Cell. Mol. Life Sci. DOI 10.1007/s00018-014-1580-9
Cellular and Molecular Life Sciences
Research Article
Chromatin composition alterations and the critical role of MeCP2 for epigenetic silencing of progesterone receptor‑B gene in endometrial cancers Yongli Chu · Yanlin Wang · Guanghua Zhang · Haibin Chen · Sean C. Dowdy · Yuning Xiong · Fengming Liu · Run Zhang · Jinping Li · Shi‑Wen Jiang
Received: 22 September 2012 / Revised: 17 January 2014 / Accepted: 28 January 2014 © Springer Basel 2014
Abstract Objective To understand the epigenetic mechanism underlying the PR-B gene silencing in endometrial cancer (EC) cells, we compared the chromatin composition between transcriptionally active and silenced PR-B genes in EC cell lines and cancer tissues. Methods Chromatin Immunoprecipitation (ChIP) assay was performed to measure MBD occupancy and histone acetylation/methylation in transcriptionally active and silenced PR-B genes. PR-B-positive/-negative, as well as epigenetic inhibitor-treated/-untreated EC cells were used as study models. Real-time polymerase chain reaction (PCR) and Western blot analysis were applied to measure the mRNA and protein levels of PR-B, MBD, and histones.
Results A close association among PR-B methylation, MBD binding and PR-B gene silencing was observed. Treatment with epigenetic inhibitors led to dynamic changes in the PR-B chromatin composition and gene expression. Increased H3/H4 acetylation and H3-K4 methylation, and decreased H3-K9 methylation were found to be associated with re-activation of silenced PR-B genes. MeCP2 knockdown resulted in a decreased MeCP2 binding to PR-B genes and an increased PR-B expression. ChIP analysis of MeCP2 binding to PR-B genes in the PR-Bpositive/-negative EC samples confirmed the significant role of MeCP2 in PR-B silencing. Conclusion PR-B gene expression is regulated by a concerted action of epigenetic factors including DNA methylation, MBD binding, and histone modifications. MeCP2 occupancy of PR-B genes plays a critical role in PR-B gene
Y. Chu, Y. Wang, G. Zhang and H. Chen contributed equally. Y. Chu Yantai Yuhuangding Hospital Affiliated to Qingdao University, Yantai 264000, China
F. Liu Department of Research and Development, Guangxi Medicinal Botanical Institute, Nanning 530024, China
Y. Wang Department of Reproductive Medicine, Binzhou Medical University Hospital, Binzhou 256603, China
R. Zhang · J. Li (*) · S.-W. Jiang (*) Department of Biomedical Science, Mercer University School of Medicine, Savannah, GA 31404, USA e-mail: [email protected]
G. Zhang Tianjin Medical University Cancer Hospital, Tianjin 300060, China H. Chen Department of Histology and Embryology, Shantou University Medical College, Guangdong, China S. C. Dowdy · Y. Xiong · J. Li · S.-W. Jiang Department of Obstetrics and Gynecology, Mayo Clinic and Mayo Medical School, Rochester, MN 55905, USA
S.‑W. Jiang e-mail: [email protected] J. Li · S.-W. Jiang Curtis & Elizabeth Anderson Cancer Institute, Memorial Health University Medical Center, 4700 Waters Avenue, Savannah, GA 31404, USA S.-W. Jiang Department of Obstetrics and Gynecology, Memorial Health University Medical Center, 4700 Waters Avenue, Savannah, GA 31404, USA
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silencing. These findings enriched our knowledge of the epigenetic regulation of PR-B expression in EC, and suggested that the epigenetic re-activation of PR-B could be explored as a potential strategy to sensitize the PR-B-negative endometrial cancers to progestational therapy. Keywords Progesterone receptor-B · Epigenetic silencing · Endometrial cancer · DNA methylation · Chromatin
Introduction One well-recognized effect of progesterone in the human uterus is the protection of the endometrium against the hyperplastic and tumorigenic activities caused by excessive levels of estrogens [1, 2]. Progestins are routinely prescribed together with estrogen in hormone replacement therapy to prevent endometrial cancers (EC) [3]. Progestational treatment represents a common hormonal therapy approach for patients with breast and gynecologic cancers. Complete reversal of endometrial hyperplasia and EC using high doses of progestins has been reported [4–6]. Progestin regimens were found to be beneficial for advanced or recurrent endometrial cancers in some, but not all clinical trials [7–9]. Improved response rates and relatively mild side effects were observed