The role of TSPYL2 on regulation of behavior and CREB-dependent gene expression

Abstract

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.