STAT92E is a positive regulator of Drosophila inhibitor of apoptosis 1 (DIAP/1) and protects against radiation-induced apoptosis

Abstract

The proapoptotic factors Reaper, Hid, Grim, and Sickle regulate apoptosis in Drosophila by inhibiting the antiapoptotic factor DIAP1 (Drosophila inhibitor of apoptosis 1). Heat, UV light, x-rays, and developmental signals can all increase the proapoptotic factors, but the control of transcription of the diap1 gene is unclear. We show that in imaginal discs the single Drosophila STAT protein (STAT92E) when activated can directly increase DIAP1 through binding to STAT DNA-binding sites in the diap1 promoter. The STAT92E contribution to DIAP1 production is required for cell survival after x-irradiation but not under unstressed conditions. Because DIAP1 prevents apoptosis after a variety of stresses, STAT92E may have a role in regulating stress responses in general.

Fig. 1.

The difference of stat92E^+/+ vs. stat92E^−/− areas is enhanced by x-irradiation and suppressed by DIAP1 overexpression. Statistically significant differences (P < 0.001) are marked with asterisks. (Aa) Developmental zones of the wing disc shown in Aa′–Aa‴′. MARCM clones with the stat92E³⁹⁷ loss-of-function (LOF) mutant. Mutant (−/−), heterozygous (+/−), and twin-spot (+/+) tissues were marked with α-STAT92E antibody (Aa′), and −/− clones were marked with GFP (Aa″). DIAP1 was expressed exclusively inside stat92E^−/− clones (Aa‴ and Aa‴′). (B) Averaged clonal and twin-spot areas from 50 third-instar wing pouches as percentage occupancy of the pouch area, color-coded as in A; the twin-spot vs. clone ratio is shown in gray. (C) As described for B, except the eye discs were scored.

Fig. 2.

STAT92E dosage in wing discs determines the degree of x-ray-induced apoptosis. α-Caspase 3 antibody (red), an antibody to STAT92E (white), distinguished stat92E^−/− clones (dark), twin spots (stat92E^+/+) (white), and (stat92E^+/−) tissue (gray). GFP marked the stat92E^−/− mutant clones. Overview (Aa) and enlarged frames from Aa (Aa′ and Aa″): stat92E LOF clones (dark areas, one with pink arrow) harbor many apoptotic cells (see Aa″), whereas apoptotic cells appear only in intermediate numbers in the heterozygous cells and very rarely in the WT twin spots (one with turquoise arrow). (Ab) Overview of identical disc as in Aa, with frame enlarged in Ab′ and Ab″. GFP-marked, clonal apoptotic cells can be seen as small dots (Ab′) and associated with the activated caspase marker (Ab″) (red), some double-labeled in yellow. (Ba) Control overexpressing DIAP1 in stat92E^−/− clonal cells (enlarged frames in Ba′ and Ba″). The clonal cells, GFP-marked in Ba′, rarely stained with activated caspase antibody (red in Ba″) because they were protected from apoptosis by high concentrations of DIAP1. (Ba″) Apoptotic cells, in red, were mostly in stat92E^+/− areas (in the white channel, not shown). (C) Quantification of activated caspase-positive areas within areas of stat92E^−/− (Cc and Cf), heterozygous (Cb and Ce), or stat92^+/+ tissue (Ca and Cd) of the type shown in A and B (n > 20 wing disc pouches of each genotype). (Ca–Cc) Genotype as in A; (Cd–Cf) genotype as in B. stat92E^−/− clones overexpressing DIAP1 in f suppress apoptosis. The asterisks mark significant differences of P < 0.001.

Fig. 3.

STAT92E contributes to DIAP1 protein expression by a tyrosine 704-dependent mechanism. DAPI is in gray; stat92E^−/− MARCM clones are shown. (A) Partial dependence of DIAP1 expression levels on STAT92E dosage. (Aa and Aa′) A reduction of DIAP1 can be seen in the pouch and the hinge in +/− regions compared with WT levels in twin spots (marked +/+) and a further, more moderate reduction of DIAP1 in −/− clones (one marked −/−). (B) en-Gal4 drives expression of uas-stat92E in the posterior engrailed domain (Ba′) resulting in increased DIAP1 levels only in the posterior domain (Ba). (C) In contrast, en-Gal4-driven uas-stat92E[Y-F] overexpression in the posterior domain (Ca′) does not increase DIAP1 (Ca).

Fig. 4.

The diap1 locus has two conserved STAT DNA-binding sites. (A) DNA alignment of seven Drosophila species with Drosophila melanogaster. The transcriptional direction is right to left, shown on the black ORF line (28). Neighboring genes are only partially shown. The STAT sites (green), located at −3.3 and −4.3 kb upstream from the most proximal conceptual transcription start site, are contained within a highly conserved cluster of ≈1.2 kb, delineated by the box. (B) DNA sequence alignment surrounding the two STAT-binding sites of the Drosophila species shown in A. (C) Transgenic diap1(1.2 kb)lacZ reporter constructs. The 1.2-kb region boxed in A was cloned into a LacZ reporter construct with WT (WTSTDpZ) (Ca) or point-mutated (MTSTDpZ) STAT-binding sites (Cb). The vector contains a basal hs promoter (arrow) and insulator elements to prevent position effects.

Fig. 5.

STAT92E directly activates the diap1 promoter via two conserved STAT DNA-binding sites. (A) Comparison of expression patterns of the transgenic diap1(1.2 kb)lacZ reporter constructs. (Aa) Expression pattern from WTSTDpZ. (Ab) LacZ expression from MTSTDpZ is almost completely lost in the hinge region (compare white arrow in Ab to star in Aa) and is partially reduced in the pouch but is unchanged in the wing margin (horizontal stripe marked by the gray arrow). DAPI is in gray. (B) MARCM stat92E clones in the background of the WTSTDpZ reporter. (Ba–Ba″) Lower power of which enlargements of white frames are shown in Bb, Bb′, Bc, and Bc′; OF, out of focus area. (Bb and Bb′) In the hinge, reporter expression levels strongly depend on STAT92E. (Bc and Bc′) In the pouch, expression was moderately reduced from WT stat92E^+/+ to stat92E^+/− tissue along with a further subtle reduction in −/− clones. The gray arrow marks the wing margin. (C) WTSTDpZ activity requires the WT STAT DNA-binding sites. Overexpressed STAT92E in the posterior wing disc activates WTSTDpZ (Ca and Ca′) but not the MTSTDpZ reporter (Cb and Cb′).

Fig. 6.

STAT92E in the eye disc suppresses x-ray-induced apoptosis. (Ab) After x-irradiation, the activated caspase antibody (red) stains apoptotic eye disc cells in the first mitotic wave. (Ab′) A clone overexpressing STAT92E[Y→F] (green in white box) crossing the line of the first mitotic wave does not inhibit apoptosis. However, when WT STAT92E is overexpressed in the same manner, apoptosis is suppressed (compare arrowhead in Aa and location of STAT92E gain-of-function (GOF) clone in Aa′) (green in white box). (B) Quantification of A and B; gray columns were set at 100%. Significant suppression in the number of cleaved caspase-positive cells can be seen inside STAT92E GOF clones (column b) compared with equivalent outside WT areas (column a) (P < 0.001; n = 50). No significant difference was seen between inside STAT92E[Y→F] GOF clones (column d) and outside areas (column c) (n = 30).