Down-regulation of Human DAB2IP Gene Expression Mediated by Polycomb Ezh2 Complex and Histone Deacetylase in Prostate Cancer
2005; Elsevier BV; Volume: 280; Issue: 23 Linguagem: Inglês
10.1074/jbc.m501379200
ISSN1083-351X
AutoresHong Chen, Szu-wei Tu, Jer‐Tsong Hsieh,
Tópico(s)Genomics and Chromatin Dynamics
ResumoHuman DAB2IP (hDAB2IP), a novel GTPase-activating protein modulating the Ras-mediated signaling and tumor necrosis factor-mediated apoptosis, is a potent growth inhibitor in human prostate cancer (PCa). Loss of hDAB2IP expression in PCa is due to altered epigenetic regulation (i.e. DNA methylation and histone modification) of its promoter region. The elevated polycomb Ezh2, a histone methyltransferase, has been associated with PCa progression. In this study, we have demonstrated that an increased Ezh2 expression in normal prostatic epithelial cells can suppress hDAB2IP gene expression. In contrast, knocking down the endogenous Ezh2 levels in PCa by a specific small interfering RNA can increase hDAB2IP expression. The association of Ezh2 complex (including Eed and Suz12) with hDAB2IP gene promoter is also detected in PCa cells but not in normal prostatic epithelial cells. Increased Ezh2 expression in normal prostatic epithelial cells by cDNA transfection facilitates the recruitment of other components of Ezh2 complex to the hDAB2IP promoter region accompanied with the increased levels of methyl histone H3 (H3) and histone deacetylase (HDAC1). Consistently, data from PCa cells transfected with Ezh2 small interfering RNA demonstrated that reduced Ezh2 levels resulted in the dissociation of Ezh2 complex accompanied with decreased levels of both methyl H3 and HDAC1 from hDAB2IP gene promoter. We further unveiled that the methylation status of Lys-27 but not Lys-9 of H3 in hDAB2IP promoter region is consistent with the hDAB2IP levels in both normal prostatic epithelial cells and PCa cells. Together, we conclude that hDAB2IP gene is a target gene of Ezh2 in prostatic epithelium, which provides an underlying mechanism of the down-regulation of hDAB2IP gene in PCa. Human DAB2IP (hDAB2IP), a novel GTPase-activating protein modulating the Ras-mediated signaling and tumor necrosis factor-mediated apoptosis, is a potent growth inhibitor in human prostate cancer (PCa). Loss of hDAB2IP expression in PCa is due to altered epigenetic regulation (i.e. DNA methylation and histone modification) of its promoter region. The elevated polycomb Ezh2, a histone methyltransferase, has been associated with PCa progression. In this study, we have demonstrated that an increased Ezh2 expression in normal prostatic epithelial cells can suppress hDAB2IP gene expression. In contrast, knocking down the endogenous Ezh2 levels in PCa by a specific small interfering RNA can increase hDAB2IP expression. The association of Ezh2 complex (including Eed and Suz12) with hDAB2IP gene promoter is also detected in PCa cells but not in normal prostatic epithelial cells. Increased Ezh2 expression in normal prostatic epithelial cells by cDNA transfection facilitates the recruitment of other components of Ezh2 complex to the hDAB2IP promoter region accompanied with the increased levels of methyl histone H3 (H3) and histone deacetylase (HDAC1). Consistently, data from PCa cells transfected with Ezh2 small interfering RNA demonstrated that reduced Ezh2 levels resulted in the dissociation of Ezh2 complex accompanied with decreased levels of both methyl H3 and HDAC1 from hDAB2IP gene promoter. We further unveiled that the methylation status of Lys-27 but not Lys-9 of H3 in hDAB2IP promoter region is consistent with the hDAB2IP levels in both normal prostatic epithelial cells and PCa cells. Together, we conclude that hDAB2IP gene is a target gene of Ezh2 in prostatic epithelium, which provides an underlying mechanism of the down-regulation of hDAB2IP gene in PCa. The human DOC-2/DAB2 interactive protein gene (hDAB2IP) located at chromosome 9q33.1-33.3 is a new member of the Ras GTPase-activating family gene (1Chen H. Pong R.C. Wang Z. Hsieh J.T. Genomics. 