SUPPLEMENTAL DATA

Breast Cancer-associated Fibroblasts Confer AKT1-mediated

Epigenetic Silencing of Cystatin M in Epithelial Cells

Huey-Jen L. Lin, Tao Zuo, Ching-Hung Lin, Chieh Ti Kuo, Sandya Liyanarachchi, Shuying Sun, Rulong Shen, Daniel E. Deatherage, Dustin Potter, Lisa Asamoto, Shili Lin, Pearlly S. Yan, Ann-Lii Cheng, Michael C. Ostrowski, and Tim H.-M. Huang

SUPPLEMENTAL TEXT

Exposure to cancer-associated fibroblasts induces promoter hypermethylation of CpG island loci in breast epithelia cells

Microarray analysis showed that the expression of 109 was concurrently down- regulated in MCF10A cells co-cultured with cancer-associated fibroblasts (C4, C12 and C15) relative to those co-cultured with normal fibroblasts (N16 and N23) and the mock control (MCF10A cells cultured in the absence of fibroblasts) (Table S2). Among the down-regulated loci, 56 have a CpG island at their 5’ regulatory region (See Figure 2 and Table S2 for the list of down-regulated genes; other related descriptions can be found in the main text).

To investigate whether repressed expression results from promoter hypermethylation, 8 genes (CST6, KLK10, RARRES1, C15orf48, ENC1, SOX9, GRHL1 and GRHL3) were randomly chosen for further analysis. Selected regions of their promoter CpG islands were amplified by methylation-specific PCR (MSP) (Figure S3). Concurrent hypermethylation was detected in three (C4, C8, C15) of five sets of MCF10A cells exposed to cancer-associated fibroblasts (Figure S3B). Except for N30, this concurrent event was not observed in the co-cultured cells exposed to normal fibroblasts. From the clinicopathological information, fibroblasts of N30 were obtained from an individual with a diagnosis of atypical ductal hyperplasia of the breast (Table S1). This pre-neoplastic condition may impose the concurrent hypermethylation state observed in MCF10A cells in their immediate surroundings. Negligible methylation was observed in monotypic fibroblasts without the co-culture treatment (right panels, Figure S3). These findings suggest that de novo methylation detected by MSP was likely the result of microenvironmental influences on MCF10A cells.

Detailed methylation analysis of the CST6 CpG islands (a 310-bp region) was further conducted in seven sets of co-cultured samples using bisulfite sequencing (Figure S4).

1 The results showed that one outer flank (right side) of this CpG island was densely methylated in these samples. To a lesser extent, the promoter and regions flanking the first also have increased methylation densities in cells co-cultured with cancer- associated fibroblasts, compared to counterparts with normal fibroblasts. The increase in methylation was generally associated with the down-regulation of CST6. This finding is in close agreement with MassARRAY and expression data shown in Figure 3 (see the main text).

SUPPLEMENTAL EXPERIMENTAL PROCEDUES

Methylation Specific-PCR (MSP) and Bisulfite Sequencing

Approximately 0.5 μg of genomic DNA was subjected to sodium bisulfite treatment, a process that converts unmethylated cytosine to uracil without affecting methylated cytosine, using the EZ DNA Methylation Kit (Zymo Research Inc., Orange, CA). The resultant DNA was amplified by MSP. The primers for CST6 and RARRES1 were obtained from previous reports (1, 2). Other primer sequences and PCR amplification conditions are listed in Table S4. The PCR products were visualized by electrophoresis in 2% agarose gel containing ethidium bromide. Sequencing of bisulfite-treated, PCR- amplified products was performed with primers described previously (1). The PCR products were cloned into pCR2.1 TOPO vector (Invitrogen) and were subjected to conventional sequencing.

2

Figure S1

Vimentin Prolyl-4-hydroxylase

Figure S1. Characterization of Primary Breast Fibroblasts. Immunofluorescence study was conducted to stain two putative fibroblast marks, i.e. vimentin and prolyl-4- hydroxylase, on primary fibroblasts isolated from human breast tissues.