with a combined regimen consisting of progesterone and tamoxifen [10, 11] or progesterone and an aromatase inhibitor in treating patients with advanced endometrial cancers [12]. The anti-cancer effects of progestins in endometrial cells are primarily mediated by the progesterone receptor (PR), a member of the nuclear receptor family with ligand-dependent transcription activities. When bound to progesterones, the carboxyl terminus of the PR undergoes conformational changes that promote PR dimerization and binding to the target DNA [13]. The activated PR interacts with a variety of co-activators/co-repressors and regulates the expression of diverse downstream genes [14]. Progesterone inhibits EC progression via the suppression of cell cycle progression and induction of apoptosis. Progesterone decreases the expression of the estrogen receptor [15], upregulates the cyclin-dependent kinase inhibitor P27 Kip1 [16], and inhibits EC cell proliferation. In addition, prolonged progestin treatment increases cellular levels of P21Cip/waf1, a key cell-cycle regulator, through activation of P53 [17]. In vivo studies indicated that induction of apoptosis is an early event during treatment of endometrial hyperplasia with progestins [18]. Unfortunately, PR expression is frequently lost in advanced EC, which is considered a most common cause for resistance to hormonal therapy [19, 20]. While the majority (72 %) of PR-positive tumors respond to progesterone treatment,
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the response rate is much lower (10–20 %) for PR-negative tumors [21]. Endometrial glandular cells express two isoforms of progesterone receptors, PR-A and PR-B. These isoforms are generated through the use of alternative promoters with differing transcription start sites. Two lines of evidence provided support for the crucial role of PR-B in the pathogenesis of EC. First, PR-B accounts for most of the inhibitory effects of progestins on cancer cell growth [22]. Second, clinical studies in endometrial cancers have shown a drastic decrease in mRNA levels of PR isoform B, but not A [23, 24]. The loss of PR-B expression has been found to be associated with cancer invasion and recurrence [25, 26]. The PR-B gene contains a typical CpG island spanning nucleotides −80 to +500 relative to the transcription start site. Aberrant DNA hypermethylation within this region is considered to be the cause of the PR-B-negative phenotype in EC cell lines, as well as in patient samples [27]. Interestingly, we observed that DNA methyltransferases (DNMT) 1 and 3B, the enzymes responsible for genomic DNA methylation, were overexpressed three- to fourfold in endometrioid cancers compared to normal tissues [28]. These findings suggested that a simultaneous upregulation of maintenance (DNMT1) and de novo (DNMT3B) methyltransferases may take a part in the aberrant DNA hypermethylation of target genes such as PR-B. The pivotal role of DNA methylation in PR-B gene inactivation is also supported by the results of in vitro studies. We and others have shown that treatment of PR-B-negative EC cell lines with DNMT inhibitors resulted in PR-B gene demethylation and a concomitant PR-B gene re-activation [27, 29]. While these observations underscored the significance of the DNA methylation-mediated pathway in PR-B silencing, the underlying mechanism(s) remain to be investigated. It is not clear, for example, what alterations in local chromatin components may be involved in the cancer-related PR-B gene inactivation. Accumulated data suggest that changes in chromatin composition contribute to transcriptional silencing [30–32]. Methyl-CpG binding domain proteins (MBDs) specifically bind to methylated DNA and recruit histone modification enzymes such as histone deacetylases (HDAC), methyltransferases (Mets) and/or histone demethylase [33–37], leading to histone modification alterations and gene silencing. In this study, we characterized the MBD binding and histone modifications in the chromatins associated with the methylated and unmethylated PR-B genes. Moreover, the relationship between chromatin composition and PR-B silencing was determined in EC cells that were pre-treated with DNMT and HDAC inhibitors as well as PR-B-negative and PR-B-positive EC tissues. These experiments have led to the identification of specific factors and mechanisms that are critical for PR-B epigenetic silencing.
