TSPYL2 (Testis-specific Y-encoded-like protein 2) is a member of the
Nucleosome Assembly Protein (NAP) superfamily. It is a nuclear protein
expressed in the cerebral cortex and the hippocampus. Our group has generated
Tspyl2 knockout (Tspyl2m) mice, which are deficit in hippocampal long-term
potentiation (LTP) with downregulation of Nr2a and Nr2b. Since Nr2a and Nr2b,
subunits of the N-Methyl-D-Aspartate receptors, and hippocampal LTP are
important in learning and memory, our Tspyl2m mice are likely to have behavioral
deficits particularly in those related to memory. TSPYL2 could also affect LTP
via CREB-dependent gene expression, since other NAP members have shown
interaction with CBP/p300 - transcriptional co-activators of CREB which are
well-known to be involved in memory formation. Furthermore, TSPYL2 may be
linked to X-linked mental retardation (XLMR), since it is located at Xp11.2, a
region with a high density of XLMR genes; and one of its interacting partners,
CASK, is a XLMR gene.
This thesis examines the three issues mentioned above. First, to characterize
the behavior of our Tspyl2m mice, a behavioral test battery including open-field
with amphetamine challenge, social interaction, prepulse inhibition and fear
conditioning were conducted. Second, to examine the role of TSPYL2 on
CREB-dependent gene expression, I first examined the subcellular localization of
HA-TSPYL2 and endogenous CBP, p300 and pCREB in HEK293 cells. Then the
interactions between TSPYL2 and CBP were tested by mammalian two-hybrid
assay and co-immunoprecipitation. Thereafter, luciferase assay was used to
measure CRE-luc activity in HEK293 and NG108-15 cells with overexpression
and knockdown of TSPYL2. Third, to investigate the potential role of TSPYL2 on
XLMR, a mutation analysis on the TSPYL2 gene was conducted with a cohort of
82 male patients with unexplained mental retardation. The analysis included
examining the methylation on the TSPYL2 upstream sequence, DNA sequencing
of the TSPYL2 exons, and in silico splice site analysis of the identified sequence
variants.
In the behavioral test battery, our Tspyl2m mice were normal in social
ability, but showed enhanced hyperlocomotion after amphetamine injection, and
deficit in prepulse inhibition and cued fear conditioning. When expressed in
HEK293 cells, HA-TSPYL2 colocalized completely with endogenous CBP, but
not with p300 and pCREB. In mammalian two-hybrid assay,
pVP16(AD)-TSPYL2 interacted with GAL4(DBD)-CBP; however, HA-TSPYL2
did not immunoprecipitate with CBP. The luciferase assay data indicated that
TSPYL2 suppressed the transcription of CREB-target genes. Lastly, no
methylation was detected in the target sites in the TSPYL2 upstream sequence.
Seven TSPYL2 sequence variations being identified were not deleterious as
predicted by splice site analysis.
To sum up, our Tspyl2m mice were deficit in cued fear memory, a form of
associative memory. Moreover, they resembled the glutamatergic
antagonist-induced schizophrenic rodent models in having enhanced
hyperlocomotion after amphetamine injection, and deficit in prepulse inhibition.
TSPYL2 interacted with CBP and suppressed the CRE-luc activity. The
importance of TSPYL2 in XLMR has yet to be determined by larger studies. I
propose that TSPYL2 represses CREB-dependent gene expression via
sequestration of CBP as one of the possible mechanisms of how TSPYL2 causes
various behavioral phenotypes. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/208426 |
| Date | January 2011 |
| Creators | Wong, Kwun-kit, 黃冠傑 |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Rights | Creative Commons: Attribution 3.0 Hong Kong License, The author retains all proprietary rights, (such as patent rights) and the right to use in future works. |
| Relation | HKU Theses Online (HKUTO) |
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