Transcription Factor KLF11 Integrates Progesterone Receptor Signaling and Proliferation in Uterine Leiomyoma Cells

Published OnlineFirst February 2, 2010; DOI: 10.1158/0008-5472.CAN-09-2612

Tumor and Stem Cell Biology Cancer Research Transcription Factor KLF11 Integrates Progesterone Receptor Signaling and Proliferation in Uterine Leiomyoma Cells

Ping Yin, Zhihong Lin, Scott Reierstad, Ju Wu, Hiroshi Ishikawa, Erica E. Marsh, Joy Innes, Youhong Cheng, Kerry Pearson, John Sayler Coon V, J. Julie Kim, Debabrata Chakravarti, and Serdar E. Bulun

Abstract Uterine leiomyoma is the most common tumor of the female genital tract and the leading cause of hysterectomy. Although progesterone stimulates the proliferation of uterine leiomyoma cells, the mechanism of progesterone action is not well understood. We used chromatin immunoprecipitation (ChIP)–cloning approach to identify progesterone receptor (PR) target genes in primary uterine leiomyoma smooth muscle cells. We identified 18 novel PR-binding sites, one of which was located 20.5 kb upstream of the transcriptional start site of the Krüppel-like transcription factor 11 (KLF11) gene. KLF11 mRNA levels were minimally downregulated by progesterone but robustly upregulated by the progesterone antagonist RU486. Luciferase reporter assays showed significant baseline and RU486-inducible promoter activity in the KLF11 basalpromoterordistal PR-binding region, both of which contained multiple Sp1-binding sequences but lacked classic progesterone response elements. RU486 stimulated recruitment of Sp1, RNA polymerase II, PR, and the coactivators SRC-1 and SRC-2 to the distal region and basal promoter. siRNA knockdown of PR increased KLF11 expression, whereas knockdown of KLF11 increased leiomyoma cell proliferation and abolished the anti- proliferative effect of RU486. In vivo, KLF11 expression was significantly lower in leiomyoma tissues compared with adjacent myometrial tissues. Taken together, using a ChIP-cloning approach, we uncovered KLF11 as an integrator of PR signaling and proliferation in uterine leiomyoma cells. Cancer Res; 70(4); 1722–30. ©2010 AACR.

Introduction tors inhibit proliferation and induce apoptosis (6–11). Indeed, RU486 and asoprisnil were shown to reduce leio- Uterine leiomyomata are the most common gynecologic myoma volume (12, 13), and antiprogestins have been used tumors in women of reproductive age. Growing evidence as a therapeutic option for patients with leiomyomata (1). from basic and clinical studies indicates that progesterone Taken together, these findings suggest that progesterone and the progesterone receptor (PR) play key roles in uterine plays an important role in leiomyoma growth; however, leiomyoma growth and development (1). Several investiga- the underlying cellular and molecular mechanisms respon- tors have shown an increased concentration of both PR iso- sible for the pathologic and therapeutic roles of proges- forms (PR-A and PR-B) in leiomyoma tissue compared with terone and its antagonists in uterine leiomyoma are not well adjacent myometrium (2, 3). Mitotic activity in leiomyoma understood. tissue was also increased relative to the adjacent myometrial Progesterone-regulated gene expression is mediated by nu- tissue during the luteal phase and after treatment with me- clear receptors PR-A and PR-B. Only a few cis-elements tar- droxyprogesterone acetate (4, 5). geted directly by PR have been identified to date. For Progesterone suppresses apoptosis and stimulates prolif- example, progesterone response elements (PRE) have been eration of leiomyoma cells in culture, whereas PR modula- identified within the basal promoter regions of the progesterone-regulated genes pepsinogen C, c-myc, insulin-like growth Authors' Affiliation: Division of Reproductive Biology Research, factor binding protein-1, and MUC1 (14–17). However, the Department of Obstetrics and Gynecology, Feinberg School of basal promoters of several other progesterone-responsive tar- Medicine at Northwestern University, Chicago, Illinois get genes lack PREs, including PR, p21, p27, glycodelin, and Note: Supplementary data for this article are available at Cancer the folate receptor type α; these promoters contain Sp1 sites Research Online (http://cancerres.aacrjournals.org/). that seem essential for PR-mediated transcription in in vitro Corresponding Author: Serdar E. Bulun, Division of Reproductive Biol- – ogy Research, Feinberg School of Medicine, Northwestern University, reporter assays (18 22). Recently, chromatin immunoprecip- 303 East Superior Street, Ste. 4-123, Chicago, IL 60611. Phone: 312- itation (ChIP) cloning and ChIP-on-chip approaches have 503-1600; Fax: 312-503-0095; E-mail: [email protected]. successfully identified a large number of estrogen receptor doi: 10.1158/0008-5472.CAN-09-2612 α (ERα)–binding sites at significant distances from the tran- ©2010 American Association for Cancer Research. scriptional start sites of estradiol-regulated genes (23–26). A

