Author Summary Rett syndrome (RTT) is a progressive neurodevelopmental disorder that affects girls early in childhood and is caused by mutations in the MECP2 gene. Loss of MeCP2 function can also lead to clinically distinct conditions characterized by autism, learning disability, and mental retardation. Remarkably, increased levels of MeCP2 leads to related neurological disorders and mental retardation as well. These data emphasize the critical importance of regulating MeCP2 protein levels for normal post-natal development and function of the nervous system. MeCP2 is a protein that associates with chromatin and is thought to modulate gene expression. We have generated Drosophila that overexpress human MeCP2 to investigate the possibility that adjusting the activity of other genes may compensate for altered levels of MeCP2. In support of this hypothesis, we found a variety of modifier genes, including chromatin remodeling genes, that are able to ameliorate and/or aggravate the consequences of MeCP2 overexpression. These findings open the possibility of therapeutic avenues for RTT and related neuropsychiatric disorders by targeting proteins that are possibly easier to manipulate than MeCP2 itself. mutation within the MBD that eliminates the protein’s ability to bind DNA [22]. The RTT R294X mutation truncates the protein within the transcriptional repression domain (TRD), but maintains the nuclear localization signal. The D166 allele completely removes the MBD and N-terminal portion of the protein. Constructs were created inserting each allele into the pUAST vector to utilize the GAL4-UAS system [23]. This system controls expression in specific cell types depending on the Gal4 driver line used, and can be modified by varying the temperature of the fly cultures – increased temperature leads to increased expression. Independent MeCP2 transgenic lines of each allele were generated and tested to ensure that any resulting phenotypes were not caused by the insertion site. Using GMR-Gal4 to drive transgene expression in the eye [24], we confirmed protein expression by western blot analysis using extracts from whole fly heads (Figure 1B). Furthermore, we found that all three MeCP2 alleles that retain amino acid S423, which corresponds to murine S421, produce protein that is specifically phosphorylated at this site (Figure 1C). This specific signal was abolished when the protein extract was treated with alkaline phosphatase (Figure 1D). Phosphorylation at this serine in mammals is brain specific, and it is required by MeCP2 to control dendritic patterning, spine morphogenesis and to regulate the BDNF target gene [25]. Therefore, this key posttranslational modification is conserved when MeCP2 is expressed in Drosophila. Association of MeCP2 with chromatin is a functional property of the mammalian protein [12], and was evaluated by promoting MeCP2 expression with the ubiquitous Actin5c-Gal4 driver. Immunofluorescent staining of squashed salivary glands demonstrated that MeCP2 localizes to the nucleus and associates with polytene chromosomes along many bands in all four alleles (Figure 1E-I’’). While there is widespread association, MeCP2 does not localize to all polytene bands, suggesting target specificity. Association with the polytene chromosomes does not solely depend upon the MBD or C-terminal regions since all four proteins behave similarly. The ability of the R106W and D166 mutants to associate with chromosomes implies that the methyl-CpG-binding domain is not required for this activity. Additional factors, possibly functioning in various protein Genetic Modifiers of MeCP2 complexes with MeCP2, may act to recruit MeCP2 to the chromatin. Phenotypes Caused by MeCP2 Overexpression in Drosophila MeCP2 overexpression in the fly eye by GMR-Gal4 was utilized as an assay for rapid genetic screening of modifiers. Overexpres- sion of multiple independent lines of all four alleles resulted in external eye phenotypes of varying degrees (Figure 2A–E). Lines expressing comparable protein levels were selected for each allele (Figure 1B). The full-length wild-type, R106W and D166 lines cause a disruption of the external structure of the eye that is recognized as a ‘‘glassy’’ effect on the surface when observed by light microscopy (Figure 2A–D). When evaluated by scanning electron microscopy, these same animals show disorganized ommatidia and partial loss of interommatidial bristles (Figure 2A’–D’). These features were enhanced in flies cultured at a higher temperature (Figure 2A’’–D’’) as a result of elevated expression levels. Of all four alleles, the full-length protein causes the strongest disruption to the external eye. While the R294X allele does not cause an obvious disruption of the external eye structure, it shows a loss of pigmentation phenotype (Figure 2E, 2E’, and data not shown), which had only been seen in one of the most strongly expressing full-length lines. Moreover, expression of the R294X allele at a higher temperature is lethal, possibly a consequence of the leaky expression of the GMR-Gal4 driver into other tissues. We also overexpressed MeCP2 in other fly tissues. Expression of the full-length protein in the wing pouch by C5-Gal4 produces extra vein tissue around the L3 and L5 wing veins (Figure 2F, G). Furthermore, neuronal expression of full-length MeCP2 by the CHA-Gal4 driver [26] leads to impaired motor function in adult flies as measured in a climbing assay (Figure 2H, Video S1). While external eye phenotypes are most practical for primary screening to identify novel genetic modifiers of MeCP2, both the wing vein and climbing phenotypes are valuable as secondary screening assays to validate genetic interactions. Genetic Modifiers of MeCP2 We rationalized that in vivo genetic modifiers of MeCP2 function might be enriched among known MeCP2 physical interactors. In support of this hypothesis we previously showed that a large proportion of the physical interactors of huntingtin (the protein that when mutant causes Huntington’s disease) are also genetic modifiers of huntingtin-induced neurodegeneration [27]. To test this hypothesis in the case of MeCP2, we evaluated Sin3A, Smrter, and crooked legs, the Drosophila homologs of Sin3a, N-CoR, and REST (Table S1) [28,29]. We found that heterozygous loss-of-function mutations in each of these three direct interacting partners alter the MeCP2 eye phenotype (Table 1, Figure 3). We then tested other candidate modifier genes that were chosen based on their functions. In addition to chromatin remodeling genes, these included a collection of kinases because MeCP2 is phosphorylated [25], and two genes implicated in Angelman syndrome, a disorder that shares clinical features with Rett syndrome. These last two candidates are the Drosophila homolog of UBE3A, the gene encoding a ubiquitin ligase misregulated in Angelman syndrome, and its target pebble [30,31]. When available, both loss-of-function and overexpression mutant Drosophila lines of each candidate were collected. A total of 584 mutant Drosophila lines were obtained and screened against the full-length MeCP2 allele; 392 lines representing 158 individual kinases, 174 lines representing 54 unique chromatin remodeling genes, and 18 lines encompassing UBE3A and pebble mutants. PLoS Genetics | www.plosgenetics.org 2 September 2008 | Volume 4 | Issue 9 | e1000179