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Department of Medicine (S.E., X.Z.), Mount Sinai Hospital and University of Toronto; Department of Pathology (S.F., S.L.A.), University Health Network and University of Toronto; and The Freeman Centre for Endocrine Oncology and The Ontario Cancer Institute (S.E., X.Z., S.F., S.L.), Toronto, Ontario, Canada M5G 2M9
Address all correspondence and requests for reprints to: Dr. Sylvia. L. Asa, University Health Network, 610 University Avenue, 8-318, Toronto, Ontario, Canada M5G 2M9. E-mail: sylvia.asa{at}uhn.on.ca.
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-stimulating polypeptide that lead to constitutive elevation of adenylyl cyclase activity in nearly one third of GH-producing pituitary adenomas (2). Mutations of the structurally related Ras proteins, particularly at the GTP-binding domain, have also been described, albeit only in rare aggressive pituitary adenomas or carcinomas (1). Moreover, overexpression of the ligand of a G protein-coupled receptor, GHRH, results in pituitary adenoma formation in older transgenic mice (3). These findings support the importance of components of a distinct signaling cascade in pituitary tumorigenesis. Another emerging theme identified in pituitary tumorigenesis has been epigenetic silencing of tumor suppressor genes (4). Despite the development of pituitary tumors in the retinoblastoma (Rb) null mouse (5), intragenic mutations of this gene have not been identified in human pituitary adenomas (6). Instead, the Rb promoter was found to be methylated at CpG islands, resulting in loss of protein expression (7). Similarly, several other components of cell cycle control including the cyclin dependent kinase (CDK) inhibitors (CDKIs) p16, p18, and p27 have been shown to be underexpressed in pituitary adenomas, and p16 expression is known to be silenced through epigenetic silencing (8). Importantly, however, the mechanisms underlying these epigenetic changes remain unknown.
We have been interested in the role of fibroblast growth factor (FGF) receptors (FGFRs) in pituitary tumorigenesis. This has been recently highlighted by the identification of a pituitary tumor-derived N-terminally truncated isoform of FGFR4 (ptd-FGFR4) that is transforming in vitro and in vivo and causes pituitary tumorigenesis in transgenic mice (9). ptd-FGFR4 is derived by alternative initiation of the Ig-like domain and part of the second domain; it localizes to the cytoplasm, where it is constitutively phosphorylated. In contrast to ptd-FGFR4, wild-type full-length FGFR4 does not result in tumor formation. To examine the mechanism(s) responsible for this altered promoter utilization, we examined the promoter of the FGFR4 gene and showed that the 5' promoter is silenced through deacetylation of histone 3 residues (10, 11, 12). We demonstrated that the zinc-finger transcription factor Ikaros (Ik) was part of an important transcriptional complex capable of recruiting multiple corepressors with histone deacetylase complex (HDAC) activity (10).
Ik is a transcription factor that binds regulatory sequences expressed mainly in lymphoid-related genes (13, 14). This founding member of a family of zinc-finger DNA-binding proteins has also been implicated in extensive chromatin remodeling (15). The Ik gene contains seven exons that can, by alternative splicing, give rise to eight known isoforms. These isoforms differ in the number of N-terminal zinc finger motifs that bind DNA, resulting in members with variable DNA-binding properties (13, 16, 17). All Ik isoforms share a common C-terminal domain that contains two zinc finger motifs that are required for hetero- or homodimerization and for interactions with other proteins (16, 18, 19). Isoforms containing the requisite three or more N-terminal zinc fingers confer high-affinity binding to an Ik-specific core DNA sequence motif in the promoters of target genes (16). All eight Ik isoforms share a common C-terminal domain that contains two zinc finger motifs that are required for hetero- or homodimerization and for interactions with other proteins (15). Only isoforms 1–3 contain the requisite three or more N-terminal zinc fingers that confer high-affinity binding to an Ik-specific core DNA sequence motif in the promoters of target genes (15).