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similar genome-wide binding pattern of PR and consequent for KLF11 was amplified by PCR using primers 5′-ATGAGG- regulation of human genes through distant binding sites have GACCAACTCGGGAAATGG-3′ (forward) and 5′-GGAGGA- been suspected but not yet shown. CAACAGGACACTGACTTT-3′ (reverse), and cloned into the To better understand the effects of progesterone on hu- pGL3 promoter vector (Promega). The proximal 5′-flanking man uterine leiomyoma growth, we sought to identify a larg- region (−549 bp/+171 bp) was amplified by PCR using er number of PR target genes. We used a ChIP-cloning primers 5′-ATGTCACCTAGAACCG-3′ (forward) and 5′- strategy for unbiased identification of regulatory targets of CGTGTGCATCGTGCAG-3′ (reverse), and cloned into the progesterone/PR in leiomyoma. Interestingly, among the pGL3 basic vector (Promega). cloned targets, endogenous PR was found to bind in intact T47D cells (American Type Culture Collection) were trans- cells to a region located 20.5 kb upstream of the transcrip- fected with 2 μg reporter plasmid and 0.2 μg PRL-TK-Luc us- tional start site of the Krüppel-like transcription factor 11 ing Fugene HD transfection reagent (Roche). Twenty-four (KLF11) gene. Here, we report that this region may interact hours after transfection, cells were treated with vehicle − with the basal KLF11 promoter through recruitment of com- (ethanol) or RU486 (10 6 mol/L) for 48 h, and cell extracts mon transcription factors in response to the antiprogestin were prepared using passive lysis buffer (Promega). Illustrat- RU486. We also showed a potential role for KLF11 in protected results are normalized to PRL-TK-Luc using a dual lucif- ing against leiomyoma growth and mechanism for the ther- erase reporter assay system (Promega). apeutic action of antiprogestins. Immunostaining for bromo-2-deoxyuridine. The effect of KLF11 on cell proliferation was determined by bromo-2- Materials and Methods deoxyuridine (BrdUrd) incorporation assay as described previously except that BrdUrd (20 μmol/L) was added during Tissue collection and cell culture. Human uterine leio- the final 4 h of cell treatment (28). The result was expressed myoma and matched myometrium were collected from 56 as the BrdUrd labeling index (percentage of BrdUrd-labeled premenopausal women undergoing hysterectomy, following cells in total cells counted). the protocol approved by the institutional review board for Small interfering RNA. To knock down the expression of Human Research of Northwestern University. The subjects endogenous PR and KLF11, LSM cells were transfected with had not received any hormonal treatment during the 6 mo PR siRNA or KLF11 siRNA (Dharmacon) using Lipofectamine before surgery. The leiomyoma smooth muscle (LSM) cells RNAiMAX (Invitrogen). Nontargeting control siRNA (Dhar- were cultured as previously described (27). Cells used in macon) was transfected as a negative control. these experiments were passaged once or twice. Immunoblotting. Cell lysates were analyzed by immuno- ChIP and ChIP cloning. We performed the ChIP-cloning blotting as described previously (29) using monoclonal an- procedure as described previously except that the chromatin ti-PR (kindly provided by Dr. Edwards, Baylor College of fragments were immunoprecipitated using anti-PR antibody Medicine, Houston, TX), anti-KLF11 (Abcam), and anti–pro- (Abcam; ref. 25). Antibodies used for standard ChIP included liferating cell nuclear antigen (PCNA; GenScript Corp.) anti- anti-phosphorylated RNA PolII, anti-Sp1, anti–SRC-2 (Ab- bodies. Equal loading was confirmed using anti–β-actin cam), and anti–SRC-1 (Santa Cruz Biotechnology). Primers antibody (Sigma-Aldrich). The intensity of bands was quan- used for ChIP on SLC7A8 and SOX8 are available upon re- tified using ImageJ software. quest. Primers used for ChIP at 20.5 kb upstream of the Statistical analysis. Differences between groups were an- KLF11 transcription start site were 5′-GCATGA- alyzed by Student's t test or one-way ANOVA analysis fol- TAGTTTCTTGGGTCAACC-3′ (forward) and 5′-GTAGGC- lowed by Fisher's protected least significance difference GATGCTATAGTCCACAG-3′ (reverse). Real-time PCR test. Significance was accepted at P < 0.05. primers used for fold enrichment of distal regulatory sites were identical, and real-time PCR primers for basal promoter Results region were 5′-GGTCAAGGACGCATTCCTAA-3′ (forward) and 5′-GAGAGGGGACCACGCTTATAG-3′ (reverse). In this Identification of PR genomic targets in human LSM instance, real-time PCR was performed using ABI SYBR cells. To identify regulatory regions that recruit PR within Green Master kit (ABI). the entire genome of human LSM cells, we performed ChIP RNA preparation and real-time quantitative PCR. Total using anti-PR antibody in LSM cells treated with progester- RNA from LSM cells and tissues of leiomyoma and matched one for 1 hour. Twenty-one cloned fragments were mapped myometrium was extracted using Tri-reagent (Sigma-Aldrich). to the genome. Among these, 17 fragments were localized cDNA was prepared with Superscript III first-Strand Synthe- within an intron of a gene; two fragments were located 5′ sis System (Invitrogen). KLF11, SOX8, SLC7A8, PR, and glyc- or 3′ to the most proximally located genes; the other two eraldehyde-3-phosphate dehydrogenase (GAPDH)mRNA fragments were mapped to an exon of a gene. Because two were amplified by real-time PCR using the ABI TaqMan Gene genomic regions were associated with multiple ChIP frag- Expression system (ABI) and the ABI 7900 Sequence Detec- ments, a total of 18 novel PR-binding sites were identified tion System. All gene expressions were normalized to (Supplementary Table S1). GAPDH. These PR-binding sites were distributed genome-wide to Plasmids, transfection, and luciferase assays. PR-bind- chromosomes 1, 2, 5, 7, 10, 14, 15, 16, 17, 19, and 20 (Supple- ing region (−19,369 bp/−21,192 bp) identified by ChIP cloning mentary Fig. S1). Interestingly, binding sites were not

