1. Transcriptional regulation and promoter analysis
Ju-Ming Wang
Ins. of Biosignal Transduction (IBT), NCKU

2. Transcriptional regulation
Promoter and transcription factors
Promoter identification
Transcription factor-binding analysis

3. Protein DNA Gene Expression analysis Introduction-
Messenger RNA
Transcription
Translation
cDNA libraries construction
Express sequence tags (ESTs)
cDNA Microarray experiments
Differential display
Subtractive hybridization
0.5 1
1.5 2
2.5
3.5 4 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
WSB1 WSB1
KNSL2 KNSL2
KIAA0013 KIAA0013
PIF1 PIF1
KNSL1 KNSL1
FLJ22624 FLJ22624
ESPL1 ESPL1
SAGE
Microarray

4. The organization of genes on a human chromosome.
Intron
Flanking region
Exon
With the publication of the “first draft” of the entire human genome in 2001 and the “finished DNA sequence” in 2004.

5. Transcriptional regulation Promoter and transcription factors
A transcription unit

6. Transcriptional regulation- Promoter and transcription factors

7. Transcriptional regulation- Promoter and transcription factors
Promoter in intron
Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread regulation of noncoding RNAs
Cell. 2004;116(4):
This mapping reveals an unexpectedly large number of transcription factor binding site (TFBS) regions, with a minimal estimate of 12,000 for Sp1, 25,000 for cMyc, and 1600 for p53 when extrapolated to the full genome. Only 22% of these TFBS regions are located at the 5' termini of protein-coding genes while 36% lie within or immediately 3' to well-characterized genes and are significantly correlated with noncoding RNAs.

8. Transcriptional regulation- Promoter and transcription factors
Chromatin remodeling
SWI/SNF
SWI5
RNA Pol II
TATA BP
GENERAL TFs
Lodish et al. Molecular Biology of the Cell (5th ed.). W.H. Freeman & Co., 2003.
Remodeling complex: use energy from ATP hydrolysis to alter nucleosome
structure or to move nucleosomes around.

9. Transcriptional regulation- Promoter and transcription factors
Graphics from the book of “Essential Cell Biology” Alberts, 1997,p.p. 268

10. How to dissect the transcriptional regulation?
Transcriptional regulation- Promoter and transcription factors
How to dissect the transcriptional regulation?
Age of One transcription
Factor on one gene

11. How to study the global gene regulation?
Transcriptional regulation- Promoter and transcription factors
How to study the global gene regulation?
One transcription factor
regulates a cluster of genes

12. Transcriptome
The is the set of all messenger RNA (mRNA) molecules, or "transcripts," produced in one or a population of cells.
Resting cells
Stimulated cells
A set of Transcriptome
A set of Transcriptome
Transcription factor regulates a cluster of genes
ATF2
CREB1
AP1
ATF1

13. Transcriptional regulation- Promoter and transcription factors
How to identify a transcription factor-mediated gene upon ER stimulation?
ER-
ER+
when use an exogenous TF-expression
system without ER existence
TF-regulated genes
ER-
ER+
when use an exogenous TF-expression
system within ER existence
TF-regulated
genes
TF-regulated
genes

14. Chromatin IP (ChIP)

15. ChIP on chip

16. How to find the promoter region?
Transcriptional regulation- Promoter and transcription factors
How to find the promoter region?
The past: Molecular Biology- Library Screen + cloning
Now: Computer cloning + PCR
Where is the transcriptional initiation site ?
How to clone the promoter region?
How to dissect the transcriptional regulation?
How to identify the global genes’ regulation?

17. How to find the promoter region?
Transcriptional regulation- Promoter and transcription factors
How to find the promoter region?
The past: Molecular Biology- Library Screen + cloning

18. Transcriptional regulation- Promoter and transcription factors
Library screen

19. How to get a promoter sequence from NCBI website

20. Promoter identification

21. Promoter identification

22. Promoter identification

23. Promoter identification

24. Promoter identification

25. Promoter identification

26. Promoter identification

27. Promoter identification

28. Promoter identification

29. Promoter identification
126490
Direction of Transcription
126001

30. Promoter identification
5’-Flanking region
Promoter region

31. Promoter identification

32. Promoter identification
EST

33. Promoter identification

35. Promoter identificationdl

36. Promoter identification

37. Promoter identification
Different promoter regions

38. Promoter identification

40. Easier way to get a promoter region

43. Promoter identification

44. Promoter identification

45. Promoter identification

46. Promoter identification

47. Prediction of Transcription factor binding sites
Transcription factor-binding analysis
Prediction of Transcription factor binding sites

48. Transcription factor-binding analysis

49. Transcription factor-binding analysis

50. Transcription factor-binding analysis

51. Transcription factor-binding analysis

54. Transcription factor-binding analysis
Identification of a cluster by a transcription factor

55. Multiple-sequences alignment of promoters

56. Q: How can you distinguish the specific targets
among C/EBPs?

58. CEBPD Dimer Binding consensus seq.
“Score” Definition of Dimer
CEBPD Dimer Binding consensus seq. A
5.06
1.81
0.12
2.53
2.89
6.63
9.88
0.48 T 10
9.64
0.6
0.36
4.82
0.24
4.22 C
1.57
8.8
0.72
2.29
3.13
3.49 G
7.59
6.39
Position 1 2 3 4 5 6 7 8 9
C A T T G C T A C G
A T C G
6.63
9.64
0.6
4.82
1.57
0.72
0.12
0.48
1.81
Score =26.39

59. 53.86

61. To analyze the conserved regulative motifs between human and mouse pin1 promoters. You can present this job in any way, but please briefly describe your steps in a flowchart.