The formation of Ik homo- and heterodimers among the DNA-binding isoforms increases their affinity for DNA, whereas heterodimers between the DNA-binding isoforms and non-DNA-binding isoforms are unable to bind DNA. Thus, Ik proteins with fewer than three N-terminal zinc fingers can negatively interfere with the activity of Ik isoforms that can bind DNA (16, 20). In the endocrine pituitary gland, Ik is abundantly expressed during development in hormone-producing corticotroph cells where it binds the proopiomelanocortin promoter to activate the endogenous gene (21). Loss of Ik in vivo results in contraction of the pituitary corticomelanotroph population, reduced circulating adrenocorticotrophic hormone levels, and adrenal glucocorticoid insufficiency (21). Ik is also expressed in pituitary GH4 mammosomatotroph cells, where it inhibits GH but activates the prolactin gene in the same cells (22). This zinc-finger protein selectively acetylates histone 3 residues on the proximal GH promoter and limits access of the pituitary transcription activator-1 (22). In contrast, Ik acetylates the proximal prolactin promoter and facilitates pituitary transcription activator-1 binding to this region in the same cells (22). Thus, the Ik system represents an example of a finely tuned system that is the consequence of complex alternative splicing mechanisms.
In the current studies, we examined the mechanism of Ik action on pituitary cell growth in AtT20 corticotrophs and GH4 mammosomatotrophs, where this factor is abundantly expressed (21, 22). In particular, forced expression of the pituitary tumor-derived dominant negative isoform Ik6 resulted in enhanced cell survival with antiapoptotic features. Because a key regulatory point in the apoptotic pathway is the ratio of pro- and antiapoptotic Bcl-2 family members, we investigated whether tumor-derived Ik6 mediated regulation of Bcl-XL expression and its effect on cell survival.
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RESULTS |
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Three independent clones expressing Ik6 in pituitary GH4 cells also demonstrated an increase (P = 0.023) in colony number (145 ± 3; n =6) compared with Ik1-transfected (100 ± 3; n =6) or empty vector-transfected cells (87 ± 2; n =6).
To extend these data, we implanted stably transfected AtT20 cells into severe combined immunodeficient (SCID) mice. Ik6 expression resulted in more rapid growth and larger tumors compared with control pcDNA-transfected cells or cells expressing Ik1 (Fig. 1
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), and TUNEL labeling indices were reduced to 8 ± 3% (P = 0.009) from 24 ± 7% in control cells and 32 ± 8% in cells expressing Ik1 (Fig. 2B).
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). These clones were also examined by immunocytochemistry, which revealed increased cytoplasmic staining for Bcl-XL (Fig. 3B). To determine whether Ik6 action on Bcl-XL is dependent on inactive heterodimer formation with Ik1, we tested the impact of Ik down-regulation by small interfering RNA (siRNA). Bcl-XL levels in independent clones of AtT20 cells stably expressing an siRNA targeted to reduce all Ik isoforms are shown in Fig. 3C. Loss of Ik (and its isoforms) could not reproduce the induction of Bcl-XL expression by Ik6. These data suggest that Ik6 acts through more than simple heterodimerization with Ik1.
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). Moreover, given the demonstrated role of p16 on pituitary tumor cell survival (23), we examined the potential impact of Ik on this CDKI. However, we found no consistent effect of Ik1 or Ik6 on p16 levels in either AtT20 (Fig. 3D) or GH4 cells (data not shown).
Ik1 and Ik6 Differentially Acetylate Histones on Bcl-XL Promoter Activity
Because Ik has been shown to recruit components of the HDAC to selected promoters in a gene and cell-specific manner (24), we sought to determine if inhibition of HDAC activity would impact Bcl-XL promoter activity. Using trichostatin-A (TSA) as a pharmacological HDAC inhibitor, we identified stimulation of Bcl-XL promoter activity in empty vector-transfected cells (Fig. 4, left). In contrast, methylation inhibition with 5'-azacytidine had no appreciable influence on this reporter.
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), consistent with the importance of HDAC in mediating Ik1 action on the Bcl-XL promoter. In contrast, Ik6 resulted in no additive effect on histone acetylation, an effect mimicking the action of the pharmacological HDAC inhibitor TSA alone.
