Supplementary Information

Promoter hypomethylation of EpCAM-regulated bone morphogenetic protein genes in advanced endometrial cancer

Ya-Ting Hsu, Fei Gu, Yi-Wen Huang, Joseph Liu, Jianhua Ruan, Rui-Lan Huang,

Chiou-Miin Wang, Chun-Liang Chen, Rohit R. Jadhav, Hung-Cheng Lai, David G.

Mutch, Paul J. Goodfellow, Ian M. Thompson, Nameer B. Kirma, and Tim Hui-Ming

Huang Tables of contents

Page Table of contents 2

Supplementary Methods 4

Supplementary Figure S1. Summarized sequencing reads and coverage of MBDCap-seq 8

Supplementary Figure S2. Reproducibility test of MBDCap-seq 10

Supplementary Figure S3. Validation of MBDCap-seq by MassARRAY analysis 11

Supplementary Figure S4. Distribution of differentially methylated regions (DMRs) in endometrial tumors relative to normal control 12

Supplementary Figure S5. Network analysis of differential methylation loci by using Steiner-tree analysis 13 Supplementary Figure S6. DNA methylation distribution in early and late stage of the

TCGA endometrial cancer cohort 14

Supplementary Figure S7. Relative expression of BMP genes with EGF treatment in the presence or absence of PI3K/AKT and Raf (MAPK) inhibitors in endometrial cancer cells 15

Supplementary Figure S8. Induction of invasion by EGF in AN3CA and HEC1A cell lines 16

Supplementary Figure S9. Knockdown expression of BMP4 and BMP7 in RL95-2 cells 17

Supplementary Figure S10. Relative expression of BMPs and BMPRs in normal endometrial cell and endometrial cancer cell lines 18

Supplementary Figure S11. Microfluidics-based PCR analysis of EMT gene panel in

RL95-2 cells with or without EGF treatment 19

Supplementary Figure S12. Knockdown expression of EpCAM by different shRNA sequences in RL95-2 cells 20

Supplementary Figure S13. Proposed model for EpICD mediated regulation of BMP genes by EGF 21

2

Supplementary Table 1. Characteristics of endometrial cancer patients 22

Supplemental Table 2. Summary of sequencing reads in endometrial tumors an normal controls 23

Supplementary Table 3. Differentially methylated promoter CpG islands in recurrent relative to non-recurrent tumors 26

Supplementary Table 4. List of primer sequences 55

Supplementary Table 5. List of siRNA and shRNA sequences 59

Supplementary References 60

3

Supplementary Methods

MassARRAY analysis

To quantify methylation levels of CpG islands in clinical samples, the high-throughput

MassARRAY platform (Sequenom) was carried out as described previously (Huang et al. 2009).

Briefly, DNA was bisulfite-converted and subjected to PCR and base-specific cleavage. The cleaved DNA was analyzed by MALDI-TOF mass spectrometry. Methylation data of HAAO,

RXFP3, and miR-129-2 CpG islands were published previously (Huang et al. 2009, 2010).

Definition of methylation types

All known transcripts were divided into 1) promoter CpG island located within 2-kb up- or downstream of its TSS and 2) promoter non-CpG island located within 2-kb windows of its TSS.

Other regions were defined as intragenic CpG island located after 2-kb downstream of a TSS and before 3’-UTR and intergenic CpG island located before >2-kb upstream of a TSS or after

3’-UTR.

Bioinformatics analysis

Sequence reads mapped to unique genome locations were normalized to the sum of unique reads and other reads (e.g., repeat sequences). Unique reads were collected after normalization and directionally extended to 200-bp. The methylation level of each bin size was calculated by accumulating the reads number if the read was located in the bin. Linear normalization method based on the unique mapped read number was used to normalize methylation enrichment:

, , /^

4

th Where , is the normalized read number of the i bin, and , is the uniquely mapped

th read number of the i bin, is the total uniquely mapped reads number. “INT” function rounds the element (in the parenthesis) to the nearest integers towards minus infinity, “^” means the power operator. For two groups A and B, the sample number is for group A, and for group

B. For a given region R (which includes m bin size, and start at the sth bin), the average methylation level is

∑ , , , ,,…,

for group A or B (if is A or B). In equation 2, , means the average methylation level of group at region , , is the methylation levels of each sample of group at region . We then used paired t-test to compare if the methylation level of the region was significantly different between those two groups. Differentially methylated loci between endometrial carcinoma samples and normal controls and between recurrent and non-recurrent tumor groups were analyzed by the above algorithm.

Network analysis of differential methylation loci by using Steiner-tree analysis

Differentially methylated loci were mapped to a human protein-protein interaction (PPI) network (Wu G, Feng X, & Stein L. 2010) to identify a small number of connector genes that can link these loci into a singly connected sub-network. These connector genes were identified with the well-known Steiner-tree algorithm (Huang SS & Fraenkel E. 2009). Formally, the Steiner- tree for an edge-weighted graph G = (V, E, W) and a subset of vertices S ⊆V is a minimum- weight connected tree T, with vertices U⊆V and edges D⊆E that spans all vertices in S. Here the vertices in S are known as terminal vertices and UnS as Steiner vertices. For an un- weighted graph G, the problem then becomes to find the minimum number of vertices that can connect all the vertices in R through a tree in G. The Steiner tree problem is NP-hard and

5 therefore a polynomial-time 2-approximation shortest path heuristic algorithm of Steiner-tree problem was used (Huang SS & Fraenkel E. 2009). To de-noise and to increase the probability of covering all core members in the pathways and alternative functional links between pathways, a simple randomized algorithm was designed to obtain multiple Steiner trees with suboptimal quality (i.e., slightly more connector genes) (Huang SS & Fraenkel E. 2009). This procedure was repeated multiple times with different random weights, until the total number of unique

Steiner vertices converges approximately. In this work, the rate of new coming Steiner vertices reduced significantly after 10 iterations. The Steiner vertices in the 10 Steiner trees were pooled to obtain connector genes, and a sub-network containing connectors and differentially methylated loci was extracted from the PPI network. The connectors were further analyzed for enriched KEGG pathways using the online functional annotation tool DAVID (Huang de W,

Sherman BT, Lempicki RA. 2009).

DNA methylation distribution and survival analysis of BMP genes in the TCGA cohort

All TCGA cohort array and clinical data were downloaded from the TCGA website

(http://www.cancergenome.nih.gov/) and the TCGA endometrial data snapshot website

(http://23.23.224.116/ucec_tcga/). To compare DNA methylation levels in recurrent/progression group with non-recurrent group, we filtered patients who have clinical information provided with recurrent/progression status, survival and DNA methylation data as the final cohort for analysis.

In this cohort, 39 patients showed primary tumors with recurrence/progression and 193 patients were non-recurrent. Recurrent rate is 16 % (39/232=0.16). We further filtered the DNA methylation data by only looking at probes located in the 2-kb region around transcription start site (TSS) with CpG island annotation or differential methylation regions identified in BMP2,

BMP3, BMP4 and BMP7 in our own cohort. For progression free survival analysis, 15 percentile was used as the cutoff to dichotomize the DNA methylation level into high (n=197) and low methylation (n=35) in each of BMPs probe. The low methylation of BMP4 and BMP7

6 showed a significant association with poor survival (Both of P < 0.01) by Kaplan–Meier curve analysis. For the methylation distribution in disease stage, stage I and stage II were combined as early stage (n=153) and stage III and IV were designated as late stage (n=33). The methylation distributions of BMP genes were illustrated as representative figures showing one probe, corresponding to the same CpG site shown in Fig. 3A. lower panel.

PI3K/AKT and Raf (MAPK) inhibitors treatment

RL95-2 cells were grown in DMEM culture medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (Life

Technologies) at 37oC. One day before the treatment, media was changed to DMEM supplemented with 10% charcoal stripped-heat inactivated (CSHI) FBS for treatments.

PI3K/AKT inhibitor LY 294002 (4 uM, EMD Chemicals, Germany) and Raf (MAPK) inhibitor

PLX-4720 (2uM, Selleck Chemicals, Houston, TX) were added with or without EGF (10 ng/mL,

Peprotech, Rocky Hill, NJ) for 72 hours. RNA was then isolated using Direct-zolTM RNA

MiniPrep according to the manufacturer’s manual (Zymo Research, Irvine, CA).

7

B

Promoter region Whole genome (2-kb centered on TSS) (1-kb sliding window) (n=21,853) (n=3,022,656)

No 1 No 1 coverage 2-4 coverage 2-4

≥50 5-9 ≥50 Endometrial normal 5-9 25-49 25-49

10-24 10-24 No coverage 1 No 1 2-4 2-4 coverage

≥50 ≥50 Endometrial tumor 5-9 5-9 25-49 25-49 10-24 10-24

Supplementary Figure S1. Summarized sequencing reads coverage of MBDCap-seq. (A) Summarized sequencing reads in normal samples (n=10) and endometrial tumors (n=67). Each bar represents an individual sample. Blue and red bars indicate unique reads and repeat sequences, respectively. (B) Genomic coverage was quantified by the number of DNA methylation measurements that overlap with gene promoters and a 1-kb tiling of the genome.

8

Each read was extended to the mean length obtained during size selection. The number of measurements was equal to the number of unique sequencing reads that fall inside each region. Promoter regions were based on the human reference sequence (hg18; NCBI Build 36.1) and covered 1-kb upstream and 1-kb downstream of the annotated transcription start site. The genomic tiling was obtained by sliding 1-kb windows through the whole genome. Data were shown for the average coverage of tumors or normal controls.

9

A B Sample ID Pearson R Sample ID: 31 Sample ID: 47 Pearson R=0.8281 Pearson R=0.8445 2 0.8289 P<0.0001 P<0.0001 3 0.6438 5 0.7612 28 0.7641 46 0.6691 51 0.7586 73 0.4846 Methylation (experiment 2) 84 0.8073 85 0.6495 Methylation (experiment 1)

C D Sample ID: 17 Sample ID: 41 Pearson R=0.9705 Pearson R=0.9679 Sample ID Pearson R P<0.0001 P<0.0001 1 0.9335 10 0.9131 11 0.9582 16 0.9734 24 0.9662

Methylation (lane 2) 33 0.9630 80 0.9761

Methylation (lane 1)

Supplementary Figure S2. Reproducibility test of MBDCap-seq. The average of DNA methylation was calculated within an 8-kb window of each promoter CpG island region, which was defined as described in the Supplemental Methods. (A) Comparison of MBDCap-seq on biological replicates of endometrial tumors to examine reproducibility. Scatter plots and Pearson’s correlation coefficients based on genome-wide methylation proportions are shown for two samples. Two experiments were at least 6 months apart. (B) Pearson’s correlation coefficient for other 9 biological replicates. (C) Scatter plots and Pearson’s correlation coefficients of technical reproducibility test from two individual lanes for each samples. (D) Pearson’s correlation coefficient for other 7 samples with technical replicates.

10

HAAO miR-129-2 Spearman R=0.7475 Spearman R=0.5901 P<0.0001 P<0.0001 MBDCap-seq

MassARRAY MassARRAY

RXFP3 SOX11 Spearman R=0.7919 Spearman R=0.4282 P<0.0001 P=0.0002 MBDCap-seq

MassARRAY MassARRAY

Supplementary Figure S3. Validation of MBDCap-seq by MassARRAY analysis. DNA methylation levels were measured by either MBDCap-seq or MassARRAY within the same region for each locus. Scatter plots are shown Spearman correlation coefficient for each locus. Methylation levels by MassARRAY for HAAO, RXFP3, and miR-129-2 CpG islands were published previously (Supplemental Methods).

11

A B C D

Supplementary Figure S4. Distribution of differentially methylated regions (DMRs) in endometrial tumors. DMRs were identified by comparing the average methylation level of tumors to that of normal controls. DMRs were found in regions within (A) promoter CpG island (B) intragenic and (C) intergenic CpG islands, and (D) non-CpG island promoters. The definition of each category and methylation types were described in the Supplemental Methods.

12

EPHA5

CDH8

EFNA1 CDH4

SUMO2 LEF1 DM genes CDH2 SMAD2 Connectors EPHB1 EP300 SRC TJP1 CTNNB1 GRB10 NCOA3 ZFYVE9 EGFR pathway CLDN7 FYN EGFR BMPR1B EPCAM GRB2 SMAD3 MAPK NCK1 BMP4 CRK Wnt PDGFA ENG TGFB1 APPL1 Gap Junction FN1 BMP7

EPHB3 BMP2

ADIPOR2 BMP3 COL2A1 ADIPOQ

CDH13

Supplementary Figure S5. Network analysis of differential methylation loci by using Steiner- tree analysis showing protein-protein network connecting BMP members to EGFR, MAPK, Wnt, Gap junction signaling pathways. DM: differentially methylated loci.

13

BMP2 BMP3 1.0 1.0 * 0.8 0.8

0.6 0.6

0.4 0.4

Methylation level 0.2 Methylation level 0.2

0.0 0.0 I + II III + IV I + II III + IV

BMP4 BMP7 1.0 0.8

0.8 0.6 0.6 0.4 0.4 0.2

Methylation level 0.2 Methylation level

0.0 0.0 I + II III + IV I + II III + IV

Supplementary Figure S6. DNA methylation distribution in early and late stage of the TCGA endometrial cancer cohort. Group I+II (n=153) represent early stage patients diagnosed with either stages I and II; group III + IV (n=33) represent late stage patients diagnosed with stages III and IV. Methylation distribution of each BMP genes is illustrated by a representative probe (shown in Fig. 3A lower panel) corresponding to a CpG site specifically located near the TSS. * if P<0.05.