Figure S2

Figure S2. Morphological assessment of MCF10A cells co-cultured with fibroblasts. Fibroblasts generally grow into monolayer sheaths adhering to the Matrigel- containing dishes. In contrast, MCF10A cells grow into aggregates and anchor on the top of fibroblastic sheaths. These MCF10A spheres could appear as either globular structures with smooth margins (left) or ragged balls with branches (right), depending on the given fibroblasts isolated from various breast tissues. Both kinds of the morphologies become prominent at the second week and their representative images are captured in phase-contrast photographs.

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Figure S3

A Co-Cultured MCF10A Samples Monotypic Stromal Fibroblasts

I

I

10A_N16 10A_N23 10A_N26 10A_N30 10A_C4 10A_C8 10A_C11 10A_C15 10A_C17 10A_Mock SSs Water N16 N23 N26 N30 C4 C8 C11 C15 C17 SSs Water M CST6 U M HOXA9 U

B Normal Cancer Normal Cancer

10A_N16 10A_N23 10A_N26 10A_N30 10A_C4 10A_C8 10A_C11 10A_C15 10A_C17 10A_Mock N16 N23 N26 N30 C4 C8 C11 C15 C17 CST6 RARRES1 SOX9 C15orf48 KLK10 GRHL3 ENC1 GRHL1 HOXA9

Figure S3. DNA Methylation Analysis of Candidate Genes in Breast Epithelial Cells Exposed to Tumor Microenvironment. A, Methylation-specific PCR analysis (MSP). Representative results of MSP analysis of CST6 in either co-cultured MCF10A or monotypic stromal fibroblasts were shown in the upper panel, while MSP of HOXA9 in co-culture samples were illustrated in lower panel. B, MSP results of 9 genes are summarized in a diagram. While black squares indicate a sample harbors detectable methylation, white boxes signify undetectable methylation.

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Figure S4

Figure S4. Bisulfite Sequencing and Quantitative RT-PCR Analyses of the CST6 Promoter. The methylation status of each CpG site at the CST6 promoter, derived from bisulfite sequencing, was determined in MCF10A cells co-cultured respectively with 2 normal fibroblasts (N23 and N30) or with 4 cancer-associated fibroblasts (C11, C4, C15 and C8). A mock control (omitting fibroblasts during co-culture) was included in the analysis. Methylated sites are symbolized with black circles; whereas the un-methylated sites are denoted with open circles. The average methylation level in each sample is shown in the middle panel and is inversely correlated with the level of CST6 expression (right panel). The landscape plots reveal an overall increase in CST6 methylation in MCF10A cells co-cultured with cancer-associated fibroblasts, compared to the counterparts exposed to normal fibroblasts.

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Figure S5

Figure S5. Quantitative ChIP-PCR Assays on Control Genes in AKT1- and Vector- Transfected MCF10A Cells. Upper left panel, Trimethyl-H3K27 was dramatically enriched at the HOXA9 locus, but not at GAPDH, in AKT1 transfected cells. This finding was consistent with the previous studies demonstrating that the repressive histone mark confers a epigenetic silencing at HOXA gene cluster loci (3, 4). In the low left and upper right panels, moderate enrichment of dimethyl-H3K9 and DNA methyltransferase 1 (DNMT1) was apparent at the HOXA9 locus in both vector and AKT1 transfected cells, while a minimal effect was observed at the GAPDH locus in both cell types. In the low right panel, acetyl-H3K9 was negligibly enriched at both the HOXA9 and GAPDH loci, but its enrichment could be detected at the FGFR1 locus, a putative AKT target gene.

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Figure S6

Figure S6. The Negligible CST6 Methyltion in Mock-Transfected Controls of MCF10A Cells. MassARRAY analysis assessed methylation levels of the CST6 CpG island in four independent replicates of mock-transfected controls of MCF10A cells.

