Supporting Information

Fang et al. 10.1073/pnas.1525619113

A qRT-PCR (SK-MEL-28) B 1 2 1.0 shRNA: shRNA: 0.8 NS MAFG-MAFG-2 NS BACH1-1BACH1- MAFG BACH1 0.6 * 0.4 * * * TUBA TUBA * * * * * * * * 0.2 * * Relative expression 0.0 1 T3B-2 2 shRNA: shRNA: NS CHD8-CHD8-2 shRNA: NS DNMT3B-1DNM CHD8-1CHD8-2 MAFG-1MAFG-2BACH1-1BACH1- DNMT3B-1DNMT3B-2 CHD8 DNMT3B

TUBA TUBA

Fig. S1. shRNA-mediated knockdown efficiency of MAFG, BACH1, CHD8, and DNMT3B in SK-MEL-28 cells. qRT-PCR (A)andimmunoblot(B) analysis monitoring shRNA-mediated knockdown efficiency for MAFG, BACH1, CHD8, and DNMT3B in SK-MEL-28 cells using two unrelated shRNAs against the same target gene. Data are represented as mean ± SD. *P ≤ 0.05; **P ≤ 0.01. NS, nonsilencing.

Fang et al. www.pnas.org/cgi/content/short/1525619113 1of9 A

Housekeeping gene Not silenced Silenced

GAPDH CIDEB AOX1 EFHD1 CACNA1G PRDM2 IRF8 SEPT9 CYP1B1

B qRT-PCR C MeDIP D qRT-PCR (SK-MEL-28) RKO RKO DMSO 30 SK-MEL-28 1.2 SK-MEL-28 1.6 5-aza * 25 * 1.0 1.4 * 1.2 20 0.8 1.0 15 0.6 0.8 0.6 10 methylation levels 0.4 * * * 0.4 5 0.2 * * * *

* * Relative expression Relative expression * 0.2 0 0 0.0 2 Relative 1 9 EB EB HD D CIDEB CID EFHD1 PRDM SEPT9 EF PRDM2 SEPT CI EFHD1PRDM2 SEPT9

Fig. S2. CIDEB, EFHD1, PRDM2, and SEPT9 are not hypermethylated and silenced in SK-MEL-28 cells. (A) Amplification plot showing Ct values for silenced genes, nonsilenced genes, and (as a control) a housekeeping gene in SK-MEL-28 cells. The results show that the Ct values for CIDEB, EFHD1, PRDM2,andSEPT9 + were 20–22, whereas the Ct values of the other derepressed genes were 28–32, suggesting the four genes were not silenced in SK-MEL-28 cells. ΔRn = Rn − Rn−, where Rn is the fluorescence emission intensity of the reporter dye divided by the fluorescence emission intensity of the passive reference dye (Rn+ is the value of the reaction containing all components and Rn− is the value of an unreacted sample). (B) qRT-PCR analysis monitoring expression of CIDEB, EFHD1, PRDM2, and SEPT9 in SK-MEL-28 cells relative to expression observed in RKO cells. The results in SK-MEL-28 cells were normalized to the results obtained in RKO cells, which were set to 1. (C) qRT-PCR analysis monitoring expression of CIDEB, EFHD1, PRDM2, and SEPT9 in SK-MEL-28 cells treated in the presence or absence of the DNA methyltransferase inhibitor 5-azacyticine (5-aza). The results show that 5-aza treatment had no effect on expression of the four genes in SK-MEL-28 cells. (D) Methylated DNA immunoprecipitation (MeDIP) analysis of CIDEB, EFHD1, PRDM2, and SEPT9 in RKO and SK-MEL-28 cells. Methylation was normalized to normalization obtained in RKO cells, which was set to 1. Data are represented as mean ± SD. *P ≤ 0.05; **P ≤ 0.01.

