PPT

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PPT

  1. 1. Analysis of protein-DNA interactions with tiling microarrays Srinivasan (Vasan) Yegnasubramanian Sidney Kimmel Comprehensive Cancer Center Oncology Dept., Genitourinary Division March 7, 2007
  2. 2. Identical genetic sequence, but very different gene expression and phenotypes… … These differences are due to Epigenetic changes.
  3. 3. Epigenetics is the study of heritable processes that alter gene expression without an accompanying change in gene sequence These processes are usually mediated by factors, such as proteins/ribonucleo-proteins, that bind genomic DNA
  4. 4. (3.4x10 -10 meters/bp) x (6x10 9 bp/genome) = ~2 meters/genome Radius of the nucleus is ~ 10 µ M !!! Klug and Cummings, 1997
  5. 5. [(6 x 10 9 bp/genome) / (195 bp/nucleosome)] = ~ 30.8 x 10 6 nucleosomes/genome ~ 5 % of nuclear volume
  6. 6. http://www.albany.edu/~achm110/solenoidchriomatin.html
  7. 7. DNA methylation occurs at CpG dinucleotides in mammalian genomes 5’…A CG T…3’ 5’…A CG T…3’ 5-me
  8. 8. DNA methylation patterns in normal and cancer cell genomes Herman and Baylin, NEJM, 2003
  9. 9. DNA methylation can lead to silencing of gene expression Robertson and Wolffe, Nat Rev Genet, 2000 >2 MDalton Complex
  10. 10. Struhl, Cell, 2004 http://www.berkeley.edu/news/features/1999/12/09_3dimage.html
  11. 11. Diameter of DNA Double helix: 20 Angstroms Diameter of Transcriptional machinery: >1,000 Angstroms
  12. 12. Developing an understanding of epigenetic processes… DNA Modifications (e.g. Methylation) Gene Transcriptional Changes DNA-Protein Interactions
  13. 13. Characteristics of Tiling Microarrays <ul><li>Microarray contains n probes of length L distributed across x base pairs on a genomic region of interest. That is, n probes are tiled across a genomic region of interest </li></ul><ul><li>The average resolution or sampling/window size, then, is R = x / n , or </li></ul>d 1 d 2 d 6 d 5 d 4 d 3 d 7
  14. 14. Affymetrix Tiling microarrays <ul><li>Human Chromosome 21/22 microarrays </li></ul><ul><ul><li>> 35 million bp of non-repetitive sequence on Chrom 21/22 represented with >1 million probe sets on three microarrays (currently on a single array). R ~ 35 bp. </li></ul></ul><ul><li>ENCODE arrays </li></ul><ul><ul><li>representation of 1% of genome corresponding with ENCODE regions at 35 bp resolution with single microarray. </li></ul></ul><ul><li>Tiled arrays of 10 human chromosomes </li></ul><ul><ul><li>74,180,611 probe pairs interrogating 30% of human genome (i.e. 10 complete chromosomes) at on >90 microarrays. R ~ 5 bp. </li></ul></ul><ul><li>Tiled arrays of whole genome </li></ul><ul><ul><li>interrogation of whole genome (1.7 Gb) on 7 microarrays (~50,000,000 PM probes only) or 14 microarrays (~50,000,000 PM + MM probe sets). R ~ 35 bp. </li></ul></ul><ul><li>Promoter Tiling arrays </li></ul><ul><ul><li>interrogation of all 5’ upstream regions of known genes on a single microarray </li></ul></ul>All probes are 25-mers
  15. 15. Strategy Label and Hybridize Samples To Tiling Microarrays Chromatin Structure ( In vivo DNA/Protein Interactions) Biostatistical Analysis to Identify Genomic Regions of Interest DNA Methylation ( In Vitro DNA/Protein Interactions) Transcriptome Analysis
  16. 16. ChIP-Chip for “ in vivo” DNA protein interactions Crosslink Lyse & Sonicate IP Reverse crosslinks Total Reverse crosslinks Amplify Amplify Label/hybridize Label/hybridize Other controls for IP (e.g., no antibody, non-specific antibody) Y
  17. 17. Current limitations for ChIP-Chip <ul><li>Process is very inefficient and requires large amounts of input material </li></ul><ul><li>Sonication step can be quite variable and cannot be easily quality controlled with small amounts of starting material </li></ul><ul><li>Currently difficult to perform on clinical specimens </li></ul><ul><li>Labor-intensive </li></ul>
  18. 18. Genome-wide, high-resolution DNA methylation detection by taking advantage of tiling arrays and DNA-protein interactions in vitro
  19. 19. Endogenous methyl-CpG binding domain proteins <ul><li>MECP2 </li></ul><ul><li>MBD1 </li></ul><ul><li>MBD2 </li></ul><ul><li>(Anti-5mC Ab) </li></ul><ul><li>MBD3 </li></ul><ul><li>MBD4 </li></ul>
  20. 20. 6His-MBD2-MBD binds symmetrically methylated oligonucleotides Yegnasubramanian et al. , Nucleic Acids Res, 2006
  21. 21. Enrich for densely methylated fragments Real-time PCR Use of 6His-MBD2-MBD for enrichment of methylated genomic DNA Yegnasubramanian et al. , Nucleic Acids Res, 2006 Fe Fragment
  22. 22. Whole-genome DNA methylation assay Fragment Enrich methylated fragments Amplify Fragment/label/hybridize Amplify Fragment/label/hybridize Total input Fe Fe Fe
  23. 23. Fragmentation techniques Sonication Restriction Enzyme
  24. 24. Middle ground Pool different restriction enzyme digests
  25. 25. Dynamics of amplification and fold enrichment… <ul><li>Fold enrichment dependent on: </li></ul><ul><ul><li>Amount of each species after enrichment </li></ul></ul><ul><ul><li>Total amount of all enriched species </li></ul></ul>Enrich Enrich Total Amplify to 20 Amplify to 20 Amplify to 20
  26. 26. Ongoing and future work DNA Modifications (e.g. Methylation) Gene Transcriptional Changes DNA-Protein Interactions Preprocessing Preprocessing Preprocessing Analysis Analysis Analysis Meta-Analysis Cancer Normal
  27. 27. End of slides

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