Two subunits specific to the PBAP chromatin remodeling complex have distinct and redundant functions during drosophila development

Abstract

Chromatin remodeling complexes control the availability of DNA binding sites to transcriptional regulators. Two distinct conserved forms of the SWI/SNF class of complexes are characterized by the presence of specific accessory subunits. In Drosophila, the core Brahma complex associates either with Osa to form the BAP complex or with Bap170 and Bap180 to form the PBAP complex. osa mutations reproduce only a subset of the developmental phenotypes caused by mutations in subunits of the core complex. To test whether the PBAP complex performs the remaining functions, we generated mutations in bap170 and bap180. Surprisingly, we found that Bap180 is not essential for viability, although it is required in ovarian follicle cells for normal eggshell development. Bap170 is necessary to stabilize the Bap180 protein, but a mutant form that retains this function is sufficient for both survival and fertility. The two subunits act redundantly to allow metamorphosis; using gene expression profiling of bap170 bap180 double mutants, we found that the PBAP complex regulates genes involved in tissue remodeling and immune system function. Finally, we generated mutants lacking Bap170, Bap180, and Osa in the germ line to demonstrate that the core Brahma complex can function in oogenesis without any of these accessory subunits.

FIG. 1.

Generating a mutation in bap180. (A) Diagram of the bap180 genomic region, the P element EY14080 used to generate imprecise excisions, and the extent of the deletion in bap180^Δ86. An asterisk indicates the P element insertion site in the 5′ UTR, dashed lines indicate the region amplified by quantitative RT-PCR, and green plus signs indicate the region used to make a fusion protein for antibody generation. (B to D) Third-instar eye imaginal discs containing bap180^Δ86 homozygous clones marked by the absence of GFP (C; green in D) and stained with Bap180 antibody (B; magenta in D). (E) Western blot of extracts from wild-type (WT) and bap180^Δ86 homozygous adults, blotted with anti-Bap180 and the loading control anti-Armadillo (Arm) as indicated. No Bap180 protein can be detected in bap180^Δ86 mutant cells or individuals.

FIG. 2.

bap180 is required for eggshell development. Eggs laid by wild-type (WT) mothers (A and D), bap180^Δ86 mutant mothers (B and E), or bap180^Δ86 mutant mothers rescued by UAS-bap180 expression with T155-GAL4 (C and F). (A to C) External views of the chorion. (D to F) Dechorionated embryos following incubation with neutral red. bap180 mutant mothers lay eggs with irregular and thin chorions and vitelline membranes that are permeable to neutral red; both defects are rescued by expressing bap180 in the follicle cells. (G and H) Stage 12 egg chambers after BrdU labeling (red). Nuclei are stained with DAPI (4′,6′-diamidino-2-phenylindole; blue). Incorporation of BrdU at sites of chorion gene amplification appears normal in bap180 mutants.

FIG. 3.

Generating a mutation in bap170. (A) Diagram of the bap170 genomic region, the P element EY10238 used to generate imprecise excisions, and the extent of the deletions in bap170^Δ65, bap170^Δ135, and bap170^Δ115. An asterisk indicates the P element insertion site in the 5′ UTR of trap1, and plus signs indicate the peptide used as an immunogen for antibody generation. Dashed lines on the upper diagram indicate the regions amplified by quantitative RT-PCR. (B) Quantification of trap1 mRNA by quantitative RT-PCR in larvae that are either wild type or homozygous for each of the indicated bap170 alleles. The bap170^Δ65 and bap170^Δ135 deletions do not affect trap1 expression. (C and D) Adult wings. Loss of bap170 results in extra wing vein material (arrows) in the bap170^Δ65 mutant. (E) Western blot of extracts from embryos laid by wild-type or bap170^Δ65 mutant parents, blotted with anti-Bap170 and the loading control anti-Arm as indicated. This antibody detects no Bap170 protein in bap170^Δ65 mutants. WT, wild type.

FIG. 4.