7 SUPPLEMENTAL TABLES

Table S1. Clinicopathological Information of Breast Tissues Used for Isolating Fibroblasts Grade Tumor Lymph Age/ Histology Progesterone Case (Bloom- marginal metast. HER2/neu Race Diagnosis receptor receptor Richardson) invasion (met./total) C4 48, Invasive ductal 3, poorly No No (0/2) Positive Positive Negative White Ca, DCIS differentiated C6 46. Invasive ductal 3, poorly No No (0/6) Negative Negative Negative White Ca differentiated C8 81, Invasive ductal 3, poorly No No (0/4) Negative Negative Negative White Ca, DCIS differentiated C11 65, Invasive ductal 3, poorly No No (0/6) Negative Negative Negative Black Ca differentiated C12 41, Mixed invasive 2 , moderately No Yes (8/24) Positive Positive Negative White ductal and lobular differentiated Ca, DCIS C15 51, Invasive lobular 1, well No Yes (5/19) Positive Positive Negative White Ca differentiated C17 54, Invasive lobular 3, poorly Yes Yes (25/25) Negative Negative Negative White Ca , lobular differentiated carcinoma in situ C22 50, Invasive lobular N/A Yes Yes (5/11) N/A N/A N/A Black Ca C26 57, Invasive ductal 3, poorly Yes Yes (5/24) Negative Negative Negative Black Ca differentiated C30 76, Invasive ductal 2 , moderately Yes Yes (6/7) Positive Positive Negative White Ca, DCIS differentiated C41 68, Invasive lobular 1, well No Yes (1/4) Positive Positive Negative White Ca differentiated C45 60, Invasive ductal 3, poorly Yes Yes (3/20) Positive Positive Positive White Ca differentiated N10 51, Benign breast N/A N/A N/A N/A N/A N/A White tissue N11 21, Benign breast N/A N/A N/A N/A N/A N/A Black tissue N13 41, Benign breast N/A N/A N/A N/A N/A N/A White tissue N14 54, Ductal N/A N/A N/A N/A N/A N/A White hyperplasia N16 51, Ductal N/A N/A N/A N/A N/A N/A White hyperplasia N23 28, Benign breast N/A N/A N/A N/A N/A N/A White tissue N26 19, Benign breast N/A N/A N/A N/A N/A N/A White tissue N30 42, Atypical ductal N/A N/A N/A N/A N/A N/A White hyperplasia

8 Table S2. A List of Down-regulated Genes Observed in MCF10A Cells Exposed to Cancer-associated Fibroblasts Fold c Fold c a b Gene Symbol Gene Name CpG 10A_CAF/ 10A_CAF/ Score (d) 10A_NF 10A_Mock LCN2 Lipocalin 2 No -7.3 -6.3 -4.25 ALDH1A3 Aldehyde dehydrogenase 1 Yes -6.5 -3.6 -3.46 family, member A3 IVL Involucrin No -6.5 -7.3 -3.13 SCEL Sciellin No -10.3 -4.4 -3.09 RARRES1 Retinoic acid receptor Yes -12.0 -16.7 -3.06 responder (tazarotene induced) 1 CEACAM1 Arcinoembryonic antigen- No -5.2 -3.1 -2.92 related cell adhesion molecule 1 C15orf48 15 open reading Yes -7.5 -3.4 -2.92 frame 48 CST6 Cystatin E/M Yes -5.2 -2.0 -2.89 AZGP1 Alpha-2-glycoprotein 1, zinc- No -6.0 -17.3 -2.85 binding ENC1 Ectodermal-neural cortex (with Yes -3.5 -3.8 -2.84 BTB-like domain) VGLL1 Vestigial like 1 (Drosophila) No -5.0 -2.2 -2.80 GPR110 G -coupled receptor 110 No -4.6 -2.3 -2.69 SPRR3 small proline-rich protein 3 No -9.4 -5.4 -2.68 KLK6 Kallikrein-related peptidase 6 No -4.5 -2.8 -2.64 A2ML1 Alpha-2-macroglobulin-like 1 Yes -3.7 -3.5 -2.62 KRT23 Keratin 23 (histone No -5.7 -6.5 -2.56 deacetylase inducible) PTGS2 Prostaglandin-endoperoxide Yes -4.6 -4.8 -2.53 synthase 2 SYTL2 Synaptotagmin-like 2 Yes -3.6 -3.6 -2.53 SPRR1A Small proline-rich protein 1A7 No -5.4 -3.4 -2.52 KIAA1946 Hypothetical protein KIAA1946 No -4.6 -3.3 -2.51 CYP4B1 Cytochrome P450, family 4, No -9.4 -3.4 -2.49 subfamily B, polypeptide 1 KRT4 Keratin 4 No -10.4 -8.3 -2.47 EPS8L1 Epidermal growth factor Yes -3.5 -1.7 -2.46 receptor pathway substrate 8 like 1 S100P S100 calcium binding protein P Yes -10.1 -8.0 -2.45 IKZF2 IKAROS family zinc finger 2 No -4.6 -2.0 -2.44 SOX9 SRY (sex determining region Yes -5.1 -14.7 -2.42 Y)-box9 MXD1 MAX dimerization protein 1 Yes -3.4 -1.8 -2.38 PSCA Prostate stem cell antigen No -8.3 -4.8 -2.36 GRHL1 Grainyhead-like-1 Yes -3.2 -3.8 -2.36 SH3TC2 SH3 domain and No -3.1 -1.5 -2.33 tetratricopeptide repeats 2

