Supplementary Materialsand Methods

Supplementary Materialsand Methods

Antibodies and Reagents

The sources of the antibodies used in this study were: anti-FLAG (Sigma); anti-ER (Santa Cruz); anti-UTX (Bethyl Laboratories); anti-JHDM1D, anti-H3, anti-H3K27ac (Abcam); anti- H3K27me3, anti-H3K27me2, anti-H3k27me1 (Merck Millipore); anti-CXCR4 (Abclonal);anti-EZH2 (BD Bioscience); anti-CBP, anti-JMJD3 (Genetex); anti-Myc tag (Cell Signaling Technology).17-estradial (E2) and AMD3100 were from Sigma. CXCL12 was from Peprotech. Tissue microarrays were purchased from Alenabio. siRNAs were synthesized from GenePharma. The siRNA sequences were: ER-1 UCUUGAGCUGCGGACGGUUC, ER-2 GCCGUGUACAACUACCCCGA; UTX-1 GACCACTCAGAUAGUGAAU, UTX-2 GUGUGAAGUCAAUAGUACC; JHDM1D UUAGAUUCCUCCUUAAAGUUGGAUG; CBP GCUCAAGGGCAGCCGAACA.

Silver Staining and Mass Spectrometry

A stable MCF-7 cell line expressing FLAG-ER was produced by transfection of the cells with FLAG-tagged ER construct and selection in medium containing 1 mg/ml of G418. MCF-7 cells stably transfected with FLAG-ERor control empty plasmid were deprived of estrogen for 3days and 100 nM E2 or vehicle was then added for 30 min. Nuclear lysates were subsequently extracted andincubated in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.3% NP40, 2 mM EDTA and 15% glycerol) for 30 min at 4 ºC before incubated with 0.2 ml anti-FLAG M2 affinity agarose beads (Sigma) for 3 h at 4 ºC. Beads were subsequently washed with lysis buffer, and protein complexes bound to the beads FLAG protein complexes were elutedand resolved on 4-12% Bis-Tris gel (Invitrogen), which was then silver stained. Specific bandswere subsequently excised and analyzed by LC-MS/MS sequencing.

Immunoprecipitation and Western Blotting

Five hundredmicrogram of nuclear extract of MCF-7 cells was incubated with suitable antibodies (2-3 μg) at 4 ºC overnight, and60 μl of 50% protein A or G agarose beads were then added for an additional 2 h. Beads were subsequently washed 5 times using lysis buffer.Immune complexes were eluted by boiling the beads for 10 min and resolved using 8% SDS-PAGE gels. Western blotting was performed using standard procedure andimmunoreactive bands were visualized withwestern blotting luminal reagent (Merck Millipore).

GST Pull-down Assay

GST fusion or His fusion constructs,cloned in pGEX4T-3 vector (GE) or pET28a vector (Novagen) respectively, were expressed in BL21 Escherichia coli cells.The in vitro transcription and translation experiments were carried outusingrabbit reticulocyte lysate (TNT systems, Promega) by standard methods. In GST pull-down assays, we mixed 4 μg GST orindicatedGST fusion proteins with appropriate amount ofin vitro transcribed/translated products (6-40 l). Otherwise, 4 μg GST or GST-fused full-length, A/B, E of ERwere incubated with4 μg His-fusedfull-length UTX fusion in the presence or absence of 1 mM E2. The incubation was done in binding buffer (75 mM NaCl, 50 mM HEPES, pH 7.9) at 4 ºC for 2h and associated proteins were detected using indicated antibodies by western blotting following previously described procedure1.

RT-PCR and Real-time RT-PCR (qPCR)

Total RNA from cellswere extractedusing TRIzol reagent (Invitrogen). First strand cDNA synthesis was then carried out using3g total RNAwith TransScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech).qPCR was performedusing Power SYBR Green PCR Master Mix (Roche) and an ABI PRISM 7500 sequence detection system. Primers used for qPCR were listed in Supplementary Table S2. RNA18S5was used as the internal controlfor normalizing the quantitation of gene transcripts.