Fang et al. www.pnas.org/cgi/content/short/1525619113 2of9 qRT-PCR (SK-MEL-28) CIMP HIM 32 * * * ** ** * **** * shRNA: * * * ** * 16 * * * ** ** NS ** ** MAFG-2 * * * 8 CHD8-2 4 * * BACH1-2 * * DNMT3B-2 2

Relative expression 1 0.5 T IRF8 AOX1 QPC p14ARFCYP1B1 CACNA1G

Fig. S3. Validation of the results of Fig. 1 using a second, unrelated shRNA. qRT-PCR analysis monitored expression of representative CIMP and HIM genes in SK-MEL-28 cells expressing a second MAFG, CHD8, BACH1, or DNMT3B shRNA unrelated to the shRNA used in Figs. 1 A and B and 2A. Data are represented as mean ± SD. *P ≤ 0.05; **P ≤ 0.01.

InputIgG MAFGBACH1 abCHD8 abDNMT3B ab ab

MAFG

BACH1

CHD8

DNMT3B

Fig. S4. MAFG, BACH1, CHD8, and DNMT3B are stably associated in SK-MEL-28 cells. Coimmunoprecipitation analysis. SK-MEL-28 cell extracts were im- munoprecipitated with a MAFG, BACH1, CHD8, DNMT3B, or control (IgG) Ab, and the immunoprecipitate (IP) was analyzed for MAFG, BACH1, CHD8, or DNMT3B by immunoblotting.

Fang et al. www.pnas.org/cgi/content/short/1525619113 3of9 SK-MEL-5

AOX1 CACNA1G IRF8 p14ARF CYP1B1 QPCT -171 -97 -272 -215 -47 +56 -154 -10 -180 +19 -48 +178

shMAFG

shCHD8

shBACH1

shDNMT3B

A375

AOX1 CACNA1G IRF8 p14ARF CYP1B1 QPCT -171 -97 -272 -215 -47 +56 -154 -10 -180 +19 -48 +178

shMAFG

shCHD8

shBACH1

shDNMT3B

Fig. S5. Bisulfite sequencing analysis of representative CIMP and HIM genes in additional human melanoma cell lines. Bisulfite sequencing analysis of representative CIMP and HIM genes in SK-MEL-5 (Upper) and A375 (Lower) cells expressing a NS, MAFG, CHD8, BACH1, or DNMT3B shRNA. (Upper) Schematic of each promoter; positions of CpGs are shown to scale by vertical lines. Each circle represents a methylated (●) or unmethylated (○) CpG dinucleotide. Each row represents a single clone.

Fang et al. www.pnas.org/cgi/content/short/1525619113 4of9 AOX1 CACNA1G IRF8 p14ARF CYP1B1 QPCT -171 -97 -272 -215 -47 +56 -154 -10 -180 +19 -48 +178

Fig. S6. Bisulfite sequencing analysis of representative CIMP and HIM genes in snap-frozen human samples. Bisulfite sequencing analysis of representative CIMP and HIM genes in snap-frozen matched adjacent normal (N) and BRAF-positive human melanoma tumor (T) samples is shown. (Upper) Schematic of each promoter; positions of CpGs are shown to scale by vertical lines. (Lower) Each circle represents a methylated (●) or unmethylated (○) CpG dinucleotide. Each row represents a single clone.

Fang et al. www.pnas.org/cgi/content/short/1525619113 5of9 AOX1 CACNA1G IRF8 p14ARF CYP1B1 QPCT -171 -97 -272 -215 -47 +56 -154 -10 -180 +19 -48 +178

Fig. S7. Bisulfite sequencing analysis of representative CIMP and HIM genes in FFPE human samples. Bisulfite sequencing analysis of representative CIMP and HIM genes in FFPE matched adjacent N and BRAF-positive human melanoma T samples. (Upper) Schematic of each promoter; positions of CpGs are shown to scale by vertical lines. (Lower) Each circle represents a methylated (●) or unmethylated (○) CpG dinucleotide. Each row represents a single clone.

