Genetic Modifiers of MeCP2 Figure 3. Known MeCP2 physical interactors are also genetic modifiers of the MeCP2 eye phenotype. A–C. SEM images of the external eye of a control, a fly expressing full-length MeCP2, and full-length MeCP2 in the presence of a heterozygous loss-of-function allele of Sin3A cultured at 27.5uC. Reduced Sin3A activity enhances the disorganization of the ommatidia in the eye. D–G. SEM images of the external eye of a control, a fly expressing full-length MeCP2, and full-length MeCP2 in the presence of either loss-of-function of crooked legs or Smrter cultured at 30uC. Both alleles suppress the ommatidial disorganization caused by MeCP2 expression in the eye. Genotypes: A. GMR-Gal4/+. B, GMR-Gal4:UAS-MeCP2 FLM119-1M/+. C, GMR-Gal4:UAS-MeCP2 FLM119-1M/Sin3AdQ4. D, GMR-Gal4/+. E, GMR-Gal4:UAS-MeCP2 FLM119-2M/+. F, GMR-Gal4:UAS-MeCP2 FLM119-2M/crold03416. G, Smre04389/ +; GMR-Gal4:UAS-MeCP2 FLM119-2M/+. doi:10.1371/journal.pgen.1000179.g003 overexpression of trc improve the climbing phenotype caused by neuronal-specific expression of MeCP2 (Figure 5F). Consistent modification of MeCP2 phenotypes in different tissues, including a behavioral phenotype caused by neural-specific expression, provides additional evidence for the capacity of theses genes to modulate MeCP2 function. Discussion We have used the Drosophila model system to facilitate the identification of genes capable of counterbalancing the conse- quences of altered levels of the human MeCP2 protein. First, we established anatomical and behavioral assays to assess the effects of expressing human MeCP2 in flies. We used an eye phenotype as a primary assay for the genetic screen, and impaired motor performance and other phenotypes as secondary assays for validating purposes. The eye phenotype has been used successfully in a variety of genetic screens including screens for enhancer/ suppressors of other neurological disease models. Although expression of a variety of ‘‘toxic’’ human proteins leads to apparently similar ‘‘rough’’ eye phenotypes, their specificity is demonstrated when comparing the genetic modifiers uncovered in the screens. For example, there is little or no overlap between the MeCP2 modifiers reported here and modifiers of the eye phenotype produced by expression of ataxin-1 [32,33] or huntingtin [27]. In contrast, we found that the majority of the modifier genes modulating the eye phenotype caused by wild-type MeCP2 similarly modulate the phenotypes caused by the R294X and D166 MeCP2 mutations. Two exceptions are Sin3A and trx, which have opposite effects on wild-type and R294X MeCP2 (Table 1, Figures 3B, C versus Figures S4A, D, and Figures 4B, I versus Figures S4A, P). MeCP2 associates with a co-repressor complex containing Sin3A through the TRD domain [14], which is partially deleted in the truncated R294X protein. This mutant also lacks the MeCP2 C-terminal region that is important for interactions with chromatin in vitro [34]. The TRD domain and/ or C-terminal region may thus be involved in the observed genetic interaction between MeCP2 and trx. It is important to note that both Sin3A and trx do modify the eye phenotype of R294X MeCP2 animals, albeit in the opposite way from the wild-type MeCP2. Thus, the TRD/C-terminal domains may play a modulating role rather than being required for the interaction. A commonly accepted model of MeCP2 function postulates that MeCP2 binds to methylated CpG islands in promoters where it recruits histone deacetylases and other co-repressors to silence gene transcription [14,35]. However, accumulating evidence PLoS Genetics | www.plosgenetics.org 6 September 2008 | Volume 4 | Issue 9 | e1000179