Epigenetic Regulation of a Novel Tumor Suppressor Gene (hDAB2IP) in Prostate Cancer Cell Lines* 210

hDAB2IP (human DAB2 (also known as DOC-2) interactive protein) is a novel GTPase-activating protein for modulating the Ras-mediated signal pathway. We demonstrate that the down-regulation of hDAB2IPmRNA in prostate cancer (PCa) cells is regulated by transcriptional levels. Analysis of the hDAB2IP promoter revealed that it is a typical TATA-less promoter containing many GC-rich sequences. In this study, we delineated the potential impact of the epigenetic control of the hDAB2IP promoter on its gene regulation in PCa. Acetylhistone H3 was associated with the hDAB2IPpromoter, and CpG islands remained almost unmethylated in normal prostatic epithelia, but not in PCa cell lines. Our data further indicated that trichostatin A (histone deacetylase inhibitor) and 5′-aza-2′-deoxycytidine (DNA hypomethylation agent) acted cooperatively in modulating hDAB2IP gene expression in PCa, whereas histone acetylation played a more significant role in this event. Moreover, a core promoter sequence from the hDAB2IP gene responsible for these treatments was identified. We therefore conclude that epigenetic regulation plays a potential role in regulating hDAB2IP expression in PCa and that these results also provide a new therapeutic strategy for PCa patients.

hDAB2IP (human DAB2 interactive protein) is a novel member of the Ras GTPase-activating family (1,2). Our recent data indicate that it interacts directly with DAB2 (Disabled-2; also known as DOC-2 for differentially expressed in ovarian carcinoma-2) (2), which appears to be a tumor suppressor in cancer types (3)(4)(5)(6)(7). Both DAB2IP and DOC-2/ DAB2 form a unique protein complex with negative regulatory activity that modulates the Ras-mediated signal pathway (2). In the prostate gland, this complex is detected in the basal cell population (2,6) and may orchestrate the differentiation and proliferation potential of these cells during gland development. In contrast, loss of expression of DOC-2/DAB2 and hDAB2IP proteins is often detected in metastatic pros-tate cancer (PCa) 1 cell lines, and increased expression of these proteins can suppress the growth of PCa (2,6).
We have demonstrated that normal prostatic epithelial cells have elevated hDAB2IP mRNA levels compared with PCa cells, which correlate with increased hDAB2IP promoter activity (1). These data indicate that transcriptional regulation of hDAB2IP is responsible for the down-regulation of hDAB2IP expression in PCa cells. However, little is known about the underlying mechanisms for the regulation of hDAB2IP gene expression in prostatic epithelial cells.
One of the hallmarks of the regulation of gene transcription is local chromatin decondensation mediated by histone acetylation, which leads to a reduced association between chromosomal DNA and histones and subsequently increases the accession of high molecular mass protein complexes of the transcription machinery. Conversely, histone deacetylation can repress transcription by increasing histone-DNA interaction (8,9). Additionally, we have found that the hDAB2IP promoter does not have a typical TATA box, but contains many GC-rich sequences (1,2). DNA hypermethylation, particularly in the GC-rich promoter region, results in transcription repression that is often associated with a number of tumor suppressor gene promoters, including Rb, p15, and p16 (10,11). In this study, we delineated the roles of histone acetylation and DNA methylation in the regulation of the hDAB2IP gene in normal prostatic epithelia and PCa cells. The data presented in this work provide strong evidence for underlying mechanisms of the down-regulation of the hDAB2IP gene mediated by epigenetic control in PCa cells.

EXPERIMENTAL PROCEDURES
Cell Cultures and Treatments-Two human prostate cancer cell lines (LNCaP and PC-3) were maintained in T medium supplemented with 5% fetal bovine serum as previously described (2). Two normal human prostate cell lines (PrEC, a primary prostatic epithelial cell line derived from a 17-year-old juvenile prostate; and PZ-HPV-7, an immortalized cell line derived from the peripheral zone of a normal prostate) were maintained in a chemically defined medium (PrEGM) purchased from BioWhittaker, Inc. (Walkersville, MD).