14

A RL95-2 AN3CA HEC1A Fo ** 8 *** Control 2.0 2.0 ld EGF ** Ch 6 1.5 1.5 an 4 1.0 1.0 ge *** * 2 0.5 0.5 0 0 0 BMP3 BMP2 BMP7 BMP4 BMP3 BMP7 BMP4 BMP2 BMP3 BMP2 BMP4 BMP7

B

BMP2 BMP3 BMP4 BMP7

8

6

4

2 Fold Change

0 − − + + − − + + − − + + − − + + EGF (10ng/mL) − + − + − + − + − + − + − + − + LY 294002 (4uM)

BMP2 BMP3 BMP4 BMP7

8

6

4

Fold Change 2

0 − − + + − − + + − − + + − − + + EGF (10ng/mL) − + − + − + − + − + − + − + − + PLX-4720 (2uM)

Supplementary Figure S7. Relative expression of BMP genes with EGF treatment in the presence or absence of PI3K/AKT and Raf (MAPK) inhibitors in endometrial cancer cells. (A) The relative expression of BMP genes with EGF treatment (10 ng/mL) for 72 hours in RL95-2, AN3CA, and HEC1A cell lines. Cells were incubated for 24 hours in growth media supplemented with 10% CSHI FBS prior to EGF treatment. (B) The relative expression of BMP

15 genes with EGF treatment (10 ng/mL) in the absence or presence of either PI3K/AKT inhibitor (LY 294002, 4 μM), upper panel, or Raf inhibitor (PLX-4720, 2 μM), lower panel. Cells were incubated for 24 hours in growth media supplemented with 10% CSHI FBS prior to treatment. EGF-induced expression of BMP3 was most responsive, and to a lesser degree BMP7, to inhibition by PI3K/AKT and Raf/MAPK inhibitors, suggesting that both these EGF intracellular pathways contribute to the expression of these BMPs. On the other hand, BMP2 was refractory to inhibition by the PI3K/AKT and Raf inhibitors, suggesting that BMP2 regulation by EGF is independent of PI3K and MAPK pathways. As described in the main text, BMP4 is not responsive to induction by EGF. * if P<0.05; **if P<0.01; *** if P<0.00.

AN3CA HEC1A

In In 350 ** 100 va 300 va 80 sio 250 sio n 200 n 60 150 40 100 50 20 0 0 Control EGF Control EGF

Supplementary Figure S8. Induction of invasion by EGF in AN3CA and HEC1A cell lines. Cells were incubate for 24 hours in growth media supplemented with 10% CSHI FBS prior to EGF (10 ng/ml) treatment. EGF treatment was carried out for 72 hours before invasion assay. Details of invasion assay are described in the Materials and Methods. While a significant increase in the invasion of AN3CA cells was observed, a slight non-significant increase was observed in HEC1A cells. **if P<0.01.

16

A

B

Supplementary Figure S9. (A) Knockdown (KD) expression of BMP4 and BMP7 in RL95-2 cells transfected with BMP4 and BMP7 siRNA singly or in combination. RL95-2 cells were transfected with either BMP4 or BMP7 siRNA (single knockdown, KD) or both (double knockdown, KD) in the absence (control) or presence of EGF. Expression of BMP4 and BMP7 is shown in bar graphs. (B) Knockdown of BMP4 and BMP7 led to decreased invasion in RL95-

17

2 cells through matrigel. Invasion assay was carried out as described in Materials and Methods. * if P<0.05; **if P<0.01; *** if P<0.001.

BMPR1A BMPR1B BMPR2 10 10 10

8 8 8

6 6 6

4 4 4 Fold Change Fold Change Fold 2 Fold Change 2 2

0 0 0 RL95-2 HEC1A RL95-2 RL95-2 HEC1A HEC1A AN3CA AN3CA AN3CA Normal endometrial Normal Normal endometrial Normal endometrial Normal

BMP2 BMP3 BMP4 BMP7

2.0 30 2.0 30

1.5 1.5 20 20 1.0 1.0

10 10

Fold Change 0.5 Fold Change 0.5 Fold Change Fold Fold ChangeFold

0.0 0 0.0 0 RL95-2 RL95-2 RL95-2 HEC1A HEC1A HEC1A AN3CA AN3CA AN3CA RL95-2 HEC1A AN3CA Normal endometrial Normal endometrial Normal endometrial Normal Normal endometrial

Supplemental Figure S10. Relative expression of BMPs and BMPRs in normal endometrial cell and endometrial cancer cell lines. Expression of BMPRs and BMPs were normalized by using normal endometrial cells as control.

18

Supplementary Figure S11. Microfluidics-based PCR analysis of EMT gene panel in RL95-2 cells with or without EGF treatment. Cells were incubated 24 hours in growth media supplemented with 10% CSHI FBS prior to EGF (10 ng/ml) treatment. Treatment was carried out for 72 hours. RT-PCR from RNA was performed in at least quadruplicates. The Biomark microfluidic system is described in Materials and Methods. EGF-induced and –suppressed genes are shown in the top and lower panels, respectively.

19

1.2

1.0

0.8

0.6

Fold Change Fold 0.4

0.2

0.0 l 1 # #2 #4 #5 tro n Mock co

Scrambled Supplementary Figure S12. Knockdown expression of EpCAM by different shRNA sequences in RL95-2 cells. Details of knockdown assay was carried out as described in Materials and Methods.

20

A B

Supplementary Figure S13. Proposed model for EpICD mediated regulation of BMP genes by EGF. (A) In tumors that subsequently recur, EGF stimulation leads to release of the EpCAM intracellular domain (EpICD). EpICD forms a transcriptional complex that translocates into the nucleus. Binding of this complex to responsive BMP loci occludes DNA methylating enzymes (DNMT) and epigenetic repressors from target promoter sites. As a result, CpG sites are left unmethylated (designated by open lollipops) and the chromatin in an active conformation, leading to transcriptional activation. (B) In non-recurrent tumors, EpCAM signaling via EpICD is not initiated. Susceptible BMP loci are engaged by DNMT and epigenetic repressors, leading to methylation of CpG sites (designated by filled ‘lollipops’) and silencing of gene expression.

21

Supplementary Table S1 Characteristics of endometrial cancer patients Characteristic Normal Recurrent Non-recurrent Age <50 6 0 2 50~59.9 2 4 19 60~69.9 2 5 15 70~79.9 0 5 11 80 and older 0 3 3 FIGO stage 1 N/A 11 46 2 N/A 4 2 3 N/A 2 2 4 N/A 0 0 Unknown N/A 0 0 Tumor grade 1 N/A 7 26 2 N/A 6 24 3 N/A 4 0 Unknown N/A 0 0 Events Alive N/A 3 39 Death N/A 14 11 Unknown N/A 0 0 Total 10 17 50

Abbreviations: FIGO, International Federation of Gynecology and Obstetrics

22

Supplementary Table S2. Summary of sequencing reads in endometrial tumors and normal controls

Number of Number of Number of Number of Unique read reads reads reads reads Subtype ID Alignment rate rate in total (total) (aligned) (unique) (multiple) reads

Non-recurrent 1 84,415,067 71,204,114 84.40% 37,731,765 33,472,349 44.70% Non-recurrent 6 75,774,571 66,586,753 87.90% 37,682,238 28,904,515 49.70% Non-recurrent 8 61,251,766 55,867,151 91.20% 34,721,044 21,146,107 56.70% Non-recurrent 9 78,689,460 67,540,901 85.80% 39,564,825 27,976,076 50.30% Non-recurrent 10 82,481,944 72,553,143 88.00% 47,082,686 25,470,457 57.10% Non-recurrent 11 91,363,017 75,493,139 82.60% 44,604,646 30,888,493 48.80% Non-recurrent 12 66,330,555 59,567,393 89.80% 35,867,043 23,700,350 54.10% Non-recurrent 13 71,589,749 62,817,240 87.70% 34,618,180 28,199,060 48.40% Non-recurrent 14 69,544,446 61,437,697 88.30% 32,696,327 28,741,370 47.00% Non-recurrent 15 77,729,214 67,959,897 87.40% 36,061,658 31,898,239 46.40% Non-recurrent 16 82,491,794 67,390,529 81.70% 38,355,527 29,035,002 46.50% Non-recurrent 17 80,951,915 68,462,251 84.60% 35,149,849 33,312,402 43.40% Non-recurrent 18 80,766,747 68,646,494 85.00% 39,064,175 29,582,319 48.40% Non-recurrent 19 70,018,435 60,472,975 86.40% 32,135,501 28,337,474 45.90% Non-recurrent 20 71,616,013 64,012,163 89.40% 36,793,544 27,218,619 51.40% Non-recurrent 21 65,250,652 59,185,366 90.70% 37,212,453 21,972,913 57.00% Non-recurrent 22 76,502,328 66,842,017 87.40% 37,167,298 29,674,719 48.60% Non-recurrent 23 75,048,842 64,961,440 86.60% 34,919,063 30,042,377 46.50% Non-recurrent 24 88,112,284 72,978,057 82.80% 40,936,958 32,041,099 46.50% Non-recurrent 25 62,045,863 55,081,865 88.80% 28,160,759 26,921,106 45.40% Non-recurrent 26 66,797,928 58,395,254 87.40% 29,770,980 28,624,274 44.60% Non-recurrent 29 71,829,061 63,041,480 87.80% 37,060,931 25,980,549 51.60% Non-recurrent 30 76,454,519 66,228,516 86.60% 37,684,383 28,544,133 49.30% Non-recurrent 32 78,260,084 69,112,915 88.30% 40,110,551 29,002,364 51.30% Non-recurrent 33 83,963,315 69,502,685 82.80% 39,405,526 30,097,159 46.90% Non-recurrent 34 39,749,694 36,314,207 91.40% 21,212,397 15,101,810 53.40% Non-recurrent 38 65,110,070 57,536,498 88.40% 32,234,641 25,301,857 49.50% Non-recurrent 39 76,694,942 66,788,154 87.10% 37,443,317 29,344,837 48.80% Non-recurrent 40 66,694,121 59,143,083 88.70% 33,455,763 25,687,320 50.20% Non-recurrent 41 84,167,228 71,321,870 84.70% 39,940,234 31,381,636 47.50% Non-recurrent 42 70,890,197 60,942,827 86.00% 35,587,394 25,355,433 50.20% Non-recurrent 43 78,364,420 67,764,138 86.50% 37,301,367 30,462,771 47.60% Non-recurrent 44 80,324,223 66,067,586 82.30% 34,037,743 32,029,843 42.40% Non-recurrent 48 78,452,235 64,249,452 81.90% 35,142,959 29,106,493 44.80% Non-recurrent 49 37,236,643 34,236,504 91.90% 21,722,873 12,513,631 58.30% Non-recurrent 50 69,690,068 59,990,442 86.10% 34,729,086 25,261,356 49.80%

23

Non-recurrent 53 57,099,954 50,930,856 89.20% 30,248,963 20,681,893 53.00% Non-recurrent 54 37,728,245 34,921,467 92.60% 20,784,114 14,137,353 55.10% Non-recurrent 55 67,579,646 59,296,849 87.70% 35,114,522 24,182,327 52.00% Non-recurrent 56 43,681,381 36,828,408 84.30% 20,983,695 15,844,713 48.00% Non-recurrent 57 69,431,714 60,581,563 87.30% 34,858,960 25,722,603 50.20% Non-recurrent 58 41,018,330 37,470,952 91.40% 21,728,470 15,742,482 53.00% Non-recurrent 59 62,567,979 55,583,593 88.80% 35,325,534 20,258,059 56.50% Non-recurrent 60 73,675,893 65,305,671 88.60% 36,436,110 28,869,561 49.50% Non-recurrent 63 70,482,529 55,832,795 79.20% 29,489,790 26,343,005 41.80% Non-recurrent 64 60,865,170 54,402,706 89.40% 32,127,233 22,275,473 52.80% Non-recurrent 65 60,426,047 47,212,885 78.10% 26,846,348 20,366,537 44.40% Non-recurrent 67 65,071,756 58,617,234 90.10% 31,408,098 27,209,136 48.30% Non-recurrent 68 66,233,482 59,229,502 89.40% 34,643,252 24,586,250 52.30% Non-recurrent 70 65,092,853 52,719,805 81.00% 30,693,109 22,026,696 47.20% Recurrent 72 79,358,161 68,596,113 86.40% 42,019,443 26,576,670 52.90% Recurrent 74 74,461,946 65,922,341 88.50% 39,707,505 26,214,836 53.30% Recurrent 75 43,230,004 34,817,157 80.50% 20,904,491 13,912,666 48.40% Recurrent 76 77,191,363 66,557,801 86.20% 34,829,899 31,727,902 45.10% Recurrent 77 72,729,613 64,582,169 88.80% 36,923,509 27,658,660 50.80% Recurrent 78 80,673,242 69,929,974 86.70% 42,718,978 27,210,996 53.00% Recurrent 79 75,545,470 65,394,529 86.60% 34,698,199 30,696,330 45.90% Recurrent 80 81,621,462 70,581,858 86.50% 35,421,511 35,160,347 43.40% Recurrent 81 42,618,570 34,028,229 79.80% 19,713,463 14,314,766 46.30% Recurrent 82 68,961,891 61,085,215 88.60% 34,283,612 26,801,603 49.70% Recurrent 83 73,556,985 62,095,023 84.40% 32,896,112 29,198,911 44.70% Recurrent 87 39,847,045 35,400,354 88.80% 23,331,413 12,068,941 58.60% Recurrent 88 41,492,958 35,998,394 86.80% 22,825,841 13,172,553 55.00% Recurrent 89 37,556,071 33,051,493 88.00% 20,127,203 12,924,290 53.60% Recurrent 90 40,992,920 36,243,935 88.40% 25,096,412 11,147,523 61.20% Recurrent 91 64,480,365 57,107,888 88.60% 31,009,608 26,098,280 48.10% Recurrent 92 69,222,447 61,572,273 88.90% 35,421,413 26,150,860 51.20% Normal 93 63,844,678 53,809,172 84.30% 28,023,776 25,785,396 43.90% Normal 94 75,936,306 66,580,993 87.70% 36,783,510 29,797,483 48.40% Normal 95 74,142,087 65,683,290 88.60% 38,647,978 27,035,312 52.10% Normal 96 74,949,129 64,280,466 85.80% 35,117,148 29,163,318 46.90% Normal 97 62,846,781 55,807,401 88.80% 33,378,196 22,429,205 53.10% Normal 98 73,553,482 61,108,173 83.10% 34,202,805 26,905,368 46.50% Normal 99 67,933,292 51,544,883 75.90% 26,799,232 24,745,651 39.40% Normal 100 38,302,918 33,813,507 88.30% 21,866,852 11,946,655 57.10% Normal 101 70,189,125 62,845,735 89.50% 39,302,904 23,542,831 56.00% Normal 102 64,665,069 57,497,411 88.90% 34,422,802 23,074,609 53.20%

