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Rett syndrome
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Drosophila genes associated with Rett syndrome
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Relevant studies of Rett syndrome

Vonhoff, F., Williams, A., Ryglewski, S. and Duch, C. (2012). Drosophila as a model for MECP2 gain of function in neurons. PLoS One 7: e31835. PubMed ID: 22363746

Methyl-CpG-binding protein 2 (MECP2) is a multi-functional regulator of gene expression. In humans, loss of MECP2 function causes classic Rett syndrome (RTT), but gain of MECP2 function also causes mental retardation. Although mouse models provide valuable insight into Mecp2 gain and loss of function, the identification of MECP2 genetic targets and interactors remains time intensive and complicated. This study takes a step toward utilizing Drosophila as a model to identify genetic targets and cellular consequences of MECP2 gain-of function mutations in neurons, the principle cell type affected in patients with Rett-related mental retardation. It was shown that heterologous expression of human MECP2 in Drosophila motoneurons causes distinct defects in dendritic structure and motor behavior, as reported with MECP2 gain of function in humans and mice. Multiple lines of evidence suggest that these defects arise from specific MECP2 function. First, neurons with MECP2-induced dendrite loss show normal membrane currents. Second, dendritic phenotypes require an intact methyl-CpG-binding domain. Third, dendritic defects are amended by reducing the dose of the chromatin remodeling protein, osa, indicating that MECP2 may act via chromatin remodeling in Drosophila. MECP2-induced motoneuron dendritic defects cause specific motor behavior defects that are easy to score in genetic screening. In sum, this study shows that some aspects of MECP2 function can be studied in the Drosophila model, thus expanding the repertoire of genetic reagents that can be used to unravel specific neural functions of MECP2. However, additional genes and signaling pathways identified through such approaches in Drosophila will require careful validation in the mouse model (Vonhoff, 2012).


  • Full-length human MECP2 specifically causes dendritic defects but does not impair normal membrane excitability in Drosophila motoneurons.
  • Dendritic defects caused by human MECP2 in Drosophila motoneurons require normal MBD function.
  • Dendritic defects in Drosophila motoneurons caused by gain-of-function of human MECP2 can be ameliorated by reducing the dose of the BAF250 homolog, osa.
  • MECP2-induced motoneuron defects result in specific motor behavioral deficiencies.

Methyl-CpG-binding protein 2 (MECP2) is a multifunctional transcriptional regulator involved in chromatin remodeling. Loss of MECP2 function mutations cause classic Rett Syndrome (RTT), an X-linked, dominant, progressive, neuro-developmental disorder. Patients with RTT suffer from cognitive, language, motor conditions, and seizures. However, MECP2 duplication is a frequent case of mental retardation and progressive neurological symptoms in males, and overexpression of MECP2 in the developing mouse brain also causes progressive neurological disorder (Vonhoff, 2012).

The MECP2 protein contains at least five distinct functional domains (NTD, ID, MBD, TRD, and CTDα) which either bind DNA autonomously or regulate MBD (methyl-CpG binding) function. Historically, MECP2 is viewed as a transcriptional repressor that localizes to chromatin by binding to CpG dinucleotides to regulate gene expression through interactions with histone deacetylases and other cofactors. However, MECP2 can also activate transcription, associate also with un-methylated DNA, has chromatin compaction and RNA splicing functions, and several MECP2 interacting proteins are known. Therefore, multiple MECP2 functions might be mediated by interactions with diverse co-factors and by binding to both methylated and non-methylated DNA, consistent with the wide range of phenotypes observed in patients with RTT (Vonhoff, 2012).

Although Mecp2 mouse models recapitulate RTT phenotypes and provide valuable mechanistic insight into neuronal defects caused by Mecp2 mis-regulation, such as axon targeting, synaptic, and dendritic defects, the identification of MECP2 functions and target genes in this system is time intensive and complicated (Vonhoff, 2012).