Cells were seeded at low density (6 ϫ 10 5 /100-mm dish) 16  Real-time Reverse Transcription-PCR Assay-Total cellular RNA was isolated from PC-3, LNCaP, and PZ-HPV-7 cells using RNAzol B (Tel-Test, Inc., Friendswood, TX) according to the manufacturer's instructions. Two micrograms of total cellular RNA were used in each reaction. cDNA was synthesized and amplified using either the hDAB2IP primer set (2 ng/l) (F-hDAB2IP, 5Ј-TGGACGATGTGCTC-TATGCC-3Ј; and R-hDAB2IP, 5Ј-GGATGGTGATGGTTTGGTAG-3Ј) or the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer set (6 ng/l) (G3P7, 5Ј-GAAGGTGGGTCGGAGTCAACG-3Ј; and G3P4, 5Ј-AGTGAGCTTCCCGTTCAGC-3Ј) in a 40-l reaction mixture containing 20 l of platinum qPCR Supermix-UDG (Invitrogen) and 4 l of SYBR Green I (final dilution of 1:10,000). The reactions were carried out on a 96-well plate, and a PCR amplification protocol was followed (95°C for 3 min and 40 cycles of amplification at 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min) using an iCycler iQ machine (Bio-Rad). A quality control was carried out using both electrophoresis analysis on a 2% NuSieve agarose gel (3:1; FMC Corp. BioProducts) and melting curve analysis performed immediately after the end of amplification at 95°C for 1 min and 55°C for 1 min and 80 cycles of 0.5°C increments beginning at 55°C. We also performed the standard curves for hDAB2IP and GAPDH to ensure the linearity and efficiency of both genes. The linear range of both genes is from 2 ϫ 10 2 to 2 ϫ 10 9 copies, and the efficiency of each reaction ranges from 92 to 97%. The relative induction of hDAB2IP mRNA was calculated as follows: ⌬C t (threshold cycle) of each sample ϭ mean of C t(hDAB2IP) Ϫ mean of C t(GAPDH) . The -fold induction of each sample ϭ 1/2 ⌬Ct(sample)Ϫ⌬Ct(control) . Construction of Reporter Gene Vectors-To analyze the promoter region of hDAB2IP, a 7.6-kb fragment from bacterial artificial chromosome (BAC) clone 298A17 (GenBank TM /EBI accession number AL365274) containing the predicted first exon and additional 5Ј-upstream sequence of the DAB2IP gene was subcloned into the EcoRI site of pBluescript SK(Ϫ) (Stratagene) (1). pGL3-1.6S, a 1.6-kb KpnI-XhoI fragment, was subcloned from this 7.6-kb element into the pGL3-Basic vector (Promega). Two putative promoter regions (P1, a 0.8-kb SfiI-XhoI fragment from ϩ229 to ϩ981; and P2, a 0.6-kb KpnI-Kpn2I fragment from Ϫ598 to ϩ44) were subcloned into the pGL3-Basic vector (see Fig. 1A).
Cell Transfection and Luciferase Reporter Assay-We plated cells at a density of 1.0 ϫ 10 5 cells/well on a six-well plate. After 16 h, we transfected the PZ-HPV-7 and PC-3 cell lines with both 0.8 g of reporter vectors and 0.2 g of ␤-galactosidase vector (pCH110) using FuGENE 6 (Roche Molecular Biochemicals). The LNCaP and PrEC cells were transfected with the same amount of DNA with Lipo-fectAMINE Plus transfection reagent (Invitrogen). Twenty-four hours after incubation, the transfected cells were treated with TSA for 24 h, 5Ј-Aza for 48 h, or a combination of both drugs by incubating with 5Ј-Aza for 24 h and then adding TSA for an additional 24 h. After washing twice with cold phosphate-buffered saline, the cells were harvested with lysis buffer (Promega). Both luciferase and ␤-galactosidase activities were assayed as previously described (1,12). The protein concentration of each extract was measured using the Bio-Rad protein assay. The relative luciferase activity (RLA) was calculated by normalizing both ␤-galactosidase and protein concentrations in each sample, and the data were averaged from RLA in triplicate.