24

Average 67,630,023 58,500,861 86.69% 33,357,503 25,143,358 49.75%

Total 5,275,141,792 4,563,067,120 2,601,885,200 1,961,181,920

25

Supplementary Table S3. Differentially methylated promoter CpG islands in recurrent relative to non-recurrent tumors Start of End of Recurrent relative to non- Gene name Chromosome DMR DMR recurrent methylation TTLL5 1 10951481103148 Hypomethylated CPSF3L 1 12459091253909 Hypomethylated GNB1 1 18083551816355 Hypomethylated TMEM52 1 18366001844600 Hypomethylated PRKCZ 1 19677681975768 Hypomethylated C1orf86 1 21120742120074 Hypomethylated MORN1 1 23088532316853 Hypomethylated TP73 1 35930953601095 Hypomethylated AJAP1 1 46109644618964 Hypomethylated GPR153 1 62396226247622 Hypomethylated THAP3 1 66038386611838 Hypomethylated PLOD1 1 1191333211921332 Hypomethylated MIIP 1 1199809812006098 Hypomethylated PDPN 1 1377883813786838 Hypomethylated TMEM82 1 1593750315945503 Hypomethylated IGSF21 1 1830282618310826 Hypomethylated ZNF436 1 2356346623571466 Hypomethylated GALE 1 2399562124003621 Hypomethylated LIN28 1 2660585526613855 Hypomethylated MAP3K6 1 2756192427569924 Hypomethylated TMEM200B 1 2931900829327008 Hypomethylated CSMD2 1 3440003034408030 Hypomethylated TEKT2 1 3631826236326262 Hypomethylated ADPRHL2 1 3632303936331039 Hypomethylated THRAP3 1 3645860336466603 Hypomethylated GRIK3 1 3726843137276431 Hypomethylated HYI 1 4368850543696505 Hypomethylated TCTEX1D4 1 4504154445049544 Hypomethylated HPDL 1 4556113145569131 Hypomethylated FOXD2 1 4767027547678275 Hypomethylated PDE4B 1 6602744366035443 Hypomethylated LHX8 1 7536270675370706 Hypomethylated ST6GALNAC5 1 7710177377109773 Hypomethylated GFI1 1 9271794492725944 Hypomethylated MIR137 1 9828031598288315 Hypomethylated SLC6A17 1 110490654110498654 Hypomethylated RIF1 1 111304089111312089 Hypomethylated SYT6 1 114493995114501995 Hypomethylated

26

MRPS21 1 148528892148536892 Hypomethylated SLC27A3 1 152010391152018391 Hypomethylated PYGO2 1 153196882153204882 Hypomethylated MIR92B 1 153427591153435591 Hypomethylated RUSC1 1 153553263153561263 Hypomethylated BCAN 1 154874363154882363 Hypomethylated NES 1 154909813154917813 Hypomethylated CADM3 1 157404000157412000 Hypomethylated LMX1A 1 163587641163595641 Hypomethylated DNM3 1 170073243170081243 Hypomethylated LHX4 1 178462064178470064 Hypomethylated NMNAT2 1 181650360181658360 Hypomethylated KCNT2 1 194840122194848122 Hypomethylated GUK1 1 226390551226398551 Hypomethylated TRIM17 1 226667206226675206 Hypomethylated EXOC8 1 229536201229544201 Hypomethylated SNTG2 2 932553940553 Hypomethylated KLF11 2 1009713210105132 Hypomethylated UCN 2 2738063427388634 Hypomethylated ZNF513 2 2745309727461097 Hypomethylated NRXN1 2 5042439850432398 Hypomethylated CRIP1 2 6839668768404687 Hypomethylated ANKRD53 2 7105508271063082 Hypomethylated EGR4 2 7337033773378337 Hypomethylated C2orf81 2 7449435274502352 Hypomethylated LBX2 2 7457995174587951 Hypomethylated TLX2 2 7459111874599118 Hypomethylated DUSP2 2 9617090696178906 Hypomethylated CNNM3 2 9684171796849717 Hypomethylated ANKRD39 2 9688348396891483 Hypomethylated SEMA4C 2 9689546296903462 Hypomethylated CHCHD5 2 113054506113062506 Hypomethylated CNTNAP5 2 124495333124503333 Hypomethylated GYPC 2 127126153127134153 Hypomethylated NXPH2 2 139250281139258281 Hypomethylated RPRM 2 154039568154047568 Hypomethylated GALNT7 2 154432671154440671 Hypomethylated KCNJ3 2 155259338155267338 Hypomethylated LOC440925 2 171275323171283323 Hypomethylated GAD1 2 171377445171385445 Hypomethylated EVX2 2 176652936176660936 Hypomethylated HOXD12 2 176668775176676775 Hypomethylated HOXD11 2 176676329176684329 Hypomethylated

27

HOXD10 2 176685737176693737 Hypomethylated HOXD9 2 176691658176699658 Hypomethylated ITGA4 2 182025863182033863 Hypomethylated TMEFF2 2 192763889192771889 Hypomethylated SATB2 2 200029500200037500 Hypomethylated FZD7 2 202603554202611554 Hypomethylated CTDSP1 2 218968721218976721 Hypomethylated DNAJB2 2 219848283219856283 Hypomethylated PAX3 2 222867944222875944 Hypomethylated IRS1 2 227367750227375750 Hypomethylated SPHKAP 2 228750605228758605 Hypomethylated B3GNT7 2 231964578231972578 Hypomethylated NMUR1 2 232099426232107426 Hypomethylated ECEL1P2 2 232955998232963998 Hypomethylated ECEL1 2 233056776233064776 Hypomethylated ESPNL 2 238669689238677689 Hypomethylated GPC1 2 241019787241027787 Hypomethylated MIR149 2 241040090241048090 Hypomethylated RNPEPL1 2 241152776241160776 Hypomethylated SNED1 2 241582927241590927 Hypomethylated BOK 2 242142864242150864 Hypomethylated CHL1 3 209649217649 Hypomethylated LRRN1 3 38121203820120 Hypomethylated IRAK2 3 1017756210185562 Hypomethylated SLC6A1 3 1100541911013419 Hypomethylated RFTN1 3 1652622616534226 Hypomethylated ELK1 3 1916102019169020 Hypomethylated COL7A1 3 4860359748611597 Hypomethylated USP19 3 4912921749137217 Hypomethylated C3orf62 3 4928551249293512 Hypomethylated GNAT1 3 5020004650208046 Hypomethylated IFRD2 3 5030103050309030 Hypomethylated EPHB1 3 135992949136000949 Hypomethylated SOX14 3 138962268138970268 Hypomethylated SGEF 3 155317838155325838 Hypomethylated MMP12 3 156276129156284129 Hypomethylated KCNAB1 3 157487469157495469 Hypomethylated SHOX2 3 159302646159310646 Hypomethylated CLCN2 3 185558085185566085 Hypomethylated FGF12 3 193605532193613532 Hypomethylated MFI2 3 198237083198245083 Hypomethylated ATP5I 4 654122662122 Hypomethylated MFSD7 4 668973676973 Hypomethylated

28

DGKQ 4 953344961344 Hypomethylated IDUA 4 966784974784 Hypomethylated SLC26A1 4 973183981183 Hypomethylated LOC100130872 4 11887501196750 Hypomethylated KIAA1530 4 13271031335103 Hypomethylated TMEM129 4 16888821696882 Hypomethylated MIR943 4 19540021962002 Hypomethylated MXD4 4 22295372237537 Hypomethylated SH3BP2 4 27605472768547 Hypomethylated MFSD10 4 29017622909762 Hypomethylated C4orf44 4 32165643224564 Hypomethylated LRPAP1 4 35000223508022 Hypomethylated OTOP1 4 42755224283522 Hypomethylated MSX1 4 49082924916292 Hypomethylated STK32B 4 51004275108427 Hypomethylated CRMP1 4 59372165945216 Hypomethylated KCNIP4 4 2155547221563472 Hypomethylated KIAA1239 4 3691908436927084 Hypomethylated KCTD8 4 4414158144149581 Hypomethylated GABRA2 4 4608281346090813 Hypomethylated PDGFRA 4 5478602054794020 Hypomethylated EPHA5 4 6621424866222248 Hypomethylated NPFFR2 4 7311238473120384 Hypomethylated PRDM8 4 8132144781329447 Hypomethylated BMP3 4 8216714282175142 Hypomethylated NKX6-1 4 8563441185642411 Hypomethylated SNCA 4 9097337390981373 Hypomethylated CYP2U1 4 109068165109076165 Hypomethylated COL25A1 4 110439248110447248 Hypomethylated PITX2 4 111759703111767703 Hypomethylated FAT4 4 126453016126461016 Hypomethylated PCDH19 4 134285919134293919 Hypomethylated LOC441046 4 144696074144704074 Hypomethylated SFRP2 4 154925678154933678 Hypomethylated NPY2R 4 156345230156353230 Hypomethylated GUCY1B3 4 156895575156903575 Hypomethylated NPY1R 4 164469198164477198 Hypomethylated NPY5R 4 164480540164488540 Hypomethylated GALNTL6 4 172967149172975149 Hypomethylated HAND2 4 174683953174691953 Hypomethylated GLRA3 4 175983040175991040 Hypomethylated GPM6A 4 177156642177164642 Hypomethylated HELT 4 186173076186181076 Hypomethylated

29

LOC25845 5 522080530080 Hypomethylated SLC6A3 5 14945431502543 Hypomethylated MRPL36 5 18489561856956 Hypomethylated ADAMTS16 5 51894425197442 Hypomethylated ADCY2 5 74453427453342 Hypomethylated RXFP3 5 3396824733976247 Hypomethylated OSMR 5 3887771638885716 Hypomethylated PLCXD3 5 4154248741550487 Hypomethylated C5orf39 5 4307220443080204 Hypomethylated ISL1 5 5071071450718714 Hypomethylated CCNO 5 5456126554569265 Hypomethylated RAB3C 5 5791069557918695 Hypomethylated PDE4D 5 5922137859229378 Hypomethylated OTP 5 7696627876974278 Hypomethylated EDIL3 5 8371236783720367 Hypomethylated FLJ42709 5 9292825592936255 Hypomethylated GPR150 5 9497773594985735 Hypomethylated PCSK1 5 9579070895798708 Hypomethylated ST8SIA4 5 100262886100270886 Hypomethylated SLCO4C1 5 101656152101664152 Hypomethylated SLC25A46 5 110098652110106652 Hypomethylated FBN2 5 127897634127905634 Hypomethylated ACSL6 5 131371248131379248 Hypomethylated PDLIM4 5 131617249131625249 Hypomethylated PITX1 5 134393863134401863 Hypomethylated LOC389333 5 138754784138762784 Hypomethylated DND1 5 140029355140037355 Hypomethylated PCDHAC1 5 140282485140290485 Hypomethylated PCDHB6 5 140506022140514022 Hypomethylated PCDHB18 5 140590121140598121 Hypomethylated ME3 5 140720081140728081 Hypomethylated PCDHGA8 5 140747666140755666 Hypomethylated PCDHGA12 5 140786341140794341 Hypomethylated POU4F3 5 145694779145702779 Hypomethylated GLRA1 5 151280590151288590 Hypomethylated EBF1 5 158455366158463366 Hypomethylated GABRB2 5 160903708160911708 Hypomethylated TLX3 5 170664892170672892 Hypomethylated PFN3 5 176756243176764243 Hypomethylated GRM6 5 178350730178358730 Hypomethylated ADAMTS2 5 178700935178708935 Hypomethylated MGAT4B 5 179158481179166481 Hypomethylated TBC1D9B 5 179263462179271462 Hypomethylated

30

RASGEF1C 5 179564736179572736 Hypomethylated FAM50B 6 37906303798630 Hypomethylated HIST1H2BE 6 2628800226296002 Hypomethylated HIST1H3E 6 2632936126337361 Hypomethylated HIST1H3G 6 2637559126383591 Hypomethylated HIST1H2BI 6 2637718226385182 Hypomethylated HIST1H2AJ 6 2788649727894497 Hypomethylated GNMT 6 4303247743040477 Hypomethylated MRPL2 6 4313122043139220 Hypomethylated COL9A1 6 7104563271053632 Hypomethylated KCNQ5 6 7338429173392291 Hypomethylated C6orf147 6 7407280974080809 Hypomethylated RIPPLY2 6 8461570384623703 Hypomethylated POU3F2 6 9938530099393300 Hypomethylated NR2E1 6 108589907108597907 Hypomethylated FAM162B 6 117189579117197579 Hypomethylated VGLL2 6 117689413117697413 Hypomethylated CLVS2 6 123355280123363280 Hypomethylated RSPO3 6 127477740127485740 Hypomethylated EYA4 6 133600187133608187 Hypomethylated FNDC1 6 159506416159514416 Hypomethylated C6orf176 6 166317517166325517 Hypomethylated ADAP1 7 956815964815 Hypomethylated UNCX 7 12351791243179 Hypomethylated NXPH1 7 84361098444109 Hypomethylated TMEM196 7 1977492919782929 Hypomethylated PDE1C 7 3207351632081516 Hypomethylated ZNF727 7 6313925563147255 Hypomethylated ZNF804B 7 8822268888230688 Hypomethylated MTERF 7 9134395291351952 Hypomethylated BRI3 7 9774491497752914 Hypomethylated NPTX2 7 9808053298088532 Hypomethylated TMEM130 7 9830160998309609 Hypomethylated TRIM4 7 9935109099359090 Hypomethylated SAP25 7 100005206100013206 Hypomethylated GIGYF1 7 100120806100128806 Hypomethylated SRRT 7 100306636100314636 Hypomethylated RELN 7 103413199103421199 Hypomethylated LHFPL3 7 103752339103760339 Hypomethylated GRM8 7 126675664126683664 Hypomethylated CHRM2 7 136200408136208408 Hypomethylated CNTNAP2 7 145440385145448385 Hypomethylated REPIN1 7 149692811149700811 Hypomethylated

