TNFa Signaling Exposes Latent Estrogen Receptor Binding Sites to Alter the Breast Cancer Cell Transcriptome

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1 Artile TNF Signling Exposes Ltent Estrogen Reeptor Binding Sites to Alter the Brest Cner Cell Trnsriptome Grphil Astrt Authors Hetor L. Frno, Anush Ngri, W. Lee Krus Correspondene In Brief Using genomi nd moleulr nlyses, Frno et l. show tht the TNF pthwy lters estrogen signling to promote the redistriution of NF-kB nd FoxA1 inding sites ross the genome. This retes ltent ER inding sites tht drive ltered ptterns of gene expression underlying linilly relevnt ellulr responses in rest ner. Highlights d d E2 + TNF promotes extensive trnsriptionl rosstlk in rest ner ells The trget genes re strongly ssoited with linil outomes in rest ners Aession Numers GSE5953 GSE59531 GSE59532 d d TNF signling drives NF-kB nd FoxA1 to ltent enhners to promote ER inding NF-kB nd FoxA1 re required for estlishing tive ER enhners t ltent sites Frno et l., 215, Moleulr Cell 58, April 2, 215 ª215 Elsevier In.

2 Moleulr Cell Artile TNF Signling Exposes Ltent Estrogen Reeptor Binding Sites to Alter the Brest Cner Cell Trnsriptome Hetor L. Frno, 1 Anush Ngri, 1 nd W. Lee Krus 1, 1 Lortory of Signling nd Gene Regultion, Ceil H. nd Id Green Center for Reprodutive Biology Sienes nd Division of Bsi Reprodutive Biology Reserh, Deprtment of Ostetris nd Gyneology, University of Texs Southwestern Medil Center, Dlls, TX 7539, USA Correspondene: lee.krus@utsouthwestern.edu SUMMARY The interply etween mitogeni nd proinflmmtory signling pthwys plys key roles in determining the phenotypes nd linil outomes of rest ners. Using GRO-seq in MCF-7 ells, we defined the immedite trnsriptionl effets of rosstlk etween estrdiol (E2) nd TNF, identifying lrge set of trget genes whose expression is rpidly ltered with omined E2 + TNF tretment, ut not with either gent lone. The pleiotropi effets on gene trnsription in response to E2 + TNF re orhestrted y extensive remodeling of the ER enhner lndspe in n NF-kB- nd FoxA1-dependent mnner. In ddition, expression of the de novo nd synergistilly regulted genes is strongly ssoited with linil outomes in rest ners. Together, our genomi nd moleulr nlyses indite tht TNF signling, ting in pthwys ulminting in the redistriution of NF-kB nd FoxA1 inding sites ross the genome, retes ltent ER inding sites tht underlie ltered ptterns of gene expression nd linilly relevnt ellulr responses. INTRODUCTION Estrogen signling through estrogen reeptor lph (ER), nuler trnsription ftor, hs potent mitogeni effets in rest ner ells. Aout two-thirds of rest ners re ER positive nd exhiit estrogen-dependent growth t the time of dignosis, mking them good trgets for nti-hormone therpies (Igntidis nd Sotiriou, 213). As suh, ER sttus serves s n importnt dignosti nd prognosti inditor in rest ner ptients (Igntidis nd Sotiriou, 213). The estrogen signling pthwy exerts its growth-promoting effets y rpidly nd trnsiently ltering nd expnding the rest ner ell trnsriptome, whih leds to the ltered expression of wide vriety of oding nd non-oding RNAs (Hh et l., 211, 213). Estrogen signling stimultes the inding of ER to mny thousnds of sites ross the genome tht re pre-estlished nd mrked y pioneer trnsription ftors, suh s FoxA1 or AP2g (Hurtdo et l., 211; Tn et l., 211), lthough ess to new sites in the genome my e promoted y stimultion of mitogeni pthwys (Ross-Innes et l., 212). One ound t these sites, ER promotes the oordinted reruitment of oregultory proteins nd hromtin-modifying enzymes tht estlish n tive enhner, leding to hromtin looping nd trget gene trnsription (Foulds et l., 213; Fullwood et l., 29; Hh et l., 213; He et l., 212; Shlyuev et l., 214). In ontrst to the potent prolifertive effets of estrogens in rest ners, proinflmmtory signling in the tumor miroenvironment n hve either prolifertive or nti-prolifertive effets, depending on the tumor ontext (Ben-Nerih nd Krin, 211; Coussens et l., 213). Proinflmmtory signling in ners derives from ute nd hroni infetions, tumor-infiltrting immune ells, nd lol dipose tissue, ll of whih re ple of supplying the tumor miroenvironment with iotive moleules tht filitte ngiogenesis, invsion, nd metstsis (Bumgrten nd Frsor, 212; Ben-Nerih nd Krin, 211). Consequently, inflmmtion is now onsidered hllmrk of ner (Hnhn nd Weinerg, 211). One of the mjor integrtors of proinflmmtory signling is the trnsription ftor NFkB, whih is found in lmost ll ell types nd is tivted y vriety of hemokines, ytokines, nd growth ftors, inluding tumor nerosis ftor lph (TNF). In the nonil NF-kB pthwy, lignded reeptors in the ell memrne initite signling sdes tht ultimtely led to the nuler trnslotion of NF-kB suunits (e.g., p65 nd p5), whih ind to NF-kB enhner elements tht ontrol trget gene expression (Ben- Nerih nd Krin, 211; Ntoli, 212). The interply etween mitogeni nd proinflmmtory signling pthwys plys key roles in determining the phenotypes nd linil outomes of rest ners. The distint, ut interrelted, responses to estrogens nd TNF (i.e., mitogeni versus proinflmmtory) hve the potentil to rete novel ntgonisti or synergisti responses tht n profoundly ffet the iology of rest ners (Bumgrten nd Frsor, 212). A numer of reent studies hve shown tht proinflmmtory signling n led to more ggressive hormone-dependent rest ners. For exmple, onstitutive tivtion of NF-kB is ssoited with more ggressive ER-positive rest ners nd the development of resistne to endorine therpy (Zhou Moleulr Cell 58, 21 34, April 2, 215 ª215 Elsevier In. 21

3 A C Veh E2 TNF E2+TNF n = 694 All Protein Coding Genes E2 +1 Rel. to Veh TNF 974 2,141 1,66 E2+TNF Tretment Up Down E2 1, TNF 1,93 3, E2+TNF 1,78 3,838 D Overll Survivl (%) B Up Regulted (Fold Chnge) Down Regulted (Fold Chnge) Proinflmmtory Genes p p= = Yers ER+ LN+ Tumors Low Expression (n = 85) High Expression (n = 84) De Novo (Gined) Genes n = 34 E T E+T n = 1,356 E T E+T Overll Survivl (%) p = Synergistilly Regulted Genes n = 59 E T E+T n = 1,595 E T E+T Proinflmmtory Genes Untreted Tumors Low Expression (n = 136) High Expression (n = 74) Yers E Overll Survivl (%) De Novo nd Synergistilly Up-Regulted Genes p p= =.1945 ER+ LN+ Tumors Low Expression (n = 77) High Expression (n = 92) Yers Overll Survivl (%) De Novo nd Synergistilly Up-Regulted Genes p = 1 x 1-5 Untreted Tumors Low Expression (n = 153) High Expression (n = 57) Yers Overll Survivl (%) De Novo nd Synergistilly Down-Regulted Genes p =.5 ER+ LN Neg Low Expression (n = 22) High Expression (n = 16) Yers Figure 1. Crosstlk etween Mitogeni nd Proinflmmtory Signling in Brest Cner Cells: Effets on mrna Expression, Cell Growth, nd Brest Cner Ptient Outomes MCF-7 ells were treted with vehile (Veh or V), estrdiol (E or E2), TNF (T or TNF), or oth E2 + TNF (E+T) nd sujeted to GRO-seq or ell prolifertion ssys. The GRO-seq dt were nlyzed for ptterns of gene expression nd relted to rest ner ptient outomes. (A) Hetmp (left) nd Venn digrm (right) showing the regultion of ll protein-oding genes under eh tretment ondition s reveled y GRO-seq. The hnges in expression shown in the hetmp were entered reltive to the ontrol (vehile) ondition nd sled reltive to the mximum solute vlue of the expression hnge for eh gene. Thus, the vlues rnge etween +1 nd 1, where +1 or 1 denote the mximum or minimum vlue, respetively. (B) Boxplots showing hnges in GRO-seq signls for genes tht re newly (de novo or gined) or synergistilly regulted y E2 + TNF. Brs mrked with different letters re signifintly different from eh other (Wiloxon rnk sum test, p < ). For exmple, rs leled with re different from those leled with or, ut not from other rs leled with. (C) MCF-7 ell prolifertion ssy. Eh dt point represents the men ± SEM, n = 3. (D nd E) Kpln-Meier survivl nlyses for different rest ner types using three distint gene sets s inputs: (1) proinflmmtory gene set defined y gene ontologies (D), (2) the E2 + TNF de novo nd synergistilly upregulted gene set (E, left nd middle), nd (3) the E2 + TNF de novo nd synergistilly (legend ontinued on next pge) 22 Moleulr Cell 58, 21 34, April 2, 215 ª215 Elsevier In.

