supplementary table 2
DESCRIPTION
Supplementary Table 2. a The #CpG indicates the number of CpG in the sequence region. b The plus(+) and minus(-) indicate the presence and absence of CpG island, respectively. c The plus(+) and minus(-) indicate the presence and absence of candidate methylated site, respectively. - PowerPoint PPT PresentationTRANSCRIPT
Supplementary Table 2.
aThe #CpG indicates the number of CpG in the sequence region. bThe plus(+) and minus(-) indicate the presence and absence of CpG island, respectively. cThe plus(+) and minus(-) indicate the presence and absence of candidate methylated site, respectively. dThe minimum P-value shows the most significant p-value in the analyzed region. eThe methylation (%) indicates the ratio of methyl CpG sites to all CpG sites.
Number Chr. Flanking genes Location Size (bp) #CpGa CGIb CMSc Minimum P-valued Methylation(%)e
1 2 DES 1st exon 188 24 + + 8.99×10-5 97.7
2 5 Intergenic 264 7 - + 1.46×10-4 87.1
3 14 BCL11B Other intron 224 12 - + 1.49×10-6 87.0
4 18 CDH2 Other intron 193 4 - + 8.52×10-3 60.0
5 5 Intergenic 277 6 - + 4.14×10-3 81.7
6 21 ITSN1 Other intron 149 6 - + 6.76×10-3 90.0
7 13 Intergenic 206 12 - + 3.09×10-6 94.2
8 13 Intergenic 207 9 - + 1.38×10-5 94.4
9 19 RPS9 3' UTR 109 4 - + 1.09×10-4 70.0
10 21 WRB 0.5kb upstream 207 4 - + 1.05×10-3 32.5
11 11 NRXN2 1st intron 251 20 + + 8.74×10-7 83.5
12 14 BCL11B 1st intron 166 23 + + 3.36×10-4 94.8
13 6 Intergenic 273 14 + + 8.66×10-6 92.9
14 22 Intergenic 136 4 - + 5.81×10-5 50.0
15 X G6PD Other intron 170 5 - + 6.21×10-6 86.0
16 8 EXT1 1st exon 233 13 - + 6.79×10-6 95.4
17 6 TFEB 5' UTR 149 11 + - 0.19 0.0
18 9 CRAT Upstream 177 17 + - 0.05 0.6
19 7 CFTR 0.3kb upstream 338 20 - + 4.98×10-9 90.0
20 13 F7 Other intron 155 6 - - 0.21 85.0
21 13 MCF2L Other intron 187 13 + - 0.08 83.7
22 6 TFEB 3' UTR 233 7 - - 0.24 73.0
23 21 Intergenic 217 16 - - 0.07 91.0
24 X FAM3A 0.7kb upstream 252 9 + - 0.07 91.1
25 18 CDH2 1kb upstream 165 10 - + 1.34×10-5 98.0
26 2 INHA 1.5kb upstream 385 56 + + 2.78×10-5 93.6
27 22 YWHAH 1st intron 185 20 + - 0.74 0.0
28 7 HOXA1 1st exon 224 18 + - 0.13 26.1
29 7 HOXA1 2nd exon 147 11 + - 0.10 91.8
30 7 HOXA2 5' UTR 155 6 + + 8.19×10-4 88.3
31 7 HOXA3 5' UTR 133 17 + + 5.52×10-5 93.3
32 7 HOXA4 1st exon 172 16 + - 0.13 0.0
33 7 HOXA5 0.1kb upstream 416 34 + + 2.09×10-9 95.0
34 7 HOXA6 1st exon 256 17 + + 3.28×10-4 88.2
35 7 HOXA7 5' UTR 254 17 + - 0.25 0.0
36 7 HOXA9 Upstream 121 21 + - 0.66 0.0
37 7 HOXA10 1st intron 157 19 + - 0.52 0.0
38 7 HOXA11 0.7kb upstream 218 15 + + 1.87×10-6 94.0
39 7 HOXA13 2nd exon 88 8 - + 2.37×10-5 93.8
40 7 HOXA13 1st exon 177 13 + - 0.51 0.0
Supplementary Table 3.
Features of candidate methylated sites are shown. aThe Obs/exp indicates the ratio of observed and expected CpG frequency. bFold-enrichment was calculated based on population of ENCODE probes extracted randomly.
