+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

hKID (human kidney water channel)Heme oxygenase 1 (HO-1, EC 1.14.99.3) GABA-B R1a receptorOPCML (opioid binding protein/cell adhesion molecule-like) Cyclin FAILIM (activation-inducible lymphocyte immunomediatory molecule)ENaC (amiloride-sensitive epithelial sodium channel) beta subunit Myogenic determining factor 3 (MYOD1)Potassium channel homolog (KCNQ3)Unknown (gb=3413939)Unknown (gb=3882154)Retinoic Acid Receptor, Beta, Isoform 1PRGP2 (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 http://medicine1.bjmu.edu.cn/department/ biochemistry/MicroarryData/index.htm or in Gene Expression Omnibus site with accession number: GSE3013.

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PCNA Ki-67b

E2

TAM

E2

TAM

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PAX2 PKC EKLF RAR1 Cyclin F rKID0

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ControlE2TAM

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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PAX2 (ng) 0 100 100 500PAX2 (ng) 0 0 500 100

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rce

nta

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cells

in S

/G2/

M p

ha

ses

a

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PAX2 (ng) 0 100 100 500PAX2 (ng) 0 0 500 100

PAX2PAX2

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 RAR1No RNAi RNAi

No in

fect

ion

Vec

tor

hKID

No inf

ectio

n

Vec

tor

Cyclin

F

No

infe

ctio

n

Vec

tor

RAR

1

hKID Cyclin F RAR1

RAR1 hKID Cyclin F

No RNAi

Vec

tor

hKID

No RNAi

Vec

tor

Cyclin

F

No RNAi

Vec

tor

RAR1

a

b

c

d

No in

fect

ion

Vec

tor

RAR1

-gal

No RNAi

Vec

tor

RAR1

-gal

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VectorhKIDCyclin FRAR-beta1

Incr

ease

in p

erce

ntag

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lls in

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pha

ses

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ease

in p

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lls in

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-actin-actin-actin

-actin-actin-actin

Supplementary Figure 5 The effect of hKID, cyclin F, or RAR1 on the growth of endometrial carcinoma cells.

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ER-alphaER-beta

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MeCP2mSin3AHDAC1

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ER-alphaER-beta

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’.

a b

c

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ten

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A c

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ten

t

Supplementary Figure 6Real time PCR quantification of ChIP DNAs