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The Journal of Immunology, 2003, 170: 218-227.
Copyright © 2003 by The American Association of Immunologists

Regulation of Mouse Mammary Tumor Virus env Transcriptional Activator Initiated Mammary Tumor Virus Superantigen Transcripts in Lymphomas of SJL/J Mice: Role of Ikaros, Demethylation, and Chromatin Structural Change in the Transcriptional Activation of Mammary Tumor Virus Superantigen1 ,2

Rajan M. Thomas, Kamran Haleem, Abu B. Siddique, William J. Simmons, Namita Sen, Da-Jun Zhang and Vincent K. Tsiagbe3

Department of Pathology and Comprehensive Kaplan Cancer Center, New York University School of Medicine, New York, NY 10016


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mammary tumor virus (Mtv29)-encoded superantigen expressed by SJL/J mouse B cell lymphomas stimulates CD4+V{beta}16+ T cells and thereby acquires T cell help necessary for lymphoma growth. Mtv29 mouse mammary tumor virus env transcriptional activator (META) env-controlled Mtv29 superantigen (vSAg29) mRNA transcripts (1.8 kb) are not expressed in normal B or other somatic cells. Real-time PCR-based assays with DNA from normal SJL liver and vSAg29- lymphoma (cNJ101), digested with methylation-sensitive enzymes, showed hypermethylation at AvaI, FspI, HpaII, ThaI, and the distal HgaI sites of the META env, but vSAg29+ lymphoma cells showed significant demethylation at AvaI, HpaII, and the distal HgaI sites. The distal HgaI site that is adjacent to an Ikaros binding site is significantly demethylated in the META env DNA from primary lymphomas. Gel shift assays showed binding of Ikaros to a sequence representing this region in the META env. SJL lymphomas expressed the Ikaros isoform Ik6 that was absent in normal B cells. vSAg29+ cells exhibited increased DNaseI accessibility to chromatin at the vSAg29 initiation site. Treatment of cNJ101 cells with a demethylating agent, 5-azacytidine, and a histone deacetylase inhibitor, trichostatin A, caused hypomethylation at AvaI, HpaII, and distal HgaI sites and led to chromatin structural change at the vSAg29 initiation site, accompanied by the expression of vSAg29 transcripts. This enabled cNJ101 cells to stimulate SJL lymphoma-responsive CD4+V{beta}16+ T hybridoma cells. Thus, demethylation at the distal HgaI site of the Mtv29 META env permits vSAg29 expression, which may have an impact on the development of germinal center-derived B cell lymphomas of SJL/J mice.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mammary tumor virus (Mtv29),4 is an endogenous retrovirus integrated in the genome of the SJL/J mouse strain, and it encodes Mtv29 superantigen (vSAg29) in its 3' long terminal repeat (LTR). vSAg29 is implicated in the pathogenesis of germinal center (GC)-derived B cell lymphomas, also called reticulum cell sarcoma (RCS), which spontaneously arise in >90% of SJL/J mice by the age of 12 mo (1, 2). In previous studies, we have shown that stimulation of CD4+V{beta}16+ T cells by vSAg29 is required for the growth of GC-derived lymphomas (3). The activated T cells secrete cytokines, such as IL-4 and IL-5, that promote lymphoma growth. In SJL lymphomas, the Mtv29 provirus shows the expression of a 1.8-kb vSAg29 mRNA transcript that is initiated from mouse mammary tumor virus (MMTV) env transcriptional activator (META) region located within the env gene (2, 4). Although META env-initiated 1.8-kb vSAg29 transcripts are overexpressed in SJL B cell lymphomas, they are not transcribed in normal B lymphocytes or in other somatic cells. We have observed, however, that META env-initiated 1.8-kb vSAg29 transcripts are expressed in Peyer’s patch B cells from 6-mo-old SJL mice (4).

Actively transcribing genes typically have a relaxed chromatin structure with enhanced accessibility to transcription factors. Inactivation (silencing) of genes is frequently due to methylation (5), which causes condensation of chromatin, due to interactions with methylcytosine binding proteins (6). Condensed and heterochromatinized gene loci are resistant to digestion with DNaseI (7). DNA methylation plays a major role in X chromosome inactivation (8) and imprinting (9, 10) and in the loss of expression of tumor suppressor and cell cycle-controlling genes in cancer cells (11, 12, 13, 14, 15). CpG islands in the promoter elements of many tumor suppressor genes are unmethylated in normal cells but hypermethylated in many different types of cancer cells (16, 17).

Proviral DNA usually exists in a hypermethylated state (18, 19). This process can cause latency of viral infection (20) and is also the reason for a lack of expression of many integrated Mtvs (21, 22, 23), even when the Mtv-LTR is used as promoter for a transgene (24). Normal methylation patterns are frequently altered in cancer cells, and de novo demethylation of methylated retroviral elements is known to activate silenced viral genes and to play a role in carcinogenesis (25).

Ikaros is a lymphocyte-specific repressor protein, reported to be associated with silenced genes (26). It is a member of the zinc finger family of proteins and is expressed in several isoforms derived from splice variants of primary transcripts. Ikaros functions in the regulation of several genes expressed in lymphocytes. Dysregulation of Ikaros isoforms has been reported in human leukemias and lymphomas (27, 28). The Ikaros proteins, master regulators of B and T cell differentiation (29), act by associating with distinct histone deacetylase, causing chromosome remodeling and maintenance of repression in specific gene loci (26, 30). Therefore, a loss of repression may lead to a gradual de-repression of the essential genes that occur over a number of mitoses.

To the best of our knowledge, there are no reports on the regulation of META env-initiated Mtv29 transcripts in SJL GC-derived B cell lymphomas. In the present study, we have investigated the roles of Ikaros, promoter demethylation, and chromatin structural changes in the transcriptional activation of META env promoter in vSAg29-transcribing lymphoma cells. Our results suggest that the expression of META env-initiated vSAg29 transcript is associated with demethylation at a CpG site in the distal HgaI site located very close to the Ikaros binding site. The dominant negative effect produced by Ikaros isoform (Ik6) expression may contribute to chromatin remodeling in the META env region, leading to demethylation at the vSAg29 initiation region.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

The restriction endonucleases and methylation-sensitive restriction endonucleases used for the experiments were purchased from New England Biolabs (Beverly, MA). The enzyme ThaI was purchased from Life Technologies (Gaithersburg, MD). Fluorescent dye SYGR green was purchased from Molecular Probes (Eugene, OR). Rabbit polyclonal C-terminal anti-Ikaros Ab (cross-reactive with mouse and human Ikaros) was a gift from Dr. S. T. Smale (Howard Hughes Medical Institute, University of California School of Medicine, Los Angeles). Custom-made oligonucleotide primers were obtained from Gene Link (Thornwood, NY). All of the reagents used for the experiments were analytical grade and were purchased from Sigma-Aldrich (St. Louis, MO).

