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Departments of Medicine (X.Z., X.M., R.H., A.D.S., S.E.) and Pathology (S.L.A.), University Health Network, The Ontario Cancer Institute (X.Z., X.M., R.H., A.D.S., S.E., S.L.A.) Toronto, Ontario, Canada M5G-2M9
Address all correspondence and requests for reprints to: Dr. Sylvia L. Asa, Ontario Cancer Institute, 610 University Avenue 8-327, Toronto, Ontario, Canada M5G 2M9. E-mail: sylvia.asa{at}uhn.on.ca.
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Abstract |
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Design: We examined the three members of the DNA methyltransferase (DNMT) enzyme family in normal and neoplastic human and mouse pituitary cells.
Setting: This study was performed at a university-affiliated cancer research institute.
Main Outcome Measures: Gene expression, promoter DNA methylation, histone modifications, and cell proliferation were determined.
Results: In contrast to DNMT1 and DNMT3a, DNMT3b was expressed at relatively higher levels in neoplastic pituitary cells. However, examination of the human DNMT3b 5' region showed uniformly low DNA methylation levels with little difference between normal and tumor samples. Through pharmacological methylation inhibition or histone deacetylation inhibition, we identified that DNMT3b gene expression is subject to histone modifications. Down-regulation of DNMT3b resulted in induction of retinoblastoma, p21, and p27, and reduction in cell proliferation. These targeted effects were associated with enhanced histone 3 acetylation and diminished histone methylation.
Conclusion: Our findings identify DNMT3b as a putative mediator of epigenetic control through histone modifications of gene expression in pituitary cells.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study we examined the expression and function of members of the DNA methyltransferase (DNMT) family of enzymes in normal and neoplastic pituitary cells. We focused on cell cycle control elements including Rb and the cyclin-dependent kinase inhibitors (CDKIs) p21 and p27 as examples of epigenetically silenced signals in pituitary cells. The data point to the importance of DNMT3b in targeting several TSGs.
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Materials and Methods |
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Mouse pituitary corticotroph AtT20 cells were obtained from the American Type Culture Collection (Rockville, MD) and grown in Ham F-10 medium supplemented with 15% horse serum and 2.5% fetal calf serum (all from Sigma-Aldrich, St. Louis, MO) with 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin (37 C, 95% humidity, 5% CO2 atmosphere incubation). Normal mouse pituitaries were obtained from 6-wk-old male animals with Institutional Protocol Approval.
Normal human pituitary specimens with no morphological abnormalities were obtained from autopsies performed within 12 h of death and with no evidence of endocrine abnormality. Primary human pituitary adenomas were obtained at surgery after informed consent. The use of all human tissue was approved by the Ethics Committee of the University Health Network. The pathology of pituitary adenomas was examined using immunohistochemistry for all cases, and tumors were classified histologically according to currently accepted Armed Forces Institute of Pathology and World Health Organization criteria (12, 13). The adenoma samples examined included six somatotroph adenomas, four lactotroph adenomas, four oncocytic null cell adenomas, three gonadotroph adenomas, two corticotroph adenomas, one mixed lactotroph-somatotroph adenoma, and one silent subtype 3 adenoma.
5-Aza-2'-deoxycytidine (5-Aza-dC) and trichostatin-A (TSA) treatments
AtT20 cells were plated at 1 x 106 cells per 10-cm dish and allowed to attach for 24 h. For assessment of the impact of DNA methylation, cells were treated with the DNMT inhibitor 5-Aza-dC (Sigma-Aldrich) at concentrations of 5 or 10 µM for 5 d. At 24-h intervals, new medium containing freshly prepared drug was added. For assessment of chromatin histone acetylation, cells were treated with 0.3 and 0.6 µM of the histone deacetylase (HDAC) inhibitor, TSA (Sigma-Aldrich), for 24 h. Each experiment was independently performed with three separate dishes in at least three independent experiments.
