Mecp2-null mice provide new neuronal targets for Rett syndrome

Abstract

Background: Rett syndrome (RTT) is a complex neurological disorder that is one of the most frequent causes of mental retardation in women. A great landmark in research in this field was the discovery of a relationship between the disease and the presence of mutations in the gene that codes for the methyl-CpG binding protein 2 (MeCP2). Currently, MeCP2 is thought to act as a transcriptional repressor that couples DNA methylation and transcriptional silencing. The present study aimed to identify new target genes regulated by Mecp2 in a mouse model of RTT.

Methodology/principal findings: We have compared the gene expression profiles of wild type (WT) and Mecp2-null (KO) mice in three regions of the brain (cortex, midbrain, and cerebellum) by using cDNA microarrays. The results obtained were confirmed by quantitative real-time PCR. Subsequent chromatin immunoprecipitation assays revealed seven direct target genes of Mecp2 bound in vivo (Fkbp5, Mobp, Plagl1, Ddc, Mllt2h, Eya2, and S100a9), and three overexpressed genes due to an indirect effect of a lack of Mecp2 (Irak1, Prodh and Dlk1). The regions bound by Mecp2 were always methylated, suggesting the involvement of the methyl-CpG binding domain of the protein in the mechanism of interaction.

Conclusions: We identified new genes that are overexpressed in Mecp2-KO mice and are excellent candidate genes for involvement in various features of the neurological disease. Our results demonstrate new targets of MeCP2 and provide us with a better understanding of the underlying mechanisms of RTT.

Figure 1. Schematic strategy used to identify new Mecp2 target genes in a mouse model of RTT.

Figure 2. Relative expression results of each gene by qRT-PCR (normalized with respect to Gapdh).

Genes confirmed to have significantly different expression in Mecp2-WT and KO samples are shown. White bars correspond to WT samples and black bars to KO. Values for each tissue are displayed separately. Data are from three independent biological replicates. Error bars indicate standard deviation (SD).

Figure 3. Results from chromatin immunoprecipitation assays.

For Mecp2-WT and KO animals a fraction of total DNA (Input), a no-antibody control (NAB), and a fraction immunoprecipitated by the antibody (Mecp2 B) were tested. Three replicates of each reaction were performed.

Figure 4. Plots representing bisulfite genomic sequencing results for the 5′-regions of the upregulated genes identified in Mecp2 null mice.

Each shows a cloned fragment and the CpGs included. Ten clones are shown for every gene in which one column represents a CpG. Given that no differences were found between tissues or samples, one representative sample is shown for each gene. White squares correspond to non-methylated CpGs and black squares to methylated CpGs.