Mice

Female SJL/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The mice were bred and maintained in an animal care facility of New York University School of Medicine, and all animal experiments were performed in accordance with institutional guidelines and with approval from the Institutional Animal Care and Use Committee. The in vitro lymphoma cell lines, cNJ117 and cRCS-X, were originally derived from aging SJL mice. A vSAg29-negative lymphoma cell line, cNJ101 (IgM+, unlike any of the typical GC-derived lymphomas of SJL origin), was derived from a 2-year-old SJL mouse that had received chronic treatments with anti-CD4 mAb from ~5 mo of age (31).

Cell culture

SJL/J-derived B cell lymphoma cell lines (cRCS-X, cNJ117, and cNJ101) were grown in RCS medium (IMDM containing 10% FBS supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 8 µg/ml insulin, 1 mM oxaloacetic acid, 0.5 mM sodium pyruvate, and 0.05 mM 2-ME). These cell lines were cultured at standard cell culture conditions. vSAg29-responsive CD4+V{beta}16+ T cell hybridoma (1D1-E7) was derived in our laboratory (3).

B cell isolation from lymphoid tissues

Cell suspensions were separately prepared from pooled Peyer’s patches and from spleens of three SJL mice at 3, 6, and 9 mo of age. Primary lymphomas from mesenteric lymph nodes were excised from SJL mice and cells were resuspended in 1x Dulbecco’s PBS. B lymphocytes were positively selected using anti-CD45R (B220)-coated immunomagnetic microbeads (Miltenyi Biotec, Auburn, CA). Lymphoma cells were depleted of T cells by incubation with a mixture containing anti-CD8 mAb (HO2.2), anti-CD4 mAb (GK1.5), and rabbit complement. Purity of the isolated B cells, examined by flow cytometric analysis, was 94–98%.

DNA preparation

Total genomic DNA was extracted from B lymphoma cell lines, purified B cells of primary SJL/J lymphomas, normal splenic and Peyer’s patch B cells, and liver of 3-, 6-, and 9-mo-old SJL/J mice, using a genomic DNA isolation kit from Promega (Madison, WI), according to the protocol provided by the manufacturer.

Quantification of methylation by PCR analysis

To quantify the degree of methylation at META env of Mtv29, we used a PCR-based assay with DNA digested with methylation-sensitive restriction endonucleases. When enzyme-resistant DNA, with methylated sites, is amplified in the PCR, the demethylation at the respective sites leads to the digestion of the template, resulting in the absence of PCR product amplification or variable amounts of PCR product, depending on the degree of methylation at the restriction endonuclease sites. Because the genome of female SJL/J mice contains another MMTV strain, Mtv8, with close sequence homology to that of MTV29, the PCR method using Mtv29-specific primers enables selective determination of a methylation pattern in the Mtv29 META env region. DNA (0.5–1 µg) from the samples described above was digested with a 5- to 10-fold excess amount of methylation-sensitive restriction endonucleases (AvaI, FspI, HgaI, HpaII, and ThaI). To verify complete digestion of DNA, in parallel with samples, an equal amount of a control plasmid DNA containing these restriction endonuclease sites was also digested and analyzed on 1% agarose gel. The control plasmid DNA showed complete digestion with each of these enzymes. To check the methylation sensitivity of these enzymes, an internal control plasmid DNA carrying an insert from META env region lacking CpG methylation was mixed with some of the DNA samples and digested with these enzymes. After digestion, the target region from the plasmid DNA was amplified using M13 forward and reverse primers (Table IGo) specific for the plasmid templates. Using the same digested samples as template, the META env region was amplified separately using Mtv29 META env specific primers. Uncut DNA was used as control. In the mixed DNA samples, the plasmid DNA target was completely digested by these enzymes and could not be amplified by PCR.


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  		Table I. List of PCR primers and oligonucleotide sequences used for experiments

 
Semiquantitative and quantitative real-time PCRs were performed with undigested (control) and digested samples. The Mtv29 META env region was amplified using LL1 and RR1 primers (Table IGo). MRPA and MRP2 primers (Table IGo) amplified the META env region containing the distal HgaI site. PCR was performed with a thermal cycler-480 (Perkin-Elmer, Foster City, CA) under the following conditions: 5 min initial denaturation at 94°C, followed by 25 cycles of 45 s of denaturation at 94°C, annealing for 1 min at 60°C, and extension for 30 s at 72°C, with a final extension step for 10 min at 72°C. The PCR products were analyzed through a 1.8% agarose gel. The intensity of the bands was determined by densitometric scanning of ethidium bromide-stained gels. The percentage of PCR products in the enzyme-digested sample was calculated in relation to undigested DNA (used as control).

Quantitative real-time PCR with SYBR green was performed for both control and digested samples using an iCycler iQ (Bio-Rad, Hercules, CA) for 40 cycles with the same conditions as described above, without a final extension step of 10 min. Electrophoresis revealed a single discrete product of the expected size. Serial dilutions of undigested DNA were used as PCR standards. The amount of intact DNA template present in the enzyme-digested samples and uncut DNA was derived from the standard plot generated with threshold cycles vs log concentrations, using a quantitation software program available with the iCycler. The percentage of enzyme-resistant DNA present in each digested sample was calculated in relation to the amount of input DNA, quantitated for undigested (control) sample.

Southern blot analysis

Southern blot analysis was performed to determine the methylation pattern in normal SJL liver and lymphoma cell DNA at AvaI and FspI sites of META env. Total genomic DNA (10 µg) from cRCS-X, cNJ117, cNJ101, and 3-mo-old SJL/J liver was double digested with EcoRI and one of the methylation-sensitive endonucleases (AvaI or FspI). EcoRI does not have a restriction site within the target META env region. After digestion, the DNA was separated through 1% agarose gel and blotted onto a nylon membrane. The membrane was hybridized with a 32P random-primed Mtv29 META env probe in a hybridization buffer (ULTRAhyb; Ambion, Woodward Austin, TX), following the manufacturer’s instructions. The filter was washed two times in wash buffer (2x SSC + 0.1% SDS) at 42°C for 5 min, followed by another two washes with buffer (0.1x SSC + 0.1% SDS) at 42°C for 15 min. The filter was exposed to x-ray film at -70°C.