31

TMEM176B 7 150124554150132554 Hypomethylated KCNH2 7 150279848150287848 Hypomethylated ACCN3 7 150372537150380537 Hypomethylated FASTK 7 150404884150412884 Hypomethylated PTPRN2 7 158069243158077243 Hypomethylated C8orf42 8 481331489331 Hypomethylated CSMD1 8 48357364843736 Hypomethylated LOC157627 8 97942499802249 Hypomethylated MIR124-1 8 97943929802392 Hypomethylated XKR6 8 1109228511100285 Hypomethylated GATA4 8 1159512511603125 Hypomethylated SGCZ 8 1513616315144163 Hypomethylated EFHA2 8 1692511716933117 Hypomethylated EPB49 8 2196427321972273 Hypomethylated HR 8 2204051022048510 Hypomethylated PDLIM2 8 2248858722496587 Hypomethylated C8orf58 8 2250906622517066 Hypomethylated DBC1 8 2251420122522201 Hypomethylated TNFRSF10C 8 2301237823020378 Hypomethylated NEFL 8 2486604824874048 Hypomethylated EBF2 8 2595430925962309 Hypomethylated PNMA2 8 2642340026431400 Hypomethylated ADRA1A 8 2677483926782839 Hypomethylated SCARA3 8 2754349527551495 Hypomethylated NRG1 8 3161280931620809 Hypomethylated UNC5D 8 3520851635216516 Hypomethylated ADRB3 8 3793934137947341 Hypomethylated NPBWR1 8 5401102054019020 Hypomethylated PENK 8 5751783657525836 Hypomethylated NKAIN3 8 6332005463328054 Hypomethylated LOC401463 8 6564837465656374 Hypomethylated BHLHE22 8 6565136765659367 Hypomethylated LOC100192378 8 7775406577762065 Hypomethylated PKIA 8 7958689079594890 Hypomethylated ADCY8 8 132118017132126017 Hypomethylated FAM135B 8 139574247139582247 Hypomethylated JRK 8 143744403143752403 Hypomethylated TOP1MT 8 144484425144492425 Hypomethylated C8orf51 8 144518180144526180 Hypomethylated ZC3H3 8 144690763144698763 Hypomethylated C8orf73 8 144722071144730071 Hypomethylated NAPRT1 8 144727656144735656 Hypomethylated EEF1D 8 144746988144754988 Hypomethylated

32

PYCRL 8 144758907144766907 Hypomethylated LOC100130274 8 144858267144866267 Hypomethylated MIR937 8 144963200144971200 Hypomethylated SCRIB 8 144965537144973537 Hypomethylated EPPK1 8 145015422145023422 Hypomethylated OPLAH 8 145183572145191572 Hypomethylated SHARPIN 8 145227128145235128 Hypomethylated KIAA1875 8 145230616145238616 Hypomethylated FBXL6 8 145548940145556940 Hypomethylated ADCK5 8 145564538145572538 Hypomethylated MIR939 8 145586253145594253 Hypomethylated SLC39A4 8 145608725145616725 Hypomethylated VPS28 8 145620735145628735 Hypomethylated NFKBIL2 8 145636620145644620 Hypomethylated GPT 8 145696272145704272 Hypomethylated MFSD3 8 145701359145709359 Hypomethylated RECQL4 8 145710018145718018 Hypomethylated ZNF7 8 146019706146027706 Hypomethylated TMEM215 9 3276949632777496 Hypomethylated GRHPR 9 3740870637416706 Hypomethylated RORB 9 7629807176306071 Hypomethylated GNA14 9 7944905279457052 Hypomethylated GABBR2 9 100507300100515300 Hypomethylated C9orf125 9 103285296103293296 Hypomethylated GRIN3A 9 103536683103544683 Hypomethylated LHX6 9 124026840124034840 Hypomethylated PTRH1 9 129513757129521757 Hypomethylated TOR2A 9 129533425129541425 Hypomethylated ENG 9 129652868129660868 Hypomethylated AK1 9 129675843129683843 Hypomethylated ST6GALNAC6 9 129697692129705692 Hypomethylated NAIF1 9 129865420129873420 Hypomethylated C9orf16 9 129958359129966359 Hypomethylated ZDHHC12 9 130522229130530229 Hypomethylated NCS1 9 131970677131978677 Hypomethylated ASS1 9 132305914132313914 Hypomethylated PRDM12 9 132525801132533801 Hypomethylated AIF1L 9 132957732132965732 Hypomethylated BARHL1 9 134443813134451813 Hypomethylated GBGT1 9 135025122135033122 Hypomethylated SURF1 9 135209182135217182 Hypomethylated NCRNA00094 9 135876381135884381 Hypomethylated RXRA 9 136354136136362136 Hypomethylated

33

OLFM1 9 137102923137110923 Hypomethylated C9orf116 9 137527582137535582 Hypomethylated NACC2 9 138122952138130952 Hypomethylated LHX3 9 138230825138238825 Hypomethylated DNLZ 9 138374062138382062 Hypomethylated SNAPC4 9 138408710138416710 Hypomethylated INPP5E 9 138450077138458077 Hypomethylated NOTCH1 9 138556059138564059 Hypomethylated MIR126 9 138680874138688874 Hypomethylated AGPAT2 9 138697732138705732 Hypomethylated TMEM141 9 138801597138809597 Hypomethylated C9orf86 9 138818201138826201 Hypomethylated FBXW5 9 138954994138962994 Hypomethylated LCN12 9 138962588138970588 Hypomethylated PTGDS 9 138987776138995776 Hypomethylated C9orf142 9 139002690139010690 Hypomethylated UAP1L1 9 139087773139095773 Hypomethylated DPP7 9 139125016139133016 Hypomethylated GRIN1 9 139149429139157429 Hypomethylated ANAPC2 9 139198878139206878 Hypomethylated COBRA1 9 139265579139273579 Hypomethylated C9orf167 9 139288100139296100 Hypomethylated NELF 9 139469607139477607 Hypomethylated WDR85 9 139589208139597208 Hypomethylated DIP2C 10 721608729608 Hypomethylated CALML3 10 55529235560923 Hypomethylated SFMBT2 10 74894567497456 Hypomethylated ECHDC3 10 1182036111828361 Hypomethylated ITGA8 10 1579777615805776 Hypomethylated CACNB2 10 1846561118473611 Hypomethylated SPAG6 10 2267040422678404 Hypomethylated KIAA1217 10 2401968024027680 Hypomethylated LOC100128811 10 2550121125509211 Hypomethylated GAD2 10 2654124126549241 Hypomethylated MKX 10 2807078428078784 Hypomethylated LOC84856 10 4228694442294944 Hypomethylated CXCL12 10 4419654844204548 Hypomethylated SYT15 10 4638660746394607 Hypomethylated GDF10 10 4805517248063172 Hypomethylated C10orf72 10 4998956549997565 Hypomethylated C10orf53 10 5055368950561689 Hypomethylated PRKG1 10 5241695052424950 Hypomethylated STOX1 10 7025329970261299 Hypomethylated

34

NEUROG3 10 7099912871007128 Hypomethylated NODAL 10 7186747171875471 Hypomethylated ZNF503 10 7682751976835519 Hypomethylated KCNMA1 10 7906358379071583 Hypomethylated NRG3 10 8362104983629049 Hypomethylated CH25H 10 9095305190961051 Hypomethylated PDLIM1 10 9703677197044771 Hypomethylated CRTAC1 10 9977657599784575 Hypomethylated HPSE2 10 100981622100989622 Hypomethylated CNNM1 10 101074845101082845 Hypomethylated PAX2 10 102491457102499457 Hypomethylated LZTS2 10 102742954102750954 Hypomethylated TLX1 10 102877050102885050 Hypomethylated PITX3 10 103987221103995221 Hypomethylated INA 10 105022909105030909 Hypomethylated GFRA1 10 118018786118026786 Hypomethylated PRLHR 10 120341150120349150 Hypomethylated TACC2 10 123909094123917094 Hypomethylated HMX2 10 124893627124901627 Hypomethylated ADAM12 10 128063117128071117 Hypomethylated DOCK1 10 128580012128588012 Hypomethylated DPYSL4 10 133846403133854403 Hypomethylated PWWP2B 10 134056691134064691 Hypomethylated GPR123 10 134747398134755398 Hypomethylated ADAM8 10 134936397134944397 Hypomethylated ZNF511 10 134968412134976412 Hypomethylated TUBGCP2 10 134968634134976634 Hypomethylated C10orf125 10 135017519135025519 Hypomethylated ECHS1 10 135032898135040898 Hypomethylated PAOX 10 135038730135046730 Hypomethylated SPRN 10 135084111135092111 Hypomethylated CYP2E1 10 135186856135194856 Hypomethylated BET1L 11 193422201422 Hypomethylated ATHL1 11 275137283137 Hypomethylated PKP3 11 380216388216 Hypomethylated SIGIRR 11 403397411397 Hypomethylated PTDSS2 11 436279444279 Hypomethylated RNH1 11 492821500821 Hypomethylated HRAS 11 521550529550 Hypomethylated C11orf35 11 546779554779 Hypomethylated MIR210 11 554198562198 Hypomethylated IRF7 11 601999609999 Hypomethylated MUPCDH 11 611007619007 Hypomethylated

35

SCT 11 613173621173 Hypomethylated DRD4 11 623304631304 Hypomethylated SPN 11 681740689740 Hypomethylated PDDC1 11 763487771487 Hypomethylated CEND1 11 776126784126 Hypomethylated LRDD 11 791245799245 Hypomethylated EFCAB4A 11 813584821584 Hypomethylated CD151 11 818951826951 Hypomethylated AP2A2 11 911840919840 Hypomethylated MUC6 11 10227061030706 Hypomethylated TOLLIP 11 12834151291415 Hypomethylated LOC402778 11 17219251729925 Hypomethylated MRPL23 11 19210771929077 Hypomethylated MIR675 11 19706371978637 Hypomethylated IGF2 11 21127802120780 Hypomethylated TSSC4 11 23760982384098 Hypomethylated ZNF215 11 69002296908229 Hypomethylated ZNF214 11 69941627002162 Hypomethylated SYT9 11 72257567233756 Hypomethylated CALCA 11 1494640814954408 Hypomethylated MYOD1 11 1769368517701685 Hypomethylated PTPN5 11 1876596518773965 Hypomethylated DBX1 11 2013444620142446 Hypomethylated SLC6A5 11 2057352120581521 Hypomethylated NELL1 11 2064369220651692 Hypomethylated ANO5 11 2216729722175297 Hypomethylated KCNA4 11 2999106429999064 Hypomethylated WT1 11 3240966332417663 Hypomethylated MIR129-2 11 4355551943563519 Hypomethylated ALX4 11 4428429244292292 Hypomethylated MDK 11 4635579446363794 Hypomethylated ARHGAP1 11 4667469646682696 Hypomethylated PACSIN3 11 4716053447168534 Hypomethylated SLC43A3 11 5694709956955099 Hypomethylated EEF1G 11 6209403662102036 Hypomethylated TUT1 11 6211168562119685 Hypomethylated B3GAT3 11 6214202462150024 Hypomethylated LRRN4CL 11 6220977662217776 Hypomethylated C11orf84 11 6333349863341498 Hypomethylated RCOR2 11 6343689263444892 Hypomethylated FERMT3 11 6372672763734727 Hypomethylated KCNK4 11 6381136863819368 Hypomethylated TRMT112 11 6383760963845609 Hypomethylated

36

RPS6KA4 11 6387920063887200 Hypomethylated MAP4K2 11 6432328964331289 Hypomethylated TM7SF2 11 6463191664639916 Hypomethylated PCNXL3 11 6513635865144358 Hypomethylated RNASEH2C 11 6524098565248985 Hypomethylated DKFZp761E198 11 6530039865308398 Hypomethylated RAB1B 11 6578863165796631 Hypomethylated RIN1 11 6585657665864576 Hypomethylated NPAS4 11 6594105065949050 Hypomethylated LRFN4 11 6637745166385451 Hypomethylated ADRBK1 11 6678648066794480 Hypomethylated SSH3 11 6682349466831494 Hypomethylated RAD9A 11 6691199866919998 Hypomethylated PPP1CA 11 6692195266929952 Hypomethylated PTPRCAP 11 6695772966965729 Hypomethylated PITPNM1 11 6702541967033419 Hypomethylated NDUFS8 11 6755066967558669 Hypomethylated TCIRG1 11 6755905867567058 Hypomethylated TPCN2 11 6856892568576925 Hypomethylated ARHGEF17 11 7269331072701310 Hypomethylated SLC36A4 11 9256674392574743 Hypomethylated C11orf70 11 101419408101427408 Hypomethylated KIAA1826 11 105394164105402164 Hypomethylated ARHGAP20 11 110084661110092661 Hypomethylated LAYN 11 110912442110920442 Hypomethylated ZBTB16 11 113431640113439640 Hypomethylated DSCAML1 11 117169186117177186 Hypomethylated BCL9L 11 118282823118290823 Hypomethylated MCAM 11 118689050118697050 Hypomethylated BSX 11 122353589122361589 Hypomethylated ROBO3 11 124236514124244514 Hypomethylated PKNOX2 11 124535768124543768 Hypomethylated ST3GAL4 11 125727305125735305 Hypomethylated BARX2 11 128747090128755090 Hypomethylated NTM 11 131281921131289921 Hypomethylated OPCML 11 132314247132322247 Hypomethylated IGSF9B 11 133328090133336090 Hypomethylated JAM3 11 133440029133448029 Hypomethylated NTF3 12 54075405415540 Hypomethylated GRIN2B 12 1402028914028289 Hypomethylated CPNE8 12 3758168737589687 Hypomethylated FAIM2 12 4857998748587987 Hypomethylated RARG 12 5189641751904417 Hypomethylated