Western blotting
Cells were lysed with radioimmunoprecipitation assay buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 100 µg/ml phenylmethylsulfonylfluoride, aprotinin, and sodium orthovanadate in PBS). Total cell lysate protein was quantified using the Bio-Rad method (Bio-Rad Laboratories, Inc., Hercules, CA). Fifty micrograms of whole lysates were separated on 10% sodium dodecyl sulfate denaturing polyacrylamide gels and transferred onto a nylon membrane (Millipore Corp., Billerica, MA) at 100 V for 1.5 h at room temperature. Blots were incubated with antibodies against DNMT3B (Imgenex Corp., San Diego, CA), Rb (4H1 Mouse Monoclonal Antibody; Cell Signaling Technology, Inc., Danvers, MA), pRb (phospho-Rb Ser780, polyclonal 1:1000; Cell Signaling Technology), the CDKIs p21 (polyclonal 1:500; BD Biosciences, Mississauga, Ontario), and p27 (monoclonal 1:1000; Transduction Laboratories, Lexington, KY), and actin (monoclonal 1:1000; Sigma-Aldrich) as a loading control. Antibodies were diluted in PBS-5% nonfat milk with 0.1% Tween 20 at 4 C overnight, followed by washing with PBS-Tween 20 four times for 10 min each at room temperature, then incubated with peroxidase-conjugated goat antirabbit IgG horseradish peroxidase (1:2000) for 1 h at room temperature with agitation. Protein bands were visualized using chemiluminescence (Amersham, Ontario, Canada). Experiments were performed on three independent occasions.
Immunocytochemistry
DNMT3b expression by AtT20 pituitary cells was examined by immunocytochemistry on 4-µm sections of formalin-fixed, paraffin-embedded cell pellets. Briefly, sections were treated with 2% hydrogen peroxide to quench endogenous peroxide for 30 min and exposed to 5 µg/ml proteinase K for 15 min at room temperature. The sections were washed extensively and exposed to equilibration buffer for 10 min. Each slide was then incubated with anti-DNMT3B antibody (IMG-184A; Imgenex) at 4 C overnight. The reaction was visualized with the avidin-biotin method and 3,3'-diaminobenzidine.
RNA extraction, semiquantitative, and quantitative real-time RT-PCR
Total RNA was isolated from human pituitary tissue using TRIZOL reagents (Invitrogen Corp., Carlsbad, CA). RNA from AtT20 cells was isolated using the RNeasy Mini kit (QIAGEN, Germantown, MD), combined with optional on-column DNase digestion by RNase-Free DNase Set (QIAGEN) according to the manufacturer’s instructions to diminish genomic DNA contamination. Approximately 1.0 µg total RNA from each sample was used to conduct RT in a 20-µl volume using TaqMan RT reagents kit (Applied Biosystems, Roche Molecular Systems Inc., Branchburg, NJ). The synthesized cDNA was used for PCR amplification or stored at –20 C for further analysis. RT-PCR primers were designed to span exons to avoid genomic DNA contamination. The primer sequences and PCR conditions are shown in Table 1
. PCR reactions were performed for 10 min at 95 C, followed by 35 cycles of 30 sec at 95 C, 30 sec at annealing temperatures, and 30 sec at 72 C, followed by a 10-min extension at 72 C. RT-PCR examinations were performed on at least three independent occasions. For quantitative assessment the cDNAs encoding DNMT3b and 18 S were amplified using the primers shown in Table 1. Equal amounts of cDNA for each sample were added to a SYBR Green PCR Master mix (Applied Biosystems, Foster City, CA). PCR reactions were performed on an ABI Prism 7700 sequence detection system (Applied Biosystems). The relative abundance of a transcript was represented by the threshold cycle of amplification (CT), which is inversely correlated to the amount of target RNA/first-strand cDNA being amplified.
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Oligonucleotides complementary to a region of the mouse DNMT3b were synthesized by Ambion, Inc. (Austin, TX). The forward strand 5'-GGAUGCUAUUGUGAAUGUGtt-3' and reverse strand 5'-CACAUUCACAAUAGCAUCCtc-3' were transfected using Lipofectamine (Invitrogen) at different concentrations as indicated.