Sodium bisulfite conversion and methylation-sensitive single nucleotide primer extension (Ms-SNuPE) analysis

Ms-SNuPE analysis using PCR product from sodium bisulfite-converted DNA was used to determine the degree of methylation at CpG sites located outside the methylation-sensitive enzyme sites. DNA (2 µg) was digested with EcoRI. NspI, an enzyme that digest the Mtv8 META env region, was also used to exclude Mtv8 in the analysis. Digested samples were purified by phenol-chloroform extraction and resuspended in 20 µl of Tris EDTA buffer. Sodium bisulfite conversion of cytosine to uracil was performed as described (32), with a slight modification as described (33). To amplify bisulfite-converted DNA, PCR primers were designed as described (34). Nested primer PCR was performed to amplify the top strand (5'–3') of the Mtv29 META env region using a first round of PCR with MP1 and MP2 primers (Table IGo). The thermal cycling was performed as follows: initial denaturation at 94°C for 5 min, 35 cycles of denaturation at 94°C for 45 s, annealing at 56°C for 1 min, extension at 72°C for 30 s, and a final extension at 72°C for 10 min. The PCR products derived from above reaction were used as template for the second round of PCR with PR1 and PR2 primers (Table IGo) and the PCR done for 35 cycles at the same conditions, except for annealing at 63°C for 1 min. The PCR products of expected size were gel purified using the QUI quick gel extraction kit (Qiagen, Valencia, CA) and used as a template for Ms-SNuPE. Some purified PCR products were cloned into pCR2.1 TOPO TA cloning vector (Invitrogen, Carlsbad, CA) and sequenced.

Degree of methylation at CpG sites located at positions 6897, 6962, and 7153 was analyzed by quantitative Ms-SNuPE analysis (35). Multiplex Ms-SNuPE analysis was performed for three CpG sites of the top strand of the Mtv29 META env region using PCR products as template that were generated by PCR with bisulfite-converted DNA. Internal primers (A, B, and C in Table IGo) were designed to analyze the methylation of CpG sites located at positions 6897, 6962, and 7153, respectively. During the reaction, the 3' terminus of oligonucleotide primer is extended by incorporation of a single [32P]dTTP (representing unmethylation) or [32P]dCTP (representing methylation) by Taq polymerase (based on the methylation of the cytosine residue at CpG sites). The reaction was performed in a 25-µl mixture containing 50 ng of gel-purified template (PCR product) and incubated in a final concentration of 1x PCR buffer, 1 µM of each primer, 1 µCi of either [32P]dCTP or [32P]dTTP, and 1 U of Taq polymerase. Conditions for primer extensions were as follows: 95°C for 1 min, 50°C for 2 min, and 72°C for 1 min. Samples were then analyzed through 12% denaturing poylacrylamide gel containing 7 M urea, and incorporated radioactivity was quantified by phosphor imaging.

Drug treatment

To examine the effects of a demethylating agent, 5-azacytidine (5-Aza), which is an inhibitor of DNA methyltransferase, and a histone deacetylase inhibitor, trichostatin A (TSA), on a vSAg29 nontranscribing lymphoma, cNJ101 cells (5 x 106/flask) were treated with different amounts of these drugs separately and in combination. The cells were grown in RCS medium in the presence and absence of these drugs for 48–72 h. In a separate set of flasks, cNJ101 cells were treated with 20 nM TSA first and then grown in RCS medium for 24 h, followed by addition of different amounts of 5-Aza and incubated for 48 h at 37°C in a cell culture incubator. The cells were harvested at different intervals and washed three times with 1x DPBS. Total RNA was extracted from drug-treated and control cells using RNA Stat-60 (Tel-Test, Friendswood, TX), followed by chloroform extraction and precipitation with isopropanol. DNA was also extracted from the aliquots of drug-treated cells as described above.

Northern blot analysis

Northern blotting was performed to examine whether drug treatment can induce the expression of the META-initiated 1.8-kb vSAg29 transcript in cNJ101 cells. Equal amounts (20 µg) of of total RNA isolated from control and drug-treated cells were electrophoresed under denaturing conditions through 1.2% agarose/formaldehyde gel, and then were transferred to nylon membrane and cross-linked by UV irradiation. The LTR-open reading frame and Mtv29 META env probes were generated as previously described (2). The membrane was prehybridized for 1 h at 42°C in ULTRAhyb buffer and hybridized overnight with random primed 32P-labeled LTR-open reading frame in ULTRAhyb buffer at 42°C. The membrane was washed as recommended by the manufacturer of the hybridization buffer. The washed filters were exposed to x-ray films. The membrane was stripped and rehybridized with 32P-labeled Mtv29 META env and GAPDH probes.

In vitro stimulation by vSAg29

The drug-treated and control cNJ101 cells were cocultured with vSAg29-responsive CD4+V{beta}16+ (ID1-E7) T hybridoma cells (105 cell/well) in flat-bottom 96-well plates (Costar, Cambridge, MA) using RPMI 1640 medium as described (3). Plate-bound anti-TCR {alpha}{beta} mAb (H57-597), anti-CD3 mAb (145-2C11), or PHA (5 µg/ml) was used as positive control stimulus. The cells were harvested after 24 h of culture, and supernatants from replicate cultures were separated and stored at -20°C. IL-2 production was determined by ELISA using mouse rIL-2 standard (Minikit KM-IL-2; Endogen, Woburn, MA).

RT-PCR

RT-PCR was performed to examine the expression of mRNA transcripts of Ikaros isoforms. cDNA was synthesized from 5 µg of RNA (pretreated with RNase-free DNaseI) using Superscript II Reverse Transcriptase and oligo(dT)12–18 primers (Invitrogen). The Ikaros primers used are indicated in Table IGo. PCR conditions were the same as described before for semiquantitative PCR, except for amplification with 40 cycles.