The Drosophila genetic model system is increasingly being used as a tool to analyze specific genetic and cellular aspects of neurodevelopmental disorders. Short generation times, high fecundity, high throughput screening techniques, facile genetic tools, and relatively low costs have provided valuable mechanistic insights into inherited diseases like Fragile-X, Angelman syndrome, and neurofibromatosis. However, despite considerable conservation in fundamental cell biological pathways, the Drosophila genome encodes only about 75 percent of human disease associated genes, and mecp2 is not among these genes. Therefore, Drosophila can not be used to study the pathophysiology resulting from loss of endogenous mecp2. Instead, the Drosophila model relies on heterologous expression of human MECP2 allele and consequential gain of MECP2 function. Although classic Rett is mostly caused by loss-of-function of MECP2, this is likely not an artificial approach since in humans and in mouse models increased levels of MECP2 also cause disease. Genetic and behavioral proof of principle for the use of the Drosophila model to address MECP2 gain-of-function has been provided earlier. In MECP2 transgenic flies, the MECP2 protein associates with chromatin, interacts with homologs of known human MECP2 interactors, modifies the transcription of multiple genes, and is phosphorylated at serine 423, as in mammals. Most importantly, reported consequences are developmental dysfunctions and motor defects, suggesting parallels with RTT phenotypes. However, previous work on MECP2 in the Drosophila CNS has not tested for cellular phenotypes resulting from MECP2 over-expression in neurons, although mouse models demonstrate that disease phenotypes result from Mecp2 mis-regulation in postmitotic neurons. This study presents the first data on cellular defects as resulting from MECP2 gain-of-function in developing postmitotic Drosophila neurons (Vonhoff, 2012).

It was demonstrated that heterologous expression of human MECP2 in Drosophila motoneurons does not affect axonal pathfinding, dendritic territory boundaries, or the neurons' electrophysiology, but it causes a significant reduction in new dendritic branch formation during development. Similarly, in the mouse model Mecp2 mis-regulation results in pyramidal neuron dendritic defects. This study provides four lines of evidence that dendritic defects in Drosophila motoneurons are caused by specific cellular functions that result from MECP2 gain-of-function, and not from non-specific over-expression or sequestering effects. First, MECP2 protein specifically localizes to the nucleus of Drosophila neurons, so that interactions of MECP2 with molecules in the cytoplasm are unlikely. Second, targeted expression of MECP2 in Drosophila motoneurons causes significant dendritic branching defects but does not affect firing responses to current injections, voltage activated potassium current, or firing frequencies during motor behavior, indicating normal regulation of electrophysiological properties. Although it was earlier demonstrated that Drosophila motoneuron dendritic structure may undergo compensatory changes in response to altered neuronal activity, and a link between motoneuron activity and dendritic growth has clearly been established, any evidence for homeostatic changes in motoneuron excitability in response to developmental defects in dendritic structure was not found in this study. Third, MECP2-induced dendritic defects require intact MBD function of the MECP2 protein because dendritic architecture is not affected following expression of MECP2 alleles with non-functional MBD. This indicates that human MECP2 exerts specific action in Drosophila neurons via chromatin remodeling. Fourth, MECP2-induced dendritic phenotypes can be ameliorated by reducing the dose of osa, a member of the SWI/SNF complex. This genetic interaction is consistent with the hypothesis that human MECP2 may exert specific action in Drosophila motoneurons via chromatin remodeling. It also indicates that MECP2 gain-of-function activates specific cell signaling pathways in Drosophila, and may not cause unspecific over-expression effects. Therefore, the study concludes that Drosophila neurons can serve as a valuable model system to identify some cellular mechanisms by which MECP2 gain-of-function affects neuronal development (Vonhoff, 2012).

It was shown that dendritic defects, as induced by heterologous expression of MECP2 in Drosophila motoneurons, require an intact MBD domain, because expression of MECP2 with a point mutated or truncated MBD domain does not affect dendritic structure. However, each UAS-MECP2 transgene is likely inserted into a unique site in the Drosophila genome, and therefore, the possibility that different UAS-MECP2 transgenes may yield different expression levels or other genetic interactions can not be excluded. The finding that dendritic defects as caused by the expression of full length UAS-MECP2, but not by the expression of UAS-MECP2 transgenes with defective MBD domain, are a result of the unique insertion sites of the UAS-MECP2 constructs into the Drosophila genome, is unlikely for two reasons. First, both UAS-transgenes with defective MBD do not cause dendritic defects. Second, similar dendritic defects are observed following the expression of the full length MECP2 construct inserted in the second or in the third chromosome (Vonhoff, 2012). 

