of its mammalian homologs, NDR2, are involved in dendritic formation [43,44], a feature also found to be affected by mutations in MeCP2. Also, modification of the MeCP2 phenotype by the E3 ligase UBE3A target pbl [31] is noteworthy due to the similarities between Rett and Angelman syndromes. Patients with Angelman- like features have been identified with MeCP2 mutations [45,46] and, while still controversial, some studies have demonstrated a decrease of UBE3A in Rett patients and Mecp2 mice [47–49]. The data presented here suggest that shared pathways may be involved in Rett and Angelman syndromes. Misregulation of neuronal genes caused by alterations in MeCP2 activity is thought to cause Rett and Rett-like syndromes [50,51]. One possible avenue for therapy is to identify the MeCP2 target genes misregulated during disease and to restore their normal regulation. This approach may prove impractical if the targets are numerous or difficult to identify due to subtle variations in expression levels in response to MeCP2 activity [52–57]. A possible future treatment based on gene therapy to restore normal levels of MeCP2 also seems improbable. The nervous systems of Rett patients are mosaic due to random X-chromosome inactivation causing some neurons expressing the normal while others expressing the mutant allele. Therefore, in the context of neurons expressing the wild-type allele, gene therapy is not possible because doubling of MeCP2 also leads to disease [5,6,58]. An alternative approach is to identify molecular mechanisms capable of compensating for the misregulation of target genes caused by MeCP2 altered levels. This study provides support for the validity of this approach. We identified specific chromatin remodeling genes of the Pc-G and Trx-G (i.e., Asx, corto, osa, and Scm) that suppress the phenotypes caused by MeCP2 overexpres- sion in Drosophila. Interestingly, both in Drosophila and mammals, mutations in genes of either Pc-G or Trx-G also suppress the body patterning abnormalities caused by mutations in members of the other group [17,19]. In conclusion, human MeCP2 protein expressed in Drosophila maintains important features observed in mammals such as phosphorylation and association with the chromatin. The novel modifiers identified in this model system point to potential therapeutic targets that might be more amenable to manipula- tion than MeCP2, and thus they provide new opportunities to develop therapies for Rett syndrome and related neurological disorders. Methods Generation of MeCP2 Constructs and Drosophila Lines Each of the MeCP2 alleles described was cloned into the pUAST vector in order to utilizing the GAL4-UAS system (Figure 1A). The full-length human cDNA of the MECP2_e2 isoform (1461 nucleotides, 486 amino acids) was subcloned into the EcoRI site of the pUAST vector. The remaining three alleles were generated by PCR mutagenesis of this initial construct. MeCP2 R294X was amplified with primers that attached a stop codon and Kpn I site to the C-terminal end. This PCR fragment was digested with EcoRI and Kpn I and then ligated between these restriction sites in pUAST. Primers amplifying the MeCP2 D166 fragment added an EcoRI site, a conserved Drosophila consensus sequence (TCGAC), and an ATG start site to the N- terminal side of the protein. Transgenic Drosophila lines were generated by injection of these constructs in embryos following standard methods. We generated eleven MeCP2 full-length lines, ten MeCP2 R106W lines, three MeCP2 R294X lines and ten MeCP2 D166 lines. Additional Drosophila lines were obtained from the Bloomington Drosophila Stock Center, the Harvard Medical Genetic Modifiers of MeCP2 School Exelixis Drosophila Stock Collection, and private investiga- tors (see acknowledgements). Western Blot Analysis and Alkaline Phosphatase Treatment Protein was collected from Drosophila heads in a solution of 5% b-mercaptoethanol in Laemmli Sample Buffer (Bio-Rad). For the alkaline phosphatase treatment, Drosophila heads were collected in protein extraction buffer (PBS with 0.1% Nonident P40 and protease inhibitors), samples were smashed and kept on ice for 1 hour, vortexing each 10 minutes in order to facilitate protein extraction. Samples were then mixed 1:1 with the calf intestinal alkaline phosphatase (CIP) treatment, 10% CIP enzyme (New England Biolabs), 30% CIP buffer in water, and incubated for 50 minutes at 37uC. Laemmli Sample Buffer was then added to these reactions. Proteins were run on SDS-PAGE gels with eight Drosophila heads per lane (except for the alkaline phosphatase experiment which had ten heads per lane). Proteins were then transferred to nitrocellulose membrane (Optitran) using 10mM CAPS with 10% methanol. Membranes were blocked in BLOTTO 5% Non-Fat Dry Milk (Bio-Rad) in TBS-T (100mM Tris-Cl pH 7.5, 150mM NaCl, 0.1% Tween 20). The following antibodies were used diluted in BLOTTO: anti-MeCP2 antibodies (1:1000, Upstate, #07-013, and Affinity, #PA1-887), anti-lamin C (1:1000, Developmental Studies Hybridoma Bank, #LC28.26), and anti-tubulin (1:5000, Developmental Studies Hybridoma Bank, #E7). Anti-phosphorylated MeCP2 S423 was diluted in 5% BSA (1:1000) [25]. Anti-rabbit and anti-mouse horseradish peroxidase-conjugated secondary antibodies (Bio-Rad) were dilut- ed 1:5000 in BLOTTO and membranes were developed using chemiluminescence with either the ECL kit (Amersham Biosci- ences) or the SuperSignal West Dura kit (Pierce). Quantification of western blots was performed on a densitometer (Molecular Dynamics) using the ImagQuant program. Scanning Electron Microscopy Experimental and control lines were crossed to flies with the eye specific GMR-Gal4 driver. Offspring were sorted by genotype and whole adult flies were sequentially dehydrated in ethanol, critically-point dried and placed on aluminum mounts. Samples were coated with a platinum alloy for a thickness of 50 nm and flash carbon coated. Drosophila heads were then analyzed with a JEOL JSM-5900 scanning electron microscope. L3 Wing Vein Assays Experimental and control lines were crossed to flies with the C5- Gal4 driver and cultured at 25uC. Once the offspring had eclosed, flies were sorted by genotype and each individual wing was scored under a light microscope for extra vein tissue near or attached to the L3 wing vein. Wings were removed from flies and mounted in DPX Mounting Medium (Electron Microscopy Sciences). Climbing Assay Experimental and control lines were crossed to flies with the CHA- Gal4 driver and cultured at 25uC. Virgins were collected of each genotype and sorted into batches of 20 flies. Flies were enclosed inside two clean, unused 9.25 cm culturing tubes that had been taped together, for a total height of 18.5 cm. Flies were tapped down to the bottom of the vial and permitted 18 seconds to climb within both tubes to the top. At the end of 18 seconds, flies were scored as to whether their final position was either above or below 7 cm. Each group was trained in this procedure for 10 trials and then tested for 10 trials. Trials were performed between 3–6 pm. PLoS Genetics | www.plosgenetics.org 9 September 2008 | Volume 4 | Issue 9 | e1000179