To further extend these observations, we used a chromatin immunoprecipitation (ChIP) assay to compare the role of acetylation vs. methylation status of histones on the 5'-Bcl-XL promoter. Pituitary AtT20 cells overexpressing Ik1 resulted in deacetylation of the Bcl-XL promoter (Fig. 5, A and B). In contrast, Ik6 resulted in enhanced acetylation of histones (Fig. 5, A and C). Figure 5C also demonstrates the dose responsiveness of the Bcl-XL promoter to TSA-mediated histone acetylation. Neither Ik1 nor Ik6 transfection measurably impacted Bcl-XL histone methylation (Fig. 5, A and B). However, it should also be noted that pharmacological treatment with the 5'-azacytidine methylation inhibitor also failed to influence the weak histone methylation as determined by ChIP assay (Fig. 5B). This promoter region contains a CpG island with 20 potential methylation sites. Bisulfite sequencing showed no evidence of DNA methylation at any of these predicted CpG sites of this promoter in control cells and in cells transfected with Ik1 or Ik6 in the absence and presence of 5'-azacytidine treatment. Figure 5D depicts a portion of the sequenced DNA region covered by the ChIP assay. Taken together, these findings are consistent with a significantly more important role for histone acetylation than histone methylation or DNA methylation in Bcl-XL control in pituitary cells.
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). Similar findings were also obtained in GH4 cells (data not shown). These findings are consistent with a more selective role for histone 3 acetylation in modulating Bcl-XL expression by Ik in pituitary cells. |
DISCUSSION |
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We have previously demonstrated that the FGFR4 promoter is subject to regulation by a multiprotein complex that includes the ubiquitous Sp transcription factors as well as the zinc finger transcription factor Ik (10). Using deletional mapping, we defined a minimal promoter in pituitary cells that included a fragment containing important binding sites for the hematopoietic zinc finger-containing transcription factor Ik flanked by binding sites for Sp and Ets-type factors. Specific Ik expression in rodent pituitary cells was identified by immunocytochemistry and confirmed in primary rodent and human pituitary adenohypophysial cells (10).
Expression of the non-DNA-binding isoform Ik6 has been identified in nearly a third of cases of human T cell acute leukemias. Ik6 has been detected in acute myeloid leukemia. where it has been shown to enhance myeloid precursor cells to activate Bcl-XL but not Bcl-2 (25). Moreover, animals heterozygous for dominant negative forms of Ik (isoforms that lack the DNA-binding domain) develop lymphoproliferative disorders, presumably by inactivation of the normal Ik allele (19). Thus, Ik is regarded as an essential molecular switch in early cell differentiation and lineage whose altered expression may play an important role in the tumorigenic processes.
Expression of the non-DNA-binding, tumor-derived Ik6 could result in dysregulated expression of target genes including growth factor receptors such as FGFR4 that are essential for normal development and maturation. In contrast to wild-type Ik, Ik6 interrupts activation of the 5'-FGFR4 promoter, consistent with a dominant negative role for Ik6 in pituitary tumors. In pituitary tumor cells, Ik6 deacetylates the 5'-FGFR4 promoter, resulting in an environment that favors utilization of a downstream cryptic promoter in intron 4 (11). Transcription from this alternative site leads to a truncated tumor-derived receptor isoform with oncogenic and antiadhesive properties (9, 26). These findings are consistent with the notion that only Ik isoforms with DNA-binding domains when bound in cis to Ik-binding sites are able to activate gene transcription (16, 20, 27). Thus, the balance between DNA-binding and non-DNA-binding Ik isoforms may contribute to a cell- and gene-specific response of either transcriptional activation or repression. Whether Ik6 has independent functions in addition to its ability to form inactive heterodimers with other Ik family members remains to be shown. For example, another truncated Ik isoform (Ik7) appears to have the capacity to regulate the adhesion molecule L-selectin and migration in a cell-specific manner independent of Ik (28). Our data suggest that Ik6 targets Bcl-XL expression in a fashion that is, at least partially, independent of its interaction with Ik1, because siRNA-mediated down-regulation of Ik, resulting in loss of endogenous Ik, did not recapitulate the effects of Ik6 on Bcl-XL.
CDKs and CDKIs play a central role in the regulation of cell cycle progression (29). The cyclin/CDK complexes cyclinD/CDK4 and cyclinE/CDK2 are catalytically active during late G1phase and have been implicated in the regulation of G1/S progression. The Rb protein is a putative substrate of the CDKs. Rb phosphorylation abrogates the ability of these proteins to inhibit transactivation of transcription factors important in cell cycle control. In turn, CDK activity is modulated by the CDKIs p27kip1, p57kip2, p16ink4A, p15ink4B, p18ink4c, and p19ink4D.