Acid Extraction of Histone and Western Analysis-Cells were scraped, centrifuged at 200 ϫ g for 10 min, and then suspended in 10 volumes of phosphate-buffered saline. Cells were spun down; pellets were suspended in 5 volumes of lysis buffer (10 mM HEPES (pH 7.9), 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM dithiothreitol, and 1.5 mM phenylmethylsulfonyl fluoride); and sulfuric acid was added to a final concentration of 0.2 M, followed by incubation on ice for 30 min. After centrifugation at 11,000 ϫ g for 10 min at 4°C, the cell supernatant containing the acid-soluble fraction was retained. The supernatant was dialyzed twice against 200 ml of 0.1 M acetic acid for 1-2 h and then dialyzed overnight against 200 ml of H 2 O using Spectrapor ® molecular porous membrane tubing (Spectrum Medical Industries, Inc., Los Angeles, CA). The protein concentration was measured, and proteins were fractionated by SDS-PAGE (15%). Western blotting was carried out with an anti-acetylhistone H3 antibody (1:3000; Upstate Biotechnology, Inc., Lake Placid, NY). The same membrane was stripped and reprobed with an anti-histone H3 antibody (1:1000; Upstate Biotechnology, Inc.).
Chromatin Immunoprecipitation (ChIP) Assay-After treatment, formaldehyde was added to the cell medium at a final concentration of 1% for cross-linking proteins to DNA. Cells were washed, scraped off with ice-cold phosphate-buffered saline, and resuspended in SDS lysis buffer containing a mixture of protease inhibitors. An equal protein concentration of cell lysate from each sample was sonicated to reduce DNA fragments between 200 and 1000 bp. Once the cell debris was removed, the supernatant was diluted in ChIP dilution buffer (1:10), and 1% of this supernatant (as input DNA) was collected, purified, and subjected to genomic PCR with the primer sets described in Table I. Samples were precleared with salmon sperm DNA/protein A-agarose slurry (Upstate Biotechnology, Inc.) and incubated overnight at 4°C with or without (as a negative control) antibody. Immune complexes were collected by adding salmon sperm DNA/protein A-agarose slurry and incubated with 20 l of 5 M NaCl at 65°C to reverse DNA-protein cross-linking. DNA was then purified by proteinase K digestion, phenol extraction, and ethanol precipitation.
The strand-specific nested PCR primers used for amplifying the hDAB2IP gene are indicated in Table I. PCR amplifications were performed in a 50-l reaction mixture containing 2 l of DNA by the addition of 2 units of ThermalAce TM DNA polymerase (Invitrogen). A hot start was performed (98°C for 3 min), followed by 30 cycles at 98°C for 30 s, 62°C for 30 s, and 72°C for 45 s. The PCR product from the PI region (ϳ110 bp) was separated on 4% E-Gel TM (Invitrogen), and that from the PII region (ϳ230 bp) was separated on a 2% NuSieve agarose gel (3:1).
Bisulfite Genomic Sequencing-High molecular mass genomic DNA was obtained from PrEC, PZ-HPV-7, LNCaP, and PC-3 cell lines and subjected to bisulfite modification (13,14). Briefly, 1-2 g (5-10 l) of genomic DNA were denatured with NaOH (final concentration of 0.2 M), 30 l of 10 mM hydroquinone (Sigma), and 520 l of 3 M sodium bisulfite (Sigma) at pH 5 for 16 h at 50°C. The modified samples were purified using Wizard DNA Clean-Up system desalting columns (Promega), followed by ethanol precipitation. Bisulfite-modified DNA (100 ng) was amplified by PCR in a 25-l reaction mixture containing the primers indicated in Table I. A hot start was performed (95°C for 5 min) by adding 0.5 unit of HotStar Taq DNA polymerase (QIAGEN Inc., Valencia, CA). The PCR products were cloned into the TA cloning vector pCR2.1-TOPO. Four to eight individual clones were sequenced using reverse and forward M13 primers.

Identification of Two hDAB2IP Promoters in Prostatic
Epithelial Cell Lines-Two putative promoters, P1 (ϩ229 to ϩ981, located within the first intron) and P2 (Ϫ598 to ϩ44, located 5Ј-upstream of exon Ia) (Fig. 1A), were identified using both the TSSW program (human PII recognition using the TRANSFAC Database) 2 and experimental deletion analysis (1) of a 1662-bp hDAB2IP locus surrounding the transcription initiation site (ϩ1). As shown in Fig. 1B, there are many GC-rich sequences, and potential transcription factor-binding sites were detected within this region using MacVector Version 6.5.3.
