Primer SequenceFHIT fwd TGTGCAGAGGGCCTGTGGCAGFHIT rev GCCTAGAAGGCTTTAGTTGGCCCC

RARB fwd CCGGCTCCTCCCCTGCTCATTTTRARB rev AAAACTCCCAGCAGCCCTGCAA

CADM1 fwd CTGCACGCCCGGTGCCTTCCCADM1 rev GGTGCAGCACAGCCGGCCTCSFRP5 fwd CCAGGCGGCCGGAGATTGGSFRP5 rev CCCCCGGCCCTGACTCTACCMBD1 fwd CACAGCTGTTCGGCGGGGAGMBD1 rev CTTCCCTCCCGCGGTCTCCA

WNT3A fwd GCGGTGCACTCGCTGCTCTTWNT3A rev TAACCCAGGGGAGACGCGGGBACH1 fwd GGGAACACCTGTCGTGGGCGBACH1 rev TTGCTGGGGTGGGGGTGTGT

DNMT3A fwd GGAGGTCACTGGCGGACTGGTDNMT3A rev TGAAGCCCACGCTTTGCCCGDNMT3B fwd CAAGGGGAGTCGGCACCGCCCCCDNMT3B rev GCTCGGAGCGTCCACGCGGGCTCDKN2A fwd TTTCGAGGGCCTTTCCTACCTGGTCCDKN2A rev TGACCCTCCGGATTCGGCGCG

DLC1 fwd AGCTGTTGGCACTGAAACAGGCTDLC1 rev AGCTGAGCTGCATTCAGTGAGATTT

KDM6B fwd GCTGGTGCAACAACATTGCCTGGAKDM6B rev GGCGCCACACCACGCCAACACROBO1 fwd TTTGCACGAGTGAGGAAAAGGAAGTROBO1 rev TGGCATGCAAAGACAAGAACGTGTSOCS3 fwd GGCTCAGCCCCAAGGACGGASOCS3 rev GCTCCCGGGACTGAGCGAGASFRP1 fwd GGGGGAGGTGGTGACTGGAGGTGSFRP1 rev TGCCCTGGAAGTCCCAAGTCAGGATIMP3 fwd GGTTGGAAGAGGGCAGCATGGGTIMP3 rev TCCACAGCTGGAGGAGGCCC

KDM6A fwd AAGCTCCCTCTCGGTGACAACCCKDM6A rev GCGGGACGCAGGCCAATTCTAG

Supplemental table 1 : List of primers used for Me-DIP coupled Q-PCR.

TP731,2 17971771 SOX11 20564139 NKX6-1 20564139TERT1,2 19513524 SLIT21 17609981 NOL4 18381458

SOCS21,2 15161694 SCGB3A11 20496080 PAX1 20564139SOCS11,2 15161694 RPRM1 15700311 PCDH10 20833385SFRP51,2 19513747 ROBO31 16700909 POU2F3 16607278

RASSF11,2 17097722 RARB1 16806674 PTEN 15196854PRDM51,2 20213097 MAL1 19662663 PTGS2 17578348

PGR1,2 14760078 IFNK1 19887612 PTPN6 19836067ONECUT11,2 18398837 CDKN2A1 20186007 ROBO1 16700909

ITGA91,2 19885927 CALCA1 16736496 SFRP1 16434596HSPA21,2 15161694 APC 20208141 SFRP2 19038436

HOXA111,2 19609949 C13orf18 19843677 SFRP4 19038436HIC11,2 12773202 C15orf48 16434596 SLIT1 16700909CHFR1,2 17971771 CCNA1 20208141 SLIT3 16700909CDH131,2 14750164 CTDSPL2 19885927 SPARC 20208141CADM11,2 20208141 DAPK1 20208141 STAC 19885927BRCA11,2 15126331 DKK3 19003969 TFPI2 20208141TIMP32 16803511 DLC1 16862168 TNFRSF10C 17894941SOCS32 17894941 ESR1 16736496 WT1 20833385PRDM22 20159667 FANCF 15126331 RRAD 16434596OPCML2 18714356 FHIT 11297252 CDH1 17894941

ZMYND101 17097722 HS3ST2 17894941 TIMP2 14712492TWIST11 15713962 IL10 18378443 MLH1 15161694

TNFRSF10D1 14999791 LHFPL4 18381458 MYOD1 14760078THBS11 17578348 LMX1A 20564139 SYK 20563729

    MGMT 11297252 MT1G 16434596

Supplemental table 2. 77 genes reported to be methylated in cervical neoplasia with a supporting PMID reference number; 1 identifies those genes with increased methylation following transfection of PHFK with episomal HPV16; 2

those with increased methylation following HPV18 transfection; and 1,2 those hypermethylated by both HPV types. Of 5837 genes with increased methylation in HPV16 or HPV18 transfected cells, 35 (0.6%) were also aberrantly methylated in cervical neoplasia as were 42 (0.3%) of those 14048 genes which were not hypermethylated following transfection with these high risk HPV types.

