Genetic Modifiers of MeCP2 UAS-MeCP2FLM119-2M/+; pbl09645/+. G, GMR-Gal4:UAS-MeCP2FLM119-2M/+; Scme01989/+. H, GMR-Gal4:UAS-MeCP2FLM119-2M/+; UAS-trcLD/+. I, GMR-Gal4:UAS- MeCP2FLM119-2M/+; trxKG04195/+. J, GMR-Gal4/+. K, GMR-Gal4:UAS-MeCP2FLM119-2M/+. L, GMR-Gal4:UAS-MeCP2FLM119-2M/+; UAS-Scm/+. M, GMR-Gal4/+. N, GMR-Gal4:UAS-MeCP2FLM119-2M/+. O, GMR-Gal4:UAS-MeCP2FLM119-2M/UAS-osa. P, GMR-Gal4/UAS-osa. Q, GMR-Gal4:UAS-MeCP2FLM119-2M/UAS-pbl. R, GMR-Gal4/UAS-pbl. doi:10.1371/journal.pgen.1000179.g004 Figure 5. Genetic modifiers of the MeCP2 eye phenotype also suppress the L3 wing vein phenotype, and the motor impairment caused by neuronal overexpression of MeCP2. A–B. Expression of MeCP2 in the wing pouch by the C5-Gal4 driver causes extra wing vein tissue (arrowheads near L3 and L5 veins) as compared to control flies. C–D. This phenotype is suppressed by genetic modifiers of the external eye phenotype including osa and Scm. E. Quantification of the L3 wing vein phenotype demonstrates that alleles of Asx, osa, Scm, and trc are all able to significantly suppress the wing vein phenotype (p,0.05 in all cases). F. Overexpression of full-length MeCP2 by the neuronal driver CHA-Gal4 leads to a motor function impairment as measured in a climbing assay that becomes more severe over time. When MeCP2 is expressed in the presence of either a loss-of-function osa00090 allele or the gain of function UAS-trcLD allele, the severity of the climbing phenotype is reduced (Repeated measures ANOVA p,0.001 at day 13 for osa00090, and for UAS-trcLD at day 10). Each sample represents an initial group of 20 virgin female flies except one control group which had 15 virgin flies. Error bars represent the standard error. Experiment was performed in duplicate yielding similar results, but only one data set is shown. doi:10.1371/journal.pgen.1000179.g005 suggests that this may be too simple a view of MeCP2 function. For example, MeCP2 binds to unmethylated DNA with affinity only 3 times weaker than to methylated DNA [36], and MeCP2 also binds [37] or requires AT sequences for binding [38]. Moreover, MeCP2 interacts with both methylated and unmethy- lated chromatin and leads to alterations in the secondary structure of both types of chromatin [34,39]. In addition, large-scale mapping of MeCP2 binding sites in chromosomal regions containing candidate MeCP2 target genes revealed that: 1) MeCP2 is absent from highly methylated promoters, 2) only ,6% of MeCP2 binding sites are in CpG islands, and 3) many MeCP2-bound promoters are actively expressed [40]. Further- more, a recent study of gene expression patterns in mice that either lack or overexpress MeCP2 suggests that many genes are activated by MeCP2 [41]. Here we show that the methyl-CpG-binding domain is not necessary for association of the MeCP2 protein with chromatin in polytene chromosomes (Figures 1H-H’’), nor is it required to produce an eye phenotype in Drosophila (Figures 2D- D’’). In this context it is interesting to note that unlike mammals, bacteria, plants, and other insects, the levels of DNA methylation are very low in Drosophila [42]. Together these data suggest that MeCP2 function may be more complex than previously thought. MeCP2 may regulate both methylated and unmethylated target genes in vivo, possibly as part of large protein complex(es) of chromatin remodelling proteins regulating gene expression both positively and negatively. Using a candidate gene approach, we provide proof of principle that modulating the activity of modifier genes can amend MeCP2 function in vivo. Among this group of genes is the kinase trc, a member of the NDR (nuclear Dbf-related) family. We could not detect alterations in the phosphorylation of MeCP2 in trc mutants (data not shown). However, there is evidence that both trc and one PLoS Genetics | www.plosgenetics.org 8 September 2008 | Volume 4 | Issue 9 | e1000179