ChIP, qChIP and Re-ChIP

ChIP, qChIP and Re-ChIP experiments were essentially performed according to the procedure described previously2, 3. qChIP were detected using Power SYBR Green PCR Master Mix (Roche)by qPCR.Primers used for ChIP and qChIP were listed in Supplementary Table S2.

Lentiviral Production and Infection

The coding sequence of UTX was introduced into pLVX-IRES-purovector (Clontech), and plasmids with coding sequence cloned or empty vector was then co-transfected into HEK293T cells with the packaging plasmids pVSVg (AddGene 8454) and psPAX2 (AddGene 12260) to produce lentiviruses. 1 × 106MCF-7 cells were seeded in each well of the 6-well plates the day before the infection. Cells were infected with adequate amount of lentivirus and culture media was supplemented with 1.5 μg/ml puromycin for four days for selection.

Cell Proliferation Assay

The proliferation of UTX-KO MCF7 cells was monitored using TransDetect Cell Counting Kit (TransGen Biotech). Either wild-type or UTX-KO MCF-7 cells were plated at 5,000 cells/well in 96-well tissue culture plate, treated with vehicle or 100 nM E2, and incubated for 5 days. Cells were switched to fresh medium every 2 days. Subsequently, 10 μl of WST-8 solution was mixed with the culture medium and the reaction mixture was incubated at 37C in a 5% CO2 atmosphere for 2 h. The optical density was measured spectrophotometrically at 450 nm. Each experiment was performed in triplicate and repeated at least three times.

Colony Formation Assay

Wild-type or UTX-KO MCF7 cells were seeded at 5,000 cells/well in a 6-well plate and treated with vehicle or 100 nM E2 for 21 days, during whichfresh medium was switched every 2 days. Cells were fixed with 4% paraformaldehyde, and crystal violet staining was performed before cell colonies were photographed.

Wound Healing Assay

MCF-7 cells were seeded onto24-well plates.A scratch was generatedwith a 200 μl pipette tipwhen the cell confluency reached approximately90%, and this was considered astime point 0, when culture medium was also replaced to DMEM/0.5% FBS. CXCL12(200 ng/mL) or AMD3100 (50M) were added tothe cells and pictures were taken at time point 0 and after 48 h. Images were analyzed and thewidth of the wound was measured for control cells and cells treated with CXCL12 or AMD3100 at 0 h and 48 h. The effectiveness of treatments in affecting migration was evaluated by the ratioof the width of the wound at48 h divided by which at 0 h.

Cell Migration Assay

Regularly cultured MCF-7 cells were starvedfor 24h in DMEM with 0.5% FCS prior to the experiments. A totalof 1 × 105 cells were plated in the upper cavity of a Falcon cell culture insert with8-m pore size(BD Biosciences) in DMEM/0.5% FBS. DMEM/2.5% FBSwas added to the lower chamberof the insert, supplied with or without AMD3100 (50μM) or CXCL12(200 ng/mL). Cells on the upper side were allowed to migrate towards lower sidefor 24 h at 37°C.Non-migrant cells remained in the upper cavity were subsequentlywiped outusing cotton swabs, and the migrant cells on the lower chamberwere stained with 0.1%crystal violet and counted under microscopy. Theexperiment was repeated at least three times.

Supplementary References

1Liang J, Zhang H, Zhang Y, Zhang Y, Shang Y (2009). GAS, a new glutamate-rich protein, interacts differentially with SRCs and is involved in oestrogen receptor function. EMBO reports 10: 51-57.

2Wang Y, Zhang H, Chen Y, Sun Y, Yang F, Yu W et al (2009). LSD1 is a subunit of the NuRD complex and targets the metastasis programs in breast cancer. Cell 138: 660-672.

3Zhang H, Yi X, Sun X, Yin N, Shi B, Wu H et al (2004). Differential gene regulation by the SRC family of coactivators. Genes & development 18: 1753-1765.

