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. Identical genetic sequence, but very different gene expression and phenotypes…
…These differences are due to Epigenetic changes.

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. (3.4x10-10 meters/bp) x (6x109 bp/genome) = ~2 meters/genome
Radius of the nucleus is ~ 10 µM !!!
Klug and Cummings, 1997

5. [(6 x 109 bp/genome) / (195 bp/nucleosome)] = ~ 30
[(6 x 109 bp/genome) / (195 bp/nucleosome)] = ~ 30.8 x 106 nucleosomes/genome
~ 5 % of nuclear volume

7. DNA methylation occurs at CpG dinucleotides in mammalian genomes
5’…ACGT…3’
5-me

8. DNA methylation patterns in normal and cancer cell genomes
Herman and Baylin, NEJM, 2003

9. DNA methylation can lead to silencing of gene expression
>2 MDalton Complex
Robertson and Wolffe, Nat Rev Genet, 2000

10. Struhl, Cell, 2004

11. Diameter of DNA Double helix: 20 Angstroms
Diameter of Transcriptional machinery: >1,000 Angstroms

12. Developing an understanding of epigenetic processes…
DNA Modifications
(e.g. Methylation)
Gene Transcriptional
Changes
DNA-Protein
Interactions

13. Characteristics of Tiling Microarraysd1 d2 d6 d5 d4 d3 d7
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

14. 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

15. Strategy DNA Methylation Transcriptome Chromatin Structure
Analysis
DNA Methylation
(In Vitro DNA/Protein
Interactions)
Chromatin Structure
(In vivo DNA/Protein Interactions)
Label and Hybridize Samples
To Tiling Microarrays
Biostatistical Analysis to
Identify Genomic Regions
of Interest

16. ChIP-Chip for “in vivo” DNA protein interactions
Total
Reverse crosslinks
Amplify
Label/hybridize
Crosslink
Lyse & Sonicate Y
Other controls for IP
(e.g., no antibody, non-specific antibody) IP
Reverse crosslinks
Amplify
Label/hybridize

17. 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

18. Genome-wide, high-resolution DNA methylation detection by taking advantage of tiling arrays and DNA-protein interactions in vitro

19. Endogenous methyl-CpG binding domain proteins
MECP2
MBD1
MBD2
(Anti-5mC Ab)
MBD3
MBD4

20. 6His-MBD2-MBD binds symmetrically methylated oligonucleotides
Yegnasubramanian et al., Nucleic Acids Res, 2006

21. Enrich for densely methylated fragments
Use of 6His-MBD2-MBD for enrichment of methylated genomic DNA
Fragment Fe
Enrich for densely methylated fragments
Real-time PCR
Yegnasubramanian et al., Nucleic Acids Res, 2006

22. Whole-genome DNA methylation assayFe
Fragment
Total input
Enrich methylated fragments
Amplify
Amplify
Fragment/label/hybridize
Fragment/label/hybridize

23. Fragmentation techniques
Restriction
Enzyme
Sonication

24. Middle ground
Pool different restriction
enzyme digests

25. Dynamics of amplification and fold enrichment…
Total
Amplify
to 20
Fold enrichment dependent on:
Amount of each species after enrichment
Total amount of all enriched species

26. Ongoing and future work
Preprocessing
Analysis
DNA Modifications
(e.g. Methylation)
Cancer
Meta-Analysis
DNA-Protein
Interactions
Preprocessing
Analysis
Normal
Gene Transcriptional
Changes
Preprocessing
Analysis

27. End of slides