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distributed evenly between the chromosomes. For example, one antagonist RU486 and measured mRNA levels. Treat- four binding sites were located on chromosome 1 and two ment with progesterone in the presence or absence of sites were located on each of chromosomes 7, 14, 17, and RU486 altered mRNA levels in 5 of 11 (45%) genes (Supple- 20, whereas no binding to 11 chromosomes was noted, sug- mentary Table S2). In particular, we observed that SLC7A8 gesting specificity for genomic distribution. mRNA levels were increased after progesterone treatment, Of the 18 novel sites, we identified the 16 genes that had a reaching statistical significance at 4 hours (Fig. 1A). This PR-binding site within an intron or an exon. We also identi- increase was blocked by RU486. SLC7A8 encodes a key ami- fied a pair of the most proximal genes that flanked each no acid transporter protein that may be an important reg- of the remaining two PR-binding sites (Supplementary ulator of leiomyoma growth (30). SOX8 mRNA levels were Table S1). One binding site was located 5′ upstream of both not affected by progesterone but were downregulated signif- KLF11 and AILT5830, which are transcribed in opposite di- icantly at 2 hours in the presence of RU486 (Fig. 1A). SOX8 rections, whereas the remaining binding site was 3′ down- encodes a transcription factor that inhibits skeletal muscle stream of both SOX8 and FLJ32252 genes, also transcribed differentiation (31). in opposite directions (Supplementary Table S1). Conversely, KLF11 mRNA levels were slightly but signifi- We next selected 11 well-characterized genes from Sup- cantly downregulated by progesterone treatment. However, plementary Table S1 and investigated their progesterone re- they were robustly and significantly increased by 2.3-fold at sponsiveness. We treated LSM cells for 2 and 4 hours with 2 hours and 3.3-fold at 4 hours in the presence of RU486 progesterone in the presence or absence of the progester- (Fig. 1A). Similarly, mRNA levels of the genes PRDM16 and

