Identification of Transcription Factor Binding Sites

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Identification of Transcription Factor Binding
Sites

• Lior Harpaz
• Ofer Shany
• 09/05/2004

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Goal - find TFBS !
input
output
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Importance

• TF regulate gene expression.
• Identification of TF can teach us
• Mapping of regulatory pathways
• Potential functions of genes

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Experimental Methods

• Footprinting
• EMSA - electrophoretic mobility shift assay

Problems

• Time consuming
• Not scaled up to whole genomes

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Computational Methods - Goals

• Identifying known TFBSs in previously unknown
  locations.
• Identifying unknown TFBSs.

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Computational Methods

• Basic idea - locate TFBS using sequence-searching

Problems

• Short sequences (5-15 bp)
• Degenerate sequences
• Location
• Biological reality

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Computational Methods

• Possible solutions

Conservation functional importance

• mRNA expression pattern
• Phylogenetic footprinting
• Network-level conservation

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Phylogenetic footprinting

• Identify ortholog genes
• Concentrate on conserved non-coding regions
  (possible regulatory regions)
• Look for conserved motifs.

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Why should it work ?

• 40 alignment between human and mice genome
• 80 of mouse genes have orthologs in human genome
• Only 1-5 of human genome encodes proteins.

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Things to consider

• Choosing genomes.

• Locating transcriptional start site.
• Alignment method.

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More things to consider

• Different evolution rates for different regions
  in the genome.
• PSSM score cut-off
• Note - TFBSs within ORFs are not detected.

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Phylogentetic footprinting in proteobacterial
genomes

• Study set of 190 genes of E.Coly with known
  TBFSs.
• Orthologs were searched in eight other bacteria.
• Motif search by Bayesian Gibbs sampling.

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Bayesian Gibbs sampling

• Algorithm for motif search.
• Each motif is assigned with a MAP value.

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Bayesian Gibbs sampling

• Parameters and extensions
• Model sequence
• Palindromic patterns
• Background pattern
• Distribution of spacing between TFBSs and
  translation start site

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Results

• Overall in 146/184 sets, motives matched known
  regulatory sequences.
• In 18 genes (with 1 ortholog) only 67 known
  sites were matched, and with low MAP value.
• In 166 sets (with gt2 orthologs) 81 of motives
  matched known regulatory sequences.

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Results

• Out of the 166 sets (with gt 2 orthologs)
• 131 corresponded to known TFBSs.
• 3 corresponded to known stem loop structures.
• 32 data sets contained predictions with large MAP
  value could be undocumentd sites !
• Documented site were found in 138 sites without
  using palindromic models.

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Identification of a new TF

• New site found near fabA, fabB yqfA
• YijC binds to these sites.
• Site location, protein structure previous
  experimental results suggests YijC is a repressor
  for the fab genes.
• Indication of yqfAs involvement in metabolism of
  fatty-acids.

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Genomic scale phylogenetic footprinting

• 2113 ORFs of E.coli used.
• 187 new sites identified as probable sites for 46
  known TFs.
• Remaining sites are expected to represent unknown
  TFBSs
• MAP Values of predicted sites were lower.

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MAP values left-shift
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Study set
Ortholog Distribution
Full set
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Conclusions

• New sites for known TF were found.
• Conservation of Regulatory stem-loops.
• New sites for unknown TF are predicted.
• New TF identified (YijC).
• Predicted gene function (yqfA).

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Network level conservation

• Each TF regulates the expression of many genes
  (20-400).
• Conservation of global gene expression requires
  the conservation of regulatory mechanisms.

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(No Transcript)
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Data analysis

• Total motifs 80,000
• P-value filter 12,000
• Low-complexity filter 7,673
• Hierarchically clustering 1,269

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Validation

• 34/48 known sites discovered.
• Large fraction of matches for significant
  p-values.

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Identification of known binding sites
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Biological Significance

• Functional coherence
• Expression coherence

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Characteristic Features

• Conservation of binding affinity
• Conservation of position orientation

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References

• Bulyk, M. Computational prediction of
  transcription-factor binding site locations.
  Genome Biol. 2003 5201
• McCue L, Thompson W, Carmack C, Ryan MP, Liu JS,
  Derbyshire V, Lawrence CE. Phylogenetic
  footprinting of transcription factor binding
  sites in proteobacterial genomes. Nucleic Acids
  Res. 2001 29774-782.
• Pritzker M, Liu YC, Beer MA, Tavazoie S.
  Whole-genome discovery transcription factor
  binding sites by network-level conservation.
  Genome Res. 2004 1499-108

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Sensitivity Vs. Specificity