4 et l., 25). Furthermore, TNF-tivted NF-kB nd estrogentivted ER n work together to potentite the expression of numer of genes ssoited with prolifertion, invsion, nd metstsis in rest ner ells (Frsor et l., 29; Ross-Innes et l., 212). As shown in gene-speifi studies, rosstlk etween the estrogen nd TNF signling pthwys is medited, in prt, y the ility of ER nd NF-kB to physilly intert nd ind together t enhners (Klitzidis nd Gilmore, 25; Prdhn et l., 212; Prdhn et l., 21). However, the funtionl reltionships nd outomes of the rosstlk etween NF-kB/proinflmmtory signling nd ER/mitogeni signling re omplex nd ontext dependent, nd they hve the potentil to promote linil outomes tht re more fvorle (Cunninghm nd Gilkeson, 211; Frsor et l., 29; Ross-Innes et l., 212). In differentited ells, ell stte-determining trnsription ftors, suh s ER, re thought to ind to predetermined or pre-estlished enhner repertoire, ensuring onstrined nd iologilly pproprite response to stimuli (Ghvi-Helm et l., 214; Heinz et l., 21; Hurtdo et l., 211; Shlyuev et l., 214). How pre-estlished enhner repertoire my e ltered y rosstlk with other signling pthwys is not well understood. In this regrd, reent studies hve identified ltent enhners, whih re formed nd tivted de novo in response to ellulr signls t genomi sites tht re not pre-estlished (Kikkonen et l., 213; Nord et l., 213; Ostuni et l., 213). In the studies desried herein, we used vriety of genomi, moleulr, nd iohemil pprohes to (1) exmine the nture nd extent of rosstlk etween the estrogen nd TNF signling pthwys in rest ner ells, (2) determine how this rosstlk lters the ER istrome, reting new ER inding sites nd opportunities for gene regultion, nd (3) understnd how the de novo trnsriptome speifies iologil outomes. RESULTS The E2-Regulted Trnsriptome nd Cellulr Mitogeni Responses Are Drmtilly Altered y TNF in Brest Cner Cells To determine the interply etween mitogeni nd proinflmmtory signling in rest ner ells t the trnsriptionl level, we monitored the diret trnsriptionl output in ER-positive MCF-7 ells treted with 17--estrdiol (E2), TNF, or oth E2 + TNF (Figure S1A) using glol run-on oupled with deep sequening (GRO-seq). By limiting the tretment times to 4 min, when oth E2 nd TNF trnsriptionl responses re mximl for most genes (Dnko et l., 213; Hh et l., 211; Luo et l., 214), our nlyses pture primry trnsriptionl outomes. Aross the genome, TNF-indued proinflmmtory signling nd E2-indued mitogeni signling onverged on mny genes, inluding mrna genes nd long non-oding RNA (lnrna) genes, leding to numerous instnes of ntgonism or enhnement of gene expression (Figures 1A, 1B, nd S1B). Notly, the immedite nd extensive E2-regulted gene expression progrm ws drmtilly ltered y the ddition of TNF, leding to the up- or downregultion of new set of genes (de novo or gined genes, inluding 1,66 mrna genes nd 773 lnrna genes) tht were unffeted y either tretment lone (Figure 1B, left; Figure S1C, left). In ddition, we oserved group of genes (synergistilly regulted genes, inluding 2,14 mrna genes nd 649 lnrna genes) whose E2-medited up- or downregultion ws synergistilly enhned y TNF (Figure 1B, right; Figure S1C, right). These responses led to n E2 + TNF-regulted trnsriptome tht is muh lrger thn those oserved with E2 or TNF lone. The genes within this unique trnsriptome re involved in moleulr nd ellulr proesses rnging from signling nd trnsription to metolism, prolifertion, nd poptosis (Tle S1). The ltered ptterns of E2-regulted gene expression in response to TNF were ssoited with orresponding hnges in the growth of MCF-7 ells. Speifilly, TNF ompletely loked the potent mitogeni response to E2 in dose-dependent mnner (Figures 1C nd S1D). The inhiitory effet of TNF ws lso oserved in two other ER-positive rest ner ell lines (i.e., ZR-75-1 nd MDA-MB-361; Figure S1E). To determine if the unique E2 + TNF-regulted trnsriptome hs roder linil signifine, we mined rest ner outome-linked gene expression dt using the Gene Expression-Bsed Outome for Brest Cner Online (GOBO) tool, expressing the outomes in Kpln-Meier survivl plots. As referene point, we determined if high expression levels of proinflmmtory gene set (defined y gene ontologies) re ssoited with good or poor outomes in rest ners. As expeted given the omplex nture of inflmmtion in rest ners, we found tht high expression levels of this gene set re ssoited with good outomes in some rest ners (Figure 1D, left; Figure S1F) nd poor outomes in others (Figure 1D, right; Figure S1F). Likewise, the expression levels of E2 + TNF de novo nd synergistilly regulted gene sets re strong preditors of linil outomes, ut good versus poor outomes vry sed on the prtiulr rest ner type (Figures 1E nd S1G). For exmple, high-level expression of the E2 + TNF upregulted gene set predits good outomes in ER-positive, lymph node-positive rest ners (Figure 1E, left) nd poor outomes in untreted rest ners (Figure 1E, middle). Interestingly, the E2 + TNF de novo nd synergistilly regulted gene sets re lrgely non-overlpping with the proinflmmtory gene set defined y gene ontologies (Figure S1H). Colletively, these results indite tht the gene expression progrm indued y o-stimultion with E2 nd TNF is tied to roust iologil outomes tht ontrol the growth of rest ner ells nd predit distint linil outomes ross rnge of rest ner types. The studies desried elow re imed t understnding downregulted gene set (E, right). The rest ner outome-linked gene expression dt were essed using the Gene Expression-Bsed Outome for Brest Cner Online (GOBO) tool. In some ses, high expression levels of the gene sets re preditive of etter outomes in prtiulr rest ner type (e.g., D, left; E, left), while in others it is preditive of poor outome (e.g., D, right; E, middle). See lso Figure S1 nd Tle S1. Moleulr Cell 58, 21 34, April 2, 215 ª215 Elsevier In. 23