Chromatin composition alterations and the critical role of MeCP2
Materials and methods Cell lines and reagents The human endometrial cancer cell lines AN3, KLE, RL95, HEC-1A, and HEC-1B were purchased from American Type Culture Collection (ATCC, Rockville, MD). These cells were grown in DMEM/F12 medium. The well-differentiated human endometrioid adenocarcinoma Ishikawa cell line was generously provided by Dr. Masato Nishida (Kasumigaura National Hospital, Japan) [38]. Ishikawa cells are maintained in MEMα medium. All the media are supplemented with 10 % fetal bovine serum (BioWhitaker, Walkersville, MD), 100 μg/ml streptomycin, 100 units/ ml penicillin, and 2 mM l-glutamine. Cells were grown to 20 % confluence in 10-cm dishes and treated with different concentrations of aza-deoxycytidine (ADC, Sigma, St. Louis, MO) or trichostatin A (TSA, Sigma, St. Louis, MO) as indicated in the figure legends. Antibodies against MeCP2, histone H3 and H4, acetylhistone H3, acetyl-histone H4, dimethyl-histone H3 at Lysine-4 (Met H3-K4), and dimethyl-histone H3 at Lysine-9 (Met H3-K9) were obtained from Upstate Biotechnology, Inc (Lake Placid, NY). Rabbit antibody for β-actin, and rabbit and goat antibodies for MBD1, MBD2, MBD3, and MBD4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) genes were measured using the following primers: PRBF: 5′-ACT GAG CTG AAG GCA AAG GGT; PRB-R: 5′GTC CTG TCC CTG GCA GGG C; MeCP2-F: 5′-CAG GCA AAG CAG AGA CAT CA-3′; MeCP2-R: 5′-GCT TAA GCT TCC GTG TCC AG-3′; GAPDH-F: 5′-GAA GGT GAA GGT CGG AGT C-3′; GAPDH-R: 5′-GAA GAT GGT GAT GGG ATT TC-3′. PCR conditions were: initial denaturing at 95 °C for 5 min, followed by 40 cycles of denaturing at 95 °C for 15 s, annealing at 56 °C for 30 s, and extension at 72 °C for 30 s. The specificity of the realtime PCR was verified by a clear, single DNA band with the predicted size of final PCR products resolved in the agarose gel electrophoresis. siRNA knockdown of MeCP2 siRNA oligonucleotides of MeCP2 (5′-GCU CUA AAG UGG AGU UGA UUU -3′) were purchased from Dharmacon Technology, Inc (Chicago, IL). KLE cells were seeded on six-well plates at 60 % confluence 24 h before the transfection. Cells were transfected with siRNA oligo at a final concentration of 100 nM using the DharmaFECT 1 transfection reagents (Dharmacon, Chicago, IL). Total RNA was isolated from the cells and reverse transcription and realtime PCR were performed to measure MeCP2 and PR-B mRNA levels. For ChIP Assay, cells were seeded on 100mm dishes and transfected with the same reagents.
Collection of tissue samples Methylation‑specific PCR PR-B-positive and -negative endometrial cancer tissue samples (four cases in each group) were collected from patients treated at the Mayo Clinic. The PR-B expression status of tissues was diagnosed by a pathologist based on the pathological review of the immunostaining results. Fresh tissue samples were snap frozen and stored at −80 °C. This study was approved by the Institutional Review Board of the Mayo Foundation. In accordance with the Minnesota Statute for the Use of Medical Information in Research, only patients who consented to the use of their cancer tissues and medical records were included in the study.
Genomic DNA was isolated from cell cultures using DNAzol reagent (Molecular Research Center, Cincinnati, OH) following the manufacturer’s instructions. The EZ DNA methylation kit (Zymo Research, Orange, CA) was used for sodium bisulfite conversion of genomic DNA. Methylation-specific PCR was performed with the primers specific for either methylated or unmethylated PR-B CpG islands as previously published [27]. 10 μl of PCR products were resolved in agarose gel electrophoresis and the DNA bands were visualized by ethidium bromide staining.
Real‑time PCR
Chromatin immunoprecipitation (ChIP) assays
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA). cDNA was synthesized with 1 μg RNA using a SuperScript™ kit (Invitrogen, Carlsbad, CA). The 20 μl products of reverse transcription were diluted to 100 μl, and 2 μl was used for each real-time polymerase chain reaction (PCR) assay. PCR was performed in a total volume of 25 μl containing 140 ng forward and backward primers, respectively, and 12.5 μl SYBR green Master Mix (Stratagene, Cedar Creek, TX). The mRNA levels of PR-B
ChIP was performed using the ChIP Assay Kit (Upstate Biotechnology, Inc., Lake Placid, NY) with some modifications on recommended protocols. Briefly, one 10-cm dish of cell culture containing approximately 5 × 106 cells, or 50–100 μg of EC tissues, were used for each ChIP assay. Protein-DNA cross-linking was carried out by exposure to 1 % formaldehyde at 37 °C for 10 min. The medium was removed and cells were washed three times with ice-cold phosphate buffered saline (PBS). Then,