9 INPP4B Inositol polyphosphate-4- Yes -3.5 -1.2 -2.32 , type II DAPP1 Dual adaptor of No -3.1 -1.8 -2.31 phosphotyrosine and3- phosphoinositides CEACAM6 Carcinoembryonic antigen- No -3.8 -4.6 -2.30 related cell adhesion molecule 6 CLIC3 Chloride intracellular channel 3 No -5.5 -1.7 -2. 28 IGFL1 IGF-like family member-1 No -3.8 -2.8 -2.27 CAMK2N1 Calcium/calmodulin-dependent Yes -4.3 -1.4 -2.26 protein kinase II inhibitor 1 SLC6A14 Solute carrier family 6 (amino No -3.0 -6.5 -2.26 acid transporter), member 14 LOC647859 occludin pseudogene No -3.1 -3.8 -2.25 APOBEC3A Apolipoprotein B mRNA editing No -2.7 -3.8 -2.25 , catalytic polypeptide- like 3A KRT13 Keratin 13 No -7.1 -5.8 -2.24 B4GALT4 UDP-Gal:betaGlcNAc beta 1,4- Yes -3.0 -1.7 -2.23 galactosyltransferase, polypeptide 4 GDPD3 Glycerophosphodiester Yes -4.0 -1.7 -2.22 domain containing 3 DUSP5 Dual specificity phosphatase 5 Yes -2.9 -2.4 -2.21 ELF3 E74-like factor 3 (ets No -3.5 -2.2 -2.21 transcription factor, epithelial- specific ) MUC16 Mucin 16, cell surface No -5.7 -4.0 -2.19 associated VSTM2L V-set and transmembrane No -3.4 -1.4 -2.19 domain containing 2 like PDK4 Pyruvate dehydrogenase Yes -8.5 -5.9 -2.18 kinase, isozyme 4 RAB11FIP1 RAB11 family interacting Yes -4.0 -3.3 -2.16 protein 1 TLR1 Toll-like receptor 1 No -3.1 -4.3 -2.15 SAMD9 Sterile alpha motif domain No -3.1 -4.7 -2.14 containing 9 MUC15 Mucin 15, cell surface No -6.3 -2.5 -2.11 associated GABRP Gamma-aminobutyric acid A No -12.3 -8.5 -2.10 receptor PLEKHA7 Pleckstrin homology domain Yes -3.3 -2.4 -2.09 containing, family A member 7 GCNT3 Glucosaminyl (N-acetyl) No -2.8 -2.4 -2.07 transferase 3 TMOD3 Tropomodulin 3 (ubiquitous) Yes -2.7 -1.2 -2.06 HRASLS3 HRAS-like suppressor 3 Yes -3.0 -1.8 -2.05 FYB FYN binding protein No -3.2 -5.8 -2.04 ARHGDIB Rho GDPdissociation inhibitor No -2.9 -2.9 -2.00 (GDI) beta

