ORIGINAL RESEARCH

The Estrogen-Regulated PITX1 Coordinates -Specific Regulation by Estrogen -Alpha in Breast Cancer Cells

Joshua D. Stender, Fabio Stossi, Cory C. Funk, Tze Howe Charn, Daniel H. Barnett, and Benita S. Katzenellenbogen Downloaded from https://academic.oup.com/mend/article/25/10/1699/2614663 by guest on 23 September 2021 Departments of Biochemistry (J.D.S.) , Molecular and Integrative Physiology (F.S., B.S.K.), Cell and Developmental Biology (C.C.F., D.H.B., B.S.K.), and Bioengineering (T.H.C.), University of Illinois at Urbana-Champaign, Urbana Illinois 61801-3704

The ␣ (ER␣) is a master regulator of and works along with cooperating transcription factors in mediating the actions of the hormone estradiol (E2) in ER- positive tissues and breast tumors. Here, we report that expression of paired-like homeodomain transcription factor (PITX1), a tumor suppressor and member of the family of tran- scription factors, is robustly up-regulated by E2 in several ER␣-positive breast cancer cell lines via ER␣-dependent interaction between the proximal promoter and an enhancer region 5Ј upstream of the PITX1 gene. Overexpression of PITX1 selectively inhibited the transcriptional activity of ER␣ and ER␤, while enhancing the activities of the and . Reduction of PITX1 by small interfering RNA enhanced ER␣-dependent transcriptional regulation of a subset of ER␣ target . The consensus PITX1 binding motif was found to be present in 28% of genome-wide ER␣ binding sites and was in close proximity to estrogen response elements in a subset of ER␣ binding sites, and E2 treatment enhanced PITX1 as well as ER␣ recruitment to these binding sites. These studies identify PITX1 as a new ER␣ transcriptional target that acts as a repressor to coordinate and fine tune target-specific, ER␣-mediated transcriptional activity in human breast cancer cells. (Molecular Endocrinology 25: 1699–1709, 2011)

strogens regulate gene expression in target cells, the basal transcriptional machinery that leads to regula- Ethereby controlling the development and optimal tion of target gene expression (5–8). Studies have now functioning of reproductive tissues and many nonrepro- identified genome-wide binding sites for the nuclear re- ductive tissues, such as bone. Estrogens, however, may ceptor ER␣ in human breast cancer cells, and have also also contribute to the growth and progression of breast identified cooperating transcription factors the expres- and uterine cancers (1, 2). Estrogen receptor (ER) ␣,a sion of which appears critical for ER␣ regulation of gene ligand-inducible transcription factor, controls the stimu- expression (9–16). lation and repression of specific gene expression in a sig- The paired-like homeodomain transcription factor 1 nal-, tissue-, and promoter-specific fashion (3, 4). (PITX1) is present throughout pituitary development Upon binding of 17␤-estradiol (E2), the receptor and regulates several pituitary-specific promoters (17). changes conformation, resulting in dimerization and dis- Despite PITX1 characterization in the pituitary and its sociation from inhibitory complexes, which enables ER␣ cell lineages, the functions of PITX1 and its regulation association with regulatory binding sites in the promoter outside the pituitary are not well understood. PITX1 is and/or enhancer regions of target genes and the recruit- present in many tissues (18), and its expression is ment of coregulatory , RNA polymerase II, and known to be critical for the development of the anterior

ISSN Print 0888-8809 ISSN Online 1944-9917 Abbreviations: ChIP, Chromatin immunoprecipitation; E2, estradiol; ER, estrogen recep- Printed in U.S.A. tor; ERE, estrogen response element; FST, follistatin; ICI, ICI 182,780; IRF, interferon- Copyright © 2011 by The Endocrine Society regulatory factor; PITX1, paired-like homeodomain transcription factor 1; Ral, raloxifene; doi: 10.1210/me.2011-0102 Received March 2, 2011. Accepted July 27, 2011. siRNA, small interfering RNA; SDS, sodium dodecyl sulfate; TFF1, trefoil factor 1; TOT, First Published Online August 25, 2011 trans-hydroxytamoxifen.

Mol Endocrinol, October 2011, 25(10):1699–1709 mend.endojournals.org 1699 1700 Stender et al. PITX1 and Estrogen Receptor Gene Regulation Mol Endocrinol, October 2011, 25(10):1699–1709

PITX1 mRNA PITX1 mRNA Results A 4 B 8 Veh * ER␣-dependent regulation of 3 E2 * 6 *

mRNA) * * ( PITX1 in breast cancer cells 2 4 * Because our genome-wide cDNA mi- PI TX1 croarray transcriptional profiling studies 1 2 04 82448 Fold Change

Fold Change (mRNA) in ER-positive MCF-7 breast cancer

0 0 cells indicated that expression of the ZR75 MCF-7 231ER+ 0 4 8 12 16 20 24 homeobox family member PITX1 was ER+ Cell Line E2 Treatment (h) stimulated by treatment with the steroid PITX1mRNA PITX1 mRNA C 4 D 4 hormone, E2 (27), we initiated studies to Downloaded from https://academic.oup.com/mend/article/25/10/1699/2614663 by guest on 23 September 2021 * * Veh Veh further explore ER regulation of PITX1 3 * E2 3 E2 and its impact in breast cancer cells. mRNA) * ( ␣ 2 2 Treatment of the ER -positive ZR-75 ange

