+E2 +TAM 21 Up-regulated genes 14 Down-regulated genes Microarray Gain of functionLoss of function PAX2 OverexpressionRNAi Cell proliferation Tumor growth ER downstream target Hypomethylated in cancer FACS Nude mice Promoter analysis; ER recruitment; etc MSP; Bisulfite sequencing Mechanism of PAX2 Hypomethylation ChIP; WB Endometrioid carcinomas Immunomagnetical purification Endometrial epithelial cells Hypomethylation-linked PAX2 Re-activation Is Associated with Tamoxifen-stimulated Endometrial Carcinogenesis Supplementary Figure 1 Schematic illustration of the project

Some E2 Genes Full Regulation Partial Regulation Unique TAM Genes Full Regulation E2 97 TAM NF I /B (nuclear factor I/B) RPIP8 (Rap2 interacting protein 8) EKLF (erythroid Kruppel-like factor) EREG1 (estrogen responsive element- associated Gene 1) MYL7 (myosin, light polypeptide 7) Cadherin-8 NTAK (neural- and thymus-derived activator for the ErbB kinase) Nebulette DLC-1 (deleted in liver cancer-1) PKC (protein kinase C) alpha TRIP9 (thyroid receptor interactor) Unknown (gb=AL109681) SH-PTP3 protein-tyrosine phosphatase Flt3 (Fms-like tyrosine kinase-3) ligand Phosphodiesterase I alpha MEA6 (meningioma-expressed antigen 6) Myelin proteolipid protein PAX2 (paired-box protein 2) IGF II (insulin-like growth factor II) Unknown (gb=AL049390) Carboxylesterase hKID (human kidney water channel) Heme oxygenase 1 (HO-1, EC ) GABA-B R1a receptor OPCML (opioid binding protein/cell adhesion molecule-like) Cyclin F AILIM (activation-inducible lymphocyte immunomediatory molecule) ENaC (amiloride-sensitive epithelial sodium channel) beta subunit Myogenic determining factor 3 (MYOD1) Potassium channel homolog (KCNQ3) Unknown (gb= ) Unknown (gb= ) Retinoic Acid Receptor, Beta, Isoform 1 PRGP2 (proline-rich Gla protein 2) BK-2 (Bradykinin receptor) Up-regulated genes Down-regulated genes Table 1. Genes Regulated by both Estrogen and Tamoxifen in Endometrial Carcinomas Supplementary Figure 2 Genomic action of tamoxifen Experimentally-supported genomic view of tamoxifen action. See text for detailed explanation. Table 1 shows a list of genes that are up- and down- regulated by oestrogen and tamoxifen in cEECs. The complete lists of the genes and the other information on microarray experiments can be seen in biochemistry/MicroarryData/index.htm or in Gene Expression Omnibus site with accession number: GSE biochemistry/MicroarryData/index.htm

Wet weight (g) LE (  M) C E2 TAM C E2 TAM a Relative levels of mRNA c PCNA Ki-67 b E2 TAM E2 TAM N N CC PAX2 PKC  EKLF RAR  1 Cyclin F rKID a: The wet weight of rat uteri under the treatment of oestrogen and tamoxifen. The ovariectomized adult female CD rats were housed for 14 days before treatment of 48 hours with 2.5 μg 17β-estradiol (E2)/rat (n = 5)/day, 500 μg tamoxifen (TAM)/rat (n = 5)/day, or vehicle only (C) (n=5). The uteri were weighed (left panel). b: Immunochemical staining for PCNA and Ki-67 in rat uteri. Uterine samples from above-treated rats were immersion-fixed in 4% formaldehyde at 4°C for 12 hours, stored at 4°C in 70% ethanol and embedded in paraffin for immunochemical staining for PCNA and Ki-67. c: Gene regulation by E2 and TAM in rat uteri. Uterine samples from above-treated rats were used for total RNA extraction, and the expression of mRNA was measured by real time RT PCR. Supplementary Figure 3 The growth stimulation of rat uteri by oestrogen and tamoxifen

aa PAX2 PAX2  Paired box Homeodomain Activation/Repression PAX2 (ng) PAX2  (ng) Increase in percentage of cells in S/G 2 /M phases a b PAX2 (ng) PAX2  (ng) PAX2 PAX2  c Supplementary Figure 4 PAX2-mediated endometrial cancer cell proliferation a: Construction of a PAX2 mutant with an activation/repression domain deletion. b: FACS analysis of ECC-1 cell proliferation under the treatment of E2 and tamoxifen. The PAX2 mutant had a dominant negative effect which could be rescued by wild type PAX2. c: Western blotting analysis of PAX2 and the PAX2 mutant expression.

 E2 TAM  E2 TAM V hKID Cyclin F RAR  1 No RNAi RNAi No infection Vector hKID No infection Vector Cyclin F No infection Vector RAR  1 hKID Cyclin F RAR  1 hKID Cyclin F No RNAi Vector hKID No RNAi Vector Cyclin F No RNAi Vector RAR  1 a b c d No infection Vector RAR  1  -gal No RNAi Vector RAR  1  -gal Increase in percentage of cells in S/G 2 /M phases Increase in percentage of cells in S/G 2 /M phases  -actin Supplementary Figure 5 The effect of hKID, cyclin F, or RAR  1 on the growth of endometrial carcinoma cells.

cEECs nEECs PAX2 promoter  E2 TAM  E2 TAM cEECs nEECs PAX2 promoter  E2 TAM  E2 TAM cEECs nEECs EREG1 promoter  E2 TAM  E2 TAM Real time PCR measurements of the PAX2 and EREG1 promoter fragments immunoprecipitated by antibodies against ER , ER , MeCP2, mSin3A, or HDAC1. The primer sequences for PAX2 promoter: forward: 5’- CGCCACCTCGGACATC-3’, reverse: 5’-CCAAGTGCTGGCGAGTTG-3’, and probe sequence: 5’-CCGGGATTGCTACTTCTCTGCCA-3’. The primer sequences for EREG1 promoter: forward: 5’-CCACTAACCGCGGGTATTAGG- 3’, reverse: 5’-CCAGAGGGACCAAGTTCACTAAGA-3’, and probe sequence: 5’-TCGATGACCCCTCCGATTCCGTC-3’. ab c Relative levels of DNA content Supplementary Figure 6 Real time PCR quantification of ChIP DNAs