Phenotypes of homozygous dUTX mutants. (A and B) Sex combs of wild-type (A) and dUTX¹/dUTX^PB (B) males. (C and D) Eyes of homozygous dUTX¹ flies (D) have a rough appearance compared to that of wild-type (w⁻) eyes (C). (E to G) Wings of dUTX¹/dUTX^PB flies have wing vein defects and display bristle patterning defects on the wing margin (arrow in F′). Mutant wings are curved downwards and sometimes form blisters (arrow in G).

dUTX regulates the cell cycle. (A) Quantification of BrdU incorporation of S2 cells without RNAi treatment (S2 control) and with pUC19 RNAi (negative control) and three different double-stranded RNAs (dsRNAs) against dUTX. (B) Assessment of dUTX mRNA knockdown of the three different dUTX dsRNAs used in the BrdU incorporation assay (A) relative to pUC19 RNAi controls. mRNA levels of rp49 (negative control) are unaffected. (C to F) Heterozygosity for E(z) (D) and Pc (E) suppresses the overrepresentation phenotype of dUTX¹ mosaics (C). Genotypes are as follows: ey-FLP; dUTX¹ FRT40A/P[w⁺] FRT40A (C), ey-FLP; dUTX¹ FRT40A/P[w⁺] FRT40A; E(z)⁷³¹/+ (D), and ey-FLP; dUTX¹ FRT40A/P[w⁺] FRT40A; Pc¹/+ (E).

Analysis of the effect of the loss and overexpression of dUTX on global levels of H3K27 and H3K4 methylation. (A to D) Eye imaginal discs were labeled with antibodies specific for the indicated histone methyl modifications. GFP fluorescence was used to identify dUTX clones. Global levels of H3K27me2 (A), H3K27me1 (B), H3K4me2 (C), and H3K4me3 (D) in dUTX mutant clones were unchanged. Left panels are the merged GFP and antibody channels. The right panels are the antibody-only channels. (E and F) Wing imaginal discs that overexpress dUTX in the posterior compartment marked by GFP (green) were labeled with antibodies specific for H3K27me3 (E) and H3K4me1 (F). Left panels are the merged GFP and antibody channels. The right panels are the antibody-only channels. Global levels of H3K27me3 and H3K4me1 are unchanged. Similar data were obtained for the catalytic mutant dUTX^Jmjc* (data not shown). Genotypes are as follows: ey-FLP; dUTX¹ FRT40A/P[ubi-GFP] FRT40A (A to D) and en-Gal4 UAS-dUTX UAS-GFP (E and F).

Genetic interaction between Notch, Rbf, and dUTX. (A) Wild-type eye from Canton S. (B) The eyeful phenotype at 18°C. The eyeful phenotype is caused by the overexpression of UAS-Delta and GS88A8 (expressing lola and pipsqueak) driven by ey-Gal4 (26). (C to E) The eyeful phenotype at 18°C is dominantly enhanced by heterozygous dUTX alleles. The enhancement ranges from mild (C) to extreme (E). Arrows indicate areas of overgrowth or ectopic outgrowth. (F) Overexpression of Delta in otherwise wild-type eyes using the MARCM system (44) causes only a mild overgrowth phenotype. (G) Overexpression of Delta in dUTX clones using the MARCM system causes massive overgrowth of the eye. (H) Overexpression of ras^V12S35 in dUTX clones using the MARCM system does not cause severe overgrowth. (I) Simultaneous overexpression of Rbf suppresses the overgrowth observed for B. (J) Heterozygosity of Rbf strongly enhances the overrepresentation phenotype of dUTX mosaics. Genotypes are as follows: Canton S (A), eyeful (ey-Gal4 UAS-Delta GS88A8) (26) (B), eyeful; dUTX¹/+ (C to E), hs-FLP UAS-CD8:GFP; P[y⁺] FRT40A/P[tub-GAL80] FRT40A; P[tub-GAL4]/UAS-Delta (F), hs-FLP UAS-CD8:GFP; dUTX¹ FRT40A/P[tub-GAL80] FRT40A; P[tub-GAL4]/UAS-Delta (G), hs-FLP UAS-CD8:GFP; dUTX¹ FRT40A/P[tub-GAL80] FRT40A; P[tub-GAL4]/UAS-UAS-ras^V12S35 (H), hs-FLP UAS-CD8:GFP;dUTX¹ FRT40A/P[tub-GAL80] FRT40A; P[tub-GAL4]/UAS-Delta UAS-Rbf (I), and ey-FLP/Rbf¹⁴; dUTX¹ FRT40A/P[ubi-GFP] FRT40A (J).