Bap170 stabilizes Bap180 protein in vivo. (A) Western blotting with anti-Bap180 and the loading control antitubulin of extracts from larvae with the indicated genotypes. (+Bap170: tub-GAL4 UAS-bap170). Bap180 protein is almost completely absent from bap170^Δ115 and bap170^Δ135 mutants but only reduced in bap170^Δ65 mutants. Bap180 levels are restored to normal by expression of bap170 from a transgene, indicating that the loss is specifically due to the absence of Bap170. (B and C) Wing discs from flies expressing UAS-bap170RNAi in the dorsal compartment (outlined) with ap-GAL4. Panel B is stained with anti-Bap170 and panel C is stained with anti-Bap180. Both proteins show strongly reduced levels in the dorsal compartment. (D and E) Quantification of bap180 mRNA and bap170 mRNA by quantitative RT-PCR in wild-type, bap170, and bap180 mutant larvae as indicated. bap180 mRNA is unaffected in bap170 mutants, suggesting that the effect is on protein stability. Using primers to the 3′ end of the gene, some bap170 mRNA is detectable in bap170^Δ65 mutants (E). WT, wild type.

FIG. 5.

Phenotype of bap170 bap180 double mutants. (A and B) Imaginal discs from bap170^Δ65 bap180^Δ86 mutant larvae. In panel A wing (W) and leg (L) discs are stained with anti-Wg (red) and anti-Cubitus interruptus (green); in panel B an eye disc is stained with anti-Elav. All markers are expressed normally, indicating no apparent defects in anterior-posterior or dorsal-ventral patterning of the wing or leg discs or in photoreceptor differentiation in the eye disc. (C and D) Pupae at 88 h APF in wild type and the bap170^Δ65 bap180^Δ86 mutant. The legs have not everted completely (arrows). (E to H) Posterior region of wild-type and bap170^Δ65 bap180^Δ86 mutant third-instar larvae. Panels E and F show expression of the drosomycin-GFP reporter in uninfected larvae, and panels G and H show crystal cells identified by melanization after heating to 65°C. Both reporter expression and crystal cell number are increased in the double mutant larvae. (I and J) Cuticle preparations of a wild-type embryo and an embryo laid by a bap170^Δ65 mother with a bap180^Δ86 germ line clone. The ventral denticle belts are reduced, especially in the anterior region (left), suggestive of dorsalization.

FIG. 6.

The PBAP complex regulates genes required for metamorphosis independently of EcR. (A) Pie chart indicates the functional classes of genes with altered expression levels in bap170^Δ65 bap180^Δ86 mutant white prepupae compared to wild type. (B) Venn diagrams indicate the overlap between genes upregulated or downregulated in bap170^Δ65 bap180^Δ86 white prepupae and in white prepupae expressing an RNAi construct directed against the EcR (5). The extent of overlap is not significant and is not restricted to one direction of change. (C to F) Expression of an EcRE-lacZ reporter detected by 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside staining in wild-type (C and E) and bap170^Δ65 bap180^Δ86 (D and F) white prepupal wing discs (C and D) and fat body (E and F). No significant differences are observed.

FIG. 7.

Target genes of the PBAP complex are not regulated in the same way by osa. (A to F) RT-PCR mRNA quantifications in wild type (WT), bap170^Δ65 bap180^Δ86 double mutants (bap170 bap180), and osa³⁰⁸/osa^4H mutants (osa) (bars are in respective order in all panels). Panels A to C show genes with decreased expression in bap170 bap180 mutants, and panels D to F show genes with increased expression in bap170 bap180 mutants. Error bars represent the coefficient of variance expressed as a percentage of the base. Genes affected in bap170 bap180 double mutants can be unaffected in osa mutants (D), affected to a lesser extent (A, C, and F), or affected in the opposite way (B and E). (G and H) Nomarski images of ovaries from females with brm² germ line clones (G) or from bap170^Δ65 females with osa³⁰⁸ bap180^Δ86 germ line clones (H). No brm² egg chambers develop beyond stage 6, the latest stage reached by ovo^D egg chambers (G); however, egg chambers lacking osa, bap170, and bap180 function can develop to stage 9 (H), and some of the resulting eggs are laid.