Genetic Modifiers of MeCP2 Function in Drosophila Holly N. Cukier1, Alma M. Perez1, Ann L. Collins1, Zhaolan Zhou2,3, Huda Y. Zoghbi1,4,5", Juan Botas1,6"* 1 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America, 2 Neurobiology Program, Children’s Hospital Boston, Massachusetts, United States of America, 3 Departments of Neurology and Neurobiology, Harvard Medical School, Boston, Massachusetts, United States of America, 4 Departments of Neuroscience and Pediatrics, Baylor College of Medicine, Houston, Texas, United States of America, 5 Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas, United States of America, 6 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, United States of America Abstract The levels of methyl-CpG–binding protein 2 (MeCP2) are critical for normal post-natal development and function of the nervous system. Loss of function of MeCP2, a transcriptional regulator involved in chromatin remodeling, causes classic Rett syndrome (RTT) as well as other related conditions characterized by autism, learning disabilities, or mental retardation. Increased dosage of MeCP2 also leads to clinically similar neurological disorders and mental retardation. To identify molecular mechanisms capable of compensating for altered MeCP2 levels, we generated transgenic Drosophila overexpressing human MeCP2. We find that MeCP2 associates with chromatin and is phosphorylated at serine 423 in Drosophila, as is found in mammals. MeCP2 overexpression leads to anatomical (i.e., disorganized eyes, ectopic wing veins) and behavioral (i.e., motor dysfunction) abnormalities. We used a candidate gene approach to identify genes that are able to compensate for abnormal phenotypes caused by MeCP2 increased activity. These genetic modifiers include other chromatin remodeling genes (Additional sex combs, corto, osa, Sex combs on midleg, and trithorax), the kinase tricornered, the UBE3A target pebble, and Drosophila homologues of the MeCP2 physical interactors Sin3a, REST, and N-CoR. These findings demonstrate that anatomical and behavioral phenotypes caused by MeCP2 activity can be ameliorated by altering other factors that might be more amenable to manipulation than MeCP2 itself. Citation: Cukier HN, Perez AM, Collins AL, Zhou Z, Zoghbi HY, et al. (2008) Genetic Modifiers of MeCP2 Function in Drosophila. PLoS Genet 4(9): e1000179. doi:10.1371/journal.pgen.1000179 Editor: Harry Orr, University of Minnesota, United States of America Received January 20, 2008; Accepted July 18, 2008; Published September 5, 2008 Copyright: 2008 Cukier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a G.E.A.R. award from the Rett Syndrome Research Foundation to JB, and by the National Institute of General Medical Sciences predoctoral fellowship to HNC (1 F31 GM067501-01A1), National Institute for Neurological Disorders and Stroke grants to JB (NS042179) and to HYZ. (NS057819), and National Institute of Child Health and Human Development grant to the Baylor College of Medicine Mental Retardation and Developmental Disabilities Research Center (HD024064). HYZ is a Howard Hughes Medical Institute investigator. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jbotas@bcm.edu " These authors are joint senior authors on this work. Introduction Research in the last decade has linked the methyl-CpG-binding protein 2 (MeCP2) with a variety of related neurological disorders [1]. Loss of MeCP2 function causes classic Rett syndrome (RTT), but can also lead to related neurological conditions with symptoms that include autism, mild or severe mental retardation with seizures, or learning disabilities [2,3]. Increased dosage of the MECP2 locus also leads to RTT-like features and severe mental retardation [4–6]. Similar phenotypes are recapitulated in mice that either lack or overexpress MECP2, thus underscoring the importance of properly regulating MeCP2 levels [7–10]. The MeCP2 protein contains a methyl-CpG-binding domain (MBD) and localizes to the heterochromatin where it is believed to regulate gene expression by recruiting histone deacetylases to alter chromatin structure [11,12]. While an ortholog for the complete MeCP2 protein does not exist in Drosophila, methyl-CpG-binding domains are conserved from flies to humans [13]. MeCP2 also interacts with other proteins involved in transcriptional repression and chromatin remodeling including Sin3a, REST and Brahma, a core component of the SWI/SNF complex [14–16]. These and other previously identified MeCP2 interactors have well conserved orthologs in Drosophila (Table S1), as do most components of the chromatin remodeling machinery. Examples of evolutionarily conserved chromatin remodeling proteins include members of the Polycomb and trithorax groups (Pc-G and trx-G, respectively), as well as proteins involved in histone tail modification [17,18]. Many of these proteins act in protein complexes that function antagonistically to promote either activation or repression of target genes [17,19–21]. Therefore, we hypothesized that genetic screening in transgenic flies expressing human MeCP2 may permit the identification of genes capable of compensating the phenotypes caused by altered MeCP2 levels. These genetic modifiers may include genes that function antagonistically to MeCP2 in chromatin remodeling, and perhaps other genes modulating MeCP2 functions or interactions. Here we report the identification of such genes. Results Expression of Human MeCP2 in Drosophila We generated transgenic flies overexpressing wild-type MeCP2 as well as three mutant alleles using the human MECP2_e2 cDNA (Figure 1A). The RTT R106W allele produces a missense PLoS Genetics | www.plosgenetics.org 1 September 2008 | Volume 4 | Issue 9 | e1000179