5 the moleulr mehnisms nd the funtionl interply etween the estrogen nd TNF signling pthwys. Proinflmmtory Signling Alters the Repertoire of ER Binding Sites nd Unveils New Sites of Moleulr Crosstlk To explore the moleulr rosstlk etween the estrogen nd TNF signling pthwys t the genomi level, we used ChIPseq for ER ( trnsription ftor meditor of the estrogen signling pthwy) nd the p65 suunit of NF-kB ( trnsription ftor meditor of the TNF signling pthwy). Although the mjority of the E2-indued ER inding events were unhnged ( mintined ) y otretment with TNF, the otretment used gin of 1,664 new ER inding sites nd loss of 1,938 ER inding sites (Figures 2A nd 2B). We onfirmed the presene of the gined sites, whih were of prtiulr interest given the trnsription results in Figure 1A, in nother ER-positive rest ner ell line, ZR-75-1, y ChIP-qPCR (Figure S2A). Aout hlf of the gined ER inding sites were lso oupied y NF-kB upon otretment with E2 nd TNF, while the lost ER inding sites were generlly not oupied y NF-kB(Figures 2A nd 2B). Interestingly, TNF-medited NF-kB inding ws enhned in the presene of E2 t the gined (nd to lesser extent, mintined) ER inding sites (Figure 2B, top nd middle). These results suggest funtionl intertions etween two signl-regulted trnsription ftors (i.e., ER nd NF-kB) ross the genome. Suh oservtions fit with studies of individul ER inding sites showing tht ER nd NF-kB n physilly intert on hromtin, llowing for hnges in gene expression (Klitzidis nd Gilmore, 25; Prdhn et l., 21, 212). DNA sequene nlyses of the gined, mintined, nd lost ER inding sites showed n expeted enrihment of ER (ESR1), NF-kB (RELA), nd FoxA1 (FOXA1) motifs, with the most prevlent motifs t the gined ER inding sites eing RELA nd ESR1 (Figure 2C). Similr to wht we oserved with the ER istrome upon tretment with TNF, we lso oserved ltertions in the TNF-indued NF-kB istrome y otretment with E2, leding to the gin or loss of NF-kB inding sites ross the genome (Figures S2B nd S2C). Colletively, these results illustrte how distint signling pthwys onverge on nuler trnsription ftors to lter their ptterns of inding, reting new istromes nd, ultimtely, trget trnsriptomes. E2-Dependent ER Binding Sites Gined upon Cotretment with TNF Produe Enhner RNAs nd Are Assoited with Trget Gene Expression Signl-regulted enhners n modulte trget gene expression from distl positions in the genome through looping events tht ring distl enhners into ontt with proximl promoters through the formtion of hromtin loops (de Lt nd Duoule, 213). Reent studies from our l nd others hve shown tht enhners with histone modifitions inditive of tivity, suh s H3K4me1 nd H3K27, re more likely to produe enhner trnsripts (ernas) nd loop to trget gene promoters (Hh et l., 213; Kieffer-Kwon et l., 213; Kim et l., 21). Thus, erna prodution is good mrker of tive enhners tht regulte trget genes (Hh et l., 213). To ssess whether the E2-dependent ER inding sites gined upon otretment with TNF re tive nd funtionl enhners, we leverged the power of GRO-seq to ssy enhner trnsription nd trget gene trnsription upon ER inding (Figure 3A). First, we ssyed enhner trnsription t the gined, mintined, nd lost ER inding sites. We oserved tht the mount of enhner trnsription in response to vehile (V), E2 (E), TNF (T), or E2 + TNF (E+T) ws positively orrelted with the extent of ER (or p65) inding under the sme onditions; in generl, more enhner trnsription ws oserved with more ER inding, s determined y the orreltion nlyses shown in Figure S3A (see lso Figure 3B, middle; ompre to the extent of ER inding in Figure 2B). For exmple, with the gined ER inding sites, the levels of enhner trnsription were gretest with the TNF or E2 + TNF tretments (Figure 3B, middle ottom), refleting the oupny of those sites y p65 (for TNF lone; Figure 2B, top right) or ER nd p65 (for E2 + TNF; Figure 2B, top). Next, we used GRO-seq to determine the levels of trnsription t the nerest neighoring gene (upstrem or downstrem) from eh ER inding site. For the genes loted nerest to the gined nd mintined ER inding sites, we oserved tht the mount of trget gene trnsription in response to the four tretment onditions ws proportionl to the extent of enhner trnsription under the sme onditions (i.e., in generl, more trget gene trnsription ws oserved with more enhner trnsription) (Figure 3B). Furthermore, we found tht gined ER inding sites re signifintly enrihed for neighoring genes whose expression is stimulted y E2 + TNF tretment (Figure S3B). Lost ER inding sites, whih showed persistent trnsription of the nerest neighoring genes with E2 + TNF tretment in spite of redued enhner trnsription, were n exeption (Figure S3C), suggesting dissoition of ER inding from gene expression in the presene of TNF for this prtiulr set of ER inding sites. Gene-speifi nlyses onfirmed tht the E2 + TNF-dependent gene regultory events require ER (Figures S3D nd S3E). Together, these nlyses link ER inding (lled y ChIPseq), enhner trnsription (lled y GRO-seq t the ER inding sites), nd nerest gene trnsription (lled y GRO-seq), whih is illustrted further using three-dimensionl oxplots of the dt (Figures 3C, 3D, nd S3F). This representtion lso shows the impt tht otretment with TNF hs on gined ER inding sites nd their trget genes, produing new ER inding events, s well s stimulting enhner nd trget gene trnsription (Figures 3C nd 3D; note the signifint displement in ll three dimensions with E2 + TNF for the gined enhners; see lso Figure S3F). Colletively, these results indite tht the gined ER inding sites re on fide enhners, representing unique induile ER istrome tht ontrols the expression of distint nd iologilly relevnt gene set. The Clssil Estrogen-Indued ER Cistrome Is Demrted Prior to Stimulus, while the De Novo ER Cistrome Requires TNF Stimultion for Chromtin Aessiility Previous models of enhner funtion suggested tht signl-tivted trnsription ftors, suh s ER, ind within predetermined repertoire of essile genomi loi tht re estlished 24 Moleulr Cell 58, 21 34, April 2, 215 ª215 Elsevier In.

6 A ER ChIP-Seq NF- B ChIP-Seq B Tretment: Veh Gined E2 TNF E+T ER Peks (1,664) Veh E2 TNF E+T Red Counts ER Red Counts NF- B Mintined ER Peks (29,23; 1% Shown) Red Counts ER d Red Counts NF- B Lost ER Peks (1,938) Red Counts ER Red Counts NF- B 5 k Pek Center C Figure 2. Co-Stimultion of the Estrogen nd TNF Signling Pthwys Alters the ER Cistrome in MCF-7 Cells (A) Left, hetmps of ER ChIP-seq reds from MCF-7 ells treted with E2, TNF, or oth. Right, hetmp of p65 ChIP-seq reds t the genomi loi where ER is ound. (B) Boxplot of the normlized red ounts for three distint sets of ER inding sites (gined, mintined, lost). Brs mrked with different letters re signifintly different (Wiloxon rnk sum test, p < ). (C) De novo motif nlyses of the three sets of ER inding sites noted in (B) were performed using MEME. The predited motifs were mthed to known motifs using TOMTOM. See lso Figure S2. Moleulr Cell 58, 21 34, April 2, 215 ª215 Elsevier In. 25

7 A B C D Mintined ER Enhners Gined ER Enhners Enhner Txn (Log2 Fold Chnge) 1 E2: E2 + TNF : E2: E2 + TNF : Mintined ER Enhners ER inding (Log2 Fold Chnge) E2 E2+TNF Reltive Displement in Eh Dimension (E2 versus E2+TNF ) Mintined ER Enhners Gined ER Enhners ER Binding ChIP-Seq Enhner Trnsription GRO-seq (± 2.5 k round pek summit) ER ER ER ER ER ER 1 Enhner Txn (Log2 Fold Chnge) upon ellulr differentition (Ghvi-Helm et l., 214; Heinz et l., 21; Hurtdo et l., 211; Shlyuev et l., 214). In ddition, reent studies hve identified ltent enhners, whih re formed nd tivted de novo in response to ellulr signls t genomi sites tht re not pre-estlished (Kikkonen et l., 213; Nord et l., 213; Ostuni et l., 213). Aessiility t preestlished enhners is mintined y linege-speifi trnsription ftors nd pioneer trnsription ftors tht funtion to mintin n open hromtin rhiteture (Heinz et l., 21; Hurtdo et l., 211; Shlyuev et l., 214). In this regrd, the Forkhed protein FoxA1, well-estlished pioneer ftor for ER, is onstitutively ound t ER enhners prior to estrogen stimultion (Hurtdo et l., 211). Thus, we sought to determine the hromtin stte in the sl (i.e., unstimulted) ondition t the min- RPKM RPKM ER Enhner Trnsription Tretment 1 1 Nerest Gene GRO-seq (+1 k to +4 k) ER inding (Log2 Fold Chnge) E2 RPKM RPKM Nerest Gene Trnsription 4 d Tretment Gined ER Enhners E2+TNF Displement p-vlues (E2 versus E2+TNF ) Dimension Mintined Gined ) ER Binding ) Enhner Trnsription ) Nerest Gene Trnsription x x x 1-1 Figure 3. ER Binding Sites Gined y Cotretment with E2 nd TNF Are Positively Correlted with Trnsription of the Enhners nd Nery Trget Genes (A) Overview of the pipeline for integrting ChIPseq dt with GRO-seq dt to link ER inding with enhner nd trget gene trnsription in MCF-7 ells. (B) Boxplots showing GRO-seq RPKM vlues for enhner trnsription nd nerest neighor upregulted gene trnsription t mintined nd gined ER inding sites. Brs mrked with different letters re signifintly different (Wiloxon rnk sum test, p <.1). (C) Three dimensionl (3D) oxplots showing the positive reltionships mong ER inding (lled y ChIP-seq; from Figure 2B), enhner trnsription (lled y GRO-seq t the ER inding sites; from B), nd nerest gene trnsription (lled y GRO-seq; from B) for the top 5% of ER inding sites (see Supplementl Experimentl Proedures). Solid lines represent the interqurtile rnge; dotted lines represent Q1 3 IQR or Q3 + 3 IQR. (D) Reltive displement (i.e., fold hnge) in E2 versus E2 + TNF (left) with ssoited p vlues (right) in eh dimension for the 3D oxplots shown in (C). See Figure S3F for the tul medin log2 fold hnge vlues, s well s the differene (D) in medin log2 fold hnge vlues for E2 versus E2 + TNF. See lso Figure S3. tined (i.e., lssil) nd gined ER enhners prior to reeptor inding. To do so, we ligned pulily ville deep sequening dt sets for severl enhner fetures (FoxA1, DNse1 essiility, hromtin mrks, nd CBP) to the mintined nd gined ER istrome (Figures 4A nd 4B). In untreted MCF-7 ells, the mintined ER enhners hve n open hromtin rhiteture (i.e., enhned DNse1 essiility), nd re enrihed in FoxA1 inding nd fetures of tive enhners (e.g., H3K27 nd CBP), prior to stimultion with E2 or TNF (Figures 4A nd 4B). These fetures suggest tht the mintined ER enhners re onstitutively essile in these ells under sl onditions, prior to signlindued ER inding (Figures 4A nd 4B). We oserved similr results for the lost ER enhners (dt not shown). In ontrst, the gined ER enhners hve less essile ( losed ) hromtin rhiteture, nd re not signifintly enrihed in FoxA1 inding or fetures of tive enhners (e.g., H3K27 nd CBP) prior to stimultion with E2 or TNF (Figures 4A nd 4B). Interestingly, tretment with TNF or TNF + E2, ut not E2 lone, drove NF-kB nd FoxA1 inding to the gined ER enhners (Figure 4C; see Figures S4A S4D for speifi exmples). In ddition, s disussed in more detil elow, we oserved some differenes in the FoxA1 responses to the tretments depending on the 26 Moleulr Cell 58, 21 34, April 2, 215 ª215 Elsevier In.