h and MCF-7 cells, and MDA-MB-231 C

1 d 1 cells stably expressing the ER␣ (denoted Fol d Change (mRNA) Fol 231ERϩ cells) with 10 nM E2 for 4 h 0 0 Veh TOT Ral ICI Actinomycin D Cycloheximide stimulated PITX1 mRNA 2- to 3-fold in SERM(1µM) Inhibit or all three cell lines (Fig. 1A). In time FIG. 1. Up-regulation of PITX1 by estrogens in ER␣-positive breast cancer cells. A, course studies, PITX1 mRNA was ele- Quantitative PCR analysis of PITX1 mRNA expression in the ER␣-positive cell lines ZR75, MCF- 7, and 231ERϩ cells treated with 0.1% ethanol vehicle or 10 nM E2 for 4 h. B, Quantitative vated by2hofE2exposure, and levels PCR analysis for PITX1 mRNA stimulation in 231ERϩ cells treated with 10 nM E2 for the times continued to increase over the 24 h of E2 indicated. Inset, Western blot analysis of PITX1 in 231ERϩ cells treated with 10 nM E2 treatment (Fig. 1B). PITX1 protein was for 0–48 h. C, Quantitative real-time PCR analysis for PITX1 mRNA in 231ERϩ cells treated also increased after4hof10nM E2 treat- with either 1 ␮M TOT, 1 ␮M Ral, or 1 ␮M ICI alone or in combination with 10 nM E2 for 4 h. D, Real-time PCR analysis to determine whether actinomycin D (5 ␮M) or cycloheximide (10 ment of 231ERϩ cells and demonstrated ␮g/ml) can block E2 stimulation of PITX1 mRNA in 231ERϩ cells. Cells were exposed to a marked, maximal increase after 24–48 actinomycin D or cycloheximide for 1 h before addition of control vehicle or 10 nM E2 for 4 h h of hormone treatment (Fig. 1B, inset). in the continued presence of the inhibitor. Values are the mean from three independent experiments Ϯ SEM.*,P Ͻ 0.05 compared with0horvehicle treatment. Veh, Vehicle. To determine the ability of the pure an- tiestrogen ICI 182,780 (ICI), and the se- lective ER modulators trans-hydroxyta- pituitary gland and hind limb morphogenesis (19, 20). moxifen (TOT) and raloxifene (Ral) to stimulate PITX1 Initially identified as an activator of the pro-opiomela- mRNA and/or block E2 stimulation of PITX1 mRNA, we nocortin gene (21), PITX1 is now recognized to syner- treated 231ERϩ cells with 1 ␮M ICI, TOT, or Ral in the gize with the transcription factors SF-1, Pit1, and the presence and absence of 10 nM E2. As shown in Fig. 1C, we basic helix-loop-helix transcription factors to regulate observed no regulation of PITX1 mRNA by ICI or Ral alone some genes in a promoter-specific manner in pituitary- (open bars), and both ligands antagonized the E2 stimula- derived cell types (22–25). PITX1 also has the ability to tion of PITX1 mRNA (solid bars). By contrast, TOT func- trans-repress the virus-induced interferon A promoter tioned as a mixed agonist/antagonist. Treatment with TOT through interaction with the interferon-regulatory fac- alone resulted in a weak stimulation of PITX1 mRNA, and tors (IRF) 3 (IRF3) and 7 (IRF7) (26). Recent reports TOT partially antagonized the E2-induced stimulation. The have shown PITX1 to suppress RAS activity through ligands showed similar effects on PITX1 mRNA expression regulation of the RASAL gene, suggesting that PITX1 in MCF-7 cells, where TOT was a mixed agonist/antagonist may also act as a tumor suppressor (18). and Ral and ICI behaved as pure antagonists (27). Here we report that PITX1 is under primary tran- We next examined whether PITX1 was under primary scriptional control by ER␣ in breast cancer cells, and regulation by the ER and whether the E2 enhancement of we show that its up-regulation involves a long-range PITX1 mRNA requires active transcription and/or new pro- interaction between a novel estrogen-responsive en- tein production. As shown in Fig. 1D, treatment of cells with hancer and the proximal promoter of PITX1. In addi- the translational inhibitor cycloheximide did not abrogate tion, we demonstrate that PITX1 binds to ER␣,is the E2 stimulation of PITX1 mRNA, whereas the transcrip- recruited along with ER␣ to a subset of ER␣ enhancers, tional inhibitor Actinomycin D completely blocked E2 stim- and modulates the transcriptional activity of this nu- ulation. These results suggest that PITX1 mRNA is under clear receptor on ER␣ target genes that are enriched in primary transcriptional control by the receptor in several PITX1 binding sites. ER␣-positive breast cancer cells. Mol Endocrinol, October 2011, 25(10):1699–1709 mend.endojournals.org 1701

ER␣ regulation of PITX1 involves interaction of the there was enhanced recruitment of both ER␣ and RNA ER-binding distal enhancer with the promoter of polymerase II to the PITX1 enhancer. E2 also increased the PITX1 gene via intrachromosomal looping ER␣ presence at the PITX1 promoter, which contains Recent studies have identified genome-wide ER bind- only an ERE half-site, although RNA polymerase II re- ing sites in MCF-7 and 231ERϩ breast cancer cells cruitment at the promoter was minimally, if at all, in- treated with E2 (10, 13, 15, 16). Because PITX1 mRNA creased by E2 (Fig. 2B). is stimulated by E2 and TOT (Fig. 1), we examined We next determined whether the PITX1 enhancer whether there was an ER binding site in proximity of the and promoter regions were estrogen responsive by PITX1 gene in these genome-wide ER␣ binding datasets cloning these regions from genomic DNA into lu- (15, 16), and we identified a strong binding site Ϫ12 kb ciferase reporters, and we conducted transient transfec- upstream of the transcription start site of PITX1 in addi- tion assays. The regions that were cloned are indicated Downloaded from https://academic.oup.com/mend/article/25/10/1699/2614663 by guest on 23 September 2021 tion to a weak ER␣ binding site in the proximal promoter as the red blocks in Fig. 2A and include 1000 bp im- (Fig. 2A). The ER binding site located Ϫ12 kb upstream mediately upstream of the transcription start site of the will herein be referred to as the PITX1 enhancer. ER␣ PITX1 gene for the promoter, and 600 bp surrounding localized to this enhancer in the presence of E2 in the Ϫ12 kb ER␣ binding site for the enhancer. Estra- 231ERϩ cells and in the presence of both E2 and TOT in diol treatment stimulated PITX1 enhancer activity MCF-7 cells (Fig. 2A). This PITX1 enhancer contains a 4-fold, while stimulating the PITX1 promoter 2-fold full estrogen response element (ERE), GGGCACATT- (Fig. 2C). These data demonstrate that ER␣ is recruited GACC, differing from the consensus ERE at only the one to and regulates both the identified PITX1 enhancer underlined position. PITX1 is the closest gene to this and promoter regions. binding site and is therefore the most likely gene to be Even though ER␣ is recruited to the PITX1 enhancer regulated by this ER binding site. To investigate this, we and this enhancer is stimulated by E2, these observations performed chromatin immunoprecipitation (ChIP) assays do not conclusively demonstrate a role for this enhancer (Fig. 2B) and observed that, at 45 min of E2 treatment, in PITX1 gene regulation by ER␣. Therefore, to implicate

A C -12 kb Scale Pr omot er 10 kb Enhancer chr5: 134400000 134405000 134410000 5 5 26 - 231-ERα-WT-E2 4 4 * 5 _ 143 - MCF7-ERα-E2 3 3 6 _ * 30 - MCF7-ERα-Tam 2 2 Fold C hange Fold Change 5 _ Primers 1 1 Promoter (-12kb Enhancer) PITX1 0 0 Veh E2 Veh E2 Tr eat ment Tr e at ment B Promoter Enhancer

ERα pol II ERα pol II 0.5 2.0 0.6 4.5 * * D * - T4Ligase +T4Ligase 4.0 0.5 * 0.4 3.5 2036bp Veh 1.5 Veh * Veh Veh 1636bp E2 E2 0.4 E2 3.0 E2 PITX1 0.3 ut

p 2.5 1.0 0.3 1018bp %Input %In %Input %Input 2.0 0.2 E2 0.2 1.5 -+ -+ * 0.5 * 0.1 1.0 0.1 0.5 * 0.0 0.0 0.0 0.0 IgG ERα IgG Pol II IgG ERα IgG Pol II FIG. 2. E2 regulation of PITX1 involves communication between upstream and proximal ER binding sites. A, Genetic locus from the UCSC genome browser showing the natural 5Ј to 3Ј orientation of the PITX1 gene with the location of the identified ER␣ binding regions, the proximal promoter and enhancer, and the location of the primers used in ChIP and conformation capture experiments. B, ChIP assays for binding of ER␣ and RNA polymerase II (pol II) to the proximal promoter and Ϫ12 kb enhancer of PITX1 in MCF-7 cells treated with vehicle or 10 nM E2 for 45 min. Values show percent input and are representative of triplicate experiments. *, P Ͻ 0.05 compared with vehicle treatment. C, Regulation of the PITX1 proximal promoter and Ϫ12 kb enhancer luciferase reporters in response to vehicle or 10 nM E2 treatment for 24 h in MDA-MB-231 cells transfected with ER␣. The regions cloned into the luciferase reporter construct are indicated as red blocks in panel A. *, P Ͻ 0.05 compared with vehicle treatment. D, Chromosome conformation capture assay examining the interaction between the proximal promoter and Ϫ12 kb enhancer of PITX1 in MCF-7 cells treated with vehicle or 10 nM E2 for 45 min. The locations of the primers used are indicated in panel A. Tam, Tamoxifen; Veh, vehicle. 1702 Stender et al. PITX1 and Estrogen Receptor Gene Regulation Mol Endocrinol, October 2011, 25(10):1699–1709

two joined genomic DNA fragments. A α C Input ER IP IgG IP PITX1 Motifs E2-dependent looping was also ob- 0.005 PITX1 served in 231ERϩ cells (data not Veh E2 Veh E2 Veh E2 0.004 shown). These data demonstrate an ER Peaks E2-ER␣-induced interaction between 0.003 Ϫ B the 12 kb enhancer and the proximal Neither (6.4%) 0.002 promoter of the PITX1 genomic locus. Motifs per basepair 0.001 PITX1 and Random Presence of PITX1 binding motifs ERE/ Peaks Half Site(24.9%) 0 within ER binding sites -400 -300 -200 -100 0 100 200 300 400 PITXonly (2.9%) Downloaded from https://academic.oup.com/mend/article/25/10/1699/2614663 by guest on 23 September 2021 Distance to Nearest ERE Previous studies have demonstrated ERE/ Half Site only (65.8%) that ER␣ and PITX1 synergize to regulate the LH␤ gene (22). This prompted us to determine whether ER␣ and PITX1 inter- D PITX1 and ERE/half Site E ERE or half Site Only 60 60 act in breast cancer cells. Coimmunopre- Veh * * Veh t cipitation assays were performed in * E2 n E2