Figure 1. A, modulation of selected PR target gene expression by progesterone. LSM cells were treated with vehicle (ethanol) and progesterone (10−6 mol/L) in the presence or absence of progesterone antagonist RU486 (10−4 mol/L) for 2 and 4 h. SLC7A8, SOX8, and KLF11 mRNA levels were measured by real-time PCR. The results are reported as fold change of vehicle-treated cells and represent the mean ± SEM for cells from three subjects. B, regulation of KLF11 expression by progesterone or R5020. The cells were treated and mRNA levels were determined as described in A. C, in vivo recruitment of PR and RNA PolII to novel PR-binding modules. LSM cells were exposed to progesterone for 1 h. ChIP was performed using nonspecific rabbit IgG, anti-PR, or anti-phosphorylated RNA PolII antibody, followed by 40 cycles of PCR. The results shown here are representative of three independent ChIP experiments.

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Figure 2. Regulation of KLF11 mRNA and protein levels by RU486 and PR in LSM cells. A, KLF11 mRNA levels were determined from LSM cells treated with vehicle or the indicated concentrations of RU486 for 6 h. The asterisks on the columns (10−8,10−7, and 10−6 mol/L) were placed to compare each dose with the vehicle. B, KLF11 protein levels were detected by immunoblotting from LSM cells treated with 10−6 mol/L RU486 for 72 h. C, PR mRNA (top) and PR protein (bottom) levels were checked from control siRNA– or PR siRNA–transfected cells to determine the knockdown efficiency. D, LSM cells were transfected with control siRNA or PR siRNA for 48 h. KLF11 mRNA expression levels were detected using real-time PCR. The mean ± SEM for three independent experiments is reported.

MAN1C1 were upregulated by RU486 (Supplementary are unknown. KLF11 is a known tumor suppressor (34) and Table S2). As shown in Supplementary Table S1, PRDM16 was significantly upregulated in the presence of RU486 as encodes a transcription factor that may play an important shown in Fig. 1A. Therefore, we focused our investigation role in the leukemogenic transformation of the cells (32), on KLF11 regulation and function in LSM cells as a possible whereas MAN1C1 plays an important role in N-glycan bio- mediator of the therapeutic effects of antiprogestins on synthesis (33). uterine leiomyoma. Cells were treated with different doses − − The mild but significant downregulation of KLF11 by pro- of RU486 (10 10–10 6 mol/L) for 6 hours and KLF11 mRNA − gesterone was also observed independently in a gene micro- levels were determined. As shown in Fig. 2A, RU486 (10 8– − array experiment using the Illumina platform; progesterone 10 6 mol/L) significantly induced KLF11 expression. RU486 − treatment for 4 hours downregulated KLF11 mRNA by at 10 6 mol/L also stimulated comparable increases in 16.5% (P < 0.05, data not shown). We also showed that KLF11 protein levels after 72 hours of treatment (Fig. 2B). R5020, a synthetic progesterone agonist, significantly de- Next, we knocked down endogenous PR using siRNA to es- creased the KLF11 mRNA level by ∼30% (Fig. 1B). R5020 tablish a functional role for PR in mediating KLF11 expres- induced a more robust reduction in KLF11 mRNA levels sion in LSM cells. Transfection of LSM cells with PR siRNA compared with progesterone (Fig. 1B). reduced PR mRNA and protein levels (Fig. 2C). PR knock- These results suggested that the distant PR-binding mod- down increased KLF11 mRNA by 2.1-fold compared with con- ules were functionally active and differentially modulated trol siRNA–transfected cells (Fig. 2D). progesterone and antiprogestin responsiveness of the genes Stimulation of KLF11 promoter activity by RU486. To de- identified in the ChIP-cloning screen. We next confirmed the termine whether the novel PR-binding region identified by recruitment of PR to the distant PR-binding sites of three se- ChIP cloning confers promoter activity, we cloned this frag- lected genes, SLC7A8, SOX8, and KLF11, and further investi- ment (−19,369 bp/−21,192 bp) into the pGL3 promoter vec- gated whether these sites interacted with RNA PolII using a tor. To determine whether the proximal 5′-flanking region conventional ChIP assay. Independent pools of immunopre- of KLF11 gene was also involved in RU486-regulated gene cipitated chromatin fragments from LSM cells treated with transcription, the −549 bp/+171 bp sequence was cloned into progesterone for 1 hour were analyzed. As shown in the pGL3 basic vector. Individual constructs were transfected Fig. 1C, PR and RNA PolII were recruited to PR-binding sites into T47D breast cancer cells because this cell line with high in the SLC7A8, SOX8, and KLF11 genes. levels of endogenous PR could be transfected reproducibly Regulation of KLF11 mRNA and protein levels by RU486 with both vectors (21). As shown in Fig. 3A, the 1,823-bp frag- in LSM cells. RU486 and other antiprogestins have been ment bearing the distal PR-binding region showed significant shown to reduce leiomyoma size in a number of clinical trials promoter-enhancing activity in the absence or presence of (12, 13). Mechanisms responsible for this therapeutic effect RU486. The basal promoter region also conferred significant