#site Length GC% Obs/expa #CGI %CGI Fold-enrichmentb
DLD1
P<10-2 869 504 50.2 0.45 543 62.5 2.8
P<10-3 466 575 52.1 0.52 349 74.9 3.4
P<10-4 338 559 53.0 0.54 264 78.1 3.5
P<10-5 240 575 53.0 0.56 182 75.8 3.4
HCT116
P<10-2 859 485 52.1 0.46 555 64.6 2.9
P<10-3 460 526 53.5 0.53 345 75.0 3.4
P<10-4 318 519 53.7 0.55 251 78.9 3.6
P<10-5 238 502 53.8 0.57 189 79.4 3.6
SW480
P<10-2 703 492 51.2 0.46 454 64.6 2.9
P<10-3 393 541 52.3 0.52 296 75.3 3.4
P<10-4 277 530 54.0 0.56 226 81.6 3.7
P<10-5 198 549 53.3 0.56 154 77.8 3.5
Average Overlap of CpG Island
Supplementary Table 4:
aThe Upstream CMS shows a location of upstream CMS, if any, by a distance from a TSS.
bThe 5Aza+ indicates GeneChip score with 5Aza-dC treatment. cThe 5Aza- indicates GeneChip socre without 5Aza-dC treatment.
Upstream
Probe Set ID Gene Symbol CMSa 5Aza+b 5Aza-c Description
214639_s_at HOXA1 Not detected 178.3 66.2 2.7 homeo box A1
214457_at HOXA2 -100~+300 bp 19.7 11.9 1.7 homeo box A2
208604_s_at HOXA3 42.1 23.9 1.8 homeo box A3
235521_at HOXA3 120.6 94.7 1.3 homeo box A3
206289_at HOXA4 Not detected 9.1 17.9 0.5 homeo box A4
213844_at HOXA5 -1.4 kb~+300 bp 207.2 131.6 1.6 homeo box A5
208557_at HOXA6 -100~+200 bp 19.2 7.3 2.6 homeo box A6
206847_s_at HOXA7 Not detected 40.9 86.7 0.5 homeo box A7
214651_s_at HOXA9 892 1598.3 0.6 homeo box A9
209905_at HOXA9 387.5 986 0.4 homeo box A9
213150_at HOXA10 292.3 250.6 1.2 homeo box A10
213147_at HOXA10 77 115 0.7 homeo box A10
213823_at HOXA11 64.9 78.1 0.8 homeo box A11
208493_at HOXA11 10.6 19.2 0.6 homeo box A11
238571_at HOXA13 13.7 22.2 0.6 Homeo box A13
231786_at HOXA13 13.4 27.8 0.5 homeo box A13
238808_at HOXA13 26.1 60 0.4 Homeo box A13
-1 kb~-500 bp
Not detected
Not detected
Not detected
GeneChip score 5Aza+/5Aza-ratio
-200~+200 bp
Supplementary Figure 1:
－
5Aza-dC 400 200 100 50 (ng)
＋
Genomic DNA and demethylated DNA were spotted on a nitrocellulose membrane at 50-
400ng. 1st antibody: anti-methycytosine antibody, 2nd antibody: HRP anti-rabbit IgG anti
body
-5 -4 -3 -2 -1 00
-1
-2
-3
-5
-4
Log 10(P)
- Log10(P)))(
(10ProbesAll
PProbeNumberLog
)(
)(
PProbeNumber
PProbeNumber
Control
MeDIP
32
16
8
4
1
2
1 2 3 4 5 0
MeDIP1 vs.. Control1 ( n = 1 )
Control1 vs. Control2 ( n = 1 )
MeDIPs vs. Controls ( n = 2 )MeDIP1 vs. Control1 ( n = 1 )MeDIPs vs. Controls ( n = 2 )
The scanned tiling array data were analyzed with Tiling Array Analysis Software (Affymetrix). The p-values analyzed in those combinations of 2 MeDIPs vs. 2 Controls, MeDIP1 vs. Control1, Control1 vs. Control2 were obtained. a Number of probes less than each threshold p-value was plotted for each result. b The rates of probe numbers due to threshold p-values were plotted.
