SUPPORTING INFORMATION
Distinct DNA-based epigenetic switches trigger transcriptional activation of silent genes in human
dermal fibroblasts
Ganesh N. Pandiana+, Junichi Taniguchib+, Syed Junethab, Shinsuke Satoa, Le Hanb, Abhijit Sahab,
Chandran AnandhaKumarb, Toshikazu Bandob, Hiroki Nagasec, d, Thangavel Vaijayanthib, Rhys D.
Taylorb and Hiroshi Sugiyamaa, b*.
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Methods
General.
Unless specified, all the reagents and solvents used in this work were purchased from standard
suppliers and used without further purification. Abbreviations used: P, N-methylpyrrole; I,
N-methylimidazole;
AF,
ammonium
formate;
Fmoc,
9-fluorenylmethoxycarbonyl;HCTU,1-[bis(dimethylamino)methylene]-5-chloro-1H-benzothiazolium
3-oxide hexafluorophosphate; DMSO, dimethylsulfoxide; DIEA, N,N-diisopropylethylamine; β,
1
H NMR spectra were
recorded on a JEOL JNM-FX 400 model NMR spectrometer. HPLC analysis was performed with a
4.6 x 150 mm column on a JASCO PU-2089 plus pump model with UV-2075 plus HPLC UV/VIS
detector and a Chemcobond 5-ODS-H 10 x 150 mm column (Chemco Scientific Co., Ltd, Osaka,
Japan) was used for the purification of SAHA-PIPs and their precursor. Electrospray Ionization
Time-of-Flight mass (ESI-TOF-MS) was recorded on BioTOF II ESI-TOF Bruker Daltonics Mass
Spectrometer (Bremen, Germany). Flash column system was performed using Combi Flash
Companion model (Teledyne Isco Inc., NE, USA). Teledyne Isco Redi Sep Rf column (40 g) was
used for the purification of 4-(8-methoxy-8-oxooctanamido)benzoic acid.
Synthesis and purification of SAHA-PIP conjugates
Synthesis and characterization of 4-(8-methoxy-8-oxooctanamido) benzoic acid and subsequent
1
conversion to SAHA-PIP conjugates were performed on a PSSM-8 peptide synthesizer (Shimadzu,
Kyoto) as mentioned before1,2. The purities of the all SAHA-PIPs (1-32) were checked by HPLC
(elution with trifluoroacetic acid and a 0-100% acetonitrile linear gradient (0-40 min) at a flow rate
of 1.0 mL min-1 under 254 nm).
Cell culture
HDF from 54-year-old Caucasian were purchased from Cell Applications, Inc. We used these cells
for the screening studies within six passages. HDFs were maintained in Dulbecco's modified eagle
medium (DMEM, Nacalai Tesque, Japan) containing 10% fetal bovine serum (FBS, Japan Serum).
Cytotoxicity assay
Colorimetric assays using WST-8 (Dojindo, Kumamoto, Japan) were carried out in 96-well plates
with various concentrations of SAHA-PIPs as mentioned before23.
Optimization of parameters for treatment of SAHA-PIP against HDF
HDF cells with in the passage P6 were trypsinized for 5 min at 37 °C, and were resuspended in the
fresh HDF medium to a concentration of 1.5 x 105 cells/ml in a 35 mm plate and were grown for 24 h
as mentioned before3. The medium was then removed and replaced with 2 ml of fresh HDF medium
followed by the addition of 1 mM of each individual SAHA-PIP (1 to 32) to achieve a final
2
polyamide concentration of 1 µM in 0.1% DMSO and then were incubated in a 5% CO2 atmosphere
at 37 °C for 48 h. 0.1% DMSO treated cells were used as the control. The effective concentration of
the SAHA-PIPs was standardized based on the initial optimization experiments and the treatment of
HDF with various concentration of PI polyamide SAHA conjugates (100 nM, 500 nM, 1 µM and 10
µM)3. Studies with varied incubation time (24 h, 48 h and 72 h) suggested that 48 h is optimal to
achieve consistent expression3. Hence, 1 µM of effectors and 48 h incubation was employed for all
studies.
3
Figure S1
Figure S1. Number of genes affected with the treatment of SAHA, individual SAHA-PIPs 1-32 in
HDFs for 48 h. a) up-regulated by more than 10-fold b) down-regulated by more than 10-fold. . Each
bar is the summary of results derived from two individually treated culture plates
4
Figure S2
Figure S2. Heat map of top-100 down-regulated genes. An unsupervised hierarchical clustering
analysis of top 100 down-regulated genes with the treatment of SAHA, SAHA-PIPs 1-32 is provided.
Each result represents the summary of analysis of data derived from two culture plates
5
Figure S3
Figure S3. Functional annotation of the SAHA-PIP a) 1, b) 7 and c) 19 induced genes in human
dermal fibroblasts. Dark gray bar indicates a particular set of biological processes that repeatedly
occur. Each result is the summary of analysis of data derived from two individual culture plates. Data
were analyzed through the use of IPA (Ingenuity® Systems, www.ingenuity.com).