To analyze the basal activity of each hDAB2IP promoter in various prostate cells, luciferase reporter vector constructs were generated. Using pGL3-1.6S, we detected the highest luciferase activity in both PrEC and PZ-HPV-7 cells, an intermediate level in LNCaP cells, and the lowest level in PC-3 cells (Fig. 1C), correlating with the steady-state levels of hDAB2IP mRNA in each cell line (1). Similar patterns of reporter gene activity were detected in these four cell lines using either the P1 or P2 promoter (Fig. 1C).
Induction of hDAB2IP Gene Expression by a Hypomethylation Agent (5Ј-Aza) and/or a Histone Deacetylase Inhibitor (TSA)-Apparently, the decreased hDAB2IP mRNA levels detected in many human PCa cells (1) could be caused by its reduced gene promoter activity (Fig. 1C). To understand the mechanism leading to the down-regulation of the hDAB2IP gene in human PCa cells, we first examined the role of epigenetic regulation of the hDAB2IP gene. The data from a ChIP assay demonstrated that the presence of acetylhistone H3 was associated with the PI and PII regions of hDAB2IP in both PrEC and PZ-HPV-7 cells, but not in PC-3 and LNCaP cells (Fig. 1D), suggesting that histone acetylation may play a role in modulating hDAB2IP gene expression. Recent data also indicate that epigenetic controls such as histone acetylation and/or DNA methylation play cooperative roles in modulating gene expression, particularly genes involved in tumor suppression (15,16). We therefore treated these cells with TSA, 5Ј-Aza, or a combination of both. The levels of hDAB2IP mRNA expression were evaluated by real-time reverse transcriptase-PCR using GAPDH as an internal control. As shown in Table II, TSA and/or 5Ј-Aza failed to elicit any elevation of hDAB2IP mRNA because the basal activity of the hDAB2IP promoter was very high in PZ-HPV-7 cells (Fig. 1C). However, in PC-3 cells (Table  II), either TSA or 5Ј-Aza was able to induce hDAB2IP mRNA expression. In contrast, the increased hDAB2IP mRNA levels in LNCaP cells treated with a single agent were lower than those in PC-3 cells (Table II) because LNCaP cells had higher endogenous hDAB2IP mRNA levels compared with PC-3 cells (1). For the combination of both agents, the level of induction exhibited an additive effect only in the PC-3 and LNCaP cell lines. In some cases, we noticed that the mRNA levels after the combination treatment were lower than those after the singleagent treatment, which was caused by the toxicity of the drug combination.
Characterization of the hDAB2IP Promoter Regulated by Histone Acetylation and DNA Methylation-To delineate which promoter could be induced by TSA, 5Ј-Aza, or a combination of both drugs and the underlying mechanism of the induction, we transiently transfected PCa cells with pGL3-P1 or pGL3-P2 under the same treatment conditions. In PC-3 cells, TSA could induce P1 promoter activity in a dose-dependent manner; however, 5Ј-Aza only slightly induced this promoter activity ( Fig.  2A). The combination treatment exhibited an additive effect only on P1 activity. In contrast, a very different induction pattern was observed in LNCaP cells transfected with pGL3-P1; only marginal induction of P1 activity was observed in these cells after the different treatments (Fig. 2B).
By transfecting pGL3-P2 into PC-3 cells, a dose-dependent induction pattern of P2 activity was observed in these cells treated with either TSA or 5Ј-Aza (Fig. 2C). In LNCaP cells, P2 activity could also be induced by either TSA or 5Ј-Aza in a dose-dependent manner (Fig. 2D), which differed from P1 activity induced by these drugs (Fig. 2B). Again, in PC-3 and LNCaP cells, the combined treatment with TSA and 5Ј-Aza exhibited an additive effect only on P2 activity. In addition, we determined both P1 and P2 activities in PC-3 and LNCaP cells with a different transfection protocol; the overall induction pattern was consistent (see Fig. 1 in the Supplemental Material). Taken together, these results indicate that the P2 promoter is responsible for both TSA-and 5Ј-Aza-induced hDAB2IP gene expression in PC-3 and LNCaP cell lines.
