Download DNA Methylation Changes During Prostate Carcinogenesis and

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…
Normal Human Brain
Normal Human Kidney Benign Human Prostate
Normal Human Liver
…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/ribonucleoproteins, that bind genomic DNA
(3.4x10-10 meters/bp) x (6x109 bp/genome) = ~2 meters/genome
Radius of the nucleus is ~ 10 µM !!!
Klug and Cummings, 1997
[(6 x 109 bp/genome) / (195 bp/nucleosome)] = ~ 30.8 x 106 nucleosomes/genome
~ 5 % of nuclear volume
http://www.albany.edu/~achm110/solenoidchriomatin.html
DNA methylation occurs at CpG dinucleotides
in mammalian genomes
5’…ACGT…3’
5-me
5’…ACGT…3’
DNA methylation patterns in normal and
cancer cell genomes
Herman and Baylin, NEJM, 2003
DNA methylation can lead to silencing of gene expression
Sin3A HDAC
MeCP2
HDAC1 RbAp46
RbAp48
68kD
MTA2
>2 MDalton Complex
MBD3 HDAC2
66kD
Mi-2
MBD2
Robertson and Wolffe, Nat Rev Genet, 2000
Struhl, Cell, 2004
http://www.berkeley.edu/ne
ws/features/1999/12/09_3
dimage.html
Diameter of DNA Double helix: 20 Angstroms
Diameter of Transcriptional machinery: >1,000 Angstroms
Developing an understanding of
epigenetic processes…
DNA-Protein
Interactions
DNA Modifications
(e.g. Methylation)
Gene Transcriptional
Changes
Characteristics of Tiling Microarrays
d1


d2
d3
d4
d5
d6
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
n 1
R
d
N
N 1
n 1
Affymetrix Tiling microarrays

Human Chromosome 21/22 microarrays


ENCODE arrays


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


representation of 1% of genome corresponding with ENCODE regions at 35 bp
resolution with single microarray.
Tiled arrays of 10 human chromosomes


> 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.
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
Transcriptome
Analysis
DNA Methylation
(In Vitro DNA/Protein
Interactions)
Label and Hybridize Samples
To Tiling Microarrays
Biostatistical Analysis to
Identify Genomic Regions
of Interest
Chromatin Structure
(In vivo DNA/Protein
Interactions)
ChIP-Chip for “in vivo” DNA protein interactions
Total
Crosslink
Reverse
crosslinks
Amplify
Label/hybridize
Amplify
Label/hybridize
Lyse & Sonicate
Y
Other controls for IP
(e.g., no antibody, nonspecific antibody)
IP
Reverse
crosslinks
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 DNAprotein interactions in vitro
Endogenous methyl-CpG binding domain
proteins

MECP2

MBD1

MBD2

(Anti-5mC Ab)
Sin3A HDAC
MeCP2
HDAC1 RbAp46
RbAp48
68kD
MTA2
MBD3 HDAC2
66kD
Mi-2
MBD2

MBD3

MBD4
6His-MBD2-MBD binds symmetrically methylated
oligonucleotides
b
160
140
140
Anisotropy
Anisotropy
c
160
120
100
120
100
EC50 = 0.222 ± 0.067 µM
-4.0
-3.0
-2.0
-1.0
0.0
1.0
2.0
-5.0
-4.0
-3.0
-2.0
-1.0
b
1.0
2.0
-5.0
-4.0
-3.0
-2.0
Anisotropy
140
120
EC50EC>>>>10
M
10 µM
100
120
EC50EC>>>>10
M
10 µM
100
50
-4.0
-3.0
-2.0
-1.0
0.0
1.0
Log [MBD2-MBD] (µM)
Yegnasubramanian et al., Nucleic Acids Res, 2006
50
2.0
-5.0
-1.0
0.0
Log [MBD2-MBD] (µM)
160
140
Anisotropy
0.0
EC50 = 519 +/- 250 nM
Log [MBD2-MBD] (µM)
160
-5.0
EC50 = 0.519 ± 0.250 µM
EC50 = 156 +/- 70 nM
Log [MBD2-MBD] (µM)
a
120
100
EC50 = 0.156 ± 0.070 µM
EC50 = 222 +/- 67 nM
-5.0
160
140
Anisotropy
a
-4.0
-3.0
-2.0
-1.0
0.0
Log [MBD2-MBD] (µM)
1.0
2.0
1.0
2.0
Use of 6His-MBD2-MBD for enrichment of methylated
genomic DNA
1.0
HpaII Digestion Only
100
100
0.1
R2 = 0.976
0.01
10
0.1
1
0.1
0.01
R2 = 0.113
0.001
0.001
0.0
0.0001
0.01
100
0.1
MBD2-MBD
100
Anti-His-Ab
1
10
Input Genomic DNA (ng)
Protein G Magnetic Beads
+
+
+
0.0001
0.01
–
+
+
MBD2-MBD Capture Only
HpaII digestion + MBD2-MBD Capture (COMPARE-MS)
100 WBC M.SssI
WBC
10
R2 = 0.982
0.2
Output DNA (ng)
R2 = 0.962
1
Relative Enrichment
Output DNA (ng)
10
Enrich for
HpaII Digestion Only
densely
100
Fe
WBC M.SssI
methylated
WBC
10
fragments
WBC
WBC M.SssI
WBC
WBC M.SssI
WBC
Fragment
HpaII digestion
+ MBD2-MBD Capture (COMPARE-MS)
WBC M.SssI
WBC M.SssI
WBC
10
– 0.1 – 1
10
Input
–
– Genomic DNA (ng)
+COMPARE-MS
– Assay Performance
100
100
R2 = 0.981R2 = 0.982
10
0.1
R2 = 0.976
0.01
Real-time
PCR
0.001
0.0001
0.01
0.1
1
10
Input Genomic DNA (ng)
100
1
0.1
0.1
0.01
0.01
0.001
0.001
0.0001 0.0001
0.01
0.01 0.1
R2 = 0.538
R2 = 0.113
100
10
1
0.1
1
10
DNA (ng)
Input Genomic
Input Genomic
DNA (ng)
MBD2-MBD Capture Only
Yegnasubramanian
et al., Nucleic Acids Res,
2006
100
WBC M.SssI
WBC
10
R2 = 0.981
10
1
0.1
0.01
COMPARE-MS Assay Performance
100
Output DNA (ng)
1
1
Output DNA (ng)
Output DNA (ng)
Output DNA (ng)
R2 = 0.985
R2 = 0.962
100
0.001
0.01
0.1
1
10
Input M.SssI treated WBC DNA (ng)
in 20 ng of untreated WBC DNA
100
Whole-genome DNA methylation assay
Fragment
Fe
Enrich methylated
fragments
Total input
Fe
Fe
Amplify
Amplify
Fragment/label/
hybridize
Fragment/label/
hybridize
Fragmentation techniques
Sonication
Restriction
Enzyme
Middle ground
Pool different restriction
enzyme digests
Dynamics of amplification and fold
enrichment…
Enrich
Amplify
to 20

Fold enrichment
dependent on:

Enrich
Amplify
to 20

Total
Amplify
to 20
Amount of each
species after
enrichment
Total amount of all
enriched species
Ongoing and future work
DNA Modifications
(e.g. Methylation)
Preprocessing
Analysis
Cancer
DNA-Protein
Interactions
Preprocessing
Analysis
Meta-Analysis
Normal
Gene Transcriptional
Changes
Preprocessing
Analysis
End of slides