Fang et al. www.pnas.org/cgi/content/short/1525619113 6of9 Table S1. List of primers used for qRT-PCR, ChIP, bisulfite sequencing, and methylated DNA immunoprecipitation Gene Forward (5′ → 3′) Reverse (5′ → 3′)

qRT-PCR ABTB2 GCTGGTCAGTTTGTTGCTGA TGGCTGAAGCAGTTCATGTC ADAMTS1 GGATGGCTGATGTTGGAACT TAATTCATGGGCTGTGGTGA ALOX12B GGCAGAGGACACCTTCTTTG GTCTGTGACGGGGAACTTGT ALX4 AGAGAGCAACAAGGGCAAGA CACGTCTGGGTAGTGGGTCT AOX1 TCACTCACGGTGGAATTGAA CAGGTGGACATTCGACATTG BACH1 TGCGATGTCACCATCTTTGT CCTGGCCTACGATTCTTGAG BNIP3 TTCCTTCCATCTCTGCTGCT ATCAAAAGGTGCTGGTGGAG C20orf197 CCACTTCCCAAGTGTTGGTT ACGAATGCTGAGAAGCCAGT CACNA1G AAGCAGACAGTGGAGCCTGT TCTGAGTCAGGCATTTCACG CDO1 GTACGCCAAGTTCGACCAGT GTCCTTCACCCCAACAGAGA CHD8 GAGCCTATCCTCCCTGAACCA AGAACTGCATGGAAGGCAAAG CHFR CCTCTGTGGCAAGTGATGAA TCCAAATCCTCCTGATCCTG CIDEB GGAACTGCAGTGGACAGTGA CACTCCTTGTAGGGCTCCAG CLDN23 TTGCCATGCAAACTCTCAAG CCATTAAGCTGCTGGCATTT COL1A2 GTGGCCCAGAAGAACTGGTA CGCCATACTCGAACTGGAAT COL4A2 AAGGAATCATGGGCTTTCCT CTCTGGCACCTTTTGCTAGG CRABP1 GCAAGTGCAGGAGTTTAGCC CACGGGTCCAGTAGGTTTTG CRHR1 CTCCTGGGCATCACCTACAT GGAAGGATTCCAGGAAGGAG CYP1B1 TGATGGACGCCTTTATCCTC GTGATAGTGGCCGGTACGTT DAPK1 AATGGAGTTGGCGATTTCAG ATGGATTGACGTGGGATCAT DDIT4L GAAACAGAGCCGTTGACCAT CAGGCTCTCTGGGTGATAGC DFNA5 GAATGAGGTCCTGTGCGTTT GATGCCACCACACTTCTCCT DNMT3B CAGGGAAAACTGCAAAGCTC TGTCTGAATTCCCGTTCTCC DUSP26 CGGTTTCTGCCACCTCTAAG CAGGCTGTCTTGCCTGTGTA EDIL3 TTGGCTGATGGTTCCTTTTC ATGGCATGGATTAGGAGTGC EFEMP1 CAGGGACGCACAACTGTAGA ATTGAAACCCAGGACTGCAC EFHD1 GATGGCTTCATCGACCTGAT GTCCTCATCCACCTCCTTGA ELMO1 CTGCTCAGCATGGAAATCAA TCATAGTTGCTGGGCTCCTT EPHB1 CCTCCCTAATGTCCCAGGAT CCTCAGACCAGAAGGCTGAC FBN2 TCCTGGATATCAGGCTACGC TGAATTTGTGCACTGGGTGT FGF4 CTACAAGTACCCCGGCATGT GAGGAAGTGGGTGACCTTCA GDF15 GAGCTGGGAAGATTCGAACA AGAGATACGCAGGTGCAGGT GJB5 GGACTGCTTCATCTCCAAGC AGATGAGCTCCACGAGGTTG GRHL2 ATGGCTACCTCACAGCCTTG TAAGTGCTCTTGCCCCAACT GRM4 GAGGGCAGCTATGGTGAGAG CCGTGGTATCTTCACCGACT HAND1 GTCCGCAGAAGGGTTAAACA GGCAAGGCTGAAAATGAGAC HLTF CAGTGGAGGGGTCAAAGAAA TGTCCAAACTGGTCAATCCA HSPB6 CCTCCTCAGGCTCCCTCTAT GAATCCAGGAGTGGGTGAGA ID4 GGGTGGGCTACTTTTCTTCC GTCGCTCTGGGTTTTACGAG IGFBP3 AGGGCACTCTGGGAACCTAT TGCAGTCATCCGAAGAATTG IGFBP7 GGCATGGAGTGCGTGAAGAG CTTGCTGACCTGGGTGATGG IGLL1 CTCCAAGCCAACAAGGCTAC TACCATCTGCCTTCCAGGTC IRF8 AGTGGCTGATCGAGCAGATT AGTGGCTGGTTCAGCTTTGT LOX ATATTCCTGGGAATGGCACA CCAGGACTCAATCCCTGTGT LRP2 AAACAATGGTGGGTGCTCTC TTCTTGCCATCACTTTGCAG LXN TGAAGGAGCTTGCGACTTTT GCTTGGGCTACTCTGGCTTA MAFG TGTGTTTTGTATGTTGGGATTGG CATGGCCCTGGTACAAAAGG MLH1 CAGAGGAAGATGGTCCCAAA CAGGTTCCCTTCCTCATCAA MT1G CCCTGCTCCCAAGTACAAAT GGAATGTAGCAAAGGGGTCA NDNF CGCTCCCTGCAGTTTAAAAG AAGTTGCTGCGAAGTGGAGT NEUROG1 GTTACTTTCCCCCTCCCCTA CTTTAAAGCTCCCGCTTCCT NPM2 TGCAGGCTGTTGCTTCATAC TCTTGTCCTCCTGGTTTGCT OVOL1 CAGGGCTTCTAATGCTCAGG AGTGCACACACACAAGCACA p14ARF CCCTCGTGCTGATGCTACTG ACCTGGTCTTCTAGGAAGCGG p16INK4A CACATTCATGTGGGCATTTC CCCCTGAGCTTCCCTAGTTC PCDHAC2 ACCTAGAGGAGGCTGGCATT GACACTTCTCCTGCCTCTGG PCSK1 CTTGCTGGGCAGTATCCAAT TGCGGGTAGTTTGTTTTTCC PENK AAGCCAAAGAGCTGCAGAAG TTCAGGAAACCTCCATACCG PPP1R3C TTGCAAGAGCGAACAGTGAC TGCTCAGTTGGAATGACAGG PPP1R14A CTGGACGTGGAGAAGTGGAT AGCAGCTCCTGGATGAAGTC PRDM2 AGGTGCCTCCAGAACTAGCA CCCCAAATCATTCACCTCAC