To evaluate the possibility of a global gene induction effect of TSA or 5Ј-Aza on PCa cells, we examined the activity of the prostate-specific antigen (PSA) gene promoter in LNCaP and PC-3 cells treated with either agent. As shown in Fig. 2E, no induction of PSA reporter activity was detected in both cell lines treated with a single agent or a combination of both agents. In contrast, androgen could induce PSA reporter activity dramatically in LNCaP cells (androgen receptor-positive), but not in PC-3 cells (androgen receptor-negative). Therefore, we believe that TSA or 5Ј-Aza has a specific effect on regulating hDAB2IP gene expression in PCa cell lines.
Increased Levels of Acetylhistone H3 in the hDAB2IP Promoter Induced by TSA-To determine whether the TSA-induced hDAB2IP gene expression correlated with the levels of histone acetylation associated with the hDAB2IP promoter region, we analyzed the steady-state levels of acetylhistone H3 in both PC-3 and LNCaP cells after TSA treatment. As shown in Fig. 3A, Western blot analysis of PC-3 cells indicated that the basal level of acetylhistone H3 was very low, whereas TSA induced a dramatic elevation of the ratio between acetylhistone H3 and total histone H3. Comparing this with the no-treatment control, TSA induced a dose-dependent (ranging from 8-to 88-fold) elevation of acetylhistone H3. In contrast, the basal level of acetylhistone H3 was very high in LNCaP cells (Fig.  3B). Therefore, we failed to detect any changes in the steadystate levels of acetylhistone H3 in LNCaP cells treated with TSA. Nevertheless, it is still possible that TSA increases the acetylhistone H3 levels associated with the hDAB2IP promoter region.
To analyze the status of acetylhistone associated with the hDAB2IP promoter, a ChIP assay was performed using the sequences corresponding to the PI (ϩ768 to ϩ873) and PII (Ϫ520 to Ϫ287) regions (Fig. 1A). Elevated levels of acetylhistone H3 were clearly associated with the PI region in both PC-3 and LNCaP cells treated with TSA or the combination, but not with 5Ј-Aza (Fig. 4A). Similarly, an accumulation of acetylhistone H3 levels associated with the PII region was also detected in both cell lines treated with TSA or the combination (Fig. 4B). Interestingly, we also found that 5Ј-Aza treatment could induce the accumulation of acetylhistone H3 in the PII region, but not in the PI region (Fig. 4B), because P2 (but not P1) activity could be induced in both PCa cell lines treated with 5Ј-Aza (Fig. 2). A similar phenomenon has also been observed in several different genes treated with 5Ј-Aza (10, 17). Also, some data suggest that DNA methylation and histone deacetylation can act cooperatively to silence tumor suppressor genes in cancer cells (18,20).
Although the ChIP assay provides a unique analysis of the specific chromatin DNA region that associates with acetylhistone proteins, the results need to be confirmed by function assays such as a reporter gene assay. Therefore, we investi- FIG. 1. Characterization of hDAB2IP gene promoters. A, schematic representation of potential hDAB2IP promoters. The transcription start site (TSS) at ϩ1 was predicted by MacVector Version 6.5.3. P1 (ϩ229 to ϩ981) is within the first intron; P2 (Ϫ598 to ϩ44) is located 5Ј-upstream of exon Ia. The depicted restriction endonucleases sites were used in subsequent cloning. PI (ϩ768 to ϩ873) and PII (Ϫ520 to Ϫ287) were used for ChIP assay. B, potential regulatory sequences of the hDAB2IP gene. Exon Ia of the hDAB2IP gene is boxed, and the putative cis-acting elements are underlined. ISRE, interferon-stimulated response element. C, differential promoter activities of the hDAB2IP gene in PrEC, PZ-HPV-7, PC-3, and LNCaP cell. The -fold RLA was calculated from the pGL3-Basic vector (taken as 1). D, determination of the levels of acetylhistone H3 associated with the hDAB2IP gene promoter in normal and malignant prostatic epithelia. The ChIP assay was carried out to determine the status of acetylhistone H3 associated with either the PI or PII region of the hDAB2IP gene promoter in PrEC, PZ-HPV-7, PC-3, and LNCaP cells. M, molecular mass markers; NC, negative control for PCR. gated the luciferase activity of two constructs, pGL3-PI (ϩ768 to ϩ873) and pGL3-PII (Ϫ520 to Ϫ287), in PC-3 and LNCaP cell lines after treatment. As shown in Fig. 5 (A and B), the basal luciferase activity of the pGL3-PI construct was much higher than that of the pGL3-P1 construct in both PC-3 and LNCaP cells (Fig. 2, A and B), suggesting that the deletion of 5Ј-and 3Ј-flanking sequences from the P1 region may contain some negative elements. Overall, we detected a slight increase in PI activity only in PC-3 cells treated with TSA or a combination of both TSA and 5Ј-Aza (Fig. 5A); however, no change in PI activity was detected in LNCaP cells after treatment (Fig. 5B).
