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

published_or_final_version / abstract / toc / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Thesis (Ph.D.) - University of St Andrews, June 2008. / Pagination differs from that of the electronic version in the Digital Research Repository.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (p. 139-154) Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (p. 139-154) Also available online.

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Bibliography: leaves 109-124. Also available online.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1966. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis supervisor: Pamela K. Geyer. Includes bibliographic references (p. 108-123).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1964. / Typescript. Vita. Description based on print version record. Includes bibliographical references.

No description available.