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transcriptional activity that was induced by RU486 (Fig. 3B). These results indicated that both the distal region and basal promoter of the KLF11 gene have transcriptional regulatory activity. Recruitment of transcription factors and steroid receptor coactivators to KLF11 basal promoter and distal PR- binding region. As the PR-binding region is located 20.5 kb upstream of the KLF11 transcription start site, we sought to elucidate whether this distal region was associated with the basal transcriptional multimeric complex. There did not seem to be any classic, palindromic PRE consensus sequences in either the basal promoter or the 20.5-kb distal PR-binding region. However, we found several half PREs in the distal region and several GC-rich Sp1 sites in both regions, and hypothesized that ligand-bound PR may associate with Sp1 and regulate KLF11 expression by mediating an interaction between the distal and basal regulatory sites. To test this hypothesis, we designed primers flanking the basal promoter and the distal region. We then evaluated simultaneous recruitment of RNA PolII, Sp1, and PR to the distal PR-binding region and the basal promoter of KLF11 using real-time PCR. We also characterized whether RU486 stimulated KLF11 expression through recruiting coactivators to its basal promoter and the distal PR-binding region. It was reported that PR action selectively required the steroid receptor coactivator SRC-3 in breast; in contrast, it required SRC-1 as a coactivator in uterus (35). SRC-1 and SRC-2 have a similar pattern Figure 3. Transcriptional activity of the distal PR-binding region and of expression that is distinct from the pattern of expression the basal promoter of the KLF11 gene. The distal PR-binding region (A) of SRC-3, indicating that these two proteins play cooperative was cloned into the pGL3 promoter vector and the basal promoter roles in regulating steroid hormone action in the murine region (B) was cloned into the pGL3 basic vector. T47D breast cancer cells were transiently transfected with each of these plasmids, exposed uterus (36). We thus evaluated the recruitment of SRC-1 −6 KLF11 to vehicle (ethanol) or 10 mol/L RU486 for 48 h, and harvested for and SRC-2 to basal promoter and the distal regulatory luciferase assay. Data shown are expressed as mean ± SEM for a region. representative triplicate experiment reproduced thrice. In the basal promoter region, RU486 significantly increased the association of RNA PolII, Sp1, PR (Fig. 4A), SRC-1, and SRC-2 (Fig. 4B) with this region of the KLF11 and general transcription factors to its basal promoter and gene. In the distal regulatory region, however, RU486 signifi- distal regulatory region. These findings also suggest that cantly induced the recruitment of only SRC-2 to this region the distal PR-binding region regulates KLF11 expression (Fig. 4B). We observed trends that RU486 enhanced the inter- through an interaction with the basal transcriptional multi- action of RNA PolII, Sp1, PR, and SRC-1 to the distal regula- meric complex. tory region, but none of them reached statistical significance Knockdown of KLF11 is associated with increased LSM (Fig. 4A and B). proliferation. We performed knockdown of endogenous To further verify that PR is necessary for the recruitment KLF11 to investigate how it may be involved in progesterone- of transcriptional complexes, we determined the effects of PR or RU486-regulated LSM cell proliferation, as indicated by knockdown on SRC-2 recruitment in the presence or absence PCNA level and the rate of BrdUrd incorporation. Real-time of RU486. As expected, RU486 induction of PR recruitment to PCR analysis confirmed that KLF11 siRNA reduced endoge- both the distal region and the basal promoter was abolished nous KLF11 expression by ∼80% (Fig. 5A). As shown in by PR siRNA (data not shown). PR knockdown significantly Fig. 5B, KLF11 siRNA or the pure progesterone agonist reduced the baseline recruitment of SRC-2 to the distal re- R5020 decreased KLF11 protein levels and increased PCNA gion but did not cause significant change of its association levels. In contrast, RU486 decreased PCNA levels and knock- with the basal promoter region (Fig. 4C). RU486-induced down of KLF11 blunted this inhibitory effect (Fig. 5C). Altera- SRC-2 recruitment to the basal promoter was decreased ro- tions in LSM cell proliferation were verified by the BrdUrd bustly by ∼2.4-fold in the presence of PR siRNA, whereas PR incorporation assay. Figure 5D shows that RU486 significant- siRNA strikingly decreased RU486-induced SRC-2 binding to ly decreased the BrdUrd labeling index from 7.1% to 3.8%. the distal region by ∼7-fold (Fig. 4C). Knockdown of KLF11 increased the BrdUrd labeling index These data suggest that RU486 regulates KLF11 expression to 17.6% (P < 0.01, compared with vehicle-treated and control through simultaneous recruitment of PR, its coactivators, siRNA–transfected cells), even in the presence of RU486

