TraFaC

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TraFaC

• Detecting Cis-Regulatory Element Clusters in
  Orthologous and Coordinately Expressed Genes

Anil Jegga Pediatric Informatics CHRF-CHMCC and UC
http//trafac.chmcc.org
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Outline..

• Basic Problem
• Background
• Methodology
• Some Applications, Some Examples
• Known Regulatory Regions
• Co-expressed Genes
• Potential Novel Regulatory Regions
• Some Bottlenecks
• Future Imminent and Long-term

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Problem and the Approach..

• Problem

• Our Approach
• Sequence Similarity Phylogenetic Footprints
• Transcription Factor (TF) Binding Sites
• Binding Site Cluster Similarity (merge 1 2)

• Identifying potential regulatory regions
• Deciphering secret passwords for cell-type
  specific gene expression
• Searching for cis-modules in genomic DNA

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Courtesy http//www.bios.co.uk
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Basic Structure of Transcription Regulatory
Machinery
DNA
Introns
mRNA
3 UTR
Transcription factors
TBP
Exons
Eukaryotic Promoter
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RNA POLYMERASE II
REPRESSOR COMPLEX 1
Transcription start site
GENE
DNA
RE
Genes regulatory region
ENHANCER COMPLEX
No. Because the DNA bends extensively.
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The Motivation
Comparative Analysis of Homologous Sequences
After gt900 Myrs of divergence essential
functional elements remain conserved! E.g. PAX6
Human Vs Puffer fish (Fugu rubripes)
http//bio.cse.psu.edu/Multipipmaker
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More Examples.
N-mycprotein Oncogene 2nd Intron
HMG-1 3UTR
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Regulation of Gene Expression An Overview

• Transcriptional
• Tissue-specific transcription factors
• Direct binding of hormones, growth factors, etc.
• Use of alternative promoters
• Post-transcriptional
• Alternative splicing
• Alternative polyadenylation
• Tissue-specific RNA editing
• Translational control mechanisms
• Epigenetic mechanisms Chromatin structure

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Some Basic Questions.

• What is Gene Expression?
• It is the ability of a gene to produce a
  biologically active protein
• What is a Promoter?
• Combination of short sequence elements to which
  RNA polymerase binds in order to Initiate
  transcription.
• What is an Enhancer?
• Set of short sequence elements which Stimulate
  transcription. Function independent of position
  or orientation.
• What is a Silencer/Repressor?
• Set of short sequence elements which Suppress
  transcription.

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Points to Remember.

• Cis-regulators are on the same chromosome as the
  gene.
• Trans-regulators are transcription factors that
  act on many different genes, on different
  chromosomes.
• Transcription factors are proteins with
  DNA-binding domains, which bind to specific DNA
  sequences.
• Transcription factors usually do not work alone.

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Methodology.
Genomic DNA Sequences
PipMaker (To find conserved regions)
MatInspector/Match (To find putative TF binding
sites)
TRAFAC
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Frequency Matrix for muscle gene MEF2 sites
10.0 0.0 0.0 0.0 22.0 0.0 6.0 2.0 3.0 4.0
22.0 10.0 0.0 2.0 12.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 9.0 20.0 2.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 10.0 3.0 0.0
8.0 22.0 0.0 22.0 16.0 20.0 19.0 18.0 0.0
2.0 a g c t a t t t
t t a a Position Weight Matrix (PWM)
0.74 -2.51 -2.51 -2.51 1.80 -2.51 0.10 -1.07
-0.68 -0.37 1.80 0.74 -2.51 -1.07 0.98 -2.51
-2.51 -2.51 -2.51 -2.51 -2.51 -2.51 -2.51 -2.51
0.61 1.67 -1.07 -2.51 -2.51 -2.51 -2.51 -2.51
-2.51 -2.51 -2.51 0.74 -0.68 -2.51 0.46 1.80
-2.51 1.80 1.36 1.67 1.60 1.52 -2.51
-1.07 Frequency Matrix is generated by aligning
the mef2 sites gt aldolase A enhancer, Hum
(X06351 1956-2016 MEF2 at 30/31) gaatgtcaggggcttc
aggtttccctaaatataggtccctgccagaggatccgtggcggg gt
aldolase A enhancer, Rat (X04260 421-480 MEF2 at
30/31) gaatgtcaggggcctcaggtttcactaaatataggtccttgcc
gcgggattcgtggtggg gt desmin enh, myotube-spec
region, Hum (M63391 2257-2316 MEF2
30/31) tgtttcccagccatgcgttctcctctataaatacccgctctgg
tatttggggttggcagc gt desmin enh, myotube-spec
region, Mouse (Z18892 89-148 MEF2
30/31) cctagctgggcctttccttctcctctataaataccagctctgg
tatttcgccttggcagc So on.22 Sequences
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(No Transcript)
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(No Transcript)
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(No Transcript)
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Functionalities of Trafac
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Cycle of Events in Identifying Potential
Regulatory Regions
mRNA ESTs
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ADA Known Regulatory Regions
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Known Regulatory Regions
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Coordinately Expressed Genes Bates et al., 2002
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Constitutionally Similar Cis-elements Clusters in
the Upstream Region
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Coordinately Expressed Genes Tend to Share
Cis-elements
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Potential Novel regulatory Regions
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Identification of Potential Regulatory Regions
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Tissue-specific Genes Share Cis-elements
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Road-Blocks.

• Unknown TF Binding Sites Analysis based on
  Transfac Library
• Pre-Trafac Analysis
• Ortholog Genomic Sequences
• Input Files

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Conclusions.

• Phylogenetically conserved non-coding regions are
  good indicators of regions of gene regulatory
  functions.
• Combinations of TF binding sites in the same
  relative order and distance can be reliable
  indicators of potential regulatory regions.
• Coordinately expressed genes tend to share
  similar TF binding sites.

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A Word of Caution.

• Trafac is NOT a solution-provider It ASSISTS in
  finding solutions. It can be an effective
  filtration tool.
• Any computationally identified regulated region
  has to be validated experimentally.
• Transcriptional regulation is NOT the only mode
  of control of gene expression.

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Present Status of Trafac Database
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• Addition of more groups of genes to the database
  A Continuous process
• Database of regulatory modules (Cis-Mols)
  Based on
• Tissue Specificity
• Gene Ontology Functionalities
• Phenotype
• Gene Expression Pattern
• Multiple Comparison of Genomic DNA Sequences
• A loci comparison instead of individual genes

Whats Next?
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The Team
Acknowledgements
Bruce Aronow Shawn Sherwood Jim Carman Andrew
Pinski Anil Jegga
NIEHS U01 ES11038 Mouse Centers Genomics
Consortium
Howard Hughes Medical Institute