Fang et al. www.pnas.org/cgi/content/short/1525619113 7of9 Table S1. Cont. Gene Forward (5′ → 3′) Reverse (5′ → 3′) QPCT AAGGGTGGAGAAGAGGGAAG GCCATCTCTCCGAGTCTGTC RASSF2 GGTCTTCCTGCACTTGAAGC GCATCTCCACACACAAGGTG SEPT9 CATCACGCACGATATTGAGG CCAGCAGTTCTCGTTGTTGA SFRP1 AAGGGAGGCTCTCTGTAGGC AATGACCAGGCCAATCAGTC SFRP2 GGGTCTGGTTGGTTGTTGTT GGGCCACAGAGAAAATTGAA SLC6A11 AGCTGGGTAGTGGTTCATGC ATCTCGGCTCACTGCAACTT SLC6A18 TGTTCCCTTACCTGGTCCTG ATGTGCATGTTGGGAGTGAA SLC18A2 GTCCCCATCATCCCAAGTTA TGTCTGAGATGGAGGCAGTG SLC30A10 ATCCACAATGTGACCATCCA CTTGGAGATGCAGGGTGAGT SOCS1 CTCCTTCCCCTTCCAGATTT CACATGGTTCCAGGCAAGTA SPON1 CACATTTGATGGGGTGACTG TGTCTTCTCGGACCAATTCC STOX2 GGCGAACTCAACTCTTGTCC TTCTTTCCCAGAGGTGATGG THBD CGGGTTGTGTGTCTGTTCAC CCTCCATGCATCTCATAGCA THBS1 ACCAAAGCCTGCAAGAAAGA TCTGTACCCCTCCTCCACAG THBS2 AAGTGTGTGAGCCCGAAAAC GTACATGGGGTCGCTGAAGT TLE4 GTTTCCGAGGTGCTGAGAAG GCTGTCATAACGAGCTGCAA TMEFF2 CAATGGGGAGAGCTACCAGA TCTGTGGCACATGATCCTTC TOLLIP CGGTGGTACAGAGAGCCTTC ACCACTTGTCCTCCACCTTG TP73-AS1 GGGTAACTCCCCACTGTTGA GGCTGAGCTGGACAAAAGAC TRIM40 TGTGCAGGTTCTGTGAGGAG AGGGCATTTTCAATGGTCAG TSPYL5 TCCCACTTTCAGCAGTCCTT CCCAGGAGAAGCTTGAGATG TUB GAGGGAGAGGAAGCACACTG ATTCACTGTGCCCCTACCTG UCHL1 AGCGTGAGCAAGGAGAAGTC TTGAAGGGAAGAGGGGAAAT VIM GGCCCAGCTGTAAGTTGGTA CCTAGCGGTTTAGGGGAAAC WNT10B GTCACTCTTGGTCCCTGGAA GTTGGGGAGAAGGCTACACA ZSCAN18 GCTTTCCTGCAGCCATTTAG TTGAAGAAAGCACTGGCAGA ChIP AOX1 ATCCTGGCTGTGGGTAACTG TATCGCTAGCGCATTCTCCT CACNA1G GTCTGGGCAGCAGTCTGATT GGAGAGAACCACAGCTGGAA CYP1B1 CTGCGACTCCAGTTGTGAGA GCAAAGTCGAGGTTTCCTCA IRF8 AATATCCAGCGCTCGTGAAG GGCCCATTAATCAGAATCCA P14ARF GTGGGTCCCAGTCTGCAGTTA CCTTTGGCACCAGAGGTGAG QPCT AAGGGTGGAGAAGAGGGAAG GCCATCTCTCCGAGTCTGTC Bisulfite sequencing CYP1B1 GTTTTTATGAAAGTTTGTTGGTAGAG CCCACTCCCACTCCAAAATC QPCT GGGGTAGGAGGGTTGTAGTTTG ACCAACAACAACAAATAAAAAATACC Methylated DNA immunoprecipitation CIDEB (−460 to −559) CTGGAAAATGAAGCCAGAGC TAGGGTCCTGTCTGGGACAC EFHD1 (−616 to −720) CCACTATGCCTGGCTGATTT GTGTTGGTGGCAGGATCTTT PRDM2 (321–428) AGGTCACTTGTCCCCTGTTG TCCAAATTGCCTGCCTATTC SEPT9 (−729 to −817) CGTTGGAAACACCGCTTTAT CAGTGGTGAGAGGATGCAAA