MBD domains recognize two key mechanisms of chromatin regulation in eukaryotes, C5 methylations of DNA at cytosines and post-translational histone modifications. Although the existence of DNA methylation was demonstrated earlier in the fly genome, methylation levels are several orders of magnitude lower than in mammals. The fly genome contains only one methylated DNA binding protein (dMBD2/3) and only one DNA methyltransferase (dDNMT2), which shows highest affinity to t-RNA. Consequently, Drosophila DNA is only sparsely methylated, so that MECP2 interactions with modified histone tails seem the more parsimonious scenario. This is consistent with the finding in this study that MECP2-dependent dendritic defects are suppressed in an osa heterozygous mutant background. Osa is a member of the SWI/SNF complex (human homolog is BAF250), a class of trithorax proteins involved in chromatin remodeling which are highly conserved between flies and humans. This indicates that human MECP2 may exert specific action in Drosophila motoneurons via chromatin remodeling. In fact, it was previously suggested that MECP2 associates with human Brahma, a catalytic component of the SWI/SNF chromatin remodeling complex to regulate gene repression, although this finding is disputed. Nevertheless, the Drosophila system provides some unique advantages to study possible interactions of MECP2 and members of the SWI/SNF chromatin remodeling complex with genetic tools (Vonhoff, 2012). 

The finding in this study that flies with MECP2 over-expression in motoneurons show normal take-off likelihoods as well as normal motoneuron firing and wing beat frequencies but can not sustain flight is in accord with specific MECP2 effects on dendrite development in otherwise normal motoneurons. In Drosophila, take-off can be mediated by the escape response neural circuitry. This circuitry bypasses flight motoneuron dendrites by synapsing directly on MN5 axon, but it relies on normal synaptic transmission and flight motoneuron physiology. Therefore, initial take-off and initial motoneuron firing are not affected by dendritic defects. In Drosophila motoneuron, firing frequencies are directly proportional to wing beat frequency, and thus, these are also not affected. By contrast, flight can not be sustained because the significantly reduced dendritic surface likely reduces the excitatory synaptic drive to motoneuron dendrites that is necessary to stay in flight. Therefore, flies with MECP2-caused motoneuron dendritic defects show a 30- to 60-fold reduction in flight duration. This behavioral phenotype is obvious, and thus, useful for screening. Although the quantification of flight durations and take-off likelihoods does not allow for rapid genetic screening, high throughput screening can easily be developed based on the observed reduction in flight duration by more than 30-fold. Moreover, high throughput assays which utilize Drosophila behavior for rapid screening have been developed by others. Such approaches may help the future identification of candidate MECP2 targets or interactors (Vonhoff, 2012).

Identification of genetic interactors and modifiers of MECP2 function in neurons will be imperative toward developing future treatment strategies. MECP2 itself is not a promising treatment target because the X-linked MECP2 gene is mosaic regulated in the human brain. Furthermore, both loss and gain of function cause disease phenotypes. The sparse methylation landscape in Drosophila may offer unique promise of identifying non-methylated DNA-dependent functions of MECP2 in neurons, the cell type that is most relevant to Rett syndrome. Since known binding partners of MECP2 are conserved in flies (e.g. YB-1, mSin3A etc.), it seems plausible that gain-of-function of human MECP2 may affect neural development via a cellular machinery that is partly conserved between flies and humans (Vonhoff, 2012).

MECP2-induced dendritic phenotypes in flight motoneurons cause a severe motor behavioral phenotype in that flight bout duration is reduced approximately 30- to 60-fold. Rapid screening assays for Drosophila behavioral phenotypes are available. Combined with the fast generation times, high fecundity and facile genetic tools available in Drosophila, this offers a powerful tool to identify molecules that interact with MECP2 in neurons. However, potential MECP2 candidate target genes or genetic modifiers of MECP2 function that can readily be identified in the Drosophila system will then have to be further evaluated in the existing mouse models of RTT (Vonhoff, 2012).

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Hess-Homeier, D.L., Fan, C.Y., Gupta, T., Chiang, A.S and Certel, S.J. (2014). Astrocyte-specific regulation of hMeCP2 expression in Drosophila. Biol Open 3: 1011-1009. PubMed ID: 25305037

