La Salle, Janine — Medical Microbiology

The discovery of mutations in the MECP2 gene in patients with Rett syndrome (RTT) has broadened the role of epigenetic gene regulation in human development. MECP2 selectively binds to methylated CpG residues and resides in the transcriptionally inactive heterochromatic regions of the nucleus. Genomic imprinting is a process by which epigenetic differences between parental chromosomal alleles are obtained in the gametes. Silencing of imprinted gene expression has been hypothesized to be due to allelic differences in DNA methylation that can be detected for most imprinted genes. Therefore, the hypothesis of this proposal is that MECP2 is involved in silencing imprinted gene expression through the recognition of allele-specific methylation. The hypothesis predicts that cells from RTT patients with MECP2 mutations will show biallelic rather than monoallelic expression of imprinted genes. RTT is a dominant X-linked disease affecting heterozygous females. RTT lymphocytes exhibit random X inactivation, meaning the MECP2 mutations are only observed in RNA transcripts from approximately half the cells. In this proposal, we plan to use the novel approach of single T cell cloning from RTT blood so that clones differentially expressing wild type (wt) and mutant MECP2 could be examined in the same individual. The role of MECP2 in silencing imprinted gene expression and maintaining allele-specific methylation will be directly determined in these T cell clones. The results of the proposed experiments are expected to be directly relevant to understanding how mutations in MECP2 cause RTT by directly investigating genes that are normally silenced through methylation.