10 VTCN1 V-set domain containing T cell No -2.5 -3.3 -1.99 activation inhibitor 1 MALL Mal, T-cell differentiation No -2.9 -2.1 -1.99 protein like C10orf54 Chromosome 10 open reading Yes -4.3 -1.9 -1.98 frame 54 EGR3 Early growth response 3 Yes -3.5 -18.2 -1.97 KRT81 Keratin 81 Yes -3.2 -2.3 -1.93 RHBDL2 Rhomboid, veinlet-like2 No -2.4 -2.2 -1.93 TACC1 Transforming, acidic coiled-coil Yes -2.9 -1.9 -1.90 containing prtein 1 CXCL17 Chemokine (C-X-C motif) No -2.9 -1.6 -1.90 ligand 17 IL1RN Interleukin 1 receptor No -2.9 -2.6 -1.89 antagonist DKK1 Dickkopf homolog 1 (Xenopus Yes -6.4 -1.4 -1.88 laevis) FAM63B Family with sequence similarity Yes -2.6 -1.5 -1.87 63, member B SNF1LK SNF1-like kinase No -2.5 -1.7 -1.86 KRT7 Keratin 7 Yes -2.8 -1.6 -1.86 MYO5B Myosin VB Yes -2.6 -2.3 -1.85 PINK1 PTEN induced putative kinase No -2.7 -1.4 -1.85 1 SLCO3A1 Solute carrier organic anion Yes -2.5 -2.8 -1.84 transporter family member 3A1 NEBL Nebulette Yes -5.1 -3.1 -1.83 KRT78 Keratin 78 No -2.6 -1.7 -1.83 FLJ20674 Hypothetical protein FLJ20674 Yes -2.7 -1.5 -1.81 SLC4A11 Solute carrier family 4, sodium Yes -2.3 -2.4 -1.81 borate transporter member 11 SLC17A5 Solute carrier family 17, Yes -2.9 -1.4 -1.79 (anion/sugar transporter) member 5 ADAMTS9 ADAM metallopeptidase with Yes -2.4 -1.2 -1.78 thrombospondin type 1 motif 9 CTGF Connective tissue growth factor Yes -2.9 -1.2 -1.78 EMP1 Epithelial membrane protein 1 No -2.5 -1.9 -1.77 EPB41L1 Erythrocyte membrane protein Yes -2.4 -1.8 -1.76 band 4.1-like 1 DSC2 Desmocollin 2 Yes -2.2 -2.3 -1.74 GGT6 Gamma-glutamyltransferase 6 No -2.6 -2.5 -1.74 homolog (rat) ANXA3 Annexin A3 No -2.8 -2.8 -1.72 KLK10 Kallikrein-related peptidase 10 Yes -4.7 -7.6 -1.72 STS (microsomal) Yes -2.8 -2.9 -1.71 isozyme S DAPK1 Death-associated protein Yes -2.9 -1.4 -1.70 kinase 1 MYH14 Myosin, heavy chain 14 Yes -2.5 -2.1 -1.69 CRCT1 Cysteine-rech C-terminal 1 No -2.8 -1.4 -1.67

11 SLC20A1 Solute carrier family 20, Yes -2.1 -1.9 -1.67 (phosphate transporter) member 1 CTSL2 Cathepsin L2 Yes -2.8 -4.2 -1.67 GRHL3 Grainyhead-like 3 Yes -2.3 -4.0 -1.66 SLC12A6 Solute carrier family 12, Yes -2.9 -1.1 -1.65 (potassium/chloride transporters) member 6 ERBB3 V-erb-b2 erythroblastic No -2.6 -1.5 -1.65 leukemia viral oncogene homolog 3 (avian) PI3 Peptidase inhibitor 3, skin- No -4.1 -5.5 -1.65 derived (SKALP) C9orf58 Chromosome 9 open reading Yes -2.1 -1.7 -1.64 frame 58 CLDN7 Claudin 7 Yes -2.5 -2.1 -1.63 C1orf74 open reading Yes -2.2 -2.3 -1.63 frame 74 TUFT1 Tuftelin 1 Yes -2.6 -1.7 -1.62 LOC283278 Hypothetical protein No -2.2 -1.6 -1.62 LOC283278 GAS2L3 Growth arrest-specific 2 like 3 Yes -2.9 -1.1 -1.62 GPRC5A G protein-coupled receptor, No -2.9 -2.4 -1.62 family C, group 5, member A CRIM1 Cysteine rich transmembrane Yes -2.4 -1.1 -1.62 BMP regulator 1 MYO6 Myosin VI Yes -2.3 -1.5 -1.61 TRIM2 Tripartite motif-containing 2 No -2.6 -5.6 -1.60 FUT1 Fucosyltransferase 1 Yes -2.3 -1.1 -1.59 (galactoside 2-alpha-L- fucosyltransferase, H blood group) AZGP1 Alpha-2-glycoprotein 1, zinc- No -2.6 -6.5 -1.59 binding Note: a) Genes are ordered according to their SAM Score (d) listed in the most right column in table; b) Score (d) is produced by an extension of a T-test statistic analysis by SAM software; c) The negative values in fold columns indicate the decreased in MCF10A co-cultured with CAF (10A_CAF), as compared to the counterparts with NF (10A_NF) or with mock control (10A_Mock, omitting fibroblasts).