Driven by the potentially pivotal role of Rb in regulating pituitary cell growth coupled with the negative findings involving the Rb gene itself, cyclins, CDKs, and CDKIs became obvious candidates for causative factors in pituitary tumor pathogenesis. Neither Rb nor p16ink4A were found to be intragenically mutated in human pituitary tumors. Instead, both genes have been found to be frequently silenced by extensive promoter methylation (4). Indeed, reexpression of p16ink4A hypophosphorylates Rb to inhibit pituitary tumor cell growth and induce G1 arrest (23). We show here, however, that tumor-derived Ik6 does not target p16 promoter histone modification through methylation or acetylation. These findings suggest a more selective set of targets in mediating Ik action on cell growth and point to a distinct mechanism explaining the deregulation of p16 in pituitary tumor formation.
Protein acetylation plays a crucial role in regulating transcriptional activity. Acetylation complexes (such as cAMP response element binding protein-binding protein/p300) or deacetylation complexes (including HDAC) are typically recruited to DNA-bound transcription factors in response to signaling pathways. Histone hyperacetylation by histone acetyl-transferases is generally associated with transcriptional activation, presumably by remodeling nucleosomal structure into an open conformation that is more accessible to transcription complexes. Conversely, HDAC recruitment is associated with transcriptional repression reversing the chromatin remodeling process. This gene repression can be cell type and promoter specific. The ability of Ik to repress gene transcription has been described to occur through the HDAC complexes containing mSin3 (24) and Mi-2 proteins (30). The earlier findings of histone underacetylation in the vicinity of Ik recruitment sites (24) are consistent with the importance of HDAC in mediating Ik action. Our data indicating that Ik abrogates the effect of the HDAC inhibitor TSA along with ChIP-derived findings are consistent with recruitment of an HDAC complex to the Bcl-XL promoter.
We show here that pituitary cells express appreciable amounts of Bcl-XL that, unlike the Bcl-2 family member, is targeted by Ik. This is consistent with a previous report showing that endogenous levels of Bcl-2 were insufficient to protect GH4 cells from chemically induced apoptosis (31). Instead, Bcl-XL, as shown in this study, appears to be a more functionally prominent member of the Bcl family in modulating pituitary cell survival.
Bcl-XL is an important integrator of diverse signaling pathways, many of which are of recognized significance in pituitary cell survival and function. Such signaling cascades include those mediated through transcription factors such as nuclear factor 
B, signal transducer and activator of transcription 5, Ets1, and Ets2, which, in turn, activate Bcl-XL itself to promote cell survival (32). The pivotal role of Bcl-XL in neoplasia has, thus far, been most extensively studied in hematological malignancies including chronic myeloid leukemia, where the BCR-ABL fusion protein activates STAT5 to induce Bcl-XL expression (32).
Subtle changes in the cellular levels of Bcl-XL can have profound effects on apoptosis. Indeed, a 2-fold reduction in Bcl-XL is sufficient to induce apoptosis after thrombopoietin withdrawal in megakaryocytic cells (33). Similarly, cells lacking one allele of Bcl-XL display increased apoptosis in low serum. Conversely, overexpression of Bcl-XL protects cells from apoptosis induced upon withdrawal of colony-stimulating factor-1 (34). Thus, the degree of induction of Bcl-XL that we observed with Ik6 in the current studies is likely sufficient to contribute to escape from apoptotic signals upon withdrawal of survival signals. This was supported by our observed effects of forced Ik6 expression after serum withdrawal on PARP degradation and TUNEL staining.
In summary, the current studies elucidate a potential mechanism by which the zinc finger transcription factor Ik contributes to neoplastic pituitary cell growth. They underscore a histone acetylation-dependent mechanism, but neither histone methylation nor DNA methylation, in targeting the Bcl-XL antiapoptotic factor. Given the established role of Ik in the immune system, it remains to be shown whether Ik targets other common or distinct factors in governing endocrine and immune cell survival.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Stably transfected Ik1 and dominant negative Ik6 clones were generated using standard selection techniques as previously described (12, 22).
