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chIP-chip For Transcriptome Research

chIP-chip For Transcriptome Research. Vidyadhar Karmarkar Genomics and Bioinformatics 414 Life Sciences Building, Huck Institute of Life Sciences. Chromosomal Packaging. Chromatin. 2.9 million bp in haploid human genome 1.5% human genome codes for proteins 20,000 human genes.

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chIP-chip For Transcriptome Research

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  1. chIP-chip For Transcriptome Research Vidyadhar Karmarkar Genomics and Bioinformatics 414 Life Sciences Building, Huck Institute of Life Sciences

  2. 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

  3. Introns 5’ UTR 3’ UTR Exons Promoter ATG Stop Poly A Signal Gene Structure

  4. Transcription – A quick review http://www.msu.edu/course/lbs/ 145/smith/s02/graphics/ campbell_17.7.gif

  5. Single TF-Multiple Responses Hanlon and Lieb (2004) Curr. Opin. Gen. & Dev. 14:697-705

  6. Transcriptome Research chIP-chip Traditional Microarrays Computational Tag-based

  7. Limitations of current methods in Transcriptome Research • Are in vitro and not in vivo • Gel-shift assays poor predictors of TF’s actual binding site • Computational approaches frustrating • DNA-footprinting and chIP-qtPCR reveals limited information -Buck and Lieb (2004) Genomics 83:349-360 • RNA level measurement - an indirect indicator • of TF activity – Hanlon and Lieb (2004) Curr. Opin. Gen. Dev. 14:697-705

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

  9. 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

  10. chIP-chip chIP Das et al (2004) Biotechniques 37(6) 961-969 Buck and Lieb (2004) Genomics 83:349-360

  11. 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. 9:38-45

  12. 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 83:349-360

  13. Choosing chip for chIP • Promoter region is not transcribed TF binding sites mapped: • Outside the predicted promoter region (Cawley et al 2002 Genome Res. 12:1749-1755; Martone et al 2003 PNAS 100:12247-12252; Euskirchen 2004 Mol. Cell. Biol. 24:3804-3814) • In coding and non-coding regions (Martone et al 2003 PNAS 100:12247-12252; Euskirchen 2004 Mol. Cell. Biol. 24:3804-3814)

  14. 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. 14:697-705

  15. Maximizing TF-target identification • Arrays that tile across an entire regulatory region of interest (Horak et al 2002 PNAS 99:2924-2929) • Comprehensive but specific to the regulatory region • Limited information • CpG island microarray (Weinmann et al 2002 Genes & Dev 16:235-244) • Less # of primers => 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

  16. Maximizing TF-target identification • ‘DNA tiling arrays’ (whole genome arrays) representing all intergenic regions and predicted coding sequences(Iyer et al 2001 Nature 409:533-538) - Successfully used in yeast(Buck and Lieb 2004 Genomics 83:349-360) • Costly and technically challenging to make in organisms with large genomes

  17. 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. 20:835-839) • MOTIF REGRESSOR (Conlon et al 2003 PNAS 100 (6):3339-3344)

  18. 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 83:349-360

  19. Possible complications with chIP-chip Differential formation of DNA-protein crosslinks Variable epitiope accessibility Legend: Hanlon and Lieb (2004) Curr. Opin. Gen. & Dev. 14:697-705

  20. 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. 14:697-705

  21. 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

  22. 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

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