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1.5 ml PBS was added and the cells were scraped off the culture dishes, and transferred into conical tubes. Cells were collected by centrifugation at 2,000 rpm for 4 min at 4 °C. The PBS was removed and 200 μl of SDS lysis buffer (1 % SDS, 10 mM EDTA, 50 mM Tris–HCl, pH 8.1) supplemented with protease inhibitors (1 mM PMSF, 1 μg/ml aprotinin, and 1 μg/ml pepstatin A) was added to re-suspend the cell pellets. Sonication was performed on ice using a sonicator (Sonic Dismembrator, Model 500, Fisher Scientific) pre-set for 10-s pulses with 10-s intervals. Four repeated sonication cycles were applied to achieve chromatin fragmentation of 200–1,000 bp. Samples were centrifuged at 13,000 rpm for 10 min and the supernatants were transferred to new tubes. The samples were diluted tenfold with ChIP dilution buffer (0.01 % SDS, 1 % Triton X-100, 2 mM EDTA, 16.7 mM Tris–HCl, pH 8.1, 150 mM NaCl). A 20-μl aliquot of the sample was removed to serve as the input control. To reduce the nonspecific background, the DNA–protein complexes were pre-cleared by incubation with 75 μl of Protein A agarose beads (50 % slurry containing salmon sperm DNA). The pre-absorption was carried out at 4 °C with constant rotation for 2 h. Specific antibodies against MeCP2, MBD1, MBD2, MBD3, MBD4, total histones, acetylated histones, and methylated histones were used for immunoprecipitation. Antibody binding was accomplished by incubation at 4 °C overnight with constant rotation. For negative controls, a non-specific antibody was used instead of specific antibodies. To collect immune complexes, 60 μl of Protein A agarose-salmon sperm DNA (50 % slurry) was added to each tube and incubation continued for 2 h at 4 °C. Agarose beads were recovered by gentle centrifugation at 2,000 rpm for 2 min. The beads were washed sequentially with 1 ml buffer for 5 min in the following order: two times with low-salt buffer (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.1, 150 mM NaCl), two times with high-salt buffer (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.1, 500 mM NaCl), once with LiCl buffer (0.25 mM LiCl, 0.5 % deoxycholic acid, 1 mM EDTA, 10 mM Tris, pH 8.1), and once with 1X TE buffer. After washing, 500 μl fresh 1 % SDS and 0.1 M NaHCO3 was added to elute the immune complexes. Formaldehyde cross-links were reversed by adding 20 μl 5 M NaCl to 500 μl eluates and heating at 65 °C for 4 h. DNA fragments were recovered by ethanol precipitation after proteinase K digestion and phenol/chloroform extraction. Following immunoprecipitation, PCR was performed with PR-B gene-specific primers: 5′-TCA GAA TAA CGG GTG GAA ATG-3′ and 5′-TCT AAC AAC GCC TCC TCC TC-3′. The 108 bp amplicon containing four CpG sites represents the CpG islands from the PR-B promoter region. PCR conditions were: 94 °C for 5 min for
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initial denaturation, followed by 30 cycles of denaturation at 94 °C for 45 s, annealing at 56 °C for 45 s, and extension at 72 °C for 1 min. The final PCR products were analyzed with 2 % agarose gels electrophoresis and ethidium bromide staining. The ChIP results were documented by Polaroid photograph with UV excitation. Western blot analysis Cell cultures were rinsed three times with cold PBS and harvested by scraping in lysis buffer (20 mM Hepes, pH 7.2, 25 % glycerol, 0.4 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride). The lysis buffer was supplemented with 1X protease inhibitor cocktail (Sigma, St. Louis, MO). Cellular proteins were quantified and resolved by SDS polyacrylamide gel electrophoresis and transferred to ImmunoBlot™ PDVF membranes (Bio-Rad Laboratories, Hercules, CA) as previously described [39]. Expression levels of MeCP2, MBD1, total H3 and H4, and acetylated H3 and H4 were determined using specific antibodies following the manufacturer’s instructions. Chemiluminescence detection was performed with the ECLplus™ Western Blotting Detection System (Amersham Corp, Arlington Heights, IL). The blots were re-probed with β-actin antibody and the results provided controls for protein loading. Data analysis Relative PR-B mRNA levels of endometrial cancer cell lines were calculated based on the real-time PCR data. The threshold cycle number (CT) for PR-B was normalized against the GAPDH internal reference gene by the formula: ΔCT = CTPR-B − CTGAPDH. The difference between PR-B and GAPDH was further converted to a relative fold (F = 2ΔCT). The PR-B mRNA level of the Ishikawa cell was arbitrarily set at 100, and PR-B mRNA levels in AN3, KLE, RL-95, HEC-1A and HEC-1B cells were expressed as a percentage of the level in Ishikawa cells. The results of the ChIP experiments were documented with an HP Q3190A scanner and analyzed by densitometry using the NIH Image program. All data groups from the real-time PCR and ChIP experiments were analyzed by a multivariate analysis of variance (ANOVA) to determine if there was a significant difference among the groups. For data groups that satisfied the initial ANOVA criterion, individual comparisons were performed with the use of post-hoc Bonferroni t tests with the assumption of two-tail distribution and two samples with equal variance. The statistical significance (p
Cellular and Molecular Life Sciences
Research Article
Chromatin composition alterations and the critical role of MeCP2 for epigenetic silencing of progesterone receptor‑B gene in endometrial cancers Yongli Chu · Yanlin Wang · Guanghua Zhang · Haibin Chen · Sean C. Dowdy · Yuning Xiong · Fengming Liu · Run Zhang · Jinping Li · Shi‑Wen Jiang
Received: 22 September 2012 / Revised: 17 January 2014 / Accepted: 28 January 2014 © Springer Basel 2014
Abstract Objective To understand the epigenetic mechanism underlying the PR-B gene silencing in endometrial cancer (EC) cells, we compared the chromatin composition between transcriptionally active and silenced PR-B genes in EC cell lines and cancer tissues. Methods Chromatin Immunoprecipitation (ChIP) assay was performed to measure MBD occupancy and histone acetylation/methylation in transcriptionally active and silenced PR-B genes. PR-B-positive/-negative, as well as epigenetic inhibitor-treated/-untreated EC cells were used as study models. Real-time polymerase chain reaction (PCR) and Western blot analysis were applied to measure the mRNA and protein levels of PR-B, MBD, and histones.