We observed a dramatic induction of PII activity in PC-3 cells after treatment (10 -18-fold increase with TSA and 14 -30-fold increase with the combination) (Fig. 5C). Using 5Ј-Aza, an ϳ3-fold induction of PII activity was detected in PC-3 cells. A similar induction profile of PII activity was detected in LNCaP cells (Fig. 5D). For example, TSA alone induced an ϳ6 -14-fold increase in PII activity, whereas the combination treatment induced an 8 -44-fold increase in reporter gene activity. An ϳ2-fold induction of PII activity was observed in LNCaP cells treated with 5 M 5Ј-Aza. In addition, we repeated these experiments with a different transfection protocol; the overall induction pattern was consistent (see Fig. 2 in the Supplemental Material). Taken together, these data suggest that PII (Ϫ520 to Ϫ287) within the hDAB2IP promoter is the core regulatory region for modulating hDAB2IP gene transcription.
Characterization of the Methylation Status of the hDAB2IP Promoters in Prostate Cell Lines-It is known that aberrant methylation (which is associated with gene silencing) in the promoters of tumor suppressor genes is commonly detected in cancer cells (18 -20). CpG islands appear to be critical sites modulated by DNA methylation (21)(22)(23). Because the 5Ј-regulatory region in the hDAB2IP promoter is GC-rich and the DNA hypomethylation agent (5Ј-Aza) can induce hDAB2IP gene expression, determining the methylation profile of the promoter region in normal and cancerous cells could provide additional evidence for the role of DNA methylation in the regulation of hDAB2IP during PCa development. In this experiment, two PCa cell lines (PC-3 and LNCaP) and two normal prostate cell lines (PrEC and PZ-HPV-7) were subjected to bisulfite genomic sequencing. With respect to the high GC content in the hDAB2IP promoter, primers were designed to avoid potential methylation sites (e.g. CpG) such that both methylated and un-methylated DNAs would be amplified equally. For the P1 region, PmI (spanning 35 CpG sites) was designed; and for the P2 region, PmIIa (spanning 30 CpG sites) and PmIIb (spanning 56 CpG sites) were designed (Fig. 6A) because we found more CpG sites in the P2 region (86 sites) than in the P1 region (35 sites). The detailed primer information is summarized in Table I. In the PmI region, PC-3 cells showed a partial methylation pattern, and LNCaP cells showed an almost completed methylation pattern. In contrast, PZ-HPV-7 cells showed a completed unmethylation pattern, and PrEC cells contained very few methylation sites (Fig. 6B). The density of methylation of this region correlated inversely with the basal activity of the P1 promoter in all cells examined (Fig. 1C).
In the PmIIa region, both normal prostate cell lines showed an almost completed unmethylated pattern. However, LNCaP cells contained low densities of methylation, whereas PC-3 showed a significantly higher degree of methylation pattern (Fig. 6C). This evidence indicated that methylation density in the PmIIa region inversely correlated with the basal activity of P2 in these cells (Fig. 1C). Interestingly, in the PmIIb region, PC-3, PZ-HPV-7, and PrEC cells showed almost completed unmethylated patterns, and LNCaP cells contained few methylation sites (data not shown). Taken together, these data clearly indicate that the PmIIa region (Ϫ522 to Ϫ285) in hDAB2IP is the key regulatory sequence operative in prostatic epithelia.