dUTX controls H3K27me3 demethylation and H3K4 monomethylation. (A to D) Eye imaginal discs were labeled with antibodies specific for the indicated histone methyl modification. GFP fluorescence was used to identify dUTX clones. Left panels are the merged GFP and antibody channels. Right panels are the antibody-only channels. To ease the identification of dUTX clones, clonal boundaries are indicated with white lines. (A and A′) Global levels of H3K27me3 are increased in dUTX mutant clones (arrows) in eye imaginal discs. dUTX mutant clones are negatively marked by the absence of GFP. (B and B′) Global levels of H3K4me1 are reduced in dUTX mutant clones (arrows) in eye imaginal discs. dUTX mutant clones are negatively marked by the absence of GFP. (C and C′) Expression of the JmjC catalytic mutant dUTX^JmjC* in dUTX mutant clones by the MARCM system rescues the H3K4me1 methylation defect. Clones are positively marked by GFP. (D and D′) Global levels of histone H3 are not altered in dUTX mutant clones in eye imaginal discs. dUTX mutant clones are negatively marked by the absence of GFP. (E) Western blot analysis of larval extracts of the indicated genotype probed with the antibodies listed at the left. Genotypes are as follows: ey-FLP; dUTX¹ FRT40A/P[ubi-GFP] FRT40A (A, B, and D) and hs-FLP UAS-CD8:GFP; dUTX¹ FRT40A UAS-dUTX^JmjC*/P[tub-GAL80] FRT40A/P[tub-GAL4] (C).

Identification of dUTX alleles as overrepresentation mutants in mosaic eyes. (A to C) Representative examples of mosaic eyes of wild-type controls (A), dUTX¹ mosaics (B), and dUTX² mosaics (C). Note the overrepresentation of the dUTX mutant tissue, marked in white, compared to the twin spots, marked in red (B and C). (D to G) Representative examples of mosaic eye-antennal imaginal discs of wild-type control (D), dUTX¹ mosaics (E), dUTX¹ mosaics expressing a UAS-dUTX⁺ rescue construct (F), and dUTX¹ mosaics expressing a UAS-dUTX^JmjC* catalytic mutant construct (G) using the MARCM system (44). Clones are positively labeled by GFP (green). Scale bars represent 100 μm. (H) Domain structure of dUTX and location of mutations. (I and I′) Mosaic eye imaginal discs of dUTX¹ were labeled with anti-dUTX antibody. (I) Merged GFP and antibody channels. (I′) Antibody-only channels. Clones are marked by the absence of GFP. The dUTX¹ allele produces no or strongly reduced levels of the dUTX protein. Note the strong overrepresentation phenotype of dUTX¹ clones in this disc. Genotypes were as follows: ey-FLP; y⁺ FRT40A/P[w⁺] FRT40A (A), ey-FLP; dUTX¹ FRT40A/P[w⁺] FRT40A (B), ey-FLP; dUTX² FRT40A/P[w⁺] FRT40A (C), hs-FLP UAS-CD8:GFP; P[y⁺] FRT40A/P[tub-GAL80] FRT40A/P[tub-GAL4] (D), hs-FLP UAS-CD8:GFP; dUTX¹ FRT40A/P[tub-GAL80] FRT40A/P[tub-GAL4] (E), hs-FLP UAS-CD8:GFP; dUTX¹ FRT40A UAS-dUTX⁺/P[tub-GAL80] FRT40A/P[tub-GAL4] (F), hs-FLP UAS-CD8:GFP; dUTX¹ FRT40A UAS-dUTX^JmjC*/P[tub-GAL80] FRT40A/P[tub-GAL4](G), and ey-FLP; dUTX¹ FRT40A/P[ubi-GFP] FRT40A (I and I′).

dUTX interacts genetically with Notch. (A) Change of mRNA levels in dUTX mutant larvae. qRT-PCR analysis of transcript levels of the indicated genes from homozygous mutant dUTX¹ and dUTX^PB third-instar larvae. The levels are normalized to 100% for wild-type larvae (red line). Note that dUTX mRNA levels in dUTX¹ and dUTX^PB mutant larvae are also reduced. (B and B′) Anti-Notch antibody labeling of dUTX¹ mosaic eye imaginal discs. The posterior is to the right, and the dorsal half is up. (B) Merged GFP and antibody channels. (B′) Antibody-only channel. dUTX clones are marked by the absence of GFP. Clones in the ventral half of the eye disc, especially in the morphogenetic furrow, contain elevated levels of the Notch protein. The anti-Notch antibody was raised against the intracellular domain. (C) Heterozygosity of Notch suppresses the overrepresentation phenotype of dUTX mosaic eyes (compare to Fig. 1B and 4C). (D and E) Wing-notching phenotype caused by the loss of one gene dose of Notch (N) (D) and Serrate (Ser) (E). (D′, D″, E′, E″, and E‴) Heterozygosity of dUTX dominantly suppresses the wing-notching phenotype of N and Ser. The suppression varies from mild (D′ and E′) to complete (D″ and E‴), which is quantified in F and G for the three dUTX alleles. Genotypes are as follows: ey-Flp; dUTX¹ FRT40A/P[ubi-GFP] FRT40A (B and B′), ey-FLP/N^264-39; dUTX¹ FRT40A/P[w⁺] FRT40A (C), N⁸/+; P[y⁺] FRT40A/+ (D), N⁸/+; dUTX¹ FRT40A/+ (D′ and D″), Ser¹/+ (E), and dUTX¹ FRT40A/+; Ser¹/+ (E′ to E‴).