37

HOXC9 12 5267614352684143 Hypomethylated AGAP2 12 5641429656422296 Hypomethylated AVPR1A 12 6182885761836857 Hypomethylated LOC283392 12 7094955670957556 Hypomethylated ACSS3 12 7999193979999939 Hypomethylated SLC6A15 12 8382673783834737 Hypomethylated ASCL1 12 101871581101879581 Hypomethylated C12orf42 12 102409879102417879 Hypomethylated EID3 12 103217678103225678 Hypomethylated WSCD2 12 107043640107051640 Hypomethylated LHX5 12 112390260112398260 Hypomethylated B3GNT4 12 121250180121258180 Hypomethylated ARL6IP4 12 122026832122034832 Hypomethylated GLT1D1 12 127900033127908033 Hypomethylated FAM123A 13 2463985724647857 Hypomethylated SHISA2 13 2551919825527198 Hypomethylated GPR12 13 2622892226236922 Hypomethylated GSX1 13 2726077927268779 Hypomethylated TRPC4 13 3733793937345939 Hypomethylated TNFSF11 13 4204229042050290 Hypomethylated PCDH9 13 6669846966706469 Hypomethylated ATXN8OS 13 6957534569583345 Hypomethylated EDNRB 13 7738696777394967 Hypomethylated POU4F1 13 7807169678079696 Hypomethylated RNF219 13 7812731578135315 Hypomethylated DZIP1 13 9509095895098958 Hypomethylated ZIC5 13 9941817999426179 Hypomethylated FGF14 13 101362996101370996 Hypomethylated SOX1 13 111765913111773913 Hypomethylated MCF2L 13 112666757112674757 Hypomethylated GRTP1 13 113062464113070464 Hypomethylated CDH24 14 2259258722600587 Hypomethylated NOVA1 14 2613280026140800 Hypomethylated C14orf23 14 2830766028315660 Hypomethylated SFTA3 14 3604874136056741 Hypomethylated NKX2-1 14 3605518136063181 Hypomethylated FOXA1 14 3713024037138240 Hypomethylated LRFN5 14 4114251341150513 Hypomethylated MDGA2 14 4720973847217738 Hypomethylated TRIM9 14 5062817250636172 Hypomethylated NID2 14 5160169651609696 Hypomethylated BMP4 14 5348702053495020 Hypomethylated PNMA1 14 7324688173254881 Hypomethylated

38

VSX2 14 7377192773779927 Hypomethylated FAM164C 14 7460205174610051 Hypomethylated VASH1 14 7629398776301987 Hypomethylated GALC 14 8752566087533660 Hypomethylated PTPN21 14 8808687688094876 Hypomethylated AHNAK2 14 104511739104519739 Hypomethylated PACS2 14 104848125104856125 Hypomethylated GABRB3 15 2456534424573344 Hypomethylated GABRA5 15 2465936524667365 Hypomethylated GJD2 15 3282998132837981 Hypomethylated LOC145845 15 3496202634970026 Hypomethylated MEIS2 15 3517379535181795 Hypomethylated TYRO3 15 3963452339642523 Hypomethylated ONECUT1 15 5086550150873501 Hypomethylated ALDH1A2 15 5614119856149198 Hypomethylated LBXCOR1 15 6590099465908994 Hypomethylated NR2E3 15 6988594769893947 Hypomethylated GRAMD2 15 7027319070281190 Hypomethylated CYP11A1 15 7244160672449606 Hypomethylated ULK3 15 7291860572926605 Hypomethylated ANKRD34C 15 7735820077366200 Hypomethylated CPEB1 15 8110978381117783 Hypomethylated BNC1 15 8174047281748472 Hypomethylated ZSCAN2 15 8294125282949252 Hypomethylated NTRK3 15 8659666586604665 Hypomethylated MIR9-3 15 8770825187716251 Hypomethylated MESP1 15 8809154488099544 Hypomethylated ANPEP 15 8815507688163076 Hypomethylated FES 15 8922466889232668 Hypomethylated RCCD1 15 8929510989303109 Hypomethylated MRPL28 16 356541364541 Hypomethylated NME4 16 383192391192 Hypomethylated DECR2 16 387858395858 Hypomethylated C16orf10 16 513408521408 Hypomethylated PIGQ 16 556004564004 Hypomethylated C16orf13 16 622348630348 Hypomethylated FAM195A 16 627849635849 Hypomethylated WDR90 16 635363643363 Hypomethylated RHOT2 16 654133662133 Hypomethylated STUB1 16 666115674115 Hypomethylated WDR24 16 676401684401 Hypomethylated HAGHL 16 712958720958 Hypomethylated MSLNL 16 768927776927 Hypomethylated

39

RPUSD1 16 774384782384 Hypomethylated SOX8 16 967808975808 Hypomethylated CACNA1H 16 11392411147241 Hypomethylated C16orf42 16 13378741345874 Hypomethylated C16orf91 16 14153461423346 Hypomethylated CCDC154 16 14304911438491 Hypomethylated C16orf38 16 14744691482469 Hypomethylated TELO2 16 14793521487352 Hypomethylated TMEM204 16 15202311528231 Hypomethylated NME3 16 17577111765711 Hypomethylated SPSB3 16 17685821776582 Hypomethylated IGFALS 16 17797351787735 Hypomethylated SEPX1 16 19292951937295 Hypomethylated TBL3 16 19580641966064 Hypomethylated ZNF598 16 19957642003764 Hypomethylated SLC9A3R2 16 20128882020888 Hypomethylated NTHL1 16 20338682041868 Hypomethylated MIR1225 16 20762862084286 Hypomethylated TRAF7 16 21417992149799 Hypomethylated MLST8 16 21914502199450 Hypomethylated PGP 16 22008232208823 Hypomethylated DNASE1L2 16 22224682230468 Hypomethylated RNPS1 16 22537982261798 Hypomethylated C16orf59 16 24461152454115 Hypomethylated ATP6V0C 16 24999532507953 Hypomethylated CEMP1 16 25174202525420 Hypomethylated ZNF75A 16 32914333299433 Hypomethylated VASN 16 43578494365849 Hypomethylated NUDT16L1 16 46797164687716 Hypomethylated ROGDI 16 47886754796675 Hypomethylated A2BP1 16 60051326013132 Hypomethylated FAM18A 16 1081612210824122 Hypomethylated SHISA9 16 1289897712906977 Hypomethylated HS3ST2 16 2272936022737360 Hypomethylated PRKCB 16 2375080023758800 Hypomethylated CACNG3 16 2417037624178376 Hypomethylated HS3ST4 16 2560684725614847 Hypomethylated SH2B1 16 2877857828786578 Hypomethylated ATP2A1 16 2879330928801309 Hypomethylated MAZ 16 2972135529729355 Hypomethylated PRRT2 16 2972690929734909 Hypomethylated CDIPT 16 2977807929786079 Hypomethylated MAPK3 16 3003813130046131 Hypomethylated

40

TBC1D10B 16 3028502330293023 Hypomethylated ZNF48 16 3031055730318557 Hypomethylated ZNF768 16 3044141130449411 Hypomethylated PRR14 16 3056574130573741 Hypomethylated SRCAP 16 3061396230621962 Hypomethylated BCKDK 16 3102316231031162 Hypomethylated PYCARD 16 3111775231125752 Hypomethylated COX6A2 16 3134322231351222 Hypomethylated TGFB1I1 16 3138697631394976 Hypomethylated IRX3 16 5287387952881879 Hypomethylated IRX5 16 5351861153526611 Hypomethylated IRX6 16 5391197153919971 Hypomethylated MT1M 16 5522003455228034 Hypomethylated MT1A 16 5522607855234078 Hypomethylated CDH8 16 6062353760631537 Hypomethylated CMTM2 16 6516685165174851 Hypomethylated HSF4 16 6575078865758788 Hypomethylated ELMO3 16 6578652865794528 Hypomethylated TPPP3 16 6598092265988922 Hypomethylated ACD 16 6624821966256219 Hypomethylated NRN1L 16 6647228166480281 Hypomethylated PSMB10 16 6652425466532254 Hypomethylated MTSS1L 16 6927345569281455 Hypomethylated ZFHX3 16 7163577571643775 Hypomethylated BCAR1 16 7383902773847027 Hypomethylated NUDT7 16 7630991176317911 Hypomethylated VAT1L 16 7637598376383983 Hypomethylated CDH13 16 8121407881222078 Hypomethylated NECAB2 16 8255573782563737 Hypomethylated WFDC1 16 8288190182889901 Hypomethylated COTL1 16 8320517083213170 Hypomethylated CRISPLD2 16 8340708783415087 Hypomethylated IRF8 16 8448627484494274 Hypomethylated FOXF1 16 8509763385105633 Hypomethylated FBXO31 16 8597089585978895 Hypomethylated JPH3 16 8618999986197999 Hypomethylated LOC100129637 16 8629279186300791 Hypomethylated IL17C 16 8722850187236501 Hypomethylated CYBA 16 8724095887248958 Hypomethylated CDT1 16 8739368687401686 Hypomethylated GALNS 16 8744687587454875 Hypomethylated ACSF3 16 8768375487691754 Hypomethylated SPG7 16 8809830588106305 Hypomethylated

41

CPNE7 16 8816567688173676 Hypomethylated NXN 17 825760833760 Hypomethylated BHLHA9 17 11166071124607 Hypomethylated MYO1C 17 13318011339801 Hypomethylated SCARF1 17 14917911499791 Hypomethylated RILP 17 14961421504142 Hypomethylated PRPF8 17 15309261538926 Hypomethylated WDR81 17 15705831578583 Hypomethylated OVCA2 17 18880261896026 Hypomethylated CAMKK1 17 37367863744786 Hypomethylated SMTNL2 17 44305824438582 Hypomethylated VMO1 17 46324694640469 Hypomethylated GLTPD2 17 46349934642993 Hypomethylated C17orf107 17 47397264747726 Hypomethylated SPAG7 17 48078564815856 Hypomethylated ZFP3 17 49184774926477 Hypomethylated LOC728392 17 53412665349266 Hypomethylated SLC13A5 17 65534646561464 Hypomethylated ALOX12 17 68361076844107 Hypomethylated C17orf49 17 68547966862796 Hypomethylated SLC16A11 17 68839666891966 Hypomethylated LLGL1 17 70600937068093 Hypomethylated GPS2 17 71553827163382 Hypomethylated NEURL4 17 71693627177362 Hypomethylated KCTD11 17 71919317199931 Hypomethylated TNK1 17 72210887229088 Hypomethylated TMEM102 17 72754857283485 Hypomethylated FGF11 17 72794127287412 Hypomethylated MPDU1 17 74236887431688 Hypomethylated SOX15 17 74302127438212 Hypomethylated TMEM88 17 76951087703108 Hypomethylated LSMD1 17 76978977705897 Hypomethylated KCNAB3 17 77694787777478 Hypomethylated TRAPPC1 17 77720427780042 Hypomethylated C17orf44 17 80640868072086 Hypomethylated RPL26 17 82232908231290 Hypomethylated SHISA6 17 1108146411089464 Hypomethylated UBB 17 1622109116229091 Hypomethylated RASD1 17 1733643217344432 Hypomethylated EPN2 17 1907728219085282 Hypomethylated TMEM11 17 2105450021062500 Hypomethylated C17orf102 17 2992650129934501 Hypomethylated TMEM132E 17 2992788029935880 Hypomethylated

42

SLFN13 17 3079596930803969 Hypomethylated LHX1 17 3236461132372611 Hypomethylated ARL5C 17 3457194034579940 Hypomethylated PNMT 17 3507403235082032 Hypomethylated C17orf37 17 3513631435144314 Hypomethylated NR1D1 17 3550649935514499 Hypomethylated GJC1 17 3577047135778471 Hypomethylated PLEKHH3 17 3807857438086574 Hypomethylated CNTNAP1 17 3808415738092157 Hypomethylated NAGS 17 3943355739441557 Hypomethylated TMEM101 17 3944387139451871 Hypomethylated C17orf104 17 4008528740093287 Hypomethylated PLCD3 17 4056141740569417 Hypomethylated HEXIM1 17 4057646640584466 Hypomethylated C17orf46 17 4069126240699262 Hypomethylated SH3D20 17 4086206540870065 Hypomethylated HOXB4 17 4400674244014742 Hypomethylated MIR10A 17 4400830844016308 Hypomethylated LOC404266 17 4402431844032318 Hypomethylated IGF2BP1 17 4442577244433772 Hypomethylated DLX4 17 4539756045405560 Hypomethylated CHAD 17 4589722645905226 Hypomethylated SPATA20 17 4597556045983560 Hypomethylated CA10 17 4758713147595131 Hypomethylated RNF126P1 17 5247383752481837 Hypomethylated C17orf82 17 5683989356847893 Hypomethylated MRC2 17 5805449358062493 Hypomethylated LOC146880 17 6020408460212084 Hypomethylated SOX9 17 6762475567632755 Hypomethylated MGC16275 17 6971705569725055 Hypomethylated TTYH2 17 6971729069725290 Hypomethylated SLC16A5 17 7059164970599649 Hypomethylated MYO15B 17 7109173371099733 Hypomethylated TRIM65 17 7140064971408649 Hypomethylated SRP68 17 7157620271584202 Hypomethylated GALR2 17 7157848671586486 Hypomethylated QRICH2 17 7181135671819356 Hypomethylated SPHK1 17 7188828471896284 Hypomethylated CYGB 17 7204137772049377 Hypomethylated MXRA7 17 7221465172222651 Hypomethylated CHMP6 17 7657623576584235 Hypomethylated FLJ90757 17 7661911476627114 Hypomethylated C17orf56 17 7682345076831450 Hypomethylated

