Abstract
BackgroundFemales with the neurological disorder Rett syndrome are heterozygous for mutations in X-linked MECP2 that encodes methyl-CpG binding protein 2 (MeCP2) thought to act as a transcriptional repressor. To identify target genes for MeCP2 modulation, we studied global gene expression in single cell-derived wild-type and mutant MECP2 expressing fibroblast clones with four common mutations (R106W, R306C, 705delG, 1155del32) and in lymphoblastoid cell lines (LCLs) that included four mutant MeCP2 (T158M, 803delG, R168X and 1159del28) expressing, and five (1159del28, R106W, R255X, 803delG, 803delG) wild-type MeCP2 expressing lines.MethodsClonality and mutation status were verified by androgen receptor methylation assays for X-inactivation and by sequencing MECP2 transcripts. Expression studies were done with oligonucleotide microarrays (Affymetrix U95) and verified with real-time quantitative RT-PCR using Sybr Green.ResultsExpression of 49 transcripts was increased, and expression of 21 transcripts was decreased, in at least 3 of 4 mutant/wild-type fibroblast comparisons. Transcript levels of 11 genes, determined by quantitative RT-PCR, were highly correlated with the microarray data. Therefore, multiple additional clones from two Rett individuals were tested by RT-PCR only. Striking expression differences were found in both mutant and wildtype MeCP2 expressing clones. Comparing expression profiles of lymphoblastoid cell lines yielded 16 differentially expressed genes.ConclusionsMeCP2 deficiency does not lead to global deregulation of gene expression. Either MeCP2's in vivo function does not involve widespread transcriptional repression, or its function is redundant in cell types that also express other methyl-CpG binding proteins. Our data suggest that clonal fibroblast strains may show substantial inter-strain variation, making them a difficult and unstable resource for genome-wide expression profiling studies.
Highlights
Females with the neurological disorder Rett syndrome are heterozygous for mutations in X-linked MECP2 that encodes methyl-CpG binding protein 2 (MeCP2) thought to act as a transcriptional repressor
Characterization of clonal cell strains Clonal mutant and wild-type MECP2 expressing fibroblast cell strains were successfully established from four Rett females with different mutations, one missense in the methyl-binding domain (MBD), one missense in the transcriptional repression domain (TRD) and two frameshift mutations leading to premature stop codons
List of abbreviations cDNA complementary deoxyribonucleic acid DNA deoxyribonucleic acid EST expressed sequence tag mRNA messenger ribonucleic acid PCR polymerase chain reaction μl microliter μm micrometer ml milliliter mm millimeter. Both fibroblast and lymphoblast data together agree in the most important positive result of our studies: MeCP2 deficiency does not result in global deregulation of gene expression
Summary
Females with the neurological disorder Rett syndrome are heterozygous for mutations in X-linked MECP2 that encodes methyl-CpG binding protein 2 (MeCP2) thought to act as a transcriptional repressor. Mutations in MECP2, the gene that encodes the DNA binding protein MeCP2 (methyl-CpG binding protein 2), have been found in approximately 65–77% of Rett syndrome patients [7,8,9,10]. The functional consequence of MECP2 mutations resulting in Rett syndrome is loss of function. Three of the four MECP2 exons encode a 486 amino acid protein that is believed to be a transcriptional repressor based on in vitro studies. MeCP2's functional structure consists of an 84 amino acid methyl-binding domain (MBD) [11] and a 104 amino acid transcriptional repression domain (TRD) [12]. Deacetylation of core histones alters chromatin structure and results in transcriptional repression [13,14]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
