MeCP2 Mediated Regulation of Small Noncoding RNA Pubblico
Szulwach, Keith Edward (2010)
Abstract
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.
Table of Contents
Chapter 1: Introduction
Part I: Molecular Genetics of Rett Syndrome and MeCP2
Pages 1-5
Part II: Noncoding RNA and RNA Interference Related Pathways in
Mental
Retardation
Pages 5-10
Part III: Adult Neurogenesis and its Modulation by Noncoding RNA
and Epigenetic
Gene Regulatory Processes
Pages 10-14
Chapter 2: Identification and characterization of microRNA
regulated by
MeCP2 in adult neural stem cells
Abstract - pgs 14-15
Introduction - pgs 15-17
Results - pgs 18-29
Discussion - pgs 29-34
Materials and Methods - pgs 34-53
Figure Legends - pgs 54-64
Chapter 4: MicroRNA miR--137 regulates neuronal
maturation by targeting
ubiquitin ligase Mind Bomb--1
Abstract - pg 65
Introduction - pgs 66-67
Results - pgs 67-76
Discussion - pgs 77-80
Materials and Methods - pgs 80-94
Figure Legends - pgs 94-98
Chapter 4: Identification of unannotated small RNA regulated by
MeCP2 -
Transcription start site associated RNAs reveal poised genes
with altered
expression in MeCP2 deficient neural stem cells
Abstract - pg 99
Introduction- pgs 100-102
Results - pgs 103-107
Discussion - pgs 107-109
Materials and Methods - pgs 109-115
Figure Legends - pgs 116-120
Chapter 5: Discussion and Future Directions
Pages 121 -126
About this Dissertation
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