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Transcriptional regulation and promoter analysis

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  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. 4 3.5 WSB1 WSB1 KNSL2 KNSL2 2.5 KIAA0013 KIAA0013 PIF1 PIF1 2 KNSL1 KNSL1 FLJ22624 FLJ22624 1.5 ESPL1 ESPL1 1 0.5 1 2 0 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Introduction- Gene Expression analysis Gene Expression analysis Protein DNA Messenger RNA Translation Transcription cDNA libraries construction Express sequence tags (ESTs) cDNA Microarray experiments Differential display Subtractive hybridization SAGE Microarray

  4. The organization of genes on a human chromosome. Intron Exon Flanking region 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 Transcriptional regulation Promoter and transcription factors A transcription unit

  6. Transcriptional regulation- Promoter and transcription factors Promoter

  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):499-509 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 geneswhile 36% lie within or immediately 3' to well-characterized genes and are significantly correlated with noncoding RNAs.

  8. SWI/SNF SWI5 RNA Pol II TATA BP GENERAL TFs Transcriptional regulation- Promoter and transcription factors Chromatin remodeling 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. Transcriptional regulation- Promoter and transcription factors How to dissect the transcriptional regulation? Age of One transcription Factor on one gene

  11. 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+ TF-regulated genes when use an exogenous TF-expression system without ER existence 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. 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. 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

  34. Promoter identification dl

  35. Promoter identification

  36. Promoter identification Different promoter regions

  37. Promoter identification http://www.bioinformatics.org/sms/rev_comp.html

  38. Easier way to get a promoter region http://rulai.cshl.edu/cgi-bin/TRED/tred.cgi?process=home

  39. Promoter identification http://rgsminer.csie.ncu.edu.tw/index.php

  40. Promoter identification

  41. Promoter identification

  42. Promoter identification

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

  44. Transcription factor-binding analysis

  45. Transcription factor-binding analysis

  46. Transcription factor-binding analysis