Transcriptional regulation and promoter analysis

Ju-Ming Wang

Ins. of Biosignal Transduction (IBT), NCKU

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•Transcriptional regulation Promoter and transcription factors

• Promoter identification

• Transcription factor-binding analysis

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DNA Messenger RNA Protein

TranslationTranscription

cDNA libraries construction

cDNA Microarray experiments

Express sequence tags (ESTs)

Gene Expression analysis

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1 2 3 4 5 6 7 8 9 101112131415161718192021222324252627

WSB1 WSB1

KNSL2 KNSL2

KIAA0013 KIAA0013

PIF1 PIF1

KNSL1 KNSL1

FLJ22624 FLJ22624

ESPL1 ESPL1

Differential display

Subtractive hybridization

SAGEMicroarray

Introduction- Gene Expression analysis

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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 2004.

Exon IntronFlanking region

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Transcriptional regulation Promoter and transcription factors

A transcription unit

Transcriptional regulation- Promoter and transcription factors

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Promoter

Transcriptional regulation- Promoter and transcription factors

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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):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.

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SWI/SNF

SWI5

RNA Pol IITATA BPGENERAL TFs

Lodish et al. Molecular Biology of the Cell (5th ed.). W.H. Freeman & Co., 2003.

Transcriptional regulation- Promoter and transcription factors

Chromatin remodeling

Remodeling complex: use energy from ATP hydrolysis to alter nucleosome structure or to move nucleosomes around.

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Transcriptional regulation- Promoter and transcription factors

Graphics from the book of “Essential Cell Biology” Alberts, 1997,p.p. 268

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Transcriptional regulation- Promoter and transcription factors

How to dissect the transcriptional regulation?

Age of One transcription Factor on one gene

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Transcriptional regulation- Promoter and transcription factors

How to study the global gene regulation?

One transcription factorregulates a cluster of genes

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The is the set of all messenger RNA (mRNA) molecules, or "transcripts," produced in one or a population of cells.

Transcriptome

Resting cells Stimulated cellsA set of Transcriptome A set of Transcriptome

Transcription factor regulates a cluster of genes

ATF1

ATF2 CREB1

AP1

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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

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Chromatin IP (ChIP)

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ChIP on chip

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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?

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Transcriptional regulation- Promoter and transcription factors

How to find the promoter region?

The past: Molecular Biology- Library Screen + cloning

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Transcriptional regulation- Promoter and transcription factors

Library screen

How to get a promoter sequence from NCBI website

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Promoter identification

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Promoter identification

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Promoter identification

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Promoter identification

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Promoter identification

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Promoter identification

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Promoter identification

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Promoter identification

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Promoter identification

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126001

Promoter identification

Dir

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scri

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5’-Flanking region

Promoter region

Promoter identification

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Promoter identification

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Promoter identification

EST

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Promoter identification

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Promoter identification

dl

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Promoter identification

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Promoter identification

Different promoter regions

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

Promoter identification

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Easier way to get a promoter region

http://rulai.cshl.edu/cgi-bin/TRED/tred.cgi?process=home

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Promoter identification

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

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Promoter identification

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Promoter identification

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Promoter identification

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Prediction of Transcription factor binding sites

Transcription factor-binding analysis

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Transcription factor-binding analysis

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Transcription factor-binding analysis

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Transcription factor-binding analysis

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Transcription factor-binding analysis

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

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Transcription factor-binding analysis

Identification of a cluster by a transcription factor

http://www.bioinfo.tsinghua.edu.cn/~zhengjsh/OTFBS/

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Multiple-sequences alignment of promoters

http://genome.lbl.gov/vista/index.shtml

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

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http://140.116.234.168/ccp/prediction/

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“Score” Definition of Dimer

A 5.06 0 0 1.81 0.12 2.53 2.89 6.63 9.88 0.48

T 0.48 10 9.64 0.6 0.6 0.36 4.82 0.24 0 4.22

C 1.57 0 0 0 8.8 0.72 2.29 3.13 0.12 3.49

G 2.89 0 0.36 7.59 0.48 6.39 0 0 0 1.81

Position 1 2 3 4 5 6 7 8 9 10

CEBPD Dimer Binding consensus seq.

0 6.63

9.64 0.6 4.82

1.57 0.72 0.12

0.48 1.81

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T

C

G

C A T T G C T A C G

Score =26.39

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53.86

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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.