8 A ER ChIP-Seq Tretment: V E E+T Gined ER Peks (1,664) Mintined ER Peks (29,23; 1% Shown) C Tretment: ER Gined Peks (1664) Red Counts 5 k Pek Center ChIP- or DNseI-Seq (Prior to Tretment) FoxA1 DNse1 H3K27 H3K4me3 CBP ER ChIP-Seq Veh E2 TNF E+T ER level of NF-kB enrihment t these sites (low versus high; Figures S4A S4C). These results suggest tht TNF or TNF + E2 is required for the moiliztion of NF-kB nd FoxA1 to new genomi loi, where they help to rete new ER inding sites tht re not prt of the lssil estrogen-indued ER istrome. These results fit well with the gene expression nlyses desried ove. The de novo sites of ER inding in response to E2 + TNF in MCF-7 ells hve the fetures of reently desried ltent enhners (Heinz et l., 21; Kikkonen et l., 213; Ostuni et l., 213). Estlishment of TNF-Indued Ltent ER Enhners Requires NF-kB nd FoxA1 The TNF-dependent inding of NF-kB nd FoxA1 t ltent ER enhners suggested tht these ftors my t to inrese B Red Counts Red Counts 1 Gined (G) ER Peks Mintined (M) ER Peks DNse1 G M H3K4me Stedy Stte Levels t ER Enhner Lotions G NF- B ChIP-Seq Veh E2 TNF E+T NF- B Red Counts Red Counts Red Counts G M H3K FoxA1 G M CBP Figure 4. Ltent ER Binding Sites Exposed y Cotretment with E2 nd TNF Hve Restritive Chromtin Stte Prior to Tretment (A) Hetmp showing gined nd mintined ER inding sites ligned with genomi dt (ChIP-seq nd DNse1-seq) for known enhner fetures from untreted MCF-7 ells (sl stte). (B) Normlized ChIP-seq or DNse1-seq red ounts for known enhner fetures t gined versus mintined ER inding sites in untreted MCF-7 ells. Brs mrked with different letters re signifintly different (Wiloxon rnk sum test, p <.1). (C) Hetmp (top) nd oxplots (elow) of NF-kB nd FoxA1 oupny t gined ER inding sites, s determined y ChIP-seq. Brs mrked with different letters re signifintly different (Wiloxon rnk sum test, p <.1). See lso Figure S4. hromtin essiility nd promote ER inding. To determine the roles of M G M NF-kB nd FoxA1 t gined/ltent ER enhners in MCF-7 ells, we seprted the enhners into those with reltively FoxA1 ChIP-Seq high NF-kB o-oupny fter stimultion Veh E2 TNF E+T nd those with reltively low NF-kB o-oupny (Figure 5A). We then determined the role of NF-kB or FoxA1 t these enhners using sirna-medited knokdown of NF-kB p65 or FoxA1, oupled with ChIP-qPCR (to ssy the inding of p65, FoxA1, nd ER) nd RT-qPCR FoxA1 (to ssy gene expression outomes for 15 neighoring genes). ER inding in the 1 presene of E2 + TNF t gined/ltent enhners with high NF-kB oupny 5 (e.g., those ner POU3F1, EFNA1, nd B4GALT1) ws drmtilly redued upon p65 knokdown (Figure 5C; Figures 5B nd S5A, middle pnels), ut ws unffeted t mintined enhners (e.g., those ner FMN1, P2RY2, nd GREB1; Figures 5B nd S5A, top pnels) or gined/ltent enhners with low NF-kB oupny (e.g., those ner, GALNTL4, nd PPP1R3C; Figures 5B nd S5A, ottom pnels). We lso oserved orresponding effet of the expression of the nerest neighoring gene in the presene of E2 + TNF, nmely tht redued ER inding orrelted with redued expression upon p65 knokdown (Figures 5D nd S5B, middle pnels; ompre to the lrgely unffeted genes in the left/right nd top/ottom pnels, respetively). These results indite tht NF-kB is required for ER inding t suset of ltent/gined enhners, s well s orresponding trget gene expression in response to E2 + TNF. As noted ove, gined/ltent enhners with low NF-kB oupny showed little dependene on NF-kB for ER inding. We hypothesized tht t these enhners FoxA1, rther thn Moleulr Cell 58, 21 34, April 2, 215 ª215 Elsevier In. 27

9 A B Figure 5. NF-kB Helps to Unveil Some Ltent ER Enhners in MCF-7 Cells fter Tretment with TNF (A) Genome rowser views of ER nd NF-kB (p65) ChIP-seq dt for different types of ER enhners. (B) NF-kB filittes ER inding t TNF-responsive enhners. ChIP-qPCR for ER nd NFkB in MCF-7 ells with or without RNAi-medited knokdown (KD) of p65, followed y tretment with E2 + TNF. The enhners re designted y the nerest neighoring gene. Eh r represents the men ± SEM, n = 3. The sterisks indite signifint differenes from the orresponding ontrol (Student s t test, p vlue <.5). (C) Western lot showing KD of p65 using sirnas. (D) NF-kB is required for the effiient expression of genes neighoring ltent E2 + TNF-responsive ER enhners. Trget gene expression with or without RNAi-medited KD of p65, with E2 + TNF tretment. Eh r represents the men ± SEM, n = 3. The sterisks indite signifint differenes from the orresponding ontrol (Student s t test, p vlue <.5). See lso Figure S5. C D NF-kB, might ply ritil role in estlishing hromtin environment permissive for ER inding. We tested this in MCF-7 ells using set of experiments similr to those desried ove for NF-kB. At mintined ER enhners, FoxA1 ws ound onstitutively (Figure 4A) nd s expeted, sirna-medited knokdown of FoxA1 drmtilly redued ER inding, s well s the expression of the nerest neighoring gene (Figure 6B; Figures 6A, 6C, S6A, nd S6B, top pnels). Likewise, t gined/ltent enhners with low NF-kB oupny, knokdown of FoxA1 drmtilly redued ER inding, s well s the expression of the nerest neighoring gene (Figures 6A, 6C, S6A, nd S6B, ottom pnels). In ontrst, t gined/ltent enhners with high NF-kB oupny, knokdown of FoxA1 hd little effet on ER inding or trget gene expression (Figures 6A, 6C, S6A, nd S6B, middle pnels). A similr pttern of FoxA1 dependene ws oserved for gined ER enhners in ZR-75-1 ells (Figures S6C S6F). These results indite tht FoxA1 is required for ER inding t suset of ltent/gined enhners with low NF-kB oupny, s well s orresponding trget gene expression in response to E2 + TNF. In ChIP-seq ssys in MCF-7 ells, we found tht ER inding t 3% of the gined enhners ws signifintly redued upon sirna-medited knokdown of FoxA1 (FDR <.5; Figures 6D nd 6E; ffeted versus unffeted y FoxA1 knokdown). These results seprted the gined/ltent ER enhners into two groups: (1) FoxA1 dependent (i.e., ffeted y FoxA1 knokdown) nd (2) FoxA1 independent (i.e., unffeted y FoxA1 knokdown). Both groups hve similr levels of ER inding with E2 + TNF (Figure S6G) nd similr levels of nerest neighoring gene trnsription (Figure S6H). With these genomi dt, we were le to test the hypothesis tht gined enhners with high NF-kB oupny do not require FoxA1. We found tht FoxA1-dependent ltent ER enhners hve lower oupny of p65 thn the FoxA1-independent enhners, s expeted (Figure 6F), nd re sensitive to FoxA1 knokdown (Figure S6E). 28 Moleulr Cell 58, 21 34, April 2, 215 ª215 Elsevier In.

