Immunofluorescence Staining of Polytene Chromosomes Experimental and control lines were crossed to flies with the ubiquitous Actin5c-Gal4 driver and cultured at 25uC. Salivary glands were dissected from third instar larvae, fixed with formaldehyde, and squashed according to standard protocols. Samples were blocked with PBT with 0.2% BSA and 5% horse serum to reduce background. Primary antibodies for MeCP2 were used (1:100, Affinity and 1:200, Upstate). The secondary immunofluorescence goat anti-rabbit Cy3 antibody was used at a 1:200 dilution. The slides were also treated with an RNase cocktail (1:1000, Ambion) and then TOTO-3 (1:2000, Molecular Probes) to stain the DNA for confocal microscopy. Slides were then mounted with a drop of Vectashield containing DAPI in order to visualize the DNA by eye. Images were collected by confocal microscopy using the AxioVision and ImageJ programs. Supporting Information Figure S1 Overexpression of the novel genetic modifier osa enhances the MeCP2 external eye phenotype. MeCP2 driven by GMR-Gal4 at 27.5uC causes increased disorganization of the ommatidia and interommatidial bristles compared to controls (A– B). This disorganization is increased when combined with an overexpression allele of the chromatin remodeling gene osa such that the overall size of the eye is smaller, ommatidia are indistinguishable, there are no interommatidial bristles, and necrotic spots are visible, as shown with arrow (C). Overexpression of osa alone by GMR-Gal4 also disrupts the external eye structure (D), but to a much milder degree as compared to co-expression of MeCP2 and osa. Genotypes: A, GMR-Gal4/+. B, GMR-Gal4: UAS-MeCP2FLM119-2M/+. C, GMR-Gal4:UAS-MeCP2FLM119-2M/ UAS-osa. D, GMR-Gal4/UAS-osa. Found at: doi:10.1371/journal.pgen.1000179.s001 (3.3 MB TIF) Figure S2 Genetic modifiers do not alter protein levels of whole MeCP2 or phosphorylated S423 MeCP2. Western blots were performed using the heads of flies expressing MeCP2 by GMR- Gal4 in the presence of modifiers involved in chromatin remodeling (A) and kinases (B). Key genetic modifiers that were found to be the most consistently validated in the external eye assay and secondary assays are marked with an asterisk. All modifiers were compared to the two MeCP2 positive controls on the same blot. Quantification by densitometry failed to find a significant alteration of MeCP2 levels in the case of modifiers as compared to the variation in the internal positive controls. Found at: doi:10.1371/journal.pgen.1000179.s002 (1.9 MB TIF) Figure S3 Genetic modifiers of the full-length MeCP2 eye phenotype modify MeCP2 D166 in a similar manner. Light microscopy images of the external eye of Drosophila expressing the MeCP2 D166 allele and the indicated modifier genes. Control is shown in A. All flies cultured at 29uC. Genotypes: A, GMR- Gal4:UAS-MeCP2 D166/+. B, GMR-Gal4: UAS-MeCP2 D166/ Sin3AdQ4. C, GMR-Gal4: UAS-MeCP2 D166/crole0407. D, Smre04389/ +; GMR-Gal4: UAS-MeCP2 D166/+. E, GMR-Gal4:UAS-MeCP2 D166/UAS-osa. F, GMR-Gal4:UAS-MeCP2 D166/UAS-pbl. G, GMR-Gal4:UAS-MeCP2 D166/+; UAS-Scm/+. H, GMR-Ga- l4:UAS-MeCP2 D166/+; trxE2/+. I, GMR-Gal4:UAS-MeCP2 D166/AsxXF23. J, GMR-Gal4:UAS-MeCP2 D166/+; cortoc03244/+. K, GMR-Gal4:UAS-MeCP2 D166/+; osa00090/+. L, GMR-Ga- l4:UAS-MeCP2 D166/+; UAS-trcLD/+. M, GMR-Gal4:UAS-MeCP2 Genetic Modifiers of MeCP2 