Western blot analysis

Western blotting was performed to analyze the Ikaros isoforms expressed in cytoplasmic and nuclear extracts of normal and lymphoma B cells. Cytoplasmic and nuclear extracts from different cell samples were prepared using a kit (Pierce, Rockford, IL) following manufacturer’s directions. A mixture containing protease inhibitors (Halt; Pierce) was added during sample preparation to avert protein degradation. Ikaros isoforms were detected using a polyclonal anti-Ikaros Ab (36), which was reactive with all Ikaros isoforms. Nuclear extracts (25 µg) were size fractionated on SDS-PAGE and transferred to nitrocellulose membrane by electroblotting. The membrane was blocked (5% dry milk powder and 0.1% Tween 20 in PBS) for 1 h at room temperature. The membrane was then incubated with appropriate dilution of anti-Ikaros primary Ab (in blocking buffer) in a sealed plastic bag for 1 h at room temperature. After this, the membrane was washed for 10 min three times with wash buffer (0.1% Tween 20 in 1x PBS). It was then incubated with appropriate dilution of peroxidase-conjugated anti-rabbit IgG (Amersham Pharmacia Biotech, Piscataway, NJ) for 1 h. The membrane was washed for 10 min three times with wash buffer and then developed using ECL (Amersham Pharmacia Biotech). After stripping, the membrane was also immunoblotted for the expression of CDK2 (control) using rabbit anti-mouse cyclin-dependent kinase 2 (CDK2) followed by peroxidase-conjugated anti-rabbit IgG.

Nuclei isolation and analysis of DNaseI accessibility to chromatin

To determine DNaseI accessibility to chromatin at a locus containing the vSAg29 initiation site, cell nuclei were isolated (37). Aliquots of isolated nuclei were incubated with 15 U of DNaseI (Promega) for 3 min at 25°C, and reaction was terminated by the addition of stop buffer. DNA was extracted from control and DNaseI-treated nuclei by phenol-chloroform extraction. The accessibility of DNaseI to a region containing the vSAg29 initiation site in the Mtv29 META env region was determined by quantitative real-time PCR using MRP1 and MRP2 primers (Table IGo) following the same conditions as described previously.

EMSA

EMSA was performed to examine the ability of Ikaros to bind to an oligonucleotide representing the sequence between positions 7267 and 7297 in the META env, which contains an Ikaros binding site adjacent to the distal HgaI site. Oligonucleotide sequences (ODN 1, 2, 3, and 4) used for the EMSA are shown in Table IGo. The duplex oligonucleotides with or without a point mutation at the Ikaros binding site were 5' end-labeled with [32P{gamma}]ATP using T4 polynucleotide kinase. The labeled probe was purified using Chromaspin 10 columns (Clontech Laboratories, Palo Alto, CA). The probe was incubated with 15 µg of nuclear or cytoplasmic extract in a binding buffer (10 mM Tris-HCl, 1 mM DTT, 75 mM KCl, 10% glycerol, 100 µM spermine, and 1 µg of sheared salmon sperm DNA) and incubated at 25°C for 30 min in a final reaction volume of 20 µl. Ab supershift was performed with incubation of probe with nuclear and cytoplasmic extract preincubated with rabbit anti-Ikaros Ab for 30 min at 25°C. The reaction products were analyzed through 6% nondenaturing polyacrylamide gel in 1x Tris-borate-EDTA buffer for 3 h at 200 V and 4°C. The bands were detected by phosphor imaging (Bio-Rad).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Methylation profile of the Mtv29 META env region

In SJL/J RCS lymphomas, 1.8-kb Mtv29 vSAg29 mRNA transcripts are initiated from the META region of the env gene, as previously characterized (4). Fig. 1Go shows the schematic representation of the Mtv29 META env region. To determine the degree of methylation at the META env region in vSAg29-negative normal cells and vSAg29-positive lymphoma cells, we digested the DNA with methylation-sensitive restriction endonucleases. Methylation at AvaI, FspI, HgaI, HpaII, and ThaI sites (Fig. 1Go) of the Mtv29 META env region was quantified by real-time PCR using Mtv29-specific primers. The degree of methylation at these sites in both normal and lymphoma cells is shown in Fig. 2Go. The normal SJL liver DNA from 3-, 6-, and 9-mo-old mice exhibited hypermethylation at all analyzed sites (Fig. 2GoA). DNA from splenic B lymphocytes of 3-, 6-, and 9-mo-old normal SJL/J mice showed hypermethylation at the distal HgaI site. In contrast, DNA from Peyer’s patch B cells of the 6- and 9-mo-old mice showed a significant decrease in methylation at the distal HgaI site (Fig. 2GoB).



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  		FIGURE 1. Schematic representation of Mtv29 META env region. CpG sites (indicated by filled circles and numbered) present between position 6596 and 7358. The analyzed CpG sites within the methylation-sensitive restriction sites (AvaI, FspI, HgaI, HpaII, and ThaI) and CpGs (outside the restriction endonuclease site) at positions 6897, 6962, and 7153 (primers A, B, and C, respectively) are also indicated. The META D+ enhancer element, Mtv29 transcription start site, and positions of primers are also indicated. Base numbering corresponds to the Mtv29 env sequence that we previously reported (2 ).

 


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  		FIGURE 2. Methylation profile of Mtv29 META env region, quantified by real-time PCR-based SYBR green assay. DNA was digested with AvaI, FspI, HgaI, HpaII, and ThaI and was used as template in PCR assays. PCR was performed for each sample in triplicate. The percentage of enzyme-resistant target site, amplified in PCR, is calculated in relation to undigested (control) DNA. Student’s t test was used to calculate significance of difference between means. A, Degree of methylation quantified for Mtv29 META liver DNA from 3-, 6-, and 9-mo-old normal SJL/J mice. Values are the means ± SEM of percent methylation at each restriction enzyme site, calculated for three mice. B, Quantification of degree of methylation at the distal HgaI site of Mtv29 META of DNA from splenic and Peyer’s patch B220+ B cells, from 3-, 6-, and 9-mo-old normal SJL mice. Values are the means ± SEM of percent methylation for three separate digestions. *, p < 0.05, compared with 3-mo-old SJL Peyer’s patch DNA; **, p < 0.01, comparison between 3-mo-old and 9-mo-old SJL Peyer’s patch B cell DNA. C, Comparison of degree of methylation at the indicated methylation sites, determined with real-time PCR and semiquantitative densitometric analysis. DNA was extracted from B cells of primary lymphomas obtained from six SJL/J mice. The digested DNA was quantified for degree of methylation at the indicated sites. Enzyme digestion was done in triplicate and each sample was PCR amplified in triplicate. The values are means ± SEM of percent methylation at each site in Mtv29 META env. ***, p < 0.001, compared with other analyzed sites in primary lymphoma DNA; a, p < 0.01, compared with distal HgaI site in Mtv29 META env of DNA from cNJ101 and from 9-mo-old normal SJL liver. D, Real-time PCR quantification of degree of methylation at the indicated sites in Mtv29 META env of DNA from vSAg29+ lymphoma cell lines (cRCS-X and cNJ117) and a vSAg29- lymphoma cell line (cNJ101). Values are the means ± SEM of percent methylation at each restriction enzyme site, calculated for three separate DNA digestions. Note the significant level of demethylation in the AvaI, FspI, HgaI, and HpaII sites of vSAg29+ SJL lymphomas (cNJ117 and cRCS-X), compared with vSAg29-negative cNJ101 DNA (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

