Transcript Document

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
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Introduction- Gene Expression analysis
Gene Expression analysis
DNA
Messenger RNA
Protein
Translation
Transcription
Express sequence tags (ESTs)
cDNA libraries construction
cDNA Microarray experiments
Differential display
Subtractive hybridization
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3.5
WSB1 WSB1
KNSL2 KNSL2
2.5
KIAA0013 KIAA0013
PIF1 PIF1
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1.5
KNSL1 KNSL1
SAGE
Microarray
FLJ22624 FLJ22624
ESPL1 ESPL1
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0.5
1 20 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
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The organization of genes on a human chromosome.
Flanking region
Exon
Intron
With the publication of the “first draft” of the entire human
genome in 2001 and the “finished DNA sequence” in 2004.
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Transcriptional regulation- Promoter and transcription factors
Transcriptional regulation
Promoter and transcription factors
A transcription unit
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Transcriptional regulation- Promoter and transcription factors
Promoter
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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 wellcharacterized genes and are significantly correlated with noncoding RNAs.
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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.
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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 factor
regulates a cluster of genes
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Transcriptome
The is the set of all messenger RNA (mRNA) molecules,
or "transcripts," produced in one or a population of cells.
Resting cells
A set of Transcriptome
Stimulated cells
A set of Transcriptome
Transcription factor regulates a cluster of genes
ATF2
CREB1
AP1
ATF1
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Transcriptional regulation- Promoter and transcription factors
How to identify a transcription factor-mediated gene upon ER stimulation?
ER-
when use an exogenous TF-expression
system without ER existence
TF-regulated genes
ERwhen use an exogenous TF-expression
system within ER existence
ER+
TF-regulated
genes
ER+
TF-regulated
genes
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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
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?
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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
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How to get a promoter sequence from NCBI website
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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Direction of Transcription
Promoter identification
126490
126001
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Promoter identification
5’-Flanking region
Promoter region
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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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Promoter identification
http://www.bioinformatics.org/sms/rev_comp.html
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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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Transcription factor-binding analysis
Prediction of Transcription factor binding sites
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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
CEBPD Dimer Binding consensus seq.
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
T
G
C
A
G
T
C
T
0
T
C
A
A
C
G
6.63
9.64
0.6
4.82
1.57
0.72
0.48
0.12
1.81
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.
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