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Table S3. Clininopathological Information of the Primary Breast Tumors a

Breast cancer Normal breast Variable n = 194 n = 28 Age a 55 ± 15 51 ± 17 Stage I 33 II 85 III 45 IV 6 Unknown 25 Grade (Bloom- Richardson) 1 22 2 92 3 63 unknown 17 Estrogen receptor Positive 132 Negative 58 unknown 4 Progesterone receptor Positive 92 Negative 91 unknown 11 HER2/neu Positive 22 Negative 50 unknown 122 Phospho-AKT1 Positive 40 Negative 32 unknown 122 Histology Invasive ductal Ca 159 Invasive lobular Ca 10 DCIS 5 Others 20 Note: a) The numerics in the table indicate the number of cases under each given category, except the “Age” in which the Means ± standard deviations of patients’ age are shown.

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Table S4. Nucleotide Sequence of PCR Primers and Amplification Conditions Primer sequence 5 ’to 3’ Size Tm a KLK10 MSP Forward AAGTTTATGGTTTTTCGTGCGC 136bp 64 ºC Reverse TACTTATTTCCGCAATACGCGAC KLK10 USP Forward GAAGTTTATGGTTTTTTGTGTGT 136bp 60 ºC Reverse CTACTTATTTCCACAATACACAAC C15orf48 MSP Forward GTCGGTAGGTAAGGAAGATCGC 146bp 68 ºC Reverse AAAACGCGAAAAACCTAACTCG C15orf48 USP Forward GGGGTTGGTAGGTAAGGAAGATTGT 146bp 64 ºC Reverse CAAAAAACACAAAAAACCTAACTCA SOX9 MSP Forward CGTATTAGTTTCGGCGAATTC 172bp 64 ºC Reverse CCGCTTCTTAATACGTAACCGA SOX9 USP Forward AGTTGTATTAGTTTTGGTGAATTT 172bp 60 ºC Reverse AACCACTTCTTAATACATAACCAAA ENC1 MSP Forward CGGTTCGTCGTTTTTTTGTTC 191bp 66.5 ºC Reverse CTAATCGAAACTCCCGAATACTCG ENC1 USP Forward GATTGGTTTGTTGTTTTTTTGTTT 191bp 61 ºC Reverse CCTCTAATCAAAACTCCCAAATACTCA GRHL1 MSP Forward TCGCGGAGTTTTAGGGATTGTC 161bp 68 ºC Reverse TCTCGATTCTCGCAAACTATCACG GRHL1 USP Forward ATATTGTGGAGTTTTAGGGATTGTT 161bp 62 ºC Reverse TCTCAATTCTCACAAACTATCACACCC GRHL3 MSP Forward TCGGGTTTTTTATGTGGTCGTAGC 131bp 68 ºC Reverse CAAACGATCGACGACGACTTC GRHL3 USP Forward GTTGGGTTTTTTATGTGGTTGTAGT 131bp 62 ºC Reverse CCAAACAATCAACAACAACTTC INPP4B PCR Forward GAAGGGCAGCACTTTCTTCCTA 172bp 60 ºC Reverse TTACGGAGATCTGCACCAGTGT CST6 Sequenom Forward AGGAAGAGAGGTTTTTTGGGTTTTTTGAATTT 403bp 56 ºC Reverse CAGTAATACGACTCACTATAGGGAGAAGGCT TTACTACCCATATTATAACTAACCAC Note: a) Tm indicates the annealing temperatures.