Alterations in the expression of Methyl-CpG-binding protein 2 (MeCP2) either by mutations or gene duplication leads to a wide spectrum of neurodevelopmental disorders including Rett Syndrome and MeCP2 duplication disorder. Common features of Rett Syndrome (RTT), MeCP2 duplication disorder, and neuropsychiatric disorders indicate that even moderate changes in MeCP2 protein levels result in functional and structural cell abnormalities. This study investigates two areas of MeCP2 pathophysiology using Drosophila as a model system: the effects of MeCP2 glial gain-of-function activity on circuits controlling sleep behavior, and the cell-type specific regulation of MeCP2 expression. The effects of elevated MeCP2 levels on microcircuits were examined by expressing human MeCP2 (hMeCP2) in astrocytes and distinct subsets of amine neurons including dopamine and octopamine (OA) neurons. Depending on the cell-type, hMeCP2 expression reduces sleep levels, alters daytime/nighttime sleep patterns, and generates sleep maintenance deficits. Next, a 498 base pair region of the MeCP2e2 isoform that is targeted for regulation in distinct subsets of astrocytes was identified. Levels of the full-length hMeCP2e2 and mutant RTT R106W protein decrease in astrocytes in a temporally and spatially regulated manner. In contrast, expression of the deletion Δ166 hMeCP2 protein is not altered in the entire astrocyte population. qPCR experiments reveal a reduction in full-length hMeCP2e2 transcript levels suggesting transgenic hMeCP2 expression is regulated at the transcriptional level. Given the phenotypic complexities that are caused by alterations in MeCP2 levels, these results provide insight into distinct cellular mechanisms that control MeCP2 expression and link microcircuit abnormalities with defined behavioral deficits (Hess-Homeier, 2014).


  • Astrocyte-expression of hMeCP2 alters sleep parameters.
  • hMeCP2 expression in reduced in a subset of Drosophila astrocytes.
  • RTT hMeCP2R106W expression is reduced in the same astrocyte subset.
  • The reduction of hMeCP2 is developmentally regulated.
  • hMeCP2Δ166 expression is detected in the entire alrm-expressing glia population.
  • Transcript levels of transgenic hMeCP2FL are reduced.

The importance of tightly controlling MeCP2 levels in the human nervous system has been underscored by numerous studies encompassing loss-of-function or overexpression conditions. Loss-of-function mutations in MeCP2 cause Rett Syndrome while duplications and/or triplications spanning the MeCP2 locus result in progressive neurological disorders characterized by autism, motor abnormalities, and seizures. As the discovery of distinct neuronal and non-neuronal cell-types that contribute to the various MeCP2-related clinical phenotypes increases, the number of molecular targets of MeCP2 increases as well. Considering the critical role of MeCP2 not only during development but also in maintaining cellular function in adulthood, MeCP2 regulation whether by transcriptional control via DNA regulatory elements or post-transcriptional mechanisms by RNA–protein, RNA–RNA, or RNA–DNA interactions is likely to continue to expand in complexity and in importance (Hess-Homeier, 2014).

This study reports on the spatial and temporal post-transcriptional regulation of human MeCP2 levels in Drosophila astrocytes and on circuit functional changes due to glial expression. Until relatively recently, astrocytes, along with other glia cell types, were believed to be structural cells that function to hold neurons together. It is now appreciated that astrocytes serve many functions, including developmental roles during synaptogenesis, maintenance of the extracellular environment and stabilization of cell–cell communications in the CNS. In addition, astrocytes are increasingly recognized as active partners in synaptic function including regulating basal synaptic transmission and synaptic efficacy leading to the proposal that normal brain output arises from the coordinated activity of a network comprising both neurons and glia. A number of earlier studies highlight the role of astrocytes in the modulation of circuit concerned with sleep and sleep-related rhythmogenesis. Glial cells regulate slow oscillations, a specific thalamocortical activity that characterizes non-REM sleep, and sleep-associated behaviors (Hess-Homeier, 2014).

In this study, it was found that hMeCP2 expression in Drosophila astrocytes causes a significant decrease in sleep with the reduction occurring at a specific time point, immediately after the day:evening transition. How potential cell or non-cell autonomous morphological or functional defects cause the distinct deficit in sleep in the Drosophila males expressing hMeCP2 in astrocytes is presently unknown. However, a recent study shows that dendritic structure defects in motor neurons are caused by MeCP2 expression and as the neurons and circuits that regulate sleep duration, initiation, and maintenance are well studied in Drosophila, further analysis of these described sleep deficits should prove fruitful. Results from the sleep paradigm in this study can also be viewed as the endpoint behavioral representation of synaptic connectivity and dysfunction of circuits in general, which is a fundamental theme in neurodevelopmental syndromes including RTT (Hess-Homeier, 2014).

