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Supporting Information
CpG Island Methylation in Lymphoma Is Driven by the Genetic
Configuration of Tumor Cells
Rene Opavsky, Shu-Huei Wang, Prashant Trikha, Aparna Raval, Yuan Huang, Yue-Zhong Wu, Benjamin Rodriguez,
Benjamin Keller, Sandya Liyanarachi, Guo Wei, Ramana V. Davuluri, Michael Weinstein, Dean Felsher, Michael
Ostrowski, Gustavo Leone and Christoph Plass
Figure S1. Methylation and Expression of RLGS Sequences
(A) Southern blot analysis of control and tumor cell DNAs using RLGS fragments 6D20, 1F21
and 3F56 as probes. Control DNA (con) or Tumor DNAs (tumor) isolated from thymocytes of
healthy EμSR-tTA mice or T cell lymphomas, respectively was digested either with EcoRV
alone (-) or with EcoRV and NotI (+). The top bands (M) indicate methylated (not digested with
NotI) and the lower bands indicate fragments digested with NotI (U).
(B) Combined bisulfite restriction analysis (COBRA) analysis of bisulfite-treated DNA isolated
from thymocytes of EμSR-tTA mice (con) or from nine different T cell lymphomas (tumors).
PCR products were digested with restriction enzyme BstUI and loaded on PAGE gel along with
100 bp marker (M) and 100 % methylated (SssI treated DNA) control (100%). Undigested and
digested fragments correspond to unmethylated and methylated DNA, respectively.
(C) Expression of genes near the RLGS fragments 6D20, 5D36 and 3F70 was quantified in
thymocytes isolated from three EμSR-tTA mice (open bars) and eight EμSR-tTA;Teto-MYC
tumors (black bars) using real-time RT-PCR. The y-axis represents fold-decrease in gene
expression relative to the levels of gene expression detected in the first control.
A
con
- +
+ +
tumors
+ + + +
con
tumors
- + + + + + + + + + +
+ +
+ + +
-M
3F56
1F21
6D20
tumors
+ + + + +
-M
-M
-U
con
- +
-U
-U
M con
tumors
M con
100%
tumors
100%
M con
100%
B
tumors
C
6D20
80
80
5D36
60
60
D
60
60
30
30
40
40
20
20
20
20
10
10
con
tumors
0
con
tumors
6
7
tumors
Figure S1
T8
5
T7
4
T6
3
T5
2
T4
1
T3
3
T2
2
con
T1
1
N3
9
T9
8
T8
7
T7
6
T6
5
T5
4
T4
3
T3
2
T2
1
T1
3
N3
2
N2
0
1
N2
0
0
N1
8
T8
7
T7
6
T6
5
T5
4
T4
3
T3
2
T2
1
T1
3
N3
2
N2
1
N1
20
20
00
3F70
80
80
40
40
40
40
digest.
fragments
100
100
50
50
60
60
N1
Fold reduction
in gene expression
4E58
2F37
2G50
undigest.
fragment
8