 
The semiquantitative densitometric analysis of PCR product amplified from methylation-sensitive enzyme-digested samples showed a pattern comparable to that generated for these sites using real-time PCR. Fig. 2GoC shows the comparison of both quantitative and semiquantitative PCR methods used for the analysis of methylation at the META env region of DNA from primary lymphomas. The results showed a significant level of demethylation at the distal HgaI site (Fig. 2GoC), whereas methylation at the AvaI, FspI, HpaII, and ThaI sites was not significantly different. DNA from RCS lymphoma cells showed heterogeneity in the methylation pattern in the META env region (Fig. 2GoD). A unique vSAg29-negative and T cell-independent SJL lymphoma cell line, cNJ101, showed hypermethylation at all of the analyzed sites in the META env region, but the vSAg29-transcribing cell lines, cNJ117 and cRCS-X, showed a significant level of demethylation at AvaI, HgaI, and HpaII sites. Whereas cNJ117 showed hypomethylation at the FspI site, cRCS-X showed no significant change in methylation at the same site. The ThaI site in the META env region showed hypermethylation in all lymphoma cell lines. Southern blot analysis was also used to examine the methylation pattern at AvaI and FspI sites of the Mtv29 META env region (Fig. 3Go). cNJ101, which does not transcribe the vSAg29 mRNA, exhibited more methylation at FspI and AvaI sites in the META env region than did the vSAg29-transcribing SJL lymphomas (cNJ117 and cRCS-X). Liver DNA from 3-mo-old SJL mice showed hypermethylation at these sites, a result consistent with data obtained by PCR analysis.



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  		FIGURE 3. Southern blot of SJL lymphoma and liver DNA, after double digestion with EcoRI (no restriction site within the target) and with methylation-sensitive restriction endonuclease (AvaI or FspI), probed with META env of Mtv29.

 
We also examined the degree of methylation at three CpG sites (positions 6897, 6962, and 7153) by multiplex Ms-SNuPE analysis (Fig. 4Go). Heterogeneity in the methylation pattern was revealed, but methylation was seen at these sites in all of the analyzed samples of both liver and lymphomas, indicating that these sites may not influence vSAg29 transcription.



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  		FIGURE 4. Multiplex Ms-SNuPE analysis for the quantitation of degree of methylation at three CpG sites located outside the methylation-sensitive restriction enzyme sites. PCR product derived from the sodium bisulfite-converted DNA from cRCS-X, normal SJL liver, and primary lymphomas (PL), PL-1 and PL-2 was used as template for Ms-SNuPE. Internal primers were as follows: A, B, and C for CpG sites at positions 6897, 6962, and 7153, respectively, extended with [32P]dCTP (representing methylation) or [32P]dTTP (indicating unmethylation) were analyzed by 12% denaturing polyacrylamide/urea gel. M, Methylation; U, unmethylation. The intensities of the C and T bands were quantified by phosphor imaging. C:T signal ratio was calculated as described (35 ).

 
Effects of 5-Aza and TSA treatments on cNJ101 cells

We subsequently analyzed the effects of demethylating agent 5-Aza (5 µM) and histone deacetylase inhibitor TSA (20 nM) on cNJ101 cells (the atypical SJL/J-derived lymphoma cell line that neither transcribes META env-initiated 1.8-kb vSAg29 transcripts nor stimulates V{beta}16+ T hybridoma cells). The changes in the degree of methylation at AvaI, FspI, HgaI, HpaII, and ThaI sites induced by drug treatment were quantified by real-time PCR. The results shown in Fig. 5Go demonstrate that cNJ101 cells treated with 5-Aza and TSA showed a significant level of demethylation at AvaI, HgaI, and HpaII sites. The FspI site and ThaI sites did not show any significant changes in methylation. These drugs showed similar effects, irrespective of their use alone or in combination.



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  		FIGURE 5. Methylation profile of Mtv29 META env region in DNA from cNJ101, after treatment with 5-Aza (5 µM) and TSA (20 nM) for 72 h. DNA extracted from drug-treated cells was digested and the degree of methylation at the indicated sites was quantified by real-time PCR. The assay was performed in triplicate. Methylation profile of untreated cNJ101 DNA is shown in Fig. 2GoD. The values are means ± SEM for three treatments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; comparison between DNA from untreated (control) cNJ101 and drug-treated cNJ101 cells.

 
Next, we performed Northern blot analysis to examine whether 5-Aza and TSA treatment can induce the expression of META env-initiated 1.8-kb vSAg transcripts in cNJ101. MMTV-LTR-probed RNA from cells treated with both 5-Aza (5 µM) and TSA (20 nM) for 72 h exhibited 1.8-kb vSAg transcripts (Fig. 6GoA). The cells treated with 5-Aza alone also expressed 1.8-kb transcripts; however, in combination, the drugs were much more effective in inducing the cells to express vSAg29 transcripts. The cells treated with 5-Aza alone or in combination with TSA also up-regulated 2.9- and 4.1-kb env transcripts in a dose-dependent manner (Fig. 6Go, A and C). TSA treatment alone also caused the expression of 1.8-kb transcripts in cNJ101. The 5-Aza-induced expression of 1.8-kb vSAg29 transcripts was remarkably increased after 48 h of treatment, when this drug was added to the cNJ101 cells pretreated with 20 nM TSA alone for 24 h (Fig. 6GoB). The 1.8-kb vSAg29 transcripts in drug-treated cNJ101 cells also hybridized with a META env probe specific for the vSAg29 initiation site (Fig. 6GoC). Removal of the drugs from the cell culture medium caused loss of drug-induced META env-initiated 1.8-kb vSAg29 expression in cNJ101 cells after 48 h of recovery (data not shown).