Although MeCP2 is ubiquitously transcribed, the expression of distinct MeCP2 isoforms is developmentally regulated and heterogeneous in neuronal subpopulations and may be impacted by DNA methylation patterns at MeCP2 regulatory elements may impact the differential expression of MeCP2/MeCP2 isoforms in brain regions. A series of separate studies suggest a role of RNAi-induced down-regulation of MeCP2 expression. In this study, human MeCP2e2 and RTT R106W transcript levels were found to significantly decrease in astrocyte subsets, while expression of the MeCP2Δ166 allele was not altered. The region absent in MeCP2Δ166 contains the MBD domain, a nuclear localizing signal, and a region designated HMG1, due to the amino acid composition similarity to high mobility group (HMG) proteins. What types of evolutionarily conserved cis-regulatory elements are located in this area of the hMeCP2-coding region? Several studies have identified microRNA target sites, AU-rich elements, and G-quadruplexes within the transcribed regions of hMeCP2. Many of these DNA regions influence DNA replication, transcription, and epigenetic mechanisms. For example expression of the microRNA, miR-483-5p, decreases MeCP2 mRNA levels through the human-specific binding site in the MeCP2 long 3′ UTR. At this point, results from this study suggest that an endogenous factor expressed in Drosophila glia targets a regulatory component or components located within the first 498 base pair region of hMeCP2. As wildtype MeCP2 levels in glial cells are essential for proper development and maturation of the brain, identifying cell-type specific mechanisms that activate or repress normal levels to achieve a controlled balance of MeCP2 expression would be useful in therapeutic considerations (Hess-Homeier, 2014).

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Kim, S., Chahrour, M., Ben-Shachar, S. and Lim, J. (2013). Ube3a/E6AP is involved in a subset of MeCP2 functions. Biochem Biophys Res Commun 437: 67-73. PubMed ID: 23791832

Rett syndrome (RTT) and Angelman syndrome (AS) are devastating neurological disorders that share many clinical features. The disease-causing mutations have been identified for both syndromes. Mutations in Methyl-CpG Binding Protein 2 (MECP2) are found in a majority of patients with classical RTT while absence of maternal allele or intragenic mutation in the maternal copy of UBE3A gene encoding the human papilloma virus E6-associated protein (E6AP) cause most cases of AS. Extensive studies have been performed to determine the cause of the neurological problems in each disease. However, the genetic and molecular basis of the overlap in phenotypes between RTT and AS remains largely unknown. This study presents evidence that the phenotypic similarities between the two syndromes might be due to the shared molecular functions between MeCP2 and E6AP in gene expression. Genetic and biochemical studies suggest that E6AP acts as an essential cofactor for a subset of MeCP2 functions. Specifically, decreased expression of Ube3a is able to rescue the cellular phenotypes induced by MECP2-overexpression in Drosophila. Biochemical assays using mice and cell culture systems show that MeCP2 and E6AP physically interact and regulate the expression of shared target genes. Together these data suggest that MeCP2 and E6AP play a role in the transcriptional control of common target gene expression and provide some insight into why RTT and AS share several neurological phenotypes. (Kim, 2013).


  • Decreased expression of Ube3a rescues MECP2-overexpression phenotypes in Drosophila.
  • E6AP and MeCP2 do not regulate each other’s expression.
  • E6AP cooperates with MeCP2 to regulate target gene expression.
  • E6AP does not regulate the expression of known corepressors for MeCP2.
  • E6AP physically associates with MeCP2.

RTT and AS display many overlapping neurological phenotypes. Extensive studies have been performed to determine the cause of neurological problems. However, the genetic and molecular basis of the overlap in phenotypes remains largely unknown. This study provides in vivo genetic and molecular evidence that MeCP2 and E6AP share functions by demonstrating that they are involved in the regulation of shared target gene expression. Overexpression of the human MECP2 gene in the Drosophila eye causes disruption in the structured pattern of the normal eye surface. Similarly, overexpression of Ube3a causes many abnormal phenotypes in Drosophila, while loss of Ube3a expression does not produce any detectable alterations in the Drosophila eye. Therefore, these Drosophila models provide an excellent opportunity to determine if loss or decreased expression of Ube3a can modify MECP2 overexpression phenotypes. It was found that a heterozygous loss of a single dUbe3a allele strongly suppresses ommatidial disorganization phenotypes, without affecting MECP2 expression, in flies expressing human MECP2. These genetic interaction data suggest that Ube3a has a crucial role in inducing or mediating MECP2-induced abnormal phenotypes in Drosophila (Kim, 2013).