40 me 40 MCF-7 cells and as seen in Fig. 3A, PITX1

* it α ruitment α ␣ c interacted with ER in immunoprecipita- e ER ER R

d 20 * 20 ld Recru tion assays but not with the IgG control. o F Fol * Interaction was consistently observed in 0 0 the presence and absence of ligand in both TFF1 FST TSKU INHBB AKAP1 SDC4 Tar get Gene Tar get Gene MCF-7 cells (Fig. 3A) and in 231ERϩcells (data not shown), although the recruit- 12 12 ␣ * Veh Veh ment of PITX1 and ER to chromatin

E2 nt 9 e 9 E2 binding sites and the regulation of gene

ruitm expression was ligand dependent, as TX1 TX1

6 I 6 I P P Recruit ment * * Rec described below. Transcription is usually ld ld o F Fo 3 3 regulated by combinatorial usage of coop-

0 0 erating transcription factors in a signal- TFF1 FST TSKU INHBB AKAP1 SDC4 Tar get Gene Tar get Gene and promoter-specific manner (7). Recent FIG. 3. PITX1 motif enrichment in genome-wide ER binding sites. A, Coimmunoprecipitation genome-wide studies have shown that ER assay for ER␣ and PITX1. MCF-7 cells were infected with adenovirus expressing full-length binding site regions in human breast can- PITX1 for 24 h before 45-min control vehicle or 10 nM E2 treatment. Cells lysates were cer cells are enriched in binding sites for ␣ immunoprecipitated with either an ER -specific antibody or IgG negative control. Western cooperating transcription factors, such as blot analysis was performed using a PITX1-specific antibody. B, Percentage of ER␣ binding sites that contain a consensus PITX1 binding motif or lack PITX1 motifs. C, Spatial distribution the forkhead factor FOXA1 (10, 13). We between the identified PITX1 motifs and EREs within random genomic sequences or ER therefore examined whether the consensus binding peaks. D and E, Recruitment, monitored by ChIP, of ER␣ (top panels) and PITX1 PITX1 binding site, TAATCC, was en- (bottom panels) to ER binding sites that contain a consensus PITX1 binding site in vehicle and 10 nM E2-treated 231ERϩ cells at 45 min (panel D) or to ER binding sites that lack a riched in genome-wide ER binding sites consensus PITX1 binding site in vehicle and 10 nM E2-treated 231ERϩ cells at 45 min (panel (Fig. 3B). We identified that 1798 (28%, P E). *, P Ͻ 0.05 compared with vehicle treatment. IP, Immunoprecipitation; Veh, vehicle. Ͻ2.2 ϫ 10Ϫ16 relative to genomic back- ground) of the 6472 ER␣binding sites pre- this enhancer in receptor regulation of PITX1, we per- viously identified in 231ERϩ cells (15) contain a consensus formed chromosome conformation capture experiments PITX1 motif. Of the total 6472 ER binding sites, 65.8% con- to determine whether ER␣ promotes chromosomal loop- tained only an ERE or half-ERE site, 24.9% contained a PITX1 ing to bring the enhancer into close proximity with the and ERE/half-ERE site, 3% contained only a PITX1 site, and promoter of PITX1 in E2-treated cells. Using the primers designated in Fig. 2A, we observed an E2 and T4 ligase- 6.4% contained neither a PITX1 nor ERE/half-ERE site. This dependent PCR amplification of an approximately analysis suggests a potential role for PITX1 in modulating gene 1550-bp band, which is the predicted size based on the regulation mediated by approximately one fourth of ER bind- distance of the primers from the BamHI restriction en- ing sites (Fig. 3B). The distribution of PITX1 motifs in the ER␣ zyme cut sites. The identity of this DNA fragment was binding sites showed a strong colocalization near the putative confirmed by DNA sequencing and demonstrated that ERE, with the majority of the PITX1 motifs residing within -/ϩ approximately 10 kb of DNA was removed between the 100 bp of an ERE sequence (Fig. 3C). Mol Endocrinol, October 2011, 25(10):1699–1709 mend.endojournals.org 1703

To better characterize the role of PITX1 in ER␣-medi- evaluate whether ER␣ and PITX1 are present in the same ated gene regulation, we performed ChIP assays to exam- multiprotein complex at these gene sites, we performed ine the recruitment of ER␣ and PITX to ER binding sites ChIP/re-ChIP experiments after 45 min of E2 treatment containing both PITX1 and ERE/half-ERE sites, and for by first immunoprecipitating using a PITX1 antibody fol- comparison, to ER binding sites with ERE/half-ERE sites lowed by immunoprecipitating with an ER␣ antibody. but lacking a PITX1 binding site. As seen in Fig. 3D, E2 This method isolates DNA where ER␣ and PITX1 are treatment of 231ERϩ cells resulted in enhanced recruit- colocalized in the same complex. As shown in Fig. 4E, ment of both ER␣ and PITX1 to the trefoil factor 1 PITX1 and ER␣ were present together, in an E2-stimu- ␣ (TFF1) promoter, and also to the follistatin (FST) and lated manner, at genes with ER and PITX1 binding sites Tsukushin enhancers, sites that contain both ERE/half- (i.e. TFF1 and FST). To determine whether PITX1 ERE sites and PITX1 binding sites. By contrast, treatment recruitment at ER binding sites was dependent on the Downloaded from https://academic.oup.com/mend/article/25/10/1699/2614663 by guest on 23 September 2021 ␣ of cells with E2 increased the recruitment of ER␣ to the presence of ER , we conducted ChIP experiments in ϩ ␣ enhancer of inhibin ␤ B, A-kinase anchor protein 1, and 231ER cells in which we knocked-down ER using Syndecan 4, but, of note, E2 failed to stimulate recruit- small interfering RNA (siRNA). As shown in Fig. 4, F and ␣ ment of PITX1 to these ER␣ binding sites that lack PITX1 G, ER knockdown was very efficient as demonstrated by the complete loss of recruitment of ER␣ after E2 treat- binding motifs (Fig. 3E). E2 treatment also promoted the ment; and, more interestingly, the loss of ER␣ caused a recruitment of PITX1 to a subset of ER␣ binding sites that concomitant loss of PITX1 recruitment at the TFF1 and contain PITX1 motifs, but not ERE or half-EREs (data FST ER binding sites, indicating that recruitment of not shown). PITX1 requires the presence of ER␣. Further, ChIP time course experiments after E2 treat- ment revealed that both transcription factors were re- PITX1 inhibits ER␣ transcriptional activity, and this cruited rapidly and with similar time patterns of elevated repression maps to the C-terminal region of PITX1 recruitment at genes, such as TFF1 and FST that contain Based on the above observations, we examined the both ER␣ and PITX1 binding motifs (Fig. 4, A–D). To possibility that PITX1 might modulate ER␣ transcrip-

