MeCP2 modulates gene expression pathways in astrocytes

Dag H Yasui,Lee-Way Jin,Janine M Lasalle,Keith W Dunaway,Huichun Xu,Izumi Maezawa

doi:10.1186/2040-2392-4-3

Abstract

BackgroundMutations in MECP2 encoding methyl-CpG-binding protein 2 (MeCP2) cause the X-linked neurodevelopmental disorder Rett syndrome. Rett syndrome patients exhibit neurological symptoms that include irregular breathing, impaired mobility, stereotypic hand movements, and loss of speech. MeCP2 protein epigenetically modulates gene expression through genome-wide binding to methylated CpG dinucleotides. While neurons have the highest level of MeCP2 expression, astrocytes and other cell types also express detectable levels of MeCP2. Recent studies suggest that astrocytes likely control the progression of Rett syndrome. Thus, the object of these studies was to identify gene targets that are affected by loss of MeCP2 binding in astrocytes.MethodsTo identify gene targets of MeCP2 in astrocytes, combined approaches of expression microarray and chromatin immunoprecipitation of MeCP2 followed by sequencing (ChIP-seq) were compared between wild-type and MeCP2-deficient astrocytes. MeCP2 gene targets were compared with genes in the top 10% of MeCP2 binding levels in gene windows either within 2 kb upstream of the transcription start site, or the ‘gene body’ that extended from transcription start to end site, or 2 kb downstream of the transcription end site.ResultsA total of 118 gene transcripts surpassed the highly significant threshold (P < 0.005, fold change > 1.2) in expression microarray analysis from triplicate cultures. The top 10% of genes with the highest levels of MeCP2 binding were identified in two independent ChIP-seq experiments. Together this integrated, genome-wide screen for MeCP2 target genes provided an overlapping list of 19 high-confidence MeCP2-responsive gene transcripts in astrocytes. Validation of candidate target gene transcripts by RT-PCR revealed that expression of Apoc2, Cdon, Csrp and Nrep were consistently responsive to MeCP2 deficiency in astrocytes.ConclusionsThe first MeCP2 ChIP-seq and gene expression microarray analysis in astrocytes reveals a set of potential MeCP2 target genes that may contribute to normal astrocyte signaling, cell division and neuronal support functions, the loss of which may contribute to the Rett syndrome phenotype.

Highlights

Mutations in MECP2 encoding methyl-CpG-binding protein 2 (MeCP2) cause the X-linked neurodevelopmental disorder Rett syndrome
Primary astrocyte cultures were prepared from postnatal day 1 (P1) mouse cerebral cortex according to previously described methods [28] for expression profiling and MeCP2 chromatin immunoprecipitation (ChIP)-seq
To identify genes affected by MeCP2 deficiency in astrocytes, expression microarray analysis was performed on RNA extracted from nearly pure (> 95%) astrocyte cultures derived from Mecp2-deficient and wild-type cortices

Summary

Introduction

Mutations in MECP2 encoding methyl-CpG-binding protein 2 (MeCP2) cause the X-linked neurodevelopmental disorder Rett syndrome. MeCP2 protein epigenetically modulates gene expression through genome-wide binding to methylated CpG dinucleotides. MeCP2 is one member of a family of DNA-binding proteins that was originally hypothesized to silence gene transcription by binding to methylated CpG dinucleotides in promoters [7]. This model predicts that MeCP2 deficiency should result in activation of normally repressed genes. Early genome-wide expression profiling studies revealed that only a few genes were significantly upregulated and surprisingly, some genes appeared to be repressed even in MECP2-knockout mice displaying overt disease symptoms [8]. MeCP2 appears to have multiple, genome-wide, gene regulatory functions

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