vidyadhar karmarkar genomics and bioinformatics 414 life sciences building, huck institute of life...
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Vidyadhar Karmarkar Genomics and Bioinformatics
414 Life Sciences Building, Huck Institute of Life Sciences
Felsenfeld and Groudine (2003) Nature 421, 448-453
Chromosomal Packaging
2.9 million bp in haploid human genome 1.5% human genome codes for proteins 20,000 human genes
Chromatin
Promoter
5’ UTR
ATG
Exons
Stop
3’ UTR
Poly A Signal
Introns
Gene Structure
Transcription – A quick review
http://www.msu.edu/course/lbs/145/smith/s02/graphics/campbell_17.7.gif
Hanlon and Lieb (2004) Curr. Opin. Gen. & Dev. 14:697-705
Single TF-Multiple Responses
Transcriptome Research
Tag-based
Microarrays
chIP-chip
Computational
Traditional
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
Basic steps in chIP
Fixation
Sonication
Immunoprecipitation
Analysis of IP-ed DNA
Das et al (2004) Biotechniques 37(6) 961-969
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
chIP-chip
Buck and Lieb (2004) Genomics 83:349-360
chIP
Das et al (2004) Biotechniques37(6) 961-969
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
Types of DNA microarrays
Types:• Mechanically
spotted cDNA/amplicons
• Mechanically spotted oligos
• In situ synthesis of oligos
Buck and Lieb (2004) Genomics 83:349-360
Most of these arrays made from transcribed genomic regions
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)
Choosing chip for chIP
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
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
• ‘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
Maximizing TF-target identification
• 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)
Computational Validation of chIP-chip data
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
Possible complications with chIP-chip
Differential formation of DNA-protein crosslinks
Variable epitiope accessibility
Hanlon and Lieb (2004) Curr. Opin. Gen. & Dev. 14:697-705
Legend:
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
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
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