A TFF1 C TFF1 E TFF1 FST 6 0.4 1 * * Veh 5 * 0.75 E2 * * 0.3 * ** PITX1

4 * t

* Inpu 3 0.2 0.5 ERα % %Input %Input 2 * * 0.25 0.1 IgG 1 IgG 0 0 0 0 25 50 75 100 125 0255075100125 ERα/PITX1ERα/IgG ERα/PITX1ERα/IgG E2 Treatment (min) E2 Treatment (min) B D F G FST FST TFF1 FST TFF1 FST 8 0.6 4 0.5 t) * Veh * * Veh

* pu 0.5 E2 * PITX1* n I 0.4 E2 6 * ERα 3 * 0.4 * * * * 0.3 4 * 0.3 * 2 %Input %Input 0.2 0.2 2 0.1 IgG 1

IgG Recruitment (% 0.1 0 0 α 0 25 50 75 100 125 0 255075100125 PITX1 Recruitment (%Input) ER 0 0 E2Treatment (min) E2 Treatment (min) siCtl siERα siCtl siERα siCtl siERα siCtl siERα FIG. 4. Recruitment of ER␣ and PITX1 to binding sites of genes with both ER␣ and PITX binding motifs. A–D, Time course of ER␣ and PITX1 recruitment to two genes, TFF1 and FST, that contain both ER␣ and PITX1 binding sites, in response to 10 nM E2 treatment. ChIP assays were carried out at the times indicated with antibodies to ER␣ or PITX1, or with IgG as a negative control. *, P Ͻ 0.05 compared with corresponding IgG treatment. E, ChIP/reChIP analysis was conducted in MDA-MB-231ERϩ cells to determine whether ER␣ and PITX1 are in the same complex. ChIP assays were first performed on 231ERϩ cell lysates that were treated with vehicle or 10 nM E2 for 45 min and then with PITX1 antibody. The beads were washed extensively, and the complex was eluted from the beads. The sample was then immunoprecipitated with the ER␣ antibody or IgG. The DNA was isolated and quantified using quantitative real-time PCR. Data are presented as % Input and is mean Ϯ range of two independent experiments. F, ChIP assays for ER␣ in 231ERϩ cells after cells were transfected with ER␣ siRNA or control GL3 siRNA for 48 h before E2 or control vehicle treatment for 45 min. G, ChIP assays for PITX1 in 231ERϩ cells treated with ER␣ siRNA or control (GL3) siRNA and control vehicle or E2 as in panel F. *, P Ͻ 0.05 compared with0horvehicle treatment. Veh, Vehicle. 1704 Stender et al. PITX1 and Estrogen Receptor Gene Regulation Mol Endocrinol, October 2011, 25(10):1699–1709 tional activity. Transient transfections were performed in nuclear receptors, such as glucocorticoid receptor and pro- HEC-1 cells with ER␣, the estrogen-responsive reporter gesterone receptor. 3xERE-luciferase, which contains both an ERE and To determine the domains of PITX1 responsible for PITX1 site, and increasing amounts of PITX1 expression PITX1 trans-repression of ER␣, we created N-terminal vector. As seen in Fig. 5, A and B, increasing the level of and C-terminal truncations of PITX1 and assessed their PITX1 inhibited ER␣ and ER␤ transcriptional activity in ability to repress ER␣-mediated transcription (Fig. 5G). a dose-dependent manner. By contrast, PITX1 did not Estradiol stimulated the ERE-driven luciferase reporter alter activity of the (Fig. 5C) or reti- gene approximately 30-fold (entry 1), and addition of noic acid receptor (Fig. 5F), whereas it enhanced the full-length PITX1 blocked the ability of E2-ER␣ to stim- activity of both the glucocorticoid receptor (Fig. 5D) ulate transcription by greater than 80% (Fig. 5G, entry 2 and progesterone receptor (Fig. 5E). These data suggest vs.1). of the first 67 amino acids of PITX1 had Downloaded from https://academic.oup.com/mend/article/25/10/1699/2614663 by guest on 23 September 2021 that PITX1 specifically represses the transcriptional activity no significant effect on repression of ER␣-mediated tran- of ER␣ and ER␤ but can enhance the activities of other scription, because PITX1 (68–314, entry 6) retained full

ABC Est rogen Receptor α Estrogen Receptor β Androgen Receptor 125 125 150 Veh Veh Veh E2 E2 DHT 100 100 100 y ty

75 75 i iv t 50 * 50 %Activity %Ac %Activit 50 ‡ ‡ ‡ 25 * * 25 * ‡ ‡ ‡ ** 0 0 0 0 100 500 1000 0 100 500 1000 0 100 500 1000 PITX1 (ng) PITX1(ng) PITX1 ( n g ) DEF Glucocorticoid Receptor Progesterone Receptor α 200 300 200 Veh * * Veh Veh DEX R5020 * * RA 150 150 200 ty i iv

100 tivity 100 Ac Act

%Activity ‡ % 100 % 50 ‡ ‡ 50 ‡ ‡ 0 0 0 0 100 500 1000 0 100 500 1000 0 100 500 1000 PITX1 (ng) PITX1(ng) PITX1 (ng) G 1 Empty Vector 1 Veh FLAG NLS HD E2 1 314 2 2 PITX1-WT * 1 89 3 3 PITX1 (1-89) * 1 150 4 PITX1 (1-150) 4

1 197 5 PITX1 (1-197) 5 * 68 314 6 6 PITX1 (68-315) * 68 150 7 7 PITX1 (68-150) * 68 197 8 PITX1 (68-197) 8 * 0 20 40 60 80 100 120 140 %E2Activity FIG. 5. Effect of PITX1 on the transcriptional activity of different nuclear hormone receptors and analysis of the regions of PITX1 important for its repression of ER␣ activity. HEC1 cells were transfected with ER␣ and a 3ERE-luciferase reporter (panel A), ER␤ and a 3ERE-luciferase reporter (panel B), AR and a 2PRE-luciferase reporter (panel C), GR and a 2PRE-luciferase reporter (panel D), PRB and a 2PRE-luciferase reporter (panel E), or RAR␣ and a RARE-luciferase reporter (panel F). Transfections also included increasing amounts of pSPORT6-PITX1 (0, 100, 500, or 1000 ng). G, Schematic of wild type PITX1 and PITX1 truncation mutants used. NLS, Nuclear localization sequence; HD, Homeodomain. HEC-1 cells were transfected with pCMV5-ER␣, pTAG-2B-PITX1, and the 3ERE-luciferase reporter vector. All transfections (panels A–G) contained a ␤-galactosidase internal control reporter to normalize for transfection efficiency. Cells were treated with vehicle or 10 nM of the corresponding receptor ligand. Values show the percent activity from triplicate experiments Ϯ SEM.*,P Ͻ 0.05 compared with 100% activity (no added PITX1). ‡, P Ͻ 0.05 compared with vehicle-treated 0 ng PITX1 samples. DEX, Dexamethasone; RA, retinoic acid; Veh, vehicle. Mol Endocrinol, October 2011, 25(10):1699–1709 mend.endojournals.org 1705 ability to suppress ER. Interestingly, deletion of the last A PITX1 mRNA E PITX1 mRNA 118 amino acids of PITX1 (PITX 1-197, entry 5) or fur- 30 5 * e 4 ther deletions in from the C terminus of PITX1 (entries 3, 20 * siGL3 AdPITX1 3 * hang

4, and 7) resulted in loss of PITX1-dependent repression C 2 10 siPITX1 AdGal ld 1 *