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  		FIGURE 6. Effect of drugs (5-Aza and TSA) on a vSAg29-negative lymphoma cell line (cNJ101), analyzed by Northern blot hybridization of RNA from drug-treated cNJ101 cells. A, cNJ101 cells treated with 5-Aza alone or together with TSA for 72 h at 37°C. RNA extracted from drug-treated cells was probed with Mtv29-LTR for the expression of 1.8-kb vSAg29 mRNA transcripts. The transcripts (2.9 and 4.1 kb) represent env. Expression of housekeeping gene GAPDH is shown at the bottom. B, cNJ101 cells were exposed to 20 nM TSA alone for 24 h and then to different amounts (100–5000 nM) of 5-Aza for 48 h. RNA extracted from treated cells was probed with Mtv29-LTR for the expression of 1.8-kb vSAg29 transcripts. C, Mtv29 META env-initiated 1.8-kb vSAg29 transcripts in drug-treated cells. The blot shown in B was stripped and then hybridized with Mtv29 META env probe (7136–7339). D, cNJ101 cells exposed to 5 µM 5-Aza and 20 nM TSA for 72 h stimulate IL-2 production in RCS-responsive CD4+V{beta}16+ T hybridoma cells. RCS stimulation is the positive control.

 
To examine whether vSAg29 induced by drug treatment can stimulate CD4+V{beta}16+ T cell hybridoma cells, we cocultured vSAg29-responsive T hybridoma cells with cNJ101 cells pretreated with 20 nM TSA and 2.5 µM 5-Aza for 72 h and measured the IL-2 secreted by the responding hybridoma cells (Fig. 6GoD). Whereas the cells treated with 5-Aza and TSA (in combination) stimulated V{beta}16+ T hybridoma cells, untreated cells did not. cNJ101 cells treated with 5-Aza or TSA alone did not induce any detectable levels of IL-2 production.

DNaseI accessibility to chromatin

Nuclei prepared from vSAg29-transcribing (cRCS-X and cNJ117) and nontranscribing (cNJ101) cells, incubated with DNaseI, were examined for DNaseI accessibility to a region in META env containing the initiation site for 1.8-kb vSAg29 mRNA by quantitative PCR using the DNA isolated from the DNaseI-treated nuclei. DNaseI treatment of nuclei isolated from normal SJL liver and splenic B cells, as well as from cNJ101 cells, resulted in partial digestion in the target DNA region, whereas the same site in vSAg29+ (cNJ117 and cRCS-X) nuclei showed significantly increased accessibility to DNaseI (Fig. 7Go). The ability of DNaseI to digest the chromatin at a locus containing the vSAg29 initiation site in META env DNA of cNJ101 cells was significantly increased after drug treatment.



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  		FIGURE 7. Real-time PCR quantification of accessibility of DNaseI to a region in META env containing vSAg29 transcription start site. A, Nuclei were isolated from normal SJL liver, splenic B cells, and lymphomas (cNJ117 and cRCS-X) and were incubated with DNaseI. DNA was extracted from control (undigested) nuclei and DNaseI-treated nuclei, and the amount of DNaseI-resistant target DNA was PCR amplified and quantified. The percentage of DNaseI accessibility was calculated in relation to DNA from control nuclei. PCR was done in triplicate. Values are the mean ± SEM of percent enzyme-resistant DNA determined for three separate digestions. Comparison is made between vSAg29- normal cells (SJL liver and splenic B cells) and vSAg29+ lymphoma (cNJ117 and cRCS-X) cells. ***, p < 0.001 (Student’s t test). B, Real-time PCR quantification of DNaseI accessibility to the above-mentioned chromatin site in cNJ101 cells, after drug treatment with 5-Aza (5 µM) and TSA (20 nM) for 72 h. ***, p < 0.001, compared with untreated cNJ101 cells.

 
Role of Ikraros

Analysis of the META env sequence by a promoter matrix analysis program (http://bimas.dcrt.nih.gov) revealed an Ikaros binding site close to the distal HgaI site. Because the distal HgaI site showed a significant level of demethylation in lymphomas, we performed EMSA to determine whether Ikaros, present in the nuclear and cytoplasmic extracts, could bind to an oligonucleotide sequence containing the distal HgaI and Ikaros binding sites of the META env promoter. EMSA results are shown in Fig. 8Go. EMSA showed two prominent closely migrating bands (Fig. 8Go, see arrows). Preincubation of nuclear and cytoplasmic extracts with anti-Ikaros Ab prevented binding of Ikaros to labeled probe, as revealed by a decrease in the intensity of bound complexes. Oligonucleotide containing a point mutation at the Ikaros binding site showed a 40–60% reduction (by densitometric analysis) in the binding efficiency of bound complexes in nuclear and cytoplasmic extracts (Fig. 8Go, A and B). The nuclear and cytoplasmic extracts from cNJ101 cells treated with 5-Aza and TSA showed ~50% reduction in the intensity of bound complexes with probe, as compared with binding in untreated cells. Cytoplasmic extracts from all analyzed samples contained relatively large amounts of Ikaros complexes, relative to nuclear extracts (Fig. 8GoB).



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  		FIGURE 8. EMSA showing binding of Ikaros from nuclear extracts to a labeled oligonucleotide duplex probe representing the sequence containing HgaI and the putative Ikaros binding site. Lane 1, Control cNJ101; lane 2, cNJ101 treated with 2.5 µM 5-Aza and 20 nM TSA for 72 h; lane 3, in vivo RCS; lane 4, cRCS-X; lanes 5 and 6, binding of probe with nuclear extract from cRCS-X, preincubated with anti-Ikaros Ab (1 and 2 µl, respectively); lane 7, normal SJL splenic B cells; lane 8, binding of probe with nuclear extract from SJL splenic B cells pretreated with 2 µl of anti-Ikaros Ab; lanes 9–12, binding of Ikaros from nuclear extract to a duplex probe with point mutation at Ikaros binding site (lane 9, splenic B cells; lane 10, cNJ101; lane 11, cRCS-X; lane 12, in vivo RCS-X). B, EMSA showing binding of Ikaros from cytoplasmic extract. Lane 1, Control cNJ101; lane 2, cNJ101 drug treated; lane 3, cRCS-X; lane 4, in vivo RCS-X; lane 5, normal SJL splenic B cells; lanes 6 and 7, binding of probe with cytoplasmic extract from cRCS-X and splenic B cells, respectively, preincubated with 2 µl of anti Ikaros Ab.

