Page 45 - Mouse Molecular Genetics

Full Abstracts
Program number is above title. Author in bold is the presenter.
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University, London, Ontario, Canada.
Maternal drinking during pregnancy causes Fetal Alcohol Spectrum Disorders (FASD). Previous research on FASD has shown
behavioural, neuro-structural, and more recently, gene expression changes. The mechanism(s) underlying these changes however,
are not known and form the focus of this research. Specifically, this research examines any involvement of epigenetic
mechanisms in a mouse model of FASD using four ethanol treatment protocols in C57BL/6J mice. Total brain from resulting
mature males (postnatal day 70) from ethanol exposed and matched controls were used to assess noncoding RNA expression
using mouse miRNA arrays, mouse gene expression arrays and qPCR. The results have revealed that a large number of
microRNAs and snoRNAs are altered, both up and down, depending on treatment paradigm. Some of the observed changes are
unique to a specific treatment protocol, while others overlap across treatments. Strikingly, approximately 20% of the altered
noncoding RNA genes map to three imprinted regions of the mouse chromosome. The first two,
Snrpn-Ube3a
(
Murine
7
qC/Human 15q11-q13) and
Dlk1-Dio3
(
Murine 12qF1/Human 14q32.2), are associated with processes involved in neuronal
plasticity and several neurodevelopmental disorders. The third cluster contains
Sfmbt2
(
Murine 2qA1) and an overlapping
antisense transcript that is unique to mice and rats. We followed these results with the assessment of DNA methylation using
methylated DNA immunoprecipitation followed by hybridization to DNA arrays (MeDIP-Chip). The results show that fetal
alcohol exposure has a genome-wide effect on DNA methylation with imprinted regions of the genome appearing to be
particularly sensitive. Ultimately, our results suggest that imprinted noncoding RNAs, many of which are known to play a critical
role in neurodevelopment and brain function, may also have a role in the long-term maintenance of cognitive deficit associated
altered gene expression in the mouse model of FASD.
32
MeCP2 Interacting Partners in Development and Rett Syndrome. Mary Donohoe
1,2,3
,
Siva Muthuswamy
1,2,3
,
Tao Wu
1,2,3
. 1)
Burke Medical Research Institute, White Plains, NY; 2) Dept of Neuroscience Weill Cornell Medical College New York, NY; 3)
Dept of Cell and Developmental Biology Weill Cornell Medical College New York, NY.
Rett Syndrome (RTT) is a neurodevelopmental disorder that is one of the leading causes of mental retardation and autistic
behavior in girls. RTT is caused by mutations in the X-linked Methyl CpG-binding protein 2 (MeCP2) gene, which accounts for
approximately 80% of sporadic and 45% familial RTT cases. MeCP2 can function as a transcriptional modifier that represses
developmental silencing through binding to methylated DNA and complexing with co-repressors. In addition, MeCP2 may
operate as a transcriptional activator, splicing factor, and in long-range chromatin looping. MeCP2 is expressed in many
mammalian tissues but disruption of this regulator in RTT has a profound effect in mature neurons. Consistent with this, MeCP2
is a key regulator of neural activity-dependent gene expression controlling learning and memory and cocaine drug addiction.
MeCP2 shares many characteristics with histones. It is highly enriched with amino acids that may be regulated by post-
translational modifications (PTMs). Phosphorylation of MeCP2 can regulate its DNA binding in a neural activity dependent-
manner. Other PTMs have not been described for MeCP2. To understand the action of MeCP2, our lab is studying its protein
interacting partners as well as PTMs in a mouse model. We show that SUMO1 and SUMO3 proteins bind and modify MeCP2.
SUMOylation of MeCP2 is greatly enhanced by neural activity. The transcriptional activity of MeCP2 is modified by SUMO
modification. Using sequential Chromatin Immunoprecipitation we show that MeCP2 is SUMOylated at specific promoters in
vivo. We have identified an interacting partner for MeCP2. Loss of this MeCP2 co-factor results in an alteration of SUMOylation
suggesting that this co-factor serves as a SUMO E3 ligase for MeCP2. We will present our ongoing studies to define the
neuroepigenetic regulatory circuit of MeCP2 in normal development and in RTT.
33
The histone H2B ubiquitin ligase Rnf20 is required for self-renewal and pluripotency in mouse ES cells. Kit Wan Ma
,
Takaho A. Endo, Jafar Sharif, Haruhiko Koseki. RCAI, RIKEN Yokohama Institute, Yokohama, Kanagawa, Japan.
Self-renewal and pluripotency are two main features of embryonic stem (ES) cells, giving rise to unlimited expansion ability
and differentiation potential into any cell types in the body. They are attained by retaining the cells at the S phase of the cell
cycle, activation of pluripotency genes and suppression of differentiation regulator genes. Therefore, a molecular mechanism that
shapes the chromatin landscape for DNA replication and gene transcription is a master regulator of the ES cell features.
However, such mechanism has not yet been identified. H2B monoubiquitination at lysine 120 (H2BK120ub1) mediated by the
Rnf20/Rnf40 complex plays a key role in the regulation of chromatin landscape by manipulating histone modifications and
chromatin structure. Previous studies showed that H2BK120ub1 is a prerequisite of H3K4 trimethylation and is responsible for
regulating the dynamics of chromatin higher-order folding. Our preliminary results demonstrated that Rnf20, possibly via H2B
ubiquitination, is indispensible for maintaining the pluripotency and self-renewal. In mouse Rnf20 knockout ES cells, the global
H2BK120ub1 decreases accompanied by growth defect and cell differentiation. Cell cycle analysis by accessing the EdU
(
thymidine analog) incorporation ability showed that DNA replication activity is severely impaired in Rnf20 knockout cells,
resulting in a loss of S phase cells concomitant with G1 phase cell cycle arrest. The blockage of G1/S transition may be due to
reduced binding of replication factors to the chromatin. We purified nascent chromatin from Rnf20 depleted cells and found that
the binding of PCNA, a loading platform for various DNA replication factors, is completely abolished. It suggests that Rnf20 is
essential for the binding of replication factors, leading to efficient DNA replication. Moreover, Rnf20 mediated H2B
ubiquitination may be involved in the regulation of the core pluripotency network. Using chromatin immunoprecipitation (ChIP)
assay, we found that several core pluripotency genes including Sox2, Nanog and Oct4 are enriched in H2BK120ub1. If Rnf20 is