Supplementary Tables

Table S1. Mass Spectrometry Analysis of ER-containing Protein Complex in E2-stimulated MCF-7 cells

Band / Identified proteins / Peptides
180 kDa / BRG1 / TLPVAQAYQDNLYR
VSQASSYPDVK
YQSSQGDIGVSQSR
LVALScHGTR
SPQLTAYR
SSSQLGSPDVSHLIR
180 kDa / TOP2B / LLQAETASNSAR
QAQLAQTLQQQEQASQGLR
DSAQTSVTQAQR
QQEQADSLER
LGHELQQAGLK
QELTSQAER
ASPAAPADGAAPAQPSIR
SGccDDSALAR
LSAALSSLQR
APAEEEVGPR
SAFLQVQPR
DLPDGHAASPR
SLDQYDFSR
AVASSPAATNSEVK
SPGPGPSQSPR
LQQLKDETGELSSADEKR
AGAKPDQIGIITPYEGQR
IPSEQEQLR
LKESQTQDNITVR
TEAANVEK
GNTSGSHIVNHLVR
IFDEILVNAADNK
ELILFSNSDNER
FLEEFITPIVK
150 kDa / NCOA3 / YLTESYGTGQDIDDR
QKEEESQQQAVLEQER
mSQSIIVSGESGAGK
LcAGASEDIREK
ILKEEQELYQK
ELFESSTNNNKDTK
IQAEVEAQLAR
SSEELLSALQK
STMMTQEQIQK
ELFESSTNNNK
ALGLNENDYK
STmMTQEQIQK
LcAGASEDIR
FAEFDQImK
STmmTQEQIQK
IQEEMEKER
FVEIHFNEK
ETDKQILQNR
NIRDDEGFIIR
VmLTTAGGTK
VPLKVEQANNAR
FAEFDQIMK
STGASFIR
IcVQGKEER
RGPAVLATK
LSFISVGNK
EEQELYQK
GPAVLATK
DDEGFIIR
LHLSSPDNFR
QKEEESQQQAVLEQERR
VNHWLTcSR
cIKPNLK
QAYEPcLER
SEVPLVDVTK
NLQTKNPNYIR
SLFPEGNPAK
YNYLSLDSAK
LQISHEAAAcITGLR
GISHVIVDEIHER
ELDALDANDELTPLGR
KVFDPVPVGVTK
TTQVPQFILDDFIQNDR
AIEPPPLDAVIEAEHTLR
QLYHLGVVEAYSGLTK
LAAQScALSLVR
VQSDGQIVLVDDWIK
QPAIISQLDPVNER
DKDDDGGEDDDANcNLIcGDEYGPETR
TPLHEIALSIK
GANLKDYYSR
KDKDDDGGEDDDANcNLIcGDEYGPETR
AAMEALVVEVTK
YQILPLHSQIPR
AAEcNIVVTQPR
SSVNcPFSSQDMK
AAmEALVVEVTK
QISRPSAAGINLmIGSTR
YPSPFFVFGEK
YTQVGPDHNR
VFDPVPVGVTK
LGGIGQFLAK
LSMSQLNEK
ISAVSVAER
MGGEEAEIR
DVVQAYPEVR
RISAVSVAER
KILTTEGR
KKEGETVEPYK
LAQFEPSQR
EGETVEPYK
NFLYAWcGK
LNQYFQK
DFVNYLVR
YDNGSGYR
VAFERGEEPGK
LNMATLR
EKIQGEYK
MTPSYEIR
YGDGPRPPK
KEGETVEPYK
LSmSQLNEK
AENNSEVGASGYGVPGPTWDR
150 kDa / UTX / ASGESEEASPSLTAEER
FGTNIDLSDDKK
ScSNTSALAAR
NGLSNSSILLDK
ESKPSGNILTVPETSR
VNTDYESSLK
TYIVHcQDcAR
cYESLILK
NQLSNSTQGLHK
ESYAIQYLQK
140 kDa / TRPS1 / GNTAEGcVHETQEK
ELEAHSAEAR
EIPSATQSPISK
AGALHAQVER
SPSLLQSGAK
HQAcLLLQK
QLMGQVLAK
YcHDLGER
ESPRPLQLPGAEGPAISDGEEGGGEPGAGGGAAGAAGAGR
FQQVPTDALANK
NALPPVLTTVNGQSPPEHSAPAK
LFGAPEPSTIAR