Ikaros siRNA plasmid construction
Exon 7, which is commonly shared by all Ik isoforms, was targeted for down-regulation. Positions 896–915 of the cDNA (GenBank accession no. 009578) were aligned to the mouse genome database in a basic local alignment search tool search to exclude homology with other unrelated genes. The siRNA oligonucleotide templates were as follows: sense strand, 5'-GATCCGGAGGATATGATGACATCCTCTGCAGGAGGATGTCATCATATCCTCCTTTTTTGGAAA-3'; and antisense strand, 5'-AGCTTTTCCAAAAAAGGAGGATATGATGACATCCTCCTGCAGAGGATGTCATCATATCCTCCG-3'. These oligonucleotides were prepared according to the manufacturer’s instructions (Ambion Inc., Austin, TX). After annealing, the duplexes were ligated into pSilencer 2.1-U6 neo vector at the BamHI/HindIII sites. The products were transformed into DH5 competent cells. Ampicillin-resistant colonies were selected, identified by restriction digestion using PstI, and confirmed by DNA sequencing. A circular pSilencer2.1-U6 neo vector that expresses a hairpin siRNA with limited homology to any known sequences in the mouse, human, and rat genomes was used as a negative control. Cell populations that stably express the siRNA were selected using neomycin (G418) at a concentration of 0.4 µg/ml. Specific reduction of Ik expression was confirmed by Western blotting.
Western Blotting
Cells were lysed with RIPA buffer (1% Nonidet P-40; 0.5% sodium deoxycholate; 0.1% sodium dodecyl sulfate; 100 µg/ml phenylmethylsulfonyl fluoride, aprotinin, and sodium orthovanadate in PBS). Total-cell lysates were quantified by the Bio-Rad (Hercules, CA) method. Forty micrograms of whole lysates were separated on 10% sodium dodecyl sulfate denaturing polyacrylamide gels and transferred onto nylon membrane (Millipore, Billerica, MA) at 100 v for 1 h at room temperature. Blots were incubated with a mouse monoclonal antibody (4E9; kindly provided by K. Georgopoulos, Harvard, Boston, MA), which recognizes the C-terminal fragments of Ik proteins (16) at 1:2000 dilution in PBS-5% nonfat milk with 0.1% Tween 20 at 4 C overnight, followed by washing with PBS-Tween 20 four times, 10 min each time, at room temperature and incubated with secondary antibody of peroxidase-conjugated goat antimouse IgG (1:2000) for 1 h at room temperature with agitation. PARP (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), Bcl-XL, Bcl-2, and p16 were used at 1:2500 (all from Santa Cruz Biotechnology) and actin was used at 1:500 (Sigma, St. Louis, MO). Protein bands were visualized using chemiluminescence (Amersham, Oakville, Ontario, Canada), and band intensities were quantified by a scanning densitometer.
Immunocytochemistry
To determine the impact of Ik isoforms on endogenous Bcl-XL expression, we stained 4-µm sections of cell pellets from Ik1, Ik6, or empty vector stably transfected AtT20 and GH4 cells for Bcl-XL (Santa Cruz Biotechnology). Briefly, sections were treated with 2% hydrogen peroxide to quench endogenous peroxide for 30 min and exposed to 5 µg/ml of 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-Bcl-XL antibody at 4 C overnight.
Cell Cycle Analysis
After trypsinization, 1–3 x 106 Ik-transfected cells were washed with PBS and fixed with cold 80% ethanol for 1 h on ice. The fixed cells were washed with staining buffer (0.2% Triton X-100 and 1 mM EDTA, pH 8.0, in PBS) and resuspended in the staining buffer containing 50 µg/ml ribonuclease A (Sigma, St. Louis, MO) and 50 µg/ml propidium iodide for 1 h. Cell cycle analysis was performed by fluorescence-activated cell sorting (FACScan; Becton Dickinson, San Jose, CA) using Cellquest analysis, and specific S-phase was analyzed using the Modfit DNA analysis program (Verity Software House Inc., Topsham, ME).
Apoptosis Analyses
Protection against apoptosis was examined by serum starvation for 3 d followed by Western blotting for PARP cleavage (1:500; Sigma).