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Figure S4
7
8
9
Figure S4. Network analysis of SAHA-PIP a) 2, b) 13, c) 17, d) 18, e) 24 and f) 25 induced genes in
human dermal fibroblasts corresponding to hematological system, nervous system, hair and skin,
respiratory, sensory system and digestive system, respectively. Four-fold induction was chosen as the
remarkable effect to account for the down-stream effect. Each result is the summary of analysis of
data derived from two individual culture plates. Data were analyzed through the use of IPA
(Ingenuity® Systems, www.ingenuity.com).
10
Figure S5
Figure S5. Heat map of SAHA-PIP induced ncRNAs. An unsupervised hierarchical clustering
analysis of top 100 down regulated non-coding RNAs (ncRNAs) with the treatment of SAHA,
SAHA-PIPs 1-32 is provided. Each result is the summary of analysis of data derived from two
individual culture plates.
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Figure S6
Figure S6. SAHA-PIPs are non-toxic to HDFs. Cytotoxicity studies carried out as mentioned
before3 suggest that at a) 1 μM concentration SAHA but none of the SAHA-PIPs killed 50% of the
cells. Even at b) 10 μM concentration none of the SAHA-PIPs were cytotoxic. Each bar represents
mean ± SD from 9 well plates.
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SUPPLEMENTARY TABLES
Table S1. Number of up-/down-regulated genes in SAHA and SAHA-PIP (1-32)-treated HDFs.
Compound
Up-regulated
Down-regulated
>2 fold
>5 fold
>10 fold
>2 fold
>5 fold
>10 fold
1
1331
656
515
927
85
61
2
1809
718
498
608
104
57
3
1399
499
289
524
124
65
4
703
225
153
992
96
61
5
1489
490
217
571
110
52
6
1314
314
156
683
115
59
7
3816
449
194
2414
219
58
8
3244
453
243
2314
195
69
9
2258
692
331
382
99
54
10
2406
257
131
1705
128
57
11
909
281
162
623
127
64
12
520
85
34
543
111
59
13
1468
693
433
626
91
45
14
1050
614
410
718
114
63
15
910
575
413
882
111
68
16
689
127
41
553
164
69
17
699
355
223
645
32
7
18
728
388
213
655
45
7
19
690
382
201
702
48
8
20
755
390
240
759
48
8
21
500
258
171
906
32
7
22
472
224
155
890
26
6
23
656
342
230
669
24
6
24
907
514
337
549
18
5
25
608
363
225
659
25
4
26
834
407
234
424
32
7
27
649
370
242
653
25
5
28
883
469
279
527
33
11
29
1201
117
12
330
69
14
30
681
80
12
334
53
11
31
870
87
5
318
42
12
32
288
38
4
243
30
8
SAHA
2002
281
47
1386
182
74
*Sum of data derived from two individual culture plates
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Table S2. Summary of common genes up regulated by more than 10-fold between individual
effector-treated HDFs.
*Sum of data derived from two individual culture plates
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Table S3. Summary of common genes down regulated by more than 10-fold between individual
effector-treated HDFs.
*Sum of data derived from two individual culture plates
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Table S4. Ct values in qRT-PCR experiment
SAHA-PIP
Gene
Ct - SAHA-PIP
Ct - SAHA
Ct - DMSO
1
GRPR
26.3 ± 1.2
39.6 ± 0.5
38.5 ± 2.0
CD24
16.7 ± 0.4
30.5 ± 0.0
31.1 ± 1.4
ACTB
12.9 ± 0.5
13.7 ± 0.7
13.5 ± 0.5
HLA-DOA
27.4 ± 2.1
38.9 ± 1.5
37.4 ± 0.3