WTSQHSNTQTLGK
SLPTTVPESPNYR
VVEIVDEKVR
HSVVAGGGGGEGR
VVEIVDEK
YYSYGLEK
LGIGQSQEMNTLFR
DFQEETVKDYEAGQLYGLEK
FSHLTSLPQQLPSQQLMSK
SVQPQSHKPQPTR
MSAELAK
LQEYLGK
NLDIDPK
130 kDa / NRIP1 / SQYEQLAEQNR
SLLEGEGSSGGGGR
LASYLDKVR
IRLENEIQTYR
LAADDFRLK
QSVEADINGLR
LKYENEVALR
VTmQNLNDR
ALEESNYELEGK
RPEPSTLENITDDKYATVSSPSK
KADTALASIPPVAEGK
GEHVHSLPEASVSSKPDEGR
DFIIYREPNVSPSQVTENNFPEK
FLNADVQGIHSENGPVLVEELGKK
EESSEDENEVSNILR
TAADVVSPGANSVDSR
DcGSVSTVDEFPEAR
FLNADVQGIHSENGPVLVEELGK
KPSTSGQNFHSDVDYR
TAAAAADVNGFKPLSGNEQLK
AIVPPYSLcQTGEDLPK
EPNVSPSQVTENNFPEK
ERVEDFDHTSR
AIVPPYSLcQTGEDLPKDK
AVAEQVLHSQSR
NNGNSTSLPLSR
RPEPSTLENITDDK
SKQFYNQTYGSR
VEDFDHTSR
ESNYFGLSPEERR
GELDLSLENLDLSK
KDFIIYREPNVSPSQVTENNFPEK
QHAFSSHSSGSQSQK
AHPPTQILNR
ESNYFGLSPEER
QFYNQTYGSR
LLYEIQNR
SRDEGYQYHR
EDATPMDAYLR
ADTALASIPPVAEGK
ETVPVELEPKR
ETVPVELEPK
NVEYEIWLK
ERETVPVELEPKR
APPPESWNTVSGK
ERETVPVELEPK
EKQAIEESR
RTIQSLK
FFFNLGVK
DGScLFR
SLNPAVYR
QNIVLPSDEKGELDLSLENLDLSK
KLGLYR
DEQAFPALSSSSVNQSASQSSNPcVQR
LLYEIQNRDEQAFPALSSSSVNQSASQSSNPcVQR
GLVAAYSGESDSEEEQER
HDYDDSSEEQSAEDSYEASPGSETQR
TEQGEEEEEEEDEEEEEK
YGGISTASVDFEQPTR
AHLSENELEALEK
GSSYGVTSTESYKETLHK
ESATADAGYAILEK
SEAEQKLPLGTR
LDQQTLPLGGR
GQLQSHGVQAR
KQGIVTPIEAQTR
TYVPALEQSADGHK
RESATADAGYAILEK
INEDWLcNK
GSSYGVTSTESYK
MLPQAATEDDIR
EKYGIPEPPEPK
DGLGSDNIGSR
ESATADAGYAILEKK
QGIVTPIEAQTR
ASNIVMLR
NSFQPISSLR
QNLEIHR
YGIPEPPEPK
HQQLSGLHK
DMASNEGSR
110 kDa / TRIM24 / GSHTDAPDTATGNcLLQR
TQDGETALQLAIR
ESGAAEQVDNK
QPGANGEGEEEAR
SVAGGFVYTYK
LTISSPLEAHK
LVNNGEKLEFLHK
IVILEYQPSK
LVYILNR
70 kDa / MTA2 / ISGLIYEETRGVLK
ISGLIYEETR
LGKDAVEDLESVGK
ENAGEDPGLAR
66 kDa / ER / HGGGGGGFGGGGFGSR
GGSSSGGGYGSGGGGSSSVK
GSSSGGGYSSGSSSYGSGGR
TAAENDFVTLKK
GGSGGGGSISGGGYGSGGGSGGR
TLNNKFASFIDKVR
TLNNKFASFIDK
LLRDYQELmNVK
GFSSGSAVVSGGSR
GFSSGSAVVSGGSRR
KYEDEINKR
RSTSSFScLSR
TSQNSELNNmQDLVEDYKK
LLEGEEcR
FLEQQNQVLQTK
TAAENDFVTLK
VDPEIQNVK
FASFIDKVR
STSSFScLSR
AAFGGSGGRGSSSGGGYSSGSSSYGSGGR
IEISELNRVIQR
AQYEEIAQR
IEISELNR
YLDGLTAER
YEDEINKR
SKEEAEALYHSK
EQIKTLNNK
NPQNSSQSADGLR