2002; 79: 573-581Crossref PubMed Scopus (68) Google Scholar, 2Chen H. Toyooka S. Gazdar A.F. Hsieh J.T. J. Biol. Chem. 2003; 278: 3121-3130Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Our recent data indicate that hDAB2IP protein is a growth inhibitor in prostate cancer (PCa) 1The abbreviations used are: PCa, prostate cancer; H3, histone 3; HDAC, histone deacetylase; siRNA, small interfering RNA; PcG, polycomb group; JNK, c-Jun NH2-terminal kinase; h, human; RT, reverse transcription; qRT-PCR, quantitative RT-PCR; ChIP, chromatin immunoprecipitation; SET, Su-(var) 3–9; E(z), Trithorax. 1The abbreviations used are: PCa, prostate cancer; H3, histone 3; HDAC, histone deacetylase; siRNA, small interfering RNA; PcG, polycomb group; JNK, c-Jun NH2-terminal kinase; h, human; RT, reverse transcription; qRT-PCR, quantitative RT-PCR; ChIP, chromatin immunoprecipitation; SET, Su-(var) 3–9; E(z), Trithorax. cells (3Wang Z. Tseng C.P. Pong R.C. Chen H. McConnell J.D. Navone N. Hsieh J.T. J. Biol. Chem. 2002; 277: 12622-12631Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). In addition, hDAB2IP protein (also named ASK-interacting protein 1 (AIP1)) is involved in the tumor necrosis factor-mediated JNK signaling pathway leading to cell apoptosis (4Zhang H. Zhang R. Luo Y. D'Alessio A. Pober J.S. Min W. J. Biol. Chem. 2004; 279: 44955-44965Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 5Zhang R. He X. Liu W. Lu M. Hsieh J.T. Min W. J. Clin. Investig. 2003; 111: 1933-1943Crossref PubMed Scopus (128) Google Scholar). We have demonstrated that normal prostatic epithelial cells express higher hDAB2IP levels than PCa cells, which is due to epigenetic alternation (i.e. aberrant DNA methylation and histone deacetylation) in the promoter region during carcinogenesis. Similarly, loss of hDAB2IP expression was also detected in breast and lung cancer specimens (6Dote H. Toyooka S. Tsukuda K. Yano M. Ouchida M. Doihara H. Suzuki M. Chen H. Hsieh J.T. Gazdar A.F. Shimizu N. Clin. 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Erdjument-Bromage H. Tempst P. Jones R.S. Zhang Y. Science. 2002; 298: 1039-1043Crossref PubMed Scopus (2780) Google Scholar, 21Czermin B. Melfi R. McCabe D. Seitz V. Imhof A. Pirrotta V. Cell. 2002; 111: 185-196Abstract Full Text Full Text PDF PubMed Scopus (1243) Google Scholar, 22Muller J. Hart C.M. Francis N.J. Vargas M.L. Sengupta A. Wild B. Miller E.L. O'Connor M.B. Kingston R.E. Simon J.A. Cell. 2002; 111: 197-208Abstract Full Text Full Text PDF PubMed Scopus (1226) Google Scholar).Recent data indicate that elevated Ezh2 levels are found in hormone-refractory, metastatic PCa (23Varambally S. Dhanasekaran S.M. Zhou M. Barrette T.R. Kumar-Sinha C. Sanda M.G. Ghosh D. Pienta K.J. Sewalt R.G. Otte A.P. Rubin M.A. Chinnaiyan A.M. Nature. 2002; 419: 624-629Crossref PubMed Scopus (2161) Google Scholar, 24Rhodes D.R. Sanda M.G. Otte A.P. Chinnaiyan A.M. Rubin M.A. J. Natl. Cancer Inst. 2003; 95: 661-668Crossref PubMed Scopus (246) Google Scholar) as well as in poorly differentiated breast carcinomas (25Kleer C.G. Cao Q. Varambally S. Shen R. Ota I. Tomlins S.A. Ghosh D. Sewalt R.G. Otte A.P. Hayes D.F. Sabel M.S. Livant D. Weiss S.J. Rubin M.A. Chinnaiyan A.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11606-11611Crossref PubMed Scopus (1338) Google Scholar, 26Raaphorst F.M. Meijer C.J. Fieret E. Blokzijl T. Mommers E. Buerger H. Packeisen J. Sewalt R.A. Otte A.P. van Diest P.J. Neoplasia. 