Fang et al. www.pnas.org/cgi/content/short/1525619113 8of9 Table S2. List of melanoma tumor samples used in this study Coding Tumor Melanoma Melanoma Mutated sequence Codon sample Diagnosis type Metastatic? Tissue location gene change change

T1 Malignant Nodular Yes Lymph node Right anterior BRAF c.1799T > TA 600V > E melanoma medial thigh T2 Malignant Nodular Yes Skin NA BRAF c.1799T > TA 600V > E melanoma T3 Malignant Nodular Yes Skin Right posterior BRAF c.1799T > TA 600V > E melanoma shoulder T4 Malignant NA Yes Skin Left brain BRAF c.1799T > TA 600V > E melanoma T5 Malignant Nodular Yes Skin Left groin lymph BRAF c.1799T > TA 600V > E melanoma T6 Malignant Nodular Yes Skin Left groin lymph BRAF c.1799T > TA 600V > E melanoma T7 Malignant NA Yes Brain NA BRAF c.1799T > TA 600V > E melanoma T8 Malignant Nodular Yes Skin Right calf BRAF c.1799T > TA 600V > E melanoma T9 Malignant Nodular No Skin Right posterior BRAF c.1799T > TA 600V > E melanoma shoulder T10 Malignant Nodular Yes Skin Right flank BRAF c.1799T > TA 600V > E melanoma T11 Malignant Superficial No Skin Left vulva BRAF c.1799T > TA 600V > E melanoma spreading T12 Malignant Superficial No Skin Left preauricular BRAF c.1799T > TA 600V > E melanoma spreading cheek T13 Malignant NA Yes Right chest Right chest BRAF c.1799T > TA 600V > E melanoma wall/flank wall/flank mass T14 Malignant Nodular Yes Lymph node Left axillary lymph BRAF c.1799T > TA 600V > E melanoma node

NA, not available.

Fang et al. www.pnas.org/cgi/content/short/1525619113 9of9