43

C17orf55 17 7689364376901643 Hypomethylated C17orf70 17 7712584877133848 Hypomethylated ARL16 17 7725735977265359 Hypomethylated MRPL12 17 7727681177284811 Hypomethylated SLC25A10 17 7728577577293775 Hypomethylated PYCR1 17 7748425977492259 Hypomethylated STRA13 17 7757006277578062 Hypomethylated LRRC45 17 7757056877578568 Hypomethylated NCOA3 17 7757882077586820 Hypomethylated RFNG 17 7759893977606939 Hypomethylated FN3K 17 7828274078290740 Hypomethylated CIDEA 18 1224031712248317 Hypomethylated ZNF521 18 2118221221190212 Hypomethylated DSC2 18 2687277926880779 Hypomethylated NOL4 18 3005344430061444 Hypomethylated DTNA 18 3032325130331251 Hypomethylated BRUNOL4 18 3339599833403998 Hypomethylated ST8SIA5 18 4258703742595037 Hypomethylated MBD1 18 4605814246066142 Hypomethylated ST8SIA3 18 5316671853174718 Hypomethylated ONECUT2 18 5324991453257914 Hypomethylated GRP 18 5503437955042379 Hypomethylated RAX 18 5508760555095605 Hypomethylated DOK6 18 6521526365223263 Hypomethylated CBLN2 18 6835870368366703 Hypomethylated KIR2DS1 19 253787261787 Hypomethylated MIER2 19 291791299791 Hypomethylated CDC34 19 478732486732 Hypomethylated FGF22 19 586925594925 Hypomethylated RNF126 19 610227618227 Hypomethylated MED16 19 840218848218 Hypomethylated ARID3A 19 873036881036 Hypomethylated ABCA7 19 987101995101 Hypomethylated SBNO2 19 11212821129282 Hypomethylated C19orf26 19 11849901192990 Hypomethylated ATP5D 19 11887481196748 Hypomethylated CIRBP 19 12162661224266 Hypomethylated C19orf24 19 12225191230519 Hypomethylated RPS15 19 13853621393362 Hypomethylated C19orf25 19 14262281434228 Hypomethylated PLK5P 19 14710771479077 Hypomethylated UQCR11 19 15524831560483 Hypomethylated ATP8B3 19 17592701767270 Hypomethylated

44

MIR1909 19 17632371771237 Hypomethylated REXO1 19 17954521803452 Hypomethylated CSNK1G2 19 18881601896160 Hypomethylated C19orf34 19 19015481909548 Hypomethylated MOBKL2A 19 20432692051269 Hypomethylated TIMM13 19 23748752382875 Hypomethylated TLE6 19 29245352932535 Hypomethylated AES 19 30093713017371 Hypomethylated GNA11 19 30414073049407 Hypomethylated NCLN 19 31328743140874 Hypomethylated FZR1 19 34532943461294 Hypomethylated HMG20B 19 35199423527942 Hypomethylated TBXA2R 19 35538313561831 Hypomethylated C19orf29 19 35738133581813 Hypomethylated PIP5K1C 19 36474453655445 Hypomethylated RAX2 19 37192193727219 Hypomethylated DAPK3 19 39168263924826 Hypomethylated PIAS4 19 39547483962748 Hypomethylated SH3GL1 19 43474714355471 Hypomethylated UBXN6 19 44047914412791 Hypomethylated C19orf70 19 56279115635911 Hypomethylated RPL36 19 56372715645271 Hypomethylated DUS3L 19 57382495746249 Hypomethylated ALKBH7 19 63194436327443 Hypomethylated KHSRP 19 63718226379822 Hypomethylated MCOLN1 19 74894957497495 Hypomethylated XAB2 19 75964397604439 Hypomethylated EVI5L 19 78133857821385 Hypomethylated SNAPC2 19 78871937895193 Hypomethylated CTXN1 19 78930517901051 Hypomethylated TIMM44 19 79105387918538 Hypomethylated NDUFA7 19 82882808296280 Hypomethylated RAB11B 19 83572048365204 Hypomethylated ZNF414 19 84810488489048 Hypomethylated ZNF177 19 93306959338695 Hypomethylated EIF3G 19 1008759910095599 Hypomethylated S1PR2 19 1019894810206948 Hypomethylated FDX1L 19 1028369110291691 Hypomethylated RAVER1 19 1030131410309314 Hypomethylated TMED1 19 1080398310811983 Hypomethylated CARM1 19 1083925210847252 Hypomethylated YIPF2 19 1089635710904357 Hypomethylated KANK2 19 1116226611170266 Hypomethylated

45

ZNF653 19 1147365411481654 Hypomethylated ACP5 19 1154659211554592 Hypomethylated ZNF833 19 1164181211649812 Hypomethylated ZNF788 19 1206007712068077 Hypomethylated TNPO2 19 1269010512698105 Hypomethylated RAD23A 19 1291365312921653 Hypomethylated LYL1 19 1307068113078681 Hypomethylated CACNA1A 19 1347427413482274 Hypomethylated MRI1 19 1373233613740336 Hypomethylated C19orf53 19 1374225613750256 Hypomethylated PTGER1 19 1444317414451174 Hypomethylated CCDC105 19 1497853814986538 Hypomethylated ILVBL 19 1509357715101577 Hypomethylated EPHX3 19 1520023115208231 Hypomethylated AKAP8 19 1534760315355603 Hypomethylated AKAP8L 19 1538683315394833 Hypomethylated USE1 19 1718315417191154 Hypomethylated GTPBP3 19 1730535517313355 Hypomethylated PGLS 19 1747943117487431 Hypomethylated GLT25D1 19 1752351017531510 Hypomethylated MAP1S 19 1768730217695302 Hypomethylated PIK3R2 19 1812101518129015 Hypomethylated MPV17L2 19 1816103918169039 Hypomethylated RAB3A 19 1817187418179874 Hypomethylated PGPEP1 19 1830840718316407 Hypomethylated ISYNA1 19 1840611118414111 Hypomethylated C19orf60 19 1855649418564494 Hypomethylated GDF1 19 1886395318871953 Hypomethylated SLC25A42 19 1903180219039802 Hypomethylated NR2C2AP 19 1917123819179238 Hypomethylated YJEFN3 19 1949671919504719 Hypomethylated CILP2 19 1950607319514073 Hypomethylated GMIP 19 1961145519619455 Hypomethylated CHST8 19 3880070038808700 Hypomethylated ZNF302 19 3985640639864406 Hypomethylated HPN 19 4021924940227249 Hypomethylated USF2 19 4044773540455735 Hypomethylated MLL4 19 4089676040904760 Hypomethylated C19orf55 19 4093688340944883 Hypomethylated SDHAF1 19 4117394041181940 Hypomethylated POLR2I 19 4129404641302046 Hypomethylated COX7A1 19 4133161141339611 Hypomethylated ZNF540 19 4273014742738147 Hypomethylated

46

ZNF781 19 4287105642879056 Hypomethylated EIF3K 19 4379756143805561 Hypomethylated PAK4 19 4430425944312259 Hypomethylated PLEKHG2 19 4459158944599589 Hypomethylated SERTAD1 19 4561977245627772 Hypomethylated NUMBL 19 4588439645892396 Hypomethylated PHLDB3 19 4869682548704825 Hypomethylated TOMM40 19 5008231650090316 Hypomethylated SIX5 19 5096033750968337 Hypomethylated DMPK 19 5097170150979701 Hypomethylated RSPH6A 19 5100644551014445 Hypomethylated PGLYRP1 19 5121416351222163 Hypomethylated PNMAL2 19 5168700951695009 Hypomethylated GLTSCR2 19 5293660452944604 Hypomethylated SEPW1 19 5296965352977653 Hypomethylated TPRX1 19 5299467353002673 Hypomethylated KCNJ14 19 5365231753660317 Hypomethylated FAM83E 19 5380450653812506 Hypomethylated RPL18 19 5381024553818245 Hypomethylated MAMSTR 19 5391078853918788 Hypomethylated BCAT2 19 5400213254010132 Hypomethylated PPP1R15A 19 5406346054071460 Hypomethylated FTL 19 5415637754164377 Hypomethylated TRPM4 19 5434886354356863 Hypomethylated RPS11 19 5468744554695445 Hypomethylated RCN3 19 5471868654726686 Hypomethylated IRF3 19 5485694454864944 Hypomethylated PRMT1 19 5486830754876307 Hypomethylated C19orf76 19 5487975354887753 Hypomethylated CPT1C 19 5488221254890212 Hypomethylated AP2A1 19 5495799154965991 Hypomethylated AKT1S1 19 5506845655076456 Hypomethylated NR1H2 19 5556749655575496 Hypomethylated POLD1 19 5557540455583404 Hypomethylated SNAR-F 19 5579603155804031 Hypomethylated CACNG6 19 5918335359191353 Hypomethylated CNOT3 19 5932926059337260 Hypomethylated LENG8 19 5964787659655876 Hypomethylated TNNI3 19 6035691260364912 Hypomethylated SUV420H2 19 6053903260547032 Hypomethylated TMEM190 19 6057601560584015 Hypomethylated NAT14 19 6068440560692405 Hypomethylated SSC5D 19 6068768160695681 Hypomethylated

47

FIZ1 19 6079870560806705 Hypomethylated ZNF524 19 6079954160807541 Hypomethylated ZNF784 19 6082375360831753 Hypomethylated CCDC106 19 6084676560854765 Hypomethylated U2AF2 19 6085322760861227 Hypomethylated ZNF667 19 6167658261684582 Hypomethylated ZIK1 19 6278343962791439 Hypomethylated ZNF154 19 6290839162916391 Hypomethylated ZNF606 19 6320252663210526 Hypomethylated ZNF274 19 6338220763390207 Hypomethylated ZNF132 19 6363940163647401 Hypomethylated OXT 20 29962653004265 Hypomethylated HSPA12B 20 36573553665355 Hypomethylated PROKR2 20 52390155247015 Hypomethylated BMP2 20 66927446700744 Hypomethylated SNAP25 20 1014347610151476 Hypomethylated PCSK2 20 1715163017159630 Hypomethylated INSM1 20 2029276420300764 Hypomethylated NKX2-2 20 2143866421446664 Hypomethylated PAX1 20 2163029621638296 Hypomethylated FOXA2 20 2250910122517101 Hypomethylated TMEM90B 20 2439383424401834 Hypomethylated PXMP4 20 3176779731775797 Hypomethylated MAP1LC3A 20 3260617932614179 Hypomethylated VSTM2L 20 3596091235968912 Hypomethylated PTPRT 20 4124797141255971 Hypomethylated RBPJL 20 4336490443372904 Hypomethylated PCIF1 20 4399272344000723 Hypomethylated CEBPB 20 4823678248244782 Hypomethylated DOK5 20 5252167252529672 Hypomethylated CBLN4 20 5400941954017419 Hypomethylated BMP7 20 5527111455279114 Hypomethylated ADRM1 20 6030742160315421 Hypomethylated CABLES2 20 6041173460419734 Hypomethylated C20orf20 20 6089424960902249 Hypomethylated OGFR 20 6090262160910621 Hypomethylated DIDO1 20 6102429061032290 Hypomethylated BHLHE23 20 6110483261112832 Hypomethylated ARFGAP1 20 6137060961378609 Hypomethylated CHRNA4 20 6145913961467139 Hypomethylated PTK6 20 6163515161643151 Hypomethylated TNFRSF6B 20 6179446461802464 Hypomethylated SLC2A4RG 20 6183765461845654 Hypomethylated

48

C20orf135 20 6195900961967009 Hypomethylated TPD52L2 20 6196303361971033 Hypomethylated ZNF512B 20 6206766262075662 Hypomethylated CHODL 21 1853502018543020 Hypomethylated NCAM2 21 2128850321296503 Hypomethylated GRIK1 21 3023015330238153 Hypomethylated TIAM1 21 3184916131857161 Hypomethylated OLIG2 21 3331610833324108 Hypomethylated C21orf33 21 4437392144381921 Hypomethylated SUMO3 21 4505847245066472 Hypomethylated POFUT2 21 4552823945536239 Hypomethylated SLC19A1 21 4578277945790779 Hypomethylated C21orf56 21 4642480146432801 Hypomethylated BID 22 1663325816641258 Hypomethylated SLC25A1 22 1754230117550301 Hypomethylated TRMT2A 22 1848076818488768 Hypomethylated ZDHHC8 22 1849536418503364 Hypomethylated RTN4R 22 1863181618639816 Hypomethylated SCARF2 22 1911814619126146 Hypomethylated GAS2L1 22 2802899628036996 Hypomethylated RASL10A 22 2803774828045748 Hypomethylated NEFH 22 2820218028210180 Hypomethylated SEC14L2 22 2911893229126932 Hypomethylated SMTN 22 2980330429811304 Hypomethylated SELM 22 2982955129837551 Hypomethylated KCTD17 22 3577372435781724 Hypomethylated LGALS1 22 3639755836405558 Hypomethylated TRIOBP 22 3646818636476186 Hypomethylated SLC16A8 22 3680511636813116 Hypomethylated NAGA 22 4079279240800792 Hypomethylated C22orf26 22 4482468844832688 Hypomethylated GRAMD4 22 4539732145405321 Hypomethylated FAM19A5 22 4725995147267951 Hypomethylated ZBED4 22 4862950048637500 Hypomethylated PIM3 22 4873614648744146 Hypomethylated PANX2 22 4894728648955286 Hypomethylated TRABD 22 4896248648970486 Hypomethylated SELO 22 4897753448985534 Hypomethylated TUBGCP6 22 4902152749029527 Hypomethylated HDAC10 22 4902796149035961 Hypomethylated MAPK12 22 4903821649046216 Hypomethylated MAPK11 22 4904690649054906 Hypomethylated LMF2 22 4928900149297001 Hypomethylated