14 Table S5. Nucleotide Sequence of Primers and PCR Condition Used in ChIP-PCR Assays. a Primer sequence 5 ’to 3’ Size Tm b CST6 ChIP PCR -1 Forward CGTCAGTTAGAAATCAGGTCTCCA 158bp 60 ºC Reverse GGGAAGAGTTAGAAGACGGACAAG CST6 ChIP PCR -2 Forward ACCATGTTTCCCTCACTCCTTATC 249bp 60 ºC Reverse GGTACCTCTGACTCCCACTGACTT CST6 ChIP PCR -3 Forward CTGACCTGTGTTTCTGACCTATGG 138bp 60 ºC Reverse CAGCCTCAGGTCTTTATTTTTGGT CST6 ChIP PCR -4 Forward AAGATTCAACCAGTGAATCAGCAG 103bp 60 ºC Reverse GAAAGGAGATCTTTCAGCAAGGAA CST6 ChIP PCR -5 Forward CACAAACTGGAGTCTGTCCCTTTA 191bp 60 ºC Reverse ATTTCCCACACAGAGACCTAGACC CST6 ChIP PCR -6 Forward GCGTTGACTACTTCAACACTGCTT 232bp 60 ºC Reverse CATAGTGAGCCGTCTCTACCAAAA CST6 ChIP PCR -7 Forward CAGCATTCCACTGAAGGCTATATG 240bp 60 ºC Reverse GTGATTGTCGGGTATGTCAGGTAA CST6 ChIP PCR -8 Forward TAAGATGGCAGAGCTGACATGACT 133bp 60 ºC Reverse GAAATAAATGGTGACACTGGCTTG CST6 ChIP PCR -9 Forward CGCTGAGCAGTAATGTGTCCTACT 186bp 60 ºC Reverse TCTTACTGTTGCCACCATTTCCTA CST6 ChIP PCR -10 Forward GGAGAACTCCGGGACCTGT 115bp 60 ºC Reverse TGTGCGTGTCTCGGAAGTAG CST6 ChIP PCR -11 Forward TTTGAGGTCCTTGTGGTTCC 197bp 58 ºC Reverse GCGAAGCACTTGGAAGAAAC CST6 ChIP PCR -12 Forward TGAGTTCCTGGGCTCTGAGT 142bp 58 ºC Reverse GAGGCAGTAACCCTGCTTTG GAPDH ChIP PCR Forward GTTGCAACCAAATTGCCAGA 200 bp 62 ºC Reverse GGCAAGGGGTAGAAGGGAAC HOXA9 ChIP PCR Forward CTCACACTTTGTCCCTGACTGACT 160 bp 62 ºC Reverse GGATTTGAAGGGAGGAGACACTTA Note: a) The order of CST6 ChIP PCR number is followed from 5’ to 3’ boundary of CST6 promoter region illustrated in Figure 6. b) Tm indicates the annealing temperatures.

15 SUPPLEMENTAL REFERENCES

1. Ai, L., Kim, W. J., Kim, T. Y., Fields, C. R., Massoll, N. A., Robertson, K. D., and Brown, K. D. Epigenetic Silencing of the Tumor Suppressor Cystatin M Occurs during Breast Cancer Progression. Cancer Res, 66: 7899-7909, 2006. 2. Youssef, E. M., Chen, X. Q., Higuchi, E., Kondo, Y., Garcia-Manero, G., Lotan, R., and Issa, J. P. Hypermethylation and silencing of the putative tumor suppressor Tazarotene-induced gene 1 in human cancers. Cancer Res, 64: 2411- 2417, 2004. 3. Cao, R., Wang, H., He, J., Erdjument-Bromage, H., Tempst, P., and Zhang, Y. Role of hPHF1 in H3K27 methylation and Hox gene silencing. Mol Cell Biol, 28: 1862-1872, 2008. 4. Rinn, J. L., Kertesz, M., Wang, J. K., Squazzo, S. L., Xu, X., Brugmann, S. A., Goodnough, L. H., Helms, J. A., Farnham, P. J., Segal, E., and Chang, H. Y. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell, 129: 1311-1323, 2007.

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