Cell proliferation assay
Cells were seeded in a 96-well plate, and labeled with MTT (Thiazolyl Blue Tetrazolium Bromide; Sigma-Aldrich) as a measure of cell proliferation. Absorbance was measured with an OPTI max microplate reader (Molecular Devices, Sunnyvale, CA) at 570 nm and reference wavelength of 650 nm.
Bisulfite sequencing (BS) and quantification of DNA methylation
One microgram of genomic DNA was bisulfite modified according to the manufacturer’s protocol (CHEMICON International, Inc., Temecula, CA) diluted in 25 µl volume. One microliter of modified DNA was used for BS. Primer location, sequence, and PCR conditions are indicated in Figs. 1 and 2 and in Table 1
. Final PCR products were cut from 1.5% agarose gels, extracted, and cloned into the TA cloning system (Invitrogen) for automated sequencing. At least 10 positive clones from each sample were sequenced.
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Chromatin immunoprecipitation (ChIP) assays
The ChIP assay was performed in accordance with the manufacturer’s recommendations (United Biotechnologies, Inc., Lake Placid, NY) and as previously described (14). Briefly, histone was cross-linked to DNA by the direct addition of 37% formaldehyde, and cells were washed with cold PBS containing protease inhibitors before lysis. The lysates were sonicated to shear DNA lengths between 200 and 1000 bp. After centrifugation, cell suspensions were further diluted, and 20 µl lysate from each sample was used to monitor the amount of DNA present (input DNA) for PCR detection. The rest of the lysate was cleared with salmon sperm DNA/protein G-agarose beads. Immunoprecipitation was performed using anti-dimethyl-histone 3 (Lys9), and anti-acetyl-Histone H3 (AcH3) antibody (all from United Biotechnologies) overnight at 4 C with agitation. Negative controls were performed with omission of antibody. For PCR analysis, the histone-DNA cross-links of eluates were reversed at 65 C, and the immunocomplexes were digested with proteinase-K for 1 h at 50 C, and DNA was finally purified by phenol extraction and used for PCR amplification. PCR primers and conditions are shown in Table 1. PCR reactions were performed in a volume of 15 µl containing 1.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphate, 0.2 mM each primer, and 0.375 U AmpliTaq Gold polymerase (Applied Biosystems). Experiments were performed on three independent occasions.
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Results |
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We used RT-PCR amplification to examine the expression of the three principal members of the DNMT family, DNMT1, DNMT3a, and DNMT3b, in primary human pituitary samples that consisted of six normal pituitaries and 21 pituitary tumors (Fig. 1A). Figure 1B displays graphically the mean relative levels of DNMT mRNA in normal compared with neoplastic pituitary samples. DNMT1 and DNMT3a were expressed at readily detectable levels with no appreciable differences in the two tissue types. In contrast, DNMT3b was expressed at significantly (P < 0.001) higher levels in neoplastic than in normal tissue. The DNMT3b overexpression in pituitary tumors was further confirmed by quantitative real-time PCR (Fig. 1B).
To explore the mechanism underlying this altered expression, we examined the human DNMT3b 5' region extending from –600 to +315, which encompasses the 5' untranslated region and exon 1 (Fig. 1C). This region contains a large CpG island with multiple CG sites. COBRA covering the –93 to +189 region revealed little evidence for DNA methylation in normal or tumor samples (Fig. 1D). Similarly, bisulfite DNA sequencing (BS) (Fig. 1E) failed to identify DNA methylation in normal pituitary samples (n = 6) or in pituitary tumor samples (n = 21). Indeed, DNA methylation of the DNMT3b 5'region was uniformly low (2.1 and 1.9%, respectively) in both sample types (Fig. 1F).