Supplementary Figure 2:
A B
A
Supplementary Figure 3:
B
C
High-resolution mapping of DNA methylation in the ENCODE region. Tracks for CMS (light green bar), -10×log10(p-value) (vertical yellow bar) and CGI (green bar) are shown. a DLD1, b HCT116, b SW480
Supplementary Figure 4:
Bisulfite sequencing of CMS or non-CMS in HCT116. a Genomic view showing CMS on INHA (top). Tracks for sequenced region (pink bar), CMS (light green bar), -10×log10 (P-value) (vertical yellow bar) and CGI (green bar) are shown. Black horizontal bar indicates P=10-2. Result of bisulfite sequencing (bottom). Closed circle and open circle indicate methylated CpG and unmethylated CpG, respectively. b TFEB, c CRAT
A
B
C
P=10-2
-10× log(P-value)
HCT116_CMS
Supplementary Figure 5:
Results of bisulfite sequencing are shown described in Supplemental Table 3. Number 16 and 19 are shown in Supplemental Fig. 7, and number 17-18 and 26 are shown in Supplemental Fig 4. Number 28-40 are shown in Fig. 4. Closed circle and open circle indicate methylated CpG and unmethylated CpG, respectively.
2320 21 2215
14
21 3
87 9
4
6
5
11
10
12 13
24 25 27
Supplementary Figure 6:
A
DLD1_CMS
DLD1_CMS
DLD1_CMS
DLD1_CMS
Supplementary Figure 6:
B
SW480_CMS
SW480_CMS
High-resolution mapping of DNA methylation on the HOXA cluster. Tracks for CMS (light green bar), -10×log10(p-value) (vertical yellow bar) and CGI (green bar) are shown. a DLD1, b SW480
DLD1 HCT116 SW480 DLD1 HCT116 SW480
Supplementary Figure 7:
A
B
Comparison of methylation patterns among three colorectal cancer cell lines. a The Venn diagram of CMS in three cell lines. If CMS overlapped at least by one base, we regard them as overlapped CMS. b and c High-resolution mapping of DNA methylation for EXT1 and CFTR, respectively. Tracks for sequenced region (blue bar). CMS and -10×log10(p-value) are shown in yellow bar for DLD1, green for HCT116, and purple for SW480. Results of sequencing are shown at the bottom.
C
DLD1
HCT116
SW480
Sequence
P<10-4
P<10-2
No methylation site
DLD1
HCT116
SW480
Sequence
P<10-4
P<10-2
No methylation site
DLD1
HCT116
SW480
Sequence
P<10-5
P<10-3
No methylation site
DLD1
HCT116
SW480
Sequence
P<10-5
P<10-3
No methylation site
391
124
79 60
173
284275
DLD1: 869 HCT116: 859
SW480: 703
Supplementary Figure 8:
MSP analysis of genes overexpressed after 5Aza-dC treatment. a and b High-resolution mapping of DNA methylation for TKTL1 and EXT1, respectively.
M U M U5Aza-dC － ＋
MSP
TKTL1
EXT1
A
B
C
CMS_P<10-5
CMS_P<10-4
-10× log(P-value)
-10× log(P-value)
TKTL1: transketolase-like 1SERPINB2: serpin peptidase inhibitor, clade B, member 2
CTAG2: cancer/testis antigen 2 CTAG1B: cancer/testis antigen 1B
PDLIM4: PDZ and LIM domain 4 CTGF: connective tissue growth factor
CDH2: N-cadherin EXT1: exostoses1
RFPL2: ret finger protein-like 2 AXIN1: axin 1
-10×log(P-value)
HCT116_CMS
-10×log(P-value)
HCT116_CMS
-10×log(P-value)
HCT116_CMS
-10×log(P-value)
HCT116_CMS
-10×log(P-value)
HCT116_CMS
-10×log(P-value)
HCT116_CMS
HCT116_CMS
-10×log(P-value)
HCT116_CMS
-10×log(P-value)
HCT116_CMS
-10×log(P-value)
HCT116_CMS
-10×log(P-value)
Supplementary Figure 9:
DNA methylation maps of genes 10 top-ranking genes overexpressed after 5Aza-dC treatment are shown. Tracks for CMS (light green bar), -10×log10(p-value) (vertical yellow bar) and CGI (green bar), Repeat Masker (black bar) are shown.