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

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Presentation on theme: "Transcriptional regulation and promoter analysis Ju-Ming Wang Ins. of Biosignal Transduction (IBT), NCKU."— Presentation transcript:

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

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

3 3 DNA Messenger RNA Protein Translation Transcription cDNA libraries construction cDNA Microarray experiments Express sequence tags (ESTs) Gene Expression analysis Differential display Subtractive hybridization SAGE Microarray Introduction- Gene Expression analysis

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

5 5 Transcriptional regulation Promoter and transcription factors A transcription unit Transcriptional regulation- Promoter and transcription factors

6 6 Promoter Transcriptional regulation- Promoter and transcription factors

7 7 Promoter in intron Transcriptional regulation- Promoter and transcription factors 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 8 SWI/SNF SWI5 RNA Pol II TATA BP GENERAL TFs Lodish et al. Molecular Biology of the Cell (5 th ed.). W.H. Freeman & Co., Transcriptional regulation- Promoter and transcription factors Chromatin remodeling Remodeling complex: use energy from ATP hydrolysis to alter nucleosome structure or to move nucleosomes around.

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

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

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

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

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

14 14 Chromatin IP (ChIP)

15 15 ChIP on chip

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

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

18 18 Transcriptional regulation- Promoter and transcription factors Library screen

19 How to get a promoter sequence from NCBI website

20 20 Promoter identification

21 21 Promoter identification

22 22 Promoter identification

23 23 Promoter identification

24 24 Promoter identification

25 25 Promoter identification

26 26 Promoter identification

27 27 Promoter identification

28 28 Promoter identification

29 Promoter identification Direction of Transcription

30 30 5’-Flanking region Promoter region Promoter identification

31 31 Promoter identification

32 32 Promoter identification EST

33 33 Promoter identification

34 34

35 35 Promoter identification dl

36 36 Promoter identification

37 37 Promoter identification Different promoter regions

38 38 Promoter identification

39 39

40 40 Easier way to get a promoter region

41 41

42 42

43 43 Promoter identification

44 44 Promoter identification

45 45 Promoter identification

46 46 Promoter identification

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

48 48 Transcription factor-binding analysis

49 49 Transcription factor-binding analysis

50 50 Transcription factor-binding analysis

51 51 Transcription factor-binding analysis

52 52

53 53

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

55 55 Multiple-sequences alignment of promoters

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

57 57

58 58 “Score” Definition of Dimer A T C G Position CEBPD Dimer Binding consensus seq ATCGATCG C A T T G C T A C G Score =26.39

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60 60

61 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.


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