Download The effects of electroconvulsive seizure on the transcription

Poster number: P.1.g.014
The effects of electroconvulsive seizure on the transcription of PAR family of bZIP genes
via changes in Egr1 expression in the rat frontal cortex
Tak Youn, Se Hyun Kim, Hong Geun Park, In-Won Chung, Yong Sik Kim
Department of Psychiatry and Institute of Clinical Psychopharmacology, Dongguk University Ilsan Hospital, Goyang, Gyeonggi, Republic of Korea.
INTRODUCTION
 Electroconvulsive therapy (ECT)
• widely used to treat psychiatric disorders such as depression, mania, and
schizophrenia. ECT affects vaious brain fucntions including affective,
cognitive, and perceptive domains, as well as physiological and molecular
functions such as excitability of neurons, neurogenesis, and cell survival in
the brain
 The PAR family of bZIP genes
• Defined by the presence of a proline and acidic amino acid–rich protein
(PAR) domain is consist of three polypeptides, D-box binding protein (Dbp),
thyrotroph embryonic factor (Tef), and hepatic leukaemia factor (Hlf)
• Dbp plays an important function in the control of brain excitability, and was
suggested as the candidate gene of mood disorder
• Tef affects transcription of prolactin genes and thyroid-stimulating hormonebeta gene
• Hlf was originally isolated from the leukemic blasts of pro-B cell acute
lympho-blastic leukemia, and plays important role in cell survival and
proliferation
• Dbp, Tef, and Hlf display similar circadian and tissue-specific expression
but have different target promoter preferences
 We hypothesized that the wide range of effects of ECT could be associated
with genes with different functions under control of common regulatory
mechanisms. In this study, we investigated the effect of a single and repeated
treatment of electroconvulsive seizure (ECS), an amimal model of ECT, on the
transcription of PAR family of bZIP genes in the rat frontal cortex.
METHODS
 Male Sprague-Dawley rats (150-200g)
 ECS treatments
• Single ECS treatment was applied to examine the acute changes. To investigate the chronic
changes, ECS and sham groups were treated once per day for 10 days at the same time
during a day (12:00-13:00) for a total of ten times (E10X). The rats were decapitated 24 h
after the last treatment.
 Quantitative real-time PCR
• mRNA expression level was examined with qPCR.
 Immunoblot analysis
• Protein level was examine with immunoblot analysis.
 Analysis of Egr1 binding motif on the promoter regions of PAR bZips
• The promoter regions of PAR bZip genes were analyzed to find Egr1 binding motif on them.
The nucleic acid sequence of each gene’s promoter region which includes 2000 bps
upstream from transcription starting site (TSS) was obtained from the web site UCSC
Genome Bioinformatics (genome.ucsc.edu) and transcription factor binding motifs were
searched by using MatInspector program (Genomtix, www.genomatix.de).
 Chromatin immunoprecipitation (ChIP)
• Egr1 occupancy of the promoter regions of examined genes were examined with ChIP anlysis
RESULTS
 Acute and chornic effects of ECS on the expression of Dbp, Tef,
and Hlf
• Single treatment of ECS (E1X) induced time-dependent changes in the
mRNA expression level of Dbp, Tef, and Hlf in rat frontal cortex
Transcription factor binding
sites analysis
• We examined the transcription
factor binding sites within the
promoter regions (encompassing
1,500 bp from transcription start
site) of Dbp, Tef, and Hlf genes.
The binding sites for early growth
response-1 (Egr1), Rev-Erbα, and
RAR-related orphan receptor alpha
(Rorα) were commonly found in the
promoter regions.
Acute and chronic effects of ECS
on Egr1 expression
• mRNA level of Egr1 was gradually
increased until 60 min and decreased
thereafter by E1X
• Dcreased at 24 h after E10X as
previously reported
• These findings suggest that Egr1 could
negatively regulate the transcription of
Dbp, Tef, and Hlf genes.
Increased Egr1 binding to the Egr1 motif on PAR bZip promoter regions
• In 2000 bps upstream from transcription starting site (TSS) on PAR bZip promoter
region, Egr1 binding motifs were observed two at Dbp promoter region, one at Hlf
promoter region, and four at the Tef promoter region.
• Egr1 binding level to the motif on promoter region of PAR bZip genes were
investigated by ChIP assay at the time when peak of Egr1 expression observed, 30
min after single ECS, in the rat frontal cortex. Compared to sham, Egr1 binding to
the motif on Dbp gene promoter region (t = 2.76, p < 0.05) was increased as well as
to the motif on Hlf (t = 2.96, p < 0.05) and Tef promoter (-34, t = 2.51, p < 0.05; -492,
t = 2.16, p = 0.07; -546, t = 2.82, p < 0.05; -767, t = 4.68, p < 0.05) region at 30 min
after ECS
Expression levels of PAR bZips mRNA at the reduced expression of Egr-1
in frontal cortex of Egr1 KO mouse
• In order to know PAR bZip expression level of mRNA in the condition of reduced
Egr1 expression, we examined the expression level of PAR bZip in frontal cortex of
egr1 KO mouse. In egr1 KO mouse frontal cortex, Dbp, Tef, and Hlf were increased
(t = 2.38, p < 0.05; t = 3.55, p < 0.05; t = 2.48, p < 0.05; respectively) compared to
wild type
• mRNA levels of three genes were significantly decreased at 60 min after
ECS. The repeated ECS treatments for 10 days (E10X) significantly upregulated mRNA levels of Dbp, Tef, and Hlf compared to those in sham
control group at 24 h after E10X in rat frontal cortex
 The expression of three genes showed same pattern of changes in
response to ECS treatment, indicating possible common regulatory
mechanisms.
CONCLUSION
 ECS treatments affects the transcription of Tef, Hlf, and Dbp, via negative
transcriptional regulation of Eg1 in the rat frontal cortex.
 The common effects of ECS on the different members of PAR family of bZIP
genes could provide understanding on the various effects of ECT on
various cognitive, mood, and physical domains, which will require further
functional studies.