49

NCAPH2 22 4928951049297510 Hypomethylated SCO2 22 4930690049314900 Hypomethylated CPT1B 22 4935974449367744 Hypomethylated ARSA 22 4940947349417473 Hypomethylated KAL1 X 8656227 8664227 Hypomethylated ARHGAP6 X 11589742 11597742 Hypomethylated FRMPD4 X 12062505 12070505 Hypomethylated CTPS2 X 16636696 16644696 Hypomethylated CDKL5 X 18349645 18357645 Hypomethylated PHKA2 X 18908401 18916401 Hypomethylated GPR64 X 19046598 19054598 Hypomethylated CNKSR2 X 21298456 21306456 Hypomethylated KLHL34 X 21582369 21590369 Hypomethylated PTCHD1 X 23258905 23266905 Hypomethylated NR0B1 X 30233416 30241416 Hypomethylated EFHC2 X 44083867 44091867 Hypomethylated RGN X 46818718 46826718 Hypomethylated HDAC6 X 48541430 48549430 Hypomethylated PQBP1 X 48636138 48644138 Hypomethylated PRAF2 X 48814606 48822606 Hypomethylated MAGIX X 48902124 48910124 Hypomethylated DGKK X 50226477 50234477 Hypomethylated KLF8 X 56271594 56279594 Hypomethylated NCRNA00182 X 73426134 73434134 Hypomethylated SLC16A2 X 73553809 73561809 Hypomethylated MAGEE1 X 75560520 75568520 Hypomethylated POU3F4 X 82645924 82653924 Hypomethylated RPS6KA6 X 83325599 83333599 Hypomethylated DACH2 X 85286110 85294110 Hypomethylated PABPC5 X 90572252 90580252 Hypomethylated NAP1L3 X 92811264 92819264 Hypomethylated DIAPH2 X 95822317 95830317 Hypomethylated GPRASP2 X 101849995 101857995 Hypomethylated BHLHB9 X 101883562 101891562 Hypomethylated ESX1 X 103382255 103390255 Hypomethylated IL1RAPL2 X 103693651 103701651 Hypomethylated NRK X 104949191 104957191 Hypomethylated TMEM164 X 109128997 109136997 Hypomethylated LONRF3 X 117988740 117996740 Hypomethylated 6-Sep X 118707361 118715361 Hypomethylated RNF113A X 118885819 118893819 Hypomethylated GRIA3 X 122141776 122149776 Hypomethylated STAG2 X 122918155 122926155 Hypomethylated

50

BCORL1 X 128940349 128948349 Hypomethylated RAB33A X 129129453 129137453 Hypomethylated GPR101 X 135937499 135945499 Hypomethylated ZIC3 X 136472011 136480011 Hypomethylated SOX3 X 139410891 139418891 Hypomethylated SLITRK4 X 142546685 142554685 Hypomethylated SLITRK2 X 144703038 144711038 Hypomethylated AFF2 X 147385830 147393830 Hypomethylated GPR50 X 150091713 150099713 Hypomethylated SRPK3 X 152695649 152703649 Hypomethylated FLNA X 153252200 153260200 Hypomethylated GDI1 X 153314452 153322452 Hypomethylated FLJ39609 1 840680848680 Hypermethylated TAL1 1 4746403047472030 Hypermethylated FAM159A 1 5286765352875653 Hypermethylated SSBP3 1 5464068054648680 Hypermethylated TCTEX1D1 1 6698672766994727 Hypermethylated C1orf230 1 149956636149964636 Hypermethylated LOC100132111 1 150073568150081568 Hypermethylated C2CD4D 1 150075657150083657 Hypermethylated S100A6 1 151771341151779341 Hypermethylated NDUFS2 1 159434560159442560 Hypermethylated TMCO1 1 164000759164008759 Hypermethylated CACYBP 1 173231193173239193 Hypermethylated C1orf220 1 176774553176782553 Hypermethylated KIAA1614 1 179144935179152935 Hypermethylated AVPR1B 1 204386905204394905 Hypermethylated LYPLAL1 1 217409814217417814 Hypermethylated HLX 1 219115365219123365 Hypermethylated MIXL1 1 224474005224482005 Hypermethylated ARF1 1 226332983226340983 Hypermethylated C1orf198 1 229066925229074925 Hypermethylated CYP1B1 2 3815282738160827 Hypermethylated SIX3 2 4501854045026540 Hypermethylated VAX2 2 7097722770985227 Hypermethylated IL1RL2 2 102165864102173864 Hypermethylated FRZB 2 183435743183443743 Hypermethylated ZSWIM2 2 187418142187426142 Hypermethylated FEV 2 219554623219562623 Hypermethylated EOMES 3 2773478927742789 Hypermethylated TRIM71 3 3283051332838513 Hypermethylated EPM2AIP1 3 3700579937013799 Hypermethylated VILL 3 3800608138014081 Hypermethylated

51

SCN5A 3 3866216738670167 Hypermethylated ABHD14B 3 5197968651987686 Hypermethylated WNT5A 3 5549237155500371 Hypermethylated PTPRG 3 6151828261526282 Hypermethylated CAPS 3 6283210462840104 Hypermethylated C3orf15 3 120900558120908558 Hypermethylated TRH 3 131171803131179803 Hypermethylated FOXL2 3 140144672140152672 Hypermethylated ZIC4 3 148588874148596874 Hypermethylated GPR149 3 155626198155634198 Hypermethylated TERC 3 170961542170969542 Hypermethylated LRRC34 3 171009006171017006 Hypermethylated FAM131A 3 185532410185540410 Hypermethylated EPHB3 3 185758280185766280 Hypermethylated GP5 3 195597284195605284 Hypermethylated UBXN7 3 197639742197647742 Hypermethylated ADRA2C 4 37340933742093 Hypermethylated NEUROG1 5 134895538134903538 Hypermethylated CXCL14 5 134938868134946868 Hypermethylated CLTB 5 175772146175780146 Hypermethylated HIST1H3I 6 2794407827952078 Hypermethylated SCUBE3 6 3528616735294167 Hypermethylated TRAM2 6 5254582152553821 Hypermethylated TBX18 6 8552661885534618 Hypermethylated SGK1 6 134533727134541727 Hypermethylated OLIG3 6 137853224137861224 Hypermethylated LFNG 7 25220042530004 Hypermethylated RADIL 7 48858614893861 Hypermethylated FSCN1 7 55949795602979 Hypermethylated FERD3L 7 1914756919155569 Hypermethylated HOXA1 7 2709815027106150 Hypermethylated HOXA2 7 2710491927112919 Hypermethylated HOXA3 7 2711614127124141 Hypermethylated HOXA4 7 2713292427140924 Hypermethylated HOXA6 7 2714989327157893 Hypermethylated NCOA6 7 2715882127166821 Hypermethylated HOXA9 7 2716767427175674 Hypermethylated MIR196B 7 2717170727179707 Hypermethylated HOXA10 7 2717648027184480 Hypermethylated EVX1 7 2724468827252688 Hypermethylated NACAD 7 4509101845099018 Hypermethylated EGFR 7 5505021855058218 Hypermethylated GTF2IRD1 7 7350205573510055 Hypermethylated

52

DYNC1I1 7 9523575395243753 Hypermethylated DLX5 7 9648807996496079 Hypermethylated ATP5J2 7 9889774498905744 Hypermethylated UFSP1 7 100321275100329275 Hypermethylated CUX1 7 101242011101250011 Hypermethylated LAMB1 7 107427040107435040 Hypermethylated CCDC136 7 128215334128223334 Hypermethylated DGKI 7 137178149137186149 Hypermethylated ZYX 7 142784481142792481 Hypermethylated DPP6 7 153211351153219351 Hypermethylated MNX1 7 156490890156498890 Hypermethylated IKBKB 8 4224398542251985 Hypermethylated EFCAB1 8 4980642349814423 Hypermethylated OPRK1 8 5432274754330747 Hypermethylated MOCOS 8 5718509557193095 Hypermethylated RRS1 8 6749981667507816 Hypermethylated ADHFE1 8 6750327167511271 Hypermethylated MSC 8 7291528572923285 Hypermethylated CA3 8 8653430786542307 Hypermethylated COX6C 8 100971071100979071 Hypermethylated RGS22 8 101183520101191520 Hypermethylated GRHL2 8 102569843102577843 Hypermethylated DMRT3 9 962963970963 Hypermethylated NFIL3 9 9322196593229965 Hypermethylated AKAP2 9 111846796111854796 Hypermethylated COL27A1 9 115954051115962051 Hypermethylated NEK6 9 126056350126064350 Hypermethylated ZNF239 10 4338607143394071 Hypermethylated DKK1 10 5374004653748046 Hypermethylated ZCCHC24 10 8087138980879389 Hypermethylated AGAP11 10 8871647788724477 Hypermethylated HHEX 10 9443566094443660 Hypermethylated SH3PXD2A 10 105601154105609154 Hypermethylated DUSP5 10 112243614112251614 Hypermethylated RBM20 10 112390144112398144 Hypermethylated ATE1 10 123673536123681536 Hypermethylated PAMR1 11 3549975235507752 Hypermethylated HRASLS5 11 6301125663019256 Hypermethylated CD248 11 6583709165845091 Hypermethylated CPT1A 11 6836197568369975 Hypermethylated MRGPRF 11 6853342668541426 Hypermethylated PHOX2A 11 7162886871636868 Hypermethylated KCNA5 12 50193455027345 Hypermethylated

53

GEFT 12 5628622956294229 Hypermethylated TBX3 12 113602352113610352 Hypermethylated TMEM132D 12 128950165128958165 Hypermethylated CCNA1 13 3589996635907966 Hypermethylated DLEU1 13 4959367849601678 Hypermethylated RASA3 13 113912197113920197 Hypermethylated NDRG2 14 2055977520567775 Hypermethylated FOXG1 14 2830203728310037 Hypermethylated NKX2-8 14 3611753736125537 Hypermethylated C14orf169 14 7302339673031396 Hypermethylated FBLN5 14 9147979991487799 Hypermethylated GSC 14 9430225294310252 Hypermethylated RCOR1 14 102124985102132985 Hypermethylated SIVA1 14 104286514104294514 Hypermethylated LCMT2 15 4140618341414183 Hypermethylated ISL2 15 7441220174420201 Hypermethylated CARHSP1 16 88657498873749 Hypermethylated TXNDC11 16 1174014911748149 Hypermethylated PGAP3 17 79924788000478 Hypermethylated ANKRD13B 17 2494065224948652 Hypermethylated METRNL 17 7862685578634855 Hypermethylated RAB31 18 96942279702227 Hypermethylated FBXO17 19 4415422044162220 Hypermethylated ZNF233 19 4945191549459915 Hypermethylated ADAM33 20 36067383614738 Hypermethylated CST3 20 2356257423570574 Hypermethylated ACSS1 20 2498361624991616 Hypermethylated HCK 20 3009971730107717 Hypermethylated ZNF334 20 4457160144579601 Hypermethylated CDH4 20 5925695359264953 Hypermethylated PCMTD2 20 6235349162361491 Hypermethylated BCOR X 39837663 39845663 Hypermethylated HCFC1 X 152886013 152894013 Hypermethylated

54

Supplementary Table S4. List of primer sequences

Gene Strand Sequence Method ACTB Forward GTCTTTCAGTCAAGAGACGCTG Fluidigm PCR Reverse GTTGTGGTCGTTCTGTGGTG Fluidigm PCR ALDH1(A1) Forward CCAGAAAGTATATGCACTGGCA Fluidigm PCR Reverse GTGCTCGTACATTTGCTTGGG Fluidigm PCR ASPA Forward TGGGAAGGACCTGATGATACA Fluidigm PCR Reverse CCCAGGGATCTCTGTGCTT Fluidigm PCR BMP7 Forward TCGGCACCCATGTTCATGC Fluidigm PCR Reverse GAGGAAATGGCTATCTTGCAGG Fluidigm PCR CD133 Forward GTTCCATAGCCAAGTATCCTGTG Fluidigm PCR Reverse GCGCAGGGAGGAAATTCTTTT Fluidigm PCR CDH1 Forward CGAGAGCTACACGTTCACGG Fluidigm PCR Reverse GGGTGTCGAGGGAAAAATAGG Fluidigm PCR CDH2 Forward TCCATCGCCAGTTATCACATCT Fluidigm PCR Reverse CTGGACCAGTAACAGGGCTG Fluidigm PCR COL1A2 Forward GGCCCTCAAGGTTTCCAAGG Fluidigm PCR Reverse CACCCTGTGGTCCAACAACTC Fluidigm PCR CXCL13 Forward GTGTGTGGACCTCCATGTTATT Fluidigm PCR Reverse TGCCCCATGACGTGATACCT Fluidigm PCR DAB2IP Forward GCACGCCAGACTTACCTGTC Fluidigm PCR Reverse CCTCCTCAGTTGCAGGATTAAAG Fluidigm PCR EEF1G Forward ACAACTTTGGTATCGTGGAAGG Fluidigm PCR Reverse GCCATCACGCCACAGTTTC Fluidigm PCR EGFR Forward AGGCACGAGTAACAAGCTCAC Fluidigm PCR Reverse ATGAGGACATAACCAGCCACC Fluidigm PCR EpCAM Forward AATCGTCAATGCCAGTGTACTT Fluidigm PCR Reverse TCTCATCGCAGTCAGGATCATAA Fluidigm PCR ESR2 Forward AGTCGGAAACTGGCAGATAGC Fluidigm PCR Reverse GGTAGTGTTGTACTGGGCCAAT Fluidigm PCR FN1 Forward AGGAAGCCGAGGTTTTAACTG Fluidigm PCR Reverse AGGACGCTCATAAGTGTCACC Fluidigm PCR FOXC2 Forward CCTCCTGGTATCTCAACCACA Fluidigm PCR Reverse GAGGGTCGAGTTCTCAATCCC Fluidigm PCR FZD4 Forward AGCGAACGCACATCAAGAC Fluidigm PCR Reverse CTGTAGGCGATCTGTTGGGG Fluidigm PCR GAPDH Forward GTACCGGGAGACCGACAAGA Fluidigm PCR Reverse GATGCGGATCATGGGTCCAG Fluidigm PCR GlI1 Forward AACGCTATACAGATCCTAGCTCG Fluidigm PCR Reverse GTGCCGTTTGGTCACATGG Fluidigm PCR GLI2 Forward CCCCTACCGATTGACATGCG Fluidigm PCR

