Transcription Factors in the Central Nervous System

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 Gene mRNA Protein Transcription 1000 X Amplication Translation 100 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):260-301 (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 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 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, 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)

33. The function of the CREB has been modeled in transgenic organisms 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. Odor Avoidance Learning (one training cycle, 2.5 min total) 100 flies 100 flies 100 flies 100 flies 100 flies Electrifiable copper grid 45 s 45 s 60 s 60 s Fresh air Fresh air 3-octanol (OCT) or 4-methylcyclohexanol (MCH) (1o odor) MCH or OCT (2o odor) In a T maze 120 s for choice n2 n1 OCT MCH n1 : n2 = ~50 : 50 for untrained flies

36. 25oC 37oC 25oC 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) 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, 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

38. 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. (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.

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