Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

Public Library of Science full text link Public Library of Science Free PMC article
Full text links

Actions

Share

.2008 Sep 5;4(9):e1000179.
doi: 10.1371/journal.pgen.1000179.

Genetic modifiers of MeCP2 function in Drosophila

Affiliations

Genetic modifiers of MeCP2 function in Drosophila

Holly N Cukier et al. PLoS Genet..

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.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. MeCP2 alleles used to generate transgenicDrosophila: protein expression, phosphorylation at serine 423, and association with polytene chromosomes.
A. Four MECP2 alleles were cloned into pUAST to generate transgenic flies. The methyl-CpG-binding domain (MBD) is represented by blue boxes and the transcription repression domain (TRD) is represented by green boxes. The nuclear localization signal (NLS) falls within the TRD. B. Western blot analysis demonstrates expression of each of the alleles when driven byGMR-Gal4. Two distinct MeCP2 antibodies were utilized in order to recognize each allele to confirm that a deletion removed an epitope region. C. Immunoblot with a phospho-specific antibody shows phosphorylation in the three alleles retaining amino acid S423. D. Immunoblot with the phospho-specific MeCP2 S423 antibody in negative control, extracts from MeCP2 expressing flies when treated with calf intestinal phosphatase and untreated MeCP2 extracts. The treated samples fail to produce a band with the phospho-specific antibody, but demonstrate MeCP2 expression with the whole MeCP2 antibody (E-I”). Immunoflourescence of squashed polytene chromosomes dissected from 3rd instar larvae raised at 25°C. Control larvae do not have MeCP2 immunoreactivity (E-E”). All MeCP2 alleles demonstrate accumulation of the MeCP2 protein in banded pattern along the polytene chromosomes (F-I”).
Figure 2
Figure 2. MeCP2 overexpression leads to eye, wing and motor performance phenotypes.
Light microscope images (A–E), and scanning electron microscope images (A'–D”) of fly eyes from controls or animals expressing MeCP2 driven byGMR-Gal4 driver at either 27.5°C or 30°C. External eyes of control flies show normal ommatidial organization, while eyes from animals expressing any of four distinct MeCP2 alleles show disruption in the structured pattern of the eye the surface. Note increased severity of the phenotypes at the higher temperature. F–G. TheC5-Gal4 driver was used to drive either UAS-lacZ or full-length MeCP2 throughout the wing pouch at 25°C. Compared to controls, MeCP2 expressing flies have extra vein tissue (arrowheads) near L3 and L5. H. The neuronal driverCHA-Gal4 was used to drive expression of either UAS-eGFP or full-length MeCP2 at 25°C. Each sample represents a group of 20 virgin females. Beginning at 3 days of age, a lower percent of MeCP2 expressing flies are able to climb to 7 cm in 18 seconds as compared to control flies (Repeated measures ANOVA p<0.001). Over time, both groups decrease in their ability to climb. Error bars represent the standard error. Genotypes: A-A”,GMR-Gal4/+. B-B”,GMR-Gal4:UAS-MeCP2FLM119-2M/+. C-C”,GMR-Gal4:UAS-MeCP2 R106W/+. D-D”,GMR-Gal4:UAS-MeCP2 Δ166/+. E-E',GMR-Gal4:UAS-MeCP2 R294X/+. F,C5-Gal4/+. G,C5-Gal4:UAS-MeCP2 FLM119-1M/+. H,CHA-Gal4/UAS-eGFP andCHA-Gal4/UAS-MeCP2 FLM119-2M.
Figure 3
Figure 3. Known MeCP2 physical interactors are also genetic modifiers of the MeCP2 eye phenotype.
A–C. SEM images of the external eye of a control, a fly expressing full-length MeCP2, and full-length MeCP2 in the presence of a heterozygous loss-of-function allele of Sin3A cultured at 27.5°C. Reduced Sin3A activity enhances the disorganization of the ommatidia in the eye. D–G. SEM images of the external eye of a control, a fly expressing full-length MeCP2, and full-length MeCP2 in the presence of either loss-of-function of crooked legs or Smrter cultured at 30°C. Both alleles suppress the ommatidial disorganization caused by MeCP2 expression in the eye. Genotypes: A.GMR-Gal4/+. B,GMR-Gal4:UAS-MeCP2 FLM119-1M/+. C,GMR-Gal4:UAS-MeCP2 FLM119-1M/Sin3AdQ4. D,GMR-Gal4/+. E,GMR-Gal4:UAS-MeCP2 FLM119-2M/+. F,GMR-Gal4:UAS-MeCP2 FLM119-2M/crold03416. G,Smre04389/+; GMR-Gal4:UAS-MeCP2 FLM119-2M/+.
Figure 4
Figure 4. Novel genetic modifiers of the MeCP2 eye phenotype.
A–B. MeCP2 expression byGMR-Gal4 at 30°C causes severe disorganization of the ommatidia and interommatidial bristles compared to controls. C–H. This phenotype is alleviated when combined with loss-of-function mutants in Asx, corto, osa, pbl, Scm, or overexpression of trc. I. In contrast, the loss-of-function trx allele enhances the external eye phenotype. J–L. When MeCP2 is driven byGMR-Gal4 at 27.5°C, the mild eye phenotype of MeCP2 is enhanced when combined with overexpression of Scm. M–R. Even though the MeCP2 flies do not show an eye phenotype at 25°C (N), when combined with either overexpression of osa (O) or pbl (Q), a strongly disrupted phenotype results that causes a loss of interommatidial bristles and, in the case of pbl, a reduction in the number of ommatidia. When osa and pbl are overexpressed alone, they have very mild phenotypes (P, R). Genotypes: A,GMR-Gal4/+. B,GMR-Gal4:UAS-MeCP2FLM119-2M/+. C,GMR-Gal4:UAS-MeCP2FLM119-2M/AsxXF23. D,GMR-Gal4:UAS-MeCP2FLM119-2M/+; cortoc03244/+. E,GMR-Gal4:UAS-MeCP2FLM119-2M/+; osa00090/+. F,GMR-Gal4: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.
Figure 5
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 theC5-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 driverCHA-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.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Chahrour M, Zoghbi HY. The story of Rett syndrome: from clinic to neurobiology. Neuron. 2007;56:422–437. - PubMed
    1. Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, et al. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet. 1999;23:185–188. - PubMed
    1. Carney RM, Wolpert CM, Ravan SA, Shahbazian M, Ashley-Koch A, et al. Identification of MeCP2 mutations in a series of females with autistic disorder. Pediatr Neurol. 2003;28:205–211. - PubMed
    1. Meins M, Lehmann J, Gerresheim F, Herchenbach J, Hagedorn M, et al. Submicroscopic duplication in Xq28 causes increased expression of the MECP2 gene in a boy with severe mental retardation and features of Rett syndrome. J Med Genet. 2005;42:e12. - PMC - PubMed
    1. Van Esch H, Bauters M, Ignatius J, Jansen M, Raynaud M, et al. Duplication of the MECP2 region is a frequent cause of severe mental retardation and progressive neurological symptoms in males. Am J Hum Genet. 2005;77:442–453. - PMC - PubMed

Publication types

MeSH terms

Substances

Related information

Grants and funding

LinkOut - more resources

Full text links
Public Library of Science full text link Public Library of Science Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2025 Movatter.jp