DNMT3b expression is positively regulated by 5'-Aza-dC and TSA treatment
To explore mechanisms other than DNA methylation that may be responsible for DNMT3b regulation in pituitary cells, we used primary mouse pituitaries and clonal mouse AtT20 cells. Figure 2A demonstrates significantly lower levels of mRNA and protein levels of DNMT3b in normal mouse pituitary compared with neoplastic AtT20 cells. To determine whether these differences are related to epigenetic silencing of DNMT3b, we examined the impact of the methylation inhibitor 5'azacytidine (AZC) and the histone deacetylation inhibitor TSA on AtT20 cells. Both compounds proved effective in enhancing DNMT3b mRNA and protein levels (Fig. 2, A and B). To determine if chromatin modifications can be implicated in this alteration, we used a ChIP assay (Fig. 2, C and D). Using anti-methyl-histone H3-Lys9 (diMeH3-K9) and AcH3 antibodies, we identified the ability of AZC and TSA to enhance H3 acetylation and diminish methylation of this histone mark. To determine the extent of involvement of DNA methylation in mediating the action of AZC on DNMT3b regulation, we performed COBRA and BS (Fig. 2D). Both techniques revealed a modest degree of DNA methylation in normal pituitary cells, with little if any in AtT20 cells (Fig. 2, E and F). These studies show that, as in the human pituitary, DNA methylation contributes little toward DNMT3b regulation. Moreover, in mouse AtT20 pituitary cells, histone modifications appear more relevant in modulating DNMT3b expression.
DNMT3b down-regulation recapitulates the effect of AZC methylation inhibition on Rb, p21, and p27
We next examined the ability of DNMT3b to modulate key targets that are known to be epigenetically silenced in pituitary tumors. First, we ensured that our selected targets, which included Rb and the CDKIs, p21, and p27, were under readily measurable control in our experimental system. Specifically, we demonstrate that AZC or TSA treatment can also effectively enhance levels of these targets (Fig. 3A). To determine whether DNMT3b is a mediator of epigenetic silencing of these targets, we used an siRNA approach targeting this enzyme. Figure 3B shows the confirmation of effective down-regulation of DNMT3b protein levels. This DNMT3b down-regulation was associated with increased protein levels of Rb, p21, and p27 with dephosphorylation of Rb. Corresponding RT-PCR examination revealed similar increases in Rb, p21, and p27 mRNA levels (Fig. 3C). Thus, although AZC or TSA treatment and DNMT3b siRNA can reproduce similar actions on cell cycle control elements, the fact that DNMT3a and fibroblast growth factor receptor (FGFR) 2 mRNA levels were not altered by DNMT3b siRNA suggests that the targets of DNMT3b action are relatively more gene specific.
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). Figure 4B shows a modest degree of DNA methylation of the Rb promoter as detected by COBRA and BS, as has been described previously (15). Down-regulation of DNMT3b resulted in near complete elimination of Rb DNA methylation (Fig. 4B, upper panels). No detectable DNA methylation changes were noted for p21 or p27 by either technique (Fig. 4B). In contrast to the DNA findings, ChIP analysis revealed increased histone 3 acetylation for all three promoters with corresponding reduction in histone (Lys 9) methylation in response to DNMT3b down-regulation (Fig. 4C). Finally, using an MTT assay, we show that cells down-regulated for DNMT3b display reduced proliferation (Fig 4D).
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Given that DNMT3b was effective at targeting several histone methylation-sensitive genes, we next asked whether it can also target its own promoter. To this end, we ensured that our DNMT3b siRNA targets the most 3' coding exon (Fig. 5A). This allowed us to examine DNMT3b mRNA expression at an upstream region. Figure 5A confirms that our DNMT3b target did not directly interfere with the stability of the mRNA upstream. Figure 5B demonstrates again the sensitivity of DNMT3b mRNA to induction by AZC or TSA treatment. That DNMT3b itself was at least partially responsible for mediating the action of these compounds is demonstrated in Fig. 5B. Down-regulation of DNMT3b abrogated the effect of AZC and of TSA on DNMT3b mRNA levels. These data support the notion that DNMT3b exhibits broad epigenetic control of pituitary gene expression.