␣ o of ER , indicating that the C-terminal region (amino Fold Change * 0 F 0 * acids 151-304) is required for the observed repression 0102030 0102030 E2 Treatment (h) E2 Treatment (h) (Fig. 5G). B F siGL3 siPITX1 AdGal AdPITX1 PITX1 controls the expression of select ER PITX1 target genes PITX1 -Actin Downloaded from https://academic.oup.com/mend/article/25/10/1699/2614663 by guest on 23 September 2021 The observation that PITX1 repressed ER activity on -+-+E2 an ER-responsive reporter construct (Fig. 5) suggested -+-+E2 that PITX1 might modulate the ability of ER␣ to regulate C G 12 TFF1 mRNA 20 TFF1 mRNA e * endogenous gene expression in breast cancer cells. There- 9 15 ng

␣ a siPITX1 fore, the ability of ER to regulate gene expression was 6 AdGal h 10 assessed in the presence of reduced or increased PITX1, * C 3 ld 5 siGL3 o Fold Change AdPITX1 using siRNA knock down or adenovirus-mediated over- 0 F 0 0102030 0102030 expression of PITX1. The use of adenovirus-expressing E2 Treatment (h) E2 Treatment (h) PITX1 greatly increased the intracellular levels of PITX1 D FST mRNA H FST mRNA 12 20 mRNA and protein (Fig. 6, A and B), which resulted in a e * 9 15 siPITX1 blunted E2-stimulated response for both TFF1 and FST AdGal 6 hang 10 * C mRNA (Fig. 6, C and D). When PITX1 mRNA was # 3 * ld 5 o Ͼ Fold Change AdPITX1 siGL3 reduced by PITX1 siRNA (by 80%) and the PITX1 0 F 0 protein became no longer detectable in 231ERϩ cells 0102030 0102030 E2 Treatment (h) E2 Treatment (h) compared with the control (siGL3 luciferase)-treated cells FIG. 6. PITX1 regulates the expression of ER␣ target genes. A, (Fig. 6, E and F), we found that this knockdown of PITX1 Quantitative real-time PCR analysis of PITX1 mRNA in 231ERϩ cells resulted in a hyperstimulation of both TFF1 and FST treated with AdGal or AdPITX1 for 24 h before 10 nM E2 treatment for Ͻ mRNA by E2 (Fig. 6, G and F). PITX1 siRNA did not the times indicated. *, P 0.05 compared with corresponding AdGal ␣ E2 treatment. B, Western blot analysis for PITX1 protein levels in affect ER expression (data not shown). Interestingly, 231ERϩ cells infected with adenovirus expressing either ␤-gal or PITX1 modulation of PITX1 cellular levels by either siRNA or and treated with vehicle or 10 nM E2 for 45 min. Quantitative real-time ϩ adenovirus overexpression did not affect the E2 regula- PCR for TFF1 mRNA (panel C) or FST mRNA (panel D) in 231ER cells treated with AdGal or AdPITX1 for 24 h before 10 nM E2 treatment tion for genes containing ER binding sites that lack the for the times indicated. *, P Ͻ 0.05; #, P Ͻ 0.1 compared with consensus PITX1-binding motif (data not shown). Col- corresponding AdGal E2 treatment. E, Quantitative real-time PCR analysis of PITX1 mRNA in 231ERϩ cells treated with siGL3 or siPITX1 lectively these results demonstrate that PITX1 functions Ͻ ␣ for 48 h before 10 nM E2 treatment for times indicated. *, P 0.05 to repress ER transcriptional activity on a subset of tar- compared with corresponding siGL3 E2 treatment. F, Western blot get genes in breast cancer cells. analysis for PITX1 or ␤-actin protein levels in 231ERϩ cells transfected with siRNA targeting either GL3 luciferase or PITX1 and treated with vehicle or 10 nM E2. Quantitative real-time PCR for TFF1 mRNA (panel G) or FST mRNA (panel H) in 231ERϩ cells treated with siGL3 or siPITX1 for Discussion 48 h before 10 nM E2 treatment for the times indicated. *, P Ͻ 0.05; #, P Ͻ 0.1 compared with corresponding siGL3 E2 treatment. In the present studies we have identified PITX1 as a novel transcriptional target of the ER in several ER␣-positive ER␣ appears to be a primary transcriptional response in breast cancer cells and show that this up-regulation of which estradiol induces ER␣-dependent interaction be- PITX1 gene expression involves hormone-induced inter- tween the proximal promoter and 5Ј-upstream enhancer action between proximal and long-distance ER␣ binding ER␣ binding sites of the PITX1 gene. Long-distance gene sites. We further show that ER␣ and PITX1 interact, and regulation by the ER␣ has been highlighted by recent we have defined a novel function of PITX1 as a selective genome-wide identification of ER binding sites (10, 13). repressor that modulates the transcriptional activity of These studies have concluded that the majority of ER␣ ER␣ target genes that have ER-binding sites containing binding sites are located further than 5 kb from the tran- PITX1-binding motifs. scriptional start site of E2-regulated genes (10, 13, 15, 16) To our knowledge, this is the first report of hormonal and that regulation by ER␣ often involves the interaction regulation of PITX1 expression. PITX1 regulation by of multiple ER binding sites located far apart (28–30). 1706 Stender et al. PITX1 and Estrogen Receptor Gene Regulation Mol Endocrinol, October 2011, 25(10):1699–1709

