1. Transcription Factors in the Central Nervous System
Jau-Song Yu
Department of Cell and Molecular Biology
Chang Gung University

2. THE TRANSCRIPTIONAL PROCESS
Formation and Regulation of the Transcriptional Complex
Cis-acting elements:
Trans-acting elements:

3. Gene expression as a biological amplifier
mRNA
Protein
Transcription X Amplication
Translation X Amplication

4. cDNA microarray
analysis
Different genes

5. Protein expression analysis by 2-D gel analysis

6. Structure of transcription factors from different families

7. C2H2 zinc finger

8. Helix-turn-helix Homeodomain
Leucine zipper
Helix-loop-helix

9. Glucocorticoid and Mineralocorticoid Receptors Are
Transcription Factors
Nuclear receptors are ligand-activated gene regulatory proteins

10. Ligand-binding domain

11. Glucocorticoid and Mineralocorticoid Receptors
Regulate Transcription in the CNS
Hypothalamic-Pituitary-Adrenal (HPA) systems in rat
de Kloet et al. Endocrine Rev. 19(3): (1998)

12. TABLE 2. Milestones in brain corticosteroid receptor research

17. Molecular mechanism of corticosteroid action on gene expression
TF: such as AP-1, NF-kB
HRE: hormone-responsive element

18. Gene-mediated steroid effects on membrane properties

20. 腎上腺切除術

21. cAMP Regulation of Transcription

22. CREB transcription factor:
Transcription factor that binds to the cAMP response
element (CRE)
CRE:
palindromic sequence TGACGTCA in the promoter region
of a gene

23. Identification of the DNA sequence that binds to a specific DNA-binding protein

24. Affinity-purification of a specific DNA-binding protein

25. CREB in rat hippocampal neurons

26. 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
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 pre-existing nuclear factors.

27. Dephosphorylation
inactivation
(phosphatases?)

28. CREM:
cAMP response element
modulator
(by homology screens,
PCR, interaction screening)

29. CREB ---- dimers, leucine zippers and DNA-binding domains

31. Mechanism for generation of activators and repressors of
cAMP-stimulated transcription

32. CREB and CREM play roles in many physiological systems
Memory and long-term potentiation
(Silva et al., Annu. Rev. Neurosci. 21, )
Circadian rhythms
(Foulkes et al., Trends Neurosci. 20, )
Pituitary function
(Struthers et al., Nature 350, )
Spermatogenesis
(Sassone-Corsi P, Sem. Cell Dev. Biol. 9, )

33. Aplysia californica (海兔)
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 non-associatively (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

34. 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

35. 4-methylcyclohexanol (MCH) n1 : n2 = ~50 : 50 for untrained flies
Odor Avoidance Learning
(one training cycle, 2.5 min total)
100
flies
100
flies
100
flies
100
flies
100
flies
Electrifiable
copper grid
60 s
45 s
60 s
45 s
3-octanol (OCT) or
4-methylcyclohexanol (MCH)
(1o odor)
Fresh air
MCH or OCT
(2o odor)
Fresh air
In a T maze
120 s for
choice n1 n2
OCT
MCH
n1 : n2 = ~50 : 50 for untrained flies

36. Induction of a dominative CREB transgene specifically blocks
long-term memory in Drosophila
Yin et al., Cell 79, (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 oC oC
HS-induced dCREB2b
can be detected in brain cells

37. 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, 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

38. while 1 day memory after massed training and learning are normal
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

39. Induction of the mutant blocker (dCREB2-mLZ) does not affect
1 day memory after spaced training
(dCREB2b)
(dCREB2-mLZ)

40. hs-dCREB2-b induction does not affect olfactory acuity
or shock reactivity

41. Induction of hs-dCREB2-b completely abolishes
7 day memory rentention

42. ( )
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.

45. Conclusion: CREB is an integrator of intracellular homeostasis,
while the glucocorticoid receptor integrates whole-body
homeostasis