PHFK HPV18 PHFK HPV18 PHFK HPV180

5

10

15

20

25ROBO1

Rela

tive

fold

chan

ge

PHFK HPV18 PHFK HPV18 PHFK HPV180.92

0.94

0.96

0.98

1

1.02

1.04

1.06

1.08SOCS3

Rela

tive

fold

chan

ge

PHFK HPV18 PHFK HPV18 PHFK HPV180

0.2

0.4

0.6

0.8

1

1.2DLC1

Rela

tive

fold

chan

ge

PHFK HPV18 PHFK HPV18 PHFK HPV180

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5 CADM1

Rela

tive

fold

chan

ge

PHFK HPV18 PHFK HPV18 PHFK HPV180

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6 FHIT

Rela

tive

fold

chan

ge

PHFK HPV18 PHFK HPV18 PHFK HPV180

0.5

1

1.5

2

2.5

3

3.5BACH 1

Rela

tive

fold

chan

ge

Supplemental figure 1. Changes in methylation status detected using Me-DIP coupled Q-PCR in 3 additional PHFK donors (A, B and C) following transfection with episomal HPV18.

A B C A B C A B C

A B C A B C

PHFK HPV18 PHFK HPV18 PHFK HPV180

0.2

0.4

0.6

0.8

1

1.2 SFRP1

Rela

tive

fold

chan

ge

A B C

A B C

A B C

A B C

PHFK HPV18 PHFK HPV18 PHFK HPV180

0.5

1

1.5

2

2.5MBD1

Rela

tive

fold

chan

ge

PHFK HPV18 PHFK HPV18 PHFK HPV180

0.2

0.4

0.6

0.8

1

1.2 SFRP5

Rela

tive

fold

chan

ge

Number of pairs

Freq

uenc

y500 550 600 650 700

050

0010

000

1500

020

000 B

Number of pairs

Freq

uenc

y

1300 1350 1400 1450 1500 1550 1600

050

0010

000

1500

020

000

2500

0 A

Number of pairs

Freq

uenc

y

250 300 350 400 450 500

050

0010

000

1500

020

000

2500

0

C

Number of pairs

Freq

uenc

y

700 750 800 850 900

050

0010

000

1500

020

000

D

Supplemental figure 2. Simulation analysis designed to determine whether the observed frequency of concordant methylation changes in adjacent genes could result from a random distribution of HPV-induced methylation changes.

Following the exclusion of 1596 genes with over-lapping transcriptional start sites; 5188 genes were found to have increased methylation following HPV16 transfection of which 3336 were located immediately adjacent to another hypermethylated gene; of 3290 genes with decreased methylation in HPV16 transfected cells, 1622 were immediately adjacent to another hypomethylated gene. Of 2599 genes found to have increased methylation following HPV18 transfection, 1305 were located immediately adjacent to another hypermethylated gene; of 3822 genes with decreased methylation in HPV18 transfected cells, 2025 were immediately adjacent to another hypomethylated gene. Histograms show the distribution of the number of adjacent pairs in 100000 simulated gene sets each containing 5188 genes (Panel A), 3290 genes (Panel B), 2599 genes (Panel C) and 3822 genes (Panel D) randomly selected from the total of 18389 genes.