2003; 5: 481-488Crossref PubMed Google Scholar). However, the underlying mechanism of Ezh2 in these cancer cells is still unknown. In this study, we found an inverse correlation between Ezh2 and hDAB2IP gene expression in either normal prostatic epithelia or PCa cells. Increased Ezh2 expression in normal prostatic epithelial cells could inhibit the hDAB2IP promoter activity and its gene expression. In contrast, knockdown of Ezh2 expression by siRNA in PCa cells resulted in an elevated hDAB2IP gene expression. These data prompted us to investigate the role of Ezh2 in modulating hDAB2IP gene expression, and we demonstrated that Ezh2 complex and histone deacetylase (HDAC) are associated with hDAB2IP promoter regions in PCa cells but not in normal prostatic epithelial cells. The outcome of this study provides an underlying mechanism of the functional role of Ezh2 in metastatic PCa.EXPERIMENTAL PROCEDURESCell Cultures—Three human prostate cancer cell lines (LNCaP, C4-2, and PC3) were maintained in T medium supplemented with 5% fetal bovine serum (27Zhou J. Hsieh J.T. J. Biol. Chem. 2001; 278: 27793-27798Abstract Full Text Full Text PDF Scopus (93) Google Scholar). MDAPCa 2a and MDAPCa 2b cell lines derived from patients with bony metastasis (28Navone N. Olive M. Ozen M. Davis R. Troncoso P. Tu S.M. Johnston D. Pollack A. Pathak S. von Eschenbach A.C. Logothetis C.J. Clin. Cancer Res. 1997; 3: 2493-2500PubMed Google Scholar) were maintained in BRFF-HPC1 medium (Biological Research Faculty and Facility, Inc., Jamsville, MD) supplemented with 20% fetal bovine serum. A VCAP cell line derived from a vertebral metastatic lesion of prostate cancer (29Korenchuk S. Lehr J.E. Mclean L. Lee Y.G. Whitney S. Vessella R. Lin D.L. Pienta K.J. In Vivo (Attiki). 2001; 15: 163-168PubMed Google Scholar) and DU145 were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. Three normal human prostate epithelial cells (PrEC1, PrEC2, and PrEC3) were maintained in chemical-defined medium (PrEGM) purchased from Cambrex. PZ-HPV-7 (an immortalized cell line derived from the peripheral zone of a normal prostate) (30Weijerman P.C. Zhang Y. Shen J. Dubbink H.J. Romijn J.C. Peehl D.M. Schroder F.H. Urology. 1998; 51: 657-662Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 31Weijerman P.C. Konig J.J. Wong S.T. Niesters H.G. Peehl D.M. Cancer Res. 1994; 54: 5579-5583PubMed Google Scholar) and three additional primary prostatic epithelial cells (SWPC1, SWPC2, and SWPC3) (derived from cancer lesions), and SWNPC2 (derived from the adjacent normal tissue) were maintained in PrEGM medium.Transient Transfection and Luciferase Reporter Gene Assay—Cells were plated at a density of 1.8 × 105 cells/well in a 6-well plate. After 24 h, PrEC1 and PZ-HPV-7 cells were co-transfected with Myc-tagged human Ezh2 expression construct (32Sewalt R.G. Lachner M. Vargas M. Hamer K.M. den Blaauwen J.L. Hendrix T. Melcher M. Schweizer D. Jennwen T. Otte A.P. Mol. Cell. Biol. 2002; 22: 5539-5553Crossref PubMed Scopus (83) Google Scholar) and a luciferase reporter vector containing the hDAB2IP promoter-pGL3-P2 (2Chen H. Toyooka S. Gazdar A.F. Hsieh J.T. J. Biol. Chem. 2003; 278: 3121-3130Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) using Lipofectamine Plus transfection reagent (Invitrogen). Luciferase assay was described previously (1Chen H. Pong R.C. Wang Z. Hsieh J.T. Genomics. 2002; 79: 573-581Crossref PubMed Scopus (68) Google Scholar, 2Chen H. Toyooka S. Gazdar A.F. Hsieh J.T. J. Biol. Chem. 2003; 278: 3121-3130Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). All experiments were repeated at least three times in triplicate. The relative luciferase activity was calculated as described previously (2Chen H. Toyooka S. Gazdar A.F. Hsieh J.T. J. Biol. Chem. 2003; 278: 3121-3130Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar).RNA Isolation and Quantitative RT-PCR (qRT-PCR)—Total cellular RNA was isolated using an RNeasy kit (Qiagen, Inc) according to the manufacturer's instructions. To measure hDAB2IP or Ezh2 mRNA levels, 1 μg of total cellular RNA from each cell line was reversibly transcribed into first strand cDNA using iScript™ cDNA synthesis kit (Bio-Rad).The first strand cDNA was further amplified by qRT-PCR using hDAB2IP primer set F-hDAB2IP, 5′-TGGACGATGTGCTCTATGCC-3′; R-hDAB2IP, 5′-GGATGGTGATGGTTTGGTAG-3′ or Ezh2 primer set F-Ezh2, 5′-GCCAGACTGGGAAGAAATCTG-3′; or R-Ezh2, 5′-TGTGCTGGAAAATCCAAGTCA-3′ in a 40-μl reaction mixture containing 20 μl of iQ™ SYBGREEN Supermix® (Bio-Rad). The reactions were carried out in a 96-well plate, and PCR amplification protocol was followed by 95 °C (3 min) and 36 cycles of amplification cycle (95 °C (30 s), 55 °C (30 s), and 72 °C (1 min)) using an iCycler iQ machine (Bio-Rad). The 18S cDNA (F-18S, 5′-GGAATTGACGGAAGGGCACCACC-3′; R-18S, 5′-GTGCAGCCCCGGACATCTAAGG-3′) was used as an internal control. All experiments have been repeated twice in duplicates. hDAB2IP or Ezh2 mRNA level was determined by normalizing with the 18 S cDNA of each sample.The quality control was carried out using both electrophoresis analyses on a 2% NuSieve agarose gel (3:1, FMC Bioproducts) and melting curve analysis performed immediately after the end of amplification using 95 °C (1 min), 55 °C (1 min), and 80 cycles of 0.5 °C increment beginning at 55 °C. We also performed standard curve for hDAB2IP and Ezh2 to ensure the linearity and efficiency of both genes.Western Blot Analysis—Cells were lysed with whole cell lysis buffer (50 mm HEPES, 150 mm NaCl, 1.5 mm MgCl2, 0.5 mm EDTA, 10% glycerol, 1% Triton X-100, 10 mm NaF, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride) and were alternately frozen and thawed three times in -80 °C to rupture the cell membranes. Samples were incubated for 30 min on ice to lyse the nuclei and then centrifuged at 4,000 × g for 5 min to pellet the cell membranes. The protein concentration of each sample was determined by a standard Bradford assay. Equal amounts of protein (20 μg) of each cell line were subjected to Western blot analysis. Antibodies used for probing were hDAB2IP (3Wang Z. Tseng C.P. Pong R.C. Chen H. McConnell J.D. Navone N. Hsieh J.T. J. Biol. Chem. 2002; 277: 12622-12631Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), DOC-2/DAB2 (BD Biosciences), Lamin A/C (Upstate Biotechnology), Ezh2 (Upstate Biotechnology), and actin (Sigma).RNA Interference—Two different 21-nucleotide duplex siRNAs for Ezh2, (5′-AAGAGGTTCAGACGAGCTGAT-3′ (23Varambally S. Dhanasekaran S.M. Zhou M. Barrette T.R. Kumar-Sinha C. Sanda M.G. Ghosh D. Pienta K.J. Sewalt R.G. Otte A.P. Rubin M.A. Chinnaiyan A.M. Nature. 2002; 419: 624-629Crossref PubMed Scopus (2161) Google Scholar), 5′-AAGACTCTGAATGCAGTTGCT-3′ (33Bracken A.P. Pasini D. Capra M. Prosperini E. Colli E. Helin K. EMBO. J. 2003; 22: 5323-5335Crossref PubMed Scopus (958) Google Scholar), or control siRNA for Lamin A/C (5′-CTGGACTTCCAGAAGAACA-3′) were synthesized by Qiagen. Twenty-four hours after plating, the cells were transfected either with both Ezh2, siRNA duplexes together (100 nmol each) or with control siRNA (200 nmol) using RNAiFect transfection reagent (Qiagen) according to the manufacturer's instructions. At various time points after transfection, the cells were harvested and subjected to Western blot analysis.Chromatin Immunoprecipitation (ChIP) Assay—This assay for hDAB2IP gene promoter was performed as described previously (2Chen H. Toyooka S. Gazdar A.F. Hsieh J.T. J. Biol. Chem. 2003; 278: 3121-3130Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). For DOC-2/DAB2 gene promoter, the same PCR condition was applied with two primer sets as follows: first PCR (5′-ACATCACTCGCAGTGGC-3′ and 5′-GGCGAGAGATATGGTTC-3′) and second PCR (5′-CTCGCGGAGCTCAGGGGAG-3′ and 5′-GGTAACTCCCCCTCAACGTG-3′). Briefly, precleared chromatin from 2 × 106 cells was used for each ChIP assay, and 5 μg of each antibody was used in this assay including Ezh2, Suz12, trimethyl H3 (Lys-27), trimethyl H3 (Lys-9), and dimethyl H3 (Lys-9) purchased from Abcam Inc.; and Eed, dimethyl H3 (Lys-27), monomethyl H3 (Lys-27), acetyl H3, and HDAC1 purchased from Upstate Biotechnology. The total input or immunoprecipitated DNA was determined by qPCR at least twice with the equation ΔCt (threshold cycle) of each sample = mean of Ct(antibody) - mean of Ct(input).RESULTSProfiling hDAB2IP and Ezh2 Expression in Various Prostatic Epithelia—To evaluate the expression profile of hDAB2IP gene and Ezh2 in the prostate cell lines, we performed qRT-PCR to document the steady-state levels of hDAB2IP (Fig. 1A) and Ezh2 (Fig. 1B) mRNA in cells derived from normal prostatic epithelium (PrEC1, PrEC2, PrEC3, PZ-HPV-7, and SWNPC2), primary PCa cells (SWPC1, SWPC2, and SWPC3), or metastatic PCa cell lines (LNCaP, C4-2, PC3, MADPC2a, MADPC2b, VCAP, and DU145). We observed a trend of decreasing hDAB2IP mRNA from normal cells to malignant cells; however, an opposite trend of Ezh2 mRNA was seen from normal cells to malignant cells. Western blot data (Fig. 1C) also confirmed the qRT-PCR results. Taken together, an inverse correlation between Ezh2 and hDAB2IP gene expression prompted us to investigate the possibility that hDAB2IP gene expression could be modulated by the Ezh2 complex particularly, since loss of hDAB2IP expression was frequently observed in metastatic PCa cells.The Inhibitory Effect of Ezh2 on hDAB2IP Gene Expression—To test whether Ezh2 is a negative regulator to modulate hDAB2IP gene expression in PCa cells, Western blot analysis was performed using whole cell extract prepared from PCa cells transfected with either a mixture of two different Ezh2 siRNAs (23Varambally S. Dhanasekaran S.M. Zhou M. Barrette T.R. Kumar-Sinha C. Sanda M.G. Ghosh D. Pienta K.J. Sewalt R.G. Otte A.P. Rubin M.A. Chinnaiyan A.M. Nature. 2002; 419: 624-629Crossref PubMed Scopus (2161) Google Scholar, 33Bracken A.P. Pasini D. Capra M. Prosperini E. Colli E. Helin K. EMBO. J. 2003; 22: 5323-5335Crossref PubMed Scopus (958) Google Scholar) or lamin A/C siRNA. The significantly decreased Ezh2 levels in PC3 (Fig. 2A) and in DU145 (Fig. 2C) were observed within 48–72 h after transfection, and the reduced Ezh2 protein became more prominent 96 h after transfection. Under this condition, the elevated levels of hDAB2IP protein were detected in both PCa cells. In contrast, Lamin A/C siRNA duplex did not alter Ezh2 or hDAB2IP levels in either cell type (Fig. 2, B and D). To demonstrate the specific effect of Ezh2 on hDAB2IP gene expression, the DOC-2/DAB2 gene (27Zhou J. Hsieh J.T. J. Biol. Chem. 2001; 278: 27793-27798Abstract Full Text Full Text PDF Scopus (93) Google Scholar), which is not regulated by Ezh2 in prostate (Fig. 2), was used as a negative control in this study.Fig. 2The effect of Ezh2 on hDAB2IP protein expression in PCa cells. The cell lysate from PC3 cells (A and B) or DU145 cells (C and D) was collected at the indicated time points after transfecting with either Ezh2 siRNA (A or C) or control Lamin A/C siRNA (B or D). The Western blot analyses were performed using antibodies against Ezh2, hDAB2IP, DOC-2/DAB2, and LaminA/C. Actin was used as an internal loading control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine the inhibitory effect of Ezh2 on hDAB2IP gene expression via the transcriptional or post-transcriptional regulation, we examined the hDAB2IP promoter activity in several normal prostatic epithelial cells by transfecting with both pGL3-P2 and Ezh2 expression vectors. We observed that an increased Ezh2 protein expression (Fig. 3A) in normal prostatic epithelial cells could inhibit the hDAB2IP promoter activity (Fig. 3, B–E) in a dose- and time-dependent manner. Using qRT-PCR, we also observed a similar inhibitory effect of Ezh2 on hDAB2IP mRNA levels in these and two other normal epithelial cells such as PrEC2 and PrEC3 (data not shown). We also generated a stable Ezh2-expressing PZ-HPV-7 transfectant and found reduction of the endogenous hDAB2IP protein levels in these cells (Fig. 3F). These data clearly indicate that the suppression of hDAB2IP gene promoter activity is mediated by Ezh2 protein.Fig. 3The inhibitory effect of EZH2 on hDAB2IP promoter activity and protein expression in normal prostatic epithelia. PZ-HPV-7 (A, B, D, and F) and PrEC1 (C and E) cells were transfected with both Myc-tagged Ezh2 expression vector and pGL3-P2 reporter gene construct. In the time course experiment, Ezh2 expression vector (0.2 μg) and pGL3-P2 reporter gene construct (0.5 μg) were used. A and F, the endogenous hDAB2IP protein levels in PZ-HPV-7 cells with transient or stable expression of Ezh2 protein. RLA, relative luciferase activity. B and C, the dose-dependent inhibition of hDAB2IP promoter activity by Ezh2. D and E, the time-dependent inhibition of hDAB2IP promoter activity by Ezh2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Association between Ezh2 Complex and HDAC1 in hDAB2IP Promoter Region in PCa Cells—To demonstrate the direct interaction of Ezh2 complex with hDAB2IP promoter region, ChIP assay was performed. In Fig. 4A, robust binding of Ezh2 as well as two other components, Eed and Suz12, to the hDAB2IP promoter was seen in PC3 cells, the moderate binding of Ezh2 complex was detected in DU145 cells, and the lowest binding of Ezh2 complex was detected in PZ-HPV-7 cells. Noticeably, the amount of Ezh2 complex associated with hDAB2IP promoter inversely correlated with hDAB2IP mRNA and protein expression patterns in these cells (Fig. 1). On the other hand, we did not observe an inverse correlation between Ezh2 and DOC-2/DAB2 levels in these cells (Fig. 1C) or a consistent association pattern between Ezh2 complex and DOC-2/DAB2 gene promoter (Fig. 4).Fig. 4The status of PcG complex, HDAC1, histone methylation, and histone acetylation on the hDAB2IP promoter region in prostatic epithelia. A ChIP assay was performed using DNA-protein complex isolated from PZ-HPV-7 (lane 1), PC3 (lane 2), and DU145 (lane 3) cells immunoprecipitated with various antibodies. The amount of endogenous hDAB2IP or DOC-2/DAB2 gene promoter was determined by qPCR using the specific primer set described previously (2Chen H. Toyooka S. Gazdar A.F. Hsieh J.T. J. Biol. Chem. 2003; 278: 3121-3130Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) and visualized with gel electrophoresis. The number under each lane representing the fold of enrichment was calculated as 1/2ΔCt (sample) - ΔCt (PZ-HPV-7).View Large Image Figure ViewerDownload Hi-res image Download (PPT)It is known that transcriptional repression mediated by human polycomb group (PcG) protein involves histone deacetylation (34van der Vlag J. Otte A.P. Nat. Genet. 1999; 23: 474-478Crossref PubMed Scopus (444) Google Scholar). We therefore determined the association of HDAC1 with the hDAB2IP promoter region. As shown in Fig. 4B, the higher levels of HDAC1 associated with hDAB2IP promoter were detected in PC3 and DU145 cells than in PZ-HPV-7 cells. In contrast, the lower levels of acetyl H3 associated with hDAB2IP promoter were seen in these two PCa cells than in PZ-HPV-7 cells (Fig. 4B). These data indicate that this repression complex associated with hDAB2IP promoter contains both Ezh2 complex and HDAC1.Furthermore, by increasing Ezh2 gene expression in PZ-HPV-7 cells, we observed that the presence of Ezh2 protein could recruit not only other components of PcG proteins such as Eed and Suz12 (Fig. 5A) but also HDAC1 to the hDAB2IP promoter region in these cells. Meanwhile, the decreased acetyl H3 level became more apparent in these cells (Fig. 5B). Taken together, Ezh2 plays a key role in recruiting other transcription repressors to the hDAB2IP promoter region in prostatic epithelia. On the other hand, we were able to show the dissociation of both PcG complex and HDAC1 from hDAB2IP gene promoter in PC3 (Fig. 6) and DU145 (Fig. 6) when their endogenous Ezh2 levels were knocked down by Ezh2 siRNA (Fig. 2). Under the same condition, the acetyl H3 levels were elevated. For DOC-2/DAB2 gene, no consistent pattern of these complexes could be observed in the promoter region (Figs. 4 and 5). Thus, these data further support the notion that hDAB2IP is an Ezh2 target gene.Fig. 5The impact of Ezh2 on the status of H3 methylation and acetylation of the hDAB2IP promoter region in PZ-HPV-7 cells after recruiting PcG complex proteins and HDAC1. A ChIP assay was performed using DNA-protein complex isolated from PZ-HPV-7 cells transfected with different amounts of Ezh2 expression vector and immunoprecipitated with various antibodies. The amount of endogenous hDAB2IP gene promoter was determined by qPCR using the specific primer set described previously (2Chen H. Toyooka S. Gazdar A.F. Hsieh J.T. J. Biol. Chem. 2003; 278: 3121-3130Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) and visualized with gel electrophoresis. The number under each lane representing the fold of enrichment was calculated as 1/2ΔCt (Ezh2) - ΔCt (control).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6The status of H3 methylation and acetylation of the hDAB2IP promoter region in PCa cells transfected with Ezh2 siRNA. A ChIP assay was performed using DNA-protein complex isolated from PC3 or DU145 cells transfected with Ezh2 siRNA for 96 h and immunoprecipitated with various antibodies. The amount of endogenous hDAB2IP gene promoter was determined by qPCR using the specific primer set described previously (2Chen H. Toyooka S. Gazdar A.F. Hsieh J.T. J. Biol. Chem. 2003; 278: 3121-3130Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) and visualized with gel electrophoresis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Methylation Status of H3-Lys-9 or H3-Lys-27 in hDAB2IP Promoter Region Modulated by Ezh2—Although the human Eed-Ezh2 complex and its Drosophila ESC-E(Z) counterpart have been shown to be HMKTase (9Cao R. Zhang Y. Curr. Opin. Genet. Dev. 2004; 14: 155-164Crossref PubMed Scopus (712) Google Scholar), its substrate specificity still remains unclear. For example, Cao et al. (11Cao R. Wang L. Wang H. Xia L. Erdjument-Bromage H. Tempst P. Jones R.S. Zhang Y. Science. 2002; 298: 1039-1043Crossref PubMed Scopus (2780) Google Scholar) and Muller et al. (22Muller J. Hart C.M. Francis N.J. Vargas M.L. Sengupta A. Wild B. Miller E.L. O'Connor M.B. Kingston R.E. Simon J.A. Cell. 2002; 111: 197-208Abstract Full Text Full Text PDF PubMed Scopus (1226) Google Scholar) showed that Lys-27 in H3 is the only amino acid methylated by Ezh2; however, other groups (10Kuzmichev A. Nishioka K. Erdjument-Bromage H. Tempst P. Reinberg D. Genes Dev. 2002; 16: 2893-2905Crossref PubMed Scopus (1246) Google Scholar, 21Czermin B. Melfi R. McCabe D. Seitz V. Imhof A. Pirrotta V. Cell. 2002; 111: 185-196Ab
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