10 Our results showing loding of FoxA1 t new sites in the genome in response to TNF tretment point to key links etween ellulr signling pthwys nd the tivity of pioneer ftors. To explore the onnetions etween TNF signling nd FoxA1 loding on hromtin in more detil, we iohemilly frtionted E2- nd TNF-treted MCF-7 ells into distint ellulr omprtments (i.e., ytoplsm, nuleoplsm, nd hromtin). As expeted, FoxA1 did not lolize to the ytoplsm, nd the tretments (i.e., Veh, E2, TNF, or E2 + TNF) did not lter the levels of totl FoxA1 protein, s determined y western lotting for FoxA1 (Figure 7A; see Whole Cell Extrt nd Cytoplsm ). Also s expeted, FoxA1 ws ound to hromtin under sl/unstimulted onditions nd showed enhned inding or stiliztion upon E2 tretment (Figure 7A; see Chromtin ). Interestingly, tretment with TNF, lone or in the presene of E2, resulted in drmti inrese in hromtin-ound FoxA1 nd orresponding derese of solule FoxA1 in the nuleoplsm (Figure 7A; see Chromtin versus Nuleoplsm ). These results indite tht TNF tretment n drive more FoxA1 to hromtin, providing new sites of essile hromtin for the inding of ER. Tken together, our dt suggest model where the mjority of the E2-regulted trget genes re ontrolled y poised ER enhners tht re loted in open regions of hromtin nd re mintined y onstitutively ound FoxA1 nd other linege-speifi trnsription ftors (Figure 7B, top left). A suset of ltent ER enhners, however, re loted in less-essile regions of the genome nd require TNF signling to promote NF-kB nd FoxA1 inding, leding to enhned hromtin essiility nd susequent ER inding (Figure 7B, top right nd ottom). DISCUSSION In the studies desried herein, we hve defined the moleulr mehnisms tht underlie rosstlk etween estrogen-medited mitogeni signling nd TNF-medited proinflmmtory signling in rest ners, s well s the funtionl onsequenes nd linil relevne of this interply. We hve defined the immedite trnsriptionl effets of rosstlk etween E2 nd TNF, identifying lrge set of trget genes whose expression is rpidly nd drmtilly ltered (upregulted or downregulted) with omined E2 + TNF tretment, ut not with either gent lone. The trget genes inlude lssil mitogeni nd proinflmmtory genes, s well s lnrna genes (Figures 1A nd S1B). The pleiotropi effets on gene trnsription in response to E2 + TNF were orhestrted y the extensive remodeling of the ER enhner lndspe (Figures 2A nd 2B) in n NF-kB- nd FoxA1-dependent mnner (Figures 5, 6, S5, nd S6). The ltered ptterns of gene regultion in the presene of TNF inhiit the estrogen-dependent growth of MCF-7 ells. In ddition, high-level expression of the de novo nd synergistilly regulted genes is ssoited with good outomes in some types of rest ner nd poor outomes in other types of rest ner. Together, our genomi nd moleulr nlyses indite tht TNF signling, ting in pthwys ulminting in the redistriution of NF-kB nd FoxA1 inding sites ross the genome, retes new E2-dependent ER inding sites tht underlie ltered ptterns of gene expression. TNF Signling Exposes Ltent ER Enhners in Brest Cner Cells: Requirement for NF-kB nd FoxA1 In the lssil pthwy for estrogen-indued gene tivtion, the estrogen-indued ER istrome is demrted y FoxA1 inding prior to hormone exposure. In these ses, FoxA1 ts s pioneer ftor tht filittes hromtin opening, providing ess to the genome for the lignded reeptor (Hurtdo et l., 211). One ound to estrogen response elements, lignded ER promotes the formtion of tive enhners, whih inludes reruitment of oregultors (e.g., SRCs nd p3/cbp, Meditor) (Foulds et l., 213), histone modifitions nd dditionl hromtin opening (He et l., 212), reruitment of RNA polymerse II nd enhner trnsription (Hh et l., 213; Wng et l., 211), nd looping to trget gene promoters (Fullwood et l., 29). Herein, we show tht the TNF signling pthwy n rediret the pioneer ftor FoxA1 to new sites in the genome tht were not estlished during the ontology of the ell (Figure 7B, right). The interfe of the signling pthwy with the trnsription ftor is evident t the genomi level, with redistriution of FoxA1 to new loi, reting new opportunities for ER inding. These results re onsistent with ltent enhner model, in whih tivtion of ellulr signling pthwys promotes the sequentil inding of stimulus-tivted nd linege-determining trnsription ftors (Heinz et l., 21; Ostuni et l., 213). Importntly, de novo or gined ER inding t these ltent enhners is lignd dependent; the sites newly demrted y FoxA1 in response to TNF nnot e essed y ER in the sene of lignd. Thus, in this se, FoxA1 retins its pioneer funtions, ut they re not determined s onsequene of development or differentition, ut rther s n endpoint of proinflmmtory signling. The TNF-indued redistriution of FoxA1 my our in response to posttrnsltionl modifition of FoxA1 s n endpoint of TNF signling pthwy, lthough further nlyses re needed to test this hypothesis. We lso oserved tht lignded ER n ess n dditionl set of ltent enhners demrted y NF-kB upon TNF signling (Figure 7B, ottom left). Reent studies hve suggested tht proinflmmtory signling, primrily through NF-kB, plys role in promoting n open hromtin stte llowing for ell plstiity in response to environmentl ues; however, the role of NF-kB s pioneer ftor hs een deted (Lee et l., 212; Molinero et l., 212; Ntoli, 212; Ndlovu et l., 29; Ro et l., 211). Nonetheless, TNF-medited NF-kB inding, lone or perhps in onjuntion with other trnsription ftors, is required for ER inding t these ltent enhners, ontriuting to extensive rosstlk etween the estrogen nd TNF signling pthwys oserved in the istrome nd the trnsriptome. The slient fetures of the ltent ER enhners re (1) limited hromtin essiility (i.e., low FoxA1 oupny nd DNse1 sensitivity) nd limited enhner tivity (i.e., low H3K27 nd CBP) prior to stimultion (Figures 4A nd 4B) nd (2) rpid nd roust enhner tivtion (s illustrted y inresed enhner trnsription) nd ER inding fter stimultion (Figure 3B). The signl-dependent unveiling of these ltent ER enhners Moleulr Cell 58, 21 34, April 2, 215 ª215 Elsevier In. 29

11 A C B D E F Figure 6. Cytokine-Indued FoxA1 Binding t Ltent Enhners Is Required for ER Binding (A) FoxA1 filittes ER inding t TNF-responsive enhners. ChIP-qPCR for ER nd FoxA1 in MCF-7 ells with or without RNAi-medited knokdown (KD) of FoxA1, followed y tretment with E2 + TNF. The enhners re designted y the nerest neighoring gene. Eh r represents the men ± SEM, n = 3. The sterisks indite signifint differenes from the orresponding ontrol (Student s t test, p vlue <.5). (B) Western lot showing KD of FoxA1 using sirnas. (C) FoxA1 is required for the effiient expression of genes neighoring ltent E2 + TNF-responsive ER enhners. Trget gene expression with or without RNAi-medited KD of FoxA1, with E2 + TNF tretment. Eh r represents the men ± SEM, n = 3. The sterisks indite signifint differenes from the orresponding ontrol (Student s t test, p vlue <.5). (D) Approximtely 3% of gined ER enhners show signifint redutions in ER inding upon FoxA1 KD ( ffeted ) (FDR <.5 using SICER). The results re from ChIP-seq of ER in MCF-7 ells with or without sirna-medited KD of FoxA1 nd treted with or without E2 + TNF. (legend ontinued on next pge) 3 Moleulr Cell 58, 21 34, April 2, 215 ª215 Elsevier In.