These findings are consistent with the involvement of an We demonstrate here several levels of cross-talk be- enhancer approximately 12 kb upstream of the PITX1 tween PITX1 and ER␣. PITX1 gene expression is up- transcription start site in the long-range transcriptional regulated by estradiol via ER␣, and PITX1 also interacts control of PITX1 gene regulation by ER␣ that we have with ER␣ to suppress ER␣ transcriptional activity on a observed. subset of ER␣ target genes. It would be of interest in Transcriptional control of gene expression by ER␣ can future experiments to determine whether PITX1 directly be by binding to DNA at EREs or through indirect inter- recruits corepressor complexes to these ER␣-binding action of the receptor with DNA via transcription factors sites, or whether PITX1 along with its known binding such as Sp1 and activator protein 1 (15). Using bioinfor- partner Brg1 (32) might modify the epigenetic landscape matic approaches, we observed that 28% of the genome- of these ER␣ binding sites resulting in the dampening of wide ER␣ binding sites also contain a PITX1-binding mo- ER␣ transcriptional activity. Downloaded from https://academic.oup.com/mend/article/25/10/1699/2614663 by guest on 23 September 2021 tif, which supports the hypothesis that PITX1 may be a Our studies have identified a novel role for PITX1 in cooperating factor for ER␣, coordinating estrogen-medi- modulating the transcriptional activity of ER␣ in breast ated transcriptional control of a subset of ER␣ target cancer cells. However, the biological impact of PITX1 genes. Examination of the distribution of these PITX1 in breast tumors or other estradiol-responsive tumors sites showed that they are mostly enriched in close prox- remains unknown. Genome-wide mRNA profiling of hu- imity to EREs. Consistent with the bioinformatic analy- man tumor samples, documented in the Oncomine data- ses, we observed hormone-stimulated and ER␣-depen- base, indicates that PITX1 mRNA is significantly over- Ϫ dent recruitment of PITX1 only to ER binding sites that expressed in ductal breast carcinomas (P Ͻ 2.7 ϫ 10 9) contain consensus PITX1 binding sites. (33) compared with normal breast, but is significantly There is good evidence that ER␣ and other nuclear underexpressed in mucinous breast carcinomas (P Ͻ receptors change chromatin architecture and can poise 0.002) (34), ovarian cystadenocarcinomas (P Ͻ 0.01) chromatin in a manner that facilitates recruitment of co- (34), and prostate carcinomas (P Ͻ 0.008 and P Ͻ 0.04) operating cofactors (31, 32). Because E2 treatment in- (35, 36). Therefore, PITX1 expression is altered in several creases the level of PITX1, and ER␣ and PITX1 bind to types of E2-responsive tumors, which might affect the func- one another, ER␣ binding to its genomic sites in target tional activities of ER␣ in these tumors. Collectively, our genes could facilitate PITX1 recruitment to nearby PITX1 findings that PITX1 is up-regulated by E2 in ER␣-contain- binding sites. Studies by others have identified the DNA ing breast cancer cells and that PITX1 modulates ER␣-de- response element for FOXA1, a forkhead factor, to be pendent transcriptional activity suggest that the altered ex- enriched in ER␣ binding sites and have defined a role for pression (overexpression or underexpression) of PITX1 in FOXA1 as a pioneer factor the function of which is to different types of breast cancers and in some ovarian and poise chromatin to facilitate recruitment of ER␣ to these prostate cancers compared with their normal tissue counter- binding sites, thus directing the regulation of target genes parts, might affect the development and/or progression and (9–11). phenotypic properties of these tumors. Future investigations Increasing the intracellular level of PITX1 selectively in human tumor specimens should provide further insights inhibited the transcriptional activity of ER␣ and ER␤ on into the role of this transcription factor in breast cancer and reporter genes, whereas PITX1 had no repressive effect on other cancers in which ER␣ has important biological four other nuclear receptors that were assessed. In fact, activities. PITX1 increased the activity of progesterone receptor and glucocorticoid receptor, both in the absence and presence of their respective ligands, whereas no change was ob- Materials and Methods served in the activity of the retinoic acid receptor ␣. Our Cell culture and transient transfection assays study of truncated forms of PITX1 indicated that the C- MCF-7 cells, and MDA-MB-231 cells stably expressing ER␣, terminal portion of the protein, amino acids 197–314, as were grown as previously described (37–39). Cells were well as the region of amino acids 150–197, was needed switched 4 d before treatment to phenol red-free tissue culture for this repression. Interestingly, amino acids 150–197 of medium containing 5% charcoal-dextran-treated calf serum. PITX1 were shown previously to be necessary for PITX1 Medium was changed ond2andd4ofculture after which cells repression of IRF3 and IRF7 (26); therefore, this region of were treated with control 0.1% ethanol vehicle, 10 nM E2, or 1 ␮ PITX1 might contain an intrinsic repression domain re- M antiestrogen alone or with 10 nM E2 for the various times indicated. sponsible for PITX1 repression of several interacting Some transfections were done in ER-negative MDA-MB-231 transcription factors, now including the nuclear hormone human breast cancer cells or human endometrial cancer HEC-1 receptor ER␣. cells that were maintained as previously described (37, 40). The Mol Endocrinol, October 2011, 25(10):1699–1709 mend.endojournals.org 1707

cells were plated in 24-well plates and transfected when approx- 65 C for 20 min. Two aliquots of the chromatin samples (2 ␮g) imately 80% confluent. Transfections were performed using 0.5 were diluted in ligation buffer containing 1% Triton-X and ␮g of the 3ERE-luciferase, 0.5 ␮g 2PRE-TK-luc, 0.5 ␮gof incubated at 37 C for 1 h. The temperature was lowered to 16 C, RARE-luc, or pGL3-control construct, 0.2 ␮g of the internal and T4 Ligase (New England Biolabs) was added and samples reference ß-galactosidase reporter plasmid pCMV5-ß-gal, 0.1 were incubated overnight. The ligated DNA was purified using ␮g ER expression vector, and various concentrations of phenol/chloroform extraction and analyzed using PCR amplifi- pSport6-PITX1. A premix containing 5 ␮l/well lipofectin, 1.6 cation. Resulting PCR products were sequenced and mapped ␮g/well transferrin, and 54 ␮l/well Hank’s balanced salt solu- back to the UCSC Genome Browser for verification. tion was mixed with DNA in Hank’s balanced salt solution (75 ␮l/well) for 15 min at room temperature. The cells were washed Coimmunoprecipitation assays with serum-free medium, after which the liquid was aspirated MCF-7 cells were infected with adenovirus containing the full- and replaced with 300 ␮l serum-free media/well. A total of 150

length PITX1 cDNA (AdPITX1) for 24 h. Cells were then treated Downloaded from https://academic.oup.com/mend/article/25/10/1699/2614663 by guest on 23 September 2021 ␮ ␮ l of the DNA/lipofectin/transferrin mixture (75 l DNA and with vehicle control or 10 nM E2 for 45 min before cell lysate ␮ 75 l lipofectin/transferrin mixture) was added to each well. collection. The lysate was cleared of insoluble material after soni- After incubation for8hat37Cina5%CO2 incubator, the cells cation and centrifugation, precleared with protein A/G agarose were washed once with medium containing 5% charcoal beads, and then subjected to immunoprecipitation using ER␣ dextran-treated calf serum and then replaced with 1 ml medium (F-10, Santa Cruz Biotechnology) or IgG control antibodies. The plus serum. Cells were treated with the indicated ligand or 0.1% immunoprecipitated complexes were collected using protein A/G ethanol control for 24 h at 37 C, and cell lysates were then agarose beads, washed four times with radioimmune precipitation harvested using reporter lysis buffer (Promega Corp., Madison, assay buffer, and eluted. Eluates were separated in SDS polyacryl- WI) and analyzed using the Luciferase Assay system (Promega) amide gels and transferred to nitrocellulose membranes. Blots were on a MLX Microtiter Plate Luminometer (Dynex Technologies, incubated in LI-COR blocking buffer (LI-COR Biosciences, Chantilly, VA). Lincoln, NE) and then with PITX1 (A300–577A, Bethyl Labora- tories) antibody, followed by detection using IRDye secondary an- Quantitative real-time PCR tibody (LI-COR Biosciences). Total RNA was reverse transcribed, and real-time PCR was performed exactly as described elsewhere (39). The fold-change ChIP assays in expression for each gene was calculated as described previ- Assays were performed essentially as described previously ously, with the ribosomal protein 36B4 mRNA as an internal (41) with a few noted modifications (28). The antibodies used in control. Real-time PCR of ChIP samples was performed in a these studies were: ER␣, HC-20 (Santa Cruz Biotechnology); similar manner with appropriate primers and data normalized RNA polymerase II, N-20 (Santa Cruz); PITX1, A300-577A to percent input. (Bethyl Laboratories). For PCR, 1 ␮lfroma50␮l DNA extrac- tion was quantified using quantitative real-time PCR. ChIP- Western blot analysis reChIP experiments were performed using PITX1 antibody for Cell protein lysates were separated in sodium dodecyl sulfate the first pulldown, followed by extensive washes and elution (SDS) polyacrylamide gels and transferred to nitrocellulose using 10 mM dithiothreitol for 30 min at 37 C. The eluate was membranes. Blots were incubated in blocking buffer (5% milk diluted in immunoprecipitation buffer, and a second pulldown in Tris-buffered saline with 0.5% Tween) and then with specific was performed with ER␣ antibody or IgG as control. The antibodies for ER␣ (HC20; Santa Cruz Biotechnology, Inc., washes, elution, DNA recovery, and analysis were done as for Santa Cruz, CA), PITX1 (A300–577A; Bethyl Laboratories, ChIP assays. Montgomery, TX), and ␤-actin (AC-15; Sigma, St. Louis, MO), followed by detection using horseradish peroxidase-conjugated siRNA and overexpression studies secondary antibodies with Supersignal West Femto Detection MDA-MB-231ERϩ cells were plated in phenol red-free me- Kit (Pierce Chemical Co., Rockford, IL), as described by the dium containing 5% charcoal-dextran-treated calf serum at manufacturer. 250,000 cells per well in six-well plates, and transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) Chromosome conformation capture assays according to the manufacturer’s instructions as described previ- MCF-7 and 231ERϩ cells were treated with vehicle or 10 nM ously (42). Synthetic RNA oligonucleotides targeting PITX1 and E2 for 45 min and fixed in 2% formaldehyde at room temper- control (GL3) luciferase were obtained from Dharmacon (Lafay- ature for 10 min. The formaldehyde was quenched with addi- ette, CO). The siRNA target sequences used were: PITX1 tion of 0.125 M glycine, and cells were lysed in lysis buffer [10 NNGCAACGTACGCACTTCACA; GL3 target sequences were mM Tris (pH 8.0), 10 mM NaCl, 0.2% Nonidet P-40, 1X Com- obtained from Dharmacon (catalog no. D-001400-01). siRNA plete Protease Inhibitors (Roche, Indianapolis, IN)] at 4 C for 90 was used at 20 nM, as recommended by the manufacturer, and cells min. The nuclei were resuspended in 1X New England Biolabs were exposed to the siRNA for 72 h and then treated with control (Beverly, MA) Buffer 2 and 0.3% SDS and incubated at 37 C for vehicle or hormone for the times indicated. 60 min while rotating. Triton X-100 was added to a final con- For PITX1 overexpression studies, adenovirus containing centration of 1.8% to sequester the SDS and incubated at 37 C full-length PITX1 (AdPITX1) was prepared using the AdEasy for 60 min with gentle rotation. The chromatin was then di- System (Stratagene, La Jolla, CA) following the manufacturer’s gested overnight using MseI (New England Biolabs) or BamHI instructions. As a control, adenovirus containing the ␤-galacto- (New England Biolabs) at 37 C with gentle rotation. SDS was sidase gene (AdGal) was prepared in parallel. The viruses were added to a final volume of 1.6%, and the samples were heated at amplified and isolated from AD-293 cells as recommended. 1708 Stender et al. PITX1 and Estrogen Receptor Gene Regulation Mol Endocrinol, October 2011, 25(10):1699–1709