Table S2.Primer List

Real-time RT-PCR Primers
Gene / Strand / Sequence
UTX / F / AAGGCTGTTCGCTGCTATGA
UTX / R / AGGCAGCATTCTTCCAGTAGTC
JHDM1D / F / GAGTCTTCCCAAGTGCCGAT
JHDM1D / R / TAGGTGAAGGGAGCCTGAGT
CBP / F / AGCAGGTGAAAATGGCTGAGA
CBP / R / CCAAAATCTGTGCTGTCATTCGC
GREB1 / F / GGCAGGACCAGCTTCTGA
GREB1 / R / CTGTTCCCACCACCTTGG
TFF1 / F / TTGTGGTTTTCCTGGTGTCA
TFF1 / R / CCGAGCTCTGGGACTAATCA
MYB / F / TGCTACCAACACAGAACCACA
MYB / R / GCGCTTTCTTCAGGTAGGGA
EBAG9 / F / GATGCACCCACCAGTGTAAAGA
EBAG9 / R / AGTCAGGTTCCAGTTGTTCCAAAG
CTSD / F / GTACATGATCCCCTGTGAGAAGGT
CTSD / R / GGGACAGCTTGTAGCCTTTGC
FOXA1 / F / CCTCTGGCGCCTCTAACCC
FOXA1 / R / TAGTGGGGGTCCCCTTTCAG
HRAS / F / CTCTCCTGACGCAGCACAA
HRAS / R / CGGTGGCATTTGGGATGTTC
SMAD3 / F / AGTTGAGGCGAAGTTTGGGC
SMAD3 / R / CTGCCCCGTCTTCTTGAGTT
KAT2B / F / CTGCAAGGCCGAGGAGTC
KAT2B / R / ATGGCTACAACTCCGACAGG
ZFPM1 / F / CAGGCGGAAACAGAGCAACC
ZFPM1 / R / AGTTAACATCTGCGCTGGGAG
PAK4 / F / CGAGCCGATGAGTAACCCG
PAK4 / R / TGATGCTGGTGGGACAGAAG
MMP9 / F / AAGGATGGGAAGTACTGGCG
MMP9 / R / GCTCCTCAAAGACCGAGTCC
CXCR4 / F / GGCAGCAGGTAGCAAAGTGA
CXCR4 / R / GCCCATTTCCTCGGTGTAGT
CLDN9 / F / GGTGCATCTGGACTGGACAA
CLDN9 / R / ATCAGGCCAAGGTCGAAAGG
CXCR7 / F / GAGGCTAGAGGCTCCTTTCTG
CXCR7 / R / GTTCTGAGGCGGGCAATCAA
JMJD3 / F / TCCAATGAGACAGGGCACAC
JMJD3 / R / CTTTCACAGCCAATTCCGGC
ESR1 / F / GGGAATGATGAAAGGTGGGAT
ESR1 / R / GGTTGGCAGCTCTCATGTCT
RNA18S5 / F / CAGCCACCCGAGATTGAGCA
RNA18S5 / R / TAGTAGCGACGGGCGGTGTG
ChIP Primers
Gene / Strand / Sequence
TFF1 promoter / F / CACCCCGTGAGCCACTGT
TFF1 promoter / R / CTGCAGAAGTGATTCATAGTGAGAGAT
GREB1 promoter / F / GGAGCCCTTCATCAGTCAACA
GREB1 promoter / R / TTCATGAACCTCCCTCGCTC
GATA3 promoter / F / GCTGTGAGCAGGAGAAGATG
GATA3 promoter / R / TCAGGGCTGAATTTCCATAT
qChIP Primers
