A, and drosomycin. At bottom, the filter was stained with methylene blue. (B) The endogenous DNA- or bacteria-induced expression of the diptericin gene. The adult flies of the double mutant for dICAD and dDNase II (double) were kept uninfected, and the wild-type flies (WT) were infected for 16 h with E. coli. Total RNA (15 μg each) was analyzed by Northern hybridization with diptericin cDNA as a probe.

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Activation of the innate immunity in Drosophila by endogenous chromosomal DNA that escaped apoptotic degradation - PubMed (original) (raw)