chIP-chip For

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chIP-chip For Transcriptome Research
Vidyadhar Karmarkar Genomics and
Bioinformatics 414 Life Sciences Building, Huck
Institute of Life Sciences
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Chromosomal Packaging
Chromatin

• 2.9 million bp in haploid human genome
• 1.5 human genome codes for proteins
• 20,000 human genes

Felsenfeld and Groudine (2003) Nature 421,
448-453
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Gene Structure
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Transcription A quick review
http//www.msu.edu/course/lbs/ 145/smith/s02/graph
ics/ campbell_17.7.gif
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Single TF-Multiple Responses
Hanlon and Lieb (2004) Curr. Opin. Gen. Dev.
14697-705
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Transcriptome Research
chIP-chip
Microarrays
Traditional
Computational
Tag-based
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Limitations of current methods in Transcriptome
Research

• Are in vitro and not in vivo
• Gel-shift assays poor predictors of TFs actual
  binding site
• Computational approaches frustrating
• DNA-footprinting and chIP-qtPCR reveals limited
  information
• -Buck and Lieb (2004) Genomics 83349-360

• RNA level measurement - an indirect indicator
• of TF activity Hanlon and Lieb (2004) Curr.
  Opin. Gen. Dev. 14697-705

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Basic steps in chIP
Fixation
Sonication
Immunoprecipitation
Analysis of IP-ed DNA
Das et al (2004) Biotechniques 37(6) 961-969
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Advantages of chIP

• Information about in vivo location of TF binding
  sites on the DNA
• Captures information from living cells
• Powerful tool in genomics when coupled to cloning
  and microarrays

Das et al (2004) Biotechniques 37(6) 961-969
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chIP-chip
chIP
Das et al (2004) Biotechniques 37(6) 961-969
Buck and Lieb (2004) Genomics 83349-360
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Summary of chIP-chip

• Employs the strategy of enriching the TF-target
  sites by immunoprecipitating followed by
  microarray to detect the level of enrichment

Sikder and Kodadek (2005) Curr. Opin. Chem. Biol.
938-45
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Types of DNA microarrays

• Types
• Mechanically spotted cDNA/amplicons
• Mechanically spotted oligos
• In situ synthesis of oligos

• Most of these arrays made from
• transcribed genomic regions

Buck and Lieb (2004) Genomics 83349-360
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Choosing chip for chIP

• Promoter region is not transcribed
• TF binding sites mapped
• Outside the predicted promoter region (Cawley et
  al 2002 Genome Res. 121749-1755 Martone et al
  2003 PNAS 10012247-12252 Euskirchen 2004 Mol.
  Cell. Biol. 243804-3814)
• In coding and non-coding regions (Martone et al
  2003 PNAS 10012247-12252 Euskirchen 2004 Mol.
  Cell. Biol. 243804-3814)

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Choosing chip for chIP

• On separate arrays enrichment at any given spot
  is relative to sequences on same array
• Whole genome arrays reveals enrichment of ORFs
  relative to intergenic regions

Hanlon and Lieb (2004) Curr. Opin. Gen. Dev.
14697-705
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Maximizing TF-target identification

• Arrays that tile across an entire regulatory
  region of interest (Horak et al 2002 PNAS
  992924-2929)
• Comprehensive but specific to the regulatory
  region
• Limited information
• CpG island microarray (Weinmann et al 2002 Genes
  Dev 16235-244)
• Less of primers gt reduced cost
• Unbiased coverage of large portion of genome
• Requires sequence information on identity of
  clones
• Low cost but highly informative option to whole
  genome arrays

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Maximizing TF-target identification

• DNA tiling arrays (whole genome arrays)
  representing all intergenic regions and predicted
  coding sequences (Iyer et al 2001 Nature
  409533-538)
• - Successfully used in yeast (Buck and Lieb 2004
  Genomics 83349-360)
• Costly and technically challenging to make in
  organisms with large genomes

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Computational Validation of chIP-chip data

• Resolution of chIP-chip within 1-2 kb and exact
  site of DNA-protein interaction unknown
• Programs to analyze chIP-chip data
• MDScan (Liu et al 2002 Nature Biotech.
  20835-839)
• MOTIF REGRESSOR (Conlon et al 2003 PNAS 100
  (6)3339-3344)

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Drawbacks of chIP-chip

• chIP is technically challenging
• Promiscuous crosslinking by formaldehyde
• Resolution dependant on
• Sheared DNA fragment size,
• length and spacing of arrayed DNA elements used
  to detect IP elements
• Cost of making arrays

Buck and Lieb (2004) Genomics 83349-360
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Possible complications with chIP-chip
Differential formation of DNA-protein crosslinks
Variable epitiope accessibility
Legend
Hanlon and Lieb (2004) Curr. Opin. Gen. Dev.
14697-705
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Normalization of chIP-chip data
Mistaking ubiquitous modification to be uniform
distribution
Mistaking promoter associated modification to be
uniform distribution
Hanlon and Lieb (2004) Curr. Opin. Gen. Dev.
14697-705
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Conclusion

• chIP-chip is efficient method for TF-target
  identification
• Computational and biochemical validation of
  chIP-chip data required to pinpoint the exact
  site of TF-DNA interaction
• chIP-CpG arrays are cost effective alternative to
  chIP-WG arrays

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Future Prospects

• Novel insights in genomics of pathogenesis,
  development, apoptosis, cell cycle, genome
  stability and epigenetic silencing, chromatin
  remodelling
• High-throughput method for genome annotation and
  cross-validation of previous data