pbl09645/+. D166/+; Scme01989/+. N, GMR-Gal4:UAS-MeCP2 D166/+; Found at: doi:10.1371/journal.pgen.1000179.s003 (9.8 MB TIF) Figure S4 Genetic modifiers of the full-length MeCP2 eye phenotype in the context of the MeCP2 R294X allele. Light microscopy images of the external eye of Drosophila expressing the MeCP2 R294X allele and the indicated modifier genes. Control is shown in A. All flies cultured at 27.5uC. Note that partial loss of function of Sin3A (panel D) suppresses the MeCP2 R294X phenotype shown in A, while it enhances the full-length MeCP2 eye phenotype (see Figures 3B and 3C). Likewise, the trxE2 allele suppresses the MeCP2 R294X phenotype (compare panels A and P), while it enhances the full-length MeCP2 eye phenotype (see Figures 4B and 3I). All other genetic modifiers have similar effects on full-length MeCP2 and MeCP2 R294X phenotypes, although the genetic interactions may be evident with some but not all alleles of each modifier genes (see Table 1). Genotypes: A, GMR- Gal4:UAS-MeCP2 R294X/+. B, GMR-Gal4: UAS-MeCP2 R294X/ crolc04670. C, Smre04377/+; GMR-Gal4: UAS-MeCP2 R294X/+. D,GMR-Gal4: UAS-MeCP2 R294X/Sin3AdQ4. E, GMR-Gal4:UAS- MeCP2 R294X/AsxXF23. F, GMR-Gal4:UAS-MeCP2 R294X/+; cortoc03244/+. G, GMR-Gal4:UAS-MeCP2 R294X/+; osa00090/+. H, GMR-Gal4:UAS-MeCP2 R294X/+; pbl5/+. I, GMR-Gal4:UAS- MeCP2 R294X/+; UAS-trcLD/+. J, GMR-Gal4:UAS-MeCP2 R294X/Asx1. K, GMR-Gal4:UAS-MeCP2 R294X/UAS-osa. L, GMR-Gal4:UAS-MeCP2 R294X/UAS-pbl. M, GMR-Gal4:UAS- MeCP2 R294X/+; pbl09645/+. N, GMR-Gal4:UAS-MeCP2 R294X/ +; ScmET50e/+. O, GMR-Gal4:UAS-MeCP2 R294X/+; trxE2/+. P, GMR-Gal4:UAS-MeCP2 R294X/+; trx1/+. Found at: doi:10.1371/journal.pgen.1000179.s004 (8.5 MB TIF) Table S1 Drosophila homologs of known MeCP2 Interactors. Found at: doi:10.1371/journal.pgen.1000179.s005 (0.07 MB DOC) Video S1 Neuronal specific overexpression of MeCP2 results in motor dysfunction. Flies aged 30 days after eclosion are shown in a climbing assay. After being tapped to the bottom of the vial, flies are given 18 seconds to climb past 7 cm, marked by a line on the tube. The control flies (left side, blue) show good climbing ability, while MeCP2 transgenic flies (right side, red) do not perform well. Found at: doi:10.1371/journal.pgen.1000179.s006 (3.0 MB MOV) Acknowledgments We are grateful to Y. He for injecting the MeCP2 constructs, C. Ward for technical help on western blots, R. Kelley for advice in staining polytene chromosomes, K. Dunner, Jr. for assistance with SEM, R. Atkinson for guidance with confocal microscopy, I. Al-Ramahi and M. Canizales- Espinosa for help with figures and statistical analysis, and members of the Zoghbi and Botas laboratories for technical assistance and many insightful discussions. Thanks to H. Bellen for sharing the UAS-pbl line, J. Simon (University of Minnesota) for providing Scm strains, P. Alder (University of Virginia) for sharing the UAS-trc lines, the Bloomington Stock Center (Indiana University) for many Drosophila lines, and the Developmental Studies Hybridoma Bank (University of Iowa) for the anti-tubulin antibody. Author Contributions Conceived and designed the experiments: HNC HYZ JB. Performed the experiments: HNC AMP. Analyzed the data: HNC HYZ JB. Contributed reagents/materials/analysis tools: ALC ZZ. Wrote the paper: HNC JB. PLoS Genetics | www.plosgenetics.org 10 September 2008 | Volume 4 | Issue 9 | e1000179