Transcript Transcription Factor binding site analysis

Promoter Analysis
TFBS Detection
Daniel Rico, PhD.
[email protected]
1. Promoters and gene regulation in Eukaryotes
2. Position Weight Matrices (PWM)
3. PWM Databases
4. TFBS prediction using PWMs
5. Pattern Discovery: Finding unknown motifs
6. Exercise: Use the human NOS2 sequence
to predict TFBS with Match and JASPAR
2
1. Promoters and gene regulation in Eukaryotes
2. Position Weight Matrices (PWM)
3. PWM Databases
4. TFBS prediction using PWMs
5. Exercise: Use the human NOS2 sequence
to predict TFBS with Match and JASPAR
Transcription Factor Binding Sites
3
Enhancer
Gene
“Proximal” promoter
(100bp-2Kb 5’ Upstream)
TSS:
Transcription Start Site
4
PROMOTERS

Promoters are DNA segments upstream of transcripts
that initiate transcription
Promoter

5’
3’
Promoter attracts RNA Polymerase to the transcription
start site
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GENES IN ENSEMBL
5’
Forward (+) strand
3’
Reverse (-) strand
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Transcription
Start Site
Transcription
Termination Site
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PROMOTER STRUCTURE IN PROKARYOTES (E.COLI)
Transcription starts
at offset 0.
• Pribnow Box (-10)
• Gilbert Box (-30)
• Ribosomal
Binding Site (+10)
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PROMOTER STRUCTURE IN EUKARYOTES
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Experimental Transcription Start Sites (TSS)
by CAGE
CAGE (Cap Analysis of Gene Expression))
detects the transcriptional activity of each promoter transcript.
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Representation of CAGE
preparation protocol adapted
to various platforms.
Now Solexa and Illumina are
preferred. 454 Life Sciences
(FLX system) is not used any
longer because
concatenation requires
additional PCR cycles and
complicated manipulation.
In the future, singlemolecule sequencing
technology will be preferred
because PCR may not be
required.
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http://www.osc.riken.jp/english/activity/cage/basic/
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http://fantom.gsc.riken.jp/4/edgeexpress/view/
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http://www.epd.isb-sib.ch/
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SEQUENCE ANALYSIS: SEARCHING
TRANSCRIPTION FACTOR BINDING SITES
(TFBS)
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TFBS: DETECTION METHODS
in vivo
Functional analysis
ChIP
in vitro on cloned fragment
Footprinting reactions
Exonuclease digests
Gel retardation (EMSA)
UV Crosslinking
in vitro on artificial DNA:
SELEX: Systematic Evolution of Ligands by Exponential
enrichment
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TRANSCRIPTION FACTORS BIND TO TFBS IN DNA
Affinity
Specificity
Nat Rev Genet. 2010 Nov;11(11):751-60. Epub 2010 Sep 28.
Determining the specificity of protein-DNA interactions.
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TF BINDING SITES

Problems:






often poorly defined consensus
Sequences not conserved within species, and even
worse between species
Examples of enhancers functionally conserved but
not sequence-conserved
Most of the TFBS sequence data comes from just a
few species
Very often in vitro experiments
2 completely different binding sites could be
merged in the same matrix/consensus
1
9
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Transcription Factor Binding Sites
1. Promoters and gene regulation in Eukaryotes
2. Position Weight Matrices (PWM)
3. PWM Databases
4. TFBS prediction using PWMs
5. Pattern Discovery: Finding unknown motifs
6. Exercise: Use the human NOS2 sequence
to predict TFBS with Match and JASPAR
20
Data collection
Probabilities can be calculated and corrected for background
Also called position-specific scoring matrices (PSSMs). In log scale.
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FROM PFM TO PWM/PSSM
Transcription Factor Binding Sites
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SEQUENCE LOGOS: The information content of a matrix column ranges from 0
(no base preference) and 2 (only 1 base used).
http://weblogo.berkeley.edu/
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http://www.lecb.ncifcrf.gov/~toms/sequencelogo.html
Summary
AAGTTC
AAGCTC
AGGCTC
AAGGTC
A
C
G
T
430000
000204
014100
000140
Consensus: ARGBTC
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Transcription Factor Binding Sites
1. Promoters and gene regulation in Eukaryotes
2. Position Weight Matrices (PWM)
3. PWM Databases
4. TFBS prediction using PWMs
5. Pattern Discovery: Finding unknown motifs
6. Exercise: Obtain mouse and human fosB promoters
and predict TFBS with Match and JASPAR
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Transfac: not free, 848 matrices, loads of information and references,
quality score based on methods used
Jaspar: open sources, 123 matrices, minimal information,
majority based on SELEX method (80%)
2
6
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TRANSFAC®
http://www.gene-regulation.com/pub/databases.html
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http://jaspar.cgb.ki.se/
http://jaspar.genereg.net/
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JASPAR EXAMPLE: PAX6
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2
9
Transcription Factor Binding Sites
1. Promoters and gene regulation in Eukaryotes
2. Position Weight Matrices (PWM)
3. PWM Databases
4. Pattern Matching: TFBS prediction using PWMs
5. Pattern Discovery: Finding unknown motifs
6. Exercise: Use the human NOS2 sequence
to predict TFBS with Match and JASPAR
Transcription Factor Binding Sites
30
Click here to
select all TFBS
31
Transcription Factor Binding Sites
1. Promoters and gene regulation in Eukaryotes
2. Position Weight Matrices (PWM)
3. PWM Databases
4. Pattern Matching: TFBS prediction using PWMs
5. Pattern Discovery: Finding unknown motifs
6. Exercise: Use the human NOS2 sequence
to predict TFBS with Match and JASPAR
32
PATTERN DISCOVERY
Reference Genome
Sequences of interest
Seq. oligo
expected
frequency
Seq. oligo
observed
frequency
AAAAAA
AAAAAC
AAAAAG
AAAAAT 0.00024
AAAACC
…
AAAAAA
AAAAAC
AAAAAG
AAAAAT 0.00018
AAAACC
…
0.00024
0.00030
0.00031
0.00028
0.00023
0.00031
0.00125
***
0.00026
3
3
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http://meme.sdsc.edu/meme/
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1. Promoters and gene regulation in Eukaryotes
2. Position Weight Matrices (PWM)
3. PWM Databases
4. Pattern Matching: TFBS prediction using PWMs
5. Pattern Discovery: Finding unknown motifs
6. Exercise: Use the human NOS2 sequence
to predict TFBS with Match and JASPAR
Transcription Factor Binding Sites
35
EXERCISE
Step by step
a. Download from UCSC or Ensembl the human NOS2 gene plus 5000
bases upstream. Select the “proximal promoter” first 1Kb: from -1000 to
TSS (hint: there is no zero position!)
b. Go to JASPAR and search for TFBS in promoter with the defaults.
c. Do the same exercise with the mouse NOS2.
d. Compare the results.
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CHROMATIN ACCESSIBILITY
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Access to experimental information
http://www.nature.com/scitable/
EUCROMATINA Y HETEROCROMATINA
Replicatión temprana (early)
Replicatión tardía (late)
Nat Rev Genet. 2011 Jul 12;12(8):554-64. doi: 10.1038/nrg3017.
Determinants and dynamics of genome accessibility.
Slides from http://www.openhelix.com/ENCODE
ENCODE: WWW.GENOME.GOV/10005107
ENCyclopedia of DNA Elements, NHGRI
 Consortium of international researchers
 UCSC is the Data Coordination Center

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ENCODE BACKGROUND
Pilot phase, or phase I: www.genome.gov/26525202
 Selected regions of the genome: 1%, 30 MB

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ENCODE PILOT DATA AND BEYOND
ENCODE portal: http://genome.ucsc.edu/ENCODE/
 Pilot ENCODE browser: genome.ucsc.edu/ENCODE/pilot.html

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ENCODE NEXT PHASE: PRODUCTION PHASE
UCSC is the DCC for human and mouse data
 The portal is available: genome.ucsc.edu/ENCODE/
 New aspects of the Production Phase projects

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chromatin
transcriptome/
genes
Copyright OpenHelix. No use or
reproduction without express written
consent
ENCODE PRODUCTION PHASE FOCUS
promoters/
regulatory sites
DNase sites
ENCODE is now genome-wide
 Specific cell types and new technologies being applied
 Project focus topics selected, then supplemented

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Data being submitted to UCSC DCC by data providers
 “Wranglers” ensure meta data is present
 Quality checks occur, data is released for use
Copyright OpenHelix. No use or
reproduction without express written
consent
ENCODE DATA IS FLOWING!

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ENCODE DATA TYPES
ENCODE
Tracks
identified
with icon

Mapping data

Genes

Expression

Regulation

Variation
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REGULATION DATA
Image from NIH
Regulation data
 Structure: modifications, open vs. closed chromatin

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REGULATION DATA II
TATA bound to DNA
Transcription factor binding sites, TFBS
 RNA binding proteins

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