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Figures
- Fig. 1.
DIGs are highly cohesive and enriched for the SWI/SNF chromatin remodelling complex. (A) Protein interaction network of 339 DIGs obtained from sysID (sysid.cmbi.umcn.nl). 235 DIGs form a single network based on annotated protein–protein interactions in BioGrid small-scale studies and the Human Protein Reference Database. DIGs have significantly more interactions and connectivity than expected by random chance (PIE algorithm). (B) GO enrichment analysis for 339 DIGs. Top ten enriched terms with a Bonferroni corrected P-value <0.05 for each GO category are shown. Terms related to gene and chromatin regulation are shown in bold.
- Fig. 2.
Experimental strategy for investigating the function of individual SWI/SNF components in memory-forming neurons of the Drosophila MB. (A,B) The MB-specific Gal4 driver R14H06-Gal4 (A) was used to express UAS-RNAi lines targeting seven different components of the Drosophila SWI/SNF complex (B). (C) SWI/SNF knockdown flies and controls were examined for defects in MB morphology and courtship memory. A schematic diagram and confocal projection showing the expression domain of R14H06-Gal4 is shown in A, and a full brain confocal stack is available in Movie 1. B shows a schematic representation of the BAP and PBAP conformations of the SWI/SNF complex. Purple, core and ATPase modules; yellow, BAP-specific subunits; red, PBAP-specific subunits. Subunits with validated RNAi lines used in this study are indicated with solid colour; other subunits are indicated by transparent colour.
- Fig. 3.
Quantification of extra-dorsal projections in SWI/SNF knockdown MBs. (A-E) The appearance of extra-dorsal projections was qualitatively classified into four categories to account for the observed variation in phenotype severity. Confocal projections show representative images for normal MB morphology (A), as well as the mild (B), moderate (C), strong (D) and severe (E) extra-dorsal projection phenotypes. Scale bars: 50 μm. Arrows indicate the location of extra-dorsal projections. (F) Bar chart showing the total percentage of brains exhibiting normal (white), mild (light grey), moderate (mid grey), strong (dark grey) and severe (black) extra-dorsal projections. The total number of MBs analysed for each genotype is indicated below the bars. ***P<0.001; Fisher's exact test, Bonferroni–Dunn test for multiple comparisons.
- Fig. 4.
Some SWI/SNF components are required for MBγ neuron remodelling. (A) Schematic diagram of MBγ neuron remodelling. APF, after pupae formation. The dashed line indicates the part of the MBγ lobe that is pruned. (B-D) Confocal projections showing MB neurons labelled with R14H06-Gal4 and UAS-mCD8::GFP. Controls expressing an RNAi against mCherry were compared to SWI/SNF knockdown RNAi lines for Bap60 (UAS-Bap6032503), Snr1 (UAS-Snr132372) and E(y)3 [UAS-e(y)332346]. Images were obtained for adults (B), third-instar larvae (C) and early pupae (D). FasII was labelled by immunohistochemistry. Scale bars: 50 µm. Arrows indicate the location of unpruned MBγ axons. For each genotype and developmental stage, we imaged a minimum of ten brains. Larval (C) and pupal (D) phenotypes were 100% penetrant. The penetrance of adult phenotypes is quantified in Fig. 3.
- Fig. 5.
Some SWI/SNF components are required for MBγ axon survival during ageing. (A-C) Confocal projections showing MB neurons labelled with R14H06-Gal4 and UAS-mCD8::GFP at 1 and 7 days after eclosion. Controls expressing an mCherry RNAi (A) are compared to flies expressing RNAi constructs targeting brm (UAS-brm31712) (B) and osa (UAS-osa7810) (C). Phenotypes shown were highly consistent in at least ten individual brains for each genotype and time point. Scale bars: 50 µm.
- Fig. 6.
SWI/SNF complex components are required in the MB for short- and long-term courtship memory. (A,B) The relative learning index (LI) of SWI/SNF RNAi knockdown flies compared to their appropriate genetic background control (see Materials and Methods) for short-term memory (A) and long-term memory (B). Purple bars represent SWI/SNF subunits from the core and ATPase modules; yellow bars represent BAP-specific subunits; and red bars represent PBAP-specific subunits. # indicates a memory defect indicated by no significant reduction in courtship index (CI) in naïve flies compared to trained flies for that genotype (Kruskal–Wallis test). Raw CI and LI data are available in Figs S6 and S7. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; randomization test, 10,000 bootstrap replicates.
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