 
RT-PCR and Western blotting were used to examine the expression of Ikaros isoforms in normal and lymphoma cells. Fig. 9Go shows the Ikaros isoforms (Ik1, 2, 3, 4, and 6) detected in the analyzed samples at transcriptional and translational levels. Although RT-PCR results showed the message for a smaller size Ikaros isoform (Ik6) in lymphoma cells, normal B cells did not show expression of Ik6 (Fig. 9GoA). Western blot analysis gave similar results (Fig. 9GoB). Normal SJL B cells expressed larger isoforms (Ik1–4), but not Ik6. In vivo SJL lymphoma cells (RCS-X in vivo) expressed relatively more Ik6 protein than did in vitro SJL lymphoma cell lines. Cytoplasmic extracts from all cells analyzed contained Ik1, 2, 3, and 4 isoforms, but no detectable Ik6 (Fig. 9GoC).



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  		FIGURE 9. Expression of Ikaros isoforms in SJL lymphomas and normal splenic B cells. vSAg29- lymphoma (cNJ101) cells were treated with 20 nM TSA and 2.5 µM 5-Aza. A, RT-PCR showing the expression of mRNA transcripts of Ikaros isoforms. Expression of housekeeping gene (GAPDH) is shown at the bottom. B, Western blot analysis showing Ikaros isoforms. Nuclear proteins from control cNJ101, drug-treated cNJ101 cells, cRCS-X, RCS-X in vivo, and splenic B cells were analyzed with anti-Ikaros Ab. The blot was also analyzed with anti-CDK2 Ab, as a control for protein load. C, Western blotting of cytoplasmic extract from the above-mentioned samples (B) with anti-Ikaros Ab and anti-CDK2 (as control).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
B cell lymphomas of SJL/J mice depend for their growth on help from CD4+V{beta}16+ T cells, which they stimulate by expressing an Mtv29-encoded proviral superantigen (vSAg29) (1, 4). Unlike most other strains of MMTVs, which use promoter elements in the 5' LTR for superantigen transcription, the Mtv29 employs a promoter, META, in the env gene for the transcriptional initiation of mRNA encoding vSAg29 (2). After splicing of a 1.2-kb intronic sequence, the META env-initiated 1.8-kb mRNA transcripts contain a part of noncoding (92 base) sequence from the META env region and a coding sequence for vSAg29 transcribed from the 3' LTR. In the present investigation, we examined the factors responsible for the regulation of META env-initiated superantigen (1.8-kb) transcripts in SJL/J lymphomas. In view of the lack of META env-initiated vSAg29 transcription in normal B and other somatic cells, we first examined whether DNA methylation of CpG sites in the META env region plays any role in the regulation of vSAg29 transcription. For this purpose, we examined the Mtv29 META env methylation profile in superantigen-transcribing and nontranscribing cells. We determined the degree of methylation at CpG sites within the methylation-sensitive restriction endonuclease sites (AvaI, FspI, HgaI, HpaII, and ThaI) by quantitative real-time PCR and also by semiquantitative densitometric analysis of PCR products, using the enzyme-digested DNA as template in the PCR. Our results (Fig. 2Go, A and B) indicate hypermethylation at all sites analyzed in the META env region of DNA isolated from liver and splenic B lymphocytes of normal SJL/J mice. In primary SJL/J lymphoma cells, we detected a significant level of demethylation at the distal HgaI site, located in the vSAg29 transcription initiation region, but observed no significant change in methylation at any of the other sites examined. Analysis of methylation at CpG sites, not within methylation-sensitive enzyme sites, by Ms-SNuPE also showed more methylation at the three analyzed CpG sites in both normal and lymphoma cells. These results suggest that demethylation of the distal HgaI site may be important for vSAg29 transcription, whereas methylation at the proximal META env region may not directly influence the transcriptional activity of the META env promoter. It has been reported that EBV latency C promoter, methylated in tumor cell lines, requires demethylation at a single CpG site for transcriptional activation (38). DNA from SJL/J lymphoma cell lines (cRCS-X and cNJ117) showed demethylation at more CpG sites (AvaI, FspI, HgaI, and HpaII) than did primary lymphomas.

Demethylation at the distal HgaI site is of interest because this site is located in proximity to an Ikaros binding site. Our results on EMSA indicate the ability of Ikaros to bind to an oligonucleotide probe representing this region in the META env of Mtv29. The efficiency of binding was drastically reduced by introducing a point mutation at the Ikaros binding site and by pretreating nuclear and cytoplasmic extracts with an anti-Ikaros Ab. Ikaros is known to cause transcriptional repression by recruitment of histone deacetylase (26). A dominant mutation in the Ikaros gene is also known to cause rapid development of leukemia and lymphoma (39). In vSAg29-nontranscribing cells, the distal HgaI site is hypermethylated and may associate with histone deacetylase recruited by Ikaros. Thus, the interaction of Ikaros at this site may lead to a local condensed chromatin structure, which may prevent the transcription of vSAg29. Lymphomas showed expression of Ik6, which is not expressed in normal SJL splenic B cells and exerts a dominant negative effect on other Ikaros isoforms that have DNA binding domains (27, 28). Due to the lack of a DNA binding domain at the NH2-terminal region, Ik6 cannot bind DNA but can form homo- or heterodimers with larger isoforms, such as Ik1, 2, and 3, through COOH-terminal zinc fingers. The dimer formation of Ik6 with larger isoforms prevents the latter from binding to DNA, thereby resulting in loss of repression by Ikaros.

DNA from 6- and 9-mo-old normal SJL/J Peyer’s patch B cells showed a significant decrease in methylation at the distal HgaI site, which correlates with transcription of META env-initiated 1.8-kb vSAg29 transcript by these cells as shown previously (4). The results strengthen our previous observation (4) that GC-derived B cell lymphomas arise in Peyer’s patches, as was originally suggested by Siegler and Rich (40). The Peyer’s patch B cells, which are under chronic antigenic stimulation from the surrounding environment, may accelerate the demethylation as a result of rapid proliferative responses of these cells.

Even though CpG methylation is a property of several silenced genes, the recent view is that methylation is a secondary event targeted to genes that are already made silent by other mechanisms (41). DNA methylation can lead to the stable silencing of genes as a result of interaction of DNA binding proteins, leading to repression of gene expression by promoting condensation of chromatin. Methylated sites on DNA bind 5-methyl cytosine binding protein (42), which exists in a complex with Sin3A and histone deacetylase (43). This complex decreases the level of histone acetylation, resulting in a compact chromatin structure (44). The methylation of cytosine bases of DNA is catalyzed by DNA 5-methyl cytosine transferase (45). This process is reported to be reversible by the activity of DNA demethylase (46). A correlation has been reported between the expression of DNA demethylase and demethylation in the promoter region of the c-erb B2 gene and exon I of the survivin gene in ovarian cancers (47). It is likely that DNA in rapidly proliferating cells is more susceptible to demethylation. During DNA replication, the impairment in the activity of DNA methyl transferase1 (DNMT1) or its access to methylated sites might contribute to a progressive decrease in methylation.