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(16.3%). These results suggest that regulation of LSM cell (40), ChIP cloning (25, 26), and ChIP-on-chip methods (24). proliferation by progesterone agonists or antagonists may Although progesterone has important and complex effects be mediated, at least in part, by KLF11. on the female reproductive tract, comparatively little is KLF11 expression in leiomyoma and matched myome- known about genome-wide PR-binding sites. Using ChIP- trial tissues. To understand the in vivo relevance of proges- cloning technology and progesterone-responsive primary terone and antiprogestin-regulated KLF11 expression in leiomyoma cells, we identified 18 novel PR-binding sites, leiomyoma, we analyzed mRNA and protein levels of KLF11 which corresponded to 20 genes proximal to these sites. Spe- in human leiomyoma and matched adjacent normal myome- cifically, we showed that RU486 enhanced recruitment of PR, trial tissues using real-time PCR and Western blot. KLF11 its coactivators SRC-1 and SRC-2, as well as key general tran- mRNA levels in 17 subjects were lower in leiomyoma than scription factors RNA PolII and Sp1, to both distal PR-bind- in myometrial tissues, and only one subject had higher ing region and basal promoter of the KLF11 gene and KLF11 mRNA levels in leiomyoma compared with myome- induced its expression. We also found that KLF11 is a robust trial tissues. As shown in Fig. 6A, the mean expression level suppressor of leiomyoma growth. of KLF11 in leiomyoma tissues was approximately half of We did not identify any PR-binding sites within a basal that of matched myometrial tissues (n = 18, P < 0.001). Sim- promoter. Five percent of PR-binding sequences were located ilarly, mean KLF11 protein level was significantly lower in in far distal 5′ regions, 5% were in 3′ regions, and 80% of leiomyoma than matched myometrial tissues from six sub- binding sites lay within an intron of a gene. Considering that jects (Fig. 6B). introns comprise ∼40% of the genome, this suggests a higher affinity of PR to coding regions of its target genes. This is Discussion consistent with our previous findings indicating that 46% of ERα binding sites were located within an intron of a gene A significant volume of work has focused on identifying in breast cancer cells (25). estrogen receptor binding sites using computational geno- None of the reported PR target genes was pulled out in this mics (37–39), promoter microarrays (26), CpG island libraries study (14–22). Some unavoidable limitations of the current