Results A close association among PR-B methylation, MBD binding and PR-B gene silencing was observed. Treatment with epigenetic inhibitors led to dynamic changes in the PR-B chromatin composition and gene expression. Increased H3/H4 acetylation and H3-K4 methylation, and decreased H3-K9 methylation were found to be associated with re-activation of silenced PR-B genes. MeCP2 knockdown resulted in a decreased MeCP2 binding to PR-B genes and an increased PR-B expression. ChIP analysis of MeCP2 binding to PR-B genes in the PR-Bpositive/-negative EC samples confirmed the significant role of MeCP2 in PR-B silencing. Conclusion PR-B gene expression is regulated by a concerted action of epigenetic factors including DNA methylation, MBD binding, and histone modifications. MeCP2 occupancy of PR-B genes plays a critical role in PR-B gene
Y. Chu, Y. Wang, G. Zhang and H. Chen contributed equally. Y. Chu Yantai Yuhuangding Hospital Affiliated to Qingdao University, Yantai 264000, China
F. Liu Department of Research and Development, Guangxi Medicinal Botanical Institute, Nanning 530024, China
Y. Wang Department of Reproductive Medicine, Binzhou Medical University Hospital, Binzhou 256603, China
R. Zhang · J. Li (*) · S.-W. Jiang (*) Department of Biomedical Science, Mercer University School of Medicine, Savannah, GA 31404, USA e-mail: [email protected]
G. Zhang Tianjin Medical University Cancer Hospital, Tianjin 300060, China H. Chen Department of Histology and Embryology, Shantou University Medical College, Guangdong, China S. C. Dowdy · Y. Xiong · J. Li · S.-W. Jiang Department of Obstetrics and Gynecology, Mayo Clinic and Mayo Medical School, Rochester, MN 55905, USA
S.‑W. Jiang e-mail: [email protected] J. Li · S.-W. Jiang Curtis & Elizabeth Anderson Cancer Institute, Memorial Health University Medical Center, 4700 Waters Avenue, Savannah, GA 31404, USA S.-W. Jiang Department of Obstetrics and Gynecology, Memorial Health University Medical Center, 4700 Waters Avenue, Savannah, GA 31404, USA
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silencing. These findings enriched our knowledge of the epigenetic regulation of PR-B expression in EC, and suggested that the epigenetic re-activation of PR-B could be explored as a potential strategy to sensitize the PR-B-negative endometrial cancers to progestational therapy. Keywords Progesterone receptor-B · Epigenetic silencing · Endometrial cancer · DNA methylation · Chromatin
Introduction One well-recognized effect of progesterone in the human uterus is the protection of the endometrium against the hyperplastic and tumorigenic activities caused by excessive levels of estrogens [1, 2]. Progestins are routinely prescribed together with estrogen in hormone replacement therapy to prevent endometrial cancers (EC) [3]. Progestational treatment represents a common hormonal therapy approach for patients with breast and gynecologic cancers. Complete reversal of endometrial hyperplasia and EC using high doses of progestins has been reported [4–6]. Progestin regimens were found to be beneficial for advanced or recurrent endometrial cancers in some, but not all clinical trials [7–9]. Improved response rates and relatively mild side effects were observed with a combined regimen consisting of progesterone and tamoxifen [10, 11] or progesterone and an aromatase inhibitor in treating patients with advanced endometrial cancers [12]. The anti-cancer effects of progestins in endometrial cells are primarily mediated by the progesterone receptor (PR), a member of the nuclear receptor family with ligand-dependent transcription activities. When bound to progesterones, the carboxyl terminus of the PR undergoes conformational changes that promote PR dimerization and binding to the target DNA [13]. The activated PR interacts