12 A Figure 7. Stimultion with TNF Drives FoxA1 to Chromtin in MCF-7 Cells (A) Western lots of different suellulr protein frtions showing derese in nuleoplsmi FoxA1 nd orresponding inrese in hromtinound FoxA1 in ells treted with TNF in the presene or sene of E2. Frtion-speifi mrkers nd loding ontrols inlude -tuulin (-Tu), histone H3 (H3), nd SNRP7. (B) A model for ytokine-indued ltent ER enhners in rest ner ells, s desried in the text. B results in the formtion of on fide tive enhners with drmti effets on trget gene trnsription (Figure 3C). The trnsition from quiesent to tivted hromtin lndspe t the ltent ER enhners is lrgely medited y TNF-indued reruitment of NF-kB or FoxA1 to these sites (Figures 5 nd 6). Distint Brest Cner Cell Trnsriptomes Are Controlled y Poised versus Ltent ER Enhners Our results show tht the mjority of E2- regulted trget genes in MCF-7 ells re ontrolled y poised ( mintined ) ER enhners tht re loted in open regions of hromtin nd re onstitutively ound y FoxA1 (or other linegespeifi trnsription ftors). The estrogen-dependent expression of the genes trgeted y these enhners is lrgely unffeted y TNF signling (Figure 3C, left). In ontrst, smller set of estrogen-dependent ltent ER enhners, whih re reted in response to TNF signling nd re dependent on the tions of NF-kB nd FoxA1, speifies distint trnsriptome tht is uniquely responsive to the omined tions of E2 nd TNF (Figure 3C, right). This unique trnsriptome, whih is reted upon the onvergene of mitogeni nd proinflmmtory signling, ontins mrna nd lnrna genes tht re newly or synergistilly regulted (either up or down) only upon the omined tions of E2 nd TNF. The genes within this unique trnsriptome, whih re involved in moleulr nd ellulr proesses rnging from signling nd trnsription to metolism, prolifertion, nd poptosis (Tle S1), re likely to ontrol the unique ellulr outomes upon omined tretment with E2 nd TNF (e.g., inhiition of the mitogeni growth of MCF-7 ells; Figure 1C). Although it is formlly possile tht TNF signling ts dominntly to E2 signling to repress ell prolifertion (E) Averge fold hnges in ER inding t gined ER inding sites ffeted versus unffeted upon FoxA1 KD. Brs mrked with different letters re signifintly different (Wiloxon rnk sum test, p <.1). (F) Boxplots showing p65 oupny t gined ER inding sites ffeted versus unffeted upon FoxA1 KD. Brs mrked with different letters re signifintly different (Wiloxon rnk sum test, p <.1). See lso Figure S6. Moleulr Cell 58, 21 34, April 2, 215 ª215 Elsevier In. 31

13 through the expression of TNF-regulted trget gene set, rther thn through the unique E2 + TNF-regulted trnsriptome desried ove, we think this is unlikely for the following resons. First, the unique E2 + TNF-regulted gene set is strong preditor of linil outomes in rest ners, independent of the lssil TNF-regulted proinflmmtory gene set (Figures 1E nd S1F). Seond, the unique E2 + TNF-regulted gene set is enrihed for gene ontologies relted to ell growth ontrol (Tle S1). Third, the FoxA1 dependene of the effets we oserved, oupled with the key role tht FoxA1 plys in ER-dependent rest ner ell growth (Hurtdo et l., 211; Ross-Innes et l., 212), supports role for positive funtionl interply etween E2/ER, TNF, nd FoxA1 in ontrolling trnsriptome tht dittes ell growth responses. Proinflmmtory Signling in Brest Cners: Unexpeted Clinil Outomes Reent studies hve identified importnt effets of proinflmmtory signling in ners, whih supplies the tumor miroenvironment with iotive moleules tht filitte ngiogenesis, invsion, nd metstsis (Bumgrten nd Frsor, 212; Ben- Nerih nd Krin, 211). In this regrd, numer of reent studies hve shown tht proinflmmtory signling n led to more ggressive hormone-dependent rest ners (Coussens et l., 213; Frsor et l., 29; Klitzidis nd Gilmore, 25; Prdhn et l., 212; Ross-Innes et l., 212; Zhou et l., 25). However, s our dt show, the funtionl reltionships nd outomes of the rosstlk etween NF-kB/proinflmmtory signling nd ER/mitogeni signling in rest ners re omplex nd ontext dependent. This is oserved t the level of the proinflmmtory trnsriptome, even in the sene of estrogen signling, where high-level expression of proinflmmtory gene set n e ssoited with good or poor linil outomes depending on the rest ner type (Figures 1D nd S1F). We hve identified unique trnsriptome, whih is reted upon the onvergene of mitogeni nd proinflmmtory signling, tht is roust preditor of linil outomes. Similr to the proinflmmtory trnsriptome, this gene set n e ssoited with good or poor linil outomes depending on the rest ner type (Figures 1D nd S1F). This vriility my e ontrolled, in prt, y differenes in the length of exposure to the proinflmmtory meditors (e.g., hroni versus ute, s in our studies herein), the type of ytokine (e.g., TNF versus IL4), nd the presene of other tivted signling pthwys (e.g., with or without lignd-tivted ER). Tken together, our results highlight the potentil vried responses nd linil outomes of rest ners upon proinflmmtory signling. EXPERIMENTAL PROCEDURES Additionl detils on the experimentl proedures n e found in the Supplementl Informtion. Cell Culture, Tretments, nd RNAi-Medited Knokdown MCF-7 rest ner ells were otined from the ATCC nd mintined in Minimum Essentil Medium (MEM) Egle supplemented with 5% lf serum. Prior to ll tretments, the ells were grown for 3 dys in phenol red-free MEM Egle supplemented with 5% hrol-dextrn-treted lf serum. All tretments were performed for 4 min with 1 nm 17--estrdiol, 25 ng/ml TNF, or oth. For sirna trnsfetions, ommerilly ville sirna oligonuleotides (Sigm) were trnsfeted t finl onentrtion of 1 nm using Lipofetmine RNAiMAX regent (Invitrogen). Antiodies The ntiodies used for oth western lotting nd ChIP were s follows: ER (rit polylonl generted in the W.L.K. l), p65 (Am 797), FoxA1 (Am 23738), SNRP7 (Am 8336), -tuulin (Am 646), nd histone H3 (Am 1791). Cell Prolifertion Assys Tretments nd ell olletions were performed t 2-dy intervls. Cells were fixed with 1% formldehyde nd stined with.1% rystl violet. Inorported rystl violet ws extrted using 1% glil eti id nd the sorne ws red t 595 nm. Kpln-Meier Anlyses Kpln-Meier estimtors (Dinse nd Lgkos, 1982) were generted using the GOBO tool ( (Ringnér et l., 211). Gene sets determined y GRO-seq were provided s inputs to ssess ptient outomes in ER-positive rest ner sutypes. GRO-seq Nulei isoltion, nuler run-on, nd lirry preprtion were performed s previously desried (Dnko et l., 213; Hh et l., 211), with modifitions (Dnko et l., 213; Luo et l., 214). The lirries were generted from two iologil replites using irulrized ligtion-sed protool for dptor ligtion used to improve the effiieny of lirry preprtion, redue sequene is, nd llow for roding (Luo et l., 214; Wng et l., 211). The GROseq dt were nlyzed using the grohmm softwre pkge desried previously (Dnko et l., 213; Hh et l., 211; Luo et l., 214). The effets of E2, TNF, nd E2 + TNF on the expression of oding nd non-oding genes were nlyzed using edger (Roinson et l., 21). Further GRO-seq dt nlyses, inluding dt visuliztion, re desried in detil in the Supplementl Informtion. ChIP ChIP ws performed s previously desried (Kininis et l., 27) with few modifitions. For ChIP involving sirna knokdown of p65 or FoxA1, ells were trnsfeted with sirna omplexes 48 hr prior to E2 nd/or TNF tretment. The enrihed genomi DNA ws nlyzed y qpcr using the enhner-speifi primers listed in the Supplementl Informtion. ChIP-seq ChIP-seq lirries were generted from two iologil replites for eh ondition. A totl of 5 ng of ChIPed DNA were used to generte lirries for sequening, s previously desried (Quil et l., 28), with some modifitions. Sequening dptors were tthed using the Illumin TruSeq DNA Smple Prep Kit nd sequened using n Illumin HiSeq 2. The ChIP-seq lirries were ligned to hg19 genome using defult prmeters of BOWTIE (Lngmed et l., 29). Uniquely mpple reds were onverted into igwig files using BEDTools for visuliztion in the UCSC genome rowser (Quinln nd Hll, 21). ER, p65, nd FoxA1 ChIP-seq smples from the untreted ondition were used s ontrols for pek lling using MACS softwre (Zhng et l., 28). Further ChIP-seq dt nlysis is desried in detil in the Supplementl Informtion. mrna Expression Anlyses y RT-qPCR Totl RNA ws isolted using Trizol Regent (Invitrogen), reverse trnsried, nd sujeted to rel-time qpcr using the gene-speifi primers listed in the Supplementl Informtion. Trget gene trnsripts were normlized to the -tin trnsript. Suellulr Frtiontion nd Anlyses Whole-ell extrts were prepred in 5 mm Tris-HCl (ph 8), 15 mm NCl, 1% NP-4,.5% sodium deoxyholte, nd.1% SDS ontining protese 32 Moleulr Cell 58, 21 34, April 2, 215 ª215 Elsevier In.