Bioinformatic analysis for PITX1 binding tor binding reveals long-range regulation requiring the forkhead site enrichment protein FoxA1. Cell 122:33–43 10. Carroll JS, Meyer CA, Song J, Li W, Geistlinger TR, Eeckhoute J, ␣ The genome-wide ER binding site database established in Brodsky AS, Keeton EK, Fertuck KC, Hall GF, Wang Q, Bekiranov 231ERϩ cells (15) was searched for the presence of the consen- S, Sementchenko V, Fox EA, Silver PA, Gingeras TR, Liu XS, sus PITX1 binding site, TAATCC. For comparison, 1,000,000 Brown M 2006 Genome-wide analysis of estrogen receptor binding random DNA sequences from the (hg18), each sites. Nat Genet 38:1289–1297 consisting of 1000 bp, were also searched for the presence of 11. Eeckhoute J, Carroll JS, Geistlinger TR, Torres-Arzayus MI, PITX1 binding sites. The P value for PITX1 enrichment in the Brown M 2006 A cell-type-specific transcriptional network re- ER binding sites was generated using the hypergeometric dis- quired for estrogen regulation of cyclin D1 and cell cycle progres- sion in breast cancer. Genes Dev 20:2513–2526 tribution test (43). The ER␣ binding sites were centered on 12. Laganie`re J, Deblois G, Lefebvre C, Bataille AR, Robert F, Gigue`re the position of the ERE, and the moving averages of PITX1 V 2005 Location analysis of estrogen receptor ␣ target promoters motif frequency were plotted in relation to distance to the reveals that FOXA1 defines a domain of the estrogen response. Proc Downloaded from https://academic.oup.com/mend/article/25/10/1699/2614663 by guest on 23 September 2021 nearest ERE. Natl Acad Sci USA 102:11651–11656 13. Lin CY, Vega VB, Thomsen JS, Zhang T, Kong SL, Xie M, Chiu KP, Lipovich L, Barnett DH, Stossi F, Yeo A, George J, Kuznetsov VA, Lee YK, Charn TH, Palanisamy N, Miller LD, Cheung E, Acknowledgments Katzenellenbogen BS, Ruan Y, Bourque G, Wei CL, Liu ET 2007 Whole-genome cartography of estrogen receptor ␣ binding sites. Address all correspondence and requests for reprints to: Dr. PLoS Genet 3:e87 Benita S. Katzenellenbogen, University of Illinois, Department 14. Madak-Erdogan Z, Lupien M, Stossi F, Brown M, Katzenellenbo- of Molecular and Integrative Physiology, 524 Burrill Hall, 407 gen BS 2011 Genomic collaboration of estrogen receptor ␣ and South Goodwin Avenue, Urbana, Illinois 61801-3704. E-mail: extracellular signal-regulated kinase 2 in regulating gene and pro- liferation programs. Mol Cell Biol 31:226–236 [email protected]. 15. Stender JD, Kim K, Charn TH, Komm B, Chang KC, Kraus WL, This work was supported by National Institutes of Health Benner C, Glass CK, Katzenellenbogen BS 2010 Genome-wide (NIH) Grant P50 AT006268 from Office of Dietary Supple- analysis of estrogen receptor ␣ DNA binding and tethering mech- ments (ODS), The National Center for Complementary and anisms identifies Runx1 as a novel tethering factor in receptor- mediated transcriptional activation. Mol Cell Biol 30:3943–3955 Alternative Medicine (NCCAM), and National Cancer Institute 16. Welboren WJ, van Driel MA, Janssen-Megens EM, van Heeringen (to B.S.K.), a grant from The Breast Cancer Research Founda- SJ, Sweep FC, Span PN, Stunnenberg HG 2009 ChIP-Seq of ER␣ tion (to B.S.K.), and NIH Training Grants NIH T32 HD07028 and RNA polymerase II defines genes differentially responding to (to J.D.S. and D.H.B.) and ES07328 (to C.C.F.). ligands. EMBO J 28:1418–1428 Disclosure Summary: The authors have nothing to disclose. 17. Tremblay JJ, Lanctoˆ t C, Drouin J 1998 The pan-pituitary activator of transcription, Ptx1 (pituitary homeobox 1), acts in synergy with SF-1 and Pit1 and is an upstream regulator of the Lim-homeodo- main gene Lim3/Lhx3. Mol Endocrinol 12:428–441 References 18. Kolfschoten IG, van Leeuwen B, Berns K, Mullenders J, Beijersber- gen RL, Bernards R, Voorhoeve PM, Agami R 2005 A genetic screen identifies PITX1 as a suppressor of RAS activity and tumor- 1. Couse JF, Korach KS 1999 Estrogen receptor null mice: what have igenicity. Cell 121:849–858 we learned and where will they lead us? Endocr Rev 20:358–417 19. Szeto DP, Rodriguez-Esteban C, Ryan AK, O’Connell SM, Liu F, 2. Deroo BJ, Korach KS 2006 Estrogen receptors and human disease. Kioussi C, Gleiberman AS, Izpisu´ a-Belmonte JC, Rosenfeld MG J Clin Invest 116:561–570 1999 Role of the Bicoid-related homeodomain factor Pitx1 in spec- 3. Katzenellenbogen BS, Katzenellenbogen JA 2002 Biomedicine. De- ifying hindlimb morphogenesis and pituitary development. Genes fining the “S” in SERMs. Science 295:2380–2381 Dev 13:484–494 4. Katzenellenbogen BS, Montano MM, Ediger TR, Sun J, Ekena K, 20. Szeto DP, Ryan AK, O’Connell SM, Rosenfeld MG 1996 P-OTX: a Lazennec G, Martini PG, McInerney EM, Delage-Mourroux R, PIT-1-interacting homeodomain factor expressed during anterior Weis K, Katzenellenbogen JA 2000 Estrogen receptors: selective pituitary gland development. Proc Natl Acad Sci USA 93:7706– ligands, partners, and distinctive pharmacology. Recent Prog Horm 7710 Res 55:163–193; discussion 194–195 21. Lamonerie T, Tremblay JJ, Lanctoˆ t C, Therrien M, Gauthier Y, 5. Hall JM, Couse JF, Korach KS 2001 The multifaceted mechanisms Drouin J 1996 Ptx1, a bicoid-related homeo box transcription fac- of estradiol and estrogen receptor signaling. J Biol Chem 276: tor involved in transcription of the pro-opiomelanocortin gene. 36869–36872 Genes Dev 10:1284–1295 6. McKenna NJ, O’Malley BW 2002 Combinatorial control of gene 22. Luo M, Koh M, Feng J, Wu Q, Melamed P 2005 Cross talk in expression by nuclear receptors and coregulators. Cell 108:465– hormonally regulated gene transcription through induction of es- 474 trogen receptor ubiquitylation. Mol Cell Biol 25:7386–7398 7. Rosenfeld MG, Lunyak VV, Glass CK 2006 Sensors and signals: a 23. Melamed P, Koh M, Preklathan P, Bei L, Hew C 2002 Multiple coactivator/corepressor/epigenetic code for integrating signal- mechanisms for Pitx-1 transactivation of a luteinizing hormone ␤ dependent programs of transcriptional response. Genes Dev 20: subunit gene. J Biol Chem 277:26200–26207 1405–1428 24. Poulin G, Lebel M, Chamberland M, Paradis FW, Drouin J 2000 8. Sanchez R, Nguyen D, Rocha W, White JH, Mader S 2002 Diversity Specific protein-protein interaction between basic helix-loop-helix in the mechanisms of gene regulation by estrogen receptors. Bioes- transcription factors and homeoproteins of the Pitx family. Mol says 24:244–254 Cell Biol 20:4826–4837 9. Carroll JS, Liu XS, Brodsky AS, Li W, Meyer CA, Szary AJ, Eeck- 25. Tremblay JJ, Marcil A, Gauthier Y, Drouin J 1999 Ptx1 regulates houte J, Shao W, Hestermann EV, Geistlinger TR, Fox EA, Silver SF-1 activity by an interaction that mimics the role of the ligand- PA, Brown M 2005 Chromosome-wide mapping of estrogen recep- binding domain. EMBO J 18:3431–3441 Mol Endocrinol, October 2011, 25(10):1699–1709 mend.endojournals.org 1709