Figure 4. Association of transcription factors and steroid receptor coactivators with the distal PR-binding region and the basal promoter of the KLF11 gene. LSM cells were exposed to RU486 (10−6 mol/L) or vehicle (ethanol) for 1 h. DNA interaction with transcription factors and coactivators was detected by immunoprecipitation with an anti-phosphorylated RNA PolII, anti-PR, anti-Sp1 (A), anti–SRC-1, or anti–SRC-2 (B) antibody. Nonspecific rabbit IgG was used as a negative control. Real-time PCR was used to detect the fold enrichment of these transcription factors and coactivators using primers flanking the distal PR-binding and proximal promoter regions. The enrichment was normalized by IgG and calculated as fold increase compared with vehicle-treated cells. The results are shown as mean ± SEM of triplicates from a representative experiment reproduced using cells from four subjects. C, the effect of PR knockdown on SRC-2 association with the distal PR-binding region and the basal promoter of the KLF11 gene. LSM cells were transfected with control siRNA or PR siRNA for 72 h, and then exposed to RU486 10−6 mol/L for 1 h. DNA interaction with SRC-2 was detected as described above. The results are shown as mean ± SEM of triplicates from a representative experiment reproduced using cells from three subjects.

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ChIP-cloning strategy, such as the affinity of the antibody used SRC-2 recruitment, which is PR dependent (Fig. 4C). Taken to pull down chromatin and the on-and-off interaction pattern together, these findings are suggestive that the major func- of transcription factors with DNA, may cause the incomplete tion of the distal PR-binding site is the stabilization of the cloning of all target genes (25, 26, 41, 42). The low endogenous RU486/PR–dependent enhancer complex at the KLF11 pro- PR levels in cultured LSM cells may also account for the low moter. It seems that an interaction between the basal pro- number of PR-binding sites cloned in this study (43). moter and the distal PR-binding region would be necessary Regulation of half of the genes located proximately to for RU486-dependent regulation of KLF11 expression. these distant PR-binding sites by progesterone or RU486 sug- We found that, at least for the target genes studied here, gested that these sequences are functionally active. PR- RU486 was a much more robust regulator compared with binding fragments located distal to genes or within introns progesterone. Knockdown of PR or treatment with RU486 might function through long-range interactions that involve significantly increased KLF11 expression, whereas the native looping of chromatin to bring the regulatory elements within progesterone hormone minimally suppressed its expression. the proximity of basal promoters. The synthetic progesterone agonist R5020, however, marked- Both proximal and distal regulatory regions of the KLF11 ly suppressed KLF11 levels (Figs. 1B and 5B). The difference gene recruited not only PR and Sp1 but also RNA PolII upon between the effects of progesterone and R5020 may be due to RU486 treatment. Various models have been proposed to ex- possible rapid metabolization of progesterone in these cells. plain how a distal enhancer communicates with a proximal Consistent with the notion that coactivators play major promoter: the looping, the tracking, and the linking models roles in determining PR activity (46), we found that, through (44, 45). With regard to the distal-proximal communication interacting with PR, RU486 enhanced the binding of SRC-1 of the KLF11 gene, our data support a looping model. We and SRC-2 to the basal promoter and the distal PR-binding plan to use a chromosome conformation capture assay to in- region, which gave rise to gene induction. The function of vestigate whether the KLF11 distal region and proximal pro- RU486 as an inducer of gene transcription is not without pre- moter physically interact in intact cells. cedent. Shatnawi and colleagues showed that RU486 can RU486 stimulated the basal promoter activity but not the stimulate expression of genes encoding folate receptor type enhancer function of the distal region. RU486 also enhanced α, p21, and p27 in a specific promoter context that has G/C- PR binding to the basal promoter but not to the distal region. rich Sp1 binding elements but no classic PRE sites (22). In The distal region, however, exhibits significant basal enhanc- line with this observation, we found that both distal PR-bind- er function, basal PR-binding activity, and RU486-induced ing region and the basal promoter of the KLF11 gene, with