Gene / Strand / Sequence
GREB1 / F / CTAGCCTAGTGCCAGGGAGA
GREB1 / R / GAACCAAATGCTGGAGTCGC
EBAG9 / F / TGTACCTCTCCAGGCCGATT
EBAG9 / R / TTCCGAAGGCGGAGTAAGGT
CTSD / F / TGGAGCAGGGAAGTGGTTTC
CTSD / R / AACTAAAGCTGCCTGTGGCT
FOXA1 / F / CCCCACTTTTGCTTCGTCAC
FOXA1 / R / CTTAGCCGCAGGTACGAGTT
TFF1 / F / CGCACTTCTCGAAGGTCTCC
TFF1 / R / AGGTAAGGCGTGCTTCTTCC
HRAS / F / CGCCCCCACTTGCTCTTAAT
HRAS / R / TCCCTTTAGCCTTTCTGCCG
SMAD3 / F / AGAGCAACATGTGGGCAAGA
SMAD3 / R / CTGTGGGAATGTCGCATCCT
KAT2B / F / CGCGCCCAATTAGCTTCTTC
KAT2B / R / CGATTGCAGAAGGGACCACT
MYB / F / AGGCAAAATGGCATGCAGTC
MYB / R / TGTGCCCAGACAACCTTACC
ZFPM1 / F / CGTGCGGAGATAGATGTGCC
ZFPM1 / R / CTATCGCCTCCGCCATCTG
PAK4 / F / GTCTAGTAGCGGTTGCCTCC
PAK4 / R / AAACGAGGTCCCCAATGGAC
MMP9 / F / GCAGGAGGGATGGATAGTGC
MMP9 / R / GATCCACGCTCCATCCTGAG
CXCR4 / F / ACTGGATAGGGCACCTTCAC
CXCR4 / R / CCGGTTGGACAACTTCAACAC
CLDN9 / F / CCTCTTATGCCCTTTCCGGG
CLDN9 / R / CCGGTTCCACATTCAGCTCT
AGTX / F / CAGATCGTGGACGAGGGAAG
AGTX / R / TGGAACACGTACTGGATGCC
EFCAB10 / F / TTTCGTGTGGGAAGCGAACC
EFCAB10 / R / CTCCAAGCCGCGGAGTATT
SEC31A / F / GACCGGCGAAGAGGACAAA
SEC31A / R / TTGGAACGGAACGTGGAGGT
GAPDH / F / TACTAGCGGTTTTACGGGCG
GAPDH / R / TCGAACAGGAGGAGCAGAGAGCGA
UTX-a / F / AACAGACAGACCTTTGGCGG
UTX-a / R / CGGAGGGAATGAAGAGTCGC
UTX-b / F / CTGACGTTGTGATAGTGCTGCTA
UTX-b / R / CGTAAGACTGTGACCTACTGAGC
UTX-c / F / TTGAAGGGTGTTGGCTCACC
UTX-c / R / ACTTGGGGAACACCAGTCCA
UTX-d / F / ACAGGAGTTGCACAGGTACG
UTX-d / R / CAGCTGTTGGCCCATTAGGA
ESR1-a / F / TGTACCTGGACAGCAGCAAG
ESR1-a / R / TCTCCAGGTAGTAGGGCACC
ESR1-b / F / CTTCCGTTGCCAGTGGGAC
ESR1-b / R / TGTTGACCTCACATGCTACTCG
ESR1-c / F / TTCAGATAATCGACGCCAGGG
ESR1-c / R / TCCCTTGTCATTGGTACTGGC
ESR1-d / F / AGACATGAGAGCTGCCAACC
ESR1-d / R / CTGCGCTTCGCATTCTTACC