with a variety of co-activators/co-repressors and regulates the expression of diverse downstream genes [14]. Progesterone inhibits EC progression via the suppression of cell cycle progression and induction of apoptosis. Progesterone decreases the expression of the estrogen receptor [15], upregulates the cyclin-dependent kinase inhibitor P27 Kip1 [16], and inhibits EC cell proliferation. In addition, prolonged progestin treatment increases cellular levels of P21Cip/waf1, a key cell-cycle regulator, through activation of P53 [17]. In vivo studies indicated that induction of apoptosis is an early event during treatment of endometrial hyperplasia with progestins [18]. Unfortunately, PR expression is frequently lost in advanced EC, which is considered a most common cause for resistance to hormonal therapy [19, 20]. While the majority (72 %) of PR-positive tumors respond to progesterone treatment,
13
the response rate is much lower (10–20 %) for PR-negative tumors [21]. Endometrial glandular cells express two isoforms of progesterone receptors, PR-A and PR-B. These isoforms are generated through the use of alternative promoters with differing transcription start sites. Two lines of evidence provided support for the crucial role of PR-B in the pathogenesis of EC. First, PR-B accounts for most of the inhibitory effects of progestins on cancer cell growth [22]. Second, clinical studies in endometrial cancers have shown a drastic decrease in mRNA levels of PR isoform B, but not A [23, 24]. The loss of PR-B expression has been found to be associated with cancer invasion and recurrence [25, 26]. The PR-B gene contains a typical CpG island spanning nucleotides −80 to +500 relative to the transcription start site. Aberrant DNA hypermethylation within this region is considered to be the cause of the PR-B-negative phenotype in EC cell lines, as well as in patient samples [27]. Interestingly, we observed that DNA methyltransferases (DNMT) 1 and 3B, the enzymes responsible for genomic DNA methylation, were overexpressed three- to fourfold in endometrioid cancers compared to normal tissues [28]. These findings suggested that a simultaneous upregulation of maintenance (DNMT1) and de novo (DNMT3B) methyltransferases may take a part in the aberrant DNA hypermethylation of target genes such as PR-B. The pivotal role of DNA methylation in PR-B gene inactivation is also supported by the results of in vitro studies. We and others have shown that treatment of PR-B-negative EC cell lines with DNMT inhibitors resulted in PR-B gene demethylation and a concomitant PR-B gene re-activation [27, 29]. While these observations underscored the significance of the DNA methylation-mediated pathway in PR-B silencing, the underlying mechanism(s) remain to be investigated. It is not clear, for example, what alterations in local chromatin components may be involved in the cancer-related PR-B gene inactivation. Accumulated data suggest that changes in chromatin composition contribute to transcriptional silencing [30–32]. Methyl-CpG binding domain proteins (MBDs) specifically bind to methylated DNA and recruit histone modification enzymes such as histone deacetylases (HDAC), methyltransferases (Mets) and/or histone demethylase [33–37], leading to histone modification alterations and gene silencing. In this study, we characterized the MBD binding and histone modifications in the chromatins associated with the methylated and unmethylated PR-B genes. Moreover, the relationship between chromatin composition and PR-B silencing was determined in EC cells that were pre-treated with DNMT and HDAC inhibitors as well as PR-B-negative and PR-B-positive EC tissues. These experiments have led to the identification of specific factors and mechanisms that are critical for PR-B epigenetic silencing.