DISCUSSION
The higher levels of hDAB2IP mRNA detected in normal prostatic epithelia compared with PCa cells are mainly regulated at the transcriptional level (1). In this study, we further demonstrated that the activity of the hDAB2IP promoter is more active in normal prostatic epithelia than in PCa cells (Fig.  1C). We also noticed that the 5Ј-upstream regulatory region of the hDAB2IP gene has GC-rich sequences, but no canonical TATA boxes. This is a typical feature of the promoters of many housekeeping genes and of ϳ40% of tissue-specific genes (24). Although various mechanisms may underlie this repression in PCa cells, our data demonstrate that histone acetylation and/or DNA methylation plays a crucial role in modulating hDAB2IP gene expression in PCa cells. The treatment of PCa cell lines such as PC-3 and LNCaP with TSA, 5Ј-Aza, or a combination of both significantly increased the steady-state levels of hDAB2IP mRNA (Table II). In contrast, TSA or 5Ј-Aza could not induce hDAB2IP gene expression in normal prostatic epithelia (Table  II). These data indicate that both DNA methylation and histone deacetylation act cooperatively to silence the hDAB2IP gene in PCa cells. Such action is presumably mediated through a complex chromatin structure in which methyl-CpG-binding proteins are associated with histone deacetylases (HDACs) (25,26).
Eukaryotic DNA is packed into a highly organized structure (27). It has become increasingly clear that gene transcription from this tightly packed DNA is regulated by chromatin-remodeling events, which can render DNA either more or less accessible to transcription factors. One of the key events in the regulation of eukaryotic gene expression is the post-translational modification of nucleosomal histones, which convert regions of chromosomes to transcriptionally active or inactive. The most well studied post-translational modification of histones is the acetylation of ⑀-amino groups on positively charged lysine residues in histone amino-terminal tail domains (7,28), which can release negatively charged DNA to interact with transcription factors. The effect of histone acetyltransferases (29) is counterbalanced by the presence of (HDACs) (30). Aberrant acetylation or deacetylation leads to such diverse disorders as leukemia, epithelial cancers, fragile X syndrome, and  (31). It is also known that HDACs can function as transcriptional corepressors and are often present in multisubunit complexes such as Sin3 and Mi2 complexes (32)(33)(34). From recent reports, HDAC-containing complexes are involved in DNA methylation-mediated transcriptional silencing of various tumor suppressor genes (15,16). Therefore, targeting HDAC activity has become a new strategy of cancer chemotherapy; several inhibitors have been developed and tested in clinical trials (35). Recent studies by several groups (36 -38) have demonstrated the existence of cellular complexes containing both HDACs and ATP-dependent nucleosome-remodeling activity, suggesting that some chromatin remodeling is mediated by cellular complexes with HDAC activity. In contrast, histone acetyltransferases such as CBP (cAMP-responsive element-binding protein-binding protein)/p300, CBP-associated factor (PCAF), and GCN5 have been identified in the protein complex of transcriptional activators (39 -41). DNA hypermethylation has been implicated in parental gene imprinting, X chromosome inactivation, and endogenous retrovirus silencing (42)(43)(44)(45)(46) as well as in the transcriptional silenc- ing of tumor suppressor genes (47,48). Hypermethylation of CpG islands is also found in the 3Ј-ends of some genes; however, the density of DNA methylation in promoter or first exon regions correlates inversely with gene transcription (22,49). It has also been shown that transcription repression mediated by methyl-CpG-binding proteins involves an HDAC complex (50,51), indicating that there is a close relationship between DNA methylation and histone deacetylation.
Regarding the potential role of histone acetylation, data from the ChIP assays indicated that acetylhistone H3 was associated with the hDAB2IP promoter in normal epithelial cell lines (PrEC and PZ-HPV-7) expressing hDAB2IP proteins (Fig. 1D). A dramatic increase in the levels of acetylhistone H3 associated with the hDAB2IP promoter was detected in PCa cells in the presence of TSA (Fig. 4, A and B). We further demonstrated that the DNA fragment identified in the ChIP assay had promoter activity and could respond to TSA treatment (Fig. 5, C  and D). Based on these results, we conclude that the status of acetylhistone in the hDAB2IP promoter is critical for its gene regulation. We also noticed that several potential transcription factor-binding sites such as AP-1, AP-2, interferon-stimulated response element, CCAAT box-binding transcription factor-nuclear factor 1 (CTF-NF1), and adenovirus early region 4 promoter transcription factor 1 (E4TF1) and a cluster of Sp1binding sites located in this region (Fig. 1B). In particular, members of the Sp1 family have been shown to act as positive or negative regulators of gene transcription. This mechanism is dependent on the competition between the transcription repressor HDAC1 and the transcription factor E2F1, which actives histone acetyltransferase (52). The presence of Sp1-bind-  Table I. The input DNA (lower panels) was used as a positive control. M, 1-kb plus marker; NC, negative control without antibody.
ing elements in the proximal hDAB2IP gene promoter could underlie the basis of gene repression mediated by histone deacetylation (53,54). Further investigation is warranted.