10p15.14 15q235 2p13.12 5q31.14

10q21.21 15q25.32 2p24.33 5q31.32

10q24.312 16p13.34 2p25.12 5q33.14

11p135 16q22.12 2q22.15 6p22.12

11p14.31 17q125 2q22.34 6p235

11p15.42 17q21.24 2q24.14 6q152

11p15.55 17q23.25 2q24.22 6q215

11q14.34 17q25.34 2q32.22 6q265

11q22.22 18q11.22 2q33.24 7p132

11q23.31 18q12.14 2q33.34 7p21.11

12q21.315 19p13.112 2q345 7q31.12

12q21.332 19p13.23 2q37.31 7q31.22

12q23.15 19q13.112 3p14.14 8p125

13q21.15 19q13.22 3p14.24 8q12.12

13q21.322 19q13.424 3p26.22 8q21.22

13q22.13 1p21.32 3q27.14 8q21.35

13q22.25 1p34.31 3q285 8q22.32

14q11.22 1p36.112 4p15.312 8q24.213

14q24.15 1q21.14 4q122 9p11.22

14q24.25 1q32.25 4q13.34 9p24.15

14q24.35 1q415 4q232 9q21.132

14q32.24 1q435 4q28.32 9q34.34

15q125 20p11.212 4q31.214 Xp22.22

15q21.12 20p12.14 4q32.32 Xp22.312

15q21.33 20q13.22 5p13.25 Xq252

22q13.14 5q155

HPV16↑ HPV18↑ HPV16↓ HPV18↓10q26.3 10q26.3 10q11.23 14q11.211p15.5 11p15.5 12q13.2 15q15.211q13.1 12q24.33 14q11.2 18q21.32

12q24.33 13q31.3 19q13.33 19q13.3313q31.3 14q32.33 19q13.41 19q13.4113q34 16p13.3 1p21.1 1p21.1

14q32.33 16q24.3 1p34.2 1p35.215q12 17p13.3 1p35.2 1q21.1

16p13.3 17q25.3 1p36.21 1q23.216q22.1 18p11.21 1q21.1 3q21.116q24.3 19p13.11 1q23.2 5q33.217q25.1 19p13.3 1q23.3 6q2617q25.3 19q13.43 1q44 7q21.1218q21.1 1p36.31 20q11.23 9p21.1

19p13.11 1p36.32 20q13.31 9p22.319p13.3 1p36.33 3q13.2 9q21.321p36.31 1q42.13 3q21.1

1p36.32 20q13.33 3q25.331p36.33 21q22.3 6p21.11q42.13 22q11.21 6p24.120p12.3 22q13.33 6p24.2

20q13.33 2p13.1 6q2622q11.21 2p16.3 7p21.222q13.33 4p16.3 9p11.2

2p25.3 5p15.33 9p13.32q31.1 5q31.3 Xq21.332q37.3 6q16.2 Xq27.2

5p15.33 7p15.2

7p15.2 7p22.27p22.2 7p22.37p22.3 7q36.38q24.3 8p23.39q34.2 8q24.39q34.3 9q21.31

Xp22.31 9q34.3Xq28

11q13.316q24.216q24.317p13.317q25.319p12

19p13.1119q122q35

2q36.12q36.32q37.14p16.14p16.24p16.3

4q31.214q35.2

CBA

Supplemental table 3. Panel A lists HPV16 and HPV18 hypermethylation and hypomethylation hotspots. Panel B lists those loci of HPV integration reported in the literature which could be mapped onto the promoter array; each locus is supported by a reference (301, 312, 323, 334, 345). Panel C and D lists those regions of chromosomal loss and gain reported in Wilting et al., 2007 (37) which could be mapped onto the promoter array.

3q13.23q21.11q44

1p21.13q25.331p35.2

D

Change in methylation

statusIncreased Decreased

CpG content*

low n = 4126

273 (6.6%)

944 (22.0%)

intermediate n = 1999

259

(13.0%)406

(20.3%)

high n = 8763

1552 (17.7%)

1773 (20.2%)

Marked by H3K4 or by

H3K4me3K27me3 in HES**

neither n =4883

396 (8.1%)

1094 (22.4%)

H3K4me3 n = 10061

1584 (15.7%)

2170 (21.6%)

H3K4me3K27me3 n = 1745

411

(23.6%)266

(15.2%)

Supplemental table 4. Change in methylation status following transfection of PHFK with episomal HPV18 is related to promoter CpG content and to the pattern of histone marking in human embryonic stem (HES) cells. Risk of increased methylation increases with increasing CpG content and is greatest for genes bivalently marked by H3K4me3 and H3K27me3 in HES cells; risk of decreased methylation is lowest for bivalently marked genes. *Analysis restricted to genes listed on Weber et al., array (2007) (42). **Analysis restricted to genes listed on Zhao et al., array (2008)(47).

ALOX5^ BMP8A* FOXD4L4**CHRNA3^ BNC1* FOXD4L1**

FBN2^ GATA4* PHOX2A**FOXE3^ IRX5* SOX21**HS6ST3^ KCNC2*  

HTR7^ KCNC4*  PENK^ KCNK12*  SOX1^ MLNR*  

SOX14^ NEFM*  SPOCK2^ TBX5*  SSTR2^ TP73*  TBX3^ CTGF*  NINL^ NPTX2*  

RAB32^ SIM1*  SH3GL2^ VGF*  

  SOX8*  

Supplemental table 5. Age-related polycomb target genes which are significantly more likely to be methylated in cervical smears taken from women with HPV associated CIN than in normal cervical cells and to have increased methylation following transfection of PHFK with episomal HPV16 and HPV18: ^increased methylation following transfection of PHFK by HPV16 alone; *increased methylation following transfection by both HPV16 and HPV18; ** increased methylation following transfection by HPV18 alone (Teschendorff et al., 2010) (48).