Chromatin composition alterations and the critical role of MeCP2
Materials and methods Cell lines and reagents The human endometrial cancer cell lines AN3, KLE, RL95, HEC-1A, and HEC-1B were purchased from American Type Culture Collection (ATCC, Rockville, MD). These cells were grown in DMEM/F12 medium. The well-differentiated human endometrioid adenocarcinoma Ishikawa cell line was generously provided by Dr. Masato Nishida (Kasumigaura National Hospital, Japan) [38]. Ishikawa cells are maintained in MEMα medium. All the media are supplemented with 10 % fetal bovine serum (BioWhitaker, Walkersville, MD), 100 μg/ml streptomycin, 100 units/ ml penicillin, and 2 mM l-glutamine. Cells were grown to 20 % confluence in 10-cm dishes and treated with different concentrations of aza-deoxycytidine (ADC, Sigma, St. Louis, MO) or trichostatin A (TSA, Sigma, St. Louis, MO) as indicated in the figure legends. Antibodies against MeCP2, histone H3 and H4, acetylhistone H3, acetyl-histone H4, dimethyl-histone H3 at Lysine-4 (Met H3-K4), and dimethyl-histone H3 at Lysine-9 (Met H3-K9) were obtained from Upstate Biotechnology, Inc (Lake Placid, NY). Rabbit antibody for β-actin, and rabbit and goat antibodies for MBD1, MBD2, MBD3, and MBD4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) genes were measured using the following primers: PRBF: 5′-ACT GAG CTG AAG GCA AAG GGT; PRB-R: 5′GTC CTG TCC CTG GCA GGG C; MeCP2-F: 5′-CAG GCA AAG CAG AGA CAT CA-3′; MeCP2-R: 5′-GCT TAA GCT TCC GTG TCC AG-3′; GAPDH-F: 5′-GAA GGT GAA GGT CGG AGT C-3′; GAPDH-R: 5′-GAA GAT GGT GAT GGG ATT TC-3′. PCR conditions were: initial denaturing at 95 °C for 5 min, followed by 40 cycles of denaturing at 95 °C for 15 s, annealing at 56 °C for 30 s, and extension at 72 °C for 30 s. The specificity of the realtime PCR was verified by a clear, single DNA band with the predicted size of final PCR products resolved in the agarose gel electrophoresis. siRNA knockdown of MeCP2 siRNA oligonucleotides of MeCP2 (5′-GCU CUA AAG UGG AGU UGA UUU -3′) were purchased from Dharmacon Technology, Inc (Chicago, IL). KLE cells were seeded on six-well plates at 60 % confluence 24 h before the transfection. Cells were transfected with siRNA oligo at a final concentration of 100 nM using the DharmaFECT 1 transfection reagents (Dharmacon, Chicago, IL). Total RNA was isolated from the cells and reverse transcription and realtime PCR were performed to measure MeCP2 and PR-B mRNA levels. For ChIP Assay, cells were seeded on 100mm dishes and transfected with the same reagents.
Collection of tissue samples Methylation‑specific PCR PR-B-positive and -negative endometrial cancer tissue samples (four cases in each group) were collected from patients treated at the Mayo Clinic. The PR-B expression status of tissues was diagnosed by a pathologist based on the pathological review of the immunostaining results. Fresh tissue samples were snap frozen and stored at −80 °C. This study was approved by the Institutional Review Board of the Mayo Foundation. In accordance with the Minnesota Statute for the Use of Medical Information in Research, only patients who consented to the use of their cancer tissues and medical records were included in the study.
Genomic DNA was isolated from cell cultures using DNAzol reagent (Molecular Research Center, Cincinnati, OH) following the manufacturer’s instructions. The EZ DNA methylation kit (Zymo Research, Orange, CA) was used for sodium bisulfite conversion of genomic DNA. Methylation-specific PCR was performed with the primers specific for either methylated or unmethylated PR-B CpG islands as previously published [27]. 10 μl of PCR products were resolved in agarose gel electrophoresis and the DNA bands were visualized by ethidium bromide staining.
Real‑time PCR
Chromatin immunoprecipitation (ChIP) assays
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA). cDNA was synthesized with 1 μg RNA using a SuperScript™ kit (Invitrogen, Carlsbad, CA). The 20 μl products of reverse transcription were diluted to 100 μl, and 2 μl was used for each real-time polymerase chain reaction (PCR) assay. PCR was performed in a total volume of 25 μl containing 140 ng forward and backward primers, respectively, and 12.5 μl SYBR green Master Mix (Stratagene, Cedar Creek, TX). The mRNA levels of PR-B
ChIP was performed using the ChIP Assay Kit (Upstate Biotechnology, Inc., Lake Placid, NY) with some modifications on recommended protocols. Briefly, one 10-cm dish of cell culture containing approximately 5 × 106 cells, or 50–100 μg of EC tissues, were used for each ChIP assay. Protein-DNA cross-linking was carried out by exposure to 1 % formaldehyde at 37 °C for 10 min. The medium was removed and cells were washed three times with ice-cold phosphate buffered saline (PBS). Then,
