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  • Analysis of protein-DNA interactions with tiling microarrays Srinivasan (Vasan) Yegnasubramanian Sidney Kimmel Comprehensive Cancer Center Oncology Dept., Genitourinary Division March 7, 2007
  • Identical genetic sequence, but very different gene expression and phenotypes… … These differences are due to Epigenetic changes.
  • 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
  • (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
  • [(6 x 10 9 bp/genome) / (195 bp/nucleosome)] = ~ 30.8 x 10 6 nucleosomes/genome ~ 5 % of nuclear volume
  • http://www.albany.edu/~achm110/solenoidchriomatin.html
  • DNA methylation occurs at CpG dinucleotides in mammalian genomes 5’…A CG T…3’ 5’…A CG T…3’ 5-me
  • DNA methylation patterns in normal and cancer cell genomes Herman and Baylin, NEJM, 2003
  • DNA methylation can lead to silencing of gene expression Robertson and Wolffe, Nat Rev Genet, 2000 >2 MDalton Complex
  • Struhl, Cell, 2004 http://www.berkeley.edu/news/features/1999/12/09_3dimage.html
  • Diameter of DNA Double helix: 20 Angstroms Diameter of Transcriptional machinery: >1,000 Angstroms
  • Developing an understanding of epigenetic processes… DNA Modifications (e.g. Methylation) Gene Transcriptional Changes DNA-Protein Interactions
  • Characteristics of Tiling Microarrays
    • 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
    • The average resolution or sampling/window size, then, is R = x / n , or
    d 1 d 2 d 6 d 5 d 4 d 3 d 7
  • Affymetrix Tiling microarrays
    • Human Chromosome 21/22 microarrays
      • > 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.
    • ENCODE arrays
      • representation of 1% of genome corresponding with ENCODE regions at 35 bp resolution with single microarray.
    • Tiled arrays of 10 human chromosomes
      • 74,180,611 probe pairs interrogating 30% of human genome (i.e. 10 complete chromosomes) at on >90 microarrays. R ~ 5 bp.
    • Tiled arrays of whole genome
      • 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.
    • Promoter Tiling arrays
      • interrogation of all 5’ upstream regions of known genes on a single microarray
    All probes are 25-mers
  • 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
  • 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
  • Current limitations for ChIP-Chip
    • Process is very inefficient and requires large amounts of input material
    • Sonication step can be quite variable and cannot be easily quality controlled with small amounts of starting material
    • Currently difficult to perform on clinical specimens
    • Labor-intensive
  • Genome-wide, high-resolution DNA methylation detection by taking advantage of tiling arrays and DNA-protein interactions in vitro
  • Endogenous methyl-CpG binding domain proteins
    • MECP2
    • MBD1
    • MBD2
    • (Anti-5mC Ab)
    • MBD3
    • MBD4
  • 6His-MBD2-MBD binds symmetrically methylated oligonucleotides Yegnasubramanian et al. , Nucleic Acids Res, 2006
  • 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
  • Whole-genome DNA methylation assay Fragment Enrich methylated fragments Amplify Fragment/label/hybridize Amplify Fragment/label/hybridize Total input Fe Fe Fe
  • Fragmentation techniques Sonication Restriction Enzyme
  • Middle ground Pool different restriction enzyme digests
  • Dynamics of amplification and fold enrichment…
    • Fold enrichment dependent on:
      • Amount of each species after enrichment
      • Total amount of all enriched species
    Enrich Enrich Total Amplify to 20 Amplify to 20 Amplify to 20
  • 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
  • End of slides