promoter analysis tfbs detection daniel rico, phd. [email protected] daniel rico, phd. [email protected]
TRANSCRIPT
Promoter AnalysisTFBS Detection
Daniel Rico, PhD.
Daniel Rico, PhD.
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
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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. Exercise: Use the human NOS2 sequence
to predict TFBS with Match and JASPAR
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Gene
Enhancer
TSS: Transcription Start Site
“Proximal” promoter(100bp-2Kb 5’ Upstream)
Promoters
Promoters are DNA segments upstream of transcripts that initiate transcription
Promoter attracts RNA Polymerase to the transcription start site
5’Promoter 3’
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GENES IN ENSEMBL
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5’ Forward (+) strand 3’
Reverse (-) strand
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Transcription Termination Site
Transcription Start Site
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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CAGE (Cap Analysis of Gene Expression))detects the transcriptional activity of each promoter transcript.
Experimental Transcription Start Sites (TSS)by CAGE
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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, single-molecule sequencing technology will be preferred because PCR may not be required.
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http://www.osc.riken.jp/english/activity/cage/basic/
13http://fantom.gsc.riken.jp/4/edgeexpress/view/
http://www.epd.isb-sib.ch/ 14
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Sequence Analysis: Searching Transcription Factor Binding Sites (TFBS)
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TFBS: Detection methods
in vivoFunctional analysisChIP
in vitro on cloned fragmentFootprinting reactionsExonuclease digestsGel retardation (EMSA)UV Crosslinking
in vitro on artificial DNA:SELEX: Systematic Evolution of Ligands by Exponential
enrichment
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Affinity
Specificity
Nat Rev Genet. 2010 Nov;11(11):751-60. Epub 2010 Sep 28.Determining the specificity of protein-DNA interactions.
Transcription Factors bind TO TFBS in DNA
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TF Binding Sites
Problems:often poorly defined consensusSequences not conserved within species, and
even worse between speciesExamples of enhancers functionally conserved
but not sequence-conservedMost of the TFBS sequence data comes from
just a few speciesVery often in vitro experiments2 completely different binding sites could be
merged in the same matrix/consensus
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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
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Data collection
Probabilities can be calculated and corrected for background
Also called position-specific scoring matrices (PSSMs). In log scale.21
From PFM to PWM/PSSM
Transcription Factor Binding Sites 22
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/ http://www.lecb.ncifcrf.gov/~toms/sequencelogo.html23
AAGTTCAAGCTCAGGCTCAAGGTC
A 430000 C 000204G 014100T 000140
Consensus: ARGBTC
Summary
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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%)
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TRANSFAC®
27http://www.gene-regulation.com/pub/databases.html
http://jaspar.cgb.ki.se/
http://jaspar.genereg.net/
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Jaspar example: Pax6
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Transcription Factor Binding Sites
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
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Click here to select all TFBSClick here to
select all TFBS
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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
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Pattern discovery
Reference Genome
Seq. oligo expectedfrequency
AAAAAA 0.00024AAAAAC 0.00030AAAAAG 0.00031AAAAAT0.00024AAAACC 0.00028…
Sequences of interest
Seq. oligo observedfrequency
AAAAAA 0.00023AAAAAC 0.00031AAAAAG 0.00125AAAAAT0.00018AAAACC 0.00026…
***
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http://meme.sdsc.edu/meme/ 34
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
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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 AccessibilityAccess to experimental information37
http://www.nature.com/scitable/
Eucromatina y Heterocromatina
Replicatión tardía (late)Replicatión temprana (early)
Nat Rev Genet. 2011 Jul 12;12(8):554-64. doi: 10.1038/nrg3017.Determinants and dynamics of genome accessibility.
ENCODE: www.genome.gov/10005107
ENCyclopedia of DNA Elements, NHGRI Consortium of international researchers UCSC is the Data Coordination Center 47
Slides from http://www.openhelix.com/ENCODE
ENCODE Background
Pilot phase, or phase I: www.genome.gov/26525202 Selected regions of the genome: 1%, 30 MB 48
ENCODE Pilot Data and Beyond
ENCODE portal: http://genome.ucsc.edu/ENCODE/ Pilot ENCODE browser: genome.ucsc.edu/ENCODE/pilot.html49
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 50
ENCODE Production Phase Focus
ENCODE is now genome-wide Specific cell types and new technologies being applied Project focus topics selected, then supplemented
Copyright O
penHelix. N
o use or reproduction w
ithout express written
consent
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chromatin
transcriptome/genes
promoters/regulatory
sites
DNase sites
ENCODE Data is Flowing!
Data being submitted to UCSC DCC by data providers “Wranglers” ensure meta data is present Quality checks occur, data is released for use
Copyright O
penHelix. N
o use or reproduction w
ithout express written
consent
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ENCODE Data Types Mapping data
Genes
Expression
Regulation
Variation
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ENCODE Tracks
identified with icon
Regulation Data
Regulation data Structure: modifications, open vs. closed chromatin 54
Image from NIH
Regulation Data II
Transcription factor binding sites, TFBS RNA binding proteins 55
TATA bound to DNA