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1.5 ml PBS was added and the cells were scraped off the culture dishes, and transferred into conical tubes. Cells were collected by centrifugation at 2,000 rpm for 4 min at 4 °C. The PBS was removed and 200 μl of SDS lysis buffer (1 % SDS, 10 mM EDTA, 50 mM Tris–HCl, pH 8.1) supplemented with protease inhibitors (1 mM PMSF, 1 μg/ml aprotinin, and 1 μg/ml pepstatin A) was added to re-suspend the cell pellets. Sonication was performed on ice using a sonicator (Sonic Dismembrator, Model 500, Fisher Scientific) pre-set for 10-s pulses with 10-s intervals. Four repeated sonication cycles were applied to achieve chromatin fragmentation of 200–1,000 bp. Samples were centrifuged at 13,000 rpm for 10 min and the supernatants were transferred to new tubes. The samples were diluted tenfold with ChIP dilution buffer (0.01 % SDS, 1 % Triton X-100, 2 mM EDTA, 16.7 mM Tris–HCl, pH 8.1, 150 mM NaCl). A 20-μl aliquot of the sample was removed to serve as the input control. To reduce the nonspecific background, the DNA–protein complexes were pre-cleared by incubation with 75 μl of Protein A agarose beads (50 % slurry containing salmon sperm DNA). The pre-absorption was carried out at 4 °C with constant rotation for 2 h. Specific antibodies against MeCP2, MBD1, MBD2, MBD3, MBD4, total histones, acetylated histones, and methylated histones were used for immunoprecipitation. Antibody binding was accomplished by incubation at 4 °C overnight with constant rotation. For negative controls, a non-specific antibody was used instead of specific antibodies. To collect immune complexes, 60 μl of Protein A agarose-salmon sperm DNA (50 % slurry) was added to each tube and incubation continued for 2 h at 4 °C. Agarose beads were recovered by gentle centrifugation at 2,000 rpm for 2 min. The beads were washed sequentially with 1 ml buffer for 5 min in the following order: two times with low-salt buffer (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.1, 150 mM NaCl), two times with high-salt buffer (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.1, 500 mM NaCl), once with LiCl buffer (0.25 mM LiCl, 0.5 % deoxycholic acid, 1 mM EDTA, 10 mM Tris, pH 8.1), and once with 1X TE buffer. After washing, 500 μl fresh 1 % SDS and 0.1 M NaHCO3 was added to elute the immune complexes. Formaldehyde cross-links were reversed by adding 20 μl 5 M NaCl to 500 μl eluates and heating at 65 °C for 4 h. DNA fragments were recovered by ethanol precipitation after proteinase K digestion and phenol/chloroform extraction. Following immunoprecipitation, PCR was performed with PR-B gene-specific primers: 5′-TCA GAA TAA CGG GTG GAA ATG-3′ and 5′-TCT AAC AAC GCC TCC TCC TC-3′. The 108 bp amplicon containing four CpG sites represents the CpG islands from the PR-B promoter region. PCR conditions were: 94 °C for 5 min for
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initial denaturation, followed by 30 cycles of denaturation at 94 °C for 45 s, annealing at 56 °C for 45 s, and extension at 72 °C for 1 min. The final PCR products were analyzed with 2 % agarose gels electrophoresis and ethidium bromide staining. The ChIP results were documented by Polaroid photograph with UV excitation. Western blot analysis Cell cultures were rinsed three times with cold PBS and harvested by scraping in lysis buffer (20 mM Hepes, pH 7.2, 25 % glycerol, 0.4 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride). The lysis buffer was supplemented with 1X protease inhibitor cocktail (Sigma, St. Louis, MO). Cellular proteins were quantified and resolved by SDS polyacrylamide gel electrophoresis and transferred to ImmunoBlot™ PDVF membranes (Bio-Rad Laboratories, Hercules, CA) as previously described [39]. Expression levels of MeCP2, MBD1, total H3 and H4, and acetylated H3 and H4 were determined using specific antibodies following the manufacturer’s instructions. Chemiluminescence detection was performed with the ECLplus™ Western Blotting Detection System (Amersham Corp, Arlington Heights, IL). The blots were re-probed with β-actin antibody and the results provided controls for protein loading. Data analysis Relative PR-B mRNA levels of endometrial cancer cell lines were calculated based on the real-time PCR data. The threshold cycle number (CT) for PR-B was normalized against the GAPDH internal reference gene by the formula: ΔCT = CTPR-B − CTGAPDH. The difference between PR-B and GAPDH was further converted to a relative fold (F = 2ΔCT). The PR-B mRNA level of the Ishikawa cell was arbitrarily set at 100, and PR-B mRNA levels in AN3, KLE, RL-95, HEC-1A and HEC-1B cells were expressed as a percentage of the level in Ishikawa cells. The results of the ChIP experiments were documented with an HP Q3190A scanner and analyzed by densitometry using the NIH Image program. All data groups from the real-time PCR and ChIP experiments were analyzed by a multivariate analysis of variance (ANOVA) to determine if there was a significant difference among the groups. For data groups that satisfied the initial ANOVA criterion, individual comparisons were performed with the use of post-hoc Bonferroni t tests with the assumption of two-tail distribution and two samples with equal variance. The statistical significance (p