Signalling Pathway

Increased methylation following transfection of PHFK with HPV16 High CpG content* H3K4me3 marked in HES cells** Marked by both H3K4Kme3 and

H3K27me3 in HES cells**

Observed Expected p value Observed Expected p value Observed Expected p value Observed Expected p value

WNT signaling pathway 142 55.65 1.43E-22 160 92 6.64E-11 153 154.65 4.68E-01 73 27.35 2.02E-13

Cadherin signaling pathway 66 22.8 1.22E-13 60 44.93 1.79E-02 52 102.43 2.47E-08 38 18.11 2.72E-05

PI3 kinase pathway 51 14.75 1.27E-13 66 42.79 5.76E-04 62 44.19 6.44E-03 24 7.81 2.35E-06

TGF-beta signaling pathway 65 23.47 1.23E-12 92 38.51 1.63E-13 76 48.2 1.26E-04 38 8.52 8.81E-14

Insulin/IGF pathway-protein kinase B signaling cascade 40 12.74 7.78E-10 46 29.95 3.79E-03 35 42.18 1.51E-01 24 7.46 1.07E-06

Alpha adrenergic receptor

signaling pathway 19 3.35 3.24E-09 25 4.28 6.33E-12 24 4.02 1.06E-11 6 0.71 9.68E-05

Angiogenesis 75 38.89 1.60E-07 107 77.02 6.71E-04 108 84.36 7.25E-03 34 14.92 1.40E-05

Muscarinic acetylcholine receptor 1 and 3 signaling pathway 31 10.73 3.38E-07 37 19.26 2.07E-04 39 20.08 1.16E-04 14 3.55 2.12E-05

Alzheimer disease-presenilin pathway 51 24.81 2.55E-06 65 40.65 2.54E-04 62 58.25 3.28E-01 28 10.3 3.55E-06Metabotropic glutamate receptor

group I pathway 18 5.36 1.35E-05 23 4.28 2.12E-10 18 10.04 1.48E-02 11 1.78 2.69E-06

Supplementary Table 6A. Ontological profile of genes with increased methylation following transfection of PHFK with episomal HPV16: pathway analysis. The top ten signalling pathways over-represented among genes with increased methylation following HPV16 transfection are enriched significantly (p < 0.001, in bold) for genes with a high CpG content and for those bivalently marked by H3K4me3 and H3K27me3 in HES cells. *Reference list for this analysis comprises genes with a low CpG content; ** and for this analysis, those unmarked by either H3K4me3 or by H3K27me3 in HES cells. Source: Panther Gene.

Supplementary Table 6B. Ontological profile of genes with increased methylation following transfection of PHFK with episomal HPV16: biological processes. All of the top ten biological processes over-represented among genes with increased methylation following HPV16 transfection are enriched significantly (p < 0.001, in bold) for genes bivalently marked by H3K4me3 and H3K27me3 in HES cells, and in all but one of these processes (ectoderm development), for genes with a high CpG content *Reference list for this analysis comprises genes with a low CpG content; ** and for this analysis, those unmarked by either H3K4me3or by H3K27me3 in HES cells. Source: Panther Gene.

Biological processIncreased methylation following transfection of PHFK with HPV16 High CpG content** H3K4me3 marked in HES cells** Marked by both H3K4Kme3 and by

H3K27me3 marked in HES cells**

Observed Expected p value Observed Expected p value Observed Expected p value Observed Expected p value

segment specification 126 32.18 2.32E-36 114 62.05 1.91E-09 46 64.27 1.02E-02 117 11.37 4.09E-76

embryonic development 221 85.83 6.02E-35 233 179.72 6.67E-05 173 224.95 1.58E-04 154 39.78 1.09E-44nucleobase, nucleoside, nucleotide and

nucleic acid metabolic process 1228 888.43 2.70E-33 2142 922.12 4.39E-301 2350 1028.33 9.17E-317 461 181.85 1.25E-78

nervous system development 466 275.58 2.55E-27 613 487.8 1.02E-08 500 554.34 8.57E-03 333 98.03 1.36E-84

