+E2 +TAM 21 Up-regulated genes 14 Down-regulated genes Microarray Gain of function Loss of function PAX2 Overexpressi on RNAi 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
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+E2 +TAM 21 Up-regulated genes 14 Down-regulated genes Microarray Gain of functionLoss of function PAX2 OverexpressionRNAi Cell proliferation Tumor growth.
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+E2 +TAM
21 Up-regulated genes14 Down-regulated genes
Microarray
Gain of function Loss of function
PAX2
Overexpression RNAi
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
FullRegulation
PartialRegulation
Unique TAM Genes
Full Regulation
E297
TAM114
35
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-8NTAK (neural- and thymus-derived activator for the ErbB kinase) NebuletteDLC-1 (deleted in liver cancer-1)PKC (protein kinase C) alphaTRIP9 (thyroid receptor interactor)Unknown (gb=AL109681)SH-PTP3 protein-tyrosine phosphataseFlt3 (Fms-like tyrosine kinase-3) ligandPhosphodiesterase I alpha MEA6 (meningioma-expressed antigen 6) Myelin proteolipid proteinPAX2 (paired-box protein 2)IGF II (insulin-like growth factor II) Unknown (gb=AL049390)Carboxylesterase
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 http://medicine1.bjmu.edu.cn/department/ biochemistry/MicroarryData/index.htm or in Gene Expression Omnibus site with accession number: GSE3013.
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 1 16 143 256 278 330 415
PAX2
PAX2
Paired box Homeodomain Activation/Repression
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PAX2PAX2
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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 RAR1No RNAi RNAi
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Supplementary Figure 5 The effect of hKID, cyclin F, or RAR1 on the growth of endometrial carcinoma cells.
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cEECs nEECsPAX2 promoter
E2 TAM E2 TAM
cEECs nEECsPAX2 promoter
E2 TAM E2 TAM
cEECs nEECsEREG1 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’.
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Supplementary Figure 6Real time PCR quantification of ChIP DNAs