Figure Legend

Supplementary Figure 1.(A) Different clones of UTX-knockout MCF-7 cells that we generated using CRISPR-Cas9 technology as examined by western blotting. The crUTX 3-2 clone was selected as the UTX-KO cell line for the subsequent experiments. Sequencing analyses of crUTX 3-2 clone were shown. (B-C) Occupancy of the representative ER target sites by ER, UTX, JHDM1D, CBP, H3K27me3, H3K27me2 and H3K27ac in wild-type (WT) MCF-7 cells and UTX-KO cells. WT cells and UTX-KO cells were deprived of estrogen prior to the treatment of vehicle or E2 for 30 min. qChIP analysis of TFF1 promoter or GREB1 promoter was performed using antibodies against ER, UTX, JHDM1D, CBP (B) or against H3K27me3, H3K27me2, and H3K27ac (C). The relative enrichment is presented as fold change over “WT/E2 (-)”. (D) Depletion of UTX compromised E2-dependent transactivation of ER target genes. WT cells or UTX-KO cells were deprived of estrogen prior to the treatment of vehicle or E2 for 6h. qRT-PCR analysis was carried out to examine the mRNA level of representative ER target genes, which was normalized against that of RNA18S5. Each bar represents the mean + S.D. for triplicate measurements. (*p0.05,two-tailed unpaired t test).

Supplementary Figure2.(A) UTX is essential for hormone-dependent transactivation of chemokine receptor CXCR4 in ER+ breast cancer cells. ZR-75-1 cells transfected with control siRNA or UTX-specific siRNAs were deprived of estrogen prior to the treatment of vehicle or E2 for 6h or 12h. qRT-PCR (left) or western blotting (right) assays were performed with extractedmRNA (6h) or total cellular lysates (12h) respectively to examine the expression of CXCR4 and CXCR7 (B) The histone demethylase activity is required for UTX to regulate ER target genes. qRT-PCR was performed to analyze the mRNA level of TFF1 and GREB1 as in Figure 5C. (C) Breast cancer cells (left, MCF-7; right, ZR-75-1) weretransfectedwith control siRNA or siRNAs specific for JHDM1D or CBP. Cells were depived of estrogen prior to the treatment of were deprived of steroids for 3 days prior to treatment vehicle or E2 for 6h. qRT-PCR was performed to analyze the expression of CXCR4 and CXCR7. The levels of mRNA were normalized against that of RNA18S5. (D) The histone demethylase activity is required for JHDM1D to regulate target genes. MCF-7 cells treated with control siRNA (siC) or siRNA specific for JHDM1D (siJ) transfected with vector, wild-type JHDM1D (JHDM1Dwt) or an enzymatic-dead mutant (H282A) of JHDM1D (JHDM1Dmut) construct. Total RNAs were extracted three days after transfection and qRT-PCR was performed to examine the expression of TFF1, GREB1 and CXCR4. (E) UTX is prerequisite of the UTX/JHDM1D/CBP complex in CXCR4 transactivation. Wild-type or UTX-KO MCF-7 cells weretransfected with vector, UTX, JHDM1D or CBP construct. Total RNAs were extracted three days after transfection and qRT-PCR was performed to examine CXCR4 expression.Each bar represents the mean + S.D. for triplicate measurements (*p0.05,two-tailed unpaired t test).

Supplementary Figure 3.(A) The expression levels of UTX and CXCR4 was confirmed by western blotting for cell lines used in cell proliferation assay and colony formation assay. (B)Wound healing assays were performed with UTX-KO cells or wild-type MCF-7 cells infected with lentiviruses overexpressing UTX or the control vector. The expression levels of UTX and CXCR4 in cells used for wound healing assay and cell migration assay under different conditions were measured by western blotting. Each bar represents the mean + S.D. for triplicate measurements (*p0.05,two-tailed unpaired t test).

Supplementary Figure 4. (A) The expression of UTX and ER is positively correlated in breast cancer patients. The mRNA levels of UTX and ERa in breast cancer patients were analyzed using public data sets (GSE19614, GSE21653, GSE 32646). The relative level of UTX was plotted against that of ER. (B) Bioinformatics analysis of UTX expression in human breast carcinoma samples with different ER status using database from Oncomine. (C) Bioinformatics analysis of UTX expression in human breast carcinoma samples with different molecular clusters or collected from different clinical subtypes using Gene Expression-Based Outcome for Breast Cancer Online (GOBO).

Supplementary Figure 5. Kaplan-Meier survival analysis using the online tool ( to examinethe relationship between UTX/CXCR4 expressionand the overall survival (OS) ofsystemically untreatedER+ or ER-breast cancer patients.

1