55

Reverse GAAAGCCGGATCAAGGAGATG Fluidigm PCR GLI3 Forward GAAGTGCTCCACTCGAACAGA Fluidigm PCR Reverse GTGGCTGCATAGTGATTGCG Fluidigm PCR GSK3B Forward AGACGCTCCCTGTGATTTATGT Fluidigm PCR Reverse CCGATGGCAGATTCCAAAGG Fluidigm PCR IGFBP4 Forward GGTGACCACCCCAACAACAG Fluidigm PCR Reverse GAATTTTGGCGAAGTGCTTCTG Fluidigm PCR ITGA5 Forward GGCTTCAACTTAGACGCGGAG Fluidigm PCR Reverse TGGCTGGTATTAGCCTTGGGT Fluidigm PCR ITGB1 Forward CCTACTTCTGCACGATGTGATG Fluidigm PCR Reverse CCTTTGCTACGGTTGGTTACATT Fluidigm PCR MMP9 Forward TGTACCGCTATGGTTACACTCG Fluidigm PCR Reverse GGCAGGGACAGTTGCTTCT Fluidigm PCR NOTCH1 Forward GAGGCGTGGCAGACTATGC Fluidigm PCR Reverse CTTGTACTCCGTCAGCGTGA Fluidigm PCR PCA-3 Forward GGTCCTGCGTCTGAGAGGT Fluidigm PCR Reverse GGCCTTCACATTTTCGATGGT Fluidigm PCR PTCH1 Forward GCCAAATCATAAACGGCGAGG Fluidigm PCR Reverse CGCCCGAGCAGAACAATTC Fluidigm PCR PTCH2 Forward GCTTGAGGTGTAGATGTGTCC Fluidigm PCR Reverse CCCACGGGGCAAGATTTGAA Fluidigm PCR PTPRC Forward ACCACAAGTTTACTAACGCAAGT Fluidigm PCR Reverse TTTGAGGGGGATTCCAGGTAAT Fluidigm PCR SHH Forward CTCGCTGCTGGTATGCTCG Fluidigm PCR Reverse ATCGCTCGGAGTTTCTGGAGA Fluidigm PCR SMAD2 Forward CGTCCATCTTGCCATTCACG Fluidigm PCR Reverse CTCAAGCTCATCTAATCGTCCTG Fluidigm PCR SNAI1 Forward TCGGAAGCCTAACTACAGCGA Fluidigm PCR Reverse AGATGAGCATTGGCAGCGAG Fluidigm PCR SNAI2 Forward CGAACTGGACACACATACAGTG Fluidigm PCR Reverse CTGAGGATCTCTGGTTGTGGT Fluidigm PCR SOX9 Forward GCTTCGTGCTTACTTCCAGGG Fluidigm PCR Reverse CATGCGGAGACCTAATGCCA Fluidigm PCR TCF3 Forward CCGACTCCTACAGTGGGCTA Fluidigm PCR Reverse CGCTGACGTGTTCTCCTCG Fluidigm PCR TGFB1 Forward GGCCAGATCCTGTCCAAGC Fluidigm PCR Reverse GTGGGTTTCCACCATTAGCAC Fluidigm PCR TGFB2 Forward CAGCACACTCGATATGGACCA Fluidigm PCR Reverse CCTCGGGCTCAGGATAGTCT Fluidigm PCR TGFB3 Forward ACTTGCACCACCTTGGACTTC Fluidigm PCR Reverse GGTCATCACCGTTGGCTCA Fluidigm PCR TWIST1 Forward GTCCGCAGTCTTACGAGGAG Fluidigm PCR Reverse GCTTGAGGGTCTGAATCTTGCT Fluidigm PCR

56

UBB Forward GGTCCTGCGTCTGAGAGGT Fluidigm PCR Reverse GGCCTTCACATTTTCGATGGT Fluidigm PCR VIM Forward AGTCCACTGAGTACCGGAGAC Fluidigm PCR Reverse CATTTCACGCATCTGGCGTTC Fluidigm PCR WNT11 Forward GGAGTCGGCCTTCGTGTATG Fluidigm PCR Reverse GCCCGTAGCTGAGGTTGTC Fluidigm PCR WNT5A Forward ATTCTTGGTGGTCGCTAGGTA Fluidigm PCR Reverse CGCCTTCTCCGATGTACTGC Fluidigm PCR WNT5B Forward CATGGCCTACATAGGGGAGG Fluidigm PCR Reverse CTGTGCTGCAATTCCACCG Fluidigm PCR ZEB1 Forward GATGATGAATGCGAGTCAGATGC Fluidigm PCR Reverse ACAGCAGTGTCTTGTTGTTGT Fluidigm PCR ZEB2 Forward CAAGAGGCGCAAACAAGCC Fluidigm PCR Reverse GGTTGGCAATACCGTCATCC Fluidigm PCR BMP2 Forward ACCCGCTGTCTTCTAGCGT RT-PCR Reverse TTTCAGGCCGAACATGCTGAG RT-PCR BMP3 Forward GCAGGGAGAGAGACCGAAG RT-PCR Reverse TGGACCGTGCTGTACCTGT RT-PCR BMP4 Forward GCCGGAGGGCCAAGCGTAGCCCTAAG RT-PCR Reverse CTGCCTGATCTCAGCGGCACCCACATC RT-PCR BMP7 Forward ATCGAGAGTTCCGGTTTGATCT RT-PCR Reverse GTCTCATTGTCGAAGCGTTCC RT-PCR BMPR1A Forward TCCTGGACTCAGCTCTATTT RT-PCR Reverse TGGTGTCTAGTGTAGCACAT RT-PCR BMPR1B Forward TACAAGCCTGCATAAGTGAAGAAGC RT-PCR Reverse TCATCGTGAAACAATATCCGTCTG RT-PCR BMPR2 Forward TCAGACAGAGGTTGGAAATC RT-PCR Reverse GGAACTTGGGTCTCTGTTT RT-PCR GAPDH Forward TGCACCACCAACTGCTTAGC RT-PCR Reverse GGCATGGACTGTGGTCATGAG RT-PCR BMP2 Forward AACGGGCCCAGCGGGGAAAT ChIP-PCR Reverse ACCCTCGCGGGGACTCTGGA ChIP-PCR Forward GGACGCGCTGGCCGAGGTGTGAT ChIP-TSS-PCR Reverse CCCCATTCGCGCTCGCCAGTTGA ChIP-TSS-PCR BMP3 Forward TCCGGGATCTTCCACACACCCT ChIP-PCR Reverse CGCCCCTCTCTCCCGCATCA ChIP-PCR Forward GCGCCGCAGCTGCTCCAATCC ChIP-TSS-PCR Reverse GGGCGCCGTCTCCACTCCA ChIP-TSS-PCR BMP4 Forward TCCCTGGGACAGACATGACACT ChIP-PCR Reverse TGGCAGGATGTCTTGGCCTTGC ChIP-PCR BMP7 Forward CCAAGACTGCACCAGGCCCTC ChIP-PCR Reverse GAGTGTGAATGCGGCTGGGCA ChIP-PCR Forward GCCCGGAGCCCCACACAGCT ChIP-TSS-PCR

57

Reverse GCGTCGAGCAGTCCTTAGTGAAAA ChIP-TSS-PCR BMP2 (site 1) Forward TGGGGGTGTATTGGAGTAAG Pyro-PCR Reverse ACCCATAACTCCAAACAACCATCCTAA Pyro-PCR N/A ATTGGAGTAAGGTAGAG sequencing BMP2 (site 2) Forward AAGGGTATTTGGTTTTAGGG Pyro-PCR Reverse CCCCCCCCATCACTCTACCTTACT Pyro-PCR N/A GGTATTTGGTTTTAGGGT sequencing BMP3 (site 1) Forward Qiagen Hs_BMP3_01 PM00112595 Pyro-PCR Reverse Qiagen Hs_BMP3_01 PM00112595 Pyro-PCR N/A Qiagen Hs_BMP3_01 PM00112595 sequencing BMP3 (site 2) Forward Qiagen Hs_BMP3_02 PM00112602 Pyro-PCR Reverse Qiagen Hs_BMP3_02 PM00112602 Pyro-PCR N/A Qiagen Hs_BMP3_02 PM00112602 sequencing BMP4 (site 1) Forward Qiagen Hs_BMP4_01 PM00056525 Pyro-PCR Reverse Qiagen Hs_BMP4_01 PM00056525 Pyro-PCR N/A Qiagen Hs_BMP4_01 PM00056525 Sequencing BMP4 (site 2) Forward TTTTTTTGGGGATGGGAGT Pyro-PCR Reverse AAAAAACCCAAACTCCCATAAAT Pyro-PCR N/A ATGGGAGTTTAGTTTATATTTT Sequencing BMP7 Forward TTTTTAGTTGTGTTTAGTGGGTTTAT Pyro-PCR Reverse ATCACCCCTTCCAAAAAACCTTCTATTT Pyro-PCR N/A GAGAGTTAGATAAATGTTTATTGAG sequencing

58

Supplementary Table S5. List of siRNA and shRNA sequences

Gene Type (clone Target sequence #)

BMP4 siRNA GAGCCAUGCUAGUUUGAUA

siRNA UAGCAAGAGUGCCGUCAUU

siRNA CGACACUUCUGCAGAUGUU

siRNA CAGGAUUAGCCGAUCGUUA

BMP7 siRNA GAGGUGCACUCGAGCUUCA

siRNA CAUCGAGAGUUCCGGUUUG

siRNA GAUCAGCGUUUAUCAGGUG

siRNA GCACAACUCGGCACCCAUG

EpCAM shRNA (#1) CCGGGCAAATGGACACAAATTACAACTCGAGTTGTAATTTGTGTCCATTTGCTTTTTG

shRNA (#2) CCGGGCCGTAAACTGCTTTGTGAATCTCGAGATTCACAAAGCAGTTTACGGCTTTTTG

shRNA (#3) CCGGGCTGGTTATTTCCAGAAAGAACTCGAGTTCTTTCTGGAAATAACCAGCTTTTTG

shRNA (#4) CCGGGCGTTATCAACTGGATCCAAACTCGAGTTTGGATCCAGTTGATAACGCTTTTTG

shRNA (#5) CCGGCGCGTTATCAACTGGATCCAACTCGAGTTGGATCCAGTTGATAACGCGTTTTTG

59

Supplementary References

Alimonti A, Carracedo A, Clohessy JG, Trotman LC, Nardella C, Egia A, Salmena L, Sampieri K, Haveman WJ, Brogi E, et al. 2010. Subtle variations in Pten dose determine cancer susceptibility. Nat Genet 42: 454-458.

Bommer GT, Feng Y, Iura A, Giordano TJ, Kuick R, Kadikoy H, Sikorski D, Wu R, Cho KR, Fearon ER. 2010. IRS1 regulation by Wnt/beta-catenin signaling and varied contribution of IRS1 to the neoplastic phenotype. J Biol Chem 285: 1928-19038.

Hendrix ND, Wu R, Kuick R, Schwartz DR, Fearon ER, Cho KR. 2006. Fibroblast growth factor 9 has oncogenic activity and is a downstream target of Wnt signaling in ovarian endometrioid adenocarcinomas. Cancer Res 66: 1354-1362.

Huang SS & Fraenkel E. 2009. Integrating proteomic, transcriptional, and interactome data reveals hidden components of signaling and regulatory networks. Sci Signal 2: ra40.

Huang de W, Sherman BT, Lempicki RA. 2009. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature protocols 4: 44-57.

Huang YW, Liu JC, Deatherage DE, Luo J, Mutch DG, Goodfellow PJ, Miller DS, Huang TH. 2009. Epigenetic repression of microRNA-129-2 leads to overexpression of SOX4 oncogene in endometrial cancer. Cancer Res 69: 9038-9046.

Huang YW, Luo J, Weng YI, Mutch DG, Goodfellow PJ, Miller DS, Huang TH. 2010. Promoter hypermethylation of CIDEA, HAAO and RXFP3 associated with microsatellite instability in endometrial carcinomas. Gyneco Oncol 117: 239-247.

Pyeon D, Newton MA, Lambert PF, den Boon JA, Sengupta S, Marsit CJ, Woodworth CD, Connor JP, Haugen TH, Smith EM, et al. 2007. Fundamental differences in cell cycle deregulation in human papillomavirus-positive and human papillomavirus-negative head/neck and cervical cancers. Cancer Res 67: 4605-4619.

Richardson AL, Wang ZC, De Nicolo A, Lu X, Brown M, Miron A, Liao X, Iglehart JD, Livingston DM, Ganesan S. 2006. X chromosomal abnormalities in basal-like human breast cancer. Cancer Cell 9: 121-132.

Talbi S, Hamilton AE, Vo KC, Tulac S, Overgaard MT, Dosiou C, Le Shay N, Nezhat CN, Kempson R, Lessey BA, et al. 2006. Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo- ovulatory women. Endocrinology 147: 1097-1121.

Wallace TA, Prueitt RL, Yi M, Howe TM, Gillespie JW, Yfantis HG, Stephens RM, Caporaso NE, Loffredo CA, Ambs S. 2008. Tumor immunobiological differences in prostate cancer between African-American and European-American men. Cancer Res 68: 927-936.

60

Wang G, Wang Y, Shen C, Huang YW, Huang K, Huang TH, Nephew KP, Li L, Liu Y. 2010. RNA Polymerase II binding patterns reveal genomic regions involved in microRNA gene regulation. PLoS One 5: e13798. doi: 10.1371/journal.pone.0013798.

Wu G, Feng X, & Stein L. 2010. A human functional protein interaction network and its application to cancer data analysis. Genome biology 11: R53.

Wu H, Chen Y, Liang J, Shi B, Wu G, Zhang Y, Wang D, Li R, Yi X, Zhang H, Sun L, Shang Y. 2005. Hypomethylation-linked activation of PAX2 mediates tamoxifen-stimulated endometrial carcinogenesis. Nature 438: 981-7.

Wurmbach E, Chen YB, Khitrov G, Zhang W, Roayaie S, Schwartz M, Fiel I, Thung S, Mazzaferro V, Bruix J, et al. 2007. Genome-wide molecular profiles of HCV-induced dysplasia and hepatocellular carcinoma. Hepatology 45: 938-947.

61