26. Island ML, Mesplede T, Darracq N, Bandu MT, Christeff N, Djian cancer microarray database and integrated data-mining platform. P, Drouin J, Navarro S 2002 Repression by homeoprotein pitx1 of Neoplasia 6:1–6 virus-induced interferon a promoters is mediated by physical inter- 35. Luo JH, Yu YP, Cieply K, Lin F, Deflavia P, Dhir R, Finkelstein S, action and trans repression of IRF3 and IRF7. Mol Cell Biol 22: Michalopoulos G, Becich M 2002 Gene expression analysis of pros- 7120–7133 tate cancers. Mol Carcinog 33:25–35 27. Frasor J, Stossi F, Danes JM, Komm B, Lyttle CR, Katzenellenbo- 36. Magee JA, Araki T, Patil S, Ehrig T, True L, Humphrey PA, Cata- gen BS 2004 Selective estrogen receptor modulators: discrimination lona WJ, Watson MA, Milbrandt J 2001 Expression profiling re- of agonistic versus antagonistic activities by gene expression profil- veals hepsin overexpression in prostate cancer. Cancer Res 61: ing in breast cancer cells. Cancer Res 64:1522–1533 5692–5696 28. Barnett DH, Sheng S, Charn TH, Waheed A, Sly WS, Lin CY, Liu 37. Ediger TR, Kraus WL, Weinman EJ, Katzenellenbogen BS 1999 ET, Katzenellenbogen BS 2008 Estrogen receptor regulation of car- Estrogen receptor regulation of the Naϩ/Hϩ exchange regulatory bonic anhydrase XII through a distal enhancer in breast cancer. factor. Endocrinology 140:2976–2982 Cancer Res 68:3505–3515 38. Frasor J, Danes JM, Komm B, Chang KC, Lyttle CR, Katzenellen- 29. Descheˆnes J, Bourdeau V, White JH, Mader S 2007 Regulation of bogen BS 2003 Profiling of estrogen up- and down-regulated gene Downloaded from https://academic.oup.com/mend/article/25/10/1699/2614663 by guest on 23 September 2021 GREB1 transcription by estrogen receptor ␣ through a multipartite expression in human breast cancer cells: insights into gene networks enhancer spread over 20 kb of upstream flanking sequences. J Biol and pathways underlying estrogenic control of proliferation and Chem 282:17335–17539 cell phenotype. Endocrinology 144:4562–4574 30. Fullwood MJ, Liu MH, Pan YF, Liu J, Xu H, Mohamed YB, Orlov 39. Stender JD, Frasor J, Komm B, Chang KC, Kraus WL, Katzenellen- YL, Velkov S, Ho A, Mei PH, Chew EG, Huang PY, Welboren WJ, bogen BS 2007 Estrogen-regulated gene networks in human breast Han Y, Ooi HS, Ariyaratne PN, Vega VB, Luo Y, Tan PY, Choy cancer cells: involvement of in the regulation of cell prolifer- PY, Wansa KD, Zhao B, Lim KS, Leow SC, Yow JS, et al. 2009 An ation. Mol Endocrinol 21:2112–2123 oestrogen-receptor-␣-bound human chromatin interactome. Na- 40. Rajendran RR, Nye AC, Frasor J, Balsara RD, Martini PG, Kat- ture 462:58–64 zenellenbogen BS 2003 Regulation of transcrip- 31. He HH, Meyer CA, Shin H, Bailey ST, Wei G, Wang Q, Zhang Y, tional activity by a novel DEAD box RNA helicase (DP97). J Biol Xu K, Ni M, Lupien M, Mieczkowski P, Lieb JD, Zhao K, Brown Chem 278:4628–4638 M, Liu XS 2010 Nucleosome dynamics define transcriptional en- 41. Shang Y, Hu X, DiRenzo J, Lazar MA, Brown M 2000 Cofactor hancers. Nat Genet 42:343–347 dynamics and sufficiency in estrogen receptor-regulated transcrip- 32. Lupien M, Eeckhoute J, Meyer CA, Wang Q, Zhang Y, Li W, tion. Cell 103:843–852 Carroll JS, Liu XS, Brown M 2008 FoxA1 translates epigenetic 42. Frasor J, Danes JM, Funk CC, Katzenellenbogen BS 2005 Estrogen signatures into enhancer-driven lineage-specific transcription. Cell down-regulation of the corepressor N-CoR: mechanism and impli- 132:958–970 cations for estrogen derepression of N-CoR-regulated genes. Proc 33. Richardson AL, Wang ZC, De Nicolo A, Lu X, Brown M, Miron A, Natl Acad Sci USA 102:13153–13157 Liao X, Iglehart JD, Livingston DM, Ganesan S 2006 X chromo- 43. Heinz S, Benner C, Spann N, Bertolino E, Lin YC, Laslo P, Cheng somal abnormalities in basal-like human breast cancer. Cancer Cell JX, Murre C, Singh H, Glass CK 2010 Simple combinations of 9:121–132 lineage-determining transcription factors prime cis-regulatory ele- 34. Rhodes DR, Yu J, Shanker K, Deshpande N, Varambally R, Ghosh ments required for macrophage and B cell identities. Mol Cell 38: D, Barrette T, Pandey A, Chinnaiyan AM 2004 ONCOMINE: a 576–589