| A core issue toward understanding the molecular etiology of Rett Syndrome is determining how mutations in MECP2, which encodes a DNA methyl-CpG binding protein, ultimately cause altered transcriptional output. I have worked toward identifying transcripts displaying altered expression in MeCP2 deficient adult neural stem cells (aNSCs), focusing on small noncoding RNA. The diverse regulatory roles that small noncoding RNA, including microRNA (miRNA), have on impacting gene expression suggests the importance of understanding their function in Rett Syndrome. As such, the central hypothesis of this work is that MeCP2 mediated regulation of a subset of small noncoding regulatory RNA in the context of neurodevelopment contributes to the pathogenesis of Rett Syndrome. Based on this, the specific subset of miRNA exhibiting altered expression in MeCP2 deficient aNSCs were identified. Functional characterization of one such miRNA, miR-137, was carried out in aNSCs at different neurodevelopmental stages, ultimately revealing the complexity in gene regulatory networks influenced by MeCP2 mediated regulation of miRNA. The effect of MeCP2 on the expression of small noncoding RNA was further evaluated using a more high-throughput Sequence by Synthesis approach. These experiments identified a unique and previously unannotated set of ∼18nt RNA that associated specifically with transcription start sites. These small RNA reflected promoter proximal regulation of transcription at stimulus responsive genes. Since MeCP2 influences activity dependent transcription in neurons, the subset of genes differentially expressing small RNA at TSSs was identified in order to find MeCP2 targets more likely to be stimulus dependent. At one such gene, Pcdh10, MeCP2 was found to bind proximal to the TSS, a result suggesting MeCP2 may directly influence promoter proximal stalling of RNA Polymerase II dependent transcription at targeted genes in aNSCs. Together, these findings demonstrate the misexpression of specific small noncoding RNA in MeCP2 deficient aNSCs and reveal diversity in the types of gene regulatory pathways that may contribute to the pathogenesis of Rett Syndrome. |