DPYSL5
25.3 ± 1.1
40 ± 0
40 ± 0
ACTB
14.0 ± 0.8
14.9 ± 0.5
14.5 ± 0.9
GPC3
21.2 ± 0.1
30.1 ± 0.5
28.8 ± 0.0
SEMA6A
23.2 ± 0.0
29.0 ± 0.0
28.7 ± 0.6
ACTB
12.9 ± 0.1
13.7 ± 0.7
13.5 ± 0.5
PRSS8
22.3 ± 0.1
34.7 ± 0.2
34.6 ± 1.3
WNK2
24.5 ± 0.1
37.8 ± 3.0
36.7 ± 1.3
ACTB
13.0 ± 0.0
14.3 ± 0.8
14.1 ± 0.4
GPRC5B
27.2 ± 0.7
33.2 ± 0.1
32.5 ± 1.2
ACTB
14.2 ± 0.2
15.6 ± 0.1
15.1 ± 1.5
PDLIM3
22.5 ± 1.1
30.5 ± 0.0
32.4 ± 1.2
ACTB
11.8 ± 0.9
13.6 ± 0.1
13.8 ± 0.3
LEFTY1
19.2 ± 0.0
31.4 ± 1.1
30.0 ± 0.8
KSR2
31.9 ± 0.7
40 ± 0
40 ± 0
ACTB
12.0 ± 0.9
13.6 ± 0.1
13.8 ± 0.3
SMOC2
24.1 ± 0.8
38.1 ± 2.6
32.6 ± 0.8
TSTD1
21.9 ± 0.2
33.5 ± 1.0
32.5 ± 0.0
ACTB
13.8 ± 0.2
13.7 ± 0.1
13.9 ± 0.3
ATCAY
22.9 ± 0.2
30.0 ± 0.6
32.1 ± 0.7
ACTB
15.0 ± 0.0
14.0 ± 0.3
14.3 ± 0.2
STRA6
28.0 ± 1.2
39.6 ± 0.5
37.7 ± 3.2
ACTB
15.2 ± 1.5
16.1 ± 0.5
15.7 ± 1.0
MYO7A
31.1 ± 0.8
38.7 ± 0.8
37.5 ± 0.9
RBFOX3
25.2 ± 0.8
37.9 ± 2.5
33.2 ± 1.2
ACTB
14.8 ± 0.8
16.1 ± 0.5
15.7 ± 1.0
A_21_P0000813
22.3 ± 0.5
28.2 ± 0.2
30.9 ± 0.4
A_21_P0000821
25.2 ± 0.8
35.5 ± 1.3
36.9 ± 4.3
A_21_P0014207
24.3 ± 0.4
38.5 ± 2.0
39.5 ± 0.5
A_19_P00319154
25.1 ± 0.6
39.5 ± 0.6
40 ± 0
ACTB
13.2 ± 0.1
13.6 ± 0.1
13.2 ± 0.1
2
7
10
13
17
18
19
23
24
25
9
Mean ± SD from 6 wells and Ct cut-off value is 40.
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Table S5. Primers for q-RT PCR analysis
Genes
Endogenous primers
Genes
Endogenous primers
hACTB
F: CAATGTGGCCGAGGACTTTG
hKSR2
F: CTTCGAGGAGATGAACCTGT
R: CATTCTCCTTAGAGAGAAGTGG
hGRPR
F: GGATCTCCCCGTGAACGATG
R: AAGCGGCCCTTTCCAATGA
hTSTD1
R: TGGAACGTTTCGCATGGACT
hCD24
F: CAGTAGTCTTGATGACCAAAGTCC
R: TTGCCCATCTGACAGAAGAAA
hSMOC2
R: TCACACACACAGTAGCTTCAA
hHLA-DOA
F: TAACCGGCTCTGGATGACTC
F: CTGTGTCAACCCCAAGACGA
hATCAY
F: GGAACGTTCATTCCCCGCTG
hSYTL1
F: ATTGACGTAAGCGGCTGTCT
hMYO7A
F: CCTTTGTGCAGCTTCGAGGA
hRBFOX3
F: ACCATTGTGCCAAATGCACC
A_21_P0000813
F: GCATGTGCCAAAGAAGAGGC
A_21_P0000821
F: CAGAAGGATTGTGCAGATTGT
R: TTTCACATAGCAGGCATTTG
hLEFTY1
F: GAACTCCCTGGACACTAGCAGAAT
R: CCCTGACACTGCTTTATTTCTAACC
A_21_P0014207
R: GGCCTGTGTTCTCACCATAGA
hPDLIM3
F: TGCTGGGCCACAAGTTCA
R: CGACAGTGTTGTCAGATTTTCTATTTAA
R: AGGGGTGGCCCTCATATTCT
hGPRC5B
F: CTTTGTCCTCTGTACAGAGC
R: TGAATGGTCACACCTTGG
R: TTTCTGCCCTGTTACTCCCAC
hWNK2
F: CATGAGTGATGGCAGTGAGAAG
R: TCAGAGTCAAATGCACCAGC
R: AAGGGTGGAAAGCAAAGGCA
hPRSS8
F: GAGTCAGCCTTCTTCCCTCACG
R: GGTCCAGTATGGGAGGACACC
R: GTCAGTGCACCAGGAAGAAGA
hSEMA6A
F: GGTTTCCCTCTGGTGACACAT
R: CCAAAGCTCATGGAGACGA
R: CTGCACCAGAACACAACGTG
hGPC3
F: CTGAGACATGCCTTGTAGAAT
R: TGTGGCTCCAAAGGCTTGAT
R: TGGCTGATGCCCTAACAGAC
hDPYSL5
F: TTGGAGAGTGCTCTGCAGAT
F: GGTTGTACTCTGGCCATTAATGTG
R: CGATTACCCAAGACCCCAGAA
A_19_P00319154
F: CAGCGTACAAACCTGCATAAAAGA
R: CACCCGGTTGAATCAAATCC
F: TGTGGAGATACTGTAACCTGAG
R: ATGCACAACCCACACTTAACC
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References
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in mouse embryonic fibroblasts. Sci. Rep. 2, e544(2012).
3. Han, L. et al. A synthetic small molecule for targeted transcriptional activation of germ cell genes
in a human somatic cell. Angew. Chem. Int. Ed., 52, 13410 –13413, (2013).
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