Transcript 投影片 1

Transcription Factors in the
Central Nervous System
Jau-Song Yu
Department of Cell and Molecular Biology
Chang Gung University
THE TRANSCRIPTIONAL PROCESS
Formation and Regulation of the Transcriptional Complex
Cis-acting elements:
Trans-acting elements:
Gene expression as a biological amplifier
Gene
Transcription
1000 X Amplication
mRNA
Translation
100 X Amplication
Protein
Different genes
cDNA microarray
analysis
Protein expression analysis by 2-D gel analysis
Structure of transcription factors from different families
C2H2 zinc finger
Helix-turn-helix Homeodomain
Leucine zipper
Helix-loop-helix
Glucocorticoid and Mineralocorticoid Receptors Are
Transcription Factors
Nuclear receptors are ligand-activated gene regulatory proteins
Ligand-binding domain
Glucocorticoid and Mineralocorticoid Receptors
Regulate Transcription in the CNS
Hypothalamic-Pituitary-Adrenal (HPA) systems in rat
de Kloet et al. Endocrine Rev. 19(3):260-301 (1998)
TABLE 2. Milestones in brain corticosteroid receptor research
Molecular mechanism of corticosteroid action on gene expression
TF: such as AP-1, NF-kB
HRE: hormone-responsive element
Gene-mediated steroid effects on membrane properties
腎上腺切除術
cAMP Regulation of Transcription
CREB transcription factor:
Transcription factor that binds to the cAMP response
element (CRE)
CRE:
palindromic sequence TGACGTCA in the promoter region
of a gene
Identification of the DNA sequence that binds to a specific DNA-binding protein
Affinity-purification of a specific DNA-binding protein
CREB in rat hippocampal neurons
Nature 1988 Aug 11;334(6182):494-8
Phosphorylation-induced binding and
transcriptional efficacy of nuclear factor CREB.
Yamamoto KK, Gonzalez GA, Biggs WH 3rd, Montminy MR.
Clayton Foundation Laboratories for Peptide Biology, Salk Institute, La Jolla,
California 92037.
A nuclear protein, CREB, has been isolated from rat brain and
shown to stimulate transcription of the cyclic AMP-responsive
gene somatostatin as a dimer. Biochemical analysis suggests
that dimerization and transcriptional efficacy of CREB protein
in vitro are regulated by phosphorylation. These findings
demonstrate that cellular signals can modulate gene
expression by regulating the covalent modification of preexisting nuclear factors.
Dephosphorylation
inactivation
(phosphatases?)
CREM:
cAMP response element
modulator
(by homology screens,
PCR, interaction screening)
CREB ---- dimers, leucine zippers and DNA-binding domains
Mechanism for generation of activators and repressors of
cAMP-stimulated transcription
CREB and CREM play roles in many physiological systems
Memory and long-term potentiation
(Silva et al., Annu. Rev. Neurosci. 21, 127-148)
Circadian rhythms
(Foulkes et al., Trends Neurosci. 20, 487-492)
Pituitary function
(Struthers et al., Nature 350, 622-624)
Spermatogenesis
(Sassone-Corsi P, Sem. Cell Dev. Biol. 9, 475-482)
The function of the CREB has been modeled in transgenic organisms
Aplysia californica (海兔)
"This sea-slug [Aplysia] is about five inches
long; and is of a dirty-yellowish colour, veined
with purple.... It feeds on the delicate
seaweeds.... This slug, when disturbed, emits
a very fine purplish-red fluid, which stains the
water for the space of a foot around."
CHARLES DARWIN, Voyage of the Beagle
Aplysia (genus Opisthobranchia) are
hermaphrodites, meaning that both partners
fertilize each others each with their sperm
during mating.
This gastropod mollusc is suited for
Neurobiology mainly because of its large
neurons - they are among the largest in the
animal kingdom. In addition, its entire nervous
system contains only a few hundred neurons,
making it less complex and, hopefully, easier
to understand than that of higher animals.
Despite its simplicity, it exhibits a variety of
behaviors and the capability to learn both nonassociatively (sensitisation, habituation) and
associatively (classical, operant conditioning).
It was Aplysia that enabled researchers to
study the molecular events in the cell during
learning. In conjunction with the geneticly
more powerful fly and mouse model systems,
Aplysia research has boosted our
understanding of the biological basis of
learning and memory.
Learning and memory stimulated
by serotonin as well as cAMP
Drosophila as a model organism to study the role of CREB
in learning and memory
Fly (wt or mutants, obtained from wt fly fed with ethylmethane sulfonate, EMS)
trained by odor avoidance learning
Behaviorally tested in an odorant-associated electric shock
Characterize mutants with learning and memory deficiency
dunce mutant: deficient in cAMP phosphodiesterase
rutabaga mutant: mutation in adenylyl cyclase
Odor Avoidance Learning
(one training cycle, 2.5 min total)
100
flies
Electrifiable
copper grid
100
flies
60 s
100
flies
45 s
Fresh air
3-octanol (OCT) or
4-methylcyclohexanol (MCH)
(1o odor)
100
flies
60 s
MCH or OCT
(2o odor)
100
flies
45 s
Fresh air
In a T maze
120 s for
choice
OCT
n1
n2
MCH
n1 : n2 = ~50 : 50 for untrained flies
Induction of a dominative CREB transgene specifically blocks
long-term memory in Drosophila
Yin et al., Cell 79, 49-58 (1994)
dCREB2:
Drosophila CREB gene
dCREB2a:
Activator for CRE
dCREB2b:
Repressor for CRE
dCREB2-mLZ:
Lost of Repressor function
2 leucine in LZ domain
valine
(dCREB-mLZ)
25oC
37oC
25oC
HS-induced dCREB2b
can be detected in brain cells
Massed training cycle:
10 consecutive cycle without
rest interval between them
Spaced training cycle:
10 consecutive cycle with a 15
min rest period between each
wt flies
+ 35 mM CXM, 12-14 hr
(before group)
Training
+ 35 mM CXM, 24 hr
(after group)
Odor avoidance exp.
Performance Index (PI):
100 means 100% avoidance of the
shock-paired odor; 0 means a 50:50
distribution in the T maze
Induction of the dCREB2-b transgene disrupts 1 day memory after spaced training,
while 1 day memory after massed training and learning are normal
Can-S: wt flies
17-2 and M11-1: hs-dCREB2-b
transgenic flies
Induction of the mutant blocker (dCREB2-mLZ) does not affect
1 day memory after spaced training
(dCREB2b)
(dCREB2-mLZ)
hs-dCREB2-b induction does not affect olfactory acuity
or shock reactivity
Induction of hs-dCREB2-b completely abolishes
7 day memory rentention
(281-285)
The CREB and CREM transcription factors are activated by
phosphorylation of a key serine residue by kinases stimulated by cyclic
AMP, Ca2+, growth factors and stress signals. Phosphorylation allows
recruitment of CREB binding protein (CBP), a large co-activator that
contacts the general transcriptional machinery. Studies of the
physiological roles played by CREB and CREM have uncovered novel
routes of transcriptional activation. For example, in male germ cells
CREM is not phosphorylated but associates with ACT, a member of the
LIM-only class of proteins that has intrinsic transcriptional activity. Thus,
in some circumstances, CREM can bypass the classical requirement
for phosphorylation and association with CBP.
Conclusion:
CREB is an integrator of intracellular homeostasis,
while the glucocorticoid receptor integrates whole-body
homeostasis