Next, it was shown that MeCP2 and E6AP do not regulate each other’s expression. Furthermore, E6AP does not affect expression levels of some of MeCP2-associated proteins in mice in vivo, which are crucial for the regulation of MeCP2-mediated target gene expression. There is still the possibility of novel unidentified MeCP2 corepressors regulated by E6AP, however. Rather, MeCP2 and E6AP can physically associate with each other and modulate expression of some common target genesin HEK293T cells. MeCP2 and E6AP may co-regulate gene expression by acting directly at the promoter and/or regulatory elements of their target genes. Alternatively, E6AP may affect MeCP2 function in organization of chromatin structures and gene expression changes might be secondary to the altered chromatin structures. Although the gene expression changes may not directly explain specific behavioral and neurological phenotypes, the molecular data are in agreement with genetic interaction data in that loss of (or decrease in) Ube3a expression suppresses MECP2 overexpression-induced phenotypes. This co-regulation of shared molecular targets between MeCP2 and E6AP may explain the similarities in disease features of RTT and AS (Kim, 2013).

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Cukier, H.N., Perez, A.M., Collins, A.L., Zhou, Z., Zoghbi, H.Y. and Botas, J. (2008). Genetic modifiers of MeCP2 function in Drosophila. PLoS Genet 4: e1000179. PubMed ID: 18773074

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. This study generated transgenic Drosophila overexpressing human MeCP2 to identify molecular mechanisms capable of compensating for altered MeCP2 levels. It was found that MeCP2 associates with chromatin and is phosphorylated at serine 423 in Drosophila, as is in mammals. MeCP2 overexpression leads to anatomical (i.e., disorganized eyes, ectopic wing veins) and behavioral (i.e., motor dysfunction) abnormalities. A candidate gene approach was used 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 (Cukier, 2008).


  • Overexpression of human MeCP2 in Drosophila leads to eye, wing and motor performance phenotypes.
  • Known MeCP2 physical interactors are also genetic modifiers of the MeCP2 eye phenotype.
  • Genetic modifiers of the MeCP2 eye phenotype also suppress the L3 wing vein phenotype, and the motor impairment caused by neuronal overexpression of MeCP2.

This study uses the Drosophila model system to facilitate the identification of genes capable of counterbalancing the consequences of altered levels of the human MeCP2 protein. First, anatomical and behavioral assays were established to assess the effects of expressing human MeCP2 in flies. An eye phenotype was used as a primary assay for the genetic screen, and impaired motor performance and other phenotypes were used 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 in this study and modifiers of the eye phenotype produced by expression of ataxin-1 or huntingtin. In contrast, it was 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 Δ166 MeCP2 mutations. Two exceptions are Sin3A and trx, which have opposite effects on wild-type and R294X MeCP2. MeCP2 associates with a co-repressor complex containing Sin3A through the TRD domain, 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. 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 (Cukier, 2008).

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. However, accumulating evidence 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, and MeCP2 also binds or requires AT sequences for binding. Moreover, MeCP2 interacts with both methylated and unmethylated chromatin and leads to alterations in the secondary structure of both types of chromatin. In addition, large-scale mapping of MeCP2 binding sites in chromosomal regions containing candidate MeCP2 target genes reveals 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. Furthermore, gene expression patterns in mice that either lack or overexpress MeCP2 suggest that many genes are activated by MeCP2. This study shows that the methyl-CpG-binding domain is not necessary for association of the MeCP2 protein with chromatin in polytene chromosomes, nor is it required to produce an eye phenotype in Drosophila. 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. 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 (Cukier, 2008).

Using a candidate gene approach, this study provides 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. Alterations in the phosphorylation of MeCP2 in trc mutants are not detected. However, there is evidence that both trc and one of its mammalian homologs, NDR2, are involved in dendritic formation, a feature also found to be affected by mutations in MeCP2. Also, modification of the MeCP2 phenotype by the E3 ligase UBE3A target pbl is noteworthy due to the similarities between Rett and Angelman syndromes. Patients with Angelman-like features have been identified with MeCP2 mutations and, while controversial, some studies show a decrease of UBE3A in Rett patients and Mecp2 null mice. The data presented in this study suggests that shared pathways may be involved in Rett and Angelman syndromes (Cukier, 2008).

Misregulation of neuronal genes caused by alterations in MeCP2 activity is thought to cause Rett and Rett-like syndromes. 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. 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. 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 and identifies 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 overexpression 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 (Cukier, 2008).

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 manipulation than MeCP2, and thus they provide new opportunities to develop therapies for Rett syndrome and related neurological disorders (Cukier, 2008).

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Gatto, C.L. and Broadie, K. (2011). Drosophila modeling of heritable neurodevelopmental disorders. Curr Opin Neurobiol 21: 834-841. PubMed ID: 21596554

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Date revised: 20 June 2015

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