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

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

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

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.

Transcriptional regulation Promoter and transcription factors A transcription unit

Transcriptional regulation- Promoter and transcription factors

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 genes while 36% lie within or immediately 3' to well-characterized genes and are significantly correlated with noncoding RNAs.

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.

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

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

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

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

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

Chromatin IP (ChIP)

ChIP on chip

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?

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

Transcriptional regulation- Promoter and transcription factors Library screen

How to get a promoter sequence from NCBI website

Promoter identification

Promoter identification

Promoter identification

Promoter identification

Promoter identification

Promoter identification

Promoter identification

Promoter identification

Promoter identification

Promoter identification 126490 Direction of Transcription 126001

Promoter identification 5’-Flanking region Promoter region

Promoter identification

Promoter identification EST

Promoter identification

Promoter identification dl

Promoter identification

Promoter identification Different promoter regions

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

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

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

Promoter identification

Promoter identification

Promoter identification

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

Transcription factor-binding analysis

Transcription factor-binding analysis

Transcription factor-binding analysis

Transcription factor-binding analysis

http://alggen.lsi.upc.es/recerca/menu_recerca.html

Transcription factor-binding analysis Identification of a cluster by a transcription factor http://www.bioinfo.tsinghua.edu.cn/~zhengjsh/OTFBS/

Multiple-sequences alignment of promoters http://genome.lbl.gov/vista/index.shtml

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

http://140.116.234.168/ccp/prediction/

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

53.86

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.