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Discussion |
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We identify here DNMT3b’s relatively unique variable expression in human pituitary tissues and tumors compared with a more constitutive expression pattern of DNMT1. These data are similar to recent findings in the breast, in which DNMT3b was overexpressed in nearly 80% of sporadic carcinomas (23). We also found that siRNA down-regulation of DNMT3b recapitulates the effect of pharmacological DNA methylation inhibition or histone deacetylation inhibition in up-regulating several candidate pituitary target genes. These included cell cycle control elements known to be of importance in pituitary tumorigenesis, Rb, p21, and p27. Using methylation-specific PCR, BS, and COBRA, we found no difference between DNMT3b methylation in normal and neoplastic human pituitary tissues. Similar findings were also obtained in mouse pituitary cells. Instead, using pharmacological and ChIP approaches, we identified evidence of histone modifications, including acetylation and demethylation, in the control of DNMT3b expression. In particular, we identified histone 3 lysine 9 (K9) methylation as a target of DNMT3b action, consistent with the recognized early critical role of this modification in silencing TSGs (24).
An emerging theme from studies of human pituitary tumorigenesis has been the evolving significance of epigenetic control rather than intragenic mutations, loss-of-heterozygosity, or gene rearrangements (5). For example, despite its well-recognized impact in genetically deficient mice (25), the Rb TSG was not mutated in primary human tumors (26). Instead, the gene has been suggested to be down-regulated through methylation at a CpG island in these tumors (6, 7, 8). Moreover, no inactivating mutations have been identified within the Rb1 promoter region in pituitary tumors that fail to express the protein (27). It should be noted that Rb1 DNA promoter methylation alone, as shown in this report, is not sufficient to explain inactivation of this gene in human pituitary tumors.
Epigenetic silencing has also been shown in other tumor suppressors that are considered to be important in pituitary. Mice lacking p27kip1 have an increased propensity to develop multiorgan neoplasia, including pituitary tumors (28). Protein levels of this CDKI are reduced in human pituitary adenomas, a feature that correlates with recurrence (29, 30), however, the p27kip1 gene is not mutated; instead, protein levels are diminished through other mechanisms. Here, we show that DNMT3b can also target p27 in pituitary cells. More significantly, the magnitude of induction of p27, p21, and Rb through DNMT3b down-regulation was sufficient to diminish pituitary cell proliferation.
It has been estimated that approximately 80% of all CpG dinucleotides in the mammalian genome are subject to methylation changes (19). The remaining unmethylated CpG residues are mostly located in the promoter regions of constitutively active genes and are referred to as CpG islands. DNA methylation has long been shown to have a transcriptional silencing function with an important role in several tumorigenic states. This is mediated by recruitment of HDACs through the methyl-DNA binding motifs of components of several HDAC-containing complexes (31). More recently, direct functional links between DNA methylation and histone methylation have been uncovered. Indeed, genetic evidence indicates that histone methylation may be a prerequisite for DNA methylation (32, 33). Our findings on the effect of AZC, classically considered as a DNA-demethylating agent, on histone demethylation of DNMT3b support this model. Furthermore, our findings are in partial agreement with other recent reports supporting the view that DNA and histone methylation may have reinforcing functions (34, 35). Moreover, the effect that we observed with TSA treatment on endogenous gene expression and histone methylation further emphasizes the role of histone modifications in the control of genes responsible for this action.
In summary, we have shown that DNMT3b is relatively unique among members of the DNMT family. Expression is higher in neoplastic pituitary cells where gene expression appears to be predominantly regulated through a histone-modifying network. Given the ability of DNMT3b to regulate multiple cell cycle control elements, including Rb, p27, and p21, our findings highlight this DNMT as a potential therapeutic target. Thus, our data support the rationale exploring the role of HDAC inhibitors in the medical treatment of pituitary tumors.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors have nothing to declare.
First Published Online June 10, 2008
Abbreviations: AcH3, Anti-acetyl-Histone H3; AZC, 5'azacytidine; 5-Aza-dC, 5-Aza-2'-deoxycytidine; BS, bisulfite sequencing; CDKI, cyclin-dependent kinase inhibitor; ChIP, chromatin immunoprecipitation; COBRA, combined bisulfite restriction analysis; diMeH3-K9, dimethyl-histone 3; DNMT, DNA methyltransferase; FGFR, fibroblast growth factor receptor; HDAC, histone deacetylase; Lys9, anti-dimethyl-histone 3; Rb, retinoblastoma; siRNA, small interfering RNA; TSA, trichostatin-A; TSG, tumor suppressor gene.
Received March 12, 2008.
Accepted June 2, 2008.
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