14 inhiitors. Cytoplsmi nd nuler extrts were mde using the CelLyti NuCLEAR Extrtion Kit from Sigm. The remining hromtin pellet ws soluilized with enzonse nulese (Sigm) nd oiled in 13 SDS smple uffer. The different suellulr frtions were nlyzed for FoxA1 y western lotting using set of frtion-speifi mrkers nd loding ontrols (-tuulin, histone H3, nd SNRP7). ACCESSION NUMBERS The GRO-seq, ER ChIP-seq, FoxA1 knokdown ER ChIP-seq, NF-kB p65 ChIP-seq, nd FoxA1 ChIP-seq dt sets for ll four tretments (Veh, E2, TNF, nd E2 + TNF) re ville from the NCBI s GEO dtse using ession numers GSE5953, GSE59531, nd GSE SUPPLEMENTAL INFORMATION Supplementl Informtion inludes six figures, one tle, nd Supplementl Experimentl Proedures nd n e found with this rtile online t dx.doi.org/1.116/j.molel ACKNOWLEDGMENTS We thnk Christopher K. Glss nd Josh D. Stender t UCSD for their helpful insights nd suggestions out this work. We lso thnk memers of the W.L.K. l for helpful disussions out this mnusript, nd Rosemry Conry for tehnil ssistne. This work ws supported y postdotorl fellowship from the Amerin Cner Soiety - Lee Ntionl Denim Dy Fellowship to H.L.F. nd grnt from the NIH/NIDDK (DK5811) to W.L.K. Reeived: July 3, 214 Revised: Novemer 18, 214 Aepted: Jnury 27, 215 Pulished: Mrh 5, 215 REFERENCES Bumgrten, S.C., nd Frsor, J. (212). Minireview: inflmmtion: n instigtor of more ggressive estrogen reeptor (ER) positive rest ners. Mol. Endorinol. 26, Ben-Nerih, Y., nd Krin, M. (211). Inflmmtion meets ner, with NF-kB s the mthmker. Nt. Immunol. 12, Coussens, L.M., Zitvogel, L., nd Pluk, A.K. (213). Neutrlizing tumor-promoting hroni inflmmtion: mgi ullet? Siene 339, Cunninghm, M., nd Gilkeson, G. (211). Estrogen reeptors in immunity nd utoimmunity. Clin. Rev. Allergy Immunol. 4, Dnko, C.G., Hh, N., Luo, X., Mrtins, A.L., Core, L., Lis, J.T., Siepel, A., nd Krus, W.L. (213). 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15 Rpid nd pervsive hnges in genome-wide enhner usge during mmmlin development. Cell 155, Ostuni, R., Piolo, V., Brozzi, I., Polletti, S., Termnini, A., Bonifio, S., Curin, A., Prosperini, E., Ghisletti, S., nd Ntoli, G. (213). Ltent enhners tivted y stimultion in differentited ells. Cell 152, Prdhn, M., Beminster, L.A., Bumgrten, S.C., nd Frsor, J. (21). Proinflmmtory ytokines enhne estrogen-dependent expression of the multidrug trnsporter gene ABCG2 through estrogen reeptor nd NFkB oopertivity t djent response elements. J. Biol. Chem. 285, Prdhn, M., Bumgrten, S.C., Beminster, L.A., nd Frsor, J. (212). CBP medites NF-kB-dependent histone etyltion nd estrogen reeptor reruitment to n estrogen response element in the BIRC3 promoter. Mol. Cell. Biol. 32, Quil, M.A., Kozrew, I., Smith, F., Slly, A., Stephens, P.J., Durin, R., Swerdlow, H., nd Turner, D.J. (28). A lrge genome enter s improvements to the Illumin sequening system. Nt. Methods 5, Quinln, A.R., nd Hll, I.M. (21). BEDTools: flexile suite of utilities for ompring genomi fetures. Bioinformtis 26, Ro, N.A., MClmn, M.T., Moulos, P., Frnoijs, K.J., Chtziionnou, A., Kolisis, F.N., Alexis, M.N., Mitsiou, D.J., nd Stunnenerg, H.G. (211). Cotivtion of GR nd NFKB lters the repertoire of their inding sites nd trget genes. Genome Res. 21, Ringnér, M., Fredlund, E., Häkkinen, J., Borg, A., nd Stf, J. (211). GOBO: gene expression-sed outome for rest ner online. PLoS ONE 6, e Roinson, M.D., MCrthy, D.J., nd Smyth, G.K. (21). edger: ioondutor pkge for differentil expression nlysis of digitl gene expression dt. Bioinformtis 26, Ross-Innes, C.S., Strk, R., Teshendorff, A.E., Holmes, K.A., Ali, H.R., Dunning, M.J., Brown, G.D., Gojis, O., Ellis, I.O., Green, A.R., et l. (212). Differentil oestrogen reeptor inding is ssoited with linil outome in rest ner. Nture 481, Shlyuev, D., Stmpfel, G., nd Strk, A. (214). Trnsriptionl enhners: from properties to genome-wide preditions. Nt. Rev. Genet. 15, Tn, S.K., Lin, Z.H., Chng, C.W., Vrng, V., Chng, K.R., Pn, Y.F., Yong, E.L., Sung, W.K., nd Cheung, E. (211). AP-2g regultes oestrogen reeptor-medited long-rnge hromtin intertion nd gene trnsription. EMBO J. 3, Wng, D., Gri-Bssets, I., Benner, C., Li, W., Su, X., Zhou, Y., Qiu, J., Liu, W., Kikkonen, M.U., Ohgi, K.A., et l. (211). Reprogrmming trnsription y distint lsses of enhners funtionlly defined y erna. Nture 474, Zhng, Y., Liu, T., Meyer, C.A., Eekhoute, J., Johnson, D.S., Bernstein, B.E., Nusum, C., Myers, R.M., Brown, M., Li, W., nd Liu, X.S. (28). Modelsed nlysis of ChIP-Seq (MACS). Genome Biol. 9, R137. Zhou, Y., Eppenerger-Cstori, S., Mrx, C., Yu, C., Sott, G.K., Eppenerger, U., nd Benz, C.C. (25). Ativtion of nuler ftor-kppb (NFkppB) identifies high-risk suset of hormone-dependent rest ners. Int. J. Biohem. Cell Biol. 37, Moleulr Cell 58, 21 34, April 2, 215 ª215 Elsevier In.