The lymphoma cell line cNJ101, which does not express META env-initiated vSAg29, showed hypermethylation and exhibited methylation patterns comparable to that observed for the META env region in liver DNA. Because the META env methylation pattern in cNJ101 was significantly different from that of vSAg29 transcribing lymphomas, this cell line was used as a suitable in vitro cell line to study the role of methylation in vSAg29 transcription. Treatment of cNJ101 cells with TSA and 5-Aza caused transcriptional activation of META env promoter and the transcriptional initiation of 1.8-kb vSAg29. The methylation profile of the drug-treated cNJ101 cells was similar to that observed for cRCS-X and cNJ117. It is interesting to note that the superantigen that was induced by drug treatment stimulated T cell hybridoma cells bearing TCR V{beta}16. These results are consistent with that of our previous findings demonstrating the ability of vSAg29 to stimulate CD4+V{beta}16+ T cells (3).

In addition to methylation, the conformation of chromatin at the locus containing the vSAg29 initiation site appears to play a major role in the transcriptional activity of META promoter. The vSAg29-negative cells showed less accessibility of chromatin at this locus to DNaseI digestion when compared with a transcriptionally active locus in cNJ117 and cRCS-X cells. The cNJ101 cells treated with TSA and 5-Aza showed similar effects of increased accessibility to DNaseI digestion at the vSAg 29 initiation locus. It is interesting to note that TSA treatment alone was sufficient to induce the expression of 1.8-kb vSAg transcripts in cNJ101 cells. This result suggests that an open chromatin conformation can lead to simultaneous decrease in methylation at the crucial sites and activation of the promoter. Although TSA is not a demethylating agent, it enhances histone acetylation (48, 49) and chromatin remodeling (50), and these changes may be responsible for the observed decrease in methylation. TSA is known to cause selective loss of DNA methylation in Neuropsora (51). Histone deacetylation plays a role in the maintenance of viral latency, and histone acetylation at the promoter of the immediate early gene, BRLF1, of EBV allows the virus to express Rta and to activate the viral lytic cycle (52). Our results also suggest that inhibition of histone deacetylase activity by TSA can activate the META env promoter in cNJ101 cells.

Both control and drug-treated cNJ101 cells expressed Ik6, as did other Mtv29+ SJL/lymphoma cells analyzed. However, nuclear and cytoplasmic extracts from drug-treated cNJ101 cells revealed a prominent decrease in the ability of Ikaros complexes to bind to oligonucleotide probe (Fig. 8Go). It is not yet clear whether Ik6 has a direct role in the modulation of transcriptional activity of the META env promoter in cNJ101 cells. Demethylation at the distal HgaI site and chromatin structural changes at the vSAg29 initiation locus could be at least two major factors responsible for induction of vSAg29 META env transcripts by drug-treated cNJ101 cells.

5-Aza can cause genome-wide hypomethylation and is reported to activate and induce the expression of several silenced cellular genes (53, 54, 55). Inhibition of DNA methyltransferase by 5-aza-2'-deocycytidine (5-Aza-CdR), treatment induces the expression of genes transcriptionally down-regulated by de novo methylation in HT29 adenocarcinoma. Indeed, microarray expression analysis of 5-Aza-CdR-treated tumor cells showed induction of STAT1, 2, and 3, which are responsive to IFN-{alpha} signaling (54). 5-Aza and 5-Aza-CdR are cytosine analogs that substitute for cytosine during DNA replication and are recognized by DNMT1 on newly synthesized DNA, with covalent trapping of DNMT, leading to its sequestration. Depletion of cellular level of DNMT1 may be responsible for drug-induced DNA hypomethylation (56). It is not clear whether 5-Aza-induced expression of the META env-initiated 1.8-kb vSAg transcript in cNJ101 cells is due to a direct effect of this drug on META env promoter or if it is an indirect effect caused by activation of transcription factors or other genes that activate the silenced genes.

Our results suggest that META env-initiated vSAg29 transcription in lymphoma cells is correlated with dysregulation of Ikaros isoforms, local hypomethylation at the distal HgaI site of META env promoter, and an alteration in chromatin structure at the vSAg29 initiation site. Thus, demethylation and a relaxed chromatin structure at the vSAg29 initiation site are crucial factors responsible for the transcription of vSAg29, an effect that is required for the development of SJL lymphomas.


   Acknowledgments
 
We are most grateful to the late Dr. G. Jeanette Thorbecke for her insightful discussions and suggestions in the conduction of these studies. We are indebted to Dr. S. T. Smale (Howard Hughes Medical Institute, University of California School of Medicine, Los Angeles) for a generous gift of anti-Ikaros Ab. We thank Dr. N. M. Ponzio (University of Medicine and Dentistry of New Jersey, Newark) for cNJ101 cell line. We also thank the late Prof. G. M. Hochwald (Department of Neurology, New York University School of Medicine, New York) for proofreading the manuscript.


   Footnotes
 
1 This work was supported by U.S. Public Health Service Grants CA-14462 and CA-09665. R.M.T. was the recipient of stipend from U.S. Public Health Services Training Grant CA-09161. The National Science Foundation is thanked for its support of bioinformatics services provided by the New York University Research Computing Resource through Grant BIR-9318128. Back

2 This work is dedicated to the memory of Prof. G. J. Thorbecke, whose untimely death occurred on November 16, 2001. Her inspirations and memory will live forever. Back

3 Address correspondence and reprint requests to Dr. Vincent K. Tsiagbe, New York University School of Medicine, Department of Pathology, Room 538 Medical Science Building, 550 First Avenue, New York, NY 10016. E-mail address: tsiagv01{at}med.nyu.edu Back

4 Abbreviations used in this paper: Mtv29, mammary tumor virus 29; vSAg29, Mtv29 superantigen; LTR, long terminal repeat; GC, germinal center; RCS, reticulum cell sarcoma; MMTV, mouse mammary tumor virus; META, MMTV env transcription activator; Ms-SNuPE, methylation-sensitive single nucleotide primer extension; 5-Aza, 5-azacytidine; TSA, trichostatin A; DNMT1, DNA methyl transferase1; CDK2, cyclin-dependent kinase 2; 5-AzaCdR, 5-Aza-2'-deoxycytidine. Back

Received for publication May 21, 2002. Accepted for publication October 25, 2002.


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 Introduction
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 Results
 Discussion
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