To examine for DNA fragmentation characteristic of apoptosis, we stained Ik-transfected cells with the TUNEL technique (ApopTag kit, Oncor; Integen Co., Purchase, NY). Briefly, cells were treated with 2% hydrogen peroxide to quench endogenous peroxide for 30 min and were then exposed to 5 mg/ml proteinase K for 15 min at room temperature. After repeated washing with equilibration buffer, cells were incubated with terminal deoxytransferase and digoxigenin-labeled terminal deoxynucleotidyl transferase at 4 C overnight followed by horseradish peroxidase-conjugated antidigoxigenin antiserum for 1 h. The peroxidase reaction was visualized using diaminobenzidine. Control sections were similarly stained but in the absence of terminal deoxytransferase, digoxigenin-conjugated terminal deoxynucleotidyl transferase, or antidigoxigenin antiserum. Positive cells were counted as a percentage of total cells to determine the TUNEL labeling index.
Growth in Soft Agar
Ik stably transfected AtT20 cells (2,500) were plated in 35-mm dishes as a single-cell suspension in 0.3% agar in Ham’s F-10 medium supplemented with 15% horse serum and 10% fetal calf serum over a layer of 0.5% agar prepared in Ham’s F-10. Colony formation was monitored daily with a light microscope, and colonies were photographed and counted 2 wk later as previously described (35).
In Vivo Tumor Growth Assay
CB-17 female SCID mice, 7–8 wk of age, were purchased from the animal facility of the Ontario Cancer Institute (Toronto, Canada) and maintained under specified pathogen-free conditions. AtT20 or GH4 cells were injected into the flank at a concentration of 2 x 106 cells as previously described (9, 22). Each mouse developed a single palpable tumor (volume, 
100 mm3) 3 wk after implantation. Tumor dimensions were measured using a vernier caliper (Fisher Scientific Ltd, Ontario, Canada). Tumor volumes were calculated as (length x width x depth)/2. The care of animals was approved by the Institutional Animal Care facilities at the Ontario Cancer Institute.
Promoter Luciferase Assays
Plasmid reporters and expression vectors were prepared by column chromatography (QIAGEN, Mississauga, Ontario) for sequencing and transfections. Cells were transfected by the lipofectamine method (Invitrogen) according to the manufacturer’s protocol. Cells were plated into six-well cluster dishes (7 x 105 cells per well) and were transfected the following day with 3 µl of lipofectamine and 1 µg of DNA per well. The total amount of transfected DNA was equalized by adding empty vector. Bcl-XL promoter activity was analyzed with reporter constructs pGL2-Bcl-XL-luc containing the 848 bp upstream from the transcription start site (GenBank accession no. AH005819; kindly provided by Dr. T. Tsukahara, Shinshu University, Japan) (36). To normalize for transfection efficiency variation within and between experiments, 20 ng/well of pSV-ß-galactoside control vector (Promega, Madison, WI) was included with each transfection. Cells were lysed in buffer containing 25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, 1% Triton X-100, and 1 mM dithiothreitol. Luciferase activity was measured for 20 sec in a luminometer. Transfection efficiency was monitored by simultaneous cotransfection with a ß-galactosidase control expression plasmid cytomegalovirus-ß-galactosidase (20 ng/well). Promoter activity of each construct was expressed as firefly luciferase/ß-galactosidase activity. Each experiment was independently performed on three separate occasions with triplicate wells in each experiment. For assessment of chromatin acetylation, transfected cells were treated with TSA (0.1–0.3 µM) for 16 h. For assessment of impact of methylation, cells were treated with 5 or 10 µM of the methylation inhibitor 5'-azacytidine (Sigma) as previously described (37). Each experiment was independently performed on three separate occasions with triplicate wells for at least three independent clones in each experiment.