Regarding the role of DNA methylation in regulating hDAB2IP gene transcription, bisulfite sequencing data indicated that CpG islands remained almost unmethylated in normal prostate cell lines (PrEC and PZ-HPV-7) expressing the transcriptionally active hDAB2IP gene (Fig. 1C) (1). However, in PCa cells (PC-3 and LNCaP), hypermethylation of CpG islands was commonly associated with the hDAB2IP promoter region (Fig. 6). 5Ј-Aza could induce the expression of hDAB2IP mRNA in PCa cells (Table II). Our results are consistent with the promoter activity determined by the reporter gene assay (Fig. 2).
Regarding the regulation of the hDAB2IP gene, our data clearly demonstrate that DNA methylation and histone deacetylation can act cooperatively in silencing the hDAB2IP gene (Table II and Fig. 4). It has been shown that DNA methyltransferases recruited by an oncogene to a gene promoter suppress the expression of this gene (55). Also, the binding of the methyl-CpG-binding protein complex (21) to methyl-CpG islands competes with transcription factors and prevents them from binding to the promoter region. Recent data indicate that the methyl-CpG-binding protein can recruit HDACs, leading to condensation of the local chromatin structure and thereby ren-dering the methylated DNA less accessible to transcription factors (25).
In the hDAB2IP gene, there are two regions with potential promoter activity: P1 and P2. In this study, we found that P2 promoter activity has a better correlation with the induction of hDAB2IP mRNA in every tested cell line (Fig. 1C). The methylation profile of the P2 promoter in each cell line exhibited a reciprocal relationship between P2 reporter gene activity (Fig.  5C) and the density of methylated cytosine residues (Fig. 6C). Furthermore, the P2 (but not P1) promoter was able to respond to both TSA and 5Ј-Aza treatment (Fig. 2). Nevertheless, TSA seemed more potent than 5Ј-Aza in eliciting P2 promoter activity (Figs. 2 and 5). Therefore, we believe that the P2 region in the hDAB2IP gene represents a core promoter in prostatic epithelia. Our results also suggest that the deacetylhistonemediated transcriptional silencing of the hDAB2IP gene may be a critical event during the carcinogenesis of PCa.
In summary, cytosine methylation and histone deacetylation in the hDAB2IP regulatory regions associated with the silencing of hDAB2IP gene expression have been observed in PCa cells (PC-3 and LNCaP). Such a phenomenon seems to be specific to cancer because it was not detected in normal prostate cells (PZ-HPV-7 and PrEC). Therefore, this gene could potentially serve as a surrogate marker for early cancer detection. The outcome of this study also indicates that histone FIG. 5. Characterization of the core promoter in the hDAB2IP gene regulated by TSA and/or 5-Aza. Constructs pGL3-PI (A and B) and pGL3-PII (C and D) derived from a ChIP assay were transfected into PC-3 or LNCaP cells under different treatment conditions, and RLA was determined as described under "Experimental Procedures." The -fold RLA was calculated using the pGL3-Basic vector (taken as 1).
FIG. 6. Characterization of the methylation status of the hDAB2IP gene promoter in human prostatic epithelial cells. A, schematic representation of the three separated positions in the hDAB2IP locus subjected to bisulfite sequencing analysis. B and C, methylation patterns in the PmI (ϩ282 to ϩ975) and PmIIa (Ϫ522 to Ϫ285) regions in human prostatic epithelial cells. High molecular mass DNA isolated from each sample was modified with sodium bisulfite and amplified by PCR using the primer sets indicated in Table I. The PCR product was subcloned, and each individual clone (horizontal rows) from every sample was sequenced. The position of each CpG dinucleotide (vertical bars) is labeled with the number representing its location in the hDAB2IP gene. E, unmethylated CpG; q, methylated CpG. deacetylase and DNA methyltransferase can be novel targets for PCa therapy.