16 Moleulr Cell, Volume 58 Supplementl Informtion TNF Signling Exposes Ltent Estrogen Reeptor Binding Sites to Alter the Brest Cner Cell Trnsriptome Hetor L. Frno, Anush Ngri, nd W. Lee Krus

17 Figure S1 (see legend on the next pge) A Cytoplsm Nuleus F Proinflmmtory Gene Set p65 ERα β-tu SNRP7 B Veh E2 Veh E2 TNFα E+T Veh E2 TNFα E+T Long Non-Coding RNA Genes TNFα Tretment Up Down +1 E TNF Distnt Metstsis Free Survivl G Overll Survivl TAM Treted PAM5 HER2 Enrihed HU ERBB2 ER+ Tumors ER+ LN Negtive Untreted Tumors PAM5 Luminl A Grde 3 Tumors HU Luminl B Grde 2 Tumors De Novo nd Synergistilly Up-Regulted Genes PAM5 Luminl B HU Norml-Like Grde 1 Tumors HU Luminl A PAM5 Bsl HU Bsl PAM5 Norml-Like LN+ Tumors -log p vlue Outome 3 Good Poor C Up Regulted (Fold Chnge) Down Regulted (Fold Chnge) D Cell Growth (As 595) n = De Novo (Gined) Genes 15 n = E T E+T n = 521 E T E+T E2 E2 E2+TNF(.1ng/ml) E2+TNF(1ng/ml) E2+TNF(5ng/ml) E2+TNF(1ng/ml) E2+TNF(25ng/ml) E2+TNF 882 1,193 Synergistilly Regulted Genes 25 n = MCF-7 Cell Prolifertion Dy Dy 6 E T E+T n = 412 E T E+T Distnt Metstsis Free Survivl Overll Survivl Distnt Metstsis Free Survivl Overll Survivl LN Pos Tumors Untreted Tumors ER+ Tumors ER+ LN Negtive HU Luminl B Grde 1 Tumors PAM5 Luminl B HU Bsl TAM Treted HU Norml-Like ER+ LN Negtive Grde 3 Tumors HU Bsl PAM5 Luminl A PAM5 HER2 Enrihed PAM5 HER2 Enrihed PAM5 Norml-Like De Novo nd Synergistilly Down-Regulted Genes PAM5 Norml-Like HU ERBB2 Grde 2 Tumors Grde 2 Tumors HU Luminl A PAM5 Luminl A ER+ Tumors HU Norml-Like PAM5 Luminl B HU Luminl A HU Luminl B Grde 1 Tumors Grde 3 Tumors TAM Treted LN Pos Tumors PAM5 Bsl -log p vlue Untreted Tumors -log p vlue Outome 2 Good Poor H Outome 3 Good Poor Pro-inflmmtory Genes De Novo nd Synergistilly Up- Regulted Genes De Novo nd Synergistilly Down- Regulted Genes E Cell Growth (As 595) ZR-75-1 Cell Prolifertion Veh E2 (1-7 M) TNFα (25 ng/ml) Cell Growth (As 595) MDA-MB-361 Cell Prolifertion Veh E2 (1-7 M) TNFα (25 ng/ml) Dys Dys

18 Figure S1, relted to Figure 1. Crosstlk Between Mitogeni nd Proinflmmtory Signling in Brest Cner Cells: Effets on LnRNA Expression, Cell Growth, nd Brest Cner Ptient Outomes. MCF-7, ZR-75-1, or MDA-MB-361 ells were treted with vehile (Veh or V), estrdiol (E or E2), TNFα (T or TNF), or oth E2 nd sujeted to Western lotting for NF-κB p65 or ERα, GRO-seq, or ell prolifertion ssys. The GRO-seq dt were nlyzed for ptterns of gene expression nd relted to rest ner ptient outomes. (A) Western lot showing the nuler trnslotion of the p65 suunit of NF-κB in response to TNFα or E2 + TNFα, nd the stiliztion of ERα in the nuleus fter tretment with E2 or E2 + TNFα. (B) Hetmp (left) nd tle (right) showing the regultion of ll long non-oding RNA genes under eh tretment ondition s reveled y GRO-seq. (C) Box plots showing hnges in GRO-seq signls for lnrna genes tht re newly regulted (de novo or gined) (left) or synergistilly regulted (right) y E2 + TNFα. Brs mrked with different letters re signifintly different (Wiloxon rnk sum test, p <.1). (D) The prolifertive response of MCF-7 ells to E2 is inhiited y inresing mounts of TNFα. Eh r represents the men + SEM, n = 3. (E) ZR-75-1 nd MDA-MB-361 ell prolifertion ssys. ZR751 ells re from invsive dutl rinom from the luminl A moleulr sutype, similr to MCF-7 ells, nd MDA-MB-361 ell re from the luminl B moleulr sutype. Eh dt point represents the men ± SEM, n = 3. (F nd G) Kpln-Meier survivl nlyses for mny rest ner types using three distint gene sets s inputs: (1) proinflmmtory gene set defined y gene ontologies (pnel E), (2) the E2 + TNFα de novo nd synergistilly up-regulted gene set (pnel F, top), nd (3) the E2 + TNFα de novo nd synergistilly down-regulted gene set (pnel F, ottom). The rest ner outomelinked gene expression dt were essed using the Gene Expression-Bsed Outome for Brest Cner Online (GOBO) tool. High levels of expression of these genes sets re ssoited with good outomes in some rest ners nd poor outomes in others. (H) Venn digrm showing the limited overlp of the E2 + TNFα de novo nd synergistillyregulted gene sets with the proinflmmtory gene set defined y gene ontologies.

19 A Enhner ChIP-qPCR / ZR-75-1 Cells B MCF-7 Cells! C MCF-7 Cells! Mintined ERα Enhner; No NF-κB Gined ERα Enhner; High NF-κB Gined ERα Enhner; Low NF-κB FMN1 Veh E2 TNFα E+T POU3F1 Veh E2 TNFα E+T No A Veh E2 TNFα E+T ERα P2RY2 EFNA1 Veh E2 TNFα E+T GALNTL4 NF-κB ChIP-Seq Tretment: Veh E2 TNFα E+T Veh E2 TNFα E+T Gined NF-κB Peks (1,68) Mintined NF-κB Peks (851; 1% Shown) Lost NF-κB Peks (1,197) 5 k Pek Center ERα ChIP-Seq Red Counts Red Counts Red Counts NF-κB 5 d NF-κB NF-κB d Red Counts Red Counts Red Counts 25 2 ERα ERα d ERα Veh E2 TNFα E+T Veh E2 TNFα E+T Figure S2, relted to Figure 2. Co-Stimultion of the Estrogen nd TNFα Signling Pthwys Promotes the Formtion of Gined ERα Binding Sites in ZR-75-1 Cells nd Alters the NF-κB Cistrome in MCF-7 Cells. (A) Confirmtion of mintined nd gined ERα inding sites t TNFα-responsive enhners in ZR-75-1 ells. ChIP-qPCR for ERα in ells with or without E2 or TNFα tretment. The enhners re designted y the nerest neighoring gene. Eh r represents the men + SEM, n = 3. The sterisks indite signifint differenes from the orresponding ontrol (Student s t- test p-vlue <.5). (B) (Left) Hetmps of NF-κB p65 ChIP-seq reds from MCF-7 ells treted with E2, TNFα, or oth. (Right) Hetmp of ERα ChIP-seq reds t the genomi loi where NF-κB p65 is ound. (C) Box plot of the normlized red ounts for three distint sets of NF-κB p65 inding sites (gined, mintined, lost). Brs mrked with different letters re signifintly different (Wiloxon rnk sum test, p <.1).

20 Figure S3 (see legend on the next pge) A Enhner Trnsription (Log2 Fold Chnge with E2) Mintined ERα Binding Sites ERα Binding (Log2 Fold Chnge with E2) Enhner Trnsription (Log2 Fold Chnge with E2 + TNFα) Gined ERα Binding Sites ERα Binding (Log2 Fold Chnge with E2 + TNFα) Person s Corr. Coeff =.43 p-vlue < 2.2 x 1-16 Person s Corr. Coeff. =.39 p-vlue < 2.2 x 1-16 B Frtion of Binding Sites D Gined ERα Enhners ERα β-tu. Gined Mintined Lost Gene Ctegories si sierα sierα CTRL (1 nm) (2 nm) C Lost ERα Enhners E Reltive Expression 2.5 ER ER E2: : FMN1 mrna NS KD E+T ERα Enhner Trnsription Nerest Gene Trnsription Tretment Tretment RPKM Reltive Expression POU3F1 mrna NS KD E+T RPKM Reltive Expression mrna NS KD E+T F Dimensions! Mintined ERα Enhners Gined ERα Enhners Medin Log2 Fold Chnge Dimension E2 E2 + TNFα Δ p-vlue ) ERα inding ) Enhner Trnsription ) Nerest Gene Trnsription ) ERα inding x 1-16 ) Enhner Trnsription x 1-16 ) Nerest Gene Trnsription x 1-1