ChIP Assays
Cells stably expressing Ik1, Ik6, or their empty control vector (pcDNA3.1) were used as indicated. The ChIP assay was performed in accordance with the manufacturer’s recommendations (Upstate Biotechnology, Lake Placid, NY) and as previously described (12). The effect of Ik was examined on the endogenous Bcl-XL and p16 promoters as indicated. Briefly, histone was cross-linked to DNA by the direct addition of 37% formaldehyde in cells, and cells were washed with cold PBS containing protease inhibitors before lysing cells, and the lysates were sonicated to shear DNA lengths between 200 and 1000 bp. After centrifugation, cell suspensions were further diluted, and 20 µl of lysate from each sample was kept and used to quantitate 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. Half of the cleared lysate was incubated with either acetylated histone 3 (AcH3) or methylated histone 3 (MetH3) antibodies (both from Upstate Biotechnology) and protein G-agarose beads overnight at 4 C with agitation, and the other non-antibody-immunoprecipitated (IP) protein was used as a negative control, both of which were examined by PCR. 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 conditions for amplification of Bcl-XL were designed to amplify a 400-bp region of the –848 proximal promoter as follows: 95 C for 4 min followed by 35 cycles of 95 C 40 sec, 55 C 40 sec, and 72 C 1 min, and finally 72 C for 7 min using the following primers: sense, 5'-GGAAGTCCCTTTAGGGTTTC-3'; antisense, 5'-GATC CTGGAAGAGAATCGCT-3'. A second round of PCR using the same Bcl-XL primers was required for detection of a product after IP with the MetH3 antibody. For p16 promoter amplification, the following primers were used: sense, 5'- GAATCCTAGCACTGATACAGC-3'; and antisense, 5'- ATGCCTGGTCACCCTTTGAC-3', where the PCR conditions were the same other than the annealing temperature, which was 53 C. This region corresponds to nucleotides 99- 472 of the mouse genomic sequence (GenBank accession no. U47018).
Bisulfite DNA Sequencing
DNA samples isolated from AtT20 cells transfected with pcDNA3.1, Ik1, or Ik6 treated or untreated with 5'-azacytidine were bisulfite modified according to the manufacturer’s protocol (Chemicon International, Temecula, CA). Nested PCR was performed to cover 20 CpG dinucleotide sites in the Bcl-XL promoter (see also Fig. 5C) using the following approach. The initial forward primer extended from position –863: 5'-TTTGAGATTTATTTGGAAGTTTTTTTAGG-3'; a nested forward primer from position –827 was 5'-AAGTTTTATTTAGGGTTGGTATTTAAATA-3'. The reverse primer extended from position –384: 5'-ACCACCACCTACATTCAAATCTATCTC-3'. Products of the predicted size (453 bp) were isolated for cloning into the TA vector (Invitrogen). At least five clones were DNA purified and sequenced using an automated sequencer.
Statistical Analysis
Data are presented as mean ± SE. Differences were assessed by Student’s paired t test. Significance level was assigned at P 
0.05.
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FOOTNOTES |
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DISCLOSURE STATEMENT: The authors have nothing to disclose.
First Published Online July 27, 2006
Abbreviations: AcH3, Acetylated histone 3; CDK, cyclin-dependent kinase; CDKI, CDK inhibitor; ChIP, chromatin immunoprecipitation; FGF, fibroblast growth factor; FGFR, FGF receptor; HDAC, histone deacetylase complex; Ik, Ikaros; IP, immunoprecipitated; MetH3, methylated histone 3; PARP, poly(ADP-ribose) polymerase; ptd-FGFR4, pituitary tumor-derived N-terminally truncated isoform of FGFR4; Rb, retinoblastoma; SCID, severe combined immunodeficient; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling; siRNA, small interfering RNA; TSA, trichostatin-A.
Received for publication June 28, 2006. Accepted for publication July 14, 2006.
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B in apoptosis-resistant T-cell transfectants with Tax. J Virol 73:7981–7987This article has been cited by other articles:
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  S. Ezzat and S. L Asa The emerging role of the Ikaros stem cell factor in the neuroendocrine system J. Mol. Endocrinol., August 1, 2008; 41(2): 45 - 51. [Abstract] [Full Text] [PDF]
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 S. Loeper, S. L. Asa, and S. Ezzat Ikaros Modulates Cholesterol Uptake: A Link between Tumor Suppression and Differentiation Cancer Res., May 15, 2008; 68(10): 3715 - 3723. [Abstract] [Full Text] [PDF]
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 X. Zhu, S. L. Asa, and S. Ezzat Ikaros Is Regulated through Multiple Histone Modifications and Deoxyribonucleic Acid Methylation in the Pituitary Mol. Endocrinol., May 1, 2007; 21(5): 1205 - 1215. [Abstract] [Full Text] [PDF]
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 X. Zhu, K. Lee, S. L. Asa, and S. Ezzat Epigenetic Silencing through DNA and Histone Methylation of Fibroblast Growth Factor Receptor 2 in Neoplastic Pituitary Cells Am. J. Pathol., May 1, 2007; 170(5): 1618 - 1628. [Abstract] [Full Text] [PDF]
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