Figure 5. Regulation of LSM cell proliferation by KLF11. A, LSM cells were mock transfected (without siRNA) or transfected with control siRNA or KLF11 siRNA for 48 h, then KLF11 mRNA expression levels were detected using real-time PCR and standardized to GAPDH mRNA. Data shown are the mean ± SEM from a representative experiment repeated in cells from four subjects. B and C, the effects of KLF11 knockdown on proliferation of cells in the absence or presence of progesterone agonist R5020 or antagonist RU486. LSM cells were transfected with control siRNA or KLF11 siRNA for 24 h, then cells were treated with vehicle (ethanol), R5020 (10−6 mol/L), or RU486 (10−6 mol/L) for 72 h. The PCNA protein level was determined using anti-PCNA antibody. Immunoblot densities were quantified with ImageJ software. D, LSM cells were treated as in C except that cells were treated with RU486 (10−6 mol/L) or vehicle for 48 h and BrdUrd (20 μmol/L) was added for the final 4 h. BrdUrd-labeled cells were detected by immunostaining. Data shown are mean ± SEM for the BrdUrd labeling index of LSM cells from three subjects.

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KLF11 as a Novel PR Target Gene

growth in vitro and in vivo, and inhibits neoplastic transformation (34). Consistent with these reports, we found that KLF11 expression was significantly downregulated in leiomyoma tissues compared with adjacent matched myometrial tissues. Moreover, KLF11 inhibited LSM cell growth. Al- though the mechanism involved in KLF11-regulated cell proliferation needs to be further clarified, our studies provide an initial insight into the mechanism by which progesterone or RU486 regulates leiomyoma growth and development. Given that progesterone exerts a number of essential functions in inhibition of estrogen-dependent endometrial carcinogenesis and promotion of breast cancer development, it is tempting to explore the regulation and function of KLF11 in these ma- lignancies (47–49). In summary, using an unbiased approach, we uncovered KLF11 as a novel PR target responsive to progesterone antagonist RU486 in LSM cells. KLF11 mediated the antiprolifera- tive effects of RU486 in leiomyoma cells. These studies reveal a potential mechanism underlying the modulation of progesterone- and antiprogestin-regulated differentiation and proliferation in LSM cells. The PR-dependent downregulation of KLF11 expression in LSM cells may represent a missing link in steroid signaling pathways that explains the pathogenesis Figure 6. mRNA and protein levels of KLF11 in human leiomyoma and matched myometrial tissues. A, 36 samples from 18 subjects were of uterine leiomyoma. Furthermore, the identification of di- analyzed for mRNA; 18 samples were obtained from leiomyomas and rect targets of KLF11 will be important to elucidate its role in 18 from adjacent myometrial tissues. To allow comparisons of data uterine leiomyoma biology. obtained from samples from different subjects, mRNA levels in the myometrial tissues were normalized to 1. B, 12 samples from six subjects were analyzed for KLF protein levels. Immunoblot densities were Disclosure of Potential Conflicts of Interest quantified with Image J software. M, myometrial tissue; L, leiomyoma tissue. S.E. Bulun: consultant, GSK, Meditrina, and Novartis. The other authors disclosed no potential conflicts of interest. multiple G/C-rich Sp1 binding sites in both regions, could recruit PR. Additionally, our data suggest that the biological Grant Support activity of PR liganded by mixed antagonist/agonist com- NIH grant P01HD057877 (S.E. Bulun). pounds is complex and may depend on the promoter context The costs of publication of this article were defrayed in part by the payment and the availability of coregulators. of page charges. This article must therefore be hereby marked advertisement in KLF11 is a transcription factor that serves as a down- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. β– stream effector of transforming growth factor dependent Received 7/13/09; revised 11/12/09; accepted 12/14/09; published signaling. It is downregulated in human cancers, inhibits cell OnlineFirst 2/2/10.

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1730 Cancer Res; 70(4) February 15, 2010 Cancer Research

Transcription Factor KLF11 Integrates Progesterone Receptor Signaling and Proliferation in Uterine Leiomyoma Cells

Ping Yin, Zhihong Lin, Scott Reierstad, et al.

Cancer Res 2010;70:1722-1730. Published OnlineFirst February 2, 2010.

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