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, ThangavelVaijayanthib,Rhys D. Taylorband 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, βalanine; γ, γaminobutyric acid; SAHA, suberoylanilide hydroxamic acid.1H 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 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 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 10µM)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 µMof effectors and 48 h incubation was employed for all studies.

Figure S1

Figure S1.Number of genes affected with the treatment of SAHA, individual SAHA-PIPs 1-32in 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 twoindividually treated culture plates

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 1-32is provided. Each result represents the summary of analysis of data derived from two culture plates

Figure S3

Figure S3.Functional annotation of the SAHA-PIP a)1, b)7 and c)19 induced genes in human dermal fibroblasts. Dark graybar indicates a particular set ofbiological 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,

Figure S4

Figure S4.Network analysis of SAHA-PIP a)2, b)13,c)17, d) 18, e) 24 andf)25induced genes in human dermal fibroblasts corresponding tohematological 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,

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 1-32is provided. Each result is the summary of analysis of data derived from two individual culture plates.

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-PIPskilled 50% of the cells. Even at b) 10 μM concentration none of the SAHA-PIPs were cytotoxic. Each bar represents mean± SD from 9 wellplates.

SUPPLEMENTARY TABLES

Table S1. Numberof 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

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*Sum of data derived from two individual culture plates

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Table S2. Summary of common genes upregulated by more than 10-fold between individual effector-treated HDFs.

*Sum of data derived from two individual culture plates

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

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
2 / 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
7 / 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
10 / 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
13 / 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
17 / 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
18 / 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
19 / 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
23 / 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
24 / 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
25 / 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
9 / 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

Mean ± SD from 6 wellsand Ct cut-off value is 40.

Table S5. Primers for q-RT PCR analysis
Genes / Endogenous primers / Genes / Endogenous primers
hACTB / F: CAATGTGGCCGAGGACTTTG
R: CATTCTCCTTAGAGAGAAGTGG / hKSR2 / F: CTTCGAGGAGATGAACCTGT
R: AAGCGGCCCTTTCCAATGA
hGRPR / F: GGATCTCCCCGTGAACGATG
R: TGGAACGTTTCGCATGGACT / hTSTD1 / F: TTGGAGAGTGCTCTGCAGAT
R: TTGCCCATCTGACAGAAGAAA
hCD24 / F: CAGTAGTCTTGATGACCAAAGTCC
R: TCACACACACAGTAGCTTCAA / hSMOC2 / F: CTGAGACATGCCTTGTAGAAT
R: TGTGGCTCCAAAGGCTTGAT
hHLA-DOA / F: TAACCGGCTCTGGATGACTC
R: TGGCTGATGCCCTAACAGAC / hATCAY / F: GGTTTCCCTCTGGTGACACAT
R: CCAAAGCTCATGGAGACGA
hDPYSL5 / F: CTGTGTCAACCCCAAGACGA
R: CTGCACCAGAACACAACGTG / hSYTL1 / F:GAGTCAGCCTTCTTCCCTCACG
R: GGTCCAGTATGGGAGGACACC
hGPC3 / F: GGAACGTTCATTCCCCGCTG
R: GTCAGTGCACCAGGAAGAAGA / hMYO7A / F: CATGAGTGATGGCAGTGAGAAG
R: TCAGAGTCAAATGCACCAGC
hSEMA6A / F: ATTGACGTAAGCGGCTGTCT
R: AAGGGTGGAAAGCAAAGGCA / hRBFOX3 / F: CTTTGTCCTCTGTACAGAGC
R: TGAATGGTCACACCTTGG
hPRSS8 / F: CCTTTGTGCAGCTTCGAGGA
R: TTTCTGCCCTGTTACTCCCAC / A_21_P0000813 / F: TGCTGGGCCACAAGTTCA
R: CGACAGTGTTGTCAGATTTTCTATTTAA
hWNK2 / F: ACCATTGTGCCAAATGCACC
R: AGGGGTGGCCCTCATATTCT / A_21_P0000821 / F: GAACTCCCTGGACACTAGCAGAAT
R: CCCTGACACTGCTTTATTTCTAACC
hGPRC5B / F: GCATGTGCCAAAGAAGAGGC
R: GGCCTGTGTTCTCACCATAGA / A_21_P0014207 / F: GGTTGTACTCTGGCCATTAATGTG
R: CGATTACCCAAGACCCCAGAA
hPDLIM3 / F: CAGAAGGATTGTGCAGATTGT
R: TTTCACATAGCAGGCATTTG / A_19_P00319154 / F: CAGCGTACAAACCTGCATAAAAGA
R: CACCCGGTTGAATCAAATCC
hLEFTY1 / F: TGTGGAGATACTGTAACCTGAG
R: ATGCACAACCCACACTTAACC

References

1. Pandian, G. N. et al. Synthetic small molecules for epigenetic activation of pluripotency genes in mouse embryonic fibroblasts. ChemBioChem12, 2822-2828, (2011).

2. Pandian, G. N. et al. A synthetic small molecule for rapid induction of multiple pluripotency genes 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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