pattern specification process 166 67.05 8.19E-25 180 132.65 4.71E-05 112 158.67 5.16E-05 140 28.06 8.27E-53

primary metabolic process 2357 2020.92 1.22E-21 4170 2843.39 1.71E-191 4658 3181.41 5.42E-208 803 562.59 5.23E-33

system development 691 487.46 2.70E-20 906 943.52 1.00E-01 762 1080.55 2.89E-27 480 191.08 4.42E-81

developmental process 976 737.56 4.77E-20 1313 1489.09 1.88E-07 1215 1709.2 5.27E-43 566 302.25 7.26E-53

metabolic process 2435 2120.83 6.08E-19 4316 2986.73 3.15E-189 4826 3360.16 3.29E-201 830 594.2 2.93E-31

ectoderm development 502 335.26 7.19E-19 655 633.29 1.90E-01 535 727.06 6.25E-15 364 128.57 3.23E-72

Supplementary Table 6C. Biological processes under-represented among genes with increased methylation following HPV 16 transfection. Of the top ten biological processes most under-represented among genes with increased methylation following HPV16 transfection, all were also found to significantly depleted (p < 0.001, in bold) among genes bivalently marked by H3K4me3 and H3K27me3 in HES cells, and all but one (ectoderm development), among genes with a high CpG content *Reference list for this analysis comprises genes with a low CpG content; ** and for this analysis, those unmarked by either H3K4me3or by H3K27me3 in HES cells. Source: Panther Gene.

Biological processes

Increased methylation following transfection of PHFK with HPV16 High CpG content* H3K4me3 marked in

HES cells**

H3K4Kme3K27me3 marked in HES cells compared with genes

marked by neither H3K4me3 nor H3K4me3 (Zhao 2008)

Observed Expected p value Observed Expected p value Observed Expected p value Observed Expected p valueantigen processing and presentation 136 246.08 3.64E-15 158 725.29 1.50E-152 19 78.33 8.36E-16 9 13.85 1.16E-01antigen processing and presentation of peptide or polysaccharide antigen

via MHC class II11 47.61 1.70E-10 18 134.79 5.06E-37 7 56.24 1.34E-16 1 9.94 5.13E-04

B cell mediated immunity 413 536.41 3.12E-09 579 1335.04 5.39E-137 71 303.28 3.02E-59 34 53.63 2.47E-03blood coagulation 18 46.94 1.15E-06 27 115.53 2.48E-23 92 331.4 5.14E-56 33 58.6 1.61E-04

cellular calcium ion homeostasis 63 107.95 1.56E-06 70 278.13 1.72E-51 24 36.15 2.10E-02 4 6.39 2.35E-01cellular defense response 55 96.55 2.53E-06 68 269.58 7.83E-50 150 423.79 5.79E-55 51 74.94 1.82E-03

complement activation 649 760.36 4.46E-06 918 1831.41 3.51E-149 39 152.64 2.53E-28 27 26.99 5.26E-01fertilization 96 146.17 4.86E-06 136 380.83 4.46E-49 46 162.69 1.48E-27 17 28.77 1.22E-02

immune response 72 105.94 2.68E-04 78 293.11 1.04E-51 177 827.49 6.31E-176 78 146.33 9.71E-11immune system process 72 105.94 2.68E-04 78 293.11 1.04E-51 979 2137.01 6.97E-209 303 377.9 4.79E-06

  Observed Expected p value

Pathway analysis

Ubiquitin proteasome pathway 24 7.74 2.09E-06PDGF signaling pathway 34 19.79 2.22E-03

Blood coagulation 5 13.34 8.51E-03

Biological processes

nucleobase, nucleoside, nucleotide and nucleic acid metabolic process 637 570.06 1.27E-03

cell motion 173 140.26 3.45E-03tricarboxylic acid cycle 11 4.3 4.81E-03

cell cycle 320 279.22 6.66E-03primary metabolic process 1363 1296.73 9.80E-03

Supplemental table 7. Ontological profiling: pathways and biological processes significantly over-represented (p < 0.01) among genes with decreased methylation following HPV16 transfection.

H & E

DNMT1

DNMT3B

CIN 3Normal cervical

epithelium

Supplementary Figure 3: Changes in the topography of DNMT1 and DNMT3B expression in HPV16 and HPV18 associated CIN3. Representative example of the pattern of DNMT expression in normal cervical epithelium and in CIN3 cases. DNMT1 is seen to be consistently up-regulated throughout CIN3, and DNMT3B expression is found in cells in all layers unlike normal epithelium where it is restricted to nuclei of basal and parabasal cells.