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Down-regulation of Human DAB2IP Gene Expression Mediated by Polycomb Ezh2 Complex and Histone Deacetylase in Prostate Cancer *

Down-regulation of Human DAB2IP Gene Expression Mediated by Polycomb Ezh2 Complex and Histone... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 23, Issue of June 10, pp. 22437–22444, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Down-regulation of Human DAB2IP Gene Expression Mediated by Polycomb Ezh2 Complex and Histone Deacetylase in Prostate Cancer* Received for publication, February 7, 2005, and in revised form, March 29, 2005 Published, JBC Papers in Press, April 6, 2005, DOI 10.1074/jbc.M501379200 Hong Chen, Szu-wei Tu, and Jer-Tsong Hsieh‡ From the Department of Urology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9110 (also named ASK-interacting protein 1 (AIP1)) is involved in Human DAB2IP (hDAB2IP), a novel GTPase-activat- ing protein modulating the Ras-mediated signaling and the tumor necrosis factor-mediated JNK signaling pathway tumor necrosis factor-mediated apoptosis, is a potent leading to cell apoptosis (4, 5). We have demonstrated that growth inhibitor in human prostate cancer (PCa). Loss normal prostatic epithelial cells express higher hDAB2IP levels of hDAB2IP expression in PCa is due to altered epige- than PCa cells, which is due to epigenetic alternation (i.e. netic regulation (i.e. DNA methylation and histone mod- aberrant DNA methylation and histone deacetylation) in the ification) of its promoter region. The elevated polycomb promoter region during carcinogenesis. Similarly, loss of Ezh2, a histone methyltransferase, has been associated hDAB2IP expression was also detected in breast and lung with PCa progression. In this study, we have demon- cancer specimens (6, 7) frequently associated with the pro- strated that an increased Ezh2 expression in normal moter hypermethylation. prostatic epithelial cells can suppress hDAB2IP gene Human enhancer of Zeste homolog (Ezh2) protein belongs to expression. In contrast, knocking down the endogenous Polycomb repressive complex 2/3 (8), which also includes Eed, Ezh2 levels in PCa by a specific small interfering RNA Suz12, and the histone-binding protein RbAp48/46 (9–13). The can increase hDAB2IP expression. The association of Ezh2 complex appears to be a transcription repressor that has Ezh2 complex (including Eed and Suz12) with hDAB2IP been shown to be involved in cellular memory system, X-inac- gene promoter is also detected in PCa cells but not in tivation, germline development, stem cell pluripotency, and normal prostatic epithelial cells. Increased Ezh2 expres- cancer metastasis (14–20). This complex exhibits an intrinsic sion in normal prostatic epithelial cells by cDNA trans- histone lysine methyltransferase activity on histone H3 Lys-27 fection facilitates the recruitment of other components and 9 or histone H1 Lys-26 mediated by the SET domain of of Ezh2 complex to the hDAB2IP promoter region ac- Ezh2 (8, 10, 11, 21, 22). companied with the increased levels of methyl histone Recent data indicate that elevated Ezh2 levels are found in H3 (H3) and histone deacetylase (HDAC1). Consistently, data from PCa cells transfected with Ezh2 small inter- hormone-refractory, metastatic PCa (23, 24) as well as in fering RNA demonstrated that reduced Ezh2 levels re- poorly differentiated breast carcinomas (25, 26). However, the sulted in the dissociation of Ezh2 complex accompanied underlying mechanism of Ezh2 in these cancer cells is still with decreased levels of both methyl H3 and HDAC1 unknown. In this study, we found an inverse correlation be- from hDAB2IP gene promoter. We further unveiled that tween Ezh2 and hDAB2IP gene expression in either normal the methylation status of Lys-27 but not Lys-9 of H3 in prostatic epithelia or PCa cells. Increased Ezh2 expression in hDAB2IP promoter region is consistent with the normal prostatic epithelial cells could inhibit the hDAB2IP hDAB2IP levels in both normal prostatic epithelial cells promoter activity and its gene expression. In contrast, knock- and PCa cells. Together, we conclude that hDAB2IP down of Ezh2 expression by siRNA in PCa cells resulted in an gene is a target gene of Ezh2 in prostatic epithelium, elevated hDAB2IP gene expression. These data prompted us to which provides an underlying mechanism of the down- investigate the role of Ezh2 in modulating hDAB2IP gene ex- regulation of hDAB2IP gene in PCa. pression, 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 human DOC-2/DAB2 interactive protein gene The outcome of this study provides an underlying mechanism (hDAB2IP) located at chromosome 9q33.1-33.3 is a new mem- of the functional role of Ezh2 in metastatic PCa. ber of the Ras GTPase-activating family gene (1, 2). Our recent data indicate that hDAB2IP protein is a growth inhibitor in EXPERIMENTAL PROCEDURES prostate cancer (PCa) cells (3). In addition, hDAB2IP protein Cell Cultures—Three human prostate cancer cell lines (LNCaP, C4-2, and PC3) were maintained in T medium supplemented with 5% fetal bovine serum (27). MDAPCa 2a and MDAPCa 2b cell lines derived from patients with bony metastasis (28) were maintained in BRFF- * This work was supported by Grant W81XWH-04-1-0222 from the HPC1 medium (Biological Research Faculty and Facility, Inc., Jams- Department of Defense. The costs of publication of this article were ville, MD) supplemented with 20% fetal bovine serum. A VCAP cell line defrayed in part by the payment of page charges. This article must derived from a vertebral metastatic lesion of prostate cancer (29) and therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. DU145 were maintained in RPMI 1640 medium supplemented with ‡ To whom correspondence should be addressed: University of Texas 10% fetal bovine serum. Three normal human prostate epithelial cells Southwestern Medical Center, Dept. of Urology, 5323 Harry Hines (PrEC1, PrEC2, and PrEC3) were maintained in chemical-defined me- Blvd., Dallas, TX 75390-9110. Tel.: 214-648-3988; Fax: 214-648-8786; dium (PrEGM) purchased from Cambrex. PZ-HPV-7 (an immortalized E-mail: [email protected]. The abbreviations used are: PCa, prostate cancer; H3, histone 3; HDAC, histone deacetylase; siRNA, small interfering RNA; PcG, poly- transcription; qRT-PCR, quantitative RT-PCR; ChIP, chromatin immu- comb group; JNK, c-Jun NH -terminal kinase; h, human; RT, reverse noprecipitation; SET, Su-(var) 3–9; E(z), Trithorax. This paper is available on line at http://www.jbc.org 22437 This is an Open Access article under the CC BY license. 22438 Ezh2 Regulation of hDAB2IP Gene Expression cell line derived from the peripheral zone of a normal prostate) (30, 31) RESULTS and three additional primary prostatic epithelial cells (SWPC1, Profiling hDAB2IP and Ezh2 Expression in Various Pros- SWPC2, and SWPC3) (derived from cancer lesions), and SWNPC2 tatic Epithelia—To evaluate the expression profile of hDAB2IP (derived from the adjacent normal tissue) were maintained in PrEGM gene and Ezh2 in the prostate cell lines, we performed qRT- medium. PCR to document the steady-state levels of hDAB2IP (Fig. 1A) Transient Transfection and Luciferase Reporter Gene Assay—Cells and Ezh2 (Fig. 1B) mRNA in cells derived from normal pros- were plated at a density of 1.8  10 cells/well in a 6-well plate. After 24 h, PrEC1 and PZ-HPV-7 cells were co-transfected with Myc-tagged human tatic epithelium (PrEC1, PrEC2, PrEC3, PZ-HPV-7, and Ezh2 expression construct (32) and a luciferase reporter vector containing SWNPC2), primary PCa cells (SWPC1, SWPC2, and SWPC3), the hDAB2IP promoter-pGL3-P2 (2) using Lipofectamine Plus transfec- or metastatic PCa cell lines (LNCaP, C4-2, PC3, MADPC2a, tion reagent (Invitrogen). Luciferase assay was described previously (1, 2). MADPC2b, VCAP, and DU145). We observed a trend of de- All experiments were repeated at least three times in triplicate. The creasing hDAB2IP mRNA from normal cells to malignant cells; relative luciferase activity was calculated as described previously (2). however, an opposite trend of Ezh2 mRNA was seen from RNA Isolation and Quantitative RT-PCR (qRT-PCR)—Total cellular normal cells to malignant cells. Western blot data (Fig. 1C) also RNA was isolated using an RNeasy kit (Qiagen, Inc) according to the confirmed the qRT-PCR results. Taken together, an inverse manufacturer’s instructions. To measure hDAB2IP or Ezh2 mRNA correlation between Ezh2 and hDAB2IP gene expression levels, 1 g of total cellular RNA from each cell line was reversibly TM transcribed into first strand cDNA using iScript cDNA synthesis kit prompted us to investigate the possibility that hDAB2IP gene (Bio-Rad). expression could be modulated by the Ezh2 complex particu- The first strand cDNA was further amplified by qRT-PCR using larly, since loss of hDAB2IP expression was frequently ob- hDAB2IP primer set F-hDAB2IP, 5-TGGACGATGTGCTCTATGCC-3; served in metastatic PCa cells. R-hDAB2IP, 5-GGATGGTGATGGTTTGGTAG-3 or Ezh2 primer set The Inhibitory Effect of Ezh2 on hDAB2IP Gene Expres- F-Ezh2, 5-GCCAGACTGGGAAGAAATCTG-3; or R-Ezh2, 5-TGT- sion—To test whether Ezh2 is a negative regulator to modulate GCTGGAAAATCCAAGTCA-3 in a 40-l reaction mixture containing TM hDAB2IP gene expression in PCa cells, Western blot analysis 20 lofiQ SYBGREEN Supermix® (Bio-Rad). The reactions were was performed using whole cell extract prepared from PCa cells 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 transfected with either a mixture of two different Ezh2 siRNAs s), 55 °C (30 s), and 72 °C (1 min)) using an iCycler iQ machine (Bio- (23, 33) or lamin A/C siRNA. The significantly decreased Ezh2 Rad). The 18S cDNA (F-18S, 5-GGAATTGACGGAAGGGCACCACC- levels in PC3 (Fig. 2A) and in DU145 (Fig. 2C) were observed 3; R-18S, 5-GTGCAGCCCCGGACATCTAAGG-3) was used as an in- within 48–72 h after transfection, and the reduced Ezh2 pro- ternal control. All experiments have been repeated twice in duplicates. tein became more prominent 96 h after transfection. Under this hDAB2IP or Ezh2 mRNA level was determined by normalizing with the condition, the elevated levels of hDAB2IP protein were de- 18 S cDNA of each sample. tected in both PCa cells. In contrast, Lamin A/C siRNA duplex The quality control was carried out using both electrophoresis anal- did not alter Ezh2 or hDAB2IP levels in either cell type (Fig. 2, yses on a 2% NuSieve agarose gel (3:1, FMC Bioproducts) and melting curve analysis performed immediately after the end of amplification B and D). To demonstrate the specific effect of Ezh2 on using 95 °C (1 min), 55 °C (1 min), and 80 cycles of 0.5 °C increment hDAB2IP gene expression, the DOC-2/DAB2 gene (27), which beginning at 55 °C. We also performed standard curve for hDAB2IP and is not regulated by Ezh2 in prostate (Fig. 2), was used as a Ezh2 to ensure the linearity and efficiency of both genes. negative control in this study. Western Blot Analysis—Cells were lysed with whole cell lysis buffer To determine the inhibitory effect of Ezh2 on hDAB2IP gene (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl , 0.5 mM EDTA, 10% expression via the transcriptional or post-transcriptional reg- glycerol, 1% Triton X-100, 10 mM NaF, 1 mM dithiothreitol, 1 mM ulation, we examined the hDAB2IP promoter activity in sev- phenylmethylsulfonyl fluoride) and were alternately frozen and thawed eral normal prostatic epithelial cells by transfecting with both three times in 80 °C to rupture the cell membranes. Samples were pGL3-P2 and Ezh2 expression vectors. We observed that an 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 concen- increased Ezh2 protein expression (Fig. 3A) in normal prostatic tration of each sample was determined by a standard Bradford assay. epithelial cells could inhibit the hDAB2IP promoter activity Equal amounts of protein (20 g) of each cell line were subjected to (Fig. 3, B–E) in a dose- and time-dependent manner. Using Western blot analysis. Antibodies used for probing were hDAB2IP (3), qRT-PCR, we also observed a similar inhibitory effect of Ezh2 DOC-2/DAB2 (BD Biosciences), Lamin A/C (Upstate Biotechnology), on hDAB2IP mRNA levels in these and two other normal Ezh2 (Upstate Biotechnology), and actin (Sigma). epithelial cells such as PrEC2 and PrEC3 (data not shown). We RNA Interference—Two different 21-nucleotide duplex siRNAs for also generated a stable Ezh2-expressing PZ-HPV-7 transfec- Ezh2, (5-AAGAGGTTCAGACGAGCTGAT-3 (23), 5-AAGACTCT- tant and found reduction of the endogenous hDAB2IP protein GAATGCAGTTGCT-3 (33), or control siRNA for Lamin A/C (5-CTG- GACTTCCAGAAGAACA-3) were synthesized by Qiagen. Twenty-four levels in these cells (Fig. 3F). These data clearly indicate that hours after plating, the cells were transfected either with both Ezh2, the suppression of hDAB2IP gene promoter activity is medi- siRNA duplexes together (100 nmol each) or with control siRNA (200 ated by Ezh2 protein. nmol) using RNAiFect transfection reagent (Qiagen) according to the The Association between Ezh2 Complex and HDAC1 in manufacturer’s instructions. At various time points after transfection, hDAB2IP Promoter Region in PCa Cells—To demonstrate the the cells were harvested and subjected to Western blot analysis. direct interaction of Ezh2 complex with hDAB2IP promoter Chromatin Immunoprecipitation (ChIP) Assay—This assay for region, ChIP assay was performed. In Fig. 4A, robust binding of hDAB2IP gene promoter was performed as described previously (2). For Ezh2 as well as two other components, Eed and Suz12, to the DOC-2/DAB2 gene promoter, the same PCR condition was applied with two primer sets as follows: first PCR (5-ACATCACTCGCAGTGGC-3 hDAB2IP promoter was seen in PC3 cells, the moderate bind- and 5-GGCGAGAGATATGGTTC-3) and second PCR (5-CTCG- ing of Ezh2 complex was detected in DU145 cells, and the CGGAGCTCAGGGGAG-3 and 5-GGTAACTCCCCCTCAACGTG-3). lowest binding of Ezh2 complex was detected in PZ-HPV-7 Briefly, precleared chromatin from 2  10 cells was used for each ChIP cells. Noticeably, the amount of Ezh2 complex associated with assay, and 5 g of each antibody was used in this assay including Ezh2, hDAB2IP promoter inversely correlated with hDAB2IP mRNA Suz12, trimethyl H3 (Lys-27), trimethyl H3 (Lys-9), and dimethyl H3 and protein expression patterns in these cells (Fig. 1). On the (Lys-9) purchased from Abcam Inc.; and Eed, dimethyl H3 (Lys-27), other hand, we did not observe an inverse correlation between monomethyl H3 (Lys-27), acetyl H3, and HDAC1 purchased from Up- Ezh2 and DOC-2/DAB2 levels in these cells (Fig. 1C)ora state Biotechnology. The total input or immunoprecipitated DNA was consistent association pattern between Ezh2 complex and determined by qPCR at least twice with the equation Ct (threshold cycle) of each sample  mean of Ct  mean of Ct . DOC-2/DAB2 gene promoter (Fig. 4). (antibody) (input) Ezh2 Regulation of hDAB2IP Gene Expression 22439 FIG.1. Profiling hDAB2IP and Ezh2 expression in prostatic epithelia. The steady-state levels of hDAB2IP (A) and Ezh2 (B) mRNA from various prostatic epithelia cells were determined by qRT- PCR. After normalizing with 18 S rRNA, the copy number of each sample (mean S.D.) performed in duplicates from two different experiments was calculated based on the standard curve. The steady- state levels of hDAB2IP, Ezh2, and DOC- 2/DAB2 protein (C) were determined by Western blot analysis using the whole cell lysates extracted from four different pros- tatic epithelial cells. It is known that transcriptional repression mediated by hu- same condition, the acetyl H3 levels were elevated. For DOC- man polycomb group (PcG) protein involves histone deacetyla- 2/DAB2 gene, no consistent pattern of these complexes could be tion (34). We therefore determined the association of HDAC1 observed in the promoter region (Figs. 4 and 5). Thus, these with the hDAB2IP promoter region. As shown in Fig. 4B, the data further support the notion that hDAB2IP is an Ezh2 higher levels of HDAC1 associated with hDAB2IP promoter target gene. were detected in PC3 and DU145 cells than in PZ-HPV-7 cells. The Methylation Status of H3-Lys-9 or H3-Lys-27 in In contrast, the lower levels of acetyl H3 associated with hDAB2IP Promoter Region Modulated by Ezh2—Although the hDAB2IP promoter were seen in these two PCa cells than in human Eed-Ezh2 complex and its Drosophila ESC-E(Z) coun- PZ-HPV-7 cells (Fig. 4B). These data indicate that this repres- terpart have been shown to be HMKTase (9), its substrate sion complex associated with hDAB2IP promoter contains both specificity still remains unclear. For example, Cao et al. (11) Ezh2 complex and HDAC1. and Muller et al. (22) showed that Lys-27 in H3 is the only Furthermore, by increasing Ezh2 gene expression in PZ- amino acid methylated by Ezh2; however, other groups (10, 21) HPV-7 cells, we observed that the presence of Ezh2 protein demonstrated that Ezh2 can also methylate H3-Lys-9. More- could recruit not only other components of PcG proteins such as over, the lysine in H3 exists in mono-, di-, and tri-methyl status Eed and Suz12 (Fig. 5A) but also HDAC1 to the hDAB2IP (35–37), and their impact on gene regulation is still unclear. promoter region in these cells. Meanwhile, the decreased acetyl For hDAB2IP promoter region, di- and tri-methyl H3-Lys-27 H3 level became more apparent in these cells (Fig. 5B). Taken were highly detectable in PC3 and DU145 cells but not in together, Ezh2 plays a key role in recruiting other transcription PZ-HPV-7 (Fig. 4C), which is consistent with the presence of repressors to the hDAB2IP promoter region in prostatic epithe- Ezh2 complex in these three cell lines (Fig. 4A). We also noticed lia. On the other hand, we were able to show the dissociation of that the levels of mono-methyl H3-Lys-27 did not vary among both PcG complex and HDAC1 from hDAB2IP gene promoter these three cell lines and did not change in the presence of in PC3 (Fig. 6) and DU145 (Fig. 6) when their endogenous Ezh2 elevated Ezh2 (Figs. 4C and 5C), suggesting that mono-methyl levels were knocked down by Ezh2 siRNA (Fig. 2). Under the H3-Lys-27 may reflect a “constant basal” methylation status of 22440 Ezh2 Regulation of hDAB2IP Gene Expression FIG.2. The 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. DISCUSSION Lys-27. Caretti et al. (38) have reported a similar observation in the study of MHCIIb and MCK gene regulation during muscle PCa is a leading cause of cancer-related death in males and differentiation. By increasing Ezh2 protein expression in PZ- is second only to lung cancer. Although effective surgical and HPV-7 cells, we demonstrated that the levels of both di- and radiation treatments are available for clinically localized PCa, tri-methyl but not mono-methyl H3-Lys-27 elevated dramati- metastatic PCa remains essentially incurable. To understand cally in a dose-dependent manner (Fig. 5C). In the presence of the biology of metastatic PCa, data from a cDNA microarray Ezh2 siRNA in either PC3 or DU145 cells, the decreased levels analysis indicate that elevated Ezh2 with histone lysine meth- of both di- and tri-methyl H3-Lys-27 could be observed (Fig. 6). yltransferase activity is often associated with metastatic PCa These data suggest that the effect of Ezh2 is to convert H3- but not with benign tissue and organ-confined tumor (23). Lys-27 into tri-methyl status in the hDAB2IP gene promoter Noticeably, clinically localized prostate cancer expressing region. higher levels of Ezh2 often has a poorer prognosis (24), sug- On the other hand, for the methylation status of H3-Lys-9, gesting that Ezh2 is not only a potential biomarker for predict- we failed to detect any good correlation among these three cell ing the relative risk of PCa progression but also a key contrib- lines tested. For example, tri-methyl H3-Lys-9 was highly de- utor for the disease progression. Here we have demonstrated tectable in these cell lines, and di-methyl Lys-9 status was not that Ezh2 mRNA and protein are more consistently elevated in consistent with hDAB2IP levels among these three cell lines metastatic PCa cells than in normal cells. The pattern of (Fig. 4D). Moreover, increased Ezh2 expression in PZ-HPV-7 hDAB2IP gene expression exhibited an inverse correlation cells was associated with a dramatic elevation of di-methyl but with that of Ezh2, which prompted us to study the role of Ezh2 not tri-methyl H3-Lys-9 (Fig. 5D), suggesting that the methy- in regulating hDAB2IP gene expression since silencing lation status of H3-Lys-9 may not be involved in modulating hDAB2IP gene expression is mainly due to epigenetic regula- hDAB2IP gene promoter activity by Ezh2. Together, we believe tion (2, 6, 7). Increased hDAB2IP levels were detected in PCa that hDAB2IP gene silencing in PCa can be mediated by the cells transfected with Ezh2 siRNAs (Fig. 2). In contrast, the elevated levels of Ezh2, which results in the hypermethylation reduced hDAB2IP gene expression was observed in normal of H3-Lys-27 and the recruitment of HDAC. This study pro- prostatic epithelial cells transfected with Ezh2 expression vec- vides an underlying mechanism of the functional role of Ezh2 tor (Fig. 3). These results provide an important mechanism of in metastatic PCa. the elevated Ezh2 expression during PCa progression. Ezh2 Regulation of hDAB2IP Gene Expression 22441 FIG.3. The 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 g) and pGL3-P2 reporter gene construct (0.5 g) 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. In Drosophila, ESC-E(Z) proteins recognize and exert their tantly, Ezh2 plays an active role in recruiting other PcG com- activity through specific DNA sequences known as polycomb ponents such as Eed and Suz12 to form a functional complex response elements (39–41). In mammals, such elements have along with HDAC1 to the promoter region of hDAB2IP gene not been identified yet. It remains unclear how mammalian (Figs. 5 and 6). Based on these results, one could predict that PcG complexes are recruited to chromatin to regulate expres- the presence of elevated Ezh2 levels in PCa signifies the down- sion of a specific target gene. Because most of the PcG proteins regulation of tumor suppressor genes via the cooperative effect do not contain a DNA-binding domain, target genes cannot be of histone methylation and deacetylation. This study provides identified by searching the consensus-binding site in the ge- further evidence to support the critical role of epigenetic gene nome (42). Thus, the ChIP assay can provide direct evidence for regulation in PCa progression. the possible interaction between the Ezh2 complex and pro- The term “epigenetic” refers to the heritable changes in gene moter region of any known gene (43). Our data indicate that the expression that are caused by mechanisms other than the levels of Ezh2 associated with the hDAB2IP promoter region alteration in the nucleotide sequence. This concept generates inversely correlate with the steady-state levels of hDAB2IP tremendous new knowledge in understanding gene expression mRNA and protein in all three cell lines tested. Most impor- in mammalian cells. In addition to DNA methylation, increas- 22442 Ezh2 Regulation of hDAB2IP Gene Expression FIG.4. The status of PcG complex, HDAC1, histone methylation, and hi- stone acetylation on the hDAB2IP promoter region in prostatic epithe- lia. 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 (2) and visualized with gel elec- trophoresis. The number under each lane representing the fold of enrichment was Ct (sample) Ct (PZ-HPV-7) calculated as 1/2  . ing studies have focused on the impact of covalent modifica- tion, has emerged as an important player in regulating gene tions of the histone core in the nucleosome structure on gene expression and chromatin function. Lysine methylation usually regulation. One of the covalent modifications, lysine methyla- occurs on the Lys-4, Lys-9, Lys-27, Lys-36, and Lys-79 of H3, Ezh2 Regulation of hDAB2IP Gene Expression 22443 FIG.5. The impact of Ezh2 on the status of H3 methylation and acetyla- tion 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 immuno- precipitated with various antibodies. The amount of endogenous hDAB2IP gene promoter was determined by qPCR using the specific primer set described previ- ously (2) and visualized with gel electro- phoresis. The number under each lane representing the fold of enrichment was Ct (Ezh2) Ct (control) calculated as 1/2  . the Lys-20 of H4, and the Lys-26 of H1 (35, 36, 44). Biochemical protein contains a SET domain, and several groups (10, 11, 21) and genetic studies indicate that methylation of different lysine have shown Ezh2 with histone lysine methyltransferase activ- residues, with the exception of H3-Lys-79, is catalyzed by dif- ity, its substrate specificity is still controversial. In addition, ferent proteins containing SET domain (45). Although Ezh2 methylated lysine in H3 can exist in mono-, di-, and tri-methyl 22444 Ezh2 Regulation of hDAB2IP Gene Expression FIG.6. The status of H3 methylation and acetylation of the hDAB2IP pro- moter region in PCa cells transfected with Ezh2 siRNA. A ChIP assay was performed using DNA-protein complex isolated from PC3 or DU145 cells trans- fected with Ezh2 siRNA for 96 h and im- munoprecipitated with various antibod- ies. The amount of endogenous hDAB2IP gene promoter was determined by qPCR using the specific primer set described previously (2) and visualized with gel electrophoresis. neuve, A. M., and Reinke, V. (2002) Development (Camb.) 129, 479–492 status (35, 36), and the functional significance of these modifi- 19. Fong, Y., Bender, L., Wang, W., and Strome, S. (2002) Science 296, 2235–2238 cations is largely unknown. It appears that both di- and tri- 20. Rossant, J. (2001) Harvey Lect. 97, 17–40 21. Czermin, B., Melfi, R., McCabe, D., Seitz, V., Imhof, A., and Pirrotta, V. (2002) methyl H3-Lys-27 are consistent with the presence of Ezh2 in Cell 111, 185–196 the promoter region of hDAB2IP gene from both normal pros- 22. Muller, J., Hart, C. M., Francis, N. J., Vargas, M. L., Sengupta, A., Wild, B., tate epithelia and PCa cells. The presence of Ezh2 can dramat- Miller, E. L., O’Connor, M. B., Kingston, R. E., and Simon, J. A. (2002) Cell 111, 197–208 ically increase the levels of di- and tri-methyl H3-Lys-27, im- 23. Varambally, S., Dhanasekaran, S. M., Zhou, M., Barrette, T. R., Kumar-Sinha, plying that hypermethylation of H3-Lys-27 could be a key C., Sanda, M. G., Ghosh, D., Pienta, K. J., Sewalt, R. G., Otte, A. P., Rubin, factor in silencing hDAB2IP gene expression. In summary, M. A., and Chinnaiyan, A. M. (2002) Nature 419, 624–629 24. Rhodes, D. R., Sanda, M.G., Otte, A. P., Chinnaiyan, A. M., and Rubin, M. A. hDAB2IP gene silencing mediated by Ezh2 complex and (2003) J. Natl. Cancer Inst. 95, 661–668 HDAC1 provides a better understanding of the functional role 25. Kleer, 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, of Ezh2 in metastatic PCa and perhaps in other cancer types S. J., Rubin, M. A., and Chinnaiyan, A. M. (2003) Proc. Natl. Acad. Sci. such as breast cancer. U. S. A. 100, 11606–11611 26. Raaphorst, F. M., Meijer, C. J., Fieret, E., Blokzijl, T., Mommers, E., Buerger, Acknowledgments—We thank Dr. Jenuwein for providing Ezh2 ex- H., Packeisen, J., Sewalt, R. A., Otte, A. P., and van Diest, P. J. (2003) pression vector, Dr. Pienta for providing VCAP cell line, and Neoplasia 5, 481–488 Jennifer Stanfield for editing this manuscript. 27. Zhou, J., and Hsieh, J. T. (2001) J. Biol. Chem. 278, 27793–27798 28. Navone, N., Olive, M., Ozen, M., Davis, R., Troncoso, P., Tu, S. M., Johnston, REFERENCES D., Pollack, A., Pathak, S., von Eschenbach, A. C., and Logothetis, C. J. (1997) Clin. Cancer Res. 3, 2493–2500 1. Chen, H., Pong, R. C., Wang, Z., and Hsieh, J. T. (2002) Genomics 79, 573–581 29. Korenchuk, S., Lehr, J. E., Mclean, L., Lee, Y. G., Whitney, S., Vessella, R., 2. Chen, H., Toyooka, S., Gazdar, A. F., and Hsieh, J. T. (2003) J. Biol. Chem. Lin, D. L., and Pienta, K. J. (2001) In Vivo (Attiki) 15, 163–168 278, 3121–3130 30. Weijerman, P. C., Zhang, Y., Shen, J., Dubbink, H. J., Romijn, J. C., Peehl, 3. Wang, Z., Tseng, C. P., Pong, R. C., Chen, H., McConnell, J. D., Navone, N., D. M., and Schroder, F. H. (1998) Urology 51, 657–662 and Hsieh, J. T. (2002) J. Biol. Chem. 277, 12622–12631 31. Weijerman, P. C., Konig, J. J., Wong, S. T., Niesters, H. G., and Peehl, D. M. 4. Zhang, H., Zhang, R., Luo, Y., D’Alessio, A., Pober, J. S., and Min, W. (2004) (1994) Cancer Res. 54, 5579–5583 J. Biol. Chem. 279, 44955–44965 32. Sewalt, R.G., Lachner, M., Vargas, M., Hamer, K. M., den Blaauwen, J. L., 5. Zhang, R., He, X., Liu, W., Lu, M., Hsieh, J. T., and Min, W. (2003) J. Clin. Hendrix, T., Melcher, M., Schweizer, D., Jennwen, T., and Otte, A. P. (2002) Investig. 111, 1933–1943 Mol. Cell. Biol. 22, 5539–5553 6. Dote, H., Toyooka, S., Tsukuda, K., Yano, M., Ouchida, M., Doihara, H., 33. Bracken, A.P., Pasini, D., Capra, M., Prosperini, E., Colli, E., and Helin, K. Suzuki, M., Chen, H., Hsieh, J. T., Gazdar, A. F., and Shimizu, N. (2004) (2003) EMBO. J. 22, 5323–5335 Clin. Cancer Res. 10, 2082–2089 34. van der Vlag, J., and Otte, A. P. (1999) Nat. Genet. 23, 474–478 7. Yano, M., Toyooka, S., Tsukuda, K., Dote, H., Ouchida, M., Hanabata, T., Aoe, 35. Peters, A. H., Kubicek, S., Mechtler, K., O’Sullivan, R. J., Derijck, A. A., M., Date, H., Gazdar, A. F., and Shimizu, N. (2005) Int. J. Cancer 113, Perez-Burgos, L., Kohlmaier, A., Opravil, S., Tachibana, M., Shinkai, Y., 59–66 Martens, J. H., and Jenuwein, T. (2003) Mol. Cell. 12, 1577–1589 8. Kuzmichev, A., Jenuwein, T., Tempst, P., and Reinberg, D. (2004) Mol. Cell. 36. Paik, W. K., and Kim, S. (1971) Science 174, 114–119 14, 183–193 37. Rice, J. C., Briggs, S. D., Ueberheide, B., Barber, C. M., Shabanowitz, J., Hunt, 9. Cao, R., and Zhang, Y. (2004) Curr. Opin. Genet. Dev. 14, 155–164 D. F., Shinkai, Y., and Allis, C. D. (2004) Mol. Cell. 12, 1591–1598 10. Kuzmichev, A., Nishioka, K., Erdjument-Bromage, H., Tempst, P., and Rein- 38. Caretti, G., Di Padova, M., Micales, B., Lyons, G. E., and Sartorelli, V. (2004) berg, D. (2002) Genes Dev. 16, 2893–2905 Genes Dev. 18, 2627–2638 11. Cao, R., Wang, L., Wang, H., Xia, L., Erdjument-Bromage, H., Tempst, P., 39. Sengupta, A. K., Kuhrs, A., and Mu¨ ller, J. (2004) Development (Camb.) 131, Jones, R. S., and Zhang, Y. (2002) Science 298, 1039–1043 1959–1965 12. Pasini, D., Bracken, A. P., Jensen, M. R., Denchi, E. L., and Helin, K. (2004) 40. Bloyer, S., Cavalli, G., Brock, H. W., and Dura, J. M. (2003) Dev. Biol. 261, EMBO. J. 23, 4061–4071 426–442 13. Cao, R., and Zhang, Y. (2004) Mol. Cell. 15, 57–67 41. Pirrotta, V., Poux, S., Melfi, R., and Pilyugin, M. (2003) Genetica (Dordr.) 117, 14. Jacobs, J.J., and van Lohuizen, M. (2002) Biochim. Biophys. Acta 1602, 191–197 151–161 42. Kirmizis, A., Bartley, S. M., Kuzmichev, A., Margueron, R., Reinberg, D., 15. O’Carroll, D., Erhardt, S., Pagani, M., Barton, S. C., Surani, M. A., and Green, R., and Farnham, P. J. (2004) Genes Dev. 18, 1592–1605 Jenuwein, T. (2001) Mol. Cell. Biol. 21, 4330–4336 16. Plath, K., Fang, J., Mlynarczyk-Evans, S. K., Cao, R., Worringer, K. A., Wang, 43. Oberley, M. J., Tsao, J., Yau, P., and Farnham, P. J. (2004) Methods Enzymol. 376, 316–335 H., de la Cruz, C. C., Otte, A. P., Panning, B., and Zhang, Y. (2003) Science 300, 131–135 44. Santos-Rosa, H., Schneider, R., Bannister, A. J., Sherriff, J., Bernstein, B.E., Emre, N. C., Schreiber, S. L., Mellor, J., and Kouzarides, T. (2002) Nature 17. Silva, J., Mak, W., Zvetkova, I., Appanah, R., Nesterova, T. B., Webster, Z., Peters, A. H., Jenuwein, T., Otte, A. P., and Brockdorff, N. (2003) Dev. Cell. 419, 407–411 4, 481–495 45. Sims, III, R. J., Nishioka, K., and Reinberg, D. (2003) Trends Genet. 19, 18. Kelly, W. G., Schaner, C. E., Dernburg, A. F., Lee, M. H., Kim, S. K., Ville- 629–639 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Down-regulation of Human DAB2IP Gene Expression Mediated by Polycomb Ezh2 Complex and Histone Deacetylase in Prostate Cancer *

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American Society for Biochemistry and Molecular Biology
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Copyright © 2005 Elsevier Inc.
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0021-9258
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10.1074/jbc.m501379200
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 23, Issue of June 10, pp. 22437–22444, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Down-regulation of Human DAB2IP Gene Expression Mediated by Polycomb Ezh2 Complex and Histone Deacetylase in Prostate Cancer* Received for publication, February 7, 2005, and in revised form, March 29, 2005 Published, JBC Papers in Press, April 6, 2005, DOI 10.1074/jbc.M501379200 Hong Chen, Szu-wei Tu, and Jer-Tsong Hsieh‡ From the Department of Urology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9110 (also named ASK-interacting protein 1 (AIP1)) is involved in Human DAB2IP (hDAB2IP), a novel GTPase-activat- ing protein modulating the Ras-mediated signaling and the tumor necrosis factor-mediated JNK signaling pathway tumor necrosis factor-mediated apoptosis, is a potent leading to cell apoptosis (4, 5). We have demonstrated that growth inhibitor in human prostate cancer (PCa). Loss normal prostatic epithelial cells express higher hDAB2IP levels of hDAB2IP expression in PCa is due to altered epige- than PCa cells, which is due to epigenetic alternation (i.e. netic regulation (i.e. DNA methylation and histone mod- aberrant DNA methylation and histone deacetylation) in the ification) of its promoter region. The elevated polycomb promoter region during carcinogenesis. Similarly, loss of Ezh2, a histone methyltransferase, has been associated hDAB2IP expression was also detected in breast and lung with PCa progression. In this study, we have demon- cancer specimens (6, 7) frequently associated with the pro- strated that an increased Ezh2 expression in normal moter hypermethylation. prostatic epithelial cells can suppress hDAB2IP gene Human enhancer of Zeste homolog (Ezh2) protein belongs to expression. In contrast, knocking down the endogenous Polycomb repressive complex 2/3 (8), which also includes Eed, Ezh2 levels in PCa by a specific small interfering RNA Suz12, and the histone-binding protein RbAp48/46 (9–13). The can increase hDAB2IP expression. The association of Ezh2 complex appears to be a transcription repressor that has Ezh2 complex (including Eed and Suz12) with hDAB2IP been shown to be involved in cellular memory system, X-inac- gene promoter is also detected in PCa cells but not in tivation, germline development, stem cell pluripotency, and normal prostatic epithelial cells. Increased Ezh2 expres- cancer metastasis (14–20). This complex exhibits an intrinsic sion in normal prostatic epithelial cells by cDNA trans- histone lysine methyltransferase activity on histone H3 Lys-27 fection facilitates the recruitment of other components and 9 or histone H1 Lys-26 mediated by the SET domain of of Ezh2 complex to the hDAB2IP promoter region ac- Ezh2 (8, 10, 11, 21, 22). companied with the increased levels of methyl histone Recent data indicate that elevated Ezh2 levels are found in H3 (H3) and histone deacetylase (HDAC1). Consistently, data from PCa cells transfected with Ezh2 small inter- hormone-refractory, metastatic PCa (23, 24) as well as in fering RNA demonstrated that reduced Ezh2 levels re- poorly differentiated breast carcinomas (25, 26). However, the sulted in the dissociation of Ezh2 complex accompanied underlying mechanism of Ezh2 in these cancer cells is still with decreased levels of both methyl H3 and HDAC1 unknown. In this study, we found an inverse correlation be- from hDAB2IP gene promoter. We further unveiled that tween Ezh2 and hDAB2IP gene expression in either normal the methylation status of Lys-27 but not Lys-9 of H3 in prostatic epithelia or PCa cells. Increased Ezh2 expression in hDAB2IP promoter region is consistent with the normal prostatic epithelial cells could inhibit the hDAB2IP hDAB2IP levels in both normal prostatic epithelial cells promoter activity and its gene expression. In contrast, knock- and PCa cells. Together, we conclude that hDAB2IP down of Ezh2 expression by siRNA in PCa cells resulted in an gene is a target gene of Ezh2 in prostatic epithelium, elevated hDAB2IP gene expression. These data prompted us to which provides an underlying mechanism of the down- investigate the role of Ezh2 in modulating hDAB2IP gene ex- regulation of hDAB2IP gene in PCa. pression, 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 human DOC-2/DAB2 interactive protein gene The outcome of this study provides an underlying mechanism (hDAB2IP) located at chromosome 9q33.1-33.3 is a new mem- of the functional role of Ezh2 in metastatic PCa. ber of the Ras GTPase-activating family gene (1, 2). Our recent data indicate that hDAB2IP protein is a growth inhibitor in EXPERIMENTAL PROCEDURES prostate cancer (PCa) cells (3). In addition, hDAB2IP protein Cell Cultures—Three human prostate cancer cell lines (LNCaP, C4-2, and PC3) were maintained in T medium supplemented with 5% fetal bovine serum (27). MDAPCa 2a and MDAPCa 2b cell lines derived from patients with bony metastasis (28) were maintained in BRFF- * This work was supported by Grant W81XWH-04-1-0222 from the HPC1 medium (Biological Research Faculty and Facility, Inc., Jams- Department of Defense. The costs of publication of this article were ville, MD) supplemented with 20% fetal bovine serum. A VCAP cell line defrayed in part by the payment of page charges. This article must derived from a vertebral metastatic lesion of prostate cancer (29) and therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. DU145 were maintained in RPMI 1640 medium supplemented with ‡ To whom correspondence should be addressed: University of Texas 10% fetal bovine serum. Three normal human prostate epithelial cells Southwestern Medical Center, Dept. of Urology, 5323 Harry Hines (PrEC1, PrEC2, and PrEC3) were maintained in chemical-defined me- Blvd., Dallas, TX 75390-9110. Tel.: 214-648-3988; Fax: 214-648-8786; dium (PrEGM) purchased from Cambrex. PZ-HPV-7 (an immortalized E-mail: [email protected]. The abbreviations used are: PCa, prostate cancer; H3, histone 3; HDAC, histone deacetylase; siRNA, small interfering RNA; PcG, poly- transcription; qRT-PCR, quantitative RT-PCR; ChIP, chromatin immu- comb group; JNK, c-Jun NH -terminal kinase; h, human; RT, reverse noprecipitation; SET, Su-(var) 3–9; E(z), Trithorax. This paper is available on line at http://www.jbc.org 22437 This is an Open Access article under the CC BY license. 22438 Ezh2 Regulation of hDAB2IP Gene Expression cell line derived from the peripheral zone of a normal prostate) (30, 31) RESULTS and three additional primary prostatic epithelial cells (SWPC1, Profiling hDAB2IP and Ezh2 Expression in Various Pros- SWPC2, and SWPC3) (derived from cancer lesions), and SWNPC2 tatic Epithelia—To evaluate the expression profile of hDAB2IP (derived from the adjacent normal tissue) were maintained in PrEGM gene and Ezh2 in the prostate cell lines, we performed qRT- medium. PCR to document the steady-state levels of hDAB2IP (Fig. 1A) Transient Transfection and Luciferase Reporter Gene Assay—Cells and Ezh2 (Fig. 1B) mRNA in cells derived from normal pros- were plated at a density of 1.8  10 cells/well in a 6-well plate. After 24 h, PrEC1 and PZ-HPV-7 cells were co-transfected with Myc-tagged human tatic epithelium (PrEC1, PrEC2, PrEC3, PZ-HPV-7, and Ezh2 expression construct (32) and a luciferase reporter vector containing SWNPC2), primary PCa cells (SWPC1, SWPC2, and SWPC3), the hDAB2IP promoter-pGL3-P2 (2) using Lipofectamine Plus transfec- or metastatic PCa cell lines (LNCaP, C4-2, PC3, MADPC2a, tion reagent (Invitrogen). Luciferase assay was described previously (1, 2). MADPC2b, VCAP, and DU145). We observed a trend of de- All experiments were repeated at least three times in triplicate. The creasing hDAB2IP mRNA from normal cells to malignant cells; relative luciferase activity was calculated as described previously (2). however, an opposite trend of Ezh2 mRNA was seen from RNA Isolation and Quantitative RT-PCR (qRT-PCR)—Total cellular normal cells to malignant cells. Western blot data (Fig. 1C) also RNA was isolated using an RNeasy kit (Qiagen, Inc) according to the confirmed the qRT-PCR results. Taken together, an inverse manufacturer’s instructions. To measure hDAB2IP or Ezh2 mRNA correlation between Ezh2 and hDAB2IP gene expression levels, 1 g of total cellular RNA from each cell line was reversibly TM transcribed into first strand cDNA using iScript cDNA synthesis kit prompted us to investigate the possibility that hDAB2IP gene (Bio-Rad). expression could be modulated by the Ezh2 complex particu- The first strand cDNA was further amplified by qRT-PCR using larly, since loss of hDAB2IP expression was frequently ob- hDAB2IP primer set F-hDAB2IP, 5-TGGACGATGTGCTCTATGCC-3; served in metastatic PCa cells. R-hDAB2IP, 5-GGATGGTGATGGTTTGGTAG-3 or Ezh2 primer set The Inhibitory Effect of Ezh2 on hDAB2IP Gene Expres- F-Ezh2, 5-GCCAGACTGGGAAGAAATCTG-3; or R-Ezh2, 5-TGT- sion—To test whether Ezh2 is a negative regulator to modulate GCTGGAAAATCCAAGTCA-3 in a 40-l reaction mixture containing TM hDAB2IP gene expression in PCa cells, Western blot analysis 20 lofiQ SYBGREEN Supermix® (Bio-Rad). The reactions were was performed using whole cell extract prepared from PCa cells 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 transfected with either a mixture of two different Ezh2 siRNAs s), 55 °C (30 s), and 72 °C (1 min)) using an iCycler iQ machine (Bio- (23, 33) or lamin A/C siRNA. The significantly decreased Ezh2 Rad). The 18S cDNA (F-18S, 5-GGAATTGACGGAAGGGCACCACC- levels in PC3 (Fig. 2A) and in DU145 (Fig. 2C) were observed 3; R-18S, 5-GTGCAGCCCCGGACATCTAAGG-3) was used as an in- within 48–72 h after transfection, and the reduced Ezh2 pro- ternal control. All experiments have been repeated twice in duplicates. tein became more prominent 96 h after transfection. Under this hDAB2IP or Ezh2 mRNA level was determined by normalizing with the condition, the elevated levels of hDAB2IP protein were de- 18 S cDNA of each sample. tected in both PCa cells. In contrast, Lamin A/C siRNA duplex The quality control was carried out using both electrophoresis anal- did not alter Ezh2 or hDAB2IP levels in either cell type (Fig. 2, yses on a 2% NuSieve agarose gel (3:1, FMC Bioproducts) and melting curve analysis performed immediately after the end of amplification B and D). To demonstrate the specific effect of Ezh2 on using 95 °C (1 min), 55 °C (1 min), and 80 cycles of 0.5 °C increment hDAB2IP gene expression, the DOC-2/DAB2 gene (27), which beginning at 55 °C. We also performed standard curve for hDAB2IP and is not regulated by Ezh2 in prostate (Fig. 2), was used as a Ezh2 to ensure the linearity and efficiency of both genes. negative control in this study. Western Blot Analysis—Cells were lysed with whole cell lysis buffer To determine the inhibitory effect of Ezh2 on hDAB2IP gene (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl , 0.5 mM EDTA, 10% expression via the transcriptional or post-transcriptional reg- glycerol, 1% Triton X-100, 10 mM NaF, 1 mM dithiothreitol, 1 mM ulation, we examined the hDAB2IP promoter activity in sev- phenylmethylsulfonyl fluoride) and were alternately frozen and thawed eral normal prostatic epithelial cells by transfecting with both three times in 80 °C to rupture the cell membranes. Samples were pGL3-P2 and Ezh2 expression vectors. We observed that an 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 concen- increased Ezh2 protein expression (Fig. 3A) in normal prostatic tration of each sample was determined by a standard Bradford assay. epithelial cells could inhibit the hDAB2IP promoter activity Equal amounts of protein (20 g) of each cell line were subjected to (Fig. 3, B–E) in a dose- and time-dependent manner. Using Western blot analysis. Antibodies used for probing were hDAB2IP (3), qRT-PCR, we also observed a similar inhibitory effect of Ezh2 DOC-2/DAB2 (BD Biosciences), Lamin A/C (Upstate Biotechnology), on hDAB2IP mRNA levels in these and two other normal Ezh2 (Upstate Biotechnology), and actin (Sigma). epithelial cells such as PrEC2 and PrEC3 (data not shown). We RNA Interference—Two different 21-nucleotide duplex siRNAs for also generated a stable Ezh2-expressing PZ-HPV-7 transfec- Ezh2, (5-AAGAGGTTCAGACGAGCTGAT-3 (23), 5-AAGACTCT- tant and found reduction of the endogenous hDAB2IP protein GAATGCAGTTGCT-3 (33), or control siRNA for Lamin A/C (5-CTG- GACTTCCAGAAGAACA-3) were synthesized by Qiagen. Twenty-four levels in these cells (Fig. 3F). These data clearly indicate that hours after plating, the cells were transfected either with both Ezh2, the suppression of hDAB2IP gene promoter activity is medi- siRNA duplexes together (100 nmol each) or with control siRNA (200 ated by Ezh2 protein. nmol) using RNAiFect transfection reagent (Qiagen) according to the The Association between Ezh2 Complex and HDAC1 in manufacturer’s instructions. At various time points after transfection, hDAB2IP Promoter Region in PCa Cells—To demonstrate the the cells were harvested and subjected to Western blot analysis. direct interaction of Ezh2 complex with hDAB2IP promoter Chromatin Immunoprecipitation (ChIP) Assay—This assay for region, ChIP assay was performed. In Fig. 4A, robust binding of hDAB2IP gene promoter was performed as described previously (2). For Ezh2 as well as two other components, Eed and Suz12, to the DOC-2/DAB2 gene promoter, the same PCR condition was applied with two primer sets as follows: first PCR (5-ACATCACTCGCAGTGGC-3 hDAB2IP promoter was seen in PC3 cells, the moderate bind- and 5-GGCGAGAGATATGGTTC-3) and second PCR (5-CTCG- ing of Ezh2 complex was detected in DU145 cells, and the CGGAGCTCAGGGGAG-3 and 5-GGTAACTCCCCCTCAACGTG-3). lowest binding of Ezh2 complex was detected in PZ-HPV-7 Briefly, precleared chromatin from 2  10 cells was used for each ChIP cells. Noticeably, the amount of Ezh2 complex associated with assay, and 5 g of each antibody was used in this assay including Ezh2, hDAB2IP promoter inversely correlated with hDAB2IP mRNA Suz12, trimethyl H3 (Lys-27), trimethyl H3 (Lys-9), and dimethyl H3 and protein expression patterns in these cells (Fig. 1). On the (Lys-9) purchased from Abcam Inc.; and Eed, dimethyl H3 (Lys-27), other hand, we did not observe an inverse correlation between monomethyl H3 (Lys-27), acetyl H3, and HDAC1 purchased from Up- Ezh2 and DOC-2/DAB2 levels in these cells (Fig. 1C)ora state Biotechnology. The total input or immunoprecipitated DNA was consistent association pattern between Ezh2 complex and determined by qPCR at least twice with the equation Ct (threshold cycle) of each sample  mean of Ct  mean of Ct . DOC-2/DAB2 gene promoter (Fig. 4). (antibody) (input) Ezh2 Regulation of hDAB2IP Gene Expression 22439 FIG.1. Profiling hDAB2IP and Ezh2 expression in prostatic epithelia. The steady-state levels of hDAB2IP (A) and Ezh2 (B) mRNA from various prostatic epithelia cells were determined by qRT- PCR. After normalizing with 18 S rRNA, the copy number of each sample (mean S.D.) performed in duplicates from two different experiments was calculated based on the standard curve. The steady- state levels of hDAB2IP, Ezh2, and DOC- 2/DAB2 protein (C) were determined by Western blot analysis using the whole cell lysates extracted from four different pros- tatic epithelial cells. It is known that transcriptional repression mediated by hu- same condition, the acetyl H3 levels were elevated. For DOC- man polycomb group (PcG) protein involves histone deacetyla- 2/DAB2 gene, no consistent pattern of these complexes could be tion (34). We therefore determined the association of HDAC1 observed in the promoter region (Figs. 4 and 5). Thus, these with the hDAB2IP promoter region. As shown in Fig. 4B, the data further support the notion that hDAB2IP is an Ezh2 higher levels of HDAC1 associated with hDAB2IP promoter target gene. were detected in PC3 and DU145 cells than in PZ-HPV-7 cells. The Methylation Status of H3-Lys-9 or H3-Lys-27 in In contrast, the lower levels of acetyl H3 associated with hDAB2IP Promoter Region Modulated by Ezh2—Although the hDAB2IP promoter were seen in these two PCa cells than in human Eed-Ezh2 complex and its Drosophila ESC-E(Z) coun- PZ-HPV-7 cells (Fig. 4B). These data indicate that this repres- terpart have been shown to be HMKTase (9), its substrate sion complex associated with hDAB2IP promoter contains both specificity still remains unclear. For example, Cao et al. (11) Ezh2 complex and HDAC1. and Muller et al. (22) showed that Lys-27 in H3 is the only Furthermore, by increasing Ezh2 gene expression in PZ- amino acid methylated by Ezh2; however, other groups (10, 21) HPV-7 cells, we observed that the presence of Ezh2 protein demonstrated that Ezh2 can also methylate H3-Lys-9. More- could recruit not only other components of PcG proteins such as over, the lysine in H3 exists in mono-, di-, and tri-methyl status Eed and Suz12 (Fig. 5A) but also HDAC1 to the hDAB2IP (35–37), and their impact on gene regulation is still unclear. promoter region in these cells. Meanwhile, the decreased acetyl For hDAB2IP promoter region, di- and tri-methyl H3-Lys-27 H3 level became more apparent in these cells (Fig. 5B). Taken were highly detectable in PC3 and DU145 cells but not in together, Ezh2 plays a key role in recruiting other transcription PZ-HPV-7 (Fig. 4C), which is consistent with the presence of repressors to the hDAB2IP promoter region in prostatic epithe- Ezh2 complex in these three cell lines (Fig. 4A). We also noticed lia. On the other hand, we were able to show the dissociation of that the levels of mono-methyl H3-Lys-27 did not vary among both PcG complex and HDAC1 from hDAB2IP gene promoter these three cell lines and did not change in the presence of in PC3 (Fig. 6) and DU145 (Fig. 6) when their endogenous Ezh2 elevated Ezh2 (Figs. 4C and 5C), suggesting that mono-methyl levels were knocked down by Ezh2 siRNA (Fig. 2). Under the H3-Lys-27 may reflect a “constant basal” methylation status of 22440 Ezh2 Regulation of hDAB2IP Gene Expression FIG.2. The 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. DISCUSSION Lys-27. Caretti et al. (38) have reported a similar observation in the study of MHCIIb and MCK gene regulation during muscle PCa is a leading cause of cancer-related death in males and differentiation. By increasing Ezh2 protein expression in PZ- is second only to lung cancer. Although effective surgical and HPV-7 cells, we demonstrated that the levels of both di- and radiation treatments are available for clinically localized PCa, tri-methyl but not mono-methyl H3-Lys-27 elevated dramati- metastatic PCa remains essentially incurable. To understand cally in a dose-dependent manner (Fig. 5C). In the presence of the biology of metastatic PCa, data from a cDNA microarray Ezh2 siRNA in either PC3 or DU145 cells, the decreased levels analysis indicate that elevated Ezh2 with histone lysine meth- of both di- and tri-methyl H3-Lys-27 could be observed (Fig. 6). yltransferase activity is often associated with metastatic PCa These data suggest that the effect of Ezh2 is to convert H3- but not with benign tissue and organ-confined tumor (23). Lys-27 into tri-methyl status in the hDAB2IP gene promoter Noticeably, clinically localized prostate cancer expressing region. higher levels of Ezh2 often has a poorer prognosis (24), sug- On the other hand, for the methylation status of H3-Lys-9, gesting that Ezh2 is not only a potential biomarker for predict- we failed to detect any good correlation among these three cell ing the relative risk of PCa progression but also a key contrib- lines tested. For example, tri-methyl H3-Lys-9 was highly de- utor for the disease progression. Here we have demonstrated tectable in these cell lines, and di-methyl Lys-9 status was not that Ezh2 mRNA and protein are more consistently elevated in consistent with hDAB2IP levels among these three cell lines metastatic PCa cells than in normal cells. The pattern of (Fig. 4D). Moreover, increased Ezh2 expression in PZ-HPV-7 hDAB2IP gene expression exhibited an inverse correlation cells was associated with a dramatic elevation of di-methyl but with that of Ezh2, which prompted us to study the role of Ezh2 not tri-methyl H3-Lys-9 (Fig. 5D), suggesting that the methy- in regulating hDAB2IP gene expression since silencing lation status of H3-Lys-9 may not be involved in modulating hDAB2IP gene expression is mainly due to epigenetic regula- hDAB2IP gene promoter activity by Ezh2. Together, we believe tion (2, 6, 7). Increased hDAB2IP levels were detected in PCa that hDAB2IP gene silencing in PCa can be mediated by the cells transfected with Ezh2 siRNAs (Fig. 2). In contrast, the elevated levels of Ezh2, which results in the hypermethylation reduced hDAB2IP gene expression was observed in normal of H3-Lys-27 and the recruitment of HDAC. This study pro- prostatic epithelial cells transfected with Ezh2 expression vec- vides an underlying mechanism of the functional role of Ezh2 tor (Fig. 3). These results provide an important mechanism of in metastatic PCa. the elevated Ezh2 expression during PCa progression. Ezh2 Regulation of hDAB2IP Gene Expression 22441 FIG.3. The 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 g) and pGL3-P2 reporter gene construct (0.5 g) 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. In Drosophila, ESC-E(Z) proteins recognize and exert their tantly, Ezh2 plays an active role in recruiting other PcG com- activity through specific DNA sequences known as polycomb ponents such as Eed and Suz12 to form a functional complex response elements (39–41). In mammals, such elements have along with HDAC1 to the promoter region of hDAB2IP gene not been identified yet. It remains unclear how mammalian (Figs. 5 and 6). Based on these results, one could predict that PcG complexes are recruited to chromatin to regulate expres- the presence of elevated Ezh2 levels in PCa signifies the down- sion of a specific target gene. Because most of the PcG proteins regulation of tumor suppressor genes via the cooperative effect do not contain a DNA-binding domain, target genes cannot be of histone methylation and deacetylation. This study provides identified by searching the consensus-binding site in the ge- further evidence to support the critical role of epigenetic gene nome (42). Thus, the ChIP assay can provide direct evidence for regulation in PCa progression. the possible interaction between the Ezh2 complex and pro- The term “epigenetic” refers to the heritable changes in gene moter region of any known gene (43). Our data indicate that the expression that are caused by mechanisms other than the levels of Ezh2 associated with the hDAB2IP promoter region alteration in the nucleotide sequence. This concept generates inversely correlate with the steady-state levels of hDAB2IP tremendous new knowledge in understanding gene expression mRNA and protein in all three cell lines tested. Most impor- in mammalian cells. In addition to DNA methylation, increas- 22442 Ezh2 Regulation of hDAB2IP Gene Expression FIG.4. The status of PcG complex, HDAC1, histone methylation, and hi- stone acetylation on the hDAB2IP promoter region in prostatic epithe- lia. 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 (2) and visualized with gel elec- trophoresis. The number under each lane representing the fold of enrichment was Ct (sample) Ct (PZ-HPV-7) calculated as 1/2  . ing studies have focused on the impact of covalent modifica- tion, has emerged as an important player in regulating gene tions of the histone core in the nucleosome structure on gene expression and chromatin function. Lysine methylation usually regulation. One of the covalent modifications, lysine methyla- occurs on the Lys-4, Lys-9, Lys-27, Lys-36, and Lys-79 of H3, Ezh2 Regulation of hDAB2IP Gene Expression 22443 FIG.5. The impact of Ezh2 on the status of H3 methylation and acetyla- tion 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 immuno- precipitated with various antibodies. The amount of endogenous hDAB2IP gene promoter was determined by qPCR using the specific primer set described previ- ously (2) and visualized with gel electro- phoresis. The number under each lane representing the fold of enrichment was Ct (Ezh2) Ct (control) calculated as 1/2  . the Lys-20 of H4, and the Lys-26 of H1 (35, 36, 44). Biochemical protein contains a SET domain, and several groups (10, 11, 21) and genetic studies indicate that methylation of different lysine have shown Ezh2 with histone lysine methyltransferase activ- residues, with the exception of H3-Lys-79, is catalyzed by dif- ity, its substrate specificity is still controversial. In addition, ferent proteins containing SET domain (45). Although Ezh2 methylated lysine in H3 can exist in mono-, di-, and tri-methyl 22444 Ezh2 Regulation of hDAB2IP Gene Expression FIG.6. The status of H3 methylation and acetylation of the hDAB2IP pro- moter region in PCa cells transfected with Ezh2 siRNA. A ChIP assay was performed using DNA-protein complex isolated from PC3 or DU145 cells trans- fected with Ezh2 siRNA for 96 h and im- munoprecipitated with various antibod- ies. The amount of endogenous hDAB2IP gene promoter was determined by qPCR using the specific primer set described previously (2) and visualized with gel electrophoresis. neuve, A. M., and Reinke, V. (2002) Development (Camb.) 129, 479–492 status (35, 36), and the functional significance of these modifi- 19. Fong, Y., Bender, L., Wang, W., and Strome, S. (2002) Science 296, 2235–2238 cations is largely unknown. It appears that both di- and tri- 20. Rossant, J. (2001) Harvey Lect. 97, 17–40 21. Czermin, B., Melfi, R., McCabe, D., Seitz, V., Imhof, A., and Pirrotta, V. (2002) methyl H3-Lys-27 are consistent with the presence of Ezh2 in Cell 111, 185–196 the promoter region of hDAB2IP gene from both normal pros- 22. Muller, J., Hart, C. M., Francis, N. J., Vargas, M. L., Sengupta, A., Wild, B., tate epithelia and PCa cells. The presence of Ezh2 can dramat- Miller, E. L., O’Connor, M. B., Kingston, R. E., and Simon, J. A. (2002) Cell 111, 197–208 ically increase the levels of di- and tri-methyl H3-Lys-27, im- 23. Varambally, S., Dhanasekaran, S. M., Zhou, M., Barrette, T. R., Kumar-Sinha, plying that hypermethylation of H3-Lys-27 could be a key C., Sanda, M. G., Ghosh, D., Pienta, K. J., Sewalt, R. G., Otte, A. P., Rubin, factor in silencing hDAB2IP gene expression. In summary, M. A., and Chinnaiyan, A. M. (2002) Nature 419, 624–629 24. Rhodes, D. R., Sanda, M.G., Otte, A. P., Chinnaiyan, A. M., and Rubin, M. A. hDAB2IP gene silencing mediated by Ezh2 complex and (2003) J. Natl. Cancer Inst. 95, 661–668 HDAC1 provides a better understanding of the functional role 25. Kleer, 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, of Ezh2 in metastatic PCa and perhaps in other cancer types S. J., Rubin, M. A., and Chinnaiyan, A. M. (2003) Proc. Natl. Acad. Sci. such as breast cancer. U. S. A. 100, 11606–11611 26. Raaphorst, F. M., Meijer, C. J., Fieret, E., Blokzijl, T., Mommers, E., Buerger, Acknowledgments—We thank Dr. Jenuwein for providing Ezh2 ex- H., Packeisen, J., Sewalt, R. A., Otte, A. P., and van Diest, P. J. (2003) pression vector, Dr. Pienta for providing VCAP cell line, and Neoplasia 5, 481–488 Jennifer Stanfield for editing this manuscript. 27. Zhou, J., and Hsieh, J. T. (2001) J. Biol. Chem. 278, 27793–27798 28. Navone, N., Olive, M., Ozen, M., Davis, R., Troncoso, P., Tu, S. M., Johnston, REFERENCES D., Pollack, A., Pathak, S., von Eschenbach, A. C., and Logothetis, C. J. (1997) Clin. Cancer Res. 3, 2493–2500 1. Chen, H., Pong, R. C., Wang, Z., and Hsieh, J. T. (2002) Genomics 79, 573–581 29. Korenchuk, S., Lehr, J. E., Mclean, L., Lee, Y. G., Whitney, S., Vessella, R., 2. Chen, H., Toyooka, S., Gazdar, A. F., and Hsieh, J. T. (2003) J. Biol. Chem. Lin, D. L., and Pienta, K. J. (2001) In Vivo (Attiki) 15, 163–168 278, 3121–3130 30. Weijerman, P. C., Zhang, Y., Shen, J., Dubbink, H. J., Romijn, J. C., Peehl, 3. Wang, Z., Tseng, C. P., Pong, R. C., Chen, H., McConnell, J. D., Navone, N., D. M., and Schroder, F. H. (1998) Urology 51, 657–662 and Hsieh, J. T. (2002) J. Biol. Chem. 277, 12622–12631 31. Weijerman, P. C., Konig, J. J., Wong, S. T., Niesters, H. G., and Peehl, D. M. 4. Zhang, H., Zhang, R., Luo, Y., D’Alessio, A., Pober, J. S., and Min, W. (2004) (1994) Cancer Res. 54, 5579–5583 J. Biol. Chem. 279, 44955–44965 32. Sewalt, R.G., Lachner, M., Vargas, M., Hamer, K. M., den Blaauwen, J. L., 5. Zhang, R., He, X., Liu, W., Lu, M., Hsieh, J. T., and Min, W. (2003) J. Clin. Hendrix, T., Melcher, M., Schweizer, D., Jennwen, T., and Otte, A. P. (2002) Investig. 111, 1933–1943 Mol. Cell. Biol. 22, 5539–5553 6. Dote, H., Toyooka, S., Tsukuda, K., Yano, M., Ouchida, M., Doihara, H., 33. Bracken, A.P., Pasini, D., Capra, M., Prosperini, E., Colli, E., and Helin, K. Suzuki, M., Chen, H., Hsieh, J. T., Gazdar, A. F., and Shimizu, N. (2004) (2003) EMBO. J. 22, 5323–5335 Clin. Cancer Res. 10, 2082–2089 34. van der Vlag, J., and Otte, A. P. (1999) Nat. Genet. 23, 474–478 7. Yano, M., Toyooka, S., Tsukuda, K., Dote, H., Ouchida, M., Hanabata, T., Aoe, 35. Peters, A. H., Kubicek, S., Mechtler, K., O’Sullivan, R. J., Derijck, A. A., M., Date, H., Gazdar, A. F., and Shimizu, N. (2005) Int. J. Cancer 113, Perez-Burgos, L., Kohlmaier, A., Opravil, S., Tachibana, M., Shinkai, Y., 59–66 Martens, J. H., and Jenuwein, T. (2003) Mol. Cell. 12, 1577–1589 8. Kuzmichev, A., Jenuwein, T., Tempst, P., and Reinberg, D. (2004) Mol. Cell. 36. Paik, W. K., and Kim, S. (1971) Science 174, 114–119 14, 183–193 37. Rice, J. C., Briggs, S. D., Ueberheide, B., Barber, C. M., Shabanowitz, J., Hunt, 9. Cao, R., and Zhang, Y. (2004) Curr. Opin. Genet. Dev. 14, 155–164 D. F., Shinkai, Y., and Allis, C. D. (2004) Mol. Cell. 12, 1591–1598 10. Kuzmichev, A., Nishioka, K., Erdjument-Bromage, H., Tempst, P., and Rein- 38. Caretti, G., Di Padova, M., Micales, B., Lyons, G. E., and Sartorelli, V. (2004) berg, D. (2002) Genes Dev. 16, 2893–2905 Genes Dev. 18, 2627–2638 11. Cao, R., Wang, L., Wang, H., Xia, L., Erdjument-Bromage, H., Tempst, P., 39. Sengupta, A. K., Kuhrs, A., and Mu¨ ller, J. (2004) Development (Camb.) 131, Jones, R. S., and Zhang, Y. (2002) Science 298, 1039–1043 1959–1965 12. Pasini, D., Bracken, A. P., Jensen, M. R., Denchi, E. L., and Helin, K. (2004) 40. Bloyer, S., Cavalli, G., Brock, H. W., and Dura, J. M. (2003) Dev. Biol. 261, EMBO. J. 23, 4061–4071 426–442 13. Cao, R., and Zhang, Y. (2004) Mol. Cell. 15, 57–67 41. Pirrotta, V., Poux, S., Melfi, R., and Pilyugin, M. (2003) Genetica (Dordr.) 117, 14. Jacobs, J.J., and van Lohuizen, M. (2002) Biochim. Biophys. Acta 1602, 191–197 151–161 42. Kirmizis, A., Bartley, S. M., Kuzmichev, A., Margueron, R., Reinberg, D., 15. O’Carroll, D., Erhardt, S., Pagani, M., Barton, S. C., Surani, M. A., and Green, R., and Farnham, P. J. (2004) Genes Dev. 18, 1592–1605 Jenuwein, T. (2001) Mol. Cell. Biol. 21, 4330–4336 16. Plath, K., Fang, J., Mlynarczyk-Evans, S. K., Cao, R., Worringer, K. A., Wang, 43. Oberley, M. J., Tsao, J., Yau, P., and Farnham, P. J. (2004) Methods Enzymol. 376, 316–335 H., de la Cruz, C. C., Otte, A. P., Panning, B., and Zhang, Y. (2003) Science 300, 131–135 44. Santos-Rosa, H., Schneider, R., Bannister, A. J., Sherriff, J., Bernstein, B.E., Emre, N. C., Schreiber, S. L., Mellor, J., and Kouzarides, T. (2002) Nature 17. Silva, J., Mak, W., Zvetkova, I., Appanah, R., Nesterova, T. B., Webster, Z., Peters, A. H., Jenuwein, T., Otte, A. P., and Brockdorff, N. (2003) Dev. Cell. 419, 407–411 4, 481–495 45. Sims, III, R. J., Nishioka, K., and Reinberg, D. (2003) Trends Genet. 19, 18. Kelly, W. G., Schaner, C. E., Dernburg, A